Etiologic Antigens in Sarcoidosis

结节病是一种病因不明的多系统肉芽肿性疾病，90%以上的患者肺部受累，可能导致终末期纤维化、肺心病和死亡。结节病的病理特征是非干酪性肉芽肿性炎症。由于皮内注射的病变组织提取物在结节病患者体内引起肉芽肿性炎症，与自发性肉芽肿(Kveim反应)难以区分，我们推测结节组织提取物含有与疾病相关的抗原。Kveim提取物中活性成分的生物物理特性包括相对热稳定性、对中性洗涤剂和蛋白酶的抵抗力以及对三级结构的依赖。这项应用的总体目标是在结节病中识别这些致病组织抗原。我们的中心假设是，结节病是由T和B细胞对微生物来源的改变蛋白聚集体的关联免疫反应引起的。与这一假设一致的是，我们的初步研究表明，存在少量抗蛋白酶、中性洗涤剂不溶的蛋白，免疫印迹分析表明，这些蛋白是结节病患者T细胞依赖性免疫球蛋白的靶标，而不是健康对照组的靶标。MALDI-TOF质谱仪和免疫印迹分析已经在结节病的这些蛋白质组分中鉴定出结核分枝杆菌(MKatG)或耻垢分枝杆菌的分枝杆菌过氧化氢酶-过氧化物酶蛋白，但不是对照组织。初步研究表明，在结节病中T细胞和B细胞都对mKatG蛋白有反应，提示mKatG蛋白在结节病中是相关的致病抗原。为了验证分枝杆菌KatG蛋白是结节病的致病抗原的假设，我们提出了利用MALDI-TOF质谱仪和蛋白质免疫印迹分析来确定分枝杆菌KatG蛋白在结节病和对照组织中的存在的研究。为了确定这些微生物蛋白是否诱导疾病特异性免疫反应，我们将确定B和T细胞对结核分枝杆菌和耻垢分枝杆菌KatG蛋白和选定多肽的免疫反应的分子基础，并确定mKatG蛋白是否优先在结节病患者和对照组中扩展表达Valpha/Vbeta的特异性T细胞。总之，这些研究提供了确定一组特定的微生物抗原参与结节病肉芽肿性炎症发病机制的可能性，从而为这种疾病的治疗提供了一个新的靶点。

Sarcoidosis is a multisystem granulomatous disorder of unknown etiology that involves the lungs in over 90 percent of affected individuals and may cause end-stage fibrosis, cor pulmonale, and death. The pathologic hallmark of sarcoidosis is non-caseating granulomatous inflammation. Since extracts of diseased tissue injected intradermally elicit a nidus of granulomatous inflammation in patients with sarcoidosis that is indistinguishable from spontaneously arising granulomas (the Kveim reaction), we postulate that sarcoid tissue extracts contain disease-relevant antigens. Biophysical properties of the active component in Kveim extracts include relative heat stability, resistance to neutral detergents and proteases, and a dependence on tertiary structure. The overall goal of this application is to identify these pathogenic tissue antigens in sarcoidosis. Our central hypothesis is that sarcoidosis is caused by linked T and B cell immune responses to aggregates of altered proteins of microbial origin. Consistent with this hypothesis, our preliminary studies demonstrate the presence of a small number of protease-resistant, neutral-detergent insoluble proteins that by immunoblot analysis are targets of T cell dependent IgG from patients with sarcoidosis but not healthy controls. MALDI-TOF mass spectrometry and immunoblot analysis has identified the mycobacterial catalase-peroxidase protein from Mycobacterium tuberculosis (mKatG) or M. smegmatis in these protein fractions from sarcoidosis but not control tissues. Preliminary studies demonstrate both T and B cell responses to mKatG proteins in sarcoidosis, suggesting the mKatG proteins are relevant, pathogenic antigens in sarcoidosis. To test the hypothesis that mycobacterial KatG proteins are pathogenic antigens in sarcoidosis, we propose studies to determine the presence of mycobacterial KatG proteins in sarcoidosis and control tissues using MALDI-TOF mass spectrometry and protein immunoblot analyses. To determine whether these microbial proteins induce disease-specific immune responses, we will determine the molecular basis of the B and T cell immune responses to both M. tuberculosis and M. smegmatis KatG proteins and selected peptides, and determine whether mKatG proteins preferentially expand specific Valpha/Vbeta expressing T cells in patients with sarcoidosis and control subjects. Together, these studies offer the potential of identifying a specific group of microbial antigens involved in the pathogenesis of granulomatous inflammation in sarcoidosis, thus providing a novel target for therapy of this disease.