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

Retinal nerve fiber layer thickness in subgroups of multiple sclerosis, measured by optical coherence tomography and scanning laser polarimetry

Optical coherence tomography (OCT) and scanning laser polarimetry (GDx ECC) are non-invasive methods used to assess retinal nerve fiber layer (RNFL) thickness, which may be a reliable tool used to monitor axonal loss in multiple sclerosis (MS). The objectives of this study are (1) to compare OCT with the GDx ECC; (2) to assess and compare the RNFL thickness in subgroups of MS. Ophthalmologic examination and RNFL assessment by OCT and GDx were performed in 65 MS patients (26 relapsing-remitting (RRMS), ten secondary-progressive (SPMS), 29 primary-progressive (PPMS)). Twenty-eight patients (43%) had a history of optic neuritis (ON). Adjustments were made for age and disease duration. RNFL thickness was reduced in eyes with previous ON (p < 0.01). No differences were found between PPMS and relapse-onset MS. OCT and GDx ECC measurements were moderately correlated (rho = 0.73, p < 0.01). Visual field-mean deviation (MD) values correlated with OCT means (r = 0.44, p < 0.01) and GDx ECC TSNIT average (r = 0.41, p < 0.01). In patients without previous ON, EDSS correlated with MD (r = -0.36, p < 0.01), visual field-pattern standard deviation (PSD) (r = 0.30, p < 0.05), OCT means (r = -0.31-0.30, p < 0.05) and macular volume (r = -0.37, p < 0.01). For MSIS-29 physical impact score, significant correlations were found with MD (r = -0.48, p < 0.01) and PSD (r = 0.48, p < 0.01). Conclusions: No differences between PPMS and relapse-onset MS subgroups were found. RNFL thickness was reduced in eyes with previous ON. Although OCT and GDx ECC findings were moderately correlated and showed significant correlations with measures of visual function in patients without previous ON, EDSS correlated significantly with visual and OCT measures, but not with GDx ECC

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

Cluster of Symptomatic Graft-to-Host Transmission of Herpes Simplex Virus Type 1 in an Endothelial Keratoplasty Setting

PURPOSE: Descemet's membrane endothelial keratoplasty (DMEK) is becoming the gold standard to treat corneal endothelial dysfunctions worldwide. Compared with conventional penetrating keratoplasty, infectious complications after DMEK are ill defined. We describe the clinical picture of 2 DMEK recipients, operated on the same day and in the same clinic, who developed atypical herpes simplex virus type 1 (HSV-1) infection in the transplant recipient eye within days post-DMEK. Because recipients received cornea tissue from 2 different donors prepared by the same eye bank, the likelihood of a common HSV-1 source was determined. DESIGN: Case series. PARTICIPANTS: Two DMEK recipients who developed atypical intraocular HSV-1 disease shortly after surgery and surplus cornea specimens of 6 donors. METHODS: Surplus cornea donor (pre-DMEK cornea remnants and conditioned cornea storage and transport media) and recipient samples (post-DMEK aqueous humor) were assayed for HSV-1 DNA and infectious virus by real-time polymerase chain reaction (RT-PCR) and cell culture, respectively. Target-enriched whole viral genome sequencing was performed on HSV-1 DNA–positive ocular specimens. MAIN OUTCOMES MEASURES: Clinical picture of atypical intraocular HSV-1 infection post-DMEK and presence and homology of HSV-1 genomes between ocular specimens of DMEK donors and recipients. RESULTS: Herpes simplex virus type 1 DNA was detected in aqueous humor and donor cornea specimens of both DMEK cases, but not in the cornea remnants of 6 randomly selected donors processed by the same eye bank. Infectious HSV-1 was isolated from the cornea remnant and corresponding culture medium of 1 cornea donor. Notably, whole-genome sequencing of virus DNA-positive specimens demonstrated exceptionally high genetic similarity between HSV-1 strains in recipient and donor specimens of both DMEK cases. CONCLUSIONS: Data indicate cross-contamination of cornea grafts during DMEK preparation with subsequent graft-to-host HSV-1 transmission that caused atypical sight-threatening herpetic eye disease shortly after DMEK. Ophthalmologists should be aware that HSV-1 transmission by DMEK is possible and can lead to atypical ocular disease, a condition that can easily be prevented by taking appropriate technical and clinical measures at both eye bank and surgical levels

Whole-cell metabolic control analysis

Since its conception some fifty years ago, metabolic control analysis (MCA) aims to understand how cells control their metabolism by adjusting the activity of their enzymes. Here we extend its scope to a whole-cell context. We consider metabolism in the evolutionary context of growth-rate maximisation by optimisation of protein concentrations. This framework allows for the prediction of flux control coefficients from proteomics data or stoichiometric modelling. Since genes compete for finite biosynthetic resources, we treat all protein concentrations as interdependent. We show that elementary flux modes (EFMs) emerge naturally as the optimal metabolic networks in the whole-cell context and we derive their control properties. In the evolutionary optimum, the number of expressed EFMs is determined by the number of protein-concentration constraints that limit growth rate. We use published glucose-limited chemostat data of S. cerevisiae to illustrate that it uses only two EFMs prior to the onset of fermentation and that it uses four EFMs during fermentation. We discuss published enzyme-titration data to show that S. cerevisiae and E. coli indeed can express proteins at growth-rate maximising concentrations. Accordingly, we extend MCA to elementary flux modes operating at an optimal state. We find that the expression of growth-unassociated proteins changes results from classical metabolic control analysis. Finally, we show how flux control coefficients can be estimated from proteomics and ribosome-profiling data. We analyse published proteomics data of E. coli to provide a whole-cell perspective of the control of metabolic enzymes on growth rate. We hope that this paper stimulates a renewed interest in metabolic control analysis, so that it can serve again the purpose it once had: to identify general principles that emerge from the biochemistry of the cell and are conserved across biological species

Three-dimensional quantitative evaluation method of nonrigid registration algorithms for adaptive radiotherapy

Purpose: Current radiotherapy is progressing to the concept of adaptive radiotherapy, which implies the adaptation of planning along the treatment course. Nonrigid registration is an essential image processing tool for adaptive radiotherapy and image guided radiotherapy, and the three-dimensional (3D) nature of the current radiotherapy techniques requires a 3D quantification of the registration error that existing evaluation methods do not cover appropriately. The authors present a method for 3D evaluation of nonrigid registration algorithms’ performance, based on organ delineations, capable of working with near-spherical volumes even in the presence of concavities. Methods: The evaluation method is composed by a volume shape description stage, developed using a new ad hoc volume reconstruction algorithm proposed by the authors, and an error quantification stage. The evaluation method is applied to the organ delineations of prostate and seminal vesicles, obtained by an automatic segmentation method over images of prostate cancer patients treated with intensity modulated radiation therapy. Results: The volume reconstruction algorithm proposed has been shown to accurately model complex 3D surfaces by the definition of clusters of control points. The quantification method, inspired by the Haussdorf–Chebysev distance, provides a measure of the largest registration error per control direction, defining a valid metric for concave-convex volumes. Summarizing, the proposed evaluation methodology presents accurate results with a high spatial resolution in a negligible computation time in comparison with the nonrigid registration time. Conclusions: Experimental results show that the metric selected for quantifying the registration error is of utmost importance in a quantitative evaluation based on measuring distances between volumes. The accuracy of the volume reconstruction algorithm is not so relevant as long as the reconstruction is tight enough on the actual volume of the organ. The new evaluation method provides a smooth and accurate volume reconstruction for both the reference and the registered organ, and a complete 3D description of nonrigid registration algorithms’ performance, resulting in a useful tool for study and comparison of registration algorithms for adaptive radiotherapy

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