Novel Antigens & T & B Cell Responses in Biliary Atresia

描述（由申请人提供）： 多种证据表明，病毒感染可能在胆道闭锁（BA）的发病机制中发挥作用。然而，对推定的病毒病原体的搜索仅限于少数选定的病毒，并且没有集中于病毒特异性免疫应答。这一假说的基础是，病毒感染在胆道闭锁的发病机制中发挥作用。本提案的目的有三个方面：1）建立临床胆道闭锁中心。包括约翰霍普金斯儿科肝脏中心在内的五个转诊中心将每年招募10-20名新的BA患者，以增加现有数据库中的73名BA患者。该数据库将收集人口统计学和临床信息以及与病毒感染假设相关的生物标本。2)BA肽计划，（3年）将采用一种强大的新技术，噬菌体肽展示，寻找新的抗原表位特异性BA。BA血清将用于“生物淘选”随机肽文库以鉴定不被对照血清识别的表位。将对每个临床中心的一个患者/对照组进行研究，以尽可能实现地理、季节和种族多样性。“BA肽”将被测序和合成。肽将用于从BA血清中免疫亲和纯化相应的单特异性抗体。将在蛋白质数据库中检索同源病毒蛋白。为了确定BA肽是否表现出与HLA-A2+限制性T细胞表位的交叉反应性，将所述肽与树突细胞、从HLA-A2+母体外周血淋巴细胞（PBL）收获的抗原呈递细胞（APC）一起孵育。母体PBL将与肽脉冲的APC一起孵育，以使用靶细胞裂解测定法鉴定肽特异性CTL。3)BA-二聚体项目（1年）将采用一种敏感的新“二聚体”技术来确定BA（MBA）患者的HLA-A2+生物学母亲是否表现出独特的肽特异性CTL。二聚体技术使用免疫球蛋白作为分子支架以产生二价肽-HLA-A2-IG复合物。将用BA或同源病毒肽加载二聚体，并使用FACS分析检测来自MBA的PBL和来自每个临床中心的对照母体的二聚体反应性。新的BA特异性肽、单特异性抗体和BA二聚体可用于早期诊断、细胞靶点和肝内T细胞的鉴定。

DESCRIPTION (provided by applicant): Multiple lines of evidence have suggested that viral infection may play a pathogenetic role in biliary atresia (BA). However, searches for putative viral pathogens have been restricted to a small number of selected viruses and have not focused on virus-specific immune responses. The hypothesis underlying this proposal is that viral infection plays an etiopathologic role in biliary atresia. The purpose of the present proposal is three-fold: 1) Establish a Clinical Biliary Atresia Center. Five referral centers including the Johns Hopkins Pediatric Liver Center will recruit 10-20 new BA patients/year to add to the 73 BA patients in the present database. The database will gather demographic and clinical information as well as biological specimens pertinent to the viral infection hypothesis. 2) BA Peptide Project, (3-years) will employ a powerful new technique, bacteriophage peptide display, to search for novel antigenic epitopes specific to BA. BA sera will be utilized to "biopan" random peptide libraries to identify epitopes not recognized by control sera. One patient/control group from each of the Clinical Centers will be studied, for the greatest possible geographic, seasonal, and ethnic diversity. "BA peptides" will be sequenced and synthesized. Peptides will be used to immunoaffinity purify the corresponding monospecific antibodies from BA sera. Protein databases will be searched for homologous viral proteins. To determine if the BA peptides exhibit cross reactivity as HLA-A2+ restricted T cell epitopes, the peptides will be incubated with dendritic cells, antigen-presenting cells (APC) harvested from HLA-A2+ maternal peripheral blood lymphocytes (PBL). Maternal PBL will be incubated with the peptide-pulsed APC to identify peptide-specific CTL using target cell lysis assays. 3) The BA-Dimer Project, (1-year) will employ a sensitive new "dimer" technique to determine if HLA-A2+ biological mothers of BA (MBA) patients exhibit unique peptide-specific CTL. The dimer technique uses immunoglobulin as a molecular scaffold to produce a divalent peptide-HLA-A2-Ig complex. Dimers will be loaded with BA or homologous viral peptides and PBL from MBA and control mothers from each Clinical Center will be tested for dimer reactivity using FACS analysis. Novel BA-specific peptides, monospecific antibodies, and BA-dimers could be used for early diagnosis, identification of cellular targets and intrahepatic T cells.