Use Of Cdna Microarrays In Gene Expression Of Uveitis Pa

眼部炎症性疾病，包括葡萄膜炎，会导致严重的视力丧失。以前的非人类研究已经确定了几种细胞类型，受体系统和代谢中间体，这些已经导致了人类患者的治疗方法。然而，关于这些步骤在炎症性疾病状态下的人类基因表达的信息是缺乏的。本无干预研究建议从其他眼炎性疾病的内部试验中获取患者的外周血和组织标本，并应用当代cDNA微阵列技术分析差异基因表达。测试结果不会报告给参与者，也不会用于诊断或治疗目的。这项研究?本研究的主要目的是利用cDNA微阵列分析技术，确定处于特定临床阶段的眼部炎症性疾病患者的独特基因表达谱和疾病相关基因。这将有助于进一步了解病理机制和潜在的治疗靶点。大约3000 - 5000个基因将从与白细胞介素（IL）蛋白及其受体和肿瘤坏死因子（TNF）相关的一组选定基因开始检查。纯化的外周血单个核细胞（或使用RNA分离程序的全血裂解物）将用于从这些样品中分离总RNA。样本将在活动性或复发性炎症疾病期间采集，并在治疗后的平静期再次采集。基因分析的微阵列工具和方法现在可以在NEI获得。与这些研究相结合的是负调节细胞的功能测定和细胞表面标记，即所谓的？抑制细胞?。T细胞活化与这些负调控信号和临床活性之间存在相关性。我们继续收集患者在葡萄膜炎活跃期和疾病静止期的数据。自2002年2月批准以来，迄今已有55名患者参加了该方案。截至2005年10月，我们招募了55名患者。在参与该方案的55名患者中，31名女性，24名男性，1名儿童。其中白人33人，黑人16人，亚洲人3人，西班牙人2人，种族背景不详1人。所有患者在加入另一NEI方案之前已被纳入该方案进行评估或治疗，并在取样前签署了同意书。对于所有55名处于疾病单期或多期的患者，从纯化的pbmc中收集了74个总RNA样本，保存了101个血清样本，并对65个多表面标记物进行了FACS分析。此外，还进行了57项增殖试验，以评估T细胞对s抗原的反应，s抗原是一种非感染性葡萄膜的自身抗原。在纳入的55名患者中，有12名患者经历了至少两个阶段的疾病（即活跃到不活跃或反之亦然），并被认为已完成临床抽样。我们使用NIA阵列膜对来自10名患者的24份RNA样本进行了48次微阵列分析。我们还使用superarray膜对11名患者和1个正常池进行了20次微阵列测试。到目前为止，我们已经确定了58个假定的基因，与正常人相比，这些基因在葡萄膜炎患者中可能特异性地上调或下调。已知一些基因会导致葡萄膜炎，比如干扰素。有些是新的基因，以前没有显示出对葡萄膜炎有重要意义。我们正计划确认从58个候选基因列表中选出的一组我们认为重要的基因。

Ocular inflammatory diseases, including uveitis, cause significant visual loss. Previous non-human investigations have identified several cell types, receptor systems and metabolic intermediates that have led to treatment approaches for human patients. However,information on the human genetic expression of these steps in defined inflammatory disease states is lacking. This non-intervention study proposes to obtain peripheral blood and tissue specimens from patients enrolled in other intramural trials for ocular inflammatory diseases and to apply contemporary cDNA microarray technologies for the analysis of differential gene expression. Test results will not be reported to participants or used for diagnostic or therapeutic purposes.The study?s primary objective is to identify unique gene expression profiles as well as disease relevant genes for patients with ocular inflammatory disease at defined clinical stages using cDNA microarray analysis. This will help provide further insight to understand the pathological mechanisms and potential targets for treatment. Some 3,000-5,000 genes will be examined starting with a selected set associated with interleukin (IL) proteins and their receptors, and with tumor necrosis factors (TNF). Purified peripheral blood mononuclear cells (or whole blood lysates using RNA isolation procedures) will be used to isolate total RNA from these samples. Samples will be taken during periods of active or recurring inflammatory disease and again during periods of quiescence after treatment. The microarray tools and methods for genetic analysis are now available at the NEI.In conjunction with these studies are functional assays and cell surface markers of negative regulatory cells, so called ?suppressor cells?. A correlation is being made between T cell activation and these negative regulatory signals and clinical activity.We continue to collect data from patients during the active phase of their uveitis and when their diesease became quiescent. To date, there have been 55 patients enrolled onto this protocol since approval in February 2002. We have recruited 55 patients until October 2005. Of the 55 patients enrolled in the protocol 31 are female, 24 are male, and 1 is considered a child. Among them, 33 are white, 16 are black, 3 are Asian, 2 are Hispanic and 1 is with unknown racial background. All the patients have been enrolled in another NEI protocol for evaluation or treatment prior to be enrolled into this protocol and signed consent form before samples were taken. For all 55 patients who have been enrolled either at a single stage of the disease or at multiple stages of the disease, 74 total RNA samples have been collected from purified PBMCs, 101 serum samples stored and 65 FACS analysis for multiple surface markers have been analyzed. In addition, 57 proliferation assays have also been performed to assess T cell responses to S-antigen, a self-antigen for non-infectious uveits. Of the 55 patients enrolled, 12 patients have undergone at least two stages of the disease (i.e., active to inactive or vice versa) and are considered completed for clinical sampling. We have done 48 microarrary analyses for 24 RNA samples from 10 patients using the NIA array membranes. We have also run 20 microarray using the SupperArray membranes for 11 patients and 1 normal pool. We have, thus far, identified 58 putative genes which may specifically up- or down-regulated in uveitis patients as compared to normal. Some of the genes are known to contribute to uveitis, such as interferon gamma. Some are new genes that have not been previously shown significant for uveitis. We are planning to confirm a selected panel of genes from that 58 candidate gene lists which we believe significant.