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，其病理特征是非干酪化性肉芽肿，最常见的是累及肺、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，在肉芽肿性炎症部位存在α + CD 4 + T细胞的寡克隆聚集，这与抗原驱动的MHC限制性过程一致。结节病具有与分枝杆菌感染相似的病理学、流行病学和免疫学特征。我们对25个结节病和25个对照石蜡包埋标本中的16 S rRNA、rpoB和IS 6110进行了PCR分析，并注意到60%的结节病肉芽肿中存在分枝杆菌核酸的证据，而对照中没有（p<0.00002，卡方）。16 S rRNA和rpoB扩增子的序列分析揭示了分枝杆菌核酸，包括一种新的分枝杆菌的存在，遗传上类似于M。结核病（MTB）（99%位置同一性）。 最近的免疫学研究也表明，分枝杆菌可能是重要的结节病免疫发病机制。Song等人注意到，48%的结节病患者血清中存在重组MTB katG的IgG抗体，而PPD阴性对照血清中为0%（p=0.0059）。使用基质辅助激光解吸/电离飞行时间质谱法，他们在75%的结节病标本中发现了MTB katG肽，而对照标本为14%（p=0.0006）;原位杂交将MTB katG和16 S rRNA DNA定位在结节病肉芽肿内。最近，我们进行了酶联免疫斑点（ELISPOT）检测结节病和对照外周血单核细胞（PBMC）的分枝杆菌katG和ESAT-6蛋白的免疫识别。我们在13名结节病患者中的10名（77%）中发现katG或ESAT-6肽的免疫识别，相比之下，11名PPD阴性健康对照中的1名（9%）（p=0.001，Fisher精确检验）和5名PPD阳性健康对照中的4名（80%）（p=1.00，Fisher精确检验）。中心假设是结节病是遗传易感宿主对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的三分子复合物的成功形成。我们建议：1）确认和扩展结节病肉芽肿中存在分枝杆菌的分子遗传学证据，2）描述急性、缓解和慢性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HLA类型与针对分枝杆菌抗原的免疫应答质量之间的关系。

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.