Bradley M. Mitchell

Herpes simplex virus-1 and varicella-zoster virus latency in ganglia

Two human alpha-herpesviruses, herpes simplex virus (HSV)-1 and varicella zoster virus (VZV), account for the most frequent and serious neurologic disease caused by any of the eight human herpesviruses. Both HSV-1 and VZV become latent in ganglia. In this review, the authors describe features of latency for these viruses, such as distribution, prevalence, abundance, and configuration of viral DNA in latently infected human ganglia, as well as transcription, translation, and cell type infected. Studies of viral latency in animal models are also discussed. For each virus, remaining questions and future studies to understand the mechanism of latency are discussed with respect to prevention of serious cutaneous, ocular, and neurologic disease produced by virus reactivation.

Pathways of corneal and ocular surface inflammation: A perspective from the Cullen symposium

The goal of this symposium was to coalesce information presented by 22 investigators in the field of corneal and ocular surface inflammation into common pathways of inflammation. The perspective elucidated in this article defines the components of the normal ocular surface immune architecture and describes the consensus reached on the mechanisms/pathways involved in 1) acute inflammation; 2) late-stage (chronic) response; and 3) allergic disease. Seven diagrams didactically illustrate mechanisms. This paper is the introductory article in a supplement containing 18 articles by the symposium participants.

Immunosuppression Affects the Severity of Experimental Fusarium solani Keratitis

We have established a mouse model of corneal fusariosis that permits the evaluation of fungal infection and pathogenesis. Corneas of immunocompetent and cyclophosphamide-treated adult BALB/c mice were topically inoculated with Fusarium solani after corneal scarification. Eyes were scored for corneal involvement daily for 8 days and at 2 weeks after infection. Eyes were enucleated at various time points for quantitative fungal recovery and histopathological examination. An inoculum-dose response was observed in cyclophosphamide-treated mice, and fungi were recovered from the infected eyes by quantitative microbial culturing. Treatment with cyclophosphamide increased disease severity and delayed fungal clearance. Fungal hyphae, inflammatory cells, and stromal edema were histologically evident within corneal tissue and correlated with disease severity. Although the mouse cornea resists fungal infections, F. solani keratitis could be induced in immunosuppressed mice after surface scarification, which resulted in infection and clinical disease that could be evaluated both in vivo and in vitro.

Expression of matrix metalloproteinases during experimental Candida albicans keratitis.

PURPOSE This study was designed to investigate the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during the inception and progression of experimental keratomycosis. METHODS Scarified corneas of adult BALB/c mice were topically inoculated with Candida albicans strain SC5314 and monitored for disease severity. Infected and mock-infected corneas were compared at 1 day post inoculation (p.i.) with a murine gene microarray. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) determined MMP and TIMP levels at 1, 3, and 7 days p.i. for infected, mock-infected, and normal corneas. Immunostaining localized target proteins at 1 day p.i. RESULTS Eyes inoculated with C. albicans developed corneal infection with a mean clinical score of 8.2+/-0.8 at 1 day p.i. Compared to controls at 1 day p.i., MMP-8, -9, -10, -12, -13, -19, and TIMP-1 were significantly upregulated from fivefold to 375-fold by microarray and from threefold to 78-fold by real-time RT-PCR. Upregulated MMPs and TIMP-1 in the corneal epithelium and stroma of infected eyes correlated with the influx of acute inflammatory cells. Neither MMP-8 nor -13 expression was affected by mechanical trauma, but both increased >100-fold during the week after the onset of fungal keratitis. TIMP-1 expression rose from 21-fold more than controls at 1 day to 46-fold at 7 days p.i. by RT-PCR. CONCLUSIONS Transcriptional and translational levels of MMP-8, -9, -13, and TIMP-1 increase during the early stages of C. albicans keratitis, confirming findings for MMP-9 and TIMP-1 in other infectious keratitis models and suggesting roles for MMP-8 and -13.

Immune Transfer Protects Severely Immunosuppressed Mice from Murine Cytomegalovirus Retinitis and Reduces the Viral Load in Ocular Tissue

Cytomegalovirus (CMV) retinitis is a sight-threatening disease that affects immunosuppressed people and is prevalent in people with AIDS. The purpose of this study was to evaluate murine CMV (MCMV) retinitis in a replenishing model with adoptive immune transfer into severely immunosuppressed animals. Adult BALB/c mice, immunosuppressed with cyclophosphamide, were infected subretinally with 5x102 plaque-forming units of MCMV. Four to six hours later, 3-4x107 donor cells were transferred by intravenous infusion. Eight days after the transfer, the eyes that had received donor cells were studied histologically, titered for infectious virus, and analyzed with polymerase chain reaction (PCR). Adoptive transfer of total MCMV-immune lymph node (LN) cells or enriched LN lymphocytes specifically and significantly protected immunosuppressed mice from retinitis even after the initiation of infection. The transfer resulted in a reduced viral load, as measured by both plaque assay and PCR. This replenishment model will be useful for determining the specific immune parameters of protection from CMV retinitis.

Molecular analysis of the pediatric ocular surface for fungi.

Purpose. To analyze the conjunctival flora of individuals 21 years of age or less for fungi using polymerase chain reaction (PCR) methodology. Methods. Before povidone-iodine antisepsis, eye-swab specimens were collected from adolescent corneal donors preceding corneal excision and from children during preparation for strabismus surgery. Nucleic acid was extracted from the specimens and analyzed by PCR using primers designed for the detection of broad-spectrum fungal DNA and of Candida albicans -specific DNA. Results. Twelve (38%) of 32 eye donor surfaces and 7 (23%) of 30 patient samples were positive for fungal DNA (P = 0.1). C. albicans DNA was detected in 6 (19%) of the decedents’ eyes but from none of the surgical patients (P = 0.04). Conclusions. Fungi were present on the normal ocular surface of children and adolescents. C. albicans was more likely to be found postmortem than pre-surgically.

The RIM101 signal transduction pathway regulates Candida albicans virulence during experimental keratomycosis.

PURPOSE To examine the role of the fungal RIM101 signal transduction pathway in the pathogenesis of Candida albicans keratitis. METHODS C. albicans wild-type strain SC5314, prototrophic mutant control DAY185, and homozygous fungal mutants for the rim8, rim13, rim20, rim101, and phr1 genes were evaluated in vitro using proliferation and filamentation assays. Scarified corneas of BALB/c and C57BL/6J mice were topically inoculated and observed daily for keratitis severity. Corneal adaptation and pathogenicity were assessed ex vivo by maintaining infected porcine corneas for 3 days in an explantation culture system for histologic evaluation of hyphal penetration. RESULTS All C. albicans strains had similar growth kinetics, and SC5314 and DAY185 demonstrated pH-induced filamentation. Fungal mutants had reduced hyphal formation at alkaline and neutral pH, but normal acidic assays ascertained that mutant strains did not have a generalized filamentation defect. SC5314 and DAY185 caused moderate to severe keratitis in mice, whereas fungal strains lacking constituents of the RIM101 pathway had significantly (P<0.05) attenuated severity in vivo. Three days after inoculation of porcine corneas, SC5314 and DAY185 produced hyphae that penetrated 28% and 25%, respectively, of the corneal thickness, and all five mutant strains showed significantly (P<0.05) less stromal penetration. CONCLUSIONS The RIM101 signal transduction pathway plays an important role in the development of C. albicans keratitis. The fungal pathway intermediates Rim8p, Rim13p, Rim20p, and Rim101p and the downstream cell-wall protein Phr1p are pivotal in the process of corneal invasion by C. albicans.

Persisting murine cytomegalovirus can reactivate and has unique transcriptional activity in ocular tissue

ABSTRACT Cytomegalovirus (CMV) retinitis is an important ocular complication in human immunodeficiency virus-infected individuals and the leading cause of blindness in those not undergoing highly active antiretroviral therapy. Murine CMV (MCMV) infection of mice has been shown to be a useful small-animal model for the study of CMV pathogenesis in the eye. The purpose of this study was to evaluate CMV persistence in ocular tissue and to determine the potential for reactivation. Following subretinal inoculation of immunocompetent BALB/c mice, tissues were tested for infectious virus by plaque assay and for the presence of viral DNA and RNA by PCR. The latent phase of the infection in mouse tissues was analyzed by plaque assay, PCR, and explantation cocultivation in both immunocompetent and cyclophosphamide-treated mice. The acute phase of the infection was resolved by 2 to 3 weeks postinfection, while viral DNA persisted beyond 12 months. Immediate-early 1 transcripts were detected in 100% of the ocular samples tested, and glycoprotein H transcripts were detected in 86% of the samples, but no difference in viral DNA or RNA levels between immunocompetent and immunosuppressed animals was measured. Irrespective of immune status, no in vivo reactivation was detected; however, reactivated virus was observed in 76 to 82% of the eyes following explantation onto a permissive cell layer. The transcriptional activity and relatively high frequency of explantation-induced reactivation in both immunocompetent and immunosuppressed mice suggest that control of MCMV latency in ocular tissue might involve other regulatory events that are not entirely dependent on intact specific immunity.

Expression of matrix metalloproteinases 2 and 9 in experimental corneal injury and fungal keratitis.

Purpose: Levels of matrix metalloproteinases (MMPs) can be modulated during corneal infection, but little is known about MMP profiles during fungal keratitis. The purpose of this study was to determine the effect of corneal trauma and immunosuppressive treatment on the expression kinetics of MMP-2 and MMP-9 during experimental keratomycosis. Methods: Corneas of immunocompetent and cyclophosphamide-treated adult BALB/c mice were topically inoculated with 1 × 105 culturable units of Fusarium solani or mock-inoculated with or without superficial corneal scarification. Eyes were scored daily for disease severity and processed for zymography after 1.5 hours, 6 hours, 1 day, 4 days, or 8 days. Gelatinase activity was densitometrically quantitated and normalized to MMP-2 and MMP-9 controls. Results: MMP-9 levels in nontraumatized eyes transiently increased at 6 hours after fungal exposure, but this increase was inhibited by cyclophosphamide treatment. Corneal injury significantly induced early MMP-9 expression that returned to baseline levels within 4 days. Cyclophosphamide pretreatment reduced and delayed MMP-9 after scarification. Fusarium exposure dampened the MMP-9 response to corneal trauma in immunocompetent and cyclophosphamide-treated animals. Ocular levels of MMP-2 were not affected by scarification, fungal exposure, or immunosuppressive treatment. Conclusions: Ocular MMP-9 levels, but not MMP-2 levels, increased soon after corneal injury. A similar, although muted, MMP-9 response occurs during early filamentous fungal keratitis, with a kinetic profile similar to corneal disease progression. The early stage of ulcerative keratitis may involve selective regulation of corneal matrix metalloproteinases, suggesting an initial opportunity for therapeutic intervention.

Mechanisms for human cytomegalovirus-induced cytoplasmic p53 sequestration in endothelial cells

Human cytomegalovirus (HCMV) infection results in endothelial dysfunction, typically known as dysregulated apoptosis, and aberrant expression and sub-cellular localization of p53, a tumor suppressor that accumulates at the late stage of infection. In this study, we examined three hypotheses that could be responsible for HCMV-induced cytoplasmic p53 accumulation at the later stage of infection: hyperactive nuclear export, cytoplasmic p53 tethering and delayed p53 degradation. Leptomycin B treatment, a nuclear export inhibitor, was unable to reduce cytoplasmic p53, thereby eliminating the hyperactive nuclear export mechanism. The findings that nascent p53 still entered nuclei after the nuclear export inhibition indicated that cytoplasmic tethering may play a minor role. Cytoplasmic p53 was still observed after the translation activities were blocked by cycloheximide. There was more than an eight-fold increase in the cytoplasmic p53 half-life with abnormal p53 ubiquitination. Taken together, these results suggest that delayed degradation could be responsible for the cytoplasmic p53 accumulation. The general slow-down of the proteasomal activity and the dysregulated p53 ubiquitination process at the later stage of infection could contribute to the reduced cytoplasmic p53 degradation and might be relevant to dysregulated endothelial apoptosis. The HCMV-induced changes in p53 dynamics could contribute to endothelial dysfunction.