Prevention of hyperacute xenograft rejection in orthotopic xenotransplantation of pig hearts into baboons using immunoadsorption of antibodies and complement factors

Abstract To prevent hyperacute xenograft rejection (HXR) caused by preformed natural antibodies (XNAb) after orthotopic heart xenotransplantation (oXHTx) of landrace pig hearts into baboons, we used immunoadsorption of immunoglobulins IgG, IgM and IgA and complement with the reusable Ig‐Therasorb column. In addition to functional data, tissue was sampled for histological, immunohistochemical and electron microscopical analysis. We performed three oXHTx of landrace pig hearts to baboons using extracorporeal circulation (ECC) connected to the immunoadsorption unit. Intraoperative treatment consisted of four cycles of immunoabsorption (IA). One oXHTx of a baboon without IA served as a control. A mismatch of donor and recipient heart size was prevented by selecting a 30‐40% lower body weight of donor pigs than recipients. Four cycles of IA removed more than 80% of IgG, IgM and IgA, 86% of anti‐pig antibodies and 66% of complement factors C3 and C4 from plasma. The graft of the control animal failed after 29 min. Orthotopic xenotransplantation with IA was selectively terminated after 100 min, 11 h and 21 h, respectively without any histological signs of HXR in light and electron microscopy. After weaning off from ECC these donor xenografts showed sufficient function with normal ECG and excellent cardiac output in echocardiography and invasive measurement (1.93 ± 0.035 l/min). The myocardium of the control xenograft demonstrated more deposits of Ig and complement components (C3, C4) than in the IA group. Baboons survive HXR after orthotopic pig heart xenotransplantation due to antibody depletion by reusable Ig‐Therasorb column treatment. Long‐term survival in an orthotopic baboon xenotransplantation model after IA, especially in combination with transgenic pig organs, could be a reliable preclinical trial for future clinical xenotransplantation programs.     

Bio artificial pancreas – the immune barrier and oxygen deficiency

The heterotopic thoracic pig‐to‐baboon heart transplantation has been established by our group as a safe preclinical model. Since the recipient's own heart remains in place, it is possible to evaluate immunological reactions of various types and the symptoms of the thrombotic microangiopathy under working heart conditions. However, these experiments are complex and costly. In order to limit the number of transplants, we tested our multi‐transgenic porcine donor hearts in an ex‐vivo perfusion model first. Ex‐vivo model: Both beating ventricles were perfused with heparinized freshly drawn human whole blood using a centrifugal pump and a membrane oxygenator. During 3 h of observation, cardiac function parameters were obtained continuously; specimens from the perfusate, coronary sinus blood, and myocardial biopsies were assessed. Various genetically modified porcine donor hearts were tested; we are currently evaluating the impact of human thrombomodulin on the porcine microcirculation; the efficacy of complement regulation is another work package. Pig‐to‐baboon heterotopic cardiac xenotransplantation: In our latest group, seven baboons received five double (Gal‐KO/hCD46), and two triple (Gal‐KO/hCD46/hTM resp./HLA‐E) transgenic pig hearts. Our immunosuppressive regimen included preoperative anti‐CD20‐antibody, bortezomib, dexamethasone and cyclophosphamide; postoperatively, ATG, tacrolimus, MMF, anti‐CD20‐antibody, bortezomib and dexamethasone were administered. Total lymph node irradiation (6 Gy) was applied on postoperative day five. The triple transgenic hearts survived 35 and 37 days respectively. Both hearts maintained function throughout the experiment. Both recipients succumbed to fungal infections. Humoral rejection was seen only once. The next barrier appears to be the complex event of thrombotic microangiopathy that needs to be addressed with the additional expression of human thrombomodulin strategies.     

IG-therasorb immunoapheresis in orthotopic xenotransplantation of baboons with landrace pig hearts

BACKGROUND: The major problem of xenotransplantation is, that hyperacute xenograft rejection (HXR) causes graft failure within minutes or a few hours because of natural antibodies and activation of the complement system. As a preclinical model we transplanted pig hearts orthotopically into baboons. To prevent HXR after orthotopic xenotransplantation (oXHTx), the immunoglobulins (Ig) and natural antibodies were adsorbed to reusable Ig-Therasorb immunoadsorption (IA) columns. METHODS: We performed three oXHTx of landrace pig hearts into baboons (19+/-6.8 kg), using extracorporeal circulation (ECC) connected to the IA unit. After separating the recipient's blood into plasma and cellular fraction by a plasma filter, plasma flow was directed to the Ig-Therasorb column coated with polyclonal sheep-antibodies against human IgG, IgM, and IgA. Intraoperative treatment consisted of 4 cycles of IA. For a control, we transplanted one pig heart into a baboon (16.9 kg) without applying IA. Perioperatively, serum concentrations of Ig, anti-pig-antibodies, complement and cardiac enzymes were determined. Tissue samples of myocardium were collected at the end of the study for immunohistochemical examinations, light microscopic examination (LM) and electron microscopic examination (EM). For cardiac monitoring after oXHTx, we used ECG, echocardiography, and invasive measurement of cardiac output. To prevent a mismatch of donor and recipient heart size, the donor pig had a 30-40% lower body weight than the recipient baboon. RESULTS: Four cycles of IA removed >80% of IgG, IgM, and IgA from plasma. The graft of the control animal failed after 29 min. The first oXHTx with IA was intentionally terminated after 100 min, the second oXHTx after 11 hr and the third oXHTx after 21 hr. All xenografts showed no histological signs of HXR. After weaning off ECC, these donor hearts worked in sinus rhythm without electrocardiographic ST-segment elevation. An excellent cardiac output was measured by echocardiography and thermodilution (2 L/min). Serological parameters indicating cardiac damage were significantly lower after IA if compared with the control experiment. Macroscopically, the xenograft of the control animal showed massive hemorrhage in comparison with the almost inconspicuous grafts after IA. The myocardium of the IA group demonstrated fewer deposits of Ig and complement components compared with the control animal. CONCLUSION: Baboons do not hyperacutely reject a porcine xenograft after antibody depletion by the Ig-Therasorb column. In our experiment only 4 cycles of immunoapheresis effectively prevented HXR after oXHTx of baboons. The Ig-Therasorb column is a reusable device, which can be handled easily in combination with the ECC. IA must be tested in oXHTx longterm survival experiments, especially in combination with transgenic pig organs, which could be a reliable preclinical approach for future clinical xenotransplantation.     

Microcirculatory alterations after orthotopic pig-to-baboon heart transplantation

Bauer A, Renz V, Baschnegger H, Abicht J‐M, Beiras‐Fernandez A, Brenner P, Thein E, Schmoeckel M, Reichart B, Christ F. Microcirculatory alterations after orthotopic pig‐to‐baboon heart transplantation. Xenotransplantation 2011; 18: 232–238. © 2011 John Wiley & Sons A/S. Abstract: Background: Whilst macrohemodynamic function of porcine xenografts transplanted into baboons has been assessed perioperatively, the ability of the xenograft to maintain systemic microcirculatory perfusion has not been investigated after pig‐to‐baboon xenotransplantation so far. Methods: We investigated the sublingual microcirculation of six baboons undergoing orthotopic transplantation of hCD46‐transgenic pig hearts using orthogonal polarization spectral imaging. Microvascular measurements were performed after induction of anesthesia, in the early phase of cardiopulmonary bypass (CPB), during reperfusion of the porcine heart and 1 h after the xenograft had resumed its life‐supporting function. Microvascular blood flow was analyzed semiquantitatively and the number of visualized cell‐to‐cell interactions was counted. Results: The proportion of continuously perfused microvessels was 97 (96 to 97) % at baseline and 95 (94 to 97) % in the early phase of CPB. It decreased significantly (P < 0.05) during CPB to 89 (84 to 91), and alterations were still present (P < 0.05) when CPB was terminated and the xenograft had taken over systemic perfusion 83 (81 to 85) %. The microcirculatory changes correlated with the lactate levels (y = 18.1–0.18 x; r² = 0.55; P < 0.001), but no correlation with macrohemodynamic parameters was found. Conclusion: Microvascular blood flow is altered after orthotopic pig‐to‐baboon heart transplantation, despite systemic hemodynamic parameters being well maintained by the porcine xenograft. These changes are moderate but persist after termination of CPB. Further studies need to elucidate whether these changes are transient or add to the mortality associated with cardiac xenotransplantation.     

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.     

Structural characterization of α1,3-galactosyltransferase knockout pig heart and kidney glycolipids and their reactivity with human and baboon antibodies

Diswall M, Ångström J, Karlsson H, Phelps CJ, Ayares D, Teneberg S, Breimer ME. Structural characterization of α1,3‐galactosyltransferase knockout pig heart and kidney glycolipids and their reactivity with human and baboon antibodies. Xenotransplantation 2010; 17: 48–60. © 2010 John Wiley & Sons A/S. Abstract: Background: α1,3‐galactosyltranferase knockout (GalT‐KO) pigs have been established to avoid hyperacute rejection in GalT‐KO pig‐to‐human xenotransplantation. GalT‐KO pig heart and kidney glycolipids were studied focusing on elimination of Gal‐antigens and whether novel antigens would appear. Non‐human primates are used as pre‐clinical transplantation experimental models. Therefore, sera from baboons transplanted with GalT‐KO hearts were compared with human serum regarding reactivity with pig glycolipids. Methods: Neutral and acidic glycolipids were isolated from GalT‐KO and WT pig hearts and kidneys. Glycolipid immune reactivity was tested on TLC plates using human affinity‐purified anti‐Gal Ig, anti‐blood group monoclonal antibodies, lectins, and human serum as well as baboon serum collected before and after GalT‐KO pig heart transplantations. Selected glycolipid fractions, isolated by HPLC, were structurally characterized by mass spectrometry and proton NMR spectroscopy. Results: GalT‐KO heart and kidney lacked α3Gal‐terminated glycolipids completely. Levels of uncapped N‐acetyllactosamine precursor compounds, blood group H type 2 core chain compounds, the P1 antigen and the x₂ antigen were increased. Human serum antibodies reacted with Gal‐antigens and N‐glycolylneuraminic acid (NeuGc) in WT organs of which only the NeuGc reactivity remained in the GalT‐KO tissues. A clear difference in reactivity between baboon and human antibodies with pig glycolipids was found. This was most pronounced for acidic, not yet identified, compounds in GalT‐KO organs which were less abundant or lacking in the corresponding WT tissues. Conclusions: GalT‐KO pig heart and kidney completely lacked Gal glycolipid antigens whilst glycolipids synthesized by competing pathways were increased. Baboon and human serum antibodies showed a different reactivity pattern to pig glycolipid antigens indicating that non‐human primates have limitations as a human pre‐clinical model for immune rejection studies.     

Cardiac xenotransplantation: recent preclinical progress with 3-month median survival

OBJECTIVES: Transplantation is limited by a lack of human organ donors. Organs derived from animals, most likely the pig, represent a potential solution to this problem. For the heart, 90-day median graft survival of life-supporting pig hearts transplanted to nonhuman primates has been considered a reasonable standard for entry into the clinical arena. Overcoming the immune barrier to successful cardiac xenotransplantation is most appropriately first explored with the non-life-supporting heterotopic model. METHODS: We performed a series of 7 heterotopic heart transplantations from CD46 transgenic pigs to baboons using a combination of therapeutic agents largely targeted at controlling the synthesis of anti-pig antibodies. Rituximab (anti-CD20) and Thymoglobulin (rabbit antithymocyte globulin [ATG]; SangStat Medical Corp, Fremont, Calif) were used as induction therapy. Baseline immunosuppression consisted of splenectomy, tacrolimus, sirolimus, steroids, and TPC (an anti-Gal antibody therapeutic). Rejection events were not treated. RESULTS: By using Kaplan-Meier analysis, median graft survival was 96 days (range, 15-137 days; 95% confidence interval, 38-99 days). Only 2 grafts were lost as a result of rejection, as defined by cessation of graft palpation. There was no evidence of a consumptive coagulopathy, infectious complications were treatable, and no posttransplantation lymphoproliferative disorders occurred. No cellular infiltration was observed. CONCLUSIONS: This study reports the longest median survival to date (96 days) of pig hearts transplanted heterotopically into baboons. Duplication of these results in the orthotopic life-supporting position could bring cardiac xenotransplantation to the threshold of clinical application.     

Combination of hDAF-transgenic pig hearts and immunoadsorption in heterotopic xenotransplantation of immunosuppressed baboons

INTRODUCTION: Hyperacute xenograft rejection (HXR) and acute vascular rejection (AVR) after xenotransplantation are triggered by xenoreactive antibodies (XAb) and an activated complement cascade. In a heterotopic (abdominal) xenotransplantation model we combined immunoadsorption (IA, Ig-Therasorb column) and a quadruple immunosuppressive drug therapy in recipient baboons with donor pig hearts transgenic for human decay accelerating factor (hDAF). METHODS: According to XAb titers between 6 and 14 cycles of IA were performed preoperatively in 4 recipient baboons (18.6 +/- 2.5 kg). Hearts of hDAF-transgenic donor pigs (6.1 +/- 1.1 kg, Imutran Ltd., a Novartis Pharma AG Company, Basel, Switzerland) were heterotopically transplanted using the abdominal technique in baboons. Immunosuppression consisted of cyclophosphamide (CyP) induction therapy, ERL080 (Novartis Pharma AG), cyclosporin A (CyA, Neoral), and steroids. Blood levels of mycophenolate, CyA, immunoglobulins (Ig), anti-pig-antibodies, complement factors, and cardiac enzymes were determined. Abdominal electrocardiography (ECG), echocardiography, and palpation were used for monitoring of the pig hearts. Myocardial tissue specimens were examined using immunohistochemistry, light microscope (LM), and electron microscope (EM). RESULTS: Ten cycles of IA alone removed 78% of XAb and accordingly IgM, IgG, IgA, complement C3, and C4. None of the xenografts was hyperacutely rejected, but xenograft failure occurred after 5.0 +/- 1.3 days (range, 2.4-8.0 days) because of an AVR associated with a rapid XAb increase within 24 hours. White blood cell count (10.3 +/- 2.2 G/L) showed a maximum of 13.1 +/- 2.1 (day 1) and constant levels (1.4 +/- 0.3-2.1 +/- 1.3 G/L) between day 3 and 6. Histology (LM/EM) showed massive hemorrhage, necrosis, and vascular thrombi as signs of AVR. CONCLUSION: Although HXR was prevented by using IA and hDAF-transgenic donor hearts, AVR was not avoided due to insufficient immunosuppressive regimen used and a missed postoperative IA treatment as a result of an inefficient control of XAb production.     

Xenogeneic orthotopic heart transplantation: Where are we now?

Cardiac xenotransplantation is a promising option for satisfying the unmet need for cardiac replacement. Mechanical hearts have been in development for over 40 years and only one device is currently available “off the shelf” for implantation in the United States. It took 39 years for this first approved device to be available. This device approval arose from outcomes from the rematch trial (2001) when survival in the ventricular assist device group was fifty‐two percent and twenty‐three percent at 1 and 2 years, compared with twenty‐five percent and eight percent in the medically treated group. Quality of life was significantly improved at 1 year in the ventricular assist device group. While mechanical support continues to improve, intrinsic disadvantages remain, such as thromboembolism, the need for careful anticoagulation, infection, lack of physiological response, durability and power supply. Pursuit of cardiac xenotransplantation therefore remains a potentially important contributor to the treatment of heart failure clinically. With the addition of human complement regulating proteins to the pig genome using a micro‐injection technique and the subsequent development of Gal knockout pigs using cloning technology, pre‐clinical median survival of heterotopic cardiac xenotransplants is now in excess of 3 months. A three months median survival of orthotopic cardiac xenotransplants has been proposed as a possible threshold for a clinical trial. Attention has therefore focused on the orthotopic cardiac xenotransplant pig to baboon model. There have been six operative survivors ranging from 2 to 57 days. Immunosuppression in these recipients consisted of ATG induction, tacrolimus, sirolimus, anti‐CD20 and a steroid taper. No anticoagulation was used. None of the animals died of rejection. The causes of failure largely related to the challenges of the model system. In four of the six cases rejection was minimal and the remaining two mild to moderate. Recipients were healthy and well during the post‐operative period. Biochemistry was stable. Challenges encountered during these studies have included early peri‐operative heart failure which has markedly improved with peri‐operative management. These studies have shown maintenance of normal cardiac function for up to 2 months following pig to baboon transplantation. In addition, hearts from the recipients who survived showed peri‐operative myocardial dysfunction that completely recovered post‐operatively within a few days. Outcomes of pre‐clinical orthotopic cardiac xenotransplantation continue to improve with this report describing the longest survivors to date. Porcine hearts can function normally in primates for at least 2 months. Peri‐operative cardiac dysfunction is likely avoidable and is recoverable. Reparative processes are intact in the xenotransplant setting. Cardiac xenograft rejection is well controlled on tolerable immunosuppressants. Model limitations remain a serious challenge. The powerful advantage of biologic cardiac replacement (complete implantability, intrinsic power supply, lack of need for anticoagulation and physiological responsiveness) justifies continued pre‐clinical studies. In the first instance the goal of clinical cardiac xenotransplantation will be to provide additional treatment alternatives for patients with end‐stage organ failure. The comparison on outcomes will be with other available treatments and not allotransplantation in the first instance. Research programme supported by NIH Grant A166310.     

Results of Gal‐Knockout Porcine Thymokidney Xenografts

Clinical transplantation for the treatment of end‐stage organ disease is limited by a shortage of donor organs. Successful xenotransplantation could immediately overcome this limitation. The development of homozygous α1,3‐galactosyltransferase knockout (GalT‐KO) pigs removed hyperacute rejection as the major immunologic hurdle to xenotransplantation. Nevertheless, GalT‐KO organs stimulate robust immunologic responses that are not prevented by immunosuppressive drugs. Murine studies show that recipient thymopoiesis in thymic xenografts induces xenotolerance. We transplanted life‐supporting composite thymokidneys (composite thymus and kidneys) prepared in GalT‐KO miniature swine to baboons in an attempt to induce tolerance in a preclinical xenotransplant model. Here, we report the results of seven xenogenic thymokidney transplants using a steroid‐free immunosuppressive regimen that eliminated whole‐body irradiation in all but one recipient. The regimen resulted in average recipient survival of over 50 days. This was associated with donor‐specific unresponsiveness in vitro and early baboon thymopoiesis in the porcine thymus tissue of these grafts, suggesting the development of T‐cell tolerance. The kidney grafts had no signs of cellular infiltration or deposition of IgG, and no grafts were lost due to rejection. These results show that xenogeneic thymus transplantation can support early primate thymopoiesis, which in turn may induce T‐cell tolerance to solid organ xenografts.     

Life-supporting human complement regulator decay accelerating factor transgenic pig liver xenograft maintains the metabolic function and coagulation in the nonhuman primate for up to 8 days

BACKGROUND: It is not known whether the pig liver is capable of functioning efficiently when transplanted into a primate, neither is there experience in transplanting a liver from a transgenic pigs expressing the human complement regulator human complement regulator decay accelerating factor (h-DAF) into a baboon. The objective of this study was to determine whether the porcine liver would support the metabolic functions of non-human primates and to establish the effect of hDAF expression in the prevention of hyperacute rejection of porcine livers transplanted into primates. METHODS: Five orthotopic liver xenotransplants from pig to baboon were carried out: three from unmodified pigs and two using livers from h-DAF transgenic pigs. FINDINGS: The three control animals transplanted with livers from unmodified pigs survived for less than 12 hr. Baboons transplanted with livers from h-DAF transgenic pigs survived for 4 and 8 days. Hyperacute rejection was not detected in the baboons transplanted with hDAF transgenic pig livers; however, it was demonstrated in the three transplants from unmodified pigs. Baboons transplanted with livers from h-DAF transgenic pigs were extubated at postoperative day 1 and were awake and able to eat and drink. In the recipients of hDAF transgenic pig livers the clotting parameters reached nearly normal levels at day 2 after transplantation and remained normal up to the end of the experiments. In these hDAF liver recipients, porcine fibrinogen was first detected in the baboon plasma 2 hr postreperfusion, and was present up to the end of the experiments. One animal was euthanized at day 8 after development of sepsis and coagulopathy, the other animal arrested at day 4, after an episode of vomiting and aspiration. The postmortem examination of the hDAF transgenic liver xenografts did not demonstrate rejection. INTERPRETATION: The livers from h-DAF transgenic pigs did not undergo hyperacute rejection after orthotopic xenotransplantation in baboons. When HAR is abrogated, the porcine liver maintains sufficient coagulation and protein levels in the baboon up to 8 days after OLT.     

Cardiac xenotransplantation in primates

Successful cardiac xenotransplantation would alleviate the severe shortage of donor organs that presently limits the availability of cardiac transplantation. Early attempts at human xenotransplantation achieved minimal success. However, the effectiveness of cyclosporine in nonhuman xenotransplant models has received little experimental investigation. We have therefore studied the effect of cyclosporine-based immunosuppression in primate cardiac xenograft models using cynomolgus monkey donors and baboon recipients. Donor hearts were transplanted heterotopically into the necks of recipients or in the orthotopic position. Recipients were treated with no immunosuppression (controls), cyclosporine and steroids, or cyclosporine, steroids, azathioprine, and antithymocyte globulin. Statistically significant prolongation of graft survival compared to the control group was observed in the heterotopic groups. Mean survival time of the cyclosporine-treated and steroid-treated heterotopic grafts was 61 days compared to 6 days for grafts in the control group (p = 0.01); the addition of azathioprine and antithymocyte globulin yielded a mean survival of 84 days (p less than 0.01). No significant increase in graft survival was noted in the orthotopic groups treated with either immunosuppressive regimen. Although long-term use of human xenografts as an alternative for heart replacement is not supported by these data, further investigation of the orthotopic model is clearly justified.     

Life supporting function for over one month of a transgenic porcine heart in a baboon.

BACKGROUND: Inhibition of hyperacute rejection (HAR) and sustained graft survival have been demonstrated in a pig-to-primate model of heterotopic cardiac xenotransplantation using pigs transgenic for human Decay Accelerating Factor (hDAF). Building on this work, an orthotopic model has been developed. This case records 39-day cardiac xenograft function in a life-supporting capacity with clinically applicable immunosuppression. METHODS: Using a heart from an hDAF transgenic pig, an orthotopic cardiac transplant was performed on an adult baboon. The immunosuppressive regimen consisted of induction with a short course of cyclophosphamide, followed by maintenance therapy with cyclosporine A, mycophenolate mofetil and a tapering course of corticosteroids. Post-operative monitoring included daily anti-pig hemolytic antibody titer surveillance and endomyocardial biopsy. RESULTS: The animal survived 39 days and was active and energetic throughout its postoperative course, remaining free of signs of cardiopulmonary failure. Endomyocardial biopsy performed on post-operative Day 36 revealed only patches of sub-endocardial fibrosis with no signs of active rejection. The baboon succumbed to an acute cardiopulmonary decompensation immediately following administration of medication via oral gavage. Post-mortem histopathology demonstrated well-preserved myocardial architecture with small foci of mild humoral rejection. CONCLUSIONS: This case documents the longest survival recorded to date of a discordant orthotopic cardiac xenograft and illustrates that the hDAF transgene combined with a clinically acceptable maintenance immunosuppressive regimen enables sustained, life-supporting function of porcine cardiac xenografts in non-human primates. The inhibition of hyperacute rejection and the subsequent control of humoral and cellular rejection for over 1 month demonstrated in this experiment represent significant progress in the development of a viable strategy for clinical xenotransplantation.     

Xenotransplantation: die ewige Zukunft der Herztransplantation?

Xenotransplantation, i.e., transfer of animal organs into man, has undergone steady experimental progress over the past 20 years. Originally, survival times of transplanted organs were measured in minutes and hours due to hyperacute rejection; however, the advent of biotechnology and cloning techniques made the production of genetically engineered pigs possible. Survival of these transgenic pig hearts transplanted heterotopically into the abdomen of baboons reached up to 6 months. In the orthotopic, thus, life-supporting position, up to 2 months were achieved. Physiologic incompatibilities, e.g., of the coagulation cascade seem to be surmountable with the production of h-thrombomodulin transgenic pigs. Transmission of endogenous porcine retroviruses (PERV) from the pig genome to the immunosuppressed recipient—potentially putting the whole population at risk—are now regarded as minimal and manageable. The production of multi-transgenic pigs will be the prerequisite to achieve long-term survival in primate models and pave the way to clinical studies. Heterotopic thoracic xenotransplantation of pig hearts may become a clinical reality in the near future.     

Pediatric cardiac xenograft growth in a rhesus monkey-to-baboon transplantation model

Abstract: It is unclear that organs from other species could grow successfully in the environment of another species. We evaluated the growth capacity of a cardiac xenograft in a rhesus monkey-to-juvenile baboon orthotopic heart transplantation model where there exists a discrepancy of the organ size and its growth rate between donor and recipient species. Eight recipient baboons who survived over 60 days were followed for body weight (BW) and the size of xenografts (left ventricular end-diastolic volume, LVEDV; left ventricular mass, LVM; and left ventricular posterior wall thickness, LVPWT) by echocardiography at 1 week and monthly after xenotransplantation. One baboon is growing along the normal growth curve. The other three baboons gained BW in parallel with normal growth curve except for a few months after surgery. LVEDV, LVM, and LVPWT increased constantly in these four baboons. In one baboon, BW increased over 1 year to 5.5 kg (which was almost the same as BW of adult rhesus monkey) and then plateaued. LVEDV, LVM, and LVPWT increased until 1 year posttransplantation, but then stopped when BW plateaued. In the other four baboons who did not gain BW because of infectious complications or side effects by immunosuppressive therapy, LVEDV, LVM, and LVPWT did not change significantly. We conclude that a cardiac xenograft from a rhesus monkey can successfully grow and support normal growth of a juvenile baboon, but whether a recipient can grow beyond the donor size is yet unknown.     

Successful cross‐breeding of cloned pigs expressing endo‐β‐galactosidase C and human decay accelerating factor

Yazaki S, Iwamoto M, Onishi A, Miwa Y, Suzuki S, Fuchimoto D, Sembon S, Furusawa T, Hashimoto M, Oishi T, Liu D, Nagasaka T, Kuzuya T, Maruyama S, Ogawa H, Kadomatsu K, Uchida K,
Nakao A, Kobayashi T. Successful cross‐breeding of cloned pigs expressing endo‐β‐galactosidase C and human decay accelerating factor. Xenotransplantation 2009; 16: 511–521. © 2009 John Wiley & Sons A/S. Abstract: Background: For successful organ xenotransplantation, genetically engineered pigs have been actively produced. Our attention has focused on (i) reduction of αGal expression by its digestion enzyme, endo‐β‐galactosidase C (EndoGalC), and (ii) inhibition of complement activation by human decay accelerating factor (hDAF). Cell sorting and nuclear transfer enabled the effective production of cloned pigs expressing transgene at high levels. We report the successful cross‐breeding of pigs expressing EndoGalC and hDAF. Methods: After hDAF and EndoGalC genes were transfected into pig fibroblasts from the fetus of Landrace × Yorkshire and Meishan, respectively, transfected cells expressing transgenes effectively were collected using a cell sorter. Cloned pigs were produced using the technology of somatic cell nuclear transfer. After cross‐breeding of cloned pigs, kidneys expressing both EndoGalC and hDAF were transplanted into baboons to examine the efficacy of gene transduction. Results: Well‐designed cloned pigs were produced by cross‐breeding. αGal expression levels in cloned pigs were reduced up to 2 to 14%, compared to that in wild‐type pigs. hDAF expression reached about 10‐ to 70‐fold, compared to that in human umbilical vein endothelial cells. No congenital deformity was observed. There was no problem of increased stillbirth rate or growth retardation. Hyperacute rejection could be avoided in such a cloned pig to baboon kidney transplantation without any treatment for anti‐pig antibody removal. However, grafts suffered from fibrin deposition as early as 1 h after transplantation, and were rejected after 1 week. Conclusions: Using a cell sorting system for effective collection of transfected cells, two types of cloned pigs were produced with a very high level of hDAF expression and a low level of αGal expression. Such genetic modification was effective in preventing hyperacute rejection, but there was an immediate lapse into procoagulation after transplantation, resulting in acute vascular rejection. Effective suppression of antibody binding to the graft would be necessary, even if a high level of hDAF is expressed.     

Changes in cardiac gene expression after pig-to-primate orthotopic xenotransplantation

Byrne GW, Du Z, Sun Z, Asmann YW, McGregor CGA. Changes in cardiac gene expression after pig‐to‐primate orthotopic xenotransplantation. Xenotransplantation 2011; 18: 14–27. © 2011 John Wiley & Sons A/S. Abstract: Background: Gene profiling methods have been widely useful for delineating changes in gene expression as an approach for gaining insight into the mechanism of rejection or disease pathology. Herein, we use gene profiling to compare changes in gene expression associated with different orthotopic cardiac xenotransplantation (OCXTx) outcomes and to identify potential effects of OCXTx on cardiac physiology. Methods: We used the Affymetrix GeneChip Porcine Genomic Array to characterize three types of orthotopic cardiac xenograft outcomes: 1) rejected hearts that underwent delayed xenograft rejection (DXR); 2) survivor hearts in which the xenograft was not rejected and recipient death was due to model complications; and 3) hearts which failed to provide sufficient circulatory support within the first 48 h of transplant, termed “perioperative cardiac xenograft dysfunction” (PCXD). Gene expression in each group was compared to control, not transplanted pig hearts, and changes in gene expression > 3 standard deviations (±3SD) from the control samples were analyzed. A bioinformatics analysis was used to identify enrichments in genes involved in Kyoto Encyclopedia of Genes and Genomes pathways and gene ontogeny molecular functions. Changes in gene expression were confirmed by quantitative RT‐PCR. Results: The ±3SD data set contained 260 probes, which minimally exhibited a 3.5‐fold change in gene expression compared to control pig hearts. Hierarchical cluster analysis segregated rejected, survivor and PCXD samples, indicating a unique change in gene expression for each group. All transplant outcomes shared a set of 21 probes with similarly altered expression, which were indicative of ongoing myocardial inflammation and injury. Some outcome‐specific changes in gene expression were identified. Bioinformatics analysis detected an enrichment of genes involved in protein, carbohydrate and branched amino acid metabolism, extracellular matrix–receptor interactions, focal adhesion, and cell communication. Conclusions: This is the first genome wide assessment of changes in cardiac gene expression after OCXTx. Hierarchical cluster analysis indicates a unique gene profile for each transplant outcome but additional samples will be required to define the unique classifier probe sets. Quantitative RT‐PCR confirmed that all transplants exhibited strong evidence of ongoing inflammation and myocardial injury consistent with the effects of cytokines and vascular antibody‐mediated inflammation. This was also consistent with bioinformatic analysis suggesting ongoing tissue repair in survivor and PCXD samples. Bioinformatics analysis suggests for the first time that xenotransplantation may affect cardiac metabolism in survivor and rejected samples. This study highlights the potential utility of molecular analysis to monitor xenograft function, to identify new molecular markers and to understand processes, which may contribute to DXR.     

hDAF porcine cardiac xenograft maintains cardiac output after orthotopic transplantation into baboon--a perioperative study

BACKGROUND: Only limited data are available on the physiological functional compatibility of cardiac xenografts after orthotopic pig to baboon transplantation (oXHTx). Thus we investigated hemodynamic parameters including cardiac output (CO) before and after oXHTx. METHODS: Orthotopic xenogeneic heart transplantation from nine hDAF transgeneic piglets to baboons was performed. We used femoral arterial thermodilution for the invasive assessment of CO and stroke volume. RESULTS: Baseline CO of the baboons after induction of anesthesia was 1.36 (1.0-1.9) l/min. 30 to 60 min after termination of the cardiopulmonary bypass, CO of the cardiac xenograft was significantly increased to 1.72 (1.3-2.1) l/min (P < 0.01). The stroke volumes of the baboon heart before transplantation and the cardiac xenograft was comparable [14.9 (11-26) vs. 11.8 (10-23) ml]. Thus the higher CO was achieved by an increase in heart rate after oXHTx [75.0 (69-110) vs. 140.0 (77-180)/min; P < 0.01]. Despite the increased CO, oxygen delivery was reduced [256 (251-354) vs. 227 (172-477) ml/min; P < 0.01] due to the inevitable hemodilution during the cardiopulmonary bypass and the blood loss caused by the surgical procedures. CONCLUSION: Our results demonstrate that in the early phase after orthotopic transplantation of hDAF pig hearts to baboons, cardiac function of the donor heart is maintained and exceeds baseline CO. However, in the early intraoperative phase this was only possible by using inotropic substances and vasopressors due to the inevitable blood loss and dilution by the priming of the bypass circuit.     

Left Ventricular Pressure Measurement by Telemetry Is an Effective Means to Evaluate Transplanted Heart Function in Experimental Heterotopic Cardiac Xenotransplantation

Evaluation of the function of heterotopic cardiac transplants has traditionally been accomplished by either manual palpation or serial biopsies. Both methods have drawbacks. Palpation can be difficult to differentiate a pulse from the graft versus a transmitted pulse from the native aorta. Serial biopsies, though accurate, require multiple laparotomies, leading to increased morbidity and possibly mortality rates. In this study we used an advanced telemetry system, consisting of an intra-abdominal implant, that was capable of continuously monitoring simultaneously several parameters of the transplanted heart and the status of the recipient. In a large animal model of heterotopic cardiac xenotransplantation (pig donor to baboon recipient), we implanted the device in 12 animals: 8 with and 4 without immunosuppression. We monitored and continuously recorded the left ventricular pressure (both peak-systolic and end-diastolic [LVEDP]), heart rate, and the electrocardiogram pattern of the transplanted heart as well as the temperature of the recipient. The left ventricular pressure proved to be the most valuable parameter to assess graft heart function. In the 4 nonimmunosuppressed cases, grafts were rejected acutely. In these cases, the end-diastolic pressure increased sharply and the heart stopped contracting when the difference between the systolic and the diastolic pressure decreased to <10 mm Hg. The earliest reproducible sign of rejection was an increased LVEDP. Among long-term survivors, the increase in diastolic pressure was gradual, indicating progressive thickening of the myocardium and decreased compliance of the ventricle. Six of 8 immunosuppressed animals died of other complications before rejecting the transplanted heart. The telemetry was also helpful to indicate early onset of fever in the recipients, thus allowing us to intervene early and prevent potentially lethal septic complications. Continuous monitoring of several parameters via telemetry allowed detection of changes associated with rejection as well as other complications at an early stage, allowing prompt intervention, treatment, and possibly reversal of rejection.     

Rejection of Cardiac Xenografts Transplanted from α1,3‐Galactosyltransferase Gene‐Knockout (GalT‐KO) Pigs to Baboons

The use of α1,3‐galactosyltransferase gene‐knockout (GalT‐KO) swine donors in discordant xenotransplantation has extended the survival of cardiac xenografts in baboons following transplantation. Eight baboons received heterotopic cardiac xenografts from GalT‐KO swine and were treated with a chronic immunosuppressive regimen. The pathologic features of acute humoral xenograft rejection (AHXR), acute cellular xenograft rejection (ACXR) and chronic rejection were assessed in the grafts. No hyperacute rejection developed and one graft survived up to 6 months after transplantation. However, all GalT‐KO heart grafts underwent graft failure with AHXR, ACXR and/or chronic rejection. AHXR was characterized by interstitial hemorrhage and multiple thrombi in vessels of various sizes. ACXR was characterized by TUNEL⁺ graft cell injury with the infiltration of T cells (including CD3 and TIA‐1⁺ cytotoxic T cells), CD4⁺ cells, CD8⁺ cells, macrophages and a small number of B and NK cells. Chronic xenograft vasculopathy, a manifestation of chronic rejection, was characterized by arterial intimal thickening with TUNEL⁺ dead cells, antibody and complement deposition, and/or cytotoxic T‐cell infiltration. In conclusion, despite the absence of the Gal epitope, acute and chronic antibody and cell‐mediated rejection developed in grafts, maintained by chronic immunosupression, presumably due to de novo responses to non‐Gal antigens.