MECHANISM OF ALTERATION OF ALLOGRAFT ANTIGENICITY

点击翻译按钮获取中文摘要

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.