Transgenic animals in experimental xenotransplantation models: orthotopic heart transplantation in the pig-to-baboon model.

Pig organs are at risk for hyperacute and acute vascular rejection mediated by anti-pig antibodies, mainly binding to the Galalpha(1,3)Gal epitope. Acute cellular rejection is characterized by progressive infiltration of mononuclear cells. There is an ongoing search for immunosuppressive regimens that provide adequate protection against all patterns of xenograft rejection, but have no severe impact on the condition of xenograft recipients. Herein orthotopic heart transplantations were performed from hDAF or hCD46 piglets to nonsplenectomized baboons. Basic immunosuppression consisted of tacrolimus, sirolimus, GAS914, steroids, and ATG. Group 1 received basic immunosuppression. Group 2 was additionally treated with rituximab and group 3 with half-dose cyclophosphamide. Group 4 received cyclophosphamide and an anti-HLA-DR antibody. Three baboons received GAS914 and TPC. Monitoring included the regular assessment of anti-porcine antibodies, blood counts, therapeutic drug monitoring, and graft histology. Two grafts failed due to technical mistakes. In group 1, baboons died after 1 and 9 days. In group 2, maximum survival was 30 hours. In group 3, baboons lived 20 hours, 25 days, and 14 days. Group 4 survival times were 9.5 hours, 5.5 hours, 4 days, 34 hours, and 3 days. An increase of non-Galalpha(1,3)Gal antibodies was observed. Depositions of immunoglobulins and complement revealed a humoral rejection process. No cellular infiltration could be observed. In conclusion, suppressing cellular rejection with half-dose cyclophosphamide together with tacrolimus and sirolimus produced longer graft survival with a good general condition. Prevention of acute xenograft rejection further needs inhibition of non-Galalpha(1,3)Gal cytotoxicity by sufficient depression of B-cell activation.

Fluorescent microspheres reveal different regional blood flow in hyperacutely rejected nontransgenic and hDAF pig hearts.

Classic features of hyperacute rejection show differential severity in the inner compared to the outer myocardium. In the present study, regional blood flow (RBF) measured by fluorescent microspheres served as a marker of the extent of hyperacute rejection. Using a working heart model, hearts of nontransgenic and hDAF transgenic pigs were perfused with human blood. Additionally, hDAF transgenic pig hearts were perfused with human blood containing GAS914 or the GPIIb/IIIa inhibitor tirofiban. Injections of fluorescent microspheres into the donor heart were performed in situ and during perfusion. Reference arterial blood samples were collected from the inferior aorta and the afterload line. Perfusion was terminated before hyperacutely rejected hearts failed to pump against the afterload column. RBF was determined in tissue samples of standardized areas of the left atrium and ventricle. Each specimen was divided into subepicardial and subendocardial tissue samples. Fluorescence intensity was measured using an automated luminescence spectrometer. At the end of perfusion with human blood, hyperacutely rejected nontransgenic pig hearts showed a higher RBF in the subendocardium. In hDAF-transgenic pig hearts perfused with unmodified human blood the subendocardial/subepicardial blood flow ratio changed in favor of the subepicardium. This ratio was not further improved by GAS914. In contrast, tirofiban was able to assimilate subepicardial and subendocardial blood flow. In conclusion, RBF of hyperacutely rejected pig hearts was inhomogeneous. Inhibition of complement activation improved the reduced subepicardial RBF, but depletion of antibodies had no positive effect. The ability of tirofiban to further increase subepicardial RBF affirms thrombosis of subepicardial veins as the defining characteristic of hyperacute rejection.