MECHANISM OF ALTERATION OF ALLOGRAFT ANTIGENICITY

我们的目标是开发高压氧培养条件， 提高移植物存活率到血管化器官移植。到 为了实现这一目标，我们提出了研究，以确定分子和 高压氧培养诱导MHC的生化机制 I类下调和延长小鼠甲状腺移植物存活。 本研究由三个相互关联的部分组成，1）发展和 用于生物化学研究的体外模型系统的表征 HOC诱导的MHC下调和免疫调节的改变 性质; 2）确定氧和温度的作用 MHC下调的依赖性和移植物存活率的增加， 小鼠甲状腺模型系统;和3）所获得的信息的应用 在第1和第2部分中，用于胰岛同种异体移植物和异种移植物。第一和第二部分将 使用Epstein-Barr转化的淋巴母细胞样细胞系完成 (LCL)和鼠甲状腺同种异体移植系统。结构修饰 将通过2D凝胶电泳检测MHC I类分子， 计算机辅助分析和β 2-微球蛋白交换率。 原位杂交，北方印迹和核径流分析将是 用于确定HOC诱导的基因组结构改变。的能力 作为细胞毒性T细胞的靶细胞和作为刺激因子， 单向混合淋巴细胞培养将用于确定高压 氧培养诱导免疫学特性的改变。的作用 MHC下调中的氧含量将通过以下方法定量测定： 体外LCL和体内甲状腺中的CELISA和流式细胞术 通过用氮气或氦气代替氧气的同种异体移植系统。类似地 MHC下调的温度依赖性和延长的同种异体移植 将在体外和体内测定存活率。 最后 高压氧培养的甲状腺刺激同种异体移植的能力 将使用有限稀释法检查体内排斥（CTL生成 分析. 第一部分和第二部分中产生的信息将用于设计HOC 保持胰岛功能完整性的系统，但导致MHC I类 分子下调和延长同种异体移植物和异种移植物存活。

Our aim is to develop hyperbaric oxygen culture conditions that provide enhanced graft survival to the transplantation of vascularized organs. To accomplish this goal, we propose studies to identify the molecular and biochemical mechanism(s) of hyperbaric oxygen culture(HOC)-induced MHC class I down regulation and prolonged murine thyroid allograft survival. The study consists of three interrelated parts, 1) the development and characterization of an in vitro model system for biochemical studies of HOC-induced MHC down regulation and alteration in the immunological properties; 2) determination of the role of oxygen and the temperature dependence of MHC down regulation and increased allograft survival in a murine thyroid model system; and 3) application of the information gained in parts 1 and 2 to islet allografts and xenografts. Parts one and two will be accomplished using an Epstein-Barr transformed lymphoblastoid cell line (LCL) and the murine thyroid allograft system. Structural modification of the MHC class I molecule will be examined by 2D gel electroporesis with computer assisted analyses and the rate of beta 2-microglobulin exchange. In situ hybridization, northern blot and nuclear runoff analysis will be used to determine HOC-induced alterations in genomic structure. The ability of the LCL to serve as targets for cytotoxic T cells and as stimulators in one way mixed lymphocyte culture will be used to determine hyperbaric oxygen culture induced modification of the immunologic properties. The role of oxygen in MHC down regulation will be determined quantitatively by CELISA and Flow cytometry in vitro with the LCL and in vivo in a thyroid allograft system by substituting nitrogen or helium for oxygen. Similarly the temperature dependence of MHC down regulation and prolonged allograft survival will be determined both in vitro and in vivo. Finally, the ability of hyperbaric oxygen cultured thyroids to stimulate allograft rejection (CTL generation) in vivo will be examined using limiting dilution analysis. The information generated in parts one and two will be used to design HOC systems that preserve islet functional integrity, yet result in MHC class I molecule down regulation and prolonged allograft and xenograft survival.