Human herpes simplex virus keratitis: the pathogenesis revisted

The aim of this thesis is to elucidate pathogenic mechanisms of different forms of human HSV keratitis. HSV infection of the corneal epithelium causes a classical dendritic shaped lesion. Many studies could explain the development and growth in dendritic keratitis, but none of these found the anatomical substrate for the linear branching pattern. The most obvious explanation would be, that the shape of dendritic ulcers corresponds with the anatomical pattern of innervating nerves of the cornea. In chapter 2 a relationship between the shape of dendritic ulcers in infectious epithelial keratitis and the subbasal nerve plexus of the corneal epithelium is postulated. Recurrence of HSV keratitis is a common complication after PKP for corneal opacities resulting from HSV infection. After PKP, for reasons unrelated to HSV keratitis, epithelial defects may still be caused by HSV. In chapter 3 the incidence of newly acquired HSV keratitis after PKP is determined and possible contributing factors are assessed. Several possibilities as to the origin of the infecting HSV exist. These include reactivation oflatent virus in the trigeminal ganglion, horizontal spread, or transmission through the donor cornea. To test the assumption of graft-to-host transmission of HSV by PKP, surplus corneal material was examined for the presence of HSV DNA. Because the amount of viral DNA available could be very limited, a new method independent of viral culture, was developed to allow distinction between different virus strains. The newly developed technique was used to test our hypothesis that graft-to-host transmission of HSV is possible. This new method was used to determine the incidence of HSV-1 superinfection in patients with recurrent HSV keratitis. Although HSK has been studied extensively in the mouse model, it is not clear what triggers the immune response and to what extent the mouse data correlate with findings in human keratitis. The most logical idea, that virus-derived proteins are the eliciting factor for the immune response, has been ruled out in the experimental HSK mouse model. Alternative sources of the keratogenic antigens, like auto-antigens, have been suggested. Data on the pathogenesis of human HSK are limited. Therefore, in chapter 4 the antigenspecificity of corneal T cells in HSK patients was investigated. Besides this, corneas of patients with HSK were examined for the presence of corneal antigen reactive T cells (auto-reactive T cells). Chapter 5 provides a concise summary of the data generated in the framework of this thesis, and concludes with an overall discussion of the data and their possible impact on current ophthalmologic practice

Herpes simplex virus-specific T cells infiltrate the cornea of patients with herpetic stromal keratitis: no evidence for autoreactive T cells

PURPOSE: Herpetic stromal keratitis (HSK) is a T-cell-mediated inflammatory disease initiated by a herpes simplex virus (HSV) infection of the cornea. Recently, studies in the HSK mouse model have shown that the immunopathogenic T cells are directed against the HSV protein UL6 cross-reacting with an unknown corneal autoantigen. Whether this type of autoimmunity plays a role in human HSK was analyzed. METHODS: T-cell lines (TCLs) were generated from corneal buttons of 12 patients with different clinical stages of HSV-induced necrotizing stromal keratitis (n = 9) or immune stromal keratitis (n = 3). The initiating virus was identified by polymerase chain reaction and immunohistology performed on the corneal buttons. Peripheral blood mononuclear cells (PBMCs) were isolated, and B cell lines (BLCLs) were generated by transformation with Epstein-Barr virus. Proliferative responses of these intracorneal TCLs were determined by culturing T cells with autologous BLCLs infected with HSV-1, HSV-2, wild-type vaccinia virus (VV-WT), or VV expressing HSV-1 UL6 (rVV-UL6). Alternatively, T cells were incubated with PBMCs pulsed with human cornea protein extract. RESULTS: Irrespective of clinical diagnosis or treatment, T cells were recovered from the corneal buttons of all the 12 HSK patients. The intracorneal TCLs of 9 of the 12 HSK patients showed HSV-specific T-cell reactivity. In none of the TCLs, T-cell reactivity against HSV-1 UL6 or human corneal antigens was detected. CONCLUSIONS: These data suggest that the potentially immunopathogenic intracorneal T-cell response in HSK patients is directed to the initiating virus and not to a human corneal autoantigen or HSV-1 UL6

Corneal herpes simplex virus type 1 superinfection in patients with recrudescent herpetic keratitis

PURPOSE: Herpetic keratitis is a common sequel of a corneal infection with herpes simplex virus (HSV)-1. Recrudescent herpetic keratitis (RHK) may result in irreversible damage to the cornea. Recurrences may be caused by reactivation of endogenous HSV-1 or reinfection with exogenous HSV-1. The objective of this study was to determine the incidence and risk factors involved of HSV-1 superinfection in patients with RHK. METHODS: From 30 patients with RHK, sequential corneal HSV-1 isolates were genotyped by PCR amplification of the hypervariable regions located within the HSV-1 genes US1, US10/11, and US12. The clinical data from the patients obtained retrospectively were: ophthalmologic history, clinical picture during recurrences, number and time points of penetrating keratoplasty (PKP), and steroid or acyclovir treatment. RESULTS: Whereas the sequential corneal HSV-1 isolates of 19 (63%) of 30 patients had the same genotype (designated as group 1), the sequential isolates of 11 patients (37%) were genetically different (designated as group 2). Among the clinical data analyzed, only the time point of PKP was significantly different between the patient groups. A

Amplification of reiterated sequences of herpes simplex virus type 1 (HSV-1) genome to discriminate between clinical HSV-1 isolates.

Herpes simplex virus type 1 (HSV-1)-related disease ranges from a localized, self-limiting illness to fatal disease in immunocompromised individuals. The corneal disease herpetic keratitis may develop after reactivation of a latent virus or reinfection with an exogenous herpesvirus. Molecular analysis of the virus involved may allow distinction between these two options. The HSV-1 genome contains several hypervariable regions that vary in numbers of reiterating regions (reiterations I to VIII [ReI to ReVIII]) between individual strains. Twenty-four HSV-1 clones, derived by subcloning of HSV-1 (strain F) twice in limiting dilutions, were tested in a PCR-based assay to analyze the stabilities of ReI, ReIII, ReIV, and ReVII. ReI and ReIII proved to vary in size upon subcloning, whereas ReIV and ReVII were stable. Subsequently, 37 unrelated isolates and 10 sequential isolates from five patients, all with HSV-1-induced keratitis, were genotyped for ReIV and ReVII. Of the 37 unrelated samples, 34 (92%) could be discriminated, while the genotypes of the viruses in sequential samples were identical for each individual. Conclusively, the data show that the approach presented allows the rapid and accurate discrimination of HSV-1 strains in studies that address the transmission and pathogenesis of HSV-1 infections

Prospective clinical evaluation of McCarey-Kaufman and organ culture cornea preservation media: 14-year follow-up

Purpose: To compare the outcome of corneal grafts preserved McCarey-Kaufman (MK) medium versus organ culture after penetrating keratoplastics. Methods: Paired corneas were stored in McCarey medium for 2-44 hours (mean 21 hours) and in organ culture (OC) for 144-240 hours (mean 192 hours). Penetrating keratoplastics were performed by 2 surgeons in 2 groups of patients with keratoconus, matched for age. Each pair was transplanted by the same surgeon using the same technique. Visual acuity, central corneal thickness, and central endothelial cell density were assessed at 166 +/- 7.8 months postoperatively. Results: Nine pairs of patients were recovered for a long-term follow-up. The mean endothelial cell densities for the MK and OC groups were 611 +/- 155 and 683 +/- 168 cells/mm(2) respectively. which were not significantly different. A first rapid cell loss rate of 2.07% and 2.52% per month and second slow of 0.78% and 0.69% per month were observed in the respective groups. Individual values of best corrected visual acuity were all the same (value 1.00) for both groups. Corneal thickness were, respectively 571 +/- 52 and 540 +/- 35 mu m and were significantly differet (P = 0.013) Conclusions: After 14 years of penetrating keratoplasties performed with corneas stored in MK versus OC, no significant differences were observed in visual acuity, endothelial cell density, and cell loss. The observed thinner grafts after OC compared with MK could not be explained