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BACKGROUND The lymphatic system plays an active role in modulating inflammation in autoimmune diseases and organ rejection. In this work, we hypothesized that the transfer of donor lymph node (LN) might be used to promote lymphangiogenesis and influence rejection in vascularized composite allotransplantation (VCA). METHODS Hindlimb transplantations were performed in which (1) recipient rats received VCA containing donor LN (D:LN+), (2) recipient rats received VCA depleted of all donor LN (D:LN-), and (3) D:LN+ transplantations were followed by lymphangiogenesis inhibition using a vascular endothelial growth factor receptor-3 (VEGFR3) blocker. RESULTS Our data show that graft rejection started significantly later in D:LN+ transplanted rats as compared to the D:LN- group. Moreover, we observed a higher level of VEGF-C and a quicker and more efficient lymphangiogenesis in the D:LN+ group as compared to the D:LN- group. The presence of donor LN within the graft was associated with reduced immunoactivation in the draining LN and increased frequency of circulating and skin-resident donor T regulatory cells. Blocking of the VEGF-C pathway using a VEGFR3 blocker disrupts the lymphangiogenesis process, accelerates rejection onset, and interferes with donor T-cell migration. CONCLUSIONS This study demonstrates that VCA LNs play a pivotal role in the regulation of graft rejection and underlines the potential of specifically targeting the LN component of a VCA to control graft rejection.

BACKGROUND Successful preclinical transplantations of porcine hearts into baboon recipients are required before commencing clinical trials. Despite years of research, over half of the orthotopic cardiac xenografts were lost during the first 48 hours after transplantation, primarily caused by perioperative cardiac xenograft dysfunction (PCXD). To decrease the rate of PCXD, we adopted a preservation technique of cold non-ischemic perfusion for our ongoing pig-to-baboon cardiac xenotransplantation project. METHODS Fourteen orthotopic cardiac xenotransplantation experiments were carried out with genetically modified juvenile pigs (GGTA1- KO/hCD46/hTBM) as donors and captive-bred baboons as recipients. Organ preservation was compared according to the two techniques applied: cold static ischemic cardioplegia (IC; n = 5) and cold non-ischemic continuous perfusion (CP; n = 9) with an oxygenated albumin-containing hyperoncotic cardioplegic solution containing nutrients, erythrocytes and hormones. Prior to surgery, we measured serum levels of preformed anti-non-Gal-antibodies. During surgery, hemodynamic parameters were monitored with transpulmonary thermodilution. Central venous blood gas analyses were taken at regular intervals to estimate oxygen extraction, as well as lactate production. After surgery, we measured troponine T and serum parameters of the recipient's kidney, liver and coagulation functions. RESULTS In porcine grafts preserved with IC, we found significantly depressed systolic cardiac function after transplantation which did not recover despite increasing inotropic support. Postoperative oxygen extraction and lactate production were significantly increased. Troponin T, creatinine, aspartate aminotransferase levels were pathologically high, whereas prothrombin ratios were abnormally low. In three of five IC experiments, PCXD developed within 24 hours. By contrast, all nine hearts preserved with CP retained fully preserved systolic function, none showed any signs of PCXD. Oxygen extraction was within normal ranges; serum lactate as well as parameters of organ functions were only mildly elevated. Preformed anti-non-Gal-antibodies were similar in recipients receiving grafts from either IC or CP preservation. CONCLUSIONS While standard ischemic cardioplegia solutions have been used with great success in human allotransplantation over many years, our data indicate that they are insufficient for preservation of porcine hearts transplanted into baboons: Ischemic storage caused severe impairment of cardiac function and decreased tissue oxygen supply, leading to multi-organ failure in more than half of the xenotransplantation experiments. In contrast, cold non-ischemic heart preservation with continuous perfusion reliably prevented early graft failure. Consistent survival in the perioperative phase is a prerequisite for preclinical long-term results after cardiac xenotransplantation.

Xenotransplantation using pig organs has achieved survival times up to 195 days in pig orthotopic heart transplantation into baboons. Here we demonstrate that in addition to an improved immunosuppressive regimen, non-ischaemic preservation with continuous perfusion and control of post-transplantation growth of the transplant, prevention of transmission of the porcine cytomegalovirus (PCMV) plays an important role in achieving long survival times. For the first time we demonstrate that PCMV transmission in orthotopic pig heart xenotransplantation was associated with a reduced survival time of the transplant and increased levels of IL-6 and TNFα were found in the transplanted baboon. Furthermore, high levels of tPA-PAI-1 complexes were found, suggesting a complete loss of the pro-fibrinolytic properties of the endothelial cells. These data show that PCMV has an important impact on transplant survival and call for elimination of PCMV from donor pigs.

Background The demand for donated human hearts far exceeds the number available. Xenotransplantation of genetically modified porcine organs provides an alternative. In 2000, an Advisory Board of the International Society for Heart and Lung Transplantation set the benchmark for commencing clinical cardiac xenotransplantation as consistent 60% survival of non-human primates after life-supporting porcine heart transplantations. Recently, we reported the stepwise optimization of pig-to-baboon orthotopic cardiac xenotransplantation finally resulting in consistent success, with 4 recipients surviving 90 (n = 2), 182, and 195 days. Here, we report on 4 additional recipients, supporting the efficacy of our procedure. Results The first 2 additional recipients succumbed to porcine cytomegalovirus (PCMV) infections on Days 15 and 27, respectively. In 2 further experiments, PCMV infections were successfully avoided, and 3-months survival was achieved. Throughout all the long-term experiments, heart, liver, and renal functions remained within normal ranges. Post-mortem cardiac diameters were slightly increased when compared with that at the time of transplantation but with no detrimental effect. There were no signs of thrombotic microangiopathy. The current regimen enabled the prolonged survival and function of orthotopic cardiac xenografts in altogether 6 of 8 baboons, of which 4 were now added. These results exceed the threshold set by the Advisory Board of the International Society for Heart and Lung Transplantation. Conclusions The results of our current and previous experimental cardiac xenotransplantations together fulfill for the first time the pre-clinical efficacy suggestions. PCMV-positive donor animals must be avoided.