Contrast in the efficacy of hDAF mouse hearts between ex vivo perfusion and transplantation into primates.

In recent experiments, in which we compared hDAF transgenic rat hearts perfused with 15% human serum in the Langendorff device and hDAF rat hearts transplanted into cynomolgus monkeys, we demonstrated that in the ex vivo heart perfusion model both homozygous and heterozygous hDAF hearts survived longer as nontransgenic controls. Surprisingly, we found that only homozygous hDAF hearts were protected against hyperacute rejection in vivo. The first aim of this study was to determine whether perfusion of mouse hearts with higher human serum concentrations or human blood might explain some of the differences found in survival time of the recently performed experiments with rat heart xenografts. Secondly, we investigated whether the observed differences in survival times of rat xenografts between in vivo and ex vivo transplantation would also hold for mouse hearts transgenic for hDAF. An ex vivo model was used to perfuse hDAF mouse hearts and controls with human serum or blood, and hDAF transgenic hearts and controls were transplanted into cynomolgus monkeys. hDAF transgenic mouse hearts survived significantly longer than their controls when perfused with 15% human serum, but no difference was found when 30% human serum was used, or when these hearts were transplanted into cynomolgus monkeys. However, in both the in vivo and ex vivo models the amount of PMNs adhering to the vascular endothelium was significantly lower in hDAF transgenes as compared with their controls. In conclusion, in the ex vivo situation, the efficacy of hDAF transgenesis in preventing HAR is limited by serum complement concentration.

Negligible role for NK cells and macrophages in delayed xenograft rejection.

Hyperacute rejection (HAR) of a discordant xenograft can be avoided by complement manipulation, but delayed xenograft rejection (DXR) still leads to graft loss. It is generally assumed that macrophages and NK cells play key roles in DXR. In the present study the survival times and cellular infiltrate following guinea pig to rat heart transplantation was analyzed in the course of DXR, following aspecific and specific manipulation of macrophages and NK cells. HAR was overcome by a single injection of cobra venom factor 1 day before heart transplantation. To aspecifically reduce the inflammatory response dominating DXR, dexamethasone (DEXA) was given. Treatment with DEXA markedly reduced infiltration by NK cells, macrophages, and granulocytes. It also led to prolonged graft survival times (median survival of 0.4 days, n = 10, P < 0.05). In the second series of experiments the specific roles of NK cells and macrophages in DXR were further assessed. Monoclonal antibody 3.2.3 was used to selectively deplete NK cells. Liposome-encapsulated dichloromethylene biphosphonate was given to achieve macrophage depletion. Neither of these specific treatments, alone or combined, led to prolonged graft survival. Immunohistology revealed that at day 2 after transplantation no NK cells or macrophages were present in grafts from the combined treatment group. Only a mild infiltration of granulocytes was observed. Collectively, these results strongly suggest that NK cells and macrophages are not likely to be pivotal cell types in DXR.

Protection against hyperacute xenograft rejection of transgenic rat hearts expressing human decay accelerating factor (DAF) transplanted into primates.

BACKGROUND: Production of transgenic pigs for multiple transgenes is part of a potential strategy to prevent immunological events involved in xenograft rejection. Use of a genetically engineerable rodent as a donor in primates could allow testing in vivo of the effects of different transgenes on controlling xenograft rejection. As a first step in the development of a donor containing multiple transgenes, transgenic rats for human decay-accelerating factor (DAF) were used as heart donors to test their resistance against complement (C)-mediated rejection by non-human primates. MATERIALS AND METHODS: Transgenic rats were generated by using a construct containing the human DAF cDNA under the transcriptional control of the endothelial cell (EC)-specific human ICAM-2 promoter. DAF expression was evaluated by immunohistology and by FACS analysis of purified ECs. Resistance of transgenic hearts against C-mediated damage was evaluated by ex vivo perfusion with human serum and by transplantation into cynomolgus monkeys. RESULTS: Immunohistological analysis of DAF expression in several organs from two transgenic lines showed uniform expression on the endothelium of all blood vessels. ECs purified from transgenic hearts showed 50% DAF expression compared to human ECs and >70% reduction of C-dependent cell lysis compared to control rat ECs. Hemizygous transgenic hearts perfused with human serum showed normal function for >60 min vs. 11. 2 +/- 1.7 min in controls. Hemi- or homozygous transgenic hearts transplanted into cynomolgus monkeys showed longer survival (15.2 +/- 7 min and >4.5 hr, respectively) than controls (5.5 +/- 1.4 min). In contrast to hyperacutely rejected control hearts, rejected homozygous DAF hearts showed signs of acute vascular rejection (AVR) characterized by edema, hemorrhage, and an intense PMN infiltration. CONCLUSIONS: We demonstrate that endothelial-specific DAF expression increased heart transplant survival in a rat-to-primate model of xenotransplantation. This will aid in the analysis of AVR and of new genes that may inhibit this form of rejection, thus helping to define strategies for the production of transgenic pigs.