Presence of Langerhans cells in the central cornea linked to the development of ocular herpes in mice.

Recurrences of herpetic stromal keratitis are believed to be initiated by reactivation of herpes simplex virus infection, probably in the trigeminal ganglion. Genetic features of the virus and the host as well as the immune status of the host influence the outcome of infection. Following infection on the snout with HSV-1, mice with normal corneas usually develop mild anterior segment disease. We studied the induction of herpetic infection in mice that had abnormal corneas, containing center due to trauma or a spontaneous dystrophy. The corneal abnormality led to more frequent herpetic stromal keratitis and more severe anterior chamber reaction. In addition, we found that snout-infected mice with dystrophic corneas had an increased risk of dying from viral infection. Our data suggest that not only the strain of virus and the genetic background of the mouse, but also the state of the cornea itself, can contribute to susceptibility to ocular herpes infection.     

Increased severity of herpes simplex virus type 1-induced keratitis in Hox A5 transgenic mice

PURPOSE: Herpes simplex virus type 1 is a major cause of stromal keratitis and blindness in humans. Understanding of the role of host genes in the pathogenesis of herpes stromal keratitis is limited. We used a transgenic mouse model to examine the effect of a host gene, Hox A5 (which binds to the TAATGARAT sequence in the promoter regions of HSV-1 immediate early genes and increases HSV-1 replication), on the pathogenesis of HSV-1 induced stromal keratitis. METHODS: Corneas of wildtype and Hox A5 transgenic mice were infected with HSV-1 strain F following corneal scarification. Clinical severity of keratitis was evaluated using slit-lamp biomicroscopy. Histologic severity of keratitis was determined by light microscopic evaluation and by computerized morphometry. Ocular viral replication was measured via plaque assay. RESULTS: Clinical lesions of stromal keratitis were more severe at 17 and 23 days post infection in Hox A5 transgenic mice than in wildtype mice. Histological evaluation and morphometric analysis confirmed that keratitis lesions were more severe in the transgenic mice. HSV-1 replication was approximately100-fold greater in the corneas of transgenic mice than in wildtype mice. CONCLUSIONS: Our results demonstrate that a host gene (Hox A5) can increase ocular replication of HSV-1 and alter the pathogenesis of herpetic stromal keratitis.     

Detection of herpes simplex virus type 1, 2 and varicella zoster virus DNA in recipient corneal buttons

AIM: To study the value of polymerase chain reaction (PCR) analysis, to detect viral DNA in recipient corneal buttons taken at the time of penetrating keratoplasty (PKP) in patients with an initial diagnosis of herpetic stromal keratitis (HSK). Since HSK has a tendency to recur, an accurate diagnosis of previous HSK could be the reason to start antiviral treatment immediately, thereby possibly decreasing the number of graft failures due to recurrent herpetic keratitis. METHODS: Recipient corneal buttons and aqueous humour (AH) samples were obtained at the time of PKP from HSK patients (n=31) and from other patients (n=78). Eye bank corneas were also used (n=23). Herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), and varicella zoster virus (VZV) infection were assessed by PCR and antibody detection. RESULTS: The clinical diagnosis HSK could be confirmed by PCR for HSV-1 in 10/31 (32%). In these corneal buttons HSV-2 DNA was detected in 1/31 (3%) and VZV DNA in 6/31 (19%). Intraocular anti-HSV antibody production was detected in 9/28 AH samples tested (32%). In the other patient derived corneas HSV-1 DNA was detected in 13/78 (17%), including eight failed corneal grafts without clinically obvious herpetic keratitis in the medical history. In clear eye bank corneas HSV-1 was detected in 1/23 (4%). CONCLUSIONS: PCR of HSV-1 on corneal buttons can be a useful diagnostic tool in addition to detection of intraocular anti-HSV antibody production. Furthermore, the results were suggestive for the involvement of corneal HSV infection during allograft failure of corneas without previous clinical characteristic signs of herpetic keratitis.     

Protective immunity against herpetic ocular disease in an outbred mouse model

Immunization of outbred mice by subdermal (footpad) inoculation with the F strain of herpes simplex virus type 1 (HSV-1) induces an immune response which protects the animals against herpetic ocular disease and encephalitis, and reduces the incidence of latent trigeminal ganglion infections following corneal challenge with the RE strain of HSV type 1. The protective effects are proportional to the dose of virus used for immunization. Heat-killed virus preparations also protected the mice against encephalitis and stromal keratitis, but failed to prevent epithelial keratitis and establishment of latency.     

Amnionmembrantransplantation bessert experimentelle herpetische Keratitis

PURPOSE: Transplantation of human amniotic membrane (AMT) accelerates the healing of experimental ulcerative herpetic keratitis. Here the expression and activity of matrix metalloproteinase (MMP)-9 was studied. METHODS: BALB/c mice were corneally infected with HSV-1. Whereas the infected corneas of mice in group 1 were covered with AM, tarsorrhaphies were performed in others (group 2). After 2 days, the appearance of corneal ulcers and stromal inflammation was judged clinically, and the corneal PMN infiltration was studied histologically. The expression of MMP-9 in the corneas was localized by immunohistochemistry and analyzed by Western-blot technique. The MMP-9 activity in the corneas was determined by zymography. RESULTS: On day 14, the ulcerating corneas had a dense PMN infiltration, the ulcers and the majority of PMNs were highly positive for MMP-9, and the active forms of MMP-9 were detected. Gelatinolytic activity was found in these corneas by zymography. Compared with the mice of group 2, ulceration, stromal inflammation and neovascularization markedly improved clinically and histologically within 2 days in mice of group 1. This was associated with a reduced expression of MMP-9 in corneal tissue and in PMNs. The gelatinolytic activity of MMP-9 was reduced after AMT. CONCLUSIONS: These observations suggest that improvement of herpetic corneal ulcers and reduced corneal neovascularization after AMT may result from a reduced expression and activity of MMP-9.     

The isolation of herpes simplex virus from rabbit corneas during latency

Herpes simplex virus type 1 (HSV-1) latency, as operationally defined, is a state in which cell-free infectious virus cannot be demonstrated in tissue at the time of sacrifice, but infectious virus can be isolated from the same tissue after prolonged cultivation. Latent HSV has been routinely detected in sensory ganglia of the infected dermatome. We have isolated HSV-1 (RE) from the corneas of 11% of infected rabbits which harbored virus in a latent state in trigeminal ganglia. HSV-1 (RE) was isolated from 10 of 88 cultures of corneal cells established following collagenase digestion of individual corneas taken from asymptomatic animals 118 days after infection. Virus was recovered only after prolonged primary culture and in some cases serial passage of corneal cells (range 5 to 26 days to initial cytopathic effect, n = 10). Virus was isolated from 68 of 68 trigeminal ganglia from the same rabbits by cocultivation of ganglion pieces with Vero cells (range 9 to 20 days to initial cytopathic effect, n = 68) while no cell-free virus was isolated from ganglia at the time of sacrifice. Virus isolation from corneas during the latent period occurred in a manner independent of prior antiviral or antiviral plus immunosuppressive therapy. Clinical evaluation of the corneas throughout the course of acute disease, stromal disease, and at the time of sacrifice provided no evidence that could be used to predict which corneas would yield virus. These data suggest that HSV-1 can remain in a nonreplicative state characteristic of latency in cells of rabbit corneas for long periods after infection and therapy of herpetic eye disease.     

Participation of T lymphocyte in corneal edema in the early stage of herpetic stromal keratitis

The participation of T lymphocytes in the immunopathogenesis of corneal opacity in herpetic stromal keratitis was investigated. In BALB/c mice infected intracorneally with herpes simplex virus type 1, corneal opacity was manifested 10 days after infection, while in athymic mice corneas remained almost clear. Histologically, all opaque corneas revealed stromal edema accompanied by the diffuse presence of polymorphonuclear cells and lymphocytes. When complement (C')-treated immune spleen cells were adoptively transferred into athymic mice 6 or 72 h after corneal infection, stromal keratitis with mild opacity was observed 10 days after transfer. The athymic mice given anti-Thy 1.2+C'-treated immune spleen cells failed to develop corneal opacity. The difference, as revealed by light and electron microscopy, was the presence or absence of lymphocytic infiltration and edema in the posterior third layers of the stroma and endothelial lesions. The endothelium was infiltrated by lymphocytes or macrophages and showed various stages of destruction. The main cause of corneal opacity in the early stage of herpetic stromal keratitis is thought to be stromal edema due to an adverse effect on the endothelium by immune T lymphocytes.     

Herpes simplex virus entry receptor nectin-1 is widely expressed in the murine eye

PURPOSE: Nectin-1 belongs to the immunoglobulin superfamily, mediates cell-cell adhesion in cadherin-based adherens junctions, and acts as a receptor for herpes simplex virus (HSV). The goals of this study were (1) to determine whether nectin-1 is expressed in ocular tissue that is an important target of HSV infections and (2) to determine whether HSV type 1 (HSV-1) infection affects nectin-1 expression in the eye. METHODS: Expression of nectin-1 and HSV-1 protein was determined by immunohistochemical analysis of ocular tissues of untreated BALB/c mice and mice that were euthanized either 7 days or 7 months after corneal inoculation of HSV-1 or sterile tissue-culture medium (mock). RESULTS: In ocular tissues derived from untreated and mock-infected mice, widespread nectin-1 expression was detected among cells of the corneal epithelium and endothelium, conjunctiva, lens epithelium, ciliary body, iris, choroid, and retina. However, fibroblasts in the corneal stroma and the sclera did not express detectable levels of nectin-1. Ocular tissues from mice euthanized 7 days after corneal inoculation of HSV-1 frequently demonstrated corneal ulceration and inflammation and HSV-1 protein expression in the corneal epithelium, stroma, endothelium, conjunctiva, iris, and ciliary body but rarely in the retina. Ocular tissues from mice euthanized 7 months after HSV-1 inoculation demonstrated corneal epithelial and stromal inflammation, but HSV-1 protein expression was not detected. HSV-1 infection did not lead to a loss of nectin-1 expression in any of the tissues examined. In contrast to uninfected corneas, the inflamed and vascularized stroma of infected corneas contained mononuclear inflammatory cells, vascular cells, and fibroblasts that stained positive for nectin-1. CONCLUSIONS: Findings report that nectin-1 is widely expressed in murine ocular tissues. Only fibroblasts in the corneal stroma and sclera of uninfected tissues were devoid of nectin-1 expression. HSV-1-infected inflamed corneas contained some stromal fibroblasts with detectable nectin-1 expression, which potentially could be targeted by the virus. Widespread nectin-1 expression in the eye suggests that this receptor may play a role in the pathogenesis of ocular HSV infections.     

In vivo antiviral efficacy of a dipeptide acyclovir prodrug,val-val-acyclovir,against HSV-1 epithelial and stromal keratitis in the rabbit eye model

PURPOSE: A dipeptide prodrug of the antiviral nucleoside acyclovir (ACV), val-val-ACV (VVACV), was evaluated in vivo as a potential drug candidate for improving antiviral efficacy against herpetic epithelial and stromal keratitis. METHODS: The effect of 1% VVACV on epithelial keratitis induced by inoculation of HSV-1 strain McKrae (25 microL of 10(5) plaque-forming units [PFU]) in the scarified rabbit cornea and stromal keratitis induced by intrastromal injection of HSV-1 strain RE (10 microL of 10(5) PFU) was compared with that of 1% trifluorothymidine (TFT) and balanced salt solution as the vehicle control. Both eyes of 10 rabbits were used in each treatment group. Lesions were evaluated by slit lamp examinations over a 2-week period after infection. Aqueous humor samples and corneas were analyzed for drug concentrations at the end of each experiment. Cytotoxicity of VVACV in comparison with val-acyclovir (VACV), ACV, and TFT was evaluated in cellular proliferation assays. RESULTS: The dipeptide prodrug VVACV demonstrated excellent activity against HSV-1 in the rabbit epithelial and stromal keratitis models: 1% VVACV was as effective as 1% TFT. The prodrug was also less cytotoxic than TFT, which is the only effective drug currently licensed and routinely used for topical treatment of ocular herpes infections in the United States. CONCLUSIONS: The less cytotoxic and highly water-soluble prodrug VVACV, which showed excellent in vivo activity against HSV-1 in rabbit epithelial and stromal keratitis, is a promising drug candidate for treatment of ocular HSV infections.     

Contribution of virus and immune factors to herpes simplex virus type I-induced corneal pathology

In vivo T-lymphocyte subpopulation depletion techniques were used to identify the roles of L3T4+ (CD4) and Lyt-2+ (CD8) T-lymphocytes in the pathogenesis of corneal stromal disease induced by two different strains of Herpes simplex virus type 1 (HSV-1). Histologic examination of infected corneas revealed significant differences in the composition of the inflammatory corneal infiltrates induced by the RE and KOS strains of HSV-1. The RE strain induced a predominantly polymorphonuclear leukocyte (PMN) infiltrate, which began approximately 1 week after infection and progressed through day 21. Depletion of CD4 cells before corneal infection with RE HSV-1 greatly reduced the incidence and severity of corneal disease; depletion of CD8 cells had no effect. The strain KOS HSV-1 induced an early PMN infiltrate that became predominantly mononuclear by day 21. Depletion of CD4 cells did not change the incidence or severity of KOS HSV-1-induced corneal stromal disease. The corneal lesions of these mice contained numerous CD8 cells. Depletion of CD8 cells before KOS HSV-1 infection of the cornea moderately reduced the incidence of stromal disease. However, in CD8-depleted mice with the disease, PMNs were the most prevalent infiltrating cells, and the disease appeared identical to that seen in RE HSV-1 infected corneas. Simultaneous depletion of CD4 and CD8 cells before KOS HSV-1 infection eliminated stromal disease. However, when T-cell depletion was discontinued in these mice, stromal disease developed in concert with the appearance of T-cells in the lymphoid organs and corneas. Thus, T-lymphocytes are a necessary component of HSV-1 corneal stromal disease. These results further suggest that RE HSV-1 preferentially activates CD4 cells in the cornea, and KOS HSV-1 preferentially activates CD8 cells in the cornea.     

Herpetic keratitis in inbred mice

The corneas of four inbred strains of mice (BALB/c, DBA/2, C3H and C57BL/6) were inoculated with the RE strain of herpes simplex virus, type 1. The corneas were examined at frequent intervals and graded on a scale of 0 (clear cornea) to +5 (severe necrotizing stromal keratitis). At 3 weeks postinfection, the mean corneal scores were: DBA/2, 4.0; BALB/c, 2.2; C3H, 0.7; and C57BL/6, 0.15. The differences between the scores are statistically significant (P less than 0.05), except for the C3H and C57BL/6 strains. The order of severity of corneal disease in these mice corresponds to the order of susceptibility to systemic infection found in these same inbred strains. Additional studies of herpetic keratitis in inbred mice should prove helpful in understanding the genetic and immunologic basis of herpetic stromal keratitis.     

Efficacy of a helicase-primase inhibitor in animal models of ocular herpes simplex virus type 1 infection

PURPOSE: The aim of this study was to evaluate the effect of BAY 57-1293, a helicase-primase inhibitor, on herpes simplex virus type 1 (HSV-1) reactivation in mice and its efficacy on established disease in rabbits. METHODS: BALB/c mice latent for McKrae-strain HSV-1 were reactivated via heat stress, treated with BAY 57-1293, and their corneas were swabbed for virus or the trigeminal ganglia (TG) obtained for quantification of viral DNA. New Zealand white rabbits were infected and treated topically or orally in comparison with trifluridine or valacyclovir. RESULTS: Oral BAY 57-1293 suppressed reactivation in HSV-1-infected mice and reduced the viral load in TG up to four orders of magnitude. In the rabbits, the therapeutic efficacies of topical BAY 57-1293 and trifluridine were similar. Once-daily oral BAY 57-1293 was significantly more effective than valacyclovir and as effective as twice a day topical trifluridine. CONCLUSIONS: BAY 57-1293 may be more effective than valacyclovir, without the cytotoxicity or potential healing retardation seen with trifluridine. Oral BAY 57-1293 may be a substitute for eye drops as an effective treatment for herpetic keratitis and might be useful in treating stromal keratitis and iritis, as well as preventing recurrences of ocular herpes.     

Detection of HSV nucleic acid sequences in the cornea during acute and latent ocular disease

This study evaluated the continued presence of herpes simplex virus (HSV) nucleic acid sequences after resolution of acute herpetic stromal keratitis in the rabbit ocular model. Forty-four rabbits were inoculated bilaterally with 10(5) plaque-forming units of RE strain HSV-1 by intrastromal injection. All eyes were cultured for the presence of HSV during acute disease and immediately before the animals were killed. Full-thickness corneal buttons were then removed and processed for in situ hybridization with a 3H-labelled HSV DNA probe representing the full-length HSV genome. HSV nucleic acid sequences were detected autoradiographically at all time intervals examined. HSV nucleic acid sequences were localized in the epithelium and the anterior stromal keratocytes during acute disease and in all corneal layers during latent infection. Retention of HSV nucleic acid sequences, either HSV DNA or HSV RNA, or both, in corneal tissues (epithelium, stroma, and endothelium) may be a contributing factor in the development of HSV-induced stromal keratitis.     

Herpes simplex virus latency in the cornea

We tried to identify herpes simplex virus type 1 (HSV-1) latency in the cornea obtained at the time of penetrating keratoplasty from patients with herpetic stromal keratitis in the non-active (subsided) stage. The subject consisted of 8 patients (3 males and 5 females; average age 42.3 years) who were diagnosed as having herpetic stromal keratitis and underwent penetrating keratoplasty during a period without active lesions (subsided stage) between August, 1984 and July, 1988. No infective virus was detected in the centrifugation supernatant following each corneal homogenization. Latent virus was detected from the culture supernatant of sections of the corneas in 4 of the 8 patients. Although the ganglion trigger theory of Hill et al. has been conventionally supported as the mechanism(s) of herpetic keratitis recurrence, our results suggest that HSV-1 proliferation from latency in the cornea (peripheral tissue) might stimulate the ganglion (ganglion and skin trigger theory). The present study is the first to demonstrate HSV-1 latency in the cornea.     

Interferon production and sensitivity of rabbit corneal epithelial and stromal cells

The induction of interferon and the ability of interferon to induce the antiviral state were studied using rabbit corneal epithelial and stromal cells which were cultured for fewer than five passages. Interferon titers in the range of 7000 units/ml were induced in epithelial cell cultures and 76,000 units/ml in stromal cell cultures treated with UV-inactivated bluetongue virus. The interferon induced was stable to pH 2.0 treatment and heating to 56 degrees C for 16 hr. Infection of epithelial and stromal cell cultures with various strains of herpes simplex virus type 1 showed that all strains tested replicated to equivalent titers in the respective cell types, and that no detectable interferon was induced in stromal cells and only trace amounts in epithelial cells. Exogenously supplied rabbit interferon induced the antiviral state in cultures of both cell types restricting the replication of not only encephalomyocarditis virus but also herpes simplex virus. Sixty to ninety units of rabbit interferon reduced HSV-1 virus replication by 50%. Human interferons had less than 27% of the antiviral activity in rabbit cells than they had in a human cell line. The data indicate that exogenously supplied interferon may act to reduce the severity of herpetic keratitis by directly inducing the antiviral state in corneal epithelial and stromal cells. However, interferon endogenously produced by rabbit corneal cells in response to HSV-1 infection probably plays a minor role in the pathogenesis of ocular HSV-1 infections.     

Application of FGF-2 to modulate herpetic stromal keratitis

PURPOSE: Herpetic stromal keratitis (SK) is a tissue destructive eye lesion caused by infection of herpes simplex virus-1 (HSV-1). One step by which HSV-1 enters the cell is through binding to surface heparan sulfate proteoglycans (HSPG), a process that can be inhibited by fibroblast growth factor 2 (FGF-2). The current study examined the effect of FGF-2 application on the outcome of ocular HSV infection. METHODS: Vero cells were infected with HSV-1 after preincubation with FGF-2 protein, and viral infectivity was determined by plaque reduction assay. In an in vivo study, mice were ocularly treated with FGF-2 before (plasmid DNA) or after (recombinant protein) HSV-1 infection, and SK lesion severity was observed. Results: Whereas FGF-2 had excellent antiviral effects in vitro, it was without significant inhibitory effects when given as plasmid DNA encoding FGF-2 (100 microg/application) onto the cornea of the susceptible mouse (BALB/c) before virus infection. Only minor antiviral effects of FGF-2 in vivo were initially observed. Interestingly, topical treatment of recombinant FGF-2 protein (50 ng, two times daily until day 10 postinfection) into HSV-1-infected corneas significantly reduced SK lesion severity and incidence, presumably by promoting epithelial ulcer healing. CONCLUSIONS: These results suggest that treatment of FGF-2 has therapeutic effects on herpetic SK progression via its role in wound healing.     

OX40-Ig融合蛋白对鼠单纯疱疹性角膜基质炎的抑制作用

目的研究OX40-Ig融合蛋白对鼠单纯疱疹性角膜基质炎(HSK)的免疫抑制作用。方法将1×106PFU的单纯疱疹病毒1型(HSV-1)Mckrae毒株接种于BALB/c鼠的角膜上建立HSK模型;分别于接种病毒的当天、接种后第2、4d将OX40-Ig融合蛋白100μg注射到鼠的腹膜下,观察OX40-Ig融合蛋白对鼠HSK的影响。结果OX40-Ig融合蛋白使鼠外周血中CD4 T细胞减少了78·2%,使鼠HSK发病率由83·3%下降到20·0%。OX40-Ig治疗组的小鼠角膜基质混浊程度较对照组明显减轻,角膜内炎性细胞浸润也明显减少,迟发型超敏反应能力显著下降。结论OX40-Ig融合蛋白能够阻断OX-40/OX-40L协同刺激途径,抑制CD4 T细胞增生,阻止HSK的发病,减轻HSK的严重程度。     

Immunomodulation of experimental murine herpes simplex keratitis: II. Glycoprotein D protection

Subcutaneous immunization with purified HSV-1 glycoprotein D (gD) protects susceptible A/J mice against keratitis and encephalitis following corneal HSV-1 challenge. Mice were immunized with gD, in complete Freund's adjuvant, 3.0 micrograms/mouse followed by two booster doses of 1.5 micrograms/mouse at weeks 2 and 4. Control groups of A/J mice were injected with either complete Freund's adjuvant (unimmunized controls) or live HSV-1 MP strain (immunized controls). All mice were challenged ocularly at week 5 with HSV-1, F strain (6.5 x 10(3) PFU) after corneal scarification. None of the 16 animals immunized with gD developed stromal keratitis; only 3 out of 12 animals immunized with live HSV-1 developed a stromal keratitis; 13 out of 16 CFA primed unimmunized mice developed severe stromal keratitis within 14 days post corneal challenge, and 3 out of 16 control CFA primed animals died within 16 days post corneal challenge. At the time of sacrifice (3 weeks post corneal challenge), the ipsilateral trigeminal ganglia were removed and assayed for latent HSV-1 using cocultivation on Vero cell monolayers. The results of these experiments indicate that immunization with gD produces protection against latent ganglionic infection in 56% of the immunized animals, and provides protection against keratitis and death following HSV corneal challenge.