Ординатура / Офтальмология / Английские материалы / Essentials in Ophthalmology Cornea and External Eye Disease_Reinhard_Larkin_2007

Due to a combination of replicating virus and severe inflammatory response the lesion tends to be destructive and often refractory to treatment, especially where there is a superadded or associated bacterial infection [100].

7.5.8 Endotheliitis

There are conflicting views on whether HSV-1 endotheliitis is primarily due to infection or a herpetically induced abnormal immune response against the endothelium. Three phenotypic forms have been identified according to the pattern of endothelial disease: disciform, diffuse, and linear. The endothelial function may take many months to recover. It may be very difficult to distinguish between stromal inflammation and stromal edema secondary to endotheliitis.

A disciform type of endotheliitis is the most common form and is seen as a discshaped area of stromal edema in the central or paracentral cornea, usually involving the entire stromal thickness, giving a ground-glass appearance (Fig. 7.6). Keratic precipitates (KP) are present underlying the areas of stromal edema and mild to moderate iritis is usually present. A secondary rise in intraocular pressure may occur, which has been attributed to associated trabeculitis [4].

Less common are diffuse endotheliitis and linear endotheliitis. In the former there is diffuse stromal edema with underlying KP and mild to moderate iritis. A dense retrocorneal plaque of inflammatory cells accompanied by a hypopyon may be present. With linear endotheliitis, a serpiginous line of KP progresses centrally from the limbus accompanied by peripheral stromal and epithelial edema between the limbus and the KP [132].

7.5.9Iridocorneal Endothelial Syndrome

Iridocorneal endothelial (ICE) syndrome is typically a unilateral condition characterized by a corneal endothelial abnormality often associated with corneal edema, angle anomalies, alteration in the iris structure (presenting as corectopia), and secondary glaucoma (Fig. 7.7).

Initially thought to be a developmental disorder, it is now thought to be of viral origin as a result of observations that disease onset occurs during the postnatal period and that the endotheliopathy resembles that seen in viral disorders with chronic inflammatory cells confined to the endothelial layer [3].

The disease manifestations are thought to be secondary to abnormal endothelial cells that can

migrate across the trabecular meshwork and iris surface. It has been proposed that HSV-1 may play a role in altering these cells by transferring some unknown genes or gene fragments [69]. Indeed, HSV-1 DNA has been isolated from the aqueous and endothelium of some patients with idiopathic corneal endotheliopathy and iridocorneal endothelial syndrome. Some of these patients also respond to antiviral therapy [3].

7.5.10 Uveitis

HSV-1 uveitis reflects uveal inflammation related to the viral infection. The uveitis is usually associated with corneal stromal disease, but around 15% of cases of HSV-1 anterior uveitis may occur in the absence of corneal involvement [116]. Clues to a diagnosis of herpetic anterior uveitis include unexplained corneal scarring, decreased corneal sensation (50% of patients), focal or diffuse patchy iris atrophy, iris transillumination defects, anterior and/or vitreous cells, granulomatous or non-granulomatous keratic precipitates, posterior iris synechiae, and elevated intraocular pressure (Fig. 7.8). Sixty-five to ninety

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percent of patients with suspected HSV uveitis have been found to have a distorted or dilated pupil. It is usually unilateral (92%) and may follow a prolonged course with recurrent exacerbations [116].

Elevated intraocular pressure during intraocular inflammation is common, occurring in 30–90% of patients in various studies, compared with 18% for anterior uveitis in general [156, 181, 198]. HSV-1 has been suggested to be involved in the pathogenesis of PosnerSchlossman syndrome (also known as glaucomatous cyclitic crisis). Yamamoto and colleagues detected evidence of HSV-1 DNA from aqueous samples of 3 patients during acute attacks of the syndrome [210]. Some authors have therefore recommended an aqueous biopsy in all presumed cases of Posner-Schlossman syndrome [198].

Visual outcome is generally favorable with 94% (29 out of 31) in one study, retaining a visual acuity of 20/32 or more [198]. Primary herpetic uveitis (without a history of previous keratitis) seems to be more severe than uveitis in patients with previous corneal recurrences and the associated glaucoma may be a devastating complica-

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tion. Absence of corneal involvement may delay diagnosis and therefore worsen visual outcome [156]. Cataract is also a common long-term complication, occurring in 20% of patients. Treatment of herpetic uveitis is discussed in Sect. 7.6.

Summary for the Clinician

■Human simplex keratitis can present with a variety of clinical manifestations secondary to live viral infection, immune and inflammatory response, and their sequelae

■Human simplex virus-1 induced keratitis can be broadly divided into epithelial, stromal, and endothelial disease each with infectious and immune elements. Anterior segment disease also includes uveitis and iridocorneal endothelial syndrome

■The incidence of primary HSV infection has two peaks: 1–5 years and again between 16 and 25 years. Primary disease is probably subclinical in approximately 50%. The disease is usually mild and stromal disease and uveitis are rare

■In neonates primary disease is usually accompanied by viremia and eye disease may be just one manifestation of disseminated disease. Active ocular infection occurs in 20–25% of neonates with HSV-1 infection

■A reasonable estimate of the recurrence rate of HSK after a single episode is 10% per year. The incidence of bilateral disease varies from 1.3 to 12% and is more common in younger patients

■In children, the incidence of bilateral disease is around 16%. They also tend to suffer more frequent and severe recurrences – approximately 50% within the first 2 years

■Herpetic stromal disease accounts for 2% of initial presentations and 20–48% of recurrent herpetic disease

7.6 Pathogenesis of HSK

7.6.1Pathogenesis

of Herpetic Keratitis

Corneal, conjunctival or lid epithelial disease is initiated by infectious virus that replicates in the epithelial cells and studies in animal models have shown the requirement of viral replication for this phase [9]. A threshold effect is observed for the inoculum size; in that once infection is initiated it proceeds to completion unless the viral load is reduced [87]. The severity of disease tends to be dependent on the age of the patient. Animal studies have shown that younger mice develop more severe herpetic eye disease [82]. As discussed in Sect. 7.4.1, disease severity is also dependent on the HSV-1 strain.

7.6.1.1 Viral Component

The persistence of HSV-1 in the cornea is important for understanding the pathogenesis of and treating HSK, in particular herpetic stromal disease. For example, it has been suggested that an epitope encoded by the UL6 gene of HSV-1 can mimic a corneal antigen (IgG2ab Ig isotype) and initiate an immune attack on corneal antigens [8, 105, 212], although there is evidence against this [48]. It is also possible that antigen expression may result from host transcription of viral DNA and class I MHC presentation of the viral protein [55, 194]. The presence, however, of an active inflammatory infiltrate in the corneas of patients with HSK, together with the finding of persistent HSV-1 DNA and antigen [193], makes either a low-grade productive infection or lowgrade reactivation from a latent virus a more likely possibility. This association between longterm persistence of HSV-1 in ocular tissues and chronic inflammation has been demonstrated in several animal studies [9, 105, 117].

The likelihood of HSV-1 persistence either as a low-grade infection, following repeated reactivation from latency or from entry into the cornea from sensory nerves justifies the use of antivirals when treating all forms of herpetic keratitis.

7.6.1.2 Immune Component

Corneal epithelial disease induces an influx of innate immune cells such as polymorphonuclear leucocytes (PMN), macrophages, natural killer cells, and other mononuclear cells into the underlying stroma. Pro-inflammatory cytokines such as Interleukin (IL)-1α, IL-1β, IL-8, IL-6, Interferon (IFN)-α, Tumor Necrosis Factor (TNF0)- α, macrophage inflammatory protein (MIP)-2, monocyte chemoattractant protein (MCP)-1, IL-12, and MIP1-α are released by the infected and neighboring cells as well as PMN [23, 176]. Langerhans cells present the HSV-1 antigens to T cells in draining lymph nodes, which are attracted by chemotactic cytokines. A transient corneal clouding may occur due to the toxic effect of cytokines on the endothelial cells. The viral reflux and PMN influx subside by days 4–5 post-infection and it is felt that PMN contribute to virus removal [184].

The importance of the immune response in stromal keratitis is apparent from the observations that in T-cell deficient mice, ocular infection with HSV-1 was not associated with the development of marked stromal disease [111, 175]. Immune response to HSV-1 infection can include T cell-mediated delayed-type hypersensitivity that may be critical to the elimination of the virus, cytotoxic T lymphocytes that may prevent viral spread, and IgG antibodies, which can neutralize the virus. The CD4+ Th1 cells are most commonly implicated and secrete IL-2, INFγ, and TNFα, which are mediators of inflammation. CD8+ cytotoxic T lymphocytes have also been linked to the increased incidence and severity of HSK.

Confusion still surrounds the role of CD4+ Th2 cells, which secrete IL-4, IL-5, IL-6, and IL-10, and have been shown to produce HSK in animal models. HSK can also result from an antibody-dependent, complement-mediated inflammatory insult. Natural killer cells display surface receptors for the Fc component of IgG and may mediate antibody-dependent-cell-me- diated-cytotoxicity (ADCC). Further support for this mechanism comes from observations that immunization of mice with glycoprotein K results in more severe keratitis upon subsequent

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infection [23, 175]. HSV-1-specific T lymphocytes may also persist in corneas and memory T and B lymphocytes specific to HSV-1 circulate in the blood and lymph armed for the next encounter with the virus.

There is evidence that a chronic low-level viral infection persists in the stromal keratocytes, providing a continuous stimulus to antigen-specific T lymphocytes that kill the keratocytes by a cytotoxic mechanism [26].

Several hypotheses exist regarding the agents that drive the CD4+ T cells to orchestrate the inflammatory process. There is a role for the bystander activation of T cells, which are dependent on continuous viral replication with the release of cytokines [44].

7.6.2Pathogenesis of Herpetic Uveitis

Eighty-five percent of cases of HSV intraocular inflammation present as iridocyclitis, while approximately 15% present as posterior segment inflammation including panuveitis and retinitis (acute retinal necrosis) [116]. Although not fully understood, the pathogenesis is thought to involve viral replication within intraocular nerves, ischemic vasculitis, and lymphocytic infiltration of the iris stroma. Epidemiological and experimental data suggest that oropharyngeal primary herpetic infection may lead to eye disease later in life.

7.6.2.1 Viral Component

As discussed above, HSV1 inoculation of the lower lip of mice has been shown to lead to latent infection of the entire trigeminal ganglion, including the ophthalmic division [191]. Recurrent disease may occur due to anterograde axonal spread of previously dormant HSV-1 virus from the trigeminal ganglion or from the cornea [80]. The presence of persistent viral infection may cause an inflammatory reaction manifested as uveitis, or can trigger the immune system itself against viral antigens, causing it to cross-react with uveal tissue and cause inflammation [98].

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Herpetic uveitis may occur in the absence of keratitis, suggesting involvement of the autonomic nerves supplying the iris and ciliary body rather than the trigeminal nerve [179]. This theory is supported by post mortem studies, which have demonstrated latent herpetic infection of the central nervous system and autonomic nerves [52, 57, 201, 202].

A study by Labetoulle et al. [90] on mice demonstrated rapid propagation of HSV1 from oral mucosa to uveal tissues and cornea within 6–8 days post oral inoculation, which appeared to occur via the sympathetic nerves and superior cervical ganglion much more rapidly than via the trigeminal nerves or parasympathetic system. At 6 days post-inoculation, the entire ipsilateral superior cervical ganglion was infected, including neurons innervating the iris and ciliary body. The observation that the iris was always infected before the cornea supported the theory that eye disease resulted from the spread of virus from the nervous system [90].

7.6.2.2 Immune Component

Herpetic intraocular inflammation is characterized by the presence of predominantly mononuclearcellsmainlycomposedofTlymphocytes with smaller numbers of macrophages and plasma cells also present. The immune privilege of the eye means that it constitutively expresses immunomodulatory cytokines such as transforming growth factor (TGF)-β and macrophage migration inhibitory factor, as well as other factors, which act to suppress T cell responses and those of other cells such as natural killer (NK) cells. These factors do not, however, affect the humoral immune responses.

A recent study has suggested that eyes with HSV uveitis contain antibodies with different virus protein targets from those found in the serum, thus exhibiting a compartmentalized B cell response to HSV ocular infection [141].

Another study found that NK cells were activated following intraocular inoculation of murine eyes with the KOS strain of HSV-1 [182]. It was suggested that NK cells play a role in preventing direct anterior to posterior spread of HSV in the eye, explaining why the ipsilateral eye did

not develop experimental acute retinal necrosis. The NK response was thought to have both a local (eye, superficial cervical and submandibular lymph nodes) and systemic response (spleen).

The reason why the ocular immunomodulatory factors do not control NK activity is thought to be due to rapid viral replication outstripping the eye’s ability to produce these factors.

7.6.2.3Anterior Chamberassociated Immune Deviation

Ocular immune privilege results in part from induction of a deviant form of systemic immunity, termed anterior chamber-associ- ated immune deviation (ACAID), which is another component that may modulate the ocular immune response to HSV infection. ACAID mitigates ocular autoimmune diseases and promotes corneal allograft survival. This is an animal model of immune tolerance, in which antigen injected into the anterior chamber of the eye is internalized by intraocular F4/80-positive antigen-presenting cells (APC) such as intraocular dendritic cells and macrophages [102], which process antigen, migrate across the trabecular meshwork and then to the thymus and spleen. In the spleen they induce the differentiation of antigen-specific B cells that act as ancillary APCs and are required for ACAID induction. These in turn induce the generation of CD4-positive and CD8-posi- tive regulatory T cells resulting in tolerance to the antigen [7]. HSV-1 and HSV-2 have been shown to induce ACAID in animal models [2]. ACAID interferes with both Ag-specific delayed type hypersensitivity (DTH) and complementfixing antibody production.

It is thought that DTH may play a role in reducing viral-induced anterior and posterior uveitis. Indeed, in the case of varicella zoster virus (VZV), it has been shown that patients with VZV-induced anterior uveitis and acute retinal necrosis (ARN) had lower skin DTH responses to VZV antigen compared with patients with VZV-induced skin disease alone (control group) [86].

7.6.3Recurrence: Reactivation and Super-infection

As mentioned previously, a reasonable estimate of the recurrence rate of HSK after epithelial or stromal disease is approximately 10% per year. There is recurrence despite acquired immunity. A variety of stimuli may lead to viral reactivation, such as local injury, physical and emotional stress, fever, menstruation, exposure to ultraviolet light, and immunosuppression, although there is some disagreement whether these stimuli lead to recurrent ocular disease [17, 43, 65]. The molecular events leading to efficient reactivation center around ICP0 and LAT, as discussed in Sect. 7.3.4.2. Reactivated virus then either produces local disease if latent in the cornea, or travels down the sensory nerve to replicate in mucocutaneous epithelia, such as the conjunctiva, cornea or lid margin.

Super-infection with a different HSV strain has been noted for both genital and ocular disease. Remeijer et al. found that a third of corneas with recurrent herpetic keratitis (RHK) were su- per-infected with a different HSV-1 strain and 63% with the same strain of RHK [148, 149]. One patient in their study with bilateral HSK had different strains in each cornea. Although it would appear that super-infection occurs in the cornea, it is not clear whether this translates to super-in- fection within the trigeminal ganglion. Expression of LAT appears to interfere with super-in- fection by other HSV-1 strains [104] and latency in the trigeminal ganglion is accompanied by a chronic immune response, which may protect from super-infection [183].

7.6.4Corneal Scarring and Vascularization

The pathogenesis of corneal scarring and vascularization in HSK is still uncertain.

7.6.4.1 Immune Component

CD 8+ or both in corneal scarring [59]. It has

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also been suggested that Th1 (IL-2) responses may protect and Th2 (IL-4) responses may enhance scarring [58]. Similar controversies exist on the role of NK cells and macrophages [60]. One of the major events after HSV infection is the production of proinflammatory cytokines and chemokines, and an invasion of the cornea by PMN. This response helps to clear the virus, but at the same time lends entry to various cytokines and angiogenic factors secreted by the PMN.

IL-1 and -6 are important mediators of inflammation and a beneficial effect of IL-ra (in- terleukin-1 receptor antagonist) has been noted on disease severity and corneal scarring [18]. IL-10 and -12 can also suppress HSK lesions in animal models, possibly by the induction of antiviral cytokines such as interferon γ, and cellular defenses like NK cells and T cell apoptosis. IL-12 also has an antiangiogenic effect [94]. Co-ordi- nated phenotypic changes, extracellular matrix (ECM) deposition, and remodeling are the key elements in the process of tissue repair, as in corneal scarring.

7.6.4.2 Healing Response

Various cytokines and growth factors are involved in corneal wound healing and the most important of these are epidermal growth factor (EGF) and transforming growth factor β (TGFβ) [158]. TGFβ up-regulates fibroblast proliferation and ECM synthesis and reduces ECM degradation after injury.

One of the major challenges has been in understanding the differences between fetal (scar-free) and adult wound healing (with scarring). It is now known that “matricellular proteins,” a group of disparate proteins expressed during development, but not in adults, are upregulated in sites of tissue re-modeling and act temporally and spatially to provide regulatory signals in cell–cell and cell–matrix interactions [19].

One matricellular protein that has been extensively studied in the corneal in vivo models is the platelet-derived glycoprotein thrombospondin (TSP). Thrombospondins are a family of five glycoproteins, two of which, TSP 1 and TSP 2, are

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involved in wound healing and are potent antiangiogenic agents [5, 20]. Given that TSP 1 and 2 play an important role in corneal scarring and vascularization the next question is, what is their source in the cornea?

One mechanism could be by invading blood vessels in the cornea, which have been shown to appear as early as 24 h after infection in vivo [95]. Scarring also develops in an avascular cornea, where fibroblasts and not platelets are the predominant cells. This has led to the search for a local reservoir of TSP in the cornea. Hiscott et al. have shown this reservoir to consist of keratocytes that express TSP 1 and 2 in an in vitro stromal wound repair model [68]. TSP 1 acts by modulating cellular responses to ECM and can also bind and activate TGFβ [20].

Corneal vascularization in HSK requires active viral replication and procedures that minimize angiogenesis may diminish the severity of HSK [95, 213, 214]. HSV-1 differs from other viruses in that it does not encode molecular mimics of any known angiogenic factors. This suggests

that HSV-1 infection may disrupt the normal equilibrium between angiogenic and anti-angio- genic stimuli, leading to an “angiogenic switch,” initiating angiogenesis [166]. HSV-1 infection can induce the production of many angiogenic factors such as vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMP) 2 and 9, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), MIP-2 and MCP-1 [95, 211, 213].

Hypoxia due to corneal edema may also serve as an angiogenic stimulus. The source of these factors seems to be mainly PMN, but VEGF is also expressed in epithelial, endothelial, and stromal cells, as well as in macrophages [213]. These angiogenic responses are balanced by antiangiogenic factors such as transforming growth factor β, tissue inhibitors of metalloproteinase (TIMP), and TSP 1 and 2 [5, 211]. As discussed earlier TSP 1 and 2 are expressed by keratocytes and can bind and inhibit the activity of βFGF, VEGF, MMP2, and MMP9 [5]. HSV-1 infection can selectively suppress matrix protein synthesis and it has been

shown that TSP 1 and 2 are selectively downregulated by infection in human keratocytes in vitro (Fig. 7.9) [30]. It has also been shown that keratocytes expressed VEGF after infection and that its production continued until after TSP 1 and 2 production had ceased (Choudhary et al., unpublished findings).

These findings support the hypothesis of alteration of the normal balance between angiogenic and antiangiogenic responses being the likely cause of corneal vascularization in HSK and may form the basis for the next generation of treatment options for this condition [80].

Summary for the Clinician

■It has been suggested that an epitope encoded by the UL6 gene of HSV-1 can mimic a corneal antigen and initiate an immune attack on corneal antigens, although low-grade infection/reactivation remains more likely

■Herpetic uveitis can occur in the absence of keratitis in approximately 15%, suggesting involvement of the autonomic nerves supplying the iris and ciliary body rather than the trigeminal nerve. Elevated IOP during herpetic anterior uveitis is common, occurring in 30–90% of patients compared with 18% for anterior uveitis in general

■Herpes simplex virus has been shown to induce anterior chamber-associated immune deviation (ACAID), which in turn inhibits both antigen-specific delayed type hypersensitivity and complementfixing antibody production. It is thought that patients with lower DTH responses are more likely to develop herpetic anterior­ uveitis

■The pathogenesis of corneal scarring and vascularization are still uncertain, but are likely to involve various cytokines and growth factors such as epidermal growth factor and transforming growth factor β

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Summary for the Clinician

■The matricellular proteins thrombospondin 1 and 2 are involved in woundhealing and are potent antiangiogenic agents thought to play an important role in modulating corneal scarring and vascularization. They have been demonstrated in keratocytes in an in vitro model of stromal wound repair

■Corneal vascularization requires active virus replication and procedures that minimize angiogenesis may diminish the severity of HSK. As HSV-I does not encode for any angiogenic factors it has been suggested that infection may disrupt the normal equilibrium between angiogenic and antiangiogenic stimuli, leading to an “angiogenic switch” initiating angiogenesis

■HSV-1 infection can induce the production of many angiogenic factors, primarily by neutrophils but also epithelial, stromal, and endothelial cells. Also, HSV-1 infection has been shown to selectively suppress matrix protein synthesis of TSP 1 and 2 in an in vitro model, thereby supporting the theory of an “angiogenic switch”

7.7 Diagnosis

7.7.1 Laboratory Diagnosis

An attempt should always be made to isolate or identify HSV-1. This may help avoid complications from misdiagnosis and inappropriate treatment. This is important not only because certain other conditions can mimic HSV-1 ocular disease, but also because it allows strain characterization and provides information regarding mutations and super-infection by other strains. Laboratory tests are aimed at cell cytology, viral antigen detection (immunoassays), viral DNA detection (polymerase chain reaction), and virus isolation (tissue culture).

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Cytology is a simple method that can complement other, more specific tests. It can be carried out using cellular material obtained from corneal/conjunctival lesions smeared on a glass slide and fixed in ethanol. These can then be stained with Papanicolaou, Giemsa or Wright stains. Presumptive diagnosis is based on the presence of intranuclear inclusions and multinucleated giant cells. It has, however, a low sensitivity (57%) and should not be used on its own [177].

Herpes virus antigen can be detected by enzymeor fluorescence-based immunohistochemical techniques. This is a quick technique with good sensitivity, but is prone to false-posi- tive and -negative results. PCR is used for the detection of HSV-1 DNA and carries a sensitivity of up to 100%. Conventional PCR is a qualitative method, very useful for detecting evidence of HSV-1 in the tear film, conjunctival, cornea, and aqueous, but does not differentiate between latent and infectious virus. Competitive PCR on the other hand is quantitative, but it is complicated and time-consuming [49, 177]. Real-time PCR has now been developed, which is a quantitative method and is quicker and more sensitive. It can provide indirect evidence of virus replica-

tion by the number of DNA copies produced and hence can also be used for evaluating the efficacy of antiviral medications [110]. Fukuda et al. described PCR results from tear samples of patients with herpetic keratitis [54].

Viral DNA isolation was 100% in epithelial disease and persistent epithelial defect, 57% in disciform keratitis, and 0% in silent stromal and endothelial disease [54].

Isolation of HSV-1 in culture is the most reliable and specific method and is considered the gold standard for the diagnosis of HSK. Clinical specimens are inoculated into cell culture systems (e.g., corneal epithelial cells, foreskin fibroblasts, Vero cells), which are then observed for cytopathic effects characteristic of virus replication (Fig. 7.10). These effects develop within 24– 48 h. Various techniques such as conventional tube culture and centrifugation enhancement of HSV replication (shell vial assay) can be used for isolation. Although viral culture carries a high specificity (100%), its sensitivity is very low. Virus isolation rates in one study were as low as 8.2% [177]. As each of the aforementioned tests detects a different component of the virus and is insufficient to confirm a diagnosis on its own, a combination of tests is recommended. PCR and