Investigation of the role of bacteria in the sarcoidosis trimolecular complex

描述(由申请人提供)： 结节病是一种病因不明的疾病，病理特征为非干酪性肉芽肿，最常见的累及肺、皮肤、淋巴结和眼睛。对结节病患者T细胞受体基因表达的研究显示，肉芽肿性炎症部位有Alphabeta+CD4+T细胞的寡克隆聚集，这与MHC限制性的抗原驱动过程一致。结节病具有与分枝杆菌感染相似的病理、流行病学和免疫学特征。我们对25例结节病和25例对照石蜡包埋标本进行了16SRRNA、rpoB和IS6110的聚合酶链式反应分析，发现60%的结节样肉芽肿和无一例对照标本中有分枝杆菌核酸的证据(P&lt；0.00002，卡方检验)。16S rRNA和rpoB扩增序列分析显示，分枝杆菌核酸包括一种新的分枝杆菌，在遗传上与结核分枝杆菌(MTB)相似(99%的位置相同)。 最近的免疫学研究也表明，分枝杆菌可能在结节病的免疫发病机制中起重要作用。Song等人在48%的结节病患者血清中发现了重组结核分枝杆菌katG抗体，而在PPD阴性对照中则为0%(p=0.0059)。使用基质辅助激光解吸/电离飞行时间质谱仪，他们在75%的结节病标本中发现了MTB katG多肽，而在对照标本中发现了14%(p=0.0006)；原位杂交将MTB katG和16S rRNADNA定位在结节病肉芽肿内。最近，我们对结节病和对照外周血单个核细胞(PBMC)进行了酶联免疫斑点(ELISPOT)检测，以检测分枝杆菌katG和ESAT-6蛋白的免疫识别。我们发现13例结节病患者中有10例(77%)有katG或ESAT-6多肽的免疫识别，而11例PPD阴性的健康对照组中有1例(9%)(p=0.001，Fisher‘s精确检验)，5例PPD阳性的健康对照中有4例(80%)(p=1.00，Fisher’s精确检验)。中心假设是，结节病是对遗传易感宿主中的分枝杆菌抗原的免疫反应。 结节病免疫发病机制的一个方面是在T细胞受体(TCR)、主要组织相容性复合体(MHC)蛋白和加工抗原之间成功地形成三分子复合体。我们建议：1)确认和扩大分枝杆菌在结节病肉芽肿中存在的分子遗传学证据，2)表征急性、缓解和慢性病结节病患者对分枝杆菌抗原的T细胞反应，3)确定与良好病程相关的人类白细胞抗原类型与针对分枝杆菌抗原的免疫反应的质量之间的关系。

DESCRIPTION (provided by applicant): Sarcoidosis is a disease of unknown etiology, characterized pathologically by noncaseating granulomas which most commonly involve the lung, skin, lymph node and eyes. Studies of T cell receptor gene expression in sarcoidosis patients reveal oligoclonal collections of alphabeta+ CD4+ T cells at sites of granulomatous inflammation, consistent with an antigen-driven process which is MHC-restricted. Sarcoidosis has similar pathologic, epidemiologic, and immunologic features to mycobacterial infections. We performed PCR analysis for 16S rRNA, rpoB and IS6110 in 25 sarcoidosis and 25 control paraffin- embedded specimens and noted evidence of mycobacterial nucleic acids in 60% of the sarcoid granulomas and none of the controls (p<0.00002, chi square). Sequence analysis of the 16S rRNA and rpoB amplicons revealed mycobacterial nucleic acids including the presence of a novel Mycobacterium, genetically similar to M. tuberculosis (MTB) (99% positional identity). Recent immunologic studies also suggest that mycobacteria may be important in sarcoidosis immunopathogenesis. Song et al noted IgG antibodies to recombinant MTB katG in sera from 48% of sarcoidosis patients compared to 0% in sera from PPD negative controls (p=0.0059). Using matrix-assisted laser desorption/ionization time of flight mass spectrometry, they found MTB katG peptides in 75% of sarcoidosis specimens compared to 14% of control specimens (p=0.0006); in situ hybridization localized MTB katG and 16S rRNA DNA inside the sarcoidosis granuloma. More recently, we performed enzyme linked immunospot (ELISPOT) assays on sarcoidosis and control peripheral blood mononuclear cells (PBMC) for immune recognition of mycobacterial katG and ESAT-6 proteins. We found immune recognition of katG or ESAT-6 peptides in 10 of 13 sarcoidosis patients (77%) compared to 1 of 11 PPD negative healthy controls (9%) (p=0.001, Fisher's exact test) and 4 of 5 PPD positive healthy controls (80 %) (p=1.00, Fisher's exact test). The central hypothesis is that sarcoidosis is an immune response to mycobacterial antigens in a genetically susceptible host. One facet of sarcoidosis immunopathogenesis is the successful formation of a trimolecular complex between the T cell receptor (TCR), major histocompatibility complex (MHC) proteins and processed antigens. We propose to 1) confirm and extend molecular genetic evidence for the presence of mycobacteria in sarcoidosis granuloma, 2) to characterize the T cell response to mycobacterial antigens among sarcoidosis patients with acute, resolved, and chronic disease, and 3) to determine the relationship between HLA types associated with a favorable disease course and the quality of the immune response directed against mycobacterial antigens.