Experimental herpetic keratitis in rabbit corneal organ cultures

An in-vitro method for maintaining rabbit corneal organ cultures for short periods of time is described. These cultures supported replication of strains of type 1 and 2 herpes simplex virus, and changes typical of herpetic infections of the cornea in vivo were observed by light microscopy, and by scanning and transmission electron microscopy. Infectious virus was readily recovered from inoculated corneal organ cultures. For practical, economic and humanitarian reasons, the full potential of this system warrants further investigation.     

Resistance to herpetic stromal keratitis in immunized B-cell-deficient mice

This study evaluates the role of antibody as an indicator of immunity to ocular challenge with herpes simplex virus (HSV). Two genotypes of mice, BALB/c or BALB/c with mu-chain knockout (muK/O; which lack functional B cells), were immunized systemically either with nonvirulent infectious virus or with a eukaryotic expression plasmid encoding glycoprotein B (gB). Whereas naive muK/O mice were 10- to 100-fold more susceptible to HSV infection than BALB/c mice, following immunization both groups showed similar levels of resistance to ocular challenge. Thus both HSV-immunized groups cleared virus within 3 days and showed no signs of ocular lesions. gB DNA-immunized mice cleared virus less rapidly (5 days), and the incidence of lesions was 10 and 25% in BALB/c and muK/O mice, respectively. Since muK/O mice failed to produce detectable anti-HSV antibody, the mechanism of rapid viral removal was assumed to have a T cell basis. However, T cells would likely not mediate any protection directly since such cells were absent in infected corneas during clearance. A likely mechanism of immunity could involve innate defenses, perhaps enhanced by the action of cytokines released from antigen-reactive CD4+ cells in vascularized tissue adjacent to the cornea. Thus an abrupt inflammatory response consisting principally of neutrophils occurred in the corneal stroma in immune mice, and this subsided when virus disappeared. These data reveal that even though the deficiency in generating antibody renders mice more susceptible to HSV infection, once primed, resistance to disease expression is mediated solely by the cellular components and their products.     

Experimental herpetic keratitis in rabbit corneal organ cultures.

An in-vitro method for maintaining rabbit corneal organ cultures for short periods of time is described. These cultures supported replication of strains of type 1 and 2 herpes simplex virus, and changes typical of herpetic infections of the cornea in vivo were observed by light microscopy, and by scanning and transmission electron microscopy. Infectious virus was readily recovered from inoculated corneal organ cultures. For practical, economic and humanitarian reasons, the full potential of this system warrants further investigation.     

Improvement of herpetic stromal keratitis with fumaric acid derivate is associated with systemic induction of T helper 2 cytokines

Fumaric acid derivates have been shown to stimulate T helper-2-cytokines (interleukin (IL)-4, -5) without affecting the T-helper-1-cytokine (IL-2, interferon (IFN)-gamma)-response. Herein, the influence of systemic treatment with the fumaric acid derivate dimethylfumarate (DMF) on the secretion of T helper-cytokines and the development of HSV-1 stromal keratitis (HSK) was studied in mice. The corneas from BALB/c mice were infected with 10(5) PFU of HSV-1 (KOS strain). While one group of mice was treated intraperitoneally with PBS, another group of mice received DMF at 15 mg/kg of body weight. Expression of IL-2, -4, -10 and IFN-gamma was analysed in HSV-1 activated lymphocytes by ELISA. The severity of epithelial and stromal herpetic keratitis was investigated clinically. Corneas were studied for the inflammatory cell infiltration, and the CD3-, CD4- and CD8-positive cells were analysed by immunohistochemistry. The IL-2, -4, 10 and IFN-gamma content was measured in the corneas. Virus replication in the eyes was analysed by a plaque-assay. The DTH-response, the HSV-specific T cell proliferation and the serum neutralizing antibody-titres were investigated. DMF increased IL-4 and IL-10, but not IL-2 and IFN-gamma, secretion in activated lymphocytes from the spleen. Incidence and severity of stromal HSV-1 keratitis was reduced in the DMF group (P < 0.01). In the corneas from DMF-treated mice, the numbers of CD3+ and CD4+ cells were decreased and IL-4 was increased. Severity of epithelial disease and the virus-clearance from the eyes did not differ between the PBS and DMF group of mice. DTH, HSV-specific T cell proliferation and the neutralizing antibody-titres were not impaired. DMF increased the T helper-2-cytokine secretion in activated lymphocytes. After corneal HSV-1 infection, corneas from DMF treated mice had increased IL-4 content. This is associated with an improvement of herpetic stromal keratitis and reduced corneal T cell infiltration. DMF did not impair the systemic antiviral response.     

Therapeutic potential of bevacizumab (Avastin) in herpetic stromal keratitis (HSK)

In the stromal keratitis caused by herpes simplex virus (HSV), the formation of new vessels is the essential step for the pathogenesis of keratitis. Inhibition of angiogenesis diminishes the formation of corneal lesion induced by HSV. Procedures which suppress angiogenesis are proposed as a valuable therapeutic approach to control HSK. The mechanism by which HSV ocular infection results in corneal angiogenesis is not understood. Recent reports identified anti-vascular endothelial growth factor (VEGF) as a molecule that is highly expressed in the HSV infected eye and clearly involved in angiogenesis. The advent of VEGF treatments marks a major advancement in the treatment of angiogenic eye disease. Off-label use of bevacizumab (Avastin), a recombinant humanized monoclonal antibody directed against VEGF, in some neovascular disorders of the eye has been associated with promising short term results. Based on these evidences herein we hypothesize topical application of bevacizumab could inhibit corneal neovascularization and also scarring in HSK. We propose this drug as a novel adjunct to current anti-inflammatory strategies in HSK.     

Role of Stat4-mediated signal transduction events in the generation of aggressor CD4+ T cells in herpetic stromal keratitis pathogenesis.

Ocular infection with herpes simplex virus (HSV) causes a vision-impairing inflammatory reaction called herpetic stromal keratitis. In murine models, herpetic stromal keratitis lesions appear to be immunopathologic, mediated by CD4(+) T cells of Th1 phenotype. To provide insight about cytokine networks and signaling events involved in the development of aggressor CD4(+) T cells, ocular HSV infection was followed in mice deficient in Stat4 (Stat4(-/-) mice), the signal transducer for the cytokine interleukin-12 (IL-12). After ocular HSV infection of Stat4(-/-) and control BALB/c mice, clinical, histologic, and immunologic analyses were carried out. Further, to evaluate the involvement of Stat4 in the development of this aggressor population, naive CD4(+) T cells from Stat4(-/-) and BALB/c mice were adoptively transferred to C.B-17 SCID mice 1 day after corneal infection. Although Stat4(-/-) mice demonstrated increased susceptibility to lethal encephalitis and facial lesions, interestingly, these mice had less severe stromal keratitis in comparison to control animals. Adoptive transfer of naive CD4(+) T cells from Stat4(-/-) mice failed to produce disease in infected SCID recipients. The data imply a significant role of Stat4-mediated signaling events in the generation of an aggressor CD4(+) T cell population in stromal keratitis pathogenesis.     

Treatment with meliacine, a plant derived antiviral, prevents the development of herpetic stromal keratitis in mice

Herpetic stromal keratitis is caused by ocular infection with herpes simplex virus type 1 (HSV-1) and constitutes a leading cause of human blindness. The effect of meliacine, an antiviral compound isolated from leaves of Melia azedarach L. that inhibits HSV-1 replication in vitro, was examined on experimental corneal HSV-1 inoculation in Balb/c mice. Mice were inoculated with HSV-1 strain KOS at their corneas after abrasion. Meliacine was administered topically 3 times a day for 4 days beginning 1 day before inoculation. Infected animals treated or not with meliacine were observed carefully for the development of stromal keratitis and the clinical scoring was done 14 days post-infection. Histological examination of corneas and viral isolation from eyes from HSV-1 infected mice treated or not with meliacine were also carried out. It was found that the treatment of HSV-1-induced ocular disease in Balb/c mice with meliacine reduced significantly the development of clinical disease, as well as the histological damage in corneas. The viral titers detected in eyes of infected and treated mice were 2-orders-of-magnitude lower than those corresponding to HSV-1 infected control animals. Mock-infected and treated mice did not reveal any corneal alteration due to the administration of the compound. Meliacine was found to exert a strong antiviral action on HSV-1-induced ocular disease in mice with no evidence of toxic effects.     

Interleukin-10 (IL-10) ameliorates corneal disease in a mouse model of recurrent herpetic keratitis

Interleukin 10 (IL-10), a moderator of Delayed Type Hypersensitivity (DTH) responses, has been demonstrated to be present late in acute HSV corneal infection and may help limit blinding inflammatory lesions there. In contrast, IL-10 is present early in the development of recurrent herpetic stromal keratitis (HSK) lesions in mice. To determine the role of IL-10 and DTH responses in recurrent HSK, we examined DTH responses and disease parameters in latently infected IL-10 knock out (KO) mice, and latently infected normal mice that were untreated or received anti-IL-10 antibodies or recombinant IL-10 following ultraviolet-B stimulated ocular HSV recurrence. Low DTH responses were associated with less severe corneal disease while high DTH responses were associated with greater corneal disease. In IL-10 KO mice, and in normal mice given anti-IL-10 antibodies, corneal opacification was increased and DTH responses were significantly prolonged. Normal mice receiving rIL-10 by ocular and intra-peritoneal routes had less severe corneal lesions. Our results indicate that IL-10 and DTH responses play an important role in the pathogenesis of recurrent HSK in mice.     

TLR2 activation in corneal stromal cells by Staphylococcus aureus‐induced keratitis

The Toll‐Like Receptor 2 (TLR2) plays an active and important role in Staphylococcus aureus‐induced chronic ocular inflammation. The aim of this study was to investigate the expression and function of TLR2 of corneal stromal cells in ex vivo rabbit model of S. aureus keratitis. Corneal buttons with sclera rims placed in an ex vivo air‐interface organ culture were assigned to two groups: corneas with epithelial and stromal abrasions. Each group was then divided into two sub‐groups exposed to UV‐killed S. aureus ATCC 6538P and S. aureus ATCC 29213, respectively. TLR2 and IL‐8 mRNA expressions were analyzed by quantitative real‐time RT‐PCR. TLR2 localization was visualized by immunofluorescence analysis. The results demonstrated that TLR2 and IL‐8 mRNA were significantly expressed in the stromal cells of the groups exposed to S. aureus strains. Moreover, it has been demonstrated that, after corneal injury, keratocytes differentiated into myofibroblasts became able to express TLR2 only when exposed to S. aureus. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation. This ex vivo model can be used to clarify the molecular events of bacterial‐corneal tissue interactions and their inflammatory consequences.     

CD4(+) and CD8(+) cells are key participants in the development of recurrent herpetic stromal keratitis in mice

Ocular herpes simplex virus (HSV) infection results in an immune-mediated inflammation of the corneal stroma known as herpetic stromal keratitis (HSK). Recurrent HSK is a common cause of virus-induced corneal blindness in humans. The role of CD4(+) and CD8(+) T cell subsets in the disease pathogenesis is ill defined and varies with the virus strain and host genetic background. To examine the contribution of T cell subsets to corneal disease, we studied the development of recurrent HSK in CD4 or CD8 gene knockout (KO) mice ocularly infected with HSV-1 McKrae strain. Following UV-B induced viral reactivation, corneal opacity in latently infected BALB/c (HSV sensitive) CD4 and CD8 KO mice was reduced compared to infected BALB/c mice with normal genotype. In contrast, opacity in C57BL/6 (HSV resistant) CD4 and CD8 KO latent mice did not differ from genetically normal latent mice. Virus-induced corneal opacity was not demonstrable in C57BL/6 CD4/CD8 double KO mice. Increased viral shedding, measured by reactivation rate, days shedding or viral titers, occurred in CD4 KO mice of both strains. Our findings indicate that both CD4(+) and CD8(+) cells play a role in the immunopathogenesis of recurrent HSK, and their role is dependent upon the host genetic profile.     

MHC II-restricted, CD4+ cytotoxic T lymphocytes specific for herpes simplex virus-1: implications for the development of herpetic stromal keratitis in mice

Herpetic stromal keratitis (HSK) appears to represent an immunopathological reaction in which CD4+ T cells play a prominent role. However, the exact immunopathological mechanism(s) utilized by CD4+ T cells during HSK remains to be elucidated. In this study, the presence of cytotoxic CD4+ T lymphocytes in the cervical and retropharyngeal lymph nodes of Balb/c mice experiencing HSK was investigated. After in vitro depletion of CD4+ or CD8+ T cells with specific monoclonal antibodies and complement treatment, the cytotoxic functions of the remaining T cell populations were assayed by using target cells expressing either MHC Class I or both Class I and Class II. Our results showed the presence of a distinct cytotoxic T lymphocyte (CTL) population which was CD4+ and demonstrated lytic activity in a Class II-restricted fashion. Furthermore, these cells were able to develop into efficient effector CTL in the absence of CD8+ T lymphocytes as assessed by in vivo depletion experiments. Immunohistochemical methods were also utilized to show the presence of both CD4+ lymphocytes and I-A+ cells in the corneal tissues during HSK. These findings support the notion that direct lysis of infected Class II-bearing corneal cells by CD4+ CTL might be one of the mechanisms leading to stromal immunopathology in herpetic infections.     

Levamisole therapy: clinical and immunological evaluation in herpetic keratitis

Thirty patients with herpetic keratitis, recurrent in 17, were examined and submitted to an immunological evaluation for cell-mediated immunity (E rosette forming cells, absolute number of T lymphocytes, skin tests). A group of 15 patients was treated with Levamisole (2.5 mg/kg body weight, 3 days a week), while the control group received no Levamisole. The observation period varied from 6 to 24 months. The clinical follow-up showed a reduction in the severity and duration of herpetic attacks and in the frequency of relapses compared with the control group. The immunological findings indicated a normalization of E rosettes in all the patients (treated and controls).     

Interaction of the hormonal and immunologic impairments in men with herpetic keratitis

Data are described in the paper on the significance of hormonal and immunological impairments and on their interrelation within the pathogenesis of herpetic keratitis in males. An evaluation of contents of testosterone, cortisol, CD8+, CD95+ lymphocytes, interleukin-6 and tumor necrosis factor-alpha (TNF-alpha) as well as definition of interrelations between them has a prognostic value for males with herpetic keratitis. A dropping concentration of testosterone as observed in males with severe herpetic keratitis is indicative of the feasibility to add some hormone-correcting drugs to the combined therapy.     

Imbalance of alpha- and gamma-interferon levels in patients with herpetic keratitis

The paper presents data on elevated serum and lacrimal fluid alpha- and gamma-interferon (INF) levels in patients with herpetic keratitis (HK) in the course of the disease depending on its clinical form and stage. A more significant increase in the levels of alpha- and gamma-INF was noted in the lacrimal fluid, which was indicative of the important role of local antiherpetic corneal and conjunctival protection. The determination of serum and lacrimal fluid IFN levels in patients with HK may be used to predict the clinical course of the disease.     

Balancing autoaggressive and protective T cell responses

A finely orchestrated balance between activating and inhibitory signals is fundamental for the ability of the immune system to effectively attack and eliminate pathogenic microbes but to not react against self-antigens. Derangements of this balance underlie the pathogenesis of autoimmune diseases. Conversely, elucidating the mechanisms of this balance may provide rational strategies for manipulating it in order to enhance the efficacy of vaccines and tumor immunotherapy. One of the clearest illustrations of precise regulation is in the generation of effector and regulatory T cells. In order to analyze the mechanisms of this regulation, we have developed a transgenic mouse model in which a single population of T cells reacts against its known cognate antigen in vivo. Here we summarize our studies with this experimental model, illustrating the sequence of T cell responses that develop and attempting to dissect the stimuli that control these responses.     

Thymic stromal lymphopoietin in normal and pathogenic T cell development and function

Thymic stromal lymphopoietin, a four helix-bundle cytokine, is expressed mainly by barrier epithelial cells and is a potent activator of several cell types, particularly myeloid dendritic cells. TSLP influences the outcome of interactions between dendritic cells and CD4+ thymocytes and T cells in many situations, such as the regulation of the positive selection of regulatory T cells, maintenance of peripheral CD4+ T cell homeostasis and induction of CD4+ T cell-mediated allergic inflammation.     

Influence of experimental influenza infection of the eye on the course of herpetic keratitis in rabbits.

Influenza virus monoinfection and combined influenza and herpes simplex virus (HSV) infection of the eye was studied in rabbits. Influenza A/Hong Kong/1/68 (H3N2) virus caused a clinically overt disease of the eye only after inoculation into the eye chamber. The combined influenza and HSV infection of the eye induced severe iridocyclitis. The combined infection of the eye with influenza virus and HSV had a more severe course than HSV monoinfection. The occurence of influenza antibody in the lacrimal fluid in the presence of the antigen in eye tissues is of diagnostic importance in determining the aetiology of the disease.