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特的，因为其与原发性胆汁性肝硬化（PBC）的高度相关性。AMA不仅高度定向，而且对主要线粒体自身抗原PDC-E2的硫辛酰结构域非常特异。用硫辛酸的特定小分子模拟物修饰该硫辛酰基结构域，导致与天然分子相比与PBC血清反应良好且通常更好的结构。我们还表明，豚鼠，现在最新的NOD.1101小鼠，免疫与外源性BSA，没有任何PDC-E2的免疫原，发展自我反应AMA和胆道特异性病理。我们认为PBC是由对PDC-E2的高度定向的多细胞谱系反应引起的，并且遗传易感宿主中PDC-E2的化学修饰开始了疾病的长期自然史。我们目前的工作为这个问题提供了一些线索，包括鉴定作为PDC-E2潜在修饰剂和PBC触发剂的化学物质。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我们的肽和异源生物质缀合物的AMA结合的分子识别要求。PDC-E2的半折叠结构对于AMA特异性是必不可少的，我们必须确定该区域的关键氨基酸。我们还认识到PDC-E2以外的肽可能参与初始AMA引发事件的可能性，我们将利用我们在异生物质组合肽技术方面的专业知识来研究表位多样性。以前，我们已经开发和研究了CD 4和CD 8克隆T细胞系，现在我们将这些观察扩展到使用未修饰的PDC-E2和异源生物质修饰的PDC-E2研究来自患者和对照的PBMC，以测试异源生物质修饰的PDC-E2比未修饰的自身抗原更有效地激活和扩增特异性T细胞并且这样的T细胞将与天然线粒体自身抗原交叉反应的论点。最后，我们将利用我们的令人兴奋的新数据，在NOD.1101小鼠与硫辛酸模拟物，2-octynamido-BSA免疫的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，这些实验的结果将证明我们的论点，PBC是由异生物质化学暴露，打破耐受性和多细胞谱系PDC-E2的反应将是这种疾病的病理效应机制诱导。 公共卫生相关性：原发性胆汁性肝硬化是一种主要影响女性的肝脏疾病，被认为是继发于患者对自身胆管的免疫攻击。这种疾病的病因是神秘的，但我们相信这种疾病开始时，一个人作出免疫反应的线粒体蛋白。我们认为这种免疫攻击始于患者自身线粒体蛋白的环境修饰。在这个应用程序中，我们将研究这个反应的化学。我们还将用这些化学物质免疫小鼠，并相信这些小鼠将发展出受折磨的人类患者的临床特征，这将使我们能够关注和定义疾病病因和未来的潜在治疗。

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.