Faculty Bibliography

An orthotopic liver transplant model in the rat was used to evaluate the role of tumor necrosis factor alpha (TNF-alpha) in liver transplant rejection. There were significantly increased levels of TNF-alpha mRNA and parallel increases in 8-hydroxy-2' deoxyguanosine (8-OHdG) indicative of oxidative DNA damage present 7 to 12 days after transplantation. Cells staining positively for 8-OHdG were localized to the cytoplasm of hepatocytes adjacent to the TNF-alpha expressing inflammatory cells in the portal areas or in patches surrounded by inflammatory cells in the hepatic sinusoids. Significantly more cells staining for 8-OHdG were found in the allogeneic grafts that were strongly rejected than in the syngeneic controls or in the grafts placed in species that accepted the allograft permanently after a rejection episode. TUNEL reactivity lagged 2 days behind peak reactivity for 8-OHdG. On day 12 after transplantation, many cells stained for both 8-OHdG and TUNEL, indicating that the cells suffering oxidative DNA injury were undergoing apoptosis or death. Oxidative injury resulted in mtDNA deletion consisting of 4,834 base-pairs. Studies of hepatocytes cultured from normal rats displayed dose-dependent relationships between TNF-alpha concentration and 8-OHdG and mtDNA mutation. Repetitive intraperitoneal injection of Enbrel, a TNF receptor blocker, significantly decreased hepatocyte 8-OHdG levels and the frequency of deleted mtDNA while greatly extending graft survival time. In conclusion, the data presented implicate TNF-alpha as being capable of causing oxidative DNA damage and mtDNA mutation in hepatocytes.

Although it was established in the 1980's that positive crossmatches associated with adverse transplant outcomes were those due to HLA specific antibodies, the cell-based assays for determining antibody specificity were time consuming, laborious, and moderately specific and sensitive. Technological advances now permit much more rapid and thorough antibody characterization. While no single assay can identify all anti-HLA antibodies in a patient's serum, the repertoire of specificities that can be defined is impressive and continues to improve. By using complimentary assays, most predominant specificities can be defined in even very highly sensitized patients. Perhaps, more critical, the sensitivity of solid phase immunoassays, which in some cases surpasses that of cell-based flow cytometry, provides a new level of assurance in the detection of humoral sensitization. The impact of these changes in antibody testing is already being felt in transplantation, providing a better evaluation of the level and breadth of sensitization prior to transplantation and greatly facilitating the success of humoral desensitization protocols. Thorough antibody identification can also provide a "virtual crossmatch" or a profile of incompatible donors defined by unacceptable antigens. An approach using the frequencies of unacceptable antigens to derive the probability of incompatible donors will likely be a feature of revised renal allocation for sensitized patients in the US. A compelling body of data is also accumulating proving the efficacy of monitoring for the recurrence or de-novo production of anti-donor HLA specific antibodies after transplantation. The possibility of early detection of humoral rejection in time for effective clinical intervention may offer a means to combat the inexorable loss of grafts to chronic rejection. Looking further into the future, as extensive antibody definition and monitoring are more universally applied, the ability to rule out HLA specific antibodies as a cause of graft loss will certainly also help determine the role of non-HLA antibodies in the outcome of solid organ and hematopoietic stem cell transplantation.