| With the uSe of immunosuppressive drugs that efficiently control acute rejection, Organtransplantation has achieved impressive development. Nevertheless, clinical transplantation stillthces some important problems: lilblong immunosuppression is associated with toxicity,oppottunistic infections and a high incidence of cancer. Donor specific organ transplantationimmune tolerance, which is defined as no medication (routine immunosuppressive agents), norejection and no infection after MHC mismatched organ transplantatlon, is the trend of organtransplantation. Solutions to these problems will come from the application of new strategies ofimmunointervefltion. The application of gene therapy techniques to transplantation has greatpotential theraPeutic advantages over systemic administration of the immunosuppressivebioreagents. Local production of immunosuppressive molecules may increase their therapeuticefficiency and reduce their systemic side-effects.HuCTLA4-lg,' one of the most potent in1n1unosuppressive molecules, is a solublerecombinant fusion protein (molecu1ar weight of approximately 92 kD) consisting of theextracellular domain of human CTLA-4 and a fragment (hinge and constant region) of the Fcportion of human IgGl. It strongly adheres to the B7 molecule to block the CD28-mediatedcostimulatory signal, resulting in inhibition of in vitro and in vivo immune response. Withadministration of CTLA4-Ig, short-term blockade of CD28-mediated corecognition resulted inprolonged graft survival and tolerance in MHC mismatched rodent models.Adenoviral-mediated transfer to graft of immunoregulatory genes is a novel strategy tomod[1latil1g in1l11une 'responses.In this study, with AdEasy vector system, the recombinant adenovirus containinghuCTLA4-Ig gene was constructed. Using ex vivo gene transfer technique, exogenous gene wasintroduced to the liver graft during cold preservation and express locally in the graft. The effectof inhibition of rejection and inducing liver graft tolerance was observed. Through this study,the possibility of achieving graft tolerance by gene transfer without routine immunosuppressivedrugs was explored.The results are as followsil.The recombinant adenovirus containing huCTLA4-Ig gene was constructed successfullyand its titer was 6 X 10l3 PFU/L. The recombinant virus AdhuCTLA4-Ig prepared in this studyefliciently inltcted L-O2 'cells, and the infected cells expressed a-nd excreted solublerccolllbina11t protein huCTLA4-Ig.2.Rat orthotopic liver trarlsplantation was perfOrmed in the strong rejecter combination ofBN (RTl' ) to LEW (RTl l). This al1ogenic rejection model was stable and the survival timewithout immunosuppression agents was less than three weeks (the average survival time wasl 6.6 I 2.5 days, n=5).3.Using clamp technique, ex vivo gene transfer into liver graft was performed during coldpreservation via perfusion of the portal vein with 5ml Ringer's solution containingreplication-defective adenovirus vector AdhuCTLA4-Ig. The expression of huCTLA4-Igprotein was detected in recipient serum and liver graft within the first seven days aftertransplantation, rapidly declifning to undetectable levels by day 28. No recipient of group A(without any treatment, n=5) or group B (treated with AdGFP, n=4) died within three weeksafter transplantation and severe acute rejection (massive periportal infiltrate, endothelilitis,damage to biliary epithelium and severe tissue destruction) was confirmed by pathologicalexamination of the graft. In contrast, all recipients of group C (treated with AdhuCTLA4-Ig,n=5) achieved lohg-term liver allograft survival (>l50d). Histological examination ofAdhuCTLA4-Ig transduced allografts demonstrated a mild to moderate peripoftal inflammationand mild injury to liver graft on day 8 posttransplant. A mild mononuclear infiltrate persisted,however, there is complete preservation of the bile ducts and no evidence of vascular injury onday l50...
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