Basic Principles of Pathology

1  Basic Principles of Pathology

The most important tool that the pathologist has at his/her disposal is meaningful communication with the patient’s clinician regarding the suspected diagnosis so that the pathologist can choose the appropriate strategy for processing whatever tissue or other samples are received. As will be seen in the discussion under Modern Molecular Pathology Diagnostic Techniques, there is a dizzying array of techniques at the pathologist’s disposal; however, it is only through communication with the clinician that the pathologist can determine which of these techniques to utilize to best serve the patient.

INFLAMMATION Definition I. Inflammation is the response of a tissue or tissues to a noxious stimulus. A. The tissue may be predominantly cellular (e.g., retina), composed mainly of extracellular materials (e.g., cornea), or a mixture of both (e.g., uvea). B. The response may be localized or generalized, and the noxious stimulus may be infectious or noninfectious. II. In a general way, inflammation is a response to a foreign stimulus that may involve specific (immunologic) or nonspecific reactions. Immune reactions arise in response to specific antigens, but they may involve other components (e.g., antibodies, T cells) or nonspecific components (e.g., natural killer [NK] cells, lymphokines). III. There is an interplay between components of the inflammatory process and blood clotting factors that shapes the inflammatory process.

Causes I. Noninfectious causes A. Exogenous causes: originate outside the eye and body, and include local ocular physical injury (e.g., perforating trauma), chemical injuries (e.g., alkali), or allergic reactions to external antigens (e.g., conjunctivitis secondary to pollen). B. Endogenous causes: sources originating in the eye and body, such as inflammation secondary to cellular immunity (phacoanaphylactic endophthalmitis [phacoantigenic uveitis]); spread from continuous structures (e.g., the sinuses); hematogenous spread (e.g., foreign particles); and conditions of unknown cause (e.g., sarcoidosis).

II. Infectious causes include viral, rickettsial, bacterial, fungal, and parasitic agents.

Phases of Inflammation (Table 1.1 lists the actions of the principal mediators of inflammation.) I. Acute (immediate or shock) phase (Fig. 1.1) A. Five cardinal signs: (1) redness (rubor) and (2) heat (calor)—both caused by increased rate and volume of blood flow; (3) mass (tumor)—caused by exudation of fluid (edema) and cells; (4) pain (dolor) and (5) loss of function (functio laesa)—both caused by outpouring of fluid and irritating chemicals. Table 1.2 lists the roles of various mediators in the different inflammatory reactions. B. The acute phase is related to histamine release from mast cells and factors released from plasma (kinin, complement, and clotting systems). 1. Histamine is found in the granules of mast cells, where it is bound to a heparin–protein complex. Serotonin (5-hydroxytryptamine), found in platelets and some neuroendocrine cells, has a similar effect to histamine. 2. The kinins are peptides formed by the enzymatic action of kallikrein on the α2-globulin kininogen. Kallikrein is activated by factor XIIa, which is the active form of the coagulation factor XII (Hageman factor). Factor XIIa converts plasma prekallikrein into kallikrein. Plasmin also can activate Hageman factor. 3. Plasmin, the proteolytic enzyme responsible for fibrinolysis, has the capacity to liberate kinins from their precursors and to activate kallikrein, which brings about the formation of plasmin from plasminogen. Plasmin cleaves C3 complement protein, resulting in the formation of C3 fragments. It also breaks down fibrin to form fibrin split products. 4. The complement system (see Table 1.3, which lists the complement molecules found in the normal eye, and Table 1.4, which lists the complement molecules found in diseased eyes) consists of almost 60 proteins present in blood plasma, on the cell surfaces, or within the cell. Its vital nature is evidenced by the fact that it has been preserved by evolution for more than a billion years. 1

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CHAPTER 1  Basic Principles of Pathology

TABLE 1.1  The Actions of the Principal Mediators of Inflammation Mediator

Principal Sources

Actions

Cell-Derived Histamine Serotonin Prostaglandins Leukotrienes

Mast cells, basophils, platelets Platelets Mast cells, leukocytes Mast cells, leukocytes

Platelet-activating factor

Leukocytes, mast cells

Reactive oxygen species Nitric oxide Cytokines (TNF, IL-1) Chemokines

Leukocytes Endothelium, macrophages Macrophages, endothelial cells, mast cells Leukocytes, activated macrophages

Vasodilation, increased vascular permeability, endothelial activation Vasodilation, increased vascular permeability Vasodilation, pain, fever Increased vascular permeability, chemotaxis, leukocyte adhesion and activation Vasodilation, increased vascular permeability, leukocyte adhesion, chemotaxis, degranulation, oxidative burst Killing of microbes, tissue damage Vascular smooth muscle relaxation, killing of microbes Local endothelial activation (expression of adhesion molecules), fever/ pain/anorexia/hypotension, decreased vascular resistance (shock) Chemotaxis, leukocyte activation

Plasma Protein-Derived Complement products (C5a, C3a, C4a)

Plasma (produced in liver)

Kinins

Plasma (produced in liver)

Proteases activated during coagulation

Plasma (produced in liver)

Leukocyte chemotaxis and activation, vasodilation (mast cell stimulation) Increased vascular permeability, smooth muscle contraction, vasodilation, pain Endothelial activation, leukocyte recruitment

IL-1, interleukin-1; MAC, membrane attack complex; TNF, tumor necrosis factor. (Reproduced from Table 2.4, Kumar R, Abbas A, DeLancey A et al.: Robbins and Cotran Pathologic Basis of Disease, 8th edn. Philadelphia, Saunders. © 2010 by Saunders, an imprint of Elsevier Inc.)

A

B

C

D

Fig. 1.1  Acute inflammation. A, Corneal ulcer with hypopyon (purulent exudate). Conjunctiva hyperemic. B, Polymorphonuclear leukocytes (PMNs) adhere to corneal endothelium and are present in the anterior chamber as a hypopyon (purulent exudate). C, Leukocytes adhere to limbal, dilated, blood-vessel wall (margination) and have emigrated through endothelial cell junctions into edematous surrounding tissue. D, PMNs in corneal stroma do not show characteristic morphology but are recognized by “bits and pieces” of nuclei lining up in a row. (C and D are thin sections from rabbit corneas six hours post-corneal abrasion.)

Inflammation

TABLE 1.2  Role of Mediators in Different

TABLE 1.3  Complement Molecules Found

Role in Inflammation

Mediators

Vasodilation

Prostaglandins Nitric oxide Histamine Histamine and serotonin C3a and C5a (by liberating vasoactive amines from mast cells, other cells) Bradykinin Leukotrienes C4, D4, E4 PAF Substance P TNF, IL-1 Chemokines C3a, C5a Leukotriene B4 (Bacterial products; e.g., N-formyl methyl peptides) IL-1, TNF Prostaglandins Prostaglandins Bradykinin Lysosomal enzymes of leukocytes Reactive oxygen species Nitric oxide

Complement Molecules Expressed in the Healthy Eye

Reactions of Inflammation

Increased vascular permeability

Chemotaxis, leukocyte recruitment and activation

Fever Pain Tissue damage

in the Normal Eye







a. Initially named because it was seen to “complement” antibody and cell-mediated immune defenses against microbes. b. Classic functions: Fig. 1.2 highlights some of the myriad functions performed by complement. 1) Removal of immune (antigen–antibody) complexes. 2) Labeling (opsonization) of foreign antigens for enhanced removal by phagocytes. 3) Recruitment and activation of nearby leukocytes. 4) Direct cytolysis of invading microorganisms. c. Performs multiple functions in addition to those “classically” ascribed to it. d. Complement achieves its effect through a cascade of the separate components working in coordination and in specific sequences leading through activation of C3. (Fig. 1.3 is a schematic representation of the three primary routes or pathways of complement cascade activation through C3.) 1) The three pathways leading to activation of C3 are: a) Classical pathway. b) Lectin pathway. c) Alternative pathway.

Eye-Associated Remarks

Complement System Activators Amyloid precursor proteins (APP) Retina C-reactive protein (CRP) Retina Complement Proteins C1q, C2, C3

C4 C5–8 C9 C5b–9 Factor B Complement Regulators Factor H

Factor H-like protein 1 (FHL-1) C1 inhibitor (C1-INH) CD46 (MCP)

IL-1, interleukin-1; PAF, platelet-activating factor; TNF, tumor necrosis factor. (Reproduced from Table 2.7, Kumar R, Abbas A, DeLancey A et al.: Robbins and Cotran Pathologic Basis of Disease, 8th edn. Philadelphia, Saunders. © 2010 by Saunders, an imprint of Elsevier Inc.)



3

CD55 (DAF)

CD59 (protectin)

Vitronectin Clusterin Complement Receptors Complement receptor-1 (CR1) C3aR C5aR

Cornea, choroid, inner retina, sclera, optic nerve, retinal pigmented epithelium (RPE) cell Sclera Cornea, scleral tissue Soft drusen from non-AMD eyes, retina, optic nerve Bruch’s membrane, increase with age in non-AMD eyes Cornea, sclera Cornea, sclera, iris, ciliary body, retina, choroidal tissue outside Bruch’s membrane, optic nerve Bruch’s membrane Cornea Cornea and corneal limbus, vitreous humor, RPE basolateral surface, photoreceptors Cornea and corneal limbus, conjunctiva, iris, ciliary body, vitreous humor, retinal nerve fiber layer (NFL) and photoreceptors Cornea and corneal limbus, conjunctiva, iris, ciliary body, choroid, vitreous humor, vessels in the inner retina Soft drusen from non-AMD eyes Soft drusen from non-AMD eyes RPE apical surface Retinal ganglion cells, NFL Inner plexiform layer (IPL), Müller cells, NFL

AMD, age-related macular degeneration; RPE, retinal pigment epithelium. (From Mohlin et al.: The link between morphology and complement in ocular disease. Mol Immunol 89:84–99, 2017. Table 1. Elsevier.)



2) Cleavage of C3 produces the active fragments C3a and C3b. a) C3a is anaphylatoxin leading to chemotactic and proinflammatory responses. b) C5a also is an anaphylatoxin. c) C3b results in opsonization of foreign surfaces. 3) Thus, C3 has a major role in complement activation and generation of immune responses.

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CHAPTER 1  Basic Principles of Pathology

TABLE 1.4  Complement Molecules Found in the Human Diseased Eye, i.e., in Age-Related

Macular Degeneration (AMD), Glaucoma, Neuromyolitis Optica (NMO) and in Uveitis Complement Molecules Expressed in the Diseased Eye

Eye Disease–Associated Remarks

Complement System Activators Amyloid precursor proteins (APP) C-reactive protein (CRP) Immunoglobulin Lipoprotein

Age-Related Macular Degeneration (AMD) Drusen Drusen, choroid Drusen Drusen

Complement Proteins/Activation Products C1q Drusen Mannose binding protein (MBL) Drusen C2a C3a, C3c, C3d, C3dg, C3b, iC3b, Bb Choroid, drusen, retinal pigmented epithelial (RPE) cell C5b–9 (MAC) and sC5b−9a Drusen, RPE, choroid, macula Factor Ba Drusen, choroid Factor Da Drusen, retina Complement Regulators Factor Ia Factor Ha FHL-1 Complement receptor 1 (CR1, CD35) CD46 (MCP) Vitronectin Clusterin Complement Anaphylatoxins C3a C5a

Drusen, inner retina Drusen, retinal pigmented epithelial (RPE) cell, choroid, macula Drusen, choroid Drusen, RPE Drusen, choroidal vessels, basolateral RPE Drusen, RPE Drusen

Complement Molecules Expressed in the Diseased Eye

Eye Disease–Associated Remarks

Complement System Activators Immunoglobulin Retina, optic nerve Complement Proteins/Activation Products C1q Retina, ganglion cells (GCL) and nerve fiber layer (NFL) C3, C3b Retina, GCL and NFL C5b-9 (MAC) Retina, GCL Complement Regulators Factor H

GCL Uveitis

Complement System Activators Immunoglobulin Ocular proteins Complement Proteins/Activation Products C3c, C3d Aqueous humor C4a Aqueous humor Factor B and Bb Aqueous humor Complement Anaphylatoxins C3a, C5a

Aqueous humor Neuromyelitis optica (NMO)

Complement System Activators Immunoglobulin Optic nerve

Aqueous humor, drusen Drusen Glaucoma

a

Complement-associated genes connected with AMD: (Adamus et al., 2017; Edwards et al., 2005; Hageman et al., 2005; Haines et al., 2005; Heckner et al., 2010; Klein et al., 2005; Gold et al., 2006; Maller et al., 2007; Park et al., 2009) and uveitis: (Thompson et al., 2013; Yang et al., 2011, 2013; Xu et al., 2015). (From Mohlin et al., The link between morphology and complement in ocular disease. Mol Immunol 89:84–99, 2017. Table 2. Elsevier.)





e. C1 has been called the “defining component” of the classical complement pathway. 1) Functions as a molecular scaffold for binding of other complement components. 2) Activates and cleaves complement components to continue the complement cascade. 3) Helps to trigger Wnt receptor signaling. 4) Participates in the process of apoptosis. 5) Cleaves MHC class I molecule and other proteins. 6) Can adapt to multiple molecular and cellular processes besides the complement system. f. Complement plays major roles in immune defense against microorganisms and in clearing damaged host components. 1) It responds to recognition of pathogenassociated molecular patterns (PAMPs) when they bind to host pattern-recognition receptors



(PRRs) and/or internally produced dangerassociated molecular patterns (DAMPs). g. Activation of complement pathways results in a proinflammatory response that includes the generation of membrane attack complexes (MACs), which mediate cell lysis, the release of chemokines to attract inflammatory cells to the site of damage, and the enhancement of capillary permeability. (See Fig. 1.3 for the steps leading to activation of MAC.) 1) Composed of five terminal complement proteins: C5b, C6, C7, C8, and C9. Multiple C9 molecules may be involved. 2) There are numerous levels regulating the activity of MAC and protecting heathy cells from attack. In fact, control of the system is the responsibility of almost half of its components.

Inflammation



Increased vascular permeability

Lysis of foreign cells

8 Lysis of bacteria

7

1

Neutrophil activation and chemotaxis

5



2

Complement 6

Smooth muscle contraction

4

3

Mast cell degranulation



Localization of complexes in germinal Opsonization centers and phagocytosis of bacteria

Fig. 1.2  Summary of the actions of complement and its role in the acute inflammatory reaction. Note how the elements of the reaction are induced. Increased vascular permeability (1) due to the action of C3a and C5a on smooth muscle (2) and mast cells (3) allows exudation of plasma protein. C3 facilitates both the localization of complexes in germinal centers (4) and the opsonization and phagocytosis of bacteria (5). Neutrophils, which are attracted to the area of inflammation by chemotaxis (6), phagocytose the opsonized microorganisms. The membrane attack complex, C5–C9, is responsible for the lysis of bacteria (7) and other cells recognized as foreign (8). (Adapted with permission from Roitt IM, Brostoff J, Male DK: Immunology, 2nd edn. London, Gower Medical. © Elsevier 1989.)







h.

i.

j.



k.



l.

a) Disorders resulting from impaired regulation of complement are termed complementopathies. Complement proteins opsonize or lyse cells. Therefore, they may injure healthy tissue, particularly when there is a defect in complement regulation. Complement is important in such diseases as macular degeneration, rheumatoid arthritis, multiple sclerosis, Alzheimer’s disease, schizophrenia, and angioedema. T cells and other cell types contain multi­ ple complement components, which have been called the “complosome” in analogy to the inflammasome, which will be discussed later in this chapter. (Fig. 1.4 provides an overview of the multiple ways in which the cell complosome and other complement components may impact key cell processes when faced with various challenges.) Other immune system cells that may produce or be involved in complement function are polymorphonuclear leukocytes, mast cells, monocytes, macrophages, dendritic cells, natural killer (NK) cells, and B cells. Plays a role in adaptive immune response involving T and B cells, and functions as a bridge between innate and adaptive immunity.









5

m. Helps maintain tissue homeostasis and cellular integrity, and functions in tissue regeneration. Also functions in early sperm–egg interactions in fertilization, regulation of epiboly and organogenesis, and in refinement of cerebral synapses. n. The complement system is implicated in multiple ocular diseases including age-related macular degeneration, glaucoma, and neuromyelitis optica (Table 1.4 lists elements of the complement system and how they may be involved in these disorders). o. Complement system, components and their genetic deficiency. 1) Deficiency of early components of the classical pathway (C1q, C1r/s, C2, C4, and C3) is associated with autoimmune diseases resulting from failure of clearance of immune complexes and apoptotic materials and impairment of humoral response. 2) Deficiencies of mannan-binding lectin and the early components of the alternative (factor D and properdin) and terminal pathways (from C3 onward components C5, C6, C7, C8, and C9) increase susceptibility to infections and to their recurrence. 3) See also the discussion of monogenic autoinflammatory syndromes later in this chapter. p. Activation of complement in the tumor microenvironment enhances tumor growth and increases metastasis. 5. Prostaglandins (prostanoids), which have both inflammatory and anti-inflammatory effects, are 20-carbon, cyclical, unsaturated fatty acids with a 5-carbon ring and two aliphatic side chains. a. They are produced by mast cells, macrophages, endothelial cells, and others. b. With leukotrienes, they are designated eicosanoids. Leukotrienes are metabolized through the lipoxygenase pathway and prostaglandins through the cyclooxygenase pathway. c. Active in vascular and systemic reactions of inflammation, oxidative stress, and physiologic functions. d. Cyclooxygenase helps catalyze the biosynthesis of prostaglandins from arachidonic acid. e. Prostaglandins, cytokines, and leukotrienes function to dilate lymphatics at a site of injury. f. Prostaglandins play an important role in nociception and pain. 6. Major histocompatibility complex (MHC), called the human leukocyte antigen (HLA) complex in humans, is critical to the immune response. a. HLAs are present on all nucleated cells of the body and platelets.

The HLA region is on autosomal chromosome 6. In practice, the blood lymphocytes are the cells tested for HLA.

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CHAPTER 1  Basic Principles of Pathology

Fig. 1.3  Schematic of the complement cascade. The three primary routes for activation of complement are: (1) the lectin pathway (LP), (2) the classical pathway (CP), and (3) the alternative pathway (AP). The LP and CP are activated when specific triggers are recognized by host pattern-recognition receptors (PRRs). The AP is constitutively active. Initial activation through the LP or CP generates a shared C3 convertase (C4b•C2a). In the AP, C3b pairs with factor B (FB) to form the AP proconvertase (C3b•B), which is processed by factor D (FD) to form the AP C3 convertase (C3b•Bb). Both types of C3 convertases cleave C3 to generate C3a and C3b. C3a is an anaphylatoxin, a substance that promotes an inflammatory response. C3b that lands on the surface of a healthy host cell is quickly inactivated; C3b that attaches to the surface of a pathogen or altered host cell triggers a rapid amplification loop to generate more C3b, resulting in opsonization. C3b also complexes with the C3 convertases to form the C5 convertases (C4b•C2a•C3b and C3b•Bb•C3b). In the terminal complement cascade, C5 convertases cleave C5 into C5a (an anaphylatoxin) and C5b. C5b combines with C6–9 to form the membrane attack complex (MAC), also referred to as the terminal complement complex (TCC). Regulatory factors act at various stages of the cascade to control complement activation via their decay accelerating activity and/or cofactor activity. Additional abbreviations: MASPs, mannose-binding lectin-associated serine proteases; MBL, mannose-binding lectin; PAMPs, pathogen-associated molecular patterns. (From Baines AC, Brodsky RA: Complementopathies. Blood Rev 31:213–223, 2017. Figure 1. Elsevier.)





b. The three genetic loci belonging to HLA class I are designated by the letters HLA-A, HLA-B, and HLA-C. Class II MHC molecules are encoded at the locus HLA-D with three subregions HLA-DP, HLA-DQ, and HLA-DR. 1) Class I MHC molecules display proteins derived from foreign antigens, which are recognized by CD8+ T lymphocytes. 2) Class II MHC molecules present antigens that are contained in intracellular vesicles and derived from foreign organisms and soluble proteins. c. A tentatively identified specificity carries the additional letter “W” (workshop) and is inserted between the locus letter and the allele number— for example, HLA-BW 15.



d. The HLA system is the main human leukocyte isoantigen system and the major human histocompatibility system. 1) HLA-B 27 is positive in a high percentage of young women who have acute anterior uveitis and in young men who have ankylosing spondylitis or Reiter’s disease. 2) HLA-B 51 is strongly associated with Behçet’s disease. 7. Nonspecific soluble mediators of the immune system include cytokines, such as interleukins, which are mediators that act between leukocytes, interferons (IFNs), colony-stimulating factors (CSFs), tumor necrosis factor (TNF), transforming growth factor-β, and lymphokines (produced by lymphocytes).

Inflammation

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7

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Fig. 1.4  Suggestions on the potential impact of complosome-derived and/or pathogen-shunted intracellular complement on key cell processes during the host/pathogen interaction. Pathogens trigger an array of responses when interacting with complement during cell infection processes – some of which are beneficial for the microbe and some of which support host protection. For example, infection of human papillomavirus (HPV) triggers globular C1q receptor signaling (gC1qR), which leads to mitochondrial dysfunction and apoptosis (1). Opsonized bacteria trigger mitochondrial antiviral signaling, which increases the expression of AP-1- and NF-κB-controlled genes and proinflammatory cytokine responses. C3-opsonized viruses, on the other hand, are targeted for degradation via the proteosome (2). Opsonized Listeria is also targeted in an intracellular complement-dependent fashion for degradation after cell entry through v-set immunoglobulin domain containing 4 (VSIG4)-driven autophagosome formation (3). Supporting viral and bacterial propagation, gC1R signaling on mitochondria was also shown to block retinoic acid-inducible gene I (RIG-I) activation in a process that promoted the replication of vesicular stomatitis virus (4), while opsonized Klebsiella and other species use vitronectin to gain entry in nonphagocytic cells (5). Although in most of these processes, complement fragments were “dragged” into the cell by microbes, we propose that there will also be (subsequent) interactions of invading intracellular pathogens with components of the complosome, for example C3 and C5 activation fragments (6). In line with the “scheme” observed for the role of serum-derived complement, we further predict that in some cases the complosome will mediate clearance of the pathogen while in other cases, it will be utilized by the pathogen to promote its survival. (From Arbore G et al.: Intracellular complement – the complosome – in immune regulation. Mol Immunol 89:2–9, 2017. Figure 2. Elsevier.)







a. The TNF ligand family encompasses a large group of secreted and cell surface proteins (e.g., TNF and lymphotoxin-α and -β) that may affect the regulation of inflammatory and immune responses. b. The actions of the TNF ligand family are somewhat of a mixed blessing in that they can protect against infection, but they can also induce shock and inflammatory disease. C. Immediately after an injury, the arterioles briefly contract (for approximately five minutes) and then gradually relax and dilate because of the chemical mediators discussed previously and from antidromic axon reflexes.

After the transient arteriolar constriction terminates, blood flow increases above the normal rate for a variable time (up to a few hours) but then diminishes to below normal (or ceases) even though the vessels are still dilated. Part of the decrease in flow is caused by increased viscosity from fluid loss through the capillary and venular wall. The release of heparin by mast cells during this period probably helps to prevent widespread coagulation in the hyperviscous intravascular blood.



D. During the early period after injury, the leukocytes (predominantly the PMNs) stick to the vessel walls, at first

8

CHAPTER 1  Basic Principles of Pathology

momentarily, but then for a more prolonged time; this is an active process called margination (see Fig. 1.1C). 1. Ameboid activity then moves the PMNs through the vessel wall (intercellular passage) and through the endothelial cell junctions (usually taking 2–12 minutes); this is an active process called emigration. 2. PMNs, small lymphocytes, macrophages, and immature erythrocytes may also pass actively across endothelium through an intracellular passage in a process called emperipolesis. 3. Mature erythrocytes escape into the surrounding tissue, pushed out of the blood vessels through openings between the endothelial cells in a passive process called diapedesis. E. Chemotaxis, a positive unidirectional response to a chemical gradient by inflammatory cells, may be initiated by lysosomal enzymes released by the complement system, thrombin, or the kinins. F. PMNs (neutrophils; Fig. 1.5) are the main inflammatory cells in the acute phase of inflammation. All blood cells originate from a small, common pool of multipotential hematopoietic stem cells. Regulation of the hematopoiesis requires locally specialized bone marrow stromal cells and a coordinated activity of a group of regulatory molecules—growth factors consisting of four distinct regulators known collectively as CSFs.

1. PMNs are born in the bone marrow and are considered “the first line of cellular defense.” 2. CSFs (glycoproteins that have a variable content of carbohydrate and a molecular mass of 18–90 kDa) control the production, maturation, and function of

A

PMNs, macrophages, and eosinophils mainly, but also of megakaryocytes and dendritic cells. 3. PMNs are the most numerous of the circulating leukocytes, making up 50–70% of the total. 4. PMNs function at an alkaline pH and are drawn to a particular area by chemotaxis (e.g., by neutrophilic chemotactic factor produced by human endothelial cells). 5. The PMNs remove noxious material and bacteria by phagocytosis and lysosomal digestion. PMNs produce highly reactive metabolites, including hydrogen peroxide, which is metabolized to hypochlorous acid and then to chlorine, chloramines, and hydroxyl radicals—all important in killing microbes. Lysosomes are saclike cytoplasmic structures containing digestive enzymes and other polypeptides. Lysosomal dysfunction or lack of function has been associated with numerous heritable storage diseases: Pompe’s disease (glycogen storage disease type 2) has been traced to a lack of the enzymes α-1,4-glucosidase in liver lysosomes (see Chapter 11); Gaucher’s disease is caused by a deficiency of the lysosomal enzyme β-glucosidase (see Chapter 11). Metachromatic leukodystrophy is caused by a deficiency of the lysosomal enzyme arylsulfatase-A (see Chapter 11). Most of the common acid mucopolysaccharide, lipid, or polysaccharide storage diseases are caused by a deficiency of a lysosomal enzyme specific for the disease (see under appropriate diseases in Chapters 8 and 11). Chédiak–Higashi syndrome may be considered a general disorder of organelle formation (see section on congenital anomalies in Chapter 11) with abnormally large and fragile leukocyte lysosomes.

B

Fig. 1.5  Polymorphonuclear leukocyte (PMN). A, Macroscopic appearance of abscess—that is, a localized collection of pus (purulent exudate)—in vitreous body. B, PMNs are recognized in abscesses by their segmented (usually three parts or trilobed) nucleus. C, Electron micrograph shows segmented nucleus of typical PMN, and its cytoplasmic spherical and oval granules (storage granules or primary lysosomes).

C

Inflammation

A

9

B

Fig. 1.6  A, Eosinophils are commonly seen in allergic conditions such as this case of vernal catarrh. B, Eosinophils are characterized by bilobed nucleus and granular, pink cytoplasm. C, Electron micrograph shows segmentation of nucleus and dense cytoplasmic crystalloids in many cytoplasmic storage granules. Some granules appear degraded.

C



6. PMNs are end cells; they die after a few days and liberate proteolytic enzymes, which produce tissue necrosis. G. Eosinophils and mast cells (basophils) may be involved in the acute phase of inflammation. 1. Eosinophils (Fig. 1.6) originate in bone marrow, constitute 1% or 2% of circulating leukocytes, increase in number in parasitic infestations and allergic reactions, and decrease in number after steroid administration or stress. They elaborate toxic lysosomal components (e.g., eosinophil peroxidase) and generate reactive oxygen metabolites. 2. Mast cells (basophils; Fig. 1.7) elaborate heparin, serotonin, and histamine, and they are imperative for the initiation of the acute inflammatory reaction.

Except for location, mast cells appear identical to basophils; mast cells are fixed-tissue cells, whereas basophils constitute approximately 1% of circulating leukocytes. Basophils are usually recognized by the presence of a segmented nucleus, whereas the nucleus of a mast cells is large and nonsegmented.



H. The acute phase is an exudative phase (i.e., an outpouring of cells and fluid from the circulation) in which the nature of the exudate often determines and characterizes an acute inflammatory reaction. 1. Serous exudate is primarily composed of protein (e.g., seen clinically in the aqueous “flare” in the anterior chamber or under the neural retina in a rhegmatogenous neural retinal detachment). 2. Fibrinous exudate (Fig. 1.8) has high fibrin content (e.g., as seen clinically in a “plastic” aqueous). 3. Purulent exudate (see Figs. 1.1 and 1.5) is composed primarily of PMNs and necrotic products (e.g., as seen in a hypopyon). The term “pus” as commonly used is synonymous with a purulent exudate.

4. Sanguineous exudate is composed primarily of erythrocytes (e.g., as in a hyphema). II. Subacute (intermediate or reactive countershock and adaptive) phase. A. The subacute phase varies greatly and is concerned with healing and restoration of normal homeostasis

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CHAPTER 1  Basic Principles of Pathology

A

B

C

D Fig. 1.7  A, Mast cell seen in center as round cell that contains slightly basophilic cytoplasm and round to oval nucleus. B, Mast cells show metachromasia (purple) with toluidine blue (upper right and left and lower right) and C, positive (blue) staining for acid mucopolysaccharides with Alcian blue. D, Electron microscopy of granules in cytoplasm of mast cell often shows typical scroll appearance.





(formation of granulation tissue and healing) or with the exhaustion of local defenses, resulting in necrosis, recurrence, or chronicity. B. PMNs at the site of injury release lysosomal enzymes into the area. 1. The enzymes directly increase capillary permeability and cause tissue destruction. 2. Indirectly, they increase inflammation by stimulating mast cells to release histamine, by activating the kiningenerating system, and by inducing the chemotaxis of mononuclear (MN) phagocytes. C. Mononuclear (MN) cells (Fig. 1.9) include lymphocytes and circulating monocytes. 1. Monocytes constitute 3%–7% of circulating leukocytes, are bone marrow-derived, and are the progenitor of a family of cells (monocyte–histiocyte–macrophage family) that have the same fundamental characteristics, including cell surface receptors for complement and the Fc portion of immunoglobulin, intracellular lysosomes, and specific enzymes; production of monokines; and phagocytic capacity. 2. Circulating monocytes may subsequently become tissue residents and change into tissue histiocytes, macrophages, epithelioid histiocytes, and inflammatory giant cells.





3. CSFs (glycoproteins that have a variable content of carbohydrate and a molecular mass of 18–90 kDa) control the production, maturation, and function of MN cells. 4. These cells are the “second line of cellular defense,” arrive after the PMN, and depend on release of chemotactic factors by the PMN for their arrival. a. Once present, MN cells can live for weeks, and in some cases even months. b. MN cells cause much less tissue damage than do PMNs, and they are more efficient phagocytes. 5. Monocytes have an enormous phagocytic capacity and are usually named for the phagocytosed material (e.g., blood-filled macrophages [erythrophagocytosis] and lipid-laden macrophages; Fig. 1.10). 6. Monocytes replace neutrophils as the predominate cell 24–48 hours after the onset of inflammation. D. Lysosomal enzymes, including collagenase, are released by PMNs, MN cells, and other cells (e.g., epithelial cells and keratocytes in corneal ulcers) and result in considerable tissue destruction. In chronic inflammation, the major degradation of collagen may be caused by collagenase produced by lymphokine-activated macrophages.

Inflammation



11

E. If the area of injury is tiny, PMNs and MN cells alone can handle and “clean up” the area with resultant healing. F. In larger injuries, granulation tissue is produced. 1. Granulation tissue (Fig. 1.11) is composed of leukocytes, proliferating blood vessels, and fibroblasts. 2. MN cells arrive after PMNs, followed by an ingrowth of capillaries that proliferate from the endothelium of pre-existing blood vessels. The new blood vessels tend to leak fluid and leukocytes, especially PMNs.

A

3. Fibroblasts (see Fig. 1.11), which arise from fibrocytes and possibly from other cells (monocytes), proliferate, lay down collagen (Table 1.5), and elaborate ground substance. 4. With time, the blood vessels involute and disappear, the leukocytes disappear, and the fibroblasts return to their resting state (fibrocytes). This involutionary process results in shrinkage of the collagenous scar and a reorientation of the remaining cells into a parallel arrangement along the long axis of the scar. 5. If the noxious agent persists, the condition may not heal as described previously, but instead may become chronic. 6. If the noxious agent that caused the inflammation is immunogenic, a similar agent introduced at a future date can start the cycle anew (recurrence).

C

B

Fig. 1.8  A, Cobweb appearances of fibrinous exudate, stained with periodic acid–Schiff. Cells use fibrin as scaffold to move and to lay down reparative materials. B, Electron micrograph shows periodicity of fibrin cut in longitudinal section. C, Fibrin cut in cross-section.

Histiocyte/macrophage

?

Activated macrophage

?

?

Multinucleated inflammatory giant cell

Langhans

Foreign body

Activated macrophages

?

Touton Epithelioid cells

A

B Fig. 1.9  A, Monocytes have lobulated, large, vesicular nuclei and moderate amounts of cytoplasm, and they are larger than the segmented polymorphonuclear leukocytes and the lymphocytes, which have round nuclei and scant cytoplasm. B, Possible origins of multinucleated inflammatory giant cells and of epithelioid cells.

12

CHAPTER 1  Basic Principles of Pathology

A

B Fig. 1.10  A, Foamy and clear lipid-laden macrophages in subneural retinal space. B, Cytoplasm of macrophages stains positively for fat with oil red-O technique.

A

B Fig. 1.11  Granulation tissue. A, Pyogenic granuloma, here in region of healing chalazion, is composed of granulation tissue. B, Three components of granulation tissue are capillaries, fibroblasts, and leukocytes.

III. Chronic phase A. The chronic phase results from a breakdown in the preceding two phases, or it may start initially as a chronic inflammation (e.g., when the resistance of the body and the inroads of an infecting agent, such as the organisms of tuberculosis or syphilis, nearly balance; or in conditions of unknown cause such as sarcoidosis). B. Chronic nongranulomatous inflammation is a proliferative inflammation characterized by a cellular infiltrate of lymphocytes and plasma cells (and sometimes PMNs or eosinophils). 1. The lymphocyte (Fig. 1.12) constitutes 15%–30% of circulating leukocytes and represents the competent immunocyte. a. All lymphocytes probably have a common stem cell origin (perhaps in the bone marrow) from which they populate the lymphoid organs: the thymus, spleen, and lymph nodes. b. Two principal types of lymphocytes are recognized: (1) The bone marrow-dependent (or bursal equivalent) B-lymphocyte is active in humoral immunity, is the source of immunoglobulin production

(Fig. 1.13), and is identified by the presence of immunoglobulin on its surface; (2) the thymusdependent T lymphocyte participates in cellular immunity, produces a variety of lymphokines, and is identified by various surface antigens. 1) Helper-inducer T lymphocytes (CD4-positive) initiate the immune response in conjunction with macrophages and interact with (helper) B lymphocytes. CD4+ T cells are activated after interaction with antigen–MHC complex and differentiate into Helper subsets. These functionally distinct T-helper subsets participate in host defense and immunoregulation. Classically, T-helper 1 (Th1) and T-helper 2 (Th2) cells secrete a distinctive suite of cytokines: Th1 express T-bet and produce interferon-γ and are involved predominantly in cellmediated immunity (e.g., cytotoxic T-cell response); Th2 express Gata3 and produce interleukins-4, -5, and -13. Regulatory T (Treg) cells also are CD4+-derived cells,

Inflammation

13

TABLE 1.5  Heterogeneity of Collagens in the Cornea* Type

Polypeptides

I

[α1(I)]2α2(I)

II

[α1(II)]3

III

[α1(III)]3

IV

[α1(IV)]2α2(IV)

V

[α1(V)]2α2(V)

VI

[α1(VI)]2α2(VI)α3(VI)

VII

[α1(VII)]3?

VIII

[α1(VIII)]2α2(VIII)?

IX

[α1(IX)]2α2(IX)α3(IX)

XII

[α1(XII)]3

Monomer

Polymer

*At least 10 genetically distinct collagens have been described in the corneas of different animal species, ages, and pathologies. Types I, II, III, and V collagens are present as fibrils in tissues. Types IV, VI, VII, and VIII form filamentous structures. Types IX and XII are fibril-associated collagens. The sizes of the structures are not completely known. Type II collagen is found only in embryonic chick collagen associated with the primary stroma. Type III collagen is found in Descemet’s membrane and in scar tissue. Types I and V form the heterotypic fibrils of lamellar stroma. Type VII has been identified with the anchoring fibrils, and type VIII is present only in Descemet’s membrane. Type IX collagen, associated with type II fibrils in the primary stroma, and type XII collagen, associated with type I/V fibrils, are part of a family of fibril-associated collagens with interrupted triple helices. Both type IX and type XII are covalently associated with a chondroitin sulfate chain. (Reproduced from Cintron C: The molecular structure of the corneal stroma in health and disease. In Podos SM, Yanoff M, eds: Textbook of Ophthalmology, vol. 8. London, Mosby. © Elsevier 1994.)

serve an immunosuppressive function, and express the master transcription factor FoxP3. There are thymic-derived natural, nTreg cells and peripherally induced iTreg cells that relate to autoimmunity. T-helper 17 (Th17) cells participate in protective tumor immunity; however, Th17-associated cytokines may be associated with tumor initiation and growth and also with autoimmune diseases. Finally, there are follicular T-helper (Tfh) cells that are in proximity to B cells in the germinal centers of lymphoid tissue. They promote class switching of B cells and express the master regulator Bc16 and the effector cytokine IL-21 as well as other surface molecules. Fig. 1.14 illustrates the complexity, flexibility and plasticity of the relationships between T-helper cells.

2) Suppressor-cytotoxic T lymphocytes (CD8positive) suppress the immune response and are capable of killing target cells (e.g., cancer cells) through cell-mediated cytotoxicity. 3) MHC molecules present antigenic peptides to CD8+ T cells, thereby providing the foundation for immune recognition. 2. The plasma cell (Fig. 1.15) is produced by the bone marrow–derived B lymphocyte, elaborates immunoglobulins (antibodies), and occurs in certain modified forms in tissue sections. After germinal center B cells undergo somatic mutation and antigen selection, they become either memory B cells or plasma cells. CD40 ligand directs the differentiation of germinal center B cells toward memory B cells rather than toward plasma cells.

14

CHAPTER 1  Basic Principles of Pathology

rbc

m

A

B

C Fig. 1.12  Lymphocyte. A, Low magnification shows cluster of many lymphocytes appearing as a deep blue infiltrate. Cluster appears blue because cytoplasm is scant and mostly nuclei are seen. B, Electron micrograph shows lymphocyte nucleus surrounded by small cytoplasmic ring containing several mitochondria, diffusely arrayed ribonucleoprotein particles, and many surface protrusions or microvilli (rbc, red blood cell). C, Lymphocytes seen as small, dark nuclei with relatively little cytoplasm. Compare with polymorphonuclear leukocytes (segmented nuclei) and with larger plasma cells (eccentric nucleus surrounded by halo and basophilic cytoplasm).

N

VL

N CL VH

C CH1

Antigenbinding sites

C CH2

CH3 C

C

N N

Heavy chain

Light chain

Fig. 1.13  The basic immunoglobulin structure. The unit consists of two identical light polypeptide chains linked together by disulfide bonds (gray). The amino-terminal end (N) of each chain is characterized by sequence variability (VL, VH), whereas the remainder of the molecule has a relatively constant structure (CL, CH1–CH3). The antigen-binding sites are located at the N-terminal end. (Adapted with permission from Roitt IM, Brostoff J, Male DK: Immunology, 2nd edn. London, Gower Medical. © Elsevier 1989.)

a. Plasmacytoid cell (Fig. 1.16A and B): This has a single eccentric nucleus and slightly eosinophilic granular cytoplasm (instead of the normal basophilic cytoplasm of the plasma cell). b. Russell body (Fig. 1.16C and D): This is an inclusion in a plasma cell whose cytoplasm is filled and enlarged with eosinophilic grapelike clusters (morular form), with single eosinophilic globular structures, or with eosinophilic crystalline structures; usually the nucleus appears as an eccentric rim or has disappeared. The eosinophilic material in plasmacytoid cells and in Russell bodies appears to be immunoglobulin that has become inspissated, as if the plasmacytoid cells can no longer release the material because of defective transport by the cells (“constipated” plasmacytoid cells).

Inflammation

C. Chronic granulomatous inflammation is a proliferative inflammation characterized by a cellular infiltrate of lymphocytes and plasma cells (and sometimes PMNs or eosinophils). 1. Epithelioid cells (Fig. 1.17) are bone marrow–derived cells in the monocyte–histiocyte–macrophage family (Fig. 1.18). a. In particular, epithelioid cells are tissue monocytes that have abundant eosinophilic cytoplasm, somewhat resembling epithelial cells. Tfh Bcl6

Plasticity Th1 Bcl6 T-bet

T-bet

T-bet RORyt

Bcl6 FoxP3 Flexibility

RORyt FoxP3 RORyt Th17

Th2 Bcl6 Gata3

Gata3



b. They are often found oriented around necrosis as large polygonal cells that contain pale nuclei and abundant eosinophilic cytoplasm whose borders blend imperceptibly with those of their neighbors in a pseudosyncytium (“palisading” histiocytes in a granuloma). c. All cells of this family interact with T lymphocytes, are capable of phagocytosis, and are identified by the presence of surface receptors for complement and the Fc portion of immunoglobulin. 2. Inflammatory giant cells, probably formed by fusion of macrophages rather than by amitotic division, predominate in three forms: a. Langhans’ giant cell (Fig. 1.19; see Fig. 1.17): This is typically found in tuberculosis, but it is also seen in many other granulomatous processes. When sectioned through its center, it shows a perfectly homogeneous, eosinophilic, central cytoplasm with a peripheral rim of nuclei.

Gata3 FoxP3

If the central portion is not homogeneous, foreign material such as fungi may be present: the cell is then not a Langhans’ giant cell but a foreign-body giant cell. When a Langhans’ giant cell is sectioned through its periphery, it simulates a foreign-body giant cell.

T-bet FoxP3 FoxP3 Tregs

Fig. 1.14  Flexibility and plasticity of helper T cells. Recent studies continue to reveal surprising flexibility in expression of “master regulator” transcription factors. In addition, there are now many examples in which helper T cell phenotypes can change their pattern of expression of signature cytokines and gene expression. Striking examples exist in which apparently fully committed “lineages” readily switch their phenotype, and there are now many circumstances in which helper T cells have been shown to express more than one master regulator. This may be advantageous in terms of host defense, but it needs to be borne in mind in thinking about effective therapies for immune-mediated disease and vaccine development. (From Nakayamada S, Takahashi H, Kanno Y et al.: Helper T cell diversity and plasticity. Curr Opin Immunol 24:297, 2012.)

A

15

b. Foreign-body giant cell (Fig. 1.20): This has its nuclei randomly distributed in its eosinophilic cytoplasm and contains foreign material. c. Touton giant cell (Fig. 1.21), frequently associated with lipid disorders such as juvenile xanthogranuloma, appears much like a Langhans’ giant cell with the addition of a rim of foamy (fat-positive) cytoplasm peripheral to the rim of nuclei. 3. Three patterns of inflammatory reaction may be found in granulomatous inflammations: a. Diffuse type (Fig. 1.22A): This typically occurs in sympathetic uveitis, disseminated histoplasmosis

B Fig. 1.15  Plasma cell. A, Plasma cells are identified by eccentrically located nucleus containing clumped chromatin and perinuclear halo in basophilic cytoplasm that attenuates opposite to nucleus. Plasma cells are larger than small lymphocytes, which contain deep blue nuclei and scant cytoplasm. B, Electron microscopy shows exceedingly prominent granular endoplasmic reticulum that accounts for cytoplasmic basophilia and surrounds nucleus. Mitochondria are also present in cytoplasm.

16

CHAPTER 1  Basic Principles of Pathology

A

B

C

D Fig. 1.16  Altered plasma cells. A, Electron micrograph shows that left plasmacytoid cell contains many small pockets of inspissated material (γ-globulin) in segments of rough endoplasmic reticulum; right cell contains large globules (γ-globulin), which would appear eosinophilic in light microscopy. B, Plasmacytoid cell in center has eosinophilic (instead of basophilic) cytoplasm that contains tiny pink globules (γ-globulin). C, Russell body appears as large anuclear sphere or D, multiple anuclear spheres.

Activated macrophage

Lymphokine

Langerhans’ cell

? Monocyte/ macrophage

Giant cell

?

Fig. 1.17  Epithelioid cells in conjunctival, sarcoidal granuloma, here forming three nodules, which are identified by eosinophilic color resembling epithelium. Giant cells, simulating Langhans’ giant cells, are seen in nodules.

?

Foreign body

? Epithelioid cell

Touton

Fig. 1.18  Proposed scheme for the terminal differentiation of cells of the monocyte/macrophage system. The pathologic changes result from the inability of the macrophage to deal effectively with the pathogen. Lymphokines from active T cells induce monocytes and macrophages to become activated macrophages. Where prolonged antigenic stimulation exists, activated macrophages may differentiate into epithelioid cells and then into giant cells in vivo, in granulomatous tissue. The multinucleated giant cell may be derived from the fusion of several epithelioid cells. (Adapted with permission from Roitt IM, Brostoff J, Male DK: Immunology, 2nd edn. London, Gower Medical. © Elsevier 1989.)

Inflammation

and other fungal infections, lepromatous leprosy, juvenile xanthogranuloma, Vogt–Koyanagi–Harada syndrome, cytomegalic inclusion disease, and toxoplasmosis. The epithelioid cells (sometimes with macrophages or inflammatory giant cells or both)

Fig. 1.19  Langhans’ giant cells have homogeneous central cytoplasm surrounded by rim of nuclei.

A

are distributed randomly against a background of lymphocytes and plasma cells. b. Discrete type (sarcoidal or tuberculocidal; see Fig. 1.22B): This typically occurs in sarcoidosis, tuberculoid leprosy, and miliary tuberculosis. An accumulation of epithelioid cells (sometimes with inflammatory giant cells) forms nodules (tubercles) surrounded by a narrow rim of lymphocytes (and perhaps plasma cells). c. Zonal type (see Fig. 1.22C): This occurs in caseation tuberculosis, some fungal infections, rheumatoid scleritis, chalazion, phacoanaphylactic (phacoantigenic) endophthalmitis, toxocara endophthalmitis, and cysticercosis. 1) A central nidus (e.g., necrosis, lens, and foreign body) is surrounded by palisaded epithelioid cells (sometimes with PMNs, inflammatory giant cells, and macrophages) that in turn are surrounded by lymphocytes and plasma cells. 2) Granulation tissue often envelops the entire inflammatory reaction.

B Fig. 1.20  A, Foreign-body giant cell (FBGC) simulating Langhans’ giant cells, except that homogeneous cytoplasm is interrupted by large, circular foreign material. B, Anterior-chamber FBGCs, here surrounding clear clefts where cholesterol had been, have nuclei randomly distributed in cytoplasm.

A

17

B Fig. 1.21  A, Touton giant cells in juvenile xanthogranuloma closely resemble Langhans’ giant cells except for the addition of peripheral rim of foamy (fat-positive) cytoplasm in the former. B, Increased magnification showing fat positivity of peripheral cytoplasm with oil red-O technique. (Case presented by Dr. M Yanoff to the Eastern Ophthalmic Pathology Society, 1993, and reported in Arch Ophthalmol 113:915, 1995.)

18

CHAPTER 1  Basic Principles of Pathology

A

B

Fig. 1.22  Patterns of granulomatous inflammation. A, Diffuse type in sympathetic uveitis. B, Discrete (sarcoidal or tuberculocidal) type in sarcoidosis. C, Zonal type in phacoanaphylactic endophthalmitis.

C

Staining Patterns of Inflammation I. Patterns of inflammation are best observed microscopically under the lowest (scanning) power. II. With the hematoxylin and eosin (H&E) stain, an infiltrate of deep blue tint (basophilia) usually represents a chronic nongranulomatous inflammation. The basophilia is produced by lymphocytes that have blue nuclei (when stained with hematoxylin) and practically no cytoplasm (if it were present, it would stain pink with eosin) and by plasma cells that have blue nuclei and blue cytoplasm. III. A deep blue infiltrate with scattered gray (pale pink) areas (“pepper and salt”) usually represents a chronic granulomatous inflammation, with the blue areas lymphocytes and plasma cells, and the gray areas islands of epithelioid cells. IV. A “dirty” gray infiltrate usually represents a purulent reaction with PMNs and necrotic material. A. If the infiltrate is diffuse (Fig. 1.23; e.g., filling the vitreous [vitreous abscess]), the cause is probably bacterial. B. If the infiltrate is localized into two or more small areas (Fig. 1.24; i.e., multiple abscesses or microabscesses), the cause is probably fungal.









IMMUNOBIOLOGY Background I. There are three levels of human defense against invading organisms:



A. Anatomical and physiological barriers 1. Examples are the skin, enzymes in secretions, mucoid surface secretions, surfactant, and gastric pH. B. Innate immunity (See also discussion of complement earlier in this chapter.) 1. This system has few receptors for antigens, but ones that are widespread among potential invaders. 2. Inflammasome (Fig. 1.25) illustrates how activation of one of the upstream sensors precipitates inflammasome formation leading to cell membrane breach and cell death through pyroptosis. a. It is a multiprotein complex composed of a sensor protein, the adapter protein ASC (apoptosisassociated speck-like protein containing caspase recruitment domain), and the inflammatory protease caspase-1. b. Downstream substrates are gasdermin D, IL-1β, and IL-18 and are responsible for an inflammatory form of cell death called pyroptosis with gasdermin D functioning as the actual instrument of cell death by forming pores in the cell membrane. 1) Following its activation, caspase-1 induces activation of IL-1β, and IL-18 thereby resulting in inflammation. c. Upstream sensors include NLRP (nucleotidebinding domain and leucine-rich repeat containing) 1, NLRP3, NLRC4, AIM2, and pyrin.

Immunobiology

A

B

19

C

Fig. 1.23  Staining patterns of inflammation. A, Macroscopic appearance of diffuse vitreous abscess. B, Diffuse abscess, here filling vitreous, characteristic of bacterial infection. C, Special stain shows Gram positivity of bacterial colonies in this vitreous abscess.

A

B

C

Fig. 1.24  Staining patterns of inflammation. A, Macroscopic appearance of multiple vitreous microabscesses, characteristic of fungal infection. B, One vitreous microabscess contiguous with detached retina. C, Septate fungal mycelia (presumably Aspergillus) from same case stained with Gomori’s methenamine silver.







1) Activated by stimuli such as infection and changes in cell homeostasis. d. Involved in monogenic autoinflammatory disorders in which there is apparently spontaneous inflammation in the absence of inciting auto-antibodies or antigen-specific T cells. (See section on autoinflammation later in this chapter). 1) Abnormal response to endogenous or exogenous factors that results in exaggerated activation of inflammation and usually mediated by the inflammasome. 2) May involve the eye in idiopathic granulomatous disorders, familial Mediterranean fever, tumor necrosis factor receptor-associated periodic syndrome, mevalonate kinase deficiency, and cryopyrin-associated periodic syndrome. e. Involved in the pathogenesis of glaucoma, agerelated macular degeneration, diabetic retinopathy, dry eye, and ocular infections. f. Activity by certain inflammasomes is associate with susceptibility to infections, autoimmunity, and tumorigenesis.







g. When the cell is in a steady state, inflammasome components are present in the cytosol, but their assembly is prevented by auto-inhibitory mechanisms mediated by chaperone protein. h. Autophagy inducers reduce symptoms of inflammasome-related diseases, while deficiencies in autophagy-related proteins may induce aberrant activation of inflammasome-mediated tissue damage. C. Adaptive immunity 1. The main components of this system are B and T lymphocytes. Their strength is in their ability to generate a response to a diverse population of potential pathogens. The immune system provides the body with a mechanism to distinguish “self” from “nonself.” The distinction, made after a complex, elaborate process, ultimately relies on receptors on the only immunologically specific cells of the immune system, the B and T lymphocytes.

20

CHAPTER 1  Basic Principles of Pathology

Fig. 1.25  Inflammasome. (From Place DE, Kanneganti TD: Recent advances in inflammasome biology. Curr Opin Immunol 50:32–38, 2018. Figure 1. Elsevier.)

II. Table 1.6 lists the major effector elements in our immunologic defense system. III. All lymphocytes in mammalian lymph nodes and spleen have a remote origin in the bone marrow. Those that have undergone an intermediate cycle of proliferation in the thymus (thymus-dependent, or T lymphocytes) mediate cellular immunity, whereas those that seed directly into lymphoid tissue (thymus-independent, or B lymphocytes) provide the precursors of cells that produce circulating antibodies. A. Thus, mediators of immune responses can be either specifically reactive lymphocytes (cell-mediated immunity) or freely diffusible antibody molecules (humoral immunity). B. Antibody-producing B cells or killer T-type cells are only activated when turned on by a specific antigen.

When an antigen (immunogen) penetrates the body, it binds to an antibody-like receptor on the surface of its corresponding lymphocyte that proliferates and generates a clone of differentiated cells. Some of the cells (large B lymphocytes and plasma cells)

secrete antibodies; T cells secrete lymphokines; and other lymphocytes circulate through blood, lymph, and tissues as an expanded reservoir of antigensensitive (memory) cells. When the immunogen encounters the memory cells months or years later, it evokes a more rapid and copious secondary anamnestic response. Other immune cells (e.g., NK) are less specific and eliminate a variety of infected or cancerous cells.

IV. T lymphocytes derive from lymphoid stem cells in the bone marrow and mature under the influence of the thymus. A. T lymphocytes are identified by surface antigens (T3, T4, T8, and T11). 1. T lymphocytes are divided into two major subsets that express either CD4 or CD8 protein on their surface. CD4+ and CD8+ T cells depend on different signaling pathways to support their development and survival. B. T lymphocytes are the predominant lymphocytes in the peripheral blood and reside in well-defined interfollicular areas in lymph nodes and spleen.

21

Immunobiology

TABLE 1.6  Host Effector Mechanisms Name Soluble Effectors Complement system Coagulation system Kinin system Antibodies

Properties

Effector Mechanisms

Proteolytic cascade, activated by antibody, directly by microbial components, or via PRRs Proteolytic cascade, activated by tissue and vascular damage Proteolytic cascade triggered by tissue damage

Direct destruction of pathogens via pore formation; recruit inflammatory cells; enhance phagocytosis and killing Prevents blood loss; bars access to bloodstream; proinflammatory Proinflammatory; causes pain response; increases vascular permeability to allow increased access to plasma proteins Directly neutralize pathogens; activate complement; opsonize pathogens to enhance phagocytosis and killing

Antigen-specific proteins produced by B cells; recognize a broad range of antigens

Cellular Effectors Monocyte/macrophage dendritic cell Neutrophil Eosinophil Basophil/mast cell NK cell

B lymphocyte T lymphocyte

Have PRRs to recognize pathogens; activated by specific T cells and chemokines Have PRRs to recognize pathogens, activated antibody and complement Recognize antibody-coated parasites Associated with IgE-mediated responses

Phagocytosis and microbial killing via multiple mechanisms; antigen presentation Phagocytosis and microbial killing via multiple mechanisms Killing of multicellular pathogens Release of granules containing histamine and other mediators of anaphylaxis Induce death of infected cells via membrane pores and induced apoptosis

Lymphocyte lacking antigen-specific reactivity; recognize PAMPs of intracellular pathogens, activated by chemokines and by membrane proteins of infected cells Recognize antigens presented by APCs; regulated by T cells and chemokines Recognize antigens presented by APCs; regulate major portions of both adaptive and innate immunity

Produce antibody Directly kill infected cells via membrane pores and induced apoptosis; activate macrophages; many other functions

APCs, antigen-presenting cells; PAMPs, pathogen-associated molecular patterns; PRRs, pattern-recognition receptors. (Reproduced from Table 3.1, Coleman WB, Tsongalis GJ, eds: Molecular Pathology. Burlington, MA, Academic Press. © 2009, Elsevier Inc. All rights reserved.)









C. The T-lymphocyte system is responsible for the recognition of antigens on cell surfaces and, thus, monitors self from nonself on live cells (Fig. 1.26). D. The MHC (HLA) system allows T cells to recognize foreign antigen in cells and then, aided by macrophages, mobilizes helper T cells to make killer T cells to destroy the antigen-containing cells. E. T lymphocytes, therefore, initiate cellular immunity (delayed hypersensitivity), are responsible for graftversus-host reactions, and initiate the reactions of the body against foreign grafts such as skin and kidneys (host-versus-graft reactions). F. When activated (by an antigen), they liberate lymphokines such as macrophage inhibition factor (MIF), macrophage activation factor (MAF), interferon (IFN), and interleukins IL-2 (previously called T-cell growth factor), IL-3, and IL-15 (Fig. 1.27). The proliferation and differentiation of T lymphocytes are regulated by cytokines that act in combination with signals induced by the engagement of the T-cell antigen receptor. A principal cytokine is IL-2, itself a product of activated T cells. IL-2 also stimulates B cells, monocytes, lymphokine-activated killer cells, and glioma cells. Another growth factor that stimulates

Thymus-derived precommitted lymphocyte

Aggregated antigen A

B

E

Macrophage

C

Lymphoblast

D

Sensitized lymphocyte

Fig. 1.26  Cellular immunity. A, The participants in the cellular immune response include the thymus-derived precommitted lymphocyte (T cell), bone marrow-derived monocyte (macrophage), and the aggregated antigens. B, Aggregated antigen is seen attaching to the surface of the macrophage. C, The T cell is shown as it attaches to the aggregated antigen. D, The substance originating in the macrophage passes into the T cell, which is attached to the antigen. E, The combined T cell, antigen, and macrophagic material causes the T cell to enlarge into a lymphoblast. Sensitized or committed T lymphocytes arise from lymphoblasts. (From Yanoff M, Fine BS: Ocular Pathology: A Color Atlas, 2nd edn. New York, Gower Medical. © Elsevier 1992.)

22

CHAPTER 1  Basic Principles of Pathology

Sensitized T lymphocyte Uncommitted lymphocyte Polymorphonuclear lymphocyte

Aggregated antigen

Thymus-derived lymphocyte

Macrophage

Bone marrowderived lymphocyte

A Monocyte

Capillary

Immunoglobulin (antibody)

Antigen Tuberculosis organisms within macrophage A

Plasma cell

B B

C

D

Fig. 1.27  Cellular immunity. A, Sensitized T lymphocytes (SL) are seen in a capillary. Along with the SL are other leukocytes, including monocytes, at an antigenic site. A macrophage, which contains tubercle bacilli and antigen, may be seen in the surrounding tissue. B, Monocytes become sensitized when cytophilic antibody from SL is transferred to them. They migrate toward the antigenic stimulus. C, Biologically active molecules, which cause the monocytes and leukocytes to travel to the area, are released by SL when they have encountered a specific antigen. D, Monocytes arriving at the site are immobilized by migration inhibitory factor (MIF), which is released by SL, which also release cytotoxin and mitogenic factor. Cytotoxin causes tissue necrosis (caseation), and mitogenic factor causes proliferation of cells. Some of these cells undergo transformation, becoming epithelioid cells, causing the formation of a tuberculoma. (From Yanoff M, Fine BS: Ocular Pathology: A Color Atlas, 2nd edn. New York, Gower Medical. © Elsevier 1992).

the proliferation of T lymphocytes, the cytokine IL-15, competes for binding with IL-2 and uses components of the IL-2 receptor. T lymphocytes will not go “into action” against an “enemy” unless they are triggered by several signals at once. When one of the signals needed is lacking, the T cell becomes “paralyzed” (anergy).



G. T lymphocytes also regulate B-cell responses to antigens by direct contact and by the release of diffusible factors that act as short-range stimulators of nearby B cells. H. Many reactions in cellular immunity are mediated by lymphocyte-derived soluble factors known collectively as lymphokines, which exert profound effects on inflammatory cells such as monocytes, neutrophils, and lymphocytes. Such action falls into three main categories: (1) effects on cell motility (migration inhibition, chemotaxis, and chemokinesis); (2) effects on cell proliferation or cellular viability; and (3) effects on cellular activation for specific specialized functions. V. The B lymphocyte also arises from lymphoid stem cells in the bone marrow, but it is not influenced by the thymus. A. It resides in follicular areas in lymphoid organs distinct from the sites of the T lymphocyte.

B. The B-lymphocyte system is characterized by an enormous variety of immunoglobulins having virtually all conceivable antigenic specificities that are capable of being recognized by at least a few B-lymphocyte clones.

After germinal center B cells undergo somatic mutation and antigen selection, they become either memory B cells or plasma cells. CD40 ligand directs the differentiation of germinal center B cells toward memory B cells rather than toward plasma cells.





C

Fig. 1.28  Humoral immunity. A and B, Four prerequisites for immunoglobulin formation are demonstrated, including thymus-derived lymphocyte (T cell), thymus-independent bone marrow-derived lymphocyte (B cell), bone marrow-derived monocyte (macrophage), and aggregated antigen. In A, aggregated antigens are seen attached to macrophages. In B, T and B cells are seen attached to different determinants on the aggregated antigen. C, Cooperative interaction that occurs between T and B cells causes the B cells to differentiate into plasma cells. (From Yanoff M, Fine BS: Ocular Pathology: A Color Atlas, 2nd edn. New York, Gower Medical. © Elsevier 1992.)

C. The system is well designed to deal with unpredictable and unforeseen microbial and toxic agents. D. The B lymphocyte can be stimulated by antigen to enlarge, divide, and differentiate to form antibody-secreting plasma cells (Fig. 1.28). In most circumstances, T lymphocytes collaborate with B lymphocytes during the induction of antibodyforming cells by the latter (see the section on humoral immunoglobulin, later).

VI. Null lymphocytes, which constitute approximately 5% of lymphocytes in peripheral blood, lack the surface markers used to identify T and B lymphocytes. A. Most null cells carry a surface receptor for the Fc portion of the immunoglobulins, can function as killer cells in antibody-dependent cell-mediated cytotoxicity, and are called NK cells.

Immunobiology



B. When stimulated, NK cells release perforin, which forms pores in the cell membrane. C. They also release substances through the pores that can precipitate apoptosis in the target cell. VII. Initially, the sheep red blood cell resetting test (especially with fixed, embedded tissue) and the immunofluorescence or immunoperoxidase techniques that demonstrate surface immunoglobulins were the principal techniques for identification of T or B lymphocytes, respectively. A. Now, monoclonal antibodies (especially with fresh tissue) are used for the localization of lymphocyte subsets in tissue sections, and their use has revolutionized research in immunology, cell biology, molecular genetics, diagnosis of infectious diseases, tumor diagnosis, drug and hormone assays, and tumor therapy. B. A myriad of different types of monoclonal antibodies now exist, and new ones are continuously being created. C. Monoclonal antibodies can be obtained against B and T lymphocytes, monocytes, Langerhans’ cells, keratins, type IV collagen, retinal proteins (e.g., human S-100), and tumor antigens (e.g., factor VIII and intermediate filaments—cytokeratins, vimentin, desmin, neurofilaments, and glial filaments—neuron-specific enolase, and glial fibrillary acidic protein; all may be found in tumors).

Cellular Immunity (Delayed Hypersensitivity) I. Two distinct cell types participate in cellular immunity: the T lymphocyte and the macrophage (histiocyte). A. Phagocytic cells of the monocytic line (monocytes, reticuloendothelial cells, macrophages, Langerhans’ dendritic cells, epithelioid cells, and inflammatory giant cells—all are different forms of the same cell) are devoid of antibody and immunologic specificity. 1. Macrophages, however, have the ability to process proteins (antigens) and activate the helper T cells. 2. Macrophages also secrete proteases, complement proteins, growth-regulating factors (e.g., IL-1), and arachidonate derivatives. B. All lymphocytes seem to be pre-committed to make only one type of antibody, which is cell-bound.

23

II. The delayed hypersensitivity reaction begins with perivenous accumulation of sensitized lymphocytes and other MN cells (i.e., monocytes, which constitute 80%–90% of the cells mobilized to the lesion). The infiltrative lesions enlarge and multiply (e.g., in tuberculosis, where the lesions take a granulomatous form), and cellular invasion and destruction of tissue occur. III. Delayed hypersensitivity is involved in transplantation immunity; in the pathogenesis of various autoimmune diseases (e.g., sympathetic uveitis); and in defense against most viral, fungal, protozoal, and some bacterial diseases (e.g., tuberculosis and leprosy). Perhaps the most important role is to act as a natural defense against cancer—that is, the immunologic rejection of vascularized tumors and immunologic surveillance of neoplastic cells.

Humoral Immunoglobulin (Antibody) I. Four distinct cell types participate in humoral immunoglobulin (antibody) formation: the T lymphocyte, the B lymphocyte, the monocyte (macrophage), and the plasma cell. A. Macrophages process antigen in the early stage of the formation of cellular immunity and secrete IL-1. B. Specifically, pre-committed cells of both T and B lymphocytes attach to different determinants of the antigen; T cells then secrete a B-cell growth factor (BCGF). C. BCGF and IL-1 evoke division of triggered B cells, which then differentiate and proliferate into plasma cells that elaborate specific immunoglobulins. All humoral immunoglobulins (antibodies) are made up of multiple polypeptide chains and are the predominant mediators of immunity in certain types of infection, such as acute bacterial infection (caused by streptococci and pneumococci) and viral diseases (hepatitis). II. The B lymphocyte, once a specific antigen causes it to become committed (sensitized) to produce an immunoglobulin, makes that immunoglobulin and none other, as does its progeny. It, or its progeny, may produce immunoglobulin or become a resting memory cell to be reactivated at an accelerated rate (anamnestic response) if confronted again by the same antigen. Table 1.7 enumerates the immunoglobulin classes and functions produced by B cells.

TABLE 1.7  Antibody Classes and Functions Class

Location*

Structure

Function

IgD IgM

Surface of B cells only Plasma

IgG

Widely distributed in extracellular fluid Mucosal tissues, surfaces, and secretions Bound to mast cells and basophils

2 κ or λ light chains, 2 δ heavy chains 2 κ or λ light chains, 2 µ heavy chains, arranged in pentamers with 1 J chain 2 κ or λ light chains, 2 γ heavy chains

Unknown; expressed early in differentiation along with IgM Activated complement; first functional immunoglobulin formed in immune response Complement activation, transfer to neonate via placenta, opsonization, neutralization of viruses and other pathogens Important in mucosal immunity; has opsonizing activity

IgA IgE

2 κ or λ light chains, 2 α heavy chains, arranged in dimers with 1 J chain 2 κ or λ light chains, 2 ε heavy chains

Binds to and activates mast cells and basophils; important in defense versus parasites

*All immunoglobin classes are found on B cells as antigen receptors. (Reproduced from Table 3.2, Coleman WB, Tsongalis GJ, eds: Molecular Pathology. Burlington, MA, Academic Press. © 2009, Elsevier Inc. All rights reserved.)

24

CHAPTER 1  Basic Principles of Pathology

Autoimmunity and Autoinflammation I. Autoimmune diseases are caused by abnormalities in adaptive immunity regulation, while autoinflammatory disorders are attributed to defects in innate immunity proteins and are characterized by the absence of pathogenic autoantibodies or autoreactive T cells. In both situations, the patient’s own immunological systems becomes a source of tissue damage rather than its protector. II. Monogenic autoinflammatory syndromes have been defined as inherited conditions caused by mutations in one or both copies of a single gene that result in over-activation of the innate immune system causing inappropriate inflammation. A. Appear to be unprovoked attacks of inflammation most commonly directed at the eye, skin, joints, and gut. B. Mechanisms by which genetic defects cause autoinflammatory disease: 1. Affect intracellular sensor function. 2. Lead to accumulation of intracellular triggers that cause cell stress and activate intracellular sensors. 3. Cause loss of a negative regulator of inflammation. 4. Affect signaling molecules that upregulate innate immune cell function. C. Mediated by IL-1 secretion stimulated by monocytes and macrophages. D. Induction of inflammation in many of these disorders is triggered by the inflammasome pathway (see discussion above regarding inflammasomes, pyroptosis, and associated monogenic ocular disorders). E. IL-1 secretion in response to Toll-like receptor stimulation, and ultimately, the triggering of NLRP3 inflammasome may occur not only in response to exogenous microbial stimulation, but also to “endogenous stress molecules” by setting off an autoinflammatory process. F. Other monogenic autoinflammatory disorders arise from perturbations in signaling by the transcription factor NF-κB, ubiquitination, cytokine signaling, protein folding, and type I interferon production, and complement activation. G. Some immunologic diseases have combined features of autoinflammation, autoimmunity, and/or immunodeficiency.

A



H. Monogenic autoinflammatory syndromes include idiopathic granulomatous diseases, familial Mediterranean fever (FMF), TNF receptor–associated periodic syndrome (TRAPS), deficiency of mevalonate kinase (MKD), cryopyrin-associated periodic fever syndrome (CAPS, consisting of familial cold autoinflammatory syndrome or FCAS, Muckle–Wells syndrome or MWS, and chronic infantile neurologic cutaneous articular syndrome or CINCA syndrome). III. The eye is considered an immunologically privileged site due to 1) absence of blood and lymphatic vessels in the anterior chamber, 2) anterior chamber–associated immune deviation (ACAID) that controls the proinflammatory milieu, and 3) retinal protection identified in phagocytosis of damaged receptors and retinal pigment epithelium, which also helps construct part of the blood–retinal barrier through its tight intercellular junctions. Another important component of the blood–retinal barrier resides in the tight junctions between retinal vascular endothelial cells. Nevertheless, autoimmune disorders do occur and may have devastating consequences. A. A particularly devastating but, fortunately, rare autoimmune disorder is sympathetic ophthalmia in which the ocular immune barriers are breached, and autoimmunity develops to uveal protein resulting in a delayed hypersensitivity reaction characterized by diffuse granulomatous inflammation involving the entire uveal tract in both the inciting and sympathizing eyes.

Immunohistochemistry I. As stated previously, monoclonal antibodies can be obtained against B and T lymphocytes, monocytes, Langerhans’ cells, keratins, type IV collagen, retinal proteins, and so forth (Figs. 1.29 and 1.30). Table 1.8 lists antibody tests that are helpful in differentiating various tumors when they lack sufficient differentiation for accurate microscopic diagnosis. II. Commonly used antibodies include the following: A. Cytokeratins (AE1/AE3, CAM 5.2, CK7, and CK20) and epithelial membrane antigen are markers for epithelia. B. Factor VIII and Ulex europaeus-1 are markers for vascular endothelia.

B Fig. 1.29  Immunocytochemistry. A, Cathepsin-D, which here stains cytoplasm of conjunctival submucosal glands (shown under increased magnification in B), is an excellent stain for lipofuscin.

Immunobiology

A

B

C

D

25

Fig. 1.30  Immunocytochemistry. A, Monoclonal antibody against desmin, one of the cytoskeletal filaments, reacts with both smooth and striated muscles, and it helps to identify tumors of muscular origin. B, Monoclonal antibody against λ chains in plasma cells. C and D, Polyclonal antibody against S-100 protein in melanocytes and Langerhans’ cells in epidermis (C) and in malignant melanoma cells (D). (From Schaumberg-Lever G, Lever WF: Color Atlas of Pathology of the Skin. Philadelphia, Lippincott, 1988, with permission.)

C. Intermediate filaments: Vimentin is a marker for mesenchymal cells, including smooth muscle, Schwann cells, histiocytes, and fibrocytes; desmin is a marker for smooth and striated muscles; cytokeratin is a marker for epithelia; neurofilament is a marker for neurons; and glial fibrillary acidic protein is a marker for astrocytes and Schwann cells. D. Neuron-specific enolase is a marker for Schwann cells, neurons, smooth muscle, and neuroendocrine cells. E. S-100 is a marker for neural crest-derived tissues including melanocytes, but only melanocytic tumors should be positive for HMB45 and Mel-A. F. Smooth muscle actin (SMA) is a marker for smooth muscles and myoepithelial cells. G. Many antibodies are available for immunophenotyping of lymphomas and leukemias, both on fresh and on paraffin-embedded tissue. Table 1.9 lists the immunohistochemical paradigm for differentiating hematolymphoid neoplasms. H. Many other markers are available, and new markers seem to appear almost weekly. 1. Useful websites for further information regarding immunohistochemical stains and techniques include the following: a. http://www.immunoquery.com

2. Throughout this textbook, appropriate key immunohistochemical markers are cited where appropriate for each histopathologic diagnosis. 3. Similarly, although genetics is not the focus of this textbook, critical genetic abnormalities are highlighted as appropriate.

Immunodeficiency Diseases I. The following are disorders associated with immunodeficiency discussed elsewhere in this textbook: A. Wiskott–Aldrich syndrome (see Chapter 6) B. Ataxia–telangiectasia (see Chapter 2) C. Chédiak–Higashi syndrome (see Chapter 11) II. Severe combined immunodeficiencies (SCIDs)—heterogeneous group of inherited disorders characterized by 1) absence or very low number of T cells (<300 CD3 T cells/ mm3) and no or very low T-cell function (<10% of the lower limit of normal) as measured by response to PHA or 2) presence of T cells of maternal origin. The presence or absence of B and NK cells has permitted clinicians to direct attention to certain genetic defects. SCIDs occur in approximately 1 : 50 000 live births, and they are more common in males because of the prevalence of X-linked SCID. III. Chronic granulomatous disease of childhood (see Chapter 4)

26

CHAPTER 1  Basic Principles of Pathology

TABLE 1.8  Antibodies Useful in Determining the Origin of Undifferentiated Tumors and

Tumors of Uncertain Primary Site Panel

Antibodies

Panel

Antibodies

Undifferentiated tumors

Pan-keratin CD45 (CLA) S-100 Vimentin Pan-keratin CK5/6 AE1 AE3 CAM5.2 MAK6 Squamous keratin (HMW) Nonsquamous keratin (LMW) Pan-keratin Vimentin S-100 Desmin CD45 (CLA) Actin (muscle/HHF-35-MSA) Actin (smooth muscle-specific—SMA) Myoglobin (skeletal muscle) LN-5 (histiocytes) Lysozyme (histiocytes) LN-6 (nonlymphoid vimentin) Factor VIII antigen (endothelial cells) CD34 (vascular antigen) CD31 (vascular antigen) Ulex (vascular antigen) O13 (Ewing’s sarcoma/PNET) BRST-2 (GCDFP) Mammaglobin Cu-18 (breast-related antigen) Lactalbumin Estrogen receptor (monoclonal) Progesterone receptor (monoclonal) Her-2/neu (c-erb B-2) p52 (luminal epithelial antigen) p53 Factor VIII antigen CK7 CK20 TTF-1 Prostate-specific antigen (PSA) Prostatic acid phosphatase (PAP) Androgen receptor 34BE12 (SK) CEA COTA CDX-2 CK7 CK20 Renal antigen CEA p53 COTA OC-125 (CA-125) Estrogen receptor Progesterone receptor

Liver panel

α-Fetoprotein α1-Antitrypsin α1-Antichymotrypsin Nonsquamous keratin Hepatitis B surface antigen Hepatitis B core antigen Squamous keratin Nonsquamous keratin CEA-negative EP4 (epithelial antigen)-negative CD15 (Leu M1)-negative Epithelial membrane antigen B72.3 Secretory component Vimentin OC-125 S-100 HMB-45 Melan A Vimentin Pan-keratin Glial fibrillary acidic protein (GFAP) Neurofilament S-100 NSE Vimentin Pan-keratin Synaptophysin NSE Chromogranin Serotonin Neuron-endocrine Synaptophysin Nonsquamous keratin CD57 (Leu 7, HMK 1) Vasointestinal peptide Adrenocorticotropic hormone (ACTH) Follicle-stimulating hormone (FSH) Growth hormone Luteinizing hormone Prolactin Thyroid-stimulating hormone (TSH) α-Subunit PIT-1 Amylase Insulin Glucagon Gastrin Somatostatin CDX-2 p63 Uroplakin

Carcinoma panel

Sarcoma panel

Breast panel

Prognosis (breast carcinoma)

Lung panel

Prostate panel

Gastrointestinal panel

Kidney/bladder panel

Ovary panel

Mesothelioma panel

Melanoma panel

Central nervous system/neural panel

Neuroendocrine panel

Pituitary hormone panel

Pancreatic panel

Urothelial panel

CEA, carcinoembryonic antigen; CLA, common leukocyte antigen; COTA, colonic ovarian tumor antigen; HMW, high molecular weight; LMW, low molecular weight; NSE, neuron-specific enolase; PNET, primitive neuroectodermal tumor. (Reproduced from Table 5.2, Weidner N, Cote RJ, Suster S et al., eds: Modern Surgical Pathology, 2nd edn. Philadelphia, Saunders. © 2009, 2003 by Saunders, an imprint of Elsevier Inc.)

Immunobiology

27

TABLE 1.9  Diagnostic Algorithm for Hematolymphoid Neoplasms First Choice Antibody Panel CD3−; CD20−/+

CD3−/CD20+

Second Choice Antibody Panel

Additional Antibodies

Consistent With Tumor Type

CD10 − + +

CD34 + + −/+

CD79a −/+ + +

Precursor B-Cell Neoplasms CD22−/+; TdT+ CD22−/+; TdT+ CD22−/+; TdT−/+

Pro-B-ALL (B-1) Common ALL (B-II, early pre-B) Pre-B-ALL (B-III, late pre-B)

CD5 + + − − − − −

CD10 − − + + −/+ − −

CD23 − + +/− − − −/+ −

CD43 + + −/+ +/− −/+ −/+ −

Mature B-Cell Neoplasms Cyclin D1+ Cyclin D1−; CD38+/− BCL2+/−; BCL6+ BCL2−; Ki67+; BCL6+ CD25+; DBA44+; TRAP+; Correlation with morphology is critical

CD45+; BCL6+/−; Bob1 or OCT2+ CD3/CD20−

CD30 +/− + + −/+

CD38 + − − −

−

−

CD45 + − R/− (focal) +/− CD68+; CD4+; CD43+ CD21+; CD23+; CD35+; S100−/+; CD4− S100+; CD4+/−; CD68−/+ +/−

CD138+; K/L (Clonal) ALK+; BCL6+/−; EMA+/−; CD4+/− CD15+; Fascin+; EBV+/−; Bob1/OCT2+ S100+; CD1a+; CD68−/+

MPO+; Lysozyme; CD34−/+; C-kit+; TdT−/+

Mantle cell lymphoma CLL/SLL Follicular cell lymphoma Burkitt’s lymphoma Hairy cell leukemia MALT lymphoma B-cell prolymphocytic leukemia Lymphoplasmacytic lymphoma NLPHD Plasma cell neoplasm Anaplastic large cell lymphoma Classic Hodgkin’s lymphoma Langerhans’ cell histiocytosis True histiocytic neoplasm Follicular dendritic cell neoplasm Dendritic cell neoplasm other than follicular Acute myeloid leukemia

(Reproduced from Figure 5.5, Weidner N, Cote RJ, Suster S et al., eds: Modern Surgical Pathology, 2nd edn. Philadelphia, Saunders. © 2009, 2003 by Saunders, an imprint of Elsevier Inc.)

IV. Acquired immunodeficiency syndrome (AIDS) (Table 1.10) A. The first five cases of what came to be called “AIDS” in the United States presented with Pneumocystis carinii (now termed Pneumocystis jiroveci) pneumonia, cytomegalovirus (CMV) infections, and candidiasis. Thus, ocular manifestations were among the presenting findings in these first AIDS patients. B. In 2013 there were 1.2 million people living with HIV and there were 40,630 new cases in the U.S. that year. In 2017, worldwide, an estimated 33 million people were HIV-positive and 30% didn’t know their status. More than two-thirds of HIV-infected individuals are in subSaharan Africa. C. From an ocular perspective, AIDS is the most important immunodeficiency disease. D. AIDS is caused by a retrovirus, the human immunodeficiency virus (HIV), which is highly lethal if not treated with HAART (highly active antiretroviral therapy). Worldwide, 77% of individuals diagnosed HIV-positive are on antiretroviral therapy. 1. Excess mortality associated with AIDS has been reduced 50% since the introduction of HAART therapy. Nevertheless, excess mortality is five times





higher in AIDS patients than in HIV-infected patients without AIDS. 2. HIV-1 and HIV-2 have an affinity for the CD4 antigen on T lymphocytes, macrophages, and other cells. HIV-2 typically is less virulent than HIV-1 and generates a more effective host response. E. HIV-1 consists of an electron-dense core surrounding a single-stranded RNA genome, both enveloped by a cell membrane. Retroviruses contain DNA polymerase (reverse transcriptase) complexed to the RNA in the viral core. Reverse transcriptase catalyzes the transcription of the RNA genome into DNA form (the provirus). The provirus migrates from the host cell’s cytoplasm to the nucleus, assumes a double-stranded circular form, integrates into the host cell DNA, and may remain throughout the life of the host cell. F. Ironically, although immunodeficiency is the hallmark of HIV-AIDS, the use of inflammatory pathways by the virus are critical to its establishment and spread within its host. 1. HIV immune activation is an essential component of HIV pathobiology by increasing proinflammatory mediators, dysfunctional regulatory T cells, and a

28

CHAPTER 1  Basic Principles of Pathology

TABLE 1.10  Human Immunodeficiency



Virus-Related Ophthalmic Disorders

I. Opportunistic infections A. Retina 1. CMV retinitis a. Complications (1) Immune recovery uveitis 2. Other retinal infections (caused by various agents, VZV, and Toxoplasma gondii being most common; most occur in less than 1% of patients with AIDS) B. Choroid (uncommon; caused by various agents, fungi and mycobacteria being most common) C. Ocular surface and adnexa (important agents include VZV, microsporidia, molluscum contagiosum virus) II. Vascular abnormalities A. Microvasculopathy 1. HIV retinopathy (cotton-wool spots, retinal hemorrhages)* B. Retinal arteriolar and venular occlusions (uncommon) III. Neoplasia† A. Kaposi sarcoma (conjunctiva, eyelids) B. Lymphoma (intraocular) C. Squamous cell carcinoma (conjunctiva) IV. Other disorders of uncertain pathogenesis A. Intraocular inflammation 1. Chronic anterior uveitis (uncommon) 2. Chronic multifocal retinal infiltrates (uncommon)‡ 3. Iatrogenic uveitis (drug related: cidofovir; rifabutin) B. Blepharitis C. Dry eye V. Neuro-ophthalmic disorders associated with orbital or intracranial disease AIDS, acquired immunodeficiency syndrome; CMV, cytomegalovirus; HIV, human immunodeficiency virus; VZV, varicella–zoster virus. *Clinical signs reflect focal ischemia, attributable to undetermined factors, on a background of the retinal microvasculopathy of HIV disease. † Infection has been shown to be involved in the pathogenesis of tumors in severely immunodeficient individuals. ‡ As described in Levinson RD, Vann R, Davis JL, et al.: Chronic multifocal retinal infiltrates in patients infected with human immunodeficiency virus. Am J Ophthalmol 125:312, 1998. (Reproduced from Holland GN: AIDS and ophthalmology: The first quarter century. Am J Ophthalmol 145:397, 2008.)



pattern of T-cell senescent phenotypes as are seen in elderly individuals. 2. Chemokines, which are small chemotactic cytokines, and their receptors contribute to the “cytokine storm” that is one of the salient features of HIV infection. 3. Apoptosis contributes significantly to T-cell depletion and this process is mediated by caspase-3. The process of pyroptosis also plays a role in CD4+ T-cell depletion with dying T cells releasing inflammatory signals that perpetuate the destructive process (see discussion of the inflammasome earlier in this chapter). G. Patients with AIDS are prone to life-threatening opportunistic infections, wasting, central nervous system dysfunction, generalized lymphadenopathy, and Kaposi’s sarcoma and other malignancies, particularly squamous neoplasia.













H. Cytomegalovirus is the most common ocular opportunistic agent in AIDS even following the advent of HAART therapy. Individuals with CD4+ T-cell counts of 50 cells/µl or less are at particular risk for ocular complications of CMV infection. The other agents include most of the viral, bacterial, fungal, and parasitic agents customarily associated with cellular immunodeficiency, with herpes simplex virus, Mycobacterium tuberculosis and Mycobacterium avium-intracellulare, cat-scratch bacillus (Bartonella henselae), Candida albicans, Cryptococcus neoformans, Pneumocystis carinii, and Toxoplasma gondii heading the list. I. HIV-associated neurologic disorders (HAND) have been found in up to 70% of HIV-infected individuals. Fortunately, new cases of HAND have decreased by 75% since the advent of HAART. From the ocular perspective, approximately 10%–15% of patients with AIDS, but without ocular opportunistic infection have a presumed neuroretinal disorder as evidenced by reduced contrast sensitivity and abnormal visual fields. 1. Abnormal contrast sensitivity is associated with increased mortality in these patients and may indicate the presence of life-threatening microvascular disease. 2. Narrowed retinal vascular caliber is present in AIDS and is associated with an increased mortality risk. 3. Neuroretinal disorders in these patients are associated with specific mitochondrial haplogroups. J. The histologic appearance depends on the site of involvement and the causative agent (see appropriate sections of this book). K. HIV also predisposes to malignancies, particularly Kaposi’s sarcoma; however, other tumors, such as conjunctival lymphoma, are increased in frequency in these patients. L. HIV-associated complications also include dyslipidemia, hyperglycemia, and loss of bone mineral density, and autoimmune disorders such as autoimmune thrombocytopenia, connective tissue disorders, spondyloarthritis, and others. M. Immune recovery uveitis (IRU). 1. Usually occurs when previously immune-deficient patients with AIDS have their immune response to CMV restored by HAART therapy. It also may be associated with other pathogens such as T. gondii, M. avium complex, and Leishmania sp. 2. Uveitis may occur within several weeks of starting HAART therapy. 3. Represents a change in inflammatory reaction rather than an absolute level of inflammation or specific complications. 4. It may develop in as many as 25% of patients with advanced immunodeficiency who start HAART therapy. a. Larger CMV retinitis lesions are associated with a greater risk of IRU. 5. Ocular findings may include anterior segment inflammation, cataract, vitritis, papillitis, cystoid macular

Cellular and Tissue Reactions

edema, epiretinal membrane, vitreous hemorrhage, retinal neovascularization, and proliferative vitreoretinopathy. Severe visual loss may result. 6. The presence of HLA-B 8–18 may be associated with IRU. Other genetic factors may modulate the impact of HIV infection on a given patient. 7. Cytomegalovirus is the most common underlying infection leading to IRU.

Transplantation Terminology I. Autograft: transplantation of tissue excised from one place and grafted to another in the same individual. II. Syngraft (isograft): transplantation of tissue excised from one individual and grafted to another who is identical genetically. III. Allograft (homograft): transplantation of tissue excised from one individual and grafted to another of the same species. IV. Xenograft (heterograft): transplantation of tissue excised from one individual and grafted to another of a different species. V. Orthotopic graft: transplantation to an anatomically correct position in the recipient. VI. Heterotopic graft: transplantation to an unnatural position.

CELLULAR AND TISSUE REACTIONS Hypertrophy Hypertrophy is an increase in size of individual cells, fibers, or tissues without an increase in the number of individual elements (e.g., retinal pigment epithelium [RPE] in RPE hypertrophy).

Hyperplasia Hyperplasia is an increase in the number of individual cells in a tissue; their size may or may not increase. Therefore, hyperplasia is cellular proliferation in excess of normal, but the growth eventually reaches an equilibrium and is never indefinitely progressive (e.g., RPE hyperplasia secondary to trauma; see section on neoplasia, later).

Aplasia Aplasia is the lack of development of a tissue during embryonic life (e.g., aplasia of the optic nerve).

29

Dysplasia Dysplasia is an abnormal growth of tissue during embryonic life (e.g., retinal dysplasia).

Neoplasia I. Neoplasia is a continuous increase in number of cells in a tissue, caused by unregulated proliferation and, in some cases, failure of mechanisms (e.g., apoptosis) that lead to cell death. A. The neoplastic proliferation is probably caused by either excessive or inappropriate activation of oncogenes or reduced activity of genes that downregulate growth (anti-oncogenes). B. It differs from hyperplasia in that its growth never attains equilibrium. C. The neoplasm may be benign or malignant. II. A malignant neoplasm differs from a benign one in being invasive (it infiltrates and actively destroys surrounding tissue), in having the ability to metastasize (develop secondary centers of neoplastic growth at a distance from the primary focus), and in showing anaplasia (histologically, the features of a malignancy that include variation from the normal structure [Fig. 1.31] or behavior in the sense of a loss of specialized or “adult” characteristics of the cell or tissue, e.g., loss of cellular or tissue polarity, or inability to form photoreceptors). Mutations in the p53 tumor suppressor gene, located on the short arm of chromosome 17 at position 17p13.1, represent the most frequent genetic alteration detected in human solid malignancies. In more than half of all human cancer cases, p53 is inactivated by mutations and other genomic alterations. Mutations in p53 also can result in altered p53 proteins and endow these mutant proteins with novel activities. The p53 gene encodes a 53-kDa nucleophosphoprotein that binds DNA, is involved in the regulation of transcription and the induction of programmed cell death (apoptosis), and negatively regulates cell division, preventing progression from G to S phase. Normally, p53 responds to a complex network of stress signals. When functioning properly, it has two responses to stress: either cell-cycle arrest

Hypoplasia Hypoplasia is the arrested development of a tissue during embryonic life (e.g., hypoplasia of the iris [aniridia]).

Metaplasia Metaplasia is the transformation of one type of adult tissue into another type (e.g., fibrous metaplasia of lens epithelium [in anterior subcapsular cataract]).

Atrophy Atrophy is a diminution of size, a shrinking of cells, fibers, or tissues that had previously reached their full development (e.g., retinal vascular atrophy in retinitis pigmentosa).

Fig. 1.31  Abnormal tripolar mitotic figure in a sebaceous gland carcinoma.

30

CHAPTER 1  Basic Principles of Pathology until DNA damage is repaired or apoptotic cell death if such repair has failed. It appears to be a marker of tumor progression (i.e., a direct correlation seems to exist between mutations at the p53 locus and increasing histologic grade).

Degeneration and Dystrophy I. A dystrophy is a primary (bilateral), inherited disorder that has distinct clinicopathologic findings. The individual dystrophies are discussed elsewhere under their individual tissues. II. A degeneration (monocular or binocular) is a secondary phenomenon resulting from previous disease. It occurs in a tissue that has reached its full growth. A. Cloudy swelling is a reversible change in cells secondary to relatively mild infections, intoxications, anemia, or circulatory disturbances. The cells are enlarged and filled with granules or fluid and probably represent an intracellular edema. B. Hydropic degeneration is a reversible change in cells also secondary to relatively mild infections, intoxications, anemia, or circulatory disturbances. The cells are enlarged and contain cytoplasmic vacuoles and probably represent an early stage of swelling of the endoplasmic reticulum. C. Fatty change results when fat accumulates in cells for unknown reasons or after damage by a variety of agents (e.g., chloroform and carbon tetrachloride). D. Glycogen infiltration results from diseases such as diabetes mellitus (e.g., lacy vacuolation of iris pigment epithelium; see Chapter 15) and from a lack of nutrition (e.g., in long-standing neural retinal detachment and in proliferating retinal pigment epithelial cells). E. Amyloid may be found in ocular tissues in primary amyloidosis (see Chapters 7, 8 and 12), such as in primary familial amyloidosis and lattice corneal dystrophy (in which case it is a dystrophic change), or in secondary amyloidosis (see Chapter 7), in which case it is a degenerative change. F. Hyaline degeneration is quite common; consists of acellular, amorphous, and eosinophilic material; and may be found in places such as the walls of arteriolosclerotic vessels or in the ciliary processes in elderly people.

Necrosis (Table 1.11) I. Necrosis occurs when cells die an “accidental” death, such as from severe and sudden injury (e.g., ischemia), sustained hyperthermia, physical or chemical trauma, complement attack, or metabolic poisons. Necrosis should be differentiated from apoptosis—see later.



A. Necrosis is accompanied by the following: 1. Swelling of the cytoplasm and organelles (especially the mitochondria) and only mild changes in the nucleus.

TABLE 1.11  Features of Necrosis and

Apoptosis Feature

Necrosis

Apoptosis

Cell size Nucleus

Enlarged (swelling) Pyknosis → karyorrhexis → karyolysis Disrupted

Reduced (shrinkage) Fragmentation into nucleosome-size fragments Intact; altered structure, especially orientation of lipids Intact; may be released in apoptotic bodies

Plasma membrane

Cellular contents

Adjacent inflammation Physiologic or pathologic role

Enzymatic digestion; may leak out of cell Frequent Invariably pathologic (culmination of irreversible cell injury)

No Often physiologic, means of eliminating unwanted cells; may be pathologic after some forms of cell injury, especially DNA damage

(Reproduced from Table 1.2, Kumar R, Abbas A, DeLancey A et al.: Robbins and Cotran Pathologic Basis of Disease, 8th edn. Philadelphia, Saunders. © 2010 by Saunders, an imprint of Elsevier Inc.)

2. Organelle dissolution and rupture of the plasma membrane. 3. Leakage of cellular contents into the extracellular space. 4. Inflammatory response to the released cellular debris. B. No inflammation occurs in apoptosis—see later. II. Coagulative necrosis: This is a firm, dry necrosis generally formed in tissue that has been shut off from its blood supply. A. The gray, opaque clinical appearance of the retina after a central retinal artery occlusion is caused by coagulative necrosis (ischemic necrosis). As seen by electron microscopy, coagulative necrosis (e.g., after a laser burn) is produced by widespread focal densification of membranes in the necrotic cell. B. Caseation, characteristic of tuberculosis, is a combination of coagulative and liquefaction (see later) necrosis. III. Hemorrhagic necrosis: This type is caused by occlusion of venous blood flow but with retention of arterial blood flow, as seen classically in central retinal vein thrombosis. IV. Liquefaction necrosis: Necrosis of this type results from autolytic (see section on Autolysis and Putrefaction, later) decomposition, usually in tissue that is rich in proteolytic enzymes (e.g., suppuration is a form of liquefaction necrosis in which rapid digestion is brought about by the proteolytic enzymes from the leukocytes, especially PMNs, present in the area). It also occurs from complete dissolution of all cell components, as in ultraviolet photocomposition. V. Fat necrosis: Necrosis causes liberation of free fatty acids and glycerol that results in a lipogranulomatous reaction.

Cellular and Tissue Reactions



Apoptosis (See also multiple references to apoptosis throughout this chapter, particularly relative to complement, the inflammasome, and HIV infections.) I. Apoptosis is “physiologic” or programmed cell death, unrelated to “accidental death” (necrosis)—see earlier. A. There are intrinsic and extrinsic pathways of apoptosis, and a substitute pathway for cell killing (see description, below, and Fig. 1.32). 1. The extrinsic pathway also, is termed the “death receptor pathway,” and is mediated by the binding of cellsurface death receptors and their natural ligands. 2. The intrinsic pathway, also termed the “mitochondrial pathway,” is strictly regulated by BCL-2 family proteins.





31

a. In the intrinsic pathway, Apaf-1-like molecules assemble into a ring-like platform known as the “apoptosome” that, in turn, activates procaspases. b. Caspase-9 is activated on the apoptosome, and then breaks up cells into apoptotic bodies (see below). 1) Inability to activate caspase-9 can lead to degenerative and developmental disorders, and even to cancer. 3. There is a third “substitute pathway” to cell killing that involves cytotoxic T-cell and natural killer cell– mediated and perforin–granzyme-dependent functions. In this pathway, granzymes A and B are involved. B. Two steps accompany apoptosis: 1. The cell undergoes nuclear and cytoplasmic condensation, eventually breaking up into a number of

Caspase-10 Initiator Caspase-8/10 Caspase-9 Cell destruction Apoptosis

Fig. 1.32  Illustration of major apoptotic signaling pathways. A, The extrinsic pathway is initiated by the interaction of death receptors (DRs) and death-inducing ligands via their FADD/TRADD domains. A deathinducing signaling complex (DISC) is formed, which then recruits inactivated initiator caspase-8 and caspase10. After that, BID is cleaved into tBID and translocates to mitochondria. B, In intrinsic apoptotic pathway, the stimuli first activate BH3-only proteins, and then pass the apoptotic signals via activating interactions between BCL-2 members, forming a mitochondrial outer membrane permeabilization (MOMP) complex and eventually releasing cytochrome c. Cytochrome c induces the assembly of the apoptosome, and caspase-9 is activated. The extrinsic and intrinsic apoptotic pathways converge in which caspase-3, caspase-6 and caspase-7 function as executioner caspases that trigger various biochemical and morphological alterations in apoptotic cells that are eventually taken up by phagocytes. Inhibitors of apoptosis (IAP) proteins inhibit both initiator and executioner caspases in apoptotic pathways. C, The substitute pathway is mediated by cytotoxic T lymphocytes or NK cells which involve perforin and granzyme. (From Wu et al.: Apoptosis signaling and BCL-2 pathways provide opportunities for novel targeted therapeutic strategies in hematologic malignancies. Blood Rev 32(1):8-28, 2018. Figure 1. Elsevier.)

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CHAPTER 1  Basic Principles of Pathology

membrane-bound fragments containing structurally intact organelles. Cells undergoing apoptosis demonstrate shrinkage, nuclear condensation associated with DNA fragmentation, a relatively intact cell membrane, loss of viability, and absence of inflammation.

2. The cell fragments, termed apoptotic bodies, are phagocytosed by neighboring cells and rapidly (within minutes) degraded. The apoptotic bodies are membrane-encapsulated, thus preventing exposure of cellular contents to the extracellular space and possible inflammatory reaction.







C. Apoptosis appears to play a major role in regulating cell populations. D. Defective apoptosis may play a role in the genesis of cancer, AIDS, autoimmune diseases, degenerative and dystrophic diseases of the central nervous system (including the neural retina), and diabetic retinopathy. E. Apoptosis is significant in the pathobiology of such ocular disorders as glaucoma, cataracts, ocular tumors, macular degeneration, and diabetic retinopathy. F. Although apoptosis frequently is viewed as a pathologic process, it is vital to successful ocular embryologic development.

Calcification I. Dystrophic (degenerative) calcification: This occurs when calcium is deposited in dead or dying tissue (e.g., in long-standing cataracts, in band keratopathy, and in retinoblastoma). II. Metastatic calcification: This type of calcification occurs when calcium is deposited in previously undamaged tissue (e.g., in the cornea of people with high serum calcium levels [hyperparathyroidism and vitamin D intoxication], where it shows as a horizontal band, and in the sclera, where it shows as a senile plaque). An unusual cause of metastatic calcification is Werner’s syndrome, a heredofamilial disorder characterized by premature graying and baldness, short stature, gracile build, and “bird face.” Ocular findings include blue sclera, bullous keratopathy, presenile posterior subcapsular cataract, degenerative corneal changes post cataract surgery, retinitis pigmentosa–like features, and paramacular degeneration.

Autolysis and Putrefaction I. Autolysis is partly the self-digestion of cells using their own cellular digestive enzymes contained in lysosomes (“suicide bags”) and partly other unknown factors.

II. When certain bacteria (especially clostridia) invade necrotic (autolytic) tissue, the changes catalyzed by destructive bacterial enzymes are called putrefaction.

Pigmentation I. In ocular histologic sections stained with H&E, some commonly found pigments may resemble each other closely: (1) melanin and lipofuscin, (2) hemosiderin, (3) exogenous iron, and (4) acid hematin. II. Melanin is found in uveal melanocytes as fine, powdery, brown granules barely resolvable with the light microscope and also in pigment epithelial cells of the retina, ciliary body, and iris as rather large, black granules. Lipofuscin occurs in aged cells and in the RPE and may be difficult to identify by conventional light microscopy, but by electron microscopy it differs considerably in structure and density from melanin. III. Hemosiderin results from intraocular hemorrhage when hemoglobin is oxidized to hemosiderin. A. It occurs as an orange–brown pigment in macrophages and, when plentiful in the eye, is called hemosiderosis bulbi. B. Systemic hemochromatosis (see Chapter 6) consists of portal cirrhosis and elevated iron content in parenchymal cells of multiple organs. When increased amounts of iron are deposited in tissues of multiple organs but cirrhosis and its complications are lacking, systemic hemosiderosis is present. C. The distribution of iron in the eye differs in local ocular disease (hemosiderosis bulbi and siderosis bulbi) and systemic disease (Table 1.12).

TABLE 1.12  Deposition of Iron in the Eye

of Local* and Systemic (Hemo) Siderosis

Tissue Corneal epithelium Trabecular meshwork Iris epithelium Iris dilator and sphincter muscles Ciliary epithelium Lens epithelium Vitreous body Sclera Blood vessels Sensory retina Retinal pigment epithelium

Local Siderosis and Hemosiderosis

Systemic Hemochromatosis

Yes Yes Yes Yes

No No No No

Yes Yes Yes No Yes Yes Yes

Yes No No Yes No No Yes

*With local iron foreign body, iron is usually deposited in all adjacent (contiguous) tissues. (Modified from Roth AM, Foos RY: Ocular pathologic changes in primary hemochromatosis. Arch Ophthalmol 87:507, 1972. © American Medical Association.)

Epigenetics and Ocular Disease

IV. Exogenous iron results from an intraocular iron foreign body. The resultant ocular iron deposition is called siderosis bulbi (see Table 1.12). V. Acid hematin is an artifact produced by action of acid fixatives, particularly formaldehyde, on hemoglobin. VI. Differentiation of the pigments: A. Only acid hematin is birefringent to polarized light. B. Only melanin bleaches with oxidizing agents, such as hydrogen peroxide. C. The cathepsin-D reaction is helpful in identifying lipofuscin. D. Only iron stains positively with the common stains for iron. Hemosiderin and exogenous iron cannot be differentiated on their staining properties and sometimes may not be differentiated on structural grounds.

Growth and Aging I. In general, ocular tissue in infants and young people is quite cellular. Cellularity decreases with aging as the collagenization of tissues increases. II. The eye is at least two-thirds of its adult size at birth, and it usually reaches full size by the end of the second decade of life. Although the eyeball reaches full size, the lens, an inverted epithelial structure, continues to grow throughout life. Nuclear cataract results from the increased density of the central (nuclear) lens cells (and other factors) and can be considered an aging change.

III. Certain chemicals may be deposited in ocular tissues during the aging process, including calcium in the insertion of the rectus muscles (senile plaque; Fig. 1.33) and in Bruch’s membrane (calcification of Bruch’s membrane), and sorbitol in the lens. IV. The important growth and aging changes of individual tissues are taken up in the appropriate sections in the remaining chapters.

A

33

EPIGENETICS AND OCULAR DISEASE I. Throughout Ocular Pathology we will provide information regarding the role of classic genetic alterations in the pathogenesis of many ocular disorders. Nevertheless, epigenetic mechanisms increasingly are being demonstrated to play a role in ocular disease processes. II. Epigenetics involves the mitotically and meiotically heritable potential for gene expression that does not involve variation in the DNA sequence. III. Chromatin consists of a DNA-protein-RNA complex. IV. Epigenetics involves chemical reactions that modulate chromatin accessibility, regulate gene expression, and the environmental factors that coordinate these chemical reactions. V. Epigenetic mechanisms may be reversible, heritable, and influenced by the environment. VI. Common epigenetic mechanisms include DNA methylation, covalent modification of histones, nucleosome remodeling, nuclear dynamics, and chromatin interaction with regulatory noncoding RNAs. A. DNA methylation 1. Catalyzed by DNA methyltransferase. 2. In most cases CpG dinucleotides (region of DNA where cytosine nucleotide is followed by a guanine nucleotide in the 5′ to 3′ direction) are methylated at the cytosine resulting in 5-methyl-cytosine. 3. Methylation silences the promoter associated with this genetic region. Conversely, hypomethylation leads to increased gene expression. 4. Epigenetic mechanisms may have a pathogenic role in ocular diseases such as corneal dystrophy, cataract, glaucoma, diabetic retinopathy, ocular neoplasia, uveitis, and age-related macular degeneration. 5. For example, in retinoblastoma the RAS-associated domain family IA (RASSFIA) tumor suppressor gene is silenced by DNA methylation in 82% of tumors. B. Histone modification 1. Histones are the primary protein components of chromatin and condense the DNA into nucleosomes that are said to form a “beads-on-a-string” conformation and based on their location along the DNA helix, the histones impact the ability of other regulatory

B Fig. 1.33  A, Scleral calcium plaques present where horizontal rectus muscles insert. Plaques appear translucent gray. B, Calcium deposited through full thickness of sclera in region of insertion of rectus muscles.

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CHAPTER 1  Basic Principles of Pathology

DNA-binding proteins to access specific sites along the DNA. 2. Can include acetylation, methylation, ubiquitination, phosphorylation, and sumoylation. a. Writers, readers, and erasers are involved in adding, interpreting, and/or removing epigenetic modifications on chromatin. b. Acetylation is a very common histone modification strategy. 1) It opens up the chromatin structure allowing recruitment and binding of the transcription factor and RNA polymerase II. 3. Methylation may facilitate or depress gene expression depending upon the target site. 4. Occur primarily within the N-terminal tails of histones protruding from the surface of the nucleosome or on its core region. C. Noncoding RNA includes long noncoding RNA, microRNA, short interference RNA, and Piwi-interacting RNA. Only long noncoding RNA and microRNA will be discussed. 1. Long noncoding RNA (LncRNA) a. RNA transcripts longer than 200 nucleotides and structurally resembling RNA but have little or no protein coding potential. b. Most are located in the nucleus. c. Termed sense or antisense if they overlap with protein coding genes. d. Intronic long noncoding RNA (lincRNA) are genes derived from intron of protein coding genes. e. Circular RNA (circRNA) are longer than 200 nucleotides. f. May impact DNA transcription through multiple mechanisms. g. May be involved in glaucoma, proliferative vitreoretinopathy, diabetic retinopathy and ocular tumors. 2. Small noncoding RNAs (micro RNAs), which are about 22 nucleotides in length, bind to untranslated regions of mRNA and cleave mRNA resulting in decreased protein synthesis and gene expression. D. Chromosome remodeling proteins modulate chromatin compaction and DNA accessibility to proteins in a non-covalent manner. They modulate DNA-histone interactions.

MODERN MOLECULAR PATHOLOGY DIAGNOSTIC TECHNIQUES The scope of molecular pathology is expanding at a rapid rate. Only a few of the most common techniques can be highlighted here. I. Fluorescence in situ hybridization (FISH) (Fig. 1.34). A. Commonly employed to determine the presence of a specific mutation, or a particular chromosomal presence, absence or rearrangement. B. Targets DNA in cells, nuclei or chromosomes.



C. Uses a DNA probe that is labeled with a nucleotide that is either conjugated to fluorescein (direct labeling) and/ or a non-fluorescent hapten (indirect labeling). 1. If a non-fluorescent hapten is used, the detection depends on the addition of a fluorescence-coupled anti-hapten reporter molecule. D. The probe binds with the specific complementary region on the DNA under investigation. E. Three basic types of probes are utilized: 1. Chromosome painting probes that contain multiple DNA fragments that are complementary with regions along the entire length of the chromosome resulting in fluorescence of the entire chromosome. 2. Repeat sequence probes are used to detect the gain or loss of specific chromosomes. a. They usually employ chromosome-specific pericentromeric and/or subtelomeric probes. 3. Unique sequence probes usually are employed to identify a gene or fragment related to a specific disease. a. Subtelomeric probes are used to identify subtelomeric deletions or rearrangements. F. One use for FISH in ophthalmic oncology has been in the cytogenetic identification of abnormalities, such as those involving chromosomes 3, 4, 6, 8, 15 and 18 in uveal melanoma. 1. Other cytogenetic techniques, such as comparative genomic hybridization (CGH) and spectral karyotyping (SKY) also may be employed for these purposes. G. It can be performed rapidly (1–2 days) and can be performed on both dividing and static cells (i.e., cells in metaphase or interphase). H. There are numerous variations on the basic FISH technique that will not be discussed here. II. Flow cytometry (Fig. 1.35). A. The flow cytometer evaluates characteristics of individual cells as they flow in fluid through a laser beam. 1. The cells usually are labeled with a fluorescently conjugated monoclonal antibody. 2. Fluorescence in the laser beam occurs in response to a specific wavelength of light stimulation, and the intensity of the fluorescence is measured. 3. By using different fluorochromes for different antibodies, flow cytometers may be capable of 5- to 10-color immunophenotypic analysis. B. Multicolor flow cytometry is often utilized in evaluating vitreous samples, particularly for the detection and immunotyping of lymphoma. 1. In one report, it has an 82.4% sensitivity and 100% specificity. C. It has been used to characterize vitreous inflammatory cells. III. Polymerase chain reaction (PCR) (Fig. 1.36) A. Tremendously amplifies very small quantities of DNA so that they can be characterized for diagnostic purposes. B. Fundamentally, a three-step process that requires: 1. A DNA template that contains the target DNA region.

Modern Molecular Pathology Diagnostic Techniques

A

B

C

D

35

Fig. 1.34  Fluorescence in situ hybridization. A, Whole chromosome paint probe highlighting the two X chromosomes in a female cell. B, Chromosomes showing hybridization with subtelomere probes. The p arm subtelomere is indicated by the green signals, and the q arm subtelomere is indicated by the red signals. Paired red and green signals are seen in the interphase nucleus to the left. C and D, Repeat sequence centromere probe for chromosome 21. C, Trisomy 21 seen at left in an interphase nucleus and at right in a metaphase (arrows). D, Normal complement of two copies of chromosome 21 (arrows) seen in interphase (left and right) and in a metaphase (center). (From Stein CK. Applications of Cytogenetics in Modern Pathology. In: McPherson RA, Pincus MR, eds: Henry’s Clinical Diagnosis and Management by Laboratory Methods. St. Louis, Elsevier, 2017. pp. 1337–1359. Figure 69.6.)



2. At least two primer pairs (the sense and antisense strands of the DNA target). 3. Thermus aquaticus DNA polymerase (Taq polymerase). 4. Various agents, such as deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP) to be amplified. C. The PCR process, itself: 1. Denaturation: The reaction mixture is heated to separate the target DNA into its two component strands. 2. Annealing: The mixture is cooled to permit the primers to anneal to the target DNA in a sequencespecific manner. 3. Extension: The DNA polymerase initiates extension each primer at its 3’ end. 4. The products of the extension of each primer are heated to separate the newly formed double-stranded DNA so that the original DNA and the newly formed



extension strands can act as templates for the next annealing and extension cycle. 5. The cycle repeats. D. Reverse-transcription PCR starts with RNA as the amplification target. Complementary DNA (cDNA) is first produced from the RNA through reverse transcription and then the cDNA is amplified by PCR.



E. PCR can be extremely valuable in identifying microorganisms from extremely small samples, such as a vitreous sample removed by vitrectomy from a patient with uveitis or endophthalmitis. IV. Microarray gene profiling A. Microarrays are a tool to achieve high throughput gene expression profiling.

36

CHAPTER 1  Basic Principles of Pathology



Fig. 1.35  Structural components and function of the flow cytometer. A, Fluorochrome-labeled monoclonal antibody solutions are added to a cell suspension from peripheral blood, bone marrow aspirate, or a lymph node. The tubes are incubated at room temperature for a short time. B, Labeled cell suspensions are passed through the flow cell of a flow cytometer. Many flow cytometers are automated, but some models require the operator to process the tubes individually. More than 10,000 cells from each tube are typically analyzed to produce statistically valid information. C, Each cell passes individually through the highly focused laser beam of the flow cytometer, a process termed single cell analysis. The fluorochrome of each labeled monoclonal antibody attached to the cell is excited by the laser light and emits light of a certain wavelength. The cells also scatter light at multiple angles. Photodetectors placed at forward and right angles to the axis of the laser beam collect the emitted or scattered light. Forward and right angle scatter signals, and as many as five fluorochrome signals can be detected from each cell (multiparametric analysis). D, The signals from each photodiode are digitized and passed to a computer for storage, display, and analysis. Typically, all data recorded from each cell are stored for possible later recall for further analysis (list mode data storage). E, A variety of histograms for visual display can be generated automatically or at the discretion of the operator. List mode data can also be transferred to a separate computer for analysis. Presently, most commercial flow cytometers utilize a standardized file format for list mode storage, and a variety of computer programs are commercially available for data analysis and display. (From Riley RS: Laboratory evaluation of the cellular immune system. In: McPherson and Pincus eds: Henry’s Clinical Diagnosis and Management by Laboratory Methods, 23rd edn, Elsevier, 2017. Chapter 45, 890–912. Figure 45-3.)

B. Microarray gene chips are comprised of a solid silica surface on which thousands of oligonucleotides are covalently linked. C. Two chips usually are sufficient to cover the entire complement of the genes and expressed sequence tags in the mammalian genome. D. This technology has been used to characterize gene expression in experimental models of eye growth. E. Microarray technology has been very useful in characterizing tumors, such as uveal malignant melanoma and in prognostication for them. V. Next generation sequencing A. Next generation sequencing (NGS) refers to wholegenome sequencing (WGS), whole-exome sequencing (WES), and RNA profiling (RNA-Seq). B. NGS encompasses many different massive parallel sequencing strategies that can process millions of reactions, producing thousands or millions of sequences concurrently. 1. Using these techniques entire genomes can be investigated. C. Genome-wide association studies (GWAS), as the name implies, correlate the presence of single nucleotide polymorphisms (SNPs) on noncoding intronic chromosome regions with relatively nearby genes suspected of being causative of a specific anomaly or disorder. The SNPs’ relationship to the disorder is inferred by being nearby to a gene known to be associated with the given pathology, but its specific involvement is not necessarily confirmed by this association. 1. GWAS compares the frequency of alleles of a group of people with a particular disease (cases) to that of another group without the disease (controls). 2. WES examines exon regions of the chromosome that code for proteins. 3. Although these exon segments represent only 1% of the genome, the vast majority of inherited diseasecausing mutations are located within these protein coding regions. Therefore, these regions are more likely to be directly related to the disease process, and are more easily and directly identified utilizing WES. 4. GWAS has been useful in identifying genes associated with age-related macular degeneration, eye color, refractive error, retinal microcirculation, central corneal thickness, and primary angle-closure glaucoma. D. NGS has been employed to identify genes mutated in inherited retinal degenerations. Similarly, WES can be helpful in this regard when prescreening of specific genes for inherited retinal disease, or when targeted NGS of a larger set of these genes does not reveal causative variants in order to reveal “novel” genes. E. WES has identified mutations associated with conjunctival melanoma, which may be targeted for therapy.

Concluding Comments

37

Fig. 1.36  Polymerase chain reaction. (Redrawn from Wolk D, Mitchell S, Patel R: Principles of molecular microbiology testing methods. Infect Dis Clin N Am 15:1157–1204, 2008. From Nolte et al.: Polymerase chain reaction and other nucleic acid amplification technology. In: McPherson and Pincus eds: Henry’s Clinical Diagnosis and Management by Laboratory Methods, 23rd edn, Elsevier, 2017. Chapter 67, 1316–1327. Figure 67-1.)



F. WES also has proven useful for studying inherited retinal diseases, genes associated with strabismus, and other disorders.

CONCLUDING COMMENTS This chapter began with an emphasis on the role that communication must play in the relationship between the pathologist

and the patient’s clinical team. There is no better example of this principle than in the field of molecular pathology. The clinician must communicate her/his concerns to the pathologist so that a diagnostic strategy can be devised based upon the patient’s particular clinical presentation and suspected diagnoses.   References available online at expertconsult.com.

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