VIRUS INFECTIONS IN THE EYE

我们对各种病毒学和免疫病理过程的研究 当病毒和寄生虫在眼睛里复制时发生 微环境包括五个方面：(1)病毒诱导的视网膜 退化过程；(2)病毒在人类中的可能作用 疾病；(3)分子诊断和发病机制 人类巨细胞病毒(CMV)感染；(4)水痘-带状疱疹 眼部VZV感染和(5)弓形虫 视网膜感染。我们已经建立了一个模型系统， 研究视网膜退行性疾病，实验性冠状病毒 视网膜病变(ECR)。该病毒能够诱发一种急性 感染时存在轻微的视网膜血管炎症。 最初的视网膜损伤之后是传染性病毒的清除 和进行性视网膜变性。这是第一个视网膜 演示病毒诱导的退化的模型，病毒 持久性，一种对病毒引起的组织损伤的遗传易感性 一种病毒引发了自身免疫反应。我们的目标是确定 其病理生理机制及相关基因的鉴定 视网膜退行性疾病。在过去的一年里，我们有 评估细胞因子的作用。体内细胞因子水平的分析 眼部，显示IL-1、IL-6、肿瘤坏死因子-α和干扰素-g升高。 ECR。干扰素-g和颗粒介导的细胞毒作用 淋巴细胞(CL)在限制病毒传播和 可能对免疫病理学有贡献。对CL进行了分析 CD8 T细胞的免疫细胞化学染色和RT-PCR检测 CL细胞毒颗粒、颗粒酶B、干扰素-γ的序列分析 视网膜内颗粒酶B基因的表达表明 这些基因在清除传染性疾病时上调。 来自视网膜的病毒。这些研究表明，细胞毒性T细胞 和T细胞产物(干扰素-g和颗粒酶B)，并存在于 视网膜并有助于视网膜病毒的清除。人类巨细胞病毒是一种 疱疹病毒是导致儿童先天失明的主要原因 先天性感染和免疫功能低下的个体。它是 很难研究人的CMV潜伏期。因此，细胞培养模型 对CMV复制和延迟的了解可以提供对基本原理的见解 用于替代治疗方式。为了更好地理解 视网膜组织趋向性的巨细胞病毒，我们延长了原来的 人视网膜色素上皮细胞中巨细胞病毒复制的研究。数据表明， 人巨细胞病毒感染hRPE细胞的调控不同于人巨细胞病毒 人成纤维细胞在病毒进入和 病毒基因组的转录。CMV-HOST的这些差异 人视网膜色素上皮细胞中的细胞相互作用可能在巨细胞病毒中起作用 在眼睛内激活、复制和扩散。在一个单独的 我们评估了诊断和治疗方法的一系列研究 巨细胞病毒感染的治疗。这些研究证明了它的实用性 该CMV RPE细胞模型系统用于评估病毒复制和 反义寡核苷酸抗病毒治疗的疗效观察。人类 眼部VZV感染可导致角膜炎、葡萄膜炎、急性 视网膜坏死或进行性视网膜外坏死。然而，a 复制这些疾病的小动物模型还没有 是存在的。我们已经证明，玻璃体内接种豚鼠 VZV导致慢性葡萄膜炎，由单核细胞组成 细胞渗入眼后段。我们有 利用该模型对VZV疫苗进行评估，发现 VZV gE和gI重组蛋白诱导的免疫效力 加速VZV清除的体液和细胞反应 DNA，并可能中和眼睛内的病毒。研究仍在继续 目的：建立VZV角膜炎的体内模型。我们已经开发出 利用聚合酶链式反应分析进行分子诊断的方法 疱疹病毒。这些化验方法被用来区分 感染性和免疫性眼后段人工晶状体 发炎。

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) Varicella - zoster virus (VZV) 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 (ECR). 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 evaluated the role of cytokines. Analysis of cytokine levels within the eye, revealed that IL-1, Il-6, TNF-a and IFN-g were elevated in ECR. IFN-g and granule mediated cytotoxicity by cytotoxic lymphocytes (CL) play a critical role in limiting viral spread and may contribute of immunopathology. CL were analyzed by immunocytochemical staining for CD8 T cells and by RT-PCR assays for the CL cytotoxic granule, granzyme B. Sequenctial anaylsis of IFN-g and granzyme B gene expression within the retina demonstrated that these genes are up-regulated at the time of clearance of infectious virus from the retina. These studies indicate that cytotoxic T cells and T cell products (IFN-g and granzyme B) and present within the retina and contribute to retinal viral clearance.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. In order to understand the retinal tissue tropism for CMV, we have extended our original studies of CMV replication in HRPE. The data indicate that regulation of HCMV infection of HRPE cells differed from that of human fibroblasts at both the levels of virus entry and transcription of the viral genome. These differences in CMV - host cell interactions in HRPE cells which may be instrumental in CMV activation, replication and spread within the eye. In a separate series of studies we have evaluated methods of diagnosis and treatment of CMV infections. These studies demonstrate the utility of this CMV RPE cell model system to evaluate virus replication and efficacy of antiviral therapy with antisense oligonucleotides. Human VZV infections of the eye can result in keratitis, uveitis, acute retinal necrosis or progressive outer retinal necrosis. However, a small animal model that replicates these diseases does not yet exist. We have shown that intravitreal inoculation of guinea pigs with VZV results in a chronic uveitis consisting of a mononuclear cell infiltrate in the posterior segment of the eye. We have utilized this model to evaluate VZV vaccines and found that immunization with VZV gE and gI recombinant proteins induced potent humoral and cellular responses that accelerated the clearance of VZV DNA and may neutralize virus within the eye. Studies are continuing to establish an in vivo modelfor VZV keratitis. We have developed molecular diagnostic methods using PCR analysis to detect herpesviruses. These assays are being used to distinguish between infectious and immunopathogenic posterior segment intraocular inflammation.