John S. Logan

Cardiac xenotransplantation: progress toward the clinic.

Background. Animal organs could satisfy the demand for solid organ transplants, which currently exceeds the limited human donor supply. Hyperacute rejection, the initial immune barrier to successful xenotransplantation, has been overcome with pig donors transgenic for human complement regulatory proteins. Delayed xenograft rejection, thought to be mediated by anti-pig antibodies predominantly to Gal antigens, is currently regarded as the major barrier to successful xenotransplantation. A median graft survival of 90 days in the life-supporting position is considered a reasonable initial standard for consideration of entry to the clinic. Methods. A series of 10 heterotopic heart transplants from CD46 transgenic pigs to baboons was completed. Immunosuppression consisted of splenectomy, Rituximab (Anti-CD20), tacrolimus, sirolimus, corticosteroids, and TPC. Thymoglobulin (Rabbit Anti-Thymocyte Globulin) was used to treat putative rejection episodes. Results. Median graft survival was 76 days (range 56–113 days, n=9). Only three grafts were lost to rejection. The remaining grafts lost were due to recipient mortality with baboon cytomegalovirus (BCMV) being the major cause (n=4). No cellular infiltrates were present as a manifestation of rejection. Three hearts showed chronic graft vasculopathy. Conclusions. The median survival of 76 days in this group of heterotopic porcine-to-baboon cardiac xenografts represents a major advance over the median 27-day survival reported in the literature. Cellular rejection may not constitute a direct major barrier to xenotransplantation. A median survival of 90 days may be achievable with better control of BCMV infection. If further studies in the orthotopic position replicate these outcomes, criteria considered appropriate for clinical application of cardiac xenotransplantation would be approached.

Proteomic identification of non-Gal antibody targets after pig-to-primate cardiac xenotransplantation.

Abstract:  Background:  Experience with non‐antigenic galactose α1,3 galactose (αGal) polymers and development of αGal deficient pigs has reduced or eliminated the significance of this antigen in xenograft rejection. Despite these advances, delayed xenograft rejection (DXR) continues to occur most likely due to antibody responses to non‐Gal endothelial cell (EC) antigens.

Increased immunosuppression, not anticoagulation, extends cardiac xenograft survival

Background. Cardiac xenograft function is lost due to delayed xenograft rejection (DXR) characterized by microvascular thrombosis and myocardial necrosis. The cause of DXR is unknown but may result from thrombosis induced by antibody-mediated activation of endothelial cells and/or by incompatibilities in thromboregulatory interactions. Methods. To examine these issues, a series (Groups 1–6) of previous transgenic CD46 pig-to-baboon heterotopic cardiac transplants were reanalyzed for baseline immunosuppressive levels, graft survival and infectious complications with and without systemic anticoagulation. Groups 1–4 received low dose tacrolimus and sirolimus maintenance therapy, with splenectomy, anti-CD20 and daily α-Gal polymer. Group 1 recipients received no anticoagulation. Groups 2–4 were anticoagulated with aspirin and Plavix, Lovenox, or Coumadin, respectively. Group 5 was treated with Lovenox and high dose tacrolimus and sirolimus maintenance therapy. Group 6 recipients received no postoperative anticoagulation but the same immunosuppression as group 5. Results. Median survival (15–22 days) within groups 1–4 was not significantly different. At rejection all tissues exhibited microvascular thrombosis, coagulative necrosis and similar levels of platelet and fibrin deposition. Groups 5 and 6 median survival (76 days) was significantly increased compared to groups 1–4. There was no significant difference in median survival between Lovenox treated recipients (68 days) and anticoagulant free recipients (96 days). Rejected tissues showed vascular antibody deposition, microvascular thrombosis, and myocyte necrosis. Conclusion. Significant prolongation in xenograft survival is achieved by improved immunosuppression. These results suggest that ongoing immune responses remain the major stimulus for DXR.

T‐cell responses during pig‐to‐primate xenotransplantation

Abstract: Xenotransplantation using porcine organs may resolve a chronic shortage of donor organs for clinical transplantation if significant immunological barriers can be overcome. To determine the potential role of T lymphocytes in Xenograft (Xg) rejection, we transplanted transgenic hCD46 porcine hearts heterotopically into baboon recipients. Methods: Recipients were treated to deplete anti‐Gal antibody with a non‐antigenic α‐Gal polyethylene glycol polymer (TPC) (n=2), TPC plus rituximab (anti‐CD20) (n=1) or were untreated (n=1). None of the recipients received T‐cell immunosuppression. Results: All Xgs failed within 7 days and showed evidence of a mixed humoral and cellular rejection process. Cellular infiltration consisting primarily of CD4+ T cells and few CD8+ T cells. Proliferation and cytotoxicity assays showed sensitization of CD4+ and CD8+ T cells that reacted with porcine IFN‐γ (pIFN‐γ)‐stimulated porcine aortic endothelial cells (PAEC). The CD4+ lymphocytes displayed greater cytotoxicity than CD8+ cells. An increased frequency of PAEC‐specific interleukin (IL) 2 and IFN‐γ‐secreting T cells was observed, suggesting a Th1 cytokine bias. An increase in the percentage of circulating CD4+CD28− cells was observed at the time of rejection and over 50% of the CD4+ cells recovered from residual pig tissue at necropsy lacked CD28 expression. Conclusions: These findings show that lymphocytes are efficiently stimulated by PAEC antigens and can mediate direct tissue destruction. These studies (1) provide an insight into the potential of cellular‐mediated cardiac Xg rejection, (2) show for the first time the induction of cytotoxic pig‐specific CD4+CD28− lymphocytes and (3) provide a rational basis for determining different modes of immunosuppression to treat Xg rejection.

Warfarin or low-molecular-weight heparin therapy does not prolong pig-to-primate cardiac xenograft function

Microvascular thrombosis is a prominent feature in cardiac delayed xenograft rejection (DXR). We investigated the impact of warfarin or low‐molecular‐weight heparin (LMWH) anti‐coagulation on xenograft function using a heterotopic pig‐to‐primate model. Donor hearts were from CD46 transgenic pigs and baboon immunosuppression included tacrolimus, sirolimus, anti‐CD20 and TPC, an α‐galactosyl‐polyethylene glycol conjugate. Three groups of animals were studied. Group 1 (n = 9) was treated with warfarin, Group 2 (n = 13) with LMWH and Group 3, received no anti‐coagulant drugs. The median duration of xenograft function was 20 days (range 3–62 days), 18 days (range 5–109 days) and 15 days (range 4–53 days) in Groups 1 to 3 respectively. Anti‐coagulation achieved the targeted international normalized prothrombin ratio (INR) and anti‐factor Xa levels consistent with effective in vivo therapy yet, no significant impact on median xenograft function was observed. At rejection, a similar histology of thrombosis and ischemia was apparent in each group and the levels of fibrin deposition and platelet thrombi in rejected tissue was the same. Anti‐coagulation with warfarin or LMWH did not have a significant impact on the onset of DXR and microvascular thrombosis. However, a role for specific anti‐coagulant strategies to achieve long‐term xenograft function cannot be excluded.

Effective antiplatelet therapy does not prolong transgenic pig to baboon cardiac xenograft survival.

Abstract:  Background:  Microvascular thrombosis is a prominent characteristic of delayed xenograft rejection, therefore the effects of antiplatelet therapy with aspirin and clopidogrel on long‐term cardiac xenograft function was investigated in a heterotopic pig‐to‐baboon cardiac transplant model.

Prevention, detection, and management of early bacterial and fungal infections in a preclinical cardiac xenotransplantation model that achieves prolonged survival

Abstract:  Background:  We analyzed bacterial and fungal infectious complications in a cohort of 16 consecutive experiments with the longest surviving cardiac xenografts to date. Methods: Transgenic, porcine‐to‐baboon, heterotopic (abdomen) cardiac xenotransplantation was performed in 16 consecutive experiments, using rapamycin, tacrolimus, corticosteroids, anti‐CD20 monoclonal antibody, and an alpha‐Gal‐PEG polymer, as immunosuppression. Prophylactic anti‐microbials included i.v. trimethoprim/sulfamethoxazole, oral ganciclovir/valganciclovir, and oral itraconazole. An episode of bacterial infection was defined as a positive blood and/or wound culture with: leukocytosis, fever >101.5°F, and/or clinical deterioration. Results: Mean graft survival was 71 ± 29 days; the longest was 113 days. There were 23 episodes of bacterial infection; 14 resolved with treatment. The mean time to the first episode of infection was 44 ± 21 days (n = 12). Eight of 16 deaths were due to infection: two bacterial‐only, two cytomegalovirus (CMV) only, four both bacterial and CMV, and none fungal. The frequency of infection was 1, 2.8, and 1.8 episodes/100 survival days, respectively, for animals whose grafts survived for 30 to 59, 60 to 89, and >90 days. CMV infection (reviewed in detail in a separate communications) was due to baboon CMV, and was associated with low serum levels of ganciclovir. Conclusion: In a cardiac xenograft model that achieved prolonged (>3 months) survival, bacteremia was common, but usually reversible, and fungal infection was prevented with prophylaxis. The level of immunosuppression required to achieve clinically meaningful xenograft survival is associated with a level of bacterial and fungal infectious complications that is manageable and similar to the early clinical experiences in human transplantation. Further research will determine if the viral infectious complications observed in these experiments can be reduced by optimizing blood levels of anti‐viral prophylaxis and monitoring viral polymerase chain reaction levels.

Endothelial function in pigs transgenic for human complement regulating factor.

Background. Expression of human complement regulating factor (hCRF) in porcine organs prevents hyperacute rejection of these organs after xenotransplantation to nonhuman primates. Experiments were designed to characterize endothelial and smooth muscle function of arteries from pigs transgenic for hCD46. Methods. Arterial blood from outbred pigs transgenic for hCD46 expression and nontransgenic animals of the same lineage was analyzed for angiotensin-converting enzyme (ACE), C-type natriuretic peptide (CNP), and nitric oxide. Aortic endothelial cells were prepared for measurement of mRNA or activity for nitric oxide synthase (NOS). Rings cut from femoral and pulmonary arteries were suspended in organ chambers for measurement of isometric tension. Results. CNP was significantly greater, ACE was similar, and nitric oxide was significantly less in plasma from transgenic compared with nontransgenic pigs. Neither mRNA nor activity of NOS differed between the groups. Endothelium-dependent relaxations to bradykinin and acetylcholine but not the calcium ionophore were shifted significantly to the left in femoral and pulmonary arteries from hCD46 transgenic pigs compared with nontransgenic pigs. The ACE-inhibitor captopril augmented relaxations similarly in both groups, but NG-monomethyl-L-arginine (L-NMMA) did not inhibit relaxations in rings from transgenic pigs. Conclusions. Data suggest that expression of hCD46 on endothelium of pigs selectively augments endothelium-dependent relaxations to bradykinin by increased release of endothelium-derived factors other than nitric oxide. There does not seem to be any change in activity of ACE or NOS with expression of the human protein. Increased relaxations to bradykinin may be beneficial in lowering vascular resistance when transgenic organs are used for xenotransplantation.