Virus Infections In The Eye

我们对病毒和寄生虫在眼睛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化过程；(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；(5)视网膜的弓形虫感染。我们已经建立了一个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)。该病毒能够在存在轻度视网膜血管炎症的情况下引发急性感染。最初的视网膜损伤之后是感染性病毒的清除和进行性的视网膜变性。这是第一个显示病毒引起的变性、病毒持久性、对病毒引起的组织损伤的遗传易感性和病毒引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退行性疾病的病理生理机制，并确定与之相关的基因。在过去的一年里，我们取得了以下重要发现。由于肿瘤坏死因子-α在免疫介导的过程中起着关键作用，我们研究了视网膜变性易感的BALB/c小鼠和变性抵抗的CD-1小鼠在疾病过程中肿瘤坏死因子-α/肿瘤坏死因子-α受体和下游信号分子一氧化氮(NO)的水平。玻璃体内注射冠状病毒后，小鼠视网膜内检测到较高水平的肿瘤坏死因子-α，而血清中肿瘤坏死因子-α和肿瘤坏死因子受体1蛋白的浓度显著升高，分别为p&lt；0.005和p&lt；0.0005，但对CD-1小鼠无影响。与对照组相比，BALB/c(p&lt；0.00005)和CD-1(p&lt；0.005)小鼠的sTNFR2蛋白浓度均升高，而BALB/c(p&lt；0.0005)小鼠的sTNFR2蛋白浓度显著升高。诱导型一氧化氮合酶基因在BALB/c小鼠中最初高表达，但在感染急性期下降，而在CD-1小鼠中表达增加。这些趋势归因于菌株之间单核细胞肿瘤坏死因子受体2的释放(p&lt；0.0005)的差异，因为sTNFR2显著降低了一氧化氮的产生水平(p&lt；0.01)。这些研究表明，病毒感染后的视网膜变性与肿瘤坏死因子-a和肿瘤坏死因子受体的释放增加以及一氧化氮的下调有关。此外，他们还表明，这些分子参与了免疫反应的变化，导致了视网膜变性易感小鼠的自身免疫反应。在动物模型系统的基础上，我们还启动了评估人类视网膜退行性疾病的研究。通过免疫细胞化学染色和免疫印迹分析，对不明原因视网膜病变患者进行自身抗体检测。用患者血清筛选大鼠视网膜表达文库，鉴定阳性克隆。其中一个克隆与晶状体上皮源性生长因子/转录共激活因子p52和p75/DSF70有88%的同源性。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受损儿童失明的主要原因。人类巨细胞病毒潜伏期的研究比较困难。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提供理论基础。趋化因子调节白细胞的运输，可能在炎症细胞重新聚集到视网膜中起重要作用。人巨细胞病毒感染hRPE细胞后，引起3种重要的趋化因子MCP-1、MCP-3和IL-8的调节。感染HCMV的hRPE中MCP-1和MCP-3的mRNA和蛋白水平均低于未感染的hRPE。相反，巨细胞病毒感染使IL-8mRNA和蛋白水平升高。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可能是视网膜炎和免疫恢复性葡萄膜炎中HCMV免疫逃避发病机制的一个可能机制。

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.