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%以上的患者累及肺部，可导致终末期纤维化、肺心病和死亡。结节病的病理特征是非干酪化肉芽肿性炎症。由于瘤样病患者皮下注射病变组织提取物会引起肉芽肿性炎症，与自发产生的肉芽肿（Kveim反应）难以区分，因此我们假设瘤样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抗性以及对三级结构的依赖性。这个应用程序的总体目标是确定这些致病性组织抗原在结节病。我们的中心假设是结节病是由T细胞和B细胞对微生物来源的改变蛋白聚集体的免疫反应引起的。与这一假设相一致，我们的初步研究表明，通过免疫印迹分析，存在少量蛋白酶抗性，中性洗涤剂不溶性蛋白质，这些蛋白质是结节病患者而不是健康对照者的T细胞依赖性IgG的靶标。MALDI-TOF质谱分析和免疫印迹分析在结节病的这些蛋白组分中发现了来自结核分枝杆菌（mKatG）或耻垢分枝杆菌的过氧化氢酶过氧化物酶蛋白，但在对照组织中没有发现。初步研究表明，在结节病中，T细胞和B细胞对mKatG蛋白均有应答，提示mKatG蛋白是结节病相关的致病抗原。为了验证分枝杆菌KatG蛋白是结节病致病抗原的假设，我们提出了使用MALDI-TOF质谱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细胞和T细胞对结核分枝杆菌和耻垢分枝杆菌KatG蛋白和选定肽的免疫反应的分子基础，并确定mKatG蛋白是否优先扩增结节病患者和对照组中表达特异性Valpha/Vbeta的T细胞。总之，这些研究提供了鉴定一组特定微生物抗原的潜力，这些抗原参与了结节病肉芽肿性炎症的发病机制，从而为这种疾病的治疗提供了新的靶点。

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.