CHARACTERIZATION OF THE PEMPHIGUS FOLIACEUS ANTIGEN

性状（改编自申请人摘要）：落叶型天疱疮 (PF)是一种自身免疫性皮肤病， 其他器官特异性抗体介导的自身免疫的简单模型 疾病 实验性被动转移动物模型可用于 这种疾病和由此产生的病变迅速产生（不到24小时） 而且很容易被监控 调查人员最近表明， 自身抗体（AAb）的单价Fab片段是致病性的， 比完整的AAb更快更有效地产生疾病， 原发性病变是阻断血管功能的直接结果。 抗原（Ag）通过AAb。 PF Ag被认为是160 kD桥粒核心蛋白，桥粒芯糖蛋白I （DG-1）。 调查人员最近报告说， 不溶性胰蛋白酶抗性Ag在皮质细胞被膜部分 人类表皮 免疫反应性可溶性50 KD木瓜蛋白酶片段， PF Ag（pf-PF）可以释放到溶液中， 免疫沉淀的PF血清。 此外，调查人员还获得了另外45个 KD片段（tf-PF）通过胰蛋白酶处理活表皮细胞， 研究人员可以通过刀豆球蛋白A亲和性部分纯化它， 层析和免疫沉淀。 这些发现表明， 存在三种不同的PF Ag合并液：一种可溶性合并液， Ag最初是由其他研究人员免疫沉淀的， 胰蛋白酶敏感池和Corpus细胞凋亡相关胰蛋白酶 抵抗池 这项拨款提案的目的是净化这些 片段，研究它们与DG-I或天然PF Ag的关系， 这些池之间的PF Ag的差异，并研究抗原的表位 它们被PF自身抗体检测到， 这种疾病的病理学。 DG-I、pf-PF和tf-PF将进行纯化和化学表征， 免疫学上 对于化学表征，氨基酸和 碳水化合物分析，肽图谱和部分测序将是 完了 对于免疫学研究，多克隆和单克隆抗体将 它们的免疫化学和可能的致病潜力 研究了 单克隆抗体将用于表位作图， 与PF AAb的竞争性研究。 抗体也将用于 制备纯的PF Ag或其蛋白水解片段， 表征并研究其潜在的致病性和治疗性 方面的影响. 为了确保足够的PF Ag供应，并帮助 为了表征免疫反应性表位，将从 角质形成细胞cDNA文库，将测试融合蛋白的 免疫反应性，探针将寻找PF Ag mRNA和完整的 长度PF cDNA。 最后，天然PF抗原或其 将片段与体外表达的抗原的片段进行比较， 全长或部分长度cDNA。 申请人认为，这些研究可能会提供重要的 引发病理学的Ag-AAb相互作用的基本信息 自身免疫性疾病 这些信息应该为 考虑PF中的特定治疗干预作为模型， 其他抗体介导的自身免疫性疾病，并将显着帮助 了解表皮的分化和细胞粘附。

DESCRIPTION (Adapted from the applicant's abstract): Pemphigus foliaceus (PF) is an autoimmune skin disease that has been recently characterized as a simple model for other organ-specific, antibody-mediated autoimmune diseases. An experimental passive transfer animal model is available for this disease and the resulting lesions are rapidly produced (less than 24h) and easily monitored. The investigators have recently shown that monovalent Fab fragments of the autoantibodies (AAb) are pathogenic and produce the disease faster and more efficiently than the intact AAb proving the primary lesion is the direct result of blocking the function of the antigen (Ag) by the AAb. The PF Ag is thought to be a 160 kD desmosomal core protein, desmoglein I (DG-1). The investigators have recently reported the presence of an insoluble trypsin resistant Ag in the cornified cell envelope fraction of human epidermis. An immunoreactive soluble 50 KD papain fragment of this PF Ag (pf-PF) could be released into solution and specifically immunoprecipitated by PF sera. Also, the investigators obtained another 45 KD fragment (tf-PF) by trypsin treatment of viable epidermal cells, and the investigators could partially purify it by concanavalin A affinity chromatography and immunoprecipitation. These findings suggested the presence of three different pools of PF Ag: a soluble pool from which PF Ag was initially immunoprecipitated by other investigators, a cell-surface trypsin-sensitive pool, and a cornified cell envelope-associated trypsin resistant pool. The aims of this grant proposal are to purify these fragments, study their relation to DG-I or the native PF Ag, determine differences between these pools of PF Ag, and study the epitopes of the Ag that are detected by PF autoantibodies and are responsible for the pathology in this disease. DG-I, pf-PF, and tf-PF will be purified and characterized chemically and immunologically. For the chemical characterization, amino acid and carbohydrate analysis, peptide mapping, and partial sequencing will be done. For immunological studies, polyclonal and monoclonal antibodies will be raised and their immunochemical and possible pathogenic potentials studied. Monoclonal antibodies will be used for epitope mapping by competitive studies with the PF AAb. Antibodies will be also used for preparation of pure PF Ag or its proteolytic fragments for further characterization and to study their potential pathogenic and therapeutic effects. To assure sufficient supply of PF Ag and to help in the characterization of the immunoreactive epitopes, cDNA will be purified from a keratinocyte cDNA library, fusion protein will be tested for immunoreactivity, and probes will be made to search for PF Ag mRNA and full length PF cDNA. Finally, immunoreactivity of the native PF antigen or its fragments will be compared to that of the antigen expressed in vitro from full or partial length cDNAs. The applicants believe that these studies will likely provide important basic information about the Ag-AAb interactions that initiate the pathology in this autoimmune disease. This information should pave the way for consideration of specific therapeutic intervention in PF as a model for other antibody-mediated autoimmune disorders, and will aid significantly in understanding differentiation and cell adhesion in the epidermis.