Herpetic Stromal Keratitis Research Articles

Pathogenesis of herpes simplex virus-induced ocular immunoinflammatory lesions in B-cell-deficient mice.

The role of B cells and humoral immunity in herpes simplex virus (HSV) ocular infections was studied in immunoglobulin mu chain gene-targeted B-cell-deficient mice (muK/O). At doses of virus well tolerated by immunocompetent mice, heightened susceptibility of muK/O mice to herpetic encephalitis as well as to herpetic stromal keratitis (HSK) was observed. An explanation was sought for the increased severity of HSK in the muK/O mice. First, the lack of antibody responses in muK/O mice resulted in longer viral persistence and dissemination to the corneal stroma, the site of inflammation. Prolonged virus expression in the corneal stroma was suggested to cause bystander activation of Th1-type CD4(+) T cells, further contributing to the severity of HSK lesion expression in muK/O mice. Second, muK/O mice generated minimal Th2 cytokine responses compared to wild-type mice. Such responses might serve to downregulate the severity of Th1-mediated HSK lesions.

Polymerase chain reaction in the diagnosis of herpetic keratitis: experience in a developing country

Background: Herpetic ocular disease is a major cause of blindness. Rapid and accurate diagnosis is essential for prompt, proper treatment. We evaluated the usefulness of detection of herpes simplex virus (HSV) DNA by polymerase chain reaction (PCR) in the laboratory diagnosis of herpetic keratitis.Methods: A retrospective study was conducted involving 234 patients who attended the cornea clinic at the Regional Ophthalmic Institute, Chennai, India, between March 1995 and September 1997. Inclusion in the study was based on clinical diagnosis of herpetic keratitis. Oligonucleotide primers directed against the HSV-I thymidine kinase gene were used, yielding a 110 base pair amplicon. The utility of PCR analysis was assessed against other diagnostic markers: HSV isolation on cell culture, HSV antigen detection by indirect immunofluorescence, detection of antiHSV IgG by enzyme-linked immunosorbent assay (ELISA) and detection of HSVspecific tear secretory IgA (sIgA) by ELISA. These tests showed overall sensitivity values of 22.4%, 39.8%, 30.4% and 20.3% respectively.Results: In epithelial keratitis all 35 specimens from which virus was cultured were positive by PCR. PCR gave a positive result in 23 (82. I %) of the 28 specimens in which HSV antigen was detected and in 4 (57. I %) of the 7 specimens that showed HSV-specific IgG. In addition, PCR detected HSV DNA in 5 of the 30 cases in which these three tests gave a negative result. PCR of two pooled tear samples (collected I week apart from the same patient) from 40 patients with stromal keratitis gave a positive result in 12 cases (30%). In stromal keratitis the sensitivity of PCR in detecting HSV DNA in tear samples was 85.7% with culture, indirect immunofluorescence and detection of anti-HSV IgG as the gold standard, and 80% with detection of sIgA as the gold standard.Interpretation: The results confirm the good correlation with the clinical picture that can be obtained with PCR analysis. They also highlight the diagnostic utility of PCR in detecting HSV DNA in tear samples. This is particularly important in herpetic stromal keratitis, in which collection of corneal scrapings is not advised and, hence, conventional techniques such as virus isolation and antigen detection become difficult.

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.

Cytokines in Aqueous Humour and Serum before and after Corneal Transplantation and during Rejection

Cytokine profiles in aqueous humour were studied in relation to corneal disease and subsequent corneal graft survival or rejection. Cytokine levels in samples obtained from eyes with clear grafts (n = 59) were all within the normal range. At the time of penetrating keratoplasty (n = 146), intraocular levels of IL-6 were increased in 38% (50/131), most markedly in eyes with previous allograft failure or herpetic stromal keratitis. The level of IL-10 was increased in 1 eye (n = 144) and of IL-4 and IFN-γ in none. During rejection (n = 10), the levels of IL-6 in aqueous humour were increased in 75% (3/4), of IL-10 in 50% (3/6), of IL-4 in none (0/4) and of IFN-γ in 40% (2/5). In conclusion, the levels of total protein and IL-6 were increased prior to penetrating keratoplasty in eyes with previous inflammation. These results could however not predict the final outcome of the graft. Increased intraocular levels of IL-6, IL-10 and IFN-γ were observed during rejection.

Bystander activation of CD4(+) T cells can represent an exclusive means of immunopathology in a virus infection.

Herpetic stromal keratitis (HSK) is an immunopathological lesion involving herpes simplex virus (HSV) infection and CD4(+) T cells of the Th1 phenotype, but the nature of the target antigens which drive HSK remains uncertain. In the present report we show that ovalbumin TCR-transgenic mice backcrossed to SCID mice unable to recognize HSV show clinical signs of HSK but die of viral encephalitis before the lesions become severe. However, passive transfer of anti-HSV serum at 24 h clears virus and affords protection from both HSK lesions and death. Adoptive transfer of CD8(+) T cells at 72 h usually conferred protection but animals developed severe corneal pathology by 3 weeks post infection. At this time viral antigens were not demonstrable in the cornea and the T cells in the inflammatory lesions were CD4(+)KJ1-26.1 idiotype positive, i. e. OVA peptide specific. These results indicate bystander activation of CD4(+) T cells in a virus-induced inflammatory milieu. This mechanism of immunoinflammation may represent an important component of any lesion which involves CD4(+) T cells.

Are cytokine patterns in aqueous humour useful in distinguishing corneal graft rejection from opacification due to herpetic stromal keratitis?

Intra-ocular cytokine profiles were determined to study the immunological mechanisms of corneal graft opacification due to rejection and/or herpetic stromal keratitis (HSK). Sera and aqueous humour (AH) were sampled shortly after the onset of corneal graft opacification, group I (n=18). In eyes with clear grafts, samples were taken 5 months after transplantation, group II (n=59). Samples of non-inflamed eyes, prior to cataract surgery, were used to determine baseline cytokine levels, group III (n=49). Total protein (TP) levels were measured with Bradford reagent and interleukin (IL)-6, IL-10, IL-4 and interferon (IFN)-gamma with ELISAs. All patients who's corneal grafts showed clinical evidence of graft opacification due to rejection and/or HSK were sampled. In the AH-samples of group I, increased levels of TP were found in 60% (9/15), IL-6 in 79% (11/14), IL-10 in 39% (7/18) and IL-4 in none (0/12). IFN-gamma was detected in 19% (3/16), in the case of HSK only. In contrast, samples obtained from patients with clear grafts in group II showed increased levels of TP in 36% (20/55), IL-6 in 14% (8/57) and IL-10, IL-4 or IFN-gamma in none (n=58). During corneal graft rejection and/or HSV-infection, increased levels of TP and IL-6 in AH confirmed anterior chamber inflammation with breakdown of the blood-aqueous barrier. Based on the data presented, cytokine patterns in the AH do not appear to distinguish corneal opacification due to graft rejection from that due to herpes keratitis.

Chemokines and ocular pathology caused by corneal infection with herpes simplex virus.

The role played by chemokines in disease process is an active area of research that continues to uncover new players. In this report we discuss the likely role of selected chemokines in the disease herpetic stromal keratitis (HSK). This lesion occurs as a sequel to herpes simplex virus infection and is currently accepted as an immunopathological process which primarily involves CD4+ T lymphocytes. In this review we discuss the events involved in HSK, the chemokine profile associated with this disease, and speculate on cellular activities and molecular events which characterize HSK as an immunopathological disease.

Herpes simplex virus replication-induced expression of chemokines and proinflammatory cytokines in the eye: implications in herpetic stromal keratitis.

On infection of the cornea with herpes simplex virus (HSV), an immunopathologic response termed herpetic stromal keratitis (HSK) ensues. This response is mediated primarily by CD4+ T cells and only occurs if mice are infected with replication-competent virus, although replication-defective mutants induce cellular immune responses following infection. To determine the consequences of HSV replication in the cornea, which is crucial for HSK manifestation, corneas infected with productive virus and replication-defective mutants were analyzed for chemokines and proinflammatory cytokine mRNA expression by RT-PCR at various times. While productive infection resulted in rapid upregulation and sustained expression of such chemokines as N51/KC, macrophage inflammatory protein-1beta (MIP-1beta), MIP-2, and monocyte chemotactic protein-1 (MCP-1) and such cytokines as interleukin-1 (IL-1), IL-6, IL-12, and tumor necrosis factor-alpha (TNF-alpha), expression of such inflammatory mediators was minimal and transient after unproductive infection. Expression of MIP-1alpha and lymphotactin along with a biphasic expression of IL-6 and MIP-2 were seen only with productive infection. Initial PMN recruitment into the cornea was approximately 50-fold greater with productive infection than with unproductive infection. These data suggest that a replication-induced proinflammatory milieu in the cornea is crucial for the subsequent progression of HSK possibly because of enhancement of the expression of corneal agonists that drive HSK manifestation.

Immunopathology of Herpetic Stromal Keratitis: Discordance in CD4+ T Cell Function Between Euthymic Host and Reconstituted SCID Recipients

Infection of the mouse cornea with herpes simplex virus (HSV) results in an immunopathologic disease of the eye termed herpetic stromal keratitis (HSK), in which the principal orchestrator is the CD4+ T cell. The mouse genotype largely determines susceptibility or resistance to HSK. BALB/c mice (H2dIgh-1a) are susceptible, while its congenic C.B-17 strain (H2dIgh-1b), which differs only in the Ig heavy chain locus, is resistant to HSK. As the magnitude and duration of viral replication as well as anti-HSV immune responses were similar in both strains, it was determined whether resistance was due to failure of CD4+ T cells to organize the immunopathologic reaction. Adoptive transfer of HSV-primed or naive CD4+ T cells from resistant C.B-17 strain into HSV-infected SCID mice resulted in HSK lesions indistinguishable from those caused by similar transfers of BALB/c CD4+ T cells. Similar results were obtained with transfers of whole T cell populations as well as with unfractionated splenocytes from the resistant mice. These results show that while intact C.B-17 mice exhibit resistance to HSK, they possess potentially pathogenic CD4+ T cells in their repertoire. The data suggest that the HSV-infected SCID mouse provides a proinflammatory microenvironment that overrides regulatory controls and/or cause activation of quiescent cells into aggressive effector T cells that orchestrate HSK.

Identification and Characterization of Herpes Simplex Virus‐Specific CD4+T Cells in Corneas of Herpetic Stromal Keratitis Patients

Herpetic stromal keratitis (HSK) is a corneal disease initiated by a herpes simplex virus (HSV) infection with a postulated T cell-mediated immunopathology. To study the antigen specificity of cornea-infiltrating T cells in HSK patients, T cells were isolated and expanded by mitogenic stimulation from corneas of 2 patients with HSV-1-mediated HSK. A substantial number of the T cell clones (TCCs) obtained from these T cell lines were HSV-specific. All HSV-specific TCCs were of the CD3+CD4+CD8- phenotype. These TCCs responded to autologous HSV-infected corneal keratocytes, which expressed HLA class II molecules following incubation with interferon-gamma. Upon HSV-specific stimulation, all TCCs secreted interleukin-4, interleukin-5, and interferon-gamma. The data presented suggest that HSV-specific CD4+ T cells play a role in the immunopathogenesis of HSK in humans and that corneal keratocytes may act as antigen-presenting cells in this local T cell response.

Control of Herpetic Stromal Keratitis Using CTLA4Ig Fusion Protein

Herpetic stromal keratitis (HSK) is an immunoinflammatory lesion in the cornea of the eye set off by infection with herpes simplex virus (HSV). The disease appears to be orchestrated by CD4+ T cells of the Th1 phenotype but the identity of target antigens involved in HSK remains unknown. In this proposal, we investigated if the inhibition of T cell activation with the fusion protein CTLA4Ig would abrogate the disease process when administered systemically. BALB/c mice infected with HSV-1 (RE strain) by corneal scarification were injected intraperitoneally on a single occasion with CTLA4Ig or L6 control (IgG Fc) given on day 2, day 5, or day 8 postinfection. Lesions in CTLA4Ig-treated mice showed markedly reduced severity judged by both slit lamp biomicroscopy and histopathology if treated on day 2 or day 5. Treated animals also expressed minimal HSV-specific splenic T cell and humoral antibody responses. Judged by the profile of T cell and IgG subset responses, inhibition by CTLA4Ig appeared more directly on the HSV-specific Th1 response, correlating with the known role of such cells in HSK. Delay of treatment until the time of disease onset (day 8) had marginal or negligible effects. The results indicate that blockade of coreceptor interaction between T cells and antigen-presenting cells during the induction phase of immune response significantly impairs onset and severity of herpetic stromal keratitis.

Immunopathogenesis of herpetic ocular disease.

Herpes viruses are among the most prevalent of human virus infections. Productive replication of herpes simplex virus (HSV) is usually confined to mucocutaneous sites by the rapid deployment of innate and adaptive immune responses. Infection invariably results in establishment of latency and in some cases results in periodic reactivation of the virus. This article focuses primarily on ocular herpes with emphasis on the pathogenesis of stromal keratitis. Herpetic stromal keratitis (HSK) is an immunopathologic disease, which indeed is one of the leading causes of blindness in the Western world. The mechanisms by which HSV infection in human beings results in HSK is not well understood but studies using the mouse model has clearly indicated the role of T-cell-mediated immune response as the cause for ocular damage. We, in this article, attempt to provide an interpretive synthesis on different aspects of HSK pathogenesis, reviewing what is currently known and speculating on mysterious issues, such as, whether HSK represents a virus-induced autoimmune disease. We also discuss aspects of remission of the disease.

Suppression of ongoing ocular inflammatory disease by topical administration of plasmid DNA encoding IL-10.

Ocular infection with herpes simplex virus leads to an inflammatory lesion in the cornea orchestrated by CD4+ Th1 lymphocytes. This immunopathologic disease, called herpetic stromal keratitis, is an important cause of impaired vision. In this study, we set out to determine whether established lesions of herpetic stromal keratitis could be controlled by topically administering naked plasmid DNA encoding cytokines to the corneal surface. A single topical administration of DNA encoding IL-10 was beneficial to the majority (75%) of treated animals, and 50% (vs 10% in controls) resolved their lesions completely over a 23-day observation period. Topical ocular application of DNA encoding foreign proteins was also shown to be an effective means of inducing systemic and mucosal immune responses. The direct application of DNA encoding cytokines may represent an additional therapeutic option for the management of immunoinflammatory disease.

Efficacy of a recombinant glycoprotein D subunit vaccine on the development of primary and recurrent ocular infection with herpes simplex virus type 1 in mice.

The protective efficacy of a glycoprotein D subunit vaccine (gD2 SB AS4) was evaluated in a mouse model of human recurrent herpetic stromal keratitis (HSK). When administered before primary infection, gD2 SB AS4 protected mice against corneal pathology, mortality, and latency resulting from ocular viral challenge with herpes simplex virus type 1 (HSV-1) McKrae strain. In addition, gD2 SB AS4 significantly decreased postreactivation corneal disease. A control vaccine, gD2 alum, protected against acute ocular infection only. When administered after primary infection, gD2 SB AS4 vaccination decreased postreactivation ocular shedding but had no other significant effects. Vaccination with gD2 SB AS4 was associated with high anti-gD antibody responses and low delayed-type hypersensitivity responses. These results have identified a prophylactic vaccine, gD2 SB AS4, with activity against acute and recurrent HSK in mice and emphasize the need for vaccine evaluation in both primary and recurrent ocular herpetic disease models.

Characterization of herpes simplex virus (HSV) specific T cells isolated from corneas of patients with herpetic stromal keratitis

Characterization of herpes simplex virus (HSV) specific T cells isolated from corneas of patients with herpetic stromal keratitis

Characterization of herpes simplex virus (HSV) specific T cells isolated from corneas of patients with herpetic stromal keratitis

Characterization of herpes simplex virus (HSV) specific T cells isolated from corneas of patients with herpetic stromal keratitis

On the essential involvement of neutrophils in the immunopathologic disease: herpetic stromal keratitis.

Corneal infection with herpes simplex virus-1 in immunocompetent mice induces an immunopathologic response termed herpetic stromal keratitis (HSK). The earliest sign of disease is neutrophil infiltration, which lasts for 48 to 72 h and then disappears. However, a secondary neutrophil infiltration, this time more massive, occurs, beginning 8 to 9 days postinfection, a time in which HSK becomes clinically evident. The role of neutrophils in HSK expression was investigated by eliminating such cells using a specific mAb (RB6-8C5). In neutrophil-depleted immunocompetent mice, virus replicated more abundantly, but no effects on HSK expression were observed, possibly because sustained neutropenia could not be maintained. However, using a severe combined immunodeficient mouse model, in which HSK does not occur unless given adoptive transfer of CD4+ T cells, the effects of neutrophil depletion were more pronounced. There were significantly less incidence and severity of HSK in CD4+ T cell-reconstituted severe combined immunodeficient mice that were depleted of neutrophils as compared with controls. Neutrophil-depleted mice displayed moderate to severe periocular skin lesions, progressively became cachetic, and developed signs of encephalitis. Virus was recovered at higher titers and for longer periods from eyes of neutrophil-depleted animals. Brain virus titers were also significantly higher on day 12 postinfection as compared with control animals. These results suggest that herpes simplex virus infection of the cornea rapidly invokes recruitment of neutrophils that may aid in viral clearance, and that neutrophils directly or indirectly serve as agonists in perpetuating a CD4+ T cell-mediated inflammatory reaction.

Topical Cyclosporin as an Adjunct to Topical Acyclovir Treatment in Herpetic Stromal Keratitis

Immunological factors play an important role in the development of herpetic stromal keratitis. T lymphocytes are the principal cells involved in this immunologic reaction. We therefore investigated the efficacy and safety of topical cyclosporin A 2 % solution in herpetic stromal keratitis. Ten patients with herpetic stromal keratitis received topical cyclosporin A 2% 4 times daily for 2 months. Acyclovir 3% ointment 5 times daily and cycloplegic eyedrops 2 times daily were used in conjunction with cyclosporin A solution in the 1st month of treatment. Topical steroids were not used. The patients were followed for 6-9 months. Treatment efficacy was evaluated according to two parameters: resolution of stromal infiltrates and neovascularization and increase in visual acuity by two or more Snellen lines. Serum cyclosporin A levels were measured with high-performance liquid chromatography. In all the patients, the stromal infiltration resolved completely after 2 months of treatment. Visual acuity increased by two or more Snellen lines in 8 out of 10 patients. In the remaining 2 cases, stromal scarring that was present before cyclosporin A treatment prevented vision increase. There were no serious complications from the cyclosporin treatment but 4 cases complained of severe burning upon instillation of the drug. This complication was temporary and did not result in discontinuance of the drug. Serum levels of the drug were always below toxicity levels. While this is a noncontrolled study, our results show that topical cyclosporin can be a valuable adjunct to acyclovir treatment in herpetic stromal keratitis. Use of topical cyclosporin in dendritic herpetic ulcers and necrotic stromal keratitis has not been investigated in this study.

The Role of the Innate Immune System in the Reconstituted SCID Mouse Model of Herpetic Stromal Keratitis

Herpetic stromal keratitis (HSK) has an immunopathological basis, thought primarily to involve a CD4+T cell-mediated immune response to viral antigen. Other cell types, however, particularly those involved in nonspecific immunity, such as natural killer (NK) cells or neutrophils, may also contribute to tissue destruction in the cornea. The reconstituted SCID mouse model of HSK provides a powerful system in which to study the interactions of the innate and adaptive immune responses to herpes simplex virus type 1 corneal infection. In the present study, reconstituted SCID mice depleted of NK cells had a reduced incidence and severity of clinical and histopathological HSK. The levels of T cell cytokine protein and message in restimulated splenocytes and cytokine message in corneas did not differ between experimental groups. However, significantly fewer neutrophils were seen within the inflamed corneas of NK-depleted SCID mice. Therefore, endogenous NK cells may indirectly influence the severity of HSK in reconstituted SCID mice by affecting neutrophil migration into the cornea.

Moderation of Herpetic Stromal Keratitis by Basic Fibroblast Growth Factor

The effect of basic fibroblast growth factor (bFGF) on the evolution of herpes simplex virus (HSV) infection in eyes of rabbits was investigated. Rabbit eyes were infected with HSV-1 by a non-invasive inoculation and treated for 7 days with an eye drop solution containing either bovine bFGF (50ng; three times daily), or bFGF diluent as control. The treatment started 2hr, 24hr or 96hr post-inoculation (p.i.). Follow-up of clinical disease parameters, such as conjunctivitis, epithelial keratitis, stromal disease, corneal neovascularization and of viral isolation continued for 17 days. The most significant difference between bFGF and control treatments was observed in the development of stromal keratitis. The incidence of stromal disease in the bFGF treated group (2/16 eyes) was significantly lower than in the control group (11/12 eyes) ( P=0.0001), when bFGF was administered 2hr or 24hr p.i. The severity of the disease developed in the bFGF treated eyes was also milder than in the control eyes (determined by serial slit-lamp clinical examinations and by histologic sections). Such effect was not demonstrated if the treatment started 96hr p.i. The same duration of viral shedding was obtained with bFGF treated eyes (2hr, 24hr, or 96hr p.i.) and control eyes. Neither HSV-1-infected, nor sham-inoculated bFGF-treated eyes demonstrated increased neovascularization of the cornea, as compared with the corresponding vehicle-treated control eyes. This study demonstrates that bFGF treatment (starting 2–24hr p.i.) decreased the occurrence and severity of herpetic stromal keratitis, without subsequent aggravation of corneal vascularization. This beneficial anti-inflammatory effect of bFGF may have future application in the treatment of the most devastating stage of herpetic corneal infection.