DNA RELEASED FROM ISCHEMIC AND REJECTING ORGANS IS AN INDICATOR OF GRAFT CELLULAR DAMAGE

Abstract

A27 Warm and cold ischemia are injurious for endothelial and parenchymal cells of the transplanted organ. “Passenger ” leucocytes and fragments of disintegrated endothelial cells are washed out from the transplant during warm revascularization period and disseminate in the recipient. Donor cellular debris contains fragments with genetic material. The question arises whether the amount of donor graft released DNA could be a measure of its damage caused by ischemia, preservation and rejection. Aim: To measure concentration of donor DNA in recipient blood, lymph nodes and spleen at various time points after heart transplantation. Methods: Male LEW (RT1l) and BN (RT1n) rats served as donors and female LEW (RT1l) as recipients. After tx, the recipient blood (B), lymph nodes (LN) and spleen (SPL) were harvested. Dendritic cells were obtained from recipient spleen after digestion, gradient separation and enrichment by EA rosetting. Genomic DNA was isolated and real time PCR with rat SRY specific primers was performed. Results: Retrieval of male DNA after 1h of PBL injection into female recipient was dose-dependent with highest accumulation in LN. The male heart wash out prior to tx after 1h ex vivo ischemia did not show any presence of DNA. 1h after tx in an allogeneic combination donor DNA was found at highest concentrations in B and some in SPL, 24h later the concentration in B was slightly lower but it became detectable in LN. 72h after tx DNA concentration in B and LN decreased in favor of SPL. 1 week after tx donor DNA was found only in SPL. In a syngeneic combination 24 h after transplantation donor DNA could be detected only in B, 72 h later only in SPL. After 1 week donor DNA reappeared in B, was present in LN reaching high values in SPL. Donor DNA was found in SPL dendritic cells after allogeneic but not syngeneic grafting, however, was present in macrophages in both combinations. Conclusions: Donor heart passenger cells do not contribute to DNA disseminated in recipient. Immediately and one day after transplantation, donor DNA is present mostly in B. Later it accumulates in SPL. Recipient blood donor DNA levels on days 1 to 3 provide insight into the degree of ischemic and post ischemic damage of the graft. Later they reflect the degree of rejection damage. High levels of donor DNA and methodological simplicity of DNA measuring justify works on a clinical test for evaluation of graft damage during its procurement for grafting.