Giant cell arteritis: lesional microbial sequences

巨细胞性动脉炎(UCA)是一种老年人特有的严重疾病，可导致高达20%的患者失明。UCA的诊断通常是非特异性的，需要一系列的临床表现或动脉活检阳性。虽然其病因尚不清楚，但GCA病变的肉芽肿性炎症和局部免疫学特征表明，一种隐蔽的微生物参与了疾病的发病机制。这项建议的主要目标是识别微生物起源的基因片段，这些基因片段物理上定位于GCA病变，并被疾病特异性免疫反应靶向。这项工作的长期目标和与健康相关的是加强对OCA发病机制的了解，并开发新的诊断方法和/或治疗策略。 将分离和鉴定与GCA病变相关的DNA序列。在实验上，这一建议利用了在检测慢性病中的隐蔽微生物方面的最新进展。一种消减克隆策略(RDA)将被用来从受GCA+影响的动脉标本的组织病理学涉及的区域中分离新的DNA序列。这些序列将被克隆，并根据与已知微生物序列的同源性进行优先排序。高优先级(候选)序列将被进一步分析，以获得它们与GCA关联的更多证据。 将测试高优先级基因序列的免疫学相关性，并通过它们与UCA的关联进一步定义序列。候选序列的开放阅读框架(ORF)将被识别并作为融合蛋白产生。融合蛋白将用于检测与疾病相关的细胞介导或体液免疫反应的证据。 将对高优先级基因序列或产品的皮损流行率和位置进行评估。动脉活检的候选序列的扩增将决定这些序列的流行率和疾病相关性。通过免疫组织化学和/或原位杂交定位候选序列将进一步验证高优先级候选序列与疾病的相关性。 重要的前期工作验证了这一方法。初步研究从GCA病变中确定了几个高优先级的候选微生物序列，这些序列似乎与GCA+涉及的动脉相关，并引发与疾病相关的体液免疫反应。针对候选序列产物的疾病相关体液免疫的检测可以形成发展GCA的血清学诊断试验的基础。为了实现本提案的目标，需要对这些候选序列进行进一步分析，并确定其他候选序列。

Giant cell arteritis (UCA) is a serious disease unique to the elderly that leads to loss of sight in up to 20% of affected individuals. The diagnosis of UCA is often nonspecific and requires a constellation of clinical findings or a positive arterial biopsy. While its etiology is unknown, the granulomatous inflammation and local immunologic features of GCA lesions suggest involvement of a cryptic microorganism in disease pathogenesis. The primary goal of this proposal is to identify gene segments of microbial origin physically localized to GCA lesions and targeted by a disease-specific immune response. The long-term objective and health relatedness of this work is to enhance the understanding of OCA pathogenesis and develop new diagnostic methods and/or therapeutic strategies. GCA lesion-associated DNA sequences will be isolated and identified. Experimentally, this proposal takes advantage of recent advances in the detection of cryptic microorganisms in chronic diseases. A subtractive cloning strategy (RDA) will be performed to isolate novel DNA sequences from areas of histopathologic involvement in GCA+ affected arterial specimens. The sequences will be cloned and prioritized based on homology to known microbial sequences. High priority (candidate) sequences will be further analyzed to obtain additional evidence for their association with GCA. The immunologic relevance of high-priority gene sequences will be tested and sequences will be further defined by their association with UCA. Open reading frames (ORFs) of candidate sequences will be identified and produced as fusion proteins. The fusion proteins will be used to detect evidence for a disease-associated cell mediated or humoral immune response. Lesional prevalence and location of the high-priority gene sequences or products will be evaluated. Amplification of candidate sequences from arterial biopsies will determine the prevalence and disease-relatedness of the sequences. Localization of candidate sequences by immunohistochemistry and/or in situ hybridization will further validate the disease-association of the high-priority candidates. Significantly preliminary work validates this approach. Initial studies identify several high-priority candidate microbial sequences from GCA lesions that appear to be correlated with GCA+ involved arteries and elicit a disease-associated humoral immune response. Detection of a disease-associated humoral immunity against a product of a candidate sequence could form the basis for development of a serological diagnostic test for GCA. Further analysis of these candidates and identification of other candidate sequences is required to achieve the goals of this proposal.