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。我们已经确认了三名锥杆变性患者，其针对神经节细胞和内核层的抗体效价很高。在已知遗传性视网膜变性患者、葡萄膜炎患者或正常人的血清中，没有检测到这些水平的抗视网膜反应。用1例视锥视杆细胞变性患者的血清，从视网膜cDNA文库中鉴定出LEDGF和ATR-X两种抗原。3例视锥-视杆细胞变性患者的血清均显示对LEDGF的反应性。识别导致视网膜细胞功能和活性改变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.