Virus Infections In The Eye

我们对病毒和寄生虫在眼部微环境中复制时发生的各种病毒学和免疫病理学过程的研究包括五个方面：（1）病毒诱导的视网膜变性过程;（2）病毒在人类疾病中的可能作用;（3）人类巨细胞病毒（CMV）感染的分子诊断和发病机制;（4）眼睛的疱疹病毒感染和（5）视网膜的弓形虫感染。我们建立了一个研究视网膜变性疾病的模型系统-实验性冠状病毒视网膜病变（ECOR）。该病毒能够在存在轻度视网膜血管炎症的情况下诱导急性感染。最初的视网膜损伤之后是感染性病毒的清除和进行性视网膜变性。这是第一个证明病毒诱导的变性、病毒持续存在、对病毒诱导的组织损伤的遗传易感性和病毒触发的自身免疫反应的视网膜模型。我们的目标是确定视网膜变性疾病的病理生理机制，并确定相关基因。在过去一年中，我们取得了以下主要成果。由于TNF-α在免疫介导的过程中起着至关重要的作用，我们评估了视网膜变性易感BALB/c和变性抗性CD-1小鼠中疾病过程中TNF-α/TNF-α受体和下游信号分子一氧化氮（NO）的水平。在玻璃体内注射冠状病毒、小鼠肝炎病毒（MHV）后，在视网膜内检测到较高水平的TNF-α mRNA，并且在BALB/c小鼠而不是CD-1小鼠的血清中，TNF-α和sTNFR 1蛋白的浓度分别显著增加，p<0.005和p< 0.0005。虽然与对照相比，sTNFR 2蛋白的浓度在BALB/c（p< 0.00005）和CD-1（p< 0.005）小鼠中均升高，但在BALB/c小鼠中浓度显著更高（p< 0.0005）。在感染的急性期，BALB/c小鼠中iNOS的基因表达最初较高，但在CD-1小鼠中增加。这些趋势归因于菌株之间单核细胞TNFR 2释放的差异（p < 0.0005），因为sTNFR 2显著降低（p < 0.01）NO产生水平。这些研究表明，病毒感染后的视网膜变性与TNF-α和TNF受体的释放增加以及NO的下调有关。此外，他们表明这些分子参与免疫应答的改变，导致在视网膜变性易感小鼠中观察到的自身免疫反应性。基于动物模型系统，我们还开始了评估人类视网膜退行性疾病的研究。用免疫细胞化学染色和westernblot分析法检测不明原因视网膜病变患者的自身抗体。我们已经确定了三个锥杆变性与高滴度的抗体针对神经节细胞和内核层的患者。在已知遗传性视网膜变性、葡萄膜炎患者或正常个体的血清中未检测到这些水平的抗视网膜反应性。两个抗原，LEDGF和ATR-X，从视网膜cDNA文库与锥-杆变性患者的血清中鉴定。所有三个锥杆变性患者的血清表现出反应LEDGF。鉴定有助于改变视网膜细胞功能和活力的特异性抗视网膜抗体可能为视网膜细胞功能障碍的机制提供新的见解。

Our studies of various virologic and immunopathologic processes that occur when viruses and parasites replicate in the ocular microenvironment comprise five 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; (4) herpesvirus infections of the eye and (5) Toxoplasma gondii infections of the retina. 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. While concentrations of sTNFR2 proteins were elevated in both BALB/c (p< 0.00005) and CD-1 (p< 0.005) mice compared to controls, concentrations were significantly higher in BALB/c mice (p< 0.0005). 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.