Virus Infections In The Eye

我们对病毒和寄生虫在眼微环境中复制时发生的各种病毒学和免疫病理过程的研究包括五个方面：(1)病毒诱导的视网膜退行性过程；(2)病毒在人类疾病中的可能作用；(3)人巨细胞病毒（CMV）感染的分子诊断与发病机制；(4)眼部疱疹病毒感染；(5)视网膜弓形虫感染。我们建立了研究视网膜退行性疾病的模型系统——实验性冠状病毒视网膜病变（ECOR）。该病毒能够在轻度视网膜血管炎症的情况下诱发急性感染。最初的视网膜损伤随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变性、病毒持久性、病毒诱导组织损伤的遗传易感性和病毒引发自身免疫反应的视网膜模型。我们的目标是确定病理生理机制和识别基因参与视网膜退行性疾病。在过去一年中，我们取得了以下主要成果。由于TNF-a在免疫介导的过程中起着至关重要的作用，我们在视网膜变性易感性BALB/c和变性抵抗性CD-1小鼠的疾病过程中评估了TNF-a/TNF-a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，视网膜内检测到更高水平的TNF-a mRNA， BALB/c小鼠血清中TNF-a和sTNFR1蛋白浓度显著升高（p<0.005和p< 0.0005），而CD-1小鼠血清中TNF-a和sTNFR1蛋白浓度显著升高（p<0.005和p< 0.0005）。与对照组相比，BALB/c小鼠和CD-1小鼠的sTNFR2蛋白浓度均升高（p< 0.00005），但BALB/c小鼠的sTNFR2蛋白浓度显著升高（p< 0.0005）。在BALB/c小鼠中，iNOS的基因表达在感染急性期降低，而在CD-1小鼠中升高。这些趋势可归因于菌株之间单核细胞TNFR2释放的差异（p < 0.0005），因为sTNFR2显著降低了NO生成水平（p < 0.01）。这些研究表明，病毒感染后的视网膜变性与TNF-a和TNF受体的释放增加以及NO的下调有关。此外，他们认为这些分子参与了导致视网膜变性易感小鼠的自身免疫反应的改变。基于动物模型系统，我们也开始了评估人类视网膜退行性疾病的研究。采用免疫细胞化学染色和免疫印迹法检测不明原因视网膜病变患者的自身抗体。利用患者血清筛选大鼠视网膜cDNA表达文库，鉴定出阳性克隆。其中一个克隆对晶状体上皮衍生生长因子/转录共激活因子p52和p75/DSF70的同源性为88%。这些数据表明，在该患者中发现的针对p75/p52的抗体可能与她的视网膜病变有关。人类巨细胞病毒是一种疱疹病毒，是先天性感染儿童和免疫功能低下个体失明的主要原因。研究人类巨细胞病毒潜伏期是困难的。因此，CMV复制和潜伏期的细胞培养模型可能为替代治疗方式的基本原理提供见解。趋化因子调节白细胞运输，并可能在炎症细胞募集到视网膜中起重要作用。HCMV感染HRPE细胞导致三种重要的趋化因子MCP-1、MCP-3和IL-8的调节。与未感染HRPE相比，HCMV感染HRPE中MCP-1和MCP-3 mRNA和蛋白水平降低。相比之下，HCMV感染增加了IL-8 mRNA和蛋白水平。由于趋化因子促进白细胞向炎症部位的迁移和激活，病毒对趋化因子产生的调节提示了HCMV在视网膜炎和免疫恢复性葡萄膜炎的免疫发病机制的免疫逃避。

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. Using patient sera, a rat retina cDNA expression library was screened and positive clones were identified. One of the clones demonstrated 88% identity to lens epithelium-derived growth factor / transcription coactivators p52 and p75/DSF70. These data suggest that antibodies directed against p75/p52 identified in this patient may contribute to her retinopathy. Human CMV is a herpesvirus that is a major cause of blindness in children born with congenital infections and in immunocompromised individuals. It is difficult to study CMV latency in man. Therefore cell culture models of CMV replication and latency may provide insight into a rationale for alternative treatment modalities. Chemokines regulate leukocyte trafficking and may be important in inflammatory cell recruitment into the retina. HCMV infection in HRPE cells resulted in modulation of three important chemokines, MCP-1, MCP-3 and IL-8. The levels of MCP-1 and MCP-3 mRNA and protein in HCMV infected HRPE were reduced in comparison to uninfected HRPE. In contrast, HCMV infection enhanced IL-8 mRNA annd protein levels. Because chemokines facilitate the migration and activation of leukocytes to the sites of inflammation, the modulation of chemokines production by the virus suggests a possible mechanism in immune evasion of immunopathogenesis for HCMV in retinitis and immune recovery uveitis.