Our studies of various virologic and immunopathologic processes that occur when viruses and parasites replicate in the ocular microenvironment comprise four areas: (1) virus induced retinal degenerative processes; (2) the possible roles of viruses in human diseases; (3) molecular diagnosis and pathogenesis of cytomegalovirus (CMV) infections in man; and (4) herpesvirus infections of the eye. We have established a model system for studying retinal degenerative diseases, experimental coronavirus retinopathy (ECOR). The virus is capable of inducing an acute infection in the presence of mild retinal vascular inflammation. Initial retinal damage is followed by clearance of infectious virus and progressive retinal degeneration. This is the first retinal model to demonstrate a virus induced degeneration, viral persistence, a genetic predisposition to virus induced tissue damage and a virus triggered autoimmune response. Our goal is to determine the pathophysiological mechanisms and to identify genes involved in the retinal degenerative disease. During the past year we have made the following key findings. Since TNF-a plays a crucial role in immune-mediated processes we evaluated the levels of TNF-a/TNF-a receptors and the downstream signaling molecule nitric oxide (NO) during the course of disease in both retinal degeneration susceptible BALB/c and degeneration resistant CD-1 mice. Following intravitreal injection with the coronavirus, mouse hepatitis virus (MHV), TNF-a mRNA was detected at higher levels within the retinas, and concentrations of TNF-a and sTNFR1 proteins were significantly increased, p<0.005 and p< 0.0005 respectively, within the serum of BALB/c but not CD-1 mice. Gene expression of iNOS while initially high in BALB/c mice decreased during the acute phase of infection, while it increased in CD-1 mice. These trends are attributable to differences in monocyte TNFR2 release (p < 0.0005) between the strains since sTNFR2 significantly decreased (p < 0.01) levels of NO production. These studies demonstrate that retinal degeneration following viral infection is associated with increased release of TNF-a and TNF receptors combined with a down-regulation of NO. Furthermore they suggest that these molecules are involved in alterations in immune response leading to the autoimmune reactivity seen in retinal degeneration susceptible mice. Based on the animal model system, we have also initiated studies to evaluate human retinal degenerative diseases. Autoantibodies were detected in patients with retinopathy of unknown origin by immunocytochemical staining and western blot analysis. We have identified three patients with cone-rod degeneration with high titers of antibody directed against the ganglion cells and inner nuclear layer. Anti-retinal reactivity at these levels were not detected in sera from patients with known genetic retinal degenerations, uveitis or from normal individuals. Two antigens, LEDGF and ATR-X, were identified from a retina cDNA library with sera from a cone-rod degeneration patient. The sera of all three cone-rod degeneration patients demonstrated reactivity to LEDGF. Identification of the specific anti-retinal antibodies that contribute to altered retinal cell function and viability may provide new insights into mechanisms of retinal cell dysfunction.? ? Corneal herpes infection elicits a robust inflammatory response and eventually leads to a vision-threatening stromal keratitis as a sequela of frequent reactivation of latent virus. The aetiology of herpetic stromal keratitis is thought to be an aberrant Th1 cytokine mediated immunopathology. We evaluated HSV infection in human corneal epithelial and corneal fibroblast cells. We found that HSV DNA resulted in augmented TLR-3 and ?9 gene expression and IL-6 release. As virus is frequently in the form of neutralized virus immune complexes, the ability of these immune complexes to interact with TLRs and trigger IL-6 production was evaluated. We found that HSV-anti-HSV IgG complexes were as potent as HSV DNA in their ability to induce IL-6. These phenomena were mediated via augmented TLR-3 and ?9 gene expression. These studies indicate that viral DNA and/or immune complexes deposited in corneal stroma may continue to trigger stromal inflammation by excessive cytokine release via TLR3 and 9 long after the initial acute viral insult has subsided.? ? Cytomegalovirus (CMV) retinitis is characterized by alterations in retinal cell function and host responses to virus replication. We evaluate the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PGE) in CMV infected human retinal pigment epithelial (RPE) cells and determined their effect on virus replication. CMV immediate early (IE) protein and COX-2 proteins were identified in RPE cells in retinal tissue sections from patients with CMV retinitis. COX-2 mRNA and protein were induced after CMV infection of human RPE cell cultures. CMV infection of RPE cells induced translocation of NF-kB from the cytoplasm to the nucleus. PGE1 and PGE2 were significantly (P<0.001) increased in human RPE cell cultures infected with CMV. Inhibition of CMV IE gene by antisense oligonucleotides abrogated induction of mRNA for COX-2 and protein synthesis of COX-2 and PGE2. PGE enhanced CMV plaque formation and Real Time PCR analysis revealed that PGE treatment significantly increased CMV DNA copy numbers. These studies demonstrate that when CMV replicates within human RPE cells, COX-2 induction augments virus replication via the PGE pathway. The induction of COX-2 and PGE during retinal CMV infection may augment virus replication and alter a variety of retinal physiological responses.
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