XENOBIOTICS AND PRIMARY BILIARY CIRRHOSIS

描述（由申请人提供）：抗线粒体抗体（AMA）在自身免疫血清学反应物中是独一无二的，因为它们与原发性胆汁性肝硬化（PBC）有着极高的相关性。AMA不仅高度定向，而且对主要线粒体自身抗原PDC-E2的脂酰结构域具有特异性。用硫辛酸的特定小分子模拟物修饰这个脂酰结构域，产生的结构与PBC血清的反应一样好，而且往往比天然分子更好。我们还表明，豚鼠和最近的NOD.1101小鼠，在免疫原中没有任何PDC-E2的情况下，用异种生物bsa免疫，会产生自反应性AMA和胆道特异性病理。我们认为PBC是由PDC-E2的高度定向多细胞谱系反应引起的，PDC-E2在遗传易感宿主中的化学修饰开始了疾病的漫长自然历史。我们目前的工作已经提供了一些关于这个问题的线索，包括确定化学物质作为PDC-E2的潜在调节剂和PBC的触发物。为了证明我们的论文，我们现在将使用一系列合成类似物来定义AMA结合肽和异种生物偶联物的分子识别要求。PDC-E2的¿-sheet结构似乎是AMA特异性的必要条件，我们必须确定该区域的关键氨基酸。我们也认识到PDC-E2以外的肽可能参与了最初的AMA引发事件，我们将利用我们在外源组合肽技术方面的专业知识来研究表位多样性。之前我们已经开发和研究了CD4和CD8克隆的T细胞系，现在我们将把这些观察扩展到使用未修饰的PDC-E2和外源修饰的PDC-E2来研究来自患者和对照组的pbmc，以验证外源修饰的PDC-E2比未修饰的自身抗原更有效地激活和扩增特异性T细胞，并且这些T细胞会与天然线粒体自身抗原交叉反应的理论。最后，我们将利用我们激动人心的新数据，在用硫辛酸模拟物2-辛酰胺- bsa免疫的NOD.1101小鼠中诱导胆道特异性病理。我们现在将扩展这些数据，以严格定义血清学、肝脏组织学、免疫生物学和疾病的自然史。我们认为，这些实验结果将证明我们的观点，即PBC是由外源化学物质暴露引起的，破坏了耐受性，多细胞谱系对PDC-E2的反应将是该疾病的病理效应机制。

DESCRIPTION (provided by applicant): Antimitochondrial antibodies (AMA) are unique among autoimmune serologic reactants because of their extremely high association with primary biliary cirrhosis (PBC). The AMA is not only highly directed, but also very specific to the lipoyl domain of PDC-E2, the major mitochondrial autoantigen. Modification of this lipoyl domain, with specific small molecular mimics of lipoic acid, results in structures that react as well as and often better to PBC sera than the native molecule. We have also shown that guinea pigs, and now most recently NOD.1101 mice, immunized with xenobiotic-BSA, without any PDC-E2 in the immunogen, develop self-reacting AMA and biliary specific pathology. We propose that PBC is caused by a highly directed multicell lineage response to PDC-E2 and that chemical modification of PDC-E2 in genetically susceptible hosts begins the long natural history of disease. Our current work has provided several clues regarding this issue, including the identification of chemicals as potential modifiers of PDC-E2 and triggers of PBC. To prove our thesis, we will now define the molecular recognition requirements of AMA binding of both our peptide and xenobiotic conjugates using a series of synthetic analogues. The ¿-sheet structure of PDC-E2 appears essential for AMA specificity and we must define the critical amino acids in this region. We also recognize the possibility that peptides other than PDC-E2 may be involved in the initial AMA eliciting event and we will take advantage of our expertise in xenobiotic combinatorial peptide technology to study epitope diversity. Previously we have developed and studied CD4 and CD8 cloned T cell lines and we will now extend these observations to study PBMCs from patients and controls using unmodified PDC-E2 and xenobiotic modified PDC-E2 to test the thesis that xenobiotic modified PDC-E2 activates and amplifies specific T cells more efficiently than unmodified autoantigen and that such T cells will cross-react with native mitochondrial autoantigen. Finally, we will take advantage of our exciting new data on the induction of biliary specific pathology in NOD.1101 mice immunized with the lipoic acid mimic, 2-octynamido-BSA. We will now expand this data to rigorously define the serology, liver histology, immunobiology and natural history of disease. We submit that the results from these experiments will prove our thesis that PBC is induced by a xenobiotic chemical exposure that breaks tolerance and that the multi-cell lineage response to PDC-E2 will be the pathologic effector mechanism of this disease. Public Health Relevance: Primary biliary cirrhosis is a liver disease that primarily affects women and is believed to be secondary to an immune attack by the patient against their own bile ducts. The etiology of this disease is enigmatic but we believe the disease begins when a person makes an immune response to mitochondrial proteins. We believe that this immune attack begins by an environmental modification of the patient's own mitochondrial proteins. In this application we will study the chemistry of this reaction. We will also immunize mice with these chemicals and believe that these mice will develop the clinical features of afflicted human patients that will allow us to focus and define disease etiology and future potential treatment.