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For many patients with terminal/advanced cardiac failure, heart transplantation is the most effective, durable treatment option, and offers the best prospects for a high quality of life. The number of potentially life-saving donated human organs is far fewer than the population who could benefit from a new heart, resulting in increasing numbers of patients awaiting replacement of their failing heart, high waitlist mortality, and frequent reliance on interim mechanical support for many of those deemed among the best candidates but who are deteriorating as they wait. Currently, mechanical assist devices supporting left ventricular or biventricular heart function are the only alternative to heart transplant that is in clinical use. Unfortunately, the complication rate with mechanical assistance remains high despite advances in device design and patient selection and management, and the quality of life of the patients even with good outcomes is only moderately improved. Cardiac xenotransplantation from genetically multi-modified (GM) organ-source pigs is an emerging new option as demonstrated by the consistent long-term success of heterotopic (non-life-supporting) abdominal and life-supporting orthotopic porcine heart transplantation in baboons, and by a recent ‘compassionate use’ transplant of the heart from a GM pig with 10 modifications into a terminally ill patient who survived for 2 months. In this review, we discuss pig heart xenotransplantation as a concept, including pathobiological aspects related to immune rejection, coagulation dysregulation, and detrimental overgrowth of the heart, as well as GM strategies in pigs to prevent or minimize these problems. Additional topics discussed include relevant results of heterotopic and orthotopic heart transplantation experiments in the pig-to-baboon model, microbiological and virologic safety concepts, and efficacy requirements for initiating formal clinical trials. An adequate regulatory and ethical framework as well as stringent criteria for the selection of patients will be critical for the safe clinical development of cardiac xenotransplantation, which we expect will be clinically tested during the next few years.

After orthotopic cardiac xenotransplantation, the combination of both the inflammatory responses to the exposure of a recipient to the xenogeneic organ and the use of cardiopulmonary bypass has been assumed to cause detrimental side effects. These have been described not only to affect the transplanted organ (heart) itself, but also the recipient's lungs. In this article, we summarize how these possible detrimental processes can be minimized or even avoided.Data from eight pig-to-baboon orthotopic cardiac xenotransplantation experiments were analyzed with a special focus on early (within the first week) postoperative organ dysfunction and systemic inflammatory responses. Non-ischemic heart preservation and the careful management of the heart-lung machine were deemed essential to guarantee not only the immediate function of the transplanted xenogeneic organ but also the prompt recovery of the recipient.After weaning from cardiopulmonary bypass, very low catecholamine amounts were needed to ensure an adequate pump function and cardiac output. Central venous oxygen saturation and serum lactate levels remained within normal ranges. All animals were successfully weaned from ventilation within the first postoperative hours. Serum parameters of the transplants and native kidneys and livers were initially slightly elevated or always normal, as were hemoglobin, LDH, and platelet measurements. Markers of systemic inflammation, C-reactive protein, and IL-6 were slightly elevated, but the reactions caused no lasting damage.Consistent short-term and long-term results were achieved after orthotopic cardiac pig-to-baboon transplantation without detrimental inflammatory responses or signs of multiorgan failure. In comparison to allogeneic procedures, non-ischemic heart preservation was important for successful immediate organ function, as was the management of the heart-lung machine. Thus, we believe that genetically modified porcine hearts are ready for use in the clinical setting.

The number of donated human organs and tissues for patients with terminal organ failure falls far short of the need. According to the United Network for Organ Sharing (www.unos.org), more than 113,000 candidates for transplant are currently on the U.S. national waiting list, but only 36,527 organ transplants could be performed in 2018. Alternative sources, such as organs and tissues from animals, are therefore urgently needed. For a number or reasons, including size, anatomical, and physiological similarities with humans, the pig is the preferred donor species (reviewed in Cooper et al., 2016). Importantly, pigs can be optimized by genetic engineering as a source of cells, tissues, and organs for xenotransplantation. Recent advances in gen(om)e editing are speeding up progress in this field. Numerous genetically (multi-)modified pig lines have been generated to prevent immune rejection of xenotransplants, to overcome physiological incompatibilities, and to reduce the risk of transmitting zoonotic pathogens (Table 1; reviewed in Kemter et al., 2018). Table 1. Selection of genetic modifications of donor pigs for xenotransplantation

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.

Heart transplantation is the only long-lasting life-saving option for patients with terminal cardiac failure. The number of available human organs is however far below the actual need, resulting in substantial mortality of patients while waiting for a human heart. Mechanical assist devices are used to support cardiac function, but are associated with a high risk of severe complications and poor quality of life for the patients. Consistent success in orthotopic transplantation of genetically modified pig hearts into baboons indicates that cardiac xenotransplantation may become a clinically applicable option for heart failure patients who cannot get a human heart transplant. In this overview, we project potential paths to clinical cardiac xenotransplantation, including the choice of genetically modified source pigs, associated requirements of microbiological, including virological, safety, optimized matching of source pig and recipient, and specific treatments of the donor heart after explantation and of the recipients. Moreover, selection of patients and the regulatory framework will be discussed.

Background: Many genetically multi-modified donor lines for xenotransplantation have a background of domestic pigs with rapid body and organ growth. The intrinsic growth potential of porcine xeno-organs may impair their long-term function after orthotopic transplantation in non-human primate models. Since growth hormone is a major stimulator of postnatal growth, we deleted its receptor (GHR-KO) to reduce the size of donor pigs in one step. Methods: Heart weight and proteome profile of myocardium were investigated in GHR-KO and control pigs. GHR-KO mutations were introduced using CRISPR/Cas9 in an α1,3-galactosyltransferase (GGTA1)-deficient background expressing the human cluster of differentiation (hCD46) and human thrombomodulin (hTHBD) to generate quadruple-modified (4GM) pigs. Results: At age 6months, GHR-KO pigs had a 61% reduced body weight and a 63% reduced heart weight compared with controls. The mean minimal diameter of cardiomyocytes was 28% reduced. A holistic proteome study of myocardium samples from the two groups did not reveal prominent differences. Two 4GM founder sows had low serum insulin-like growth factor 1 (IGF1) levels (24±1ng/mL) and reached body weights of 70.3 and 73.4kg at 9months. Control pigs with IGF1 levels of 228±24ng/mL reached this weight range three months earlier. The 4GM sows showed normal sexual development and were mated with genetically multi-modified boars. Offspring revealed the expected Mendelian transmission of the genetic modifications and consistent expression of the transgenes. Conclusion: GHR-KO donor pigs can be used at an age beyond the steepest phase of their growth curve, potentially reducing the problem of xeno-organ overgrowth in preclinical studies.

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.

The activation of the endothelial surface in xenografts is still a poorly understood process and the consequences are unpredictable. The role of Ca2+ -messaging during the activation of endothelial cells is well recognized and routinely measured by synthetic Ca2+ -sensitive fluorophors. However, these compounds require fresh loading immediately before each experiment and in particular when grown in state-of-the-art 3D cell culture systems, endothelial cells are difficult to access with such sensors. Therefore, we developed transgenic pigs expressing a Ca2+ -sensitive protein and examined its principal characteristics. Primary transgenic endothelial cells stimulated by ATP showed a definite and short influx of Ca2+ into the cytosol, whereas exposure to human serum resulted in a more intense and sustained response. Surprisingly, not all endothelial cells reacted identically to a stimulus, rather activation took place in adjacent cells in a timely decelerated way and with distinct intensities. This effect was again more pronounced when cells were stimulated with human serum. Finally, we show clear evidence that antibody binding alone significantly activated endothelial cells, whereas antibody depletion dramatically reduced the stimulatory potential of serum. Transgenic porcine endothelial cells expressing a Ca2+ -sensor represent an interesting tool to dissect factors inducing activation of porcine endothelial cells after exposure to human blood or serum.

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.

BACKGROUND Transpulmonary thermodilution is well established as a tool for in-depth hemodynamic monitoring of critically ill patients during surgical procedures and intensive care. It permits easy assessment of graft function following cardiac transplantation and guides post-operative volume and catecholamine therapy. Since no pulmonary catheter is needed, transpulmonary thermodilution could be useful in experimental cardiac pig-to-baboon xenotransplantation. However, normal values for healthy animals have not yet been reported. Here, we present data from piglets and baboons before xenotransplantation experiments and highlight differences between the two species and human reference values. METHODS Transpulmonary thermodilution from baboons (body weight 10-34 kg) and piglets (body weight 10-38kg) were analyzed. Measurements were taken in steady state after induction of general anesthesia before surgical procedures commenced. Cardiac index (CI), mean arterial pressure (MAP), systemic vascular resistance index (SVRI), parameters quantifying cardiac filling (global end-diastolic volume index, GEDI), and pulmonary edema (extravascular lung water, ELWI) were assessed. RESULTS Preload, afterload, and contractility parameters clearly correlated with total body weight or body surface area. Baboons had lower CI values than weight-matched piglets (4.2 ± 0.9l/min/m2 vs 5.3 ± 1.0/min/m2 , P

BACKGROUND Perioperative monitoring and hemodynamic management after heterotopic thoracic cardiac xenotransplantation is challenging due to 2 independently beating hearts. Telemetry allows continuous monitoring of hemodynamic parameters of both the donor and recipient hearts. We describe our experience and report on the validity of a telemetric system during and after surgery. MATERIAL AND METHODS Wireless telemetry transmitters were implanted in 3 baboons receiving porcine donor hearts. Left ventricular pressure and ECG were assessed from the donor heart, whereas aortic pressure and temperature were assessed from the recipient. Telemetric data were validated with invasive blood pressure measurements. RESULTS Telemetric blood pressure was lower than invasive blood pressure. Intraoperatively, the probe in the graft's left ventricle measured negative end-diastolic pressures. Telemetry allowed simple discrimination between donor's and recipient's heart rates. Body temperature was underestimated by telemetry. Telemetric monitoring facilitates recognition of graft arrhythmias and volume demand. CONCLUSIONS In heterotopic thoracic cardiac xenotransplantation, telemetric implants are useful tools to continuously monitor the animals' hemodynamic parameters and to discriminate donor and recipient organs. Accuracy is sufficient for systemic pressure measurement, but perioperative use of left ventricular end-diastolic pressure as a surrogate parameter for graft function is not advisable. Temperature measurements by telemetry do not reflect body core temperature.

The activation of the endothelial surface in xenografts is still a poorly understood process and the consequences are unpredictable. The role of Ca

Porcine circovirus 3 (PCV3) is a newly described member of the virus family Circoviridae. PCV3 is highly distributed among pigs and wild boars worldwide. A sudden introduction of PCV3 was recently observed in a herd of triple genetically modified pigs generated for xenotransplantation. These animals were used as donor pigs for orthotopic heart transplantation into baboons. In four cases, PCV3-positive hearts were transplanted, and transmission of PCV3 to the recipient was observed. PCV3 was found in all organs of the recipient baboons and a higher virus load was found in animals with a longer survival time of the transplant, indicating replication of the virus. This is the first report showing trans-species transmission of PCV3 to baboons by transplantation of a heart from a PCV3-positive donor pig. Sequence analysis showed that PCV3a and PCV3b were present in the infected pigs and were transmitted. Experiments to infect human 293 cells with PCV3 failed.

The global prevalence of insulin-dependent diabetes mellitus is rapidly increasing. In spite of major improvements in insulin treatment regimens and diabetes technology (e.g., artificial pancreas devices), glucose control remains problematic in a substantial proportion of diabetic patients. Those patients may benefit from beta-cell replacement therapies. Allotransplantation of pancreas or isolated pancreatic islets is limited by the small number of organ donors. Thus, alternative sources of beta-cells are being developed and tested. These include endocrine progenitor cells or mature beta-cells derived from pluripotent human stem cells and attempts to derive human pancreas tissue in animal hosts by interspecific chimeric complementation experiments. Xenotransplantation of porcine islets is a realistic alternative option. Immune rejection of xenoislets can be prevented by immunosuppression of the recipient or by encapsulation of the islets in microdevices or macrodevices. Using precise and efficient genetic engineering of donor pigs, immune-protected xenoislets with improved functionality can be generated.

Background Existing large-animal, ex vivo, cardiac perfusion models are restricted in their ability to establish an ischemia/reperfusion condition as seen in cardiac surgery or transplantation. Other working heart systems only challenge one ventricle or require a substantially larger priming volume. We describe a novel biventricular cardiac perfusion system with reduced priming volume. Methods Juvenile pig hearts were cardiopleged, explanted, and reperfused ex vivo after 150 minutes of cold ischemia. Autologous whole blood was used as perfusate (minimal priming volume 350 mL). After 15 minutes of Langendorff perfusion (LM), the system was switched into a biventricular working mode (WM) and studied for 3 hours. Results During reperfusion, complete unloading of both ventricles and constant-pressure coronary perfusion was achieved. During working mode perfusion, the preload and afterload pressure of both ventricles was controlled within the targeted physiologic range. Functional parameters such as left ventricular work index were reduced in ex vivo working mode (in vivo: 787 ± 186 vs. 1 h WM 498 ± 66 mm Hg·mL/g·min; p Conclusion In the ex vivo perfusion system, stable hemodynamic and metabolic conditions can be established for a period of 3 hours while functional and blood parameters are easily accessible. Moreover, because of the minimal priming volume, the novel ex vivo cardiac perfusion circuit allows for autologous perfusion, using the limited amount of blood available from the organ donating animal.

Significant progress in understanding and overcoming cardiac xenograft rejection using a clinically relevant large animal pig-to-baboon model has accelerated in recent years. This advancement is based on improved immune suppression, which attained more effective regulation of B lymphocytes and possibly newer donor genetics. These improvements have enhanced heterotopic cardiac xenograft survival from a few weeks to over 2 years, achieved intrathoracic heterotopic cardiac xenograft survival of 50 days and orthotopic survival of 57 days. This encouraging progress has rekindled interest in xenotransplantation research and refocused efforts on preclinical orthotopic cardiac xenotransplantation.

Purpose Major hurdles in oXHTx are the delayed xenograft rejection, the early perioperative cardiac xenograft dysfunction (PCXD) and the pig heart overgrowth, which were solved in this study with a costimulation blockade, a new non-ischemic cold preservation and a growth inhibition by anti-proliferative drugs. Aim was to achieve a 90-days-survival of minimal 60% (6 of 10 baboons) in this life-supporting orthotopic pig-to-baboon model (oXHTx), because this is the recommendation of the ISHLT to begin a clinical cardiac XT program. Methods We transplanted 8 GalKO/hCD46/hTM transgenic (tg) pig hearts orthotopically into baboons with using a basic immunosuppression consisting of ATG, rituximab, mycophenolate (MMF), cortisone and a costimulation blockade CD40mAb (high dose: 50 mg/kg). To prevent PCXD, we used instead of the crystalloid solution a new non-ischemic 8°C cold perfusion technique with oxygenated erythrocytes. Additional antihypertensive drugs and an mTOR inhibitor (temsirolimus) were applied to inhibit pig xenograft growth and hypertrophy. Results In comparison to our previous group with crystalloid cardioplegia (Langin et al. Nature. 2018;564:430-433) in this group with cold perfusion preservation (non-ischemic) no PCXD was found. One baboon died of a pancreatitis on day 14, another of sepsis on day 26. By using the antiproliferative therapy, 6 of 8 recipient baboons reached the end of study, were long-term surviving (4 were actively terminated after 90 days according to the guidelines of our government). With special permit two further experiments could be prolonged to half a year and the animals were terminated on day 182 and 195. All baboons lived under excellent physical conditions and no hyperacute rejection or DXR occurred. Conclusion First time in a life-supporting oXHT of multi-tg pig hearts here was a consistent reproduceable long-term survival of 3 - 6 months achieved, which is a major progress after 25 years of research. This is an essential milestone and breakthrough and meets the prerequisite according to the ISHLT to begin a clinical phase I study with patients in terminal heart failure. This paves the way to clinical cardiac XT in the next 2 to 5 years.

For clinical xenotransplantation, transplants must be free of porcine cytomegalovirus (PCMV). Piglets become infected primarily in the perinatal period by the mother sow. While individual donor animals can be protected from infection by isolation husbandry, success is not guaranteed and this strategy poses the risk of undetected infections and raises animal welfare questions. Here, we present the establishment of a completely PCMV-negative pig herd for breeding donor animals for xenotransplantation. Eleven pregnant DanAvl Basic hybrid sows were purchased from a designated pathogen-free (DPF), PCMV-positive colony and transferred to a new pig facility at the Centre for Innovative Medical Models (CiMM) 4 weeks prior to farrowing. At the age of 24 hours, piglets were early-weaned and transferred to a commercially available Rescue Deck system dedicated to motherless rearing of piglets. Sows were removed from the facility. The PCMV status of F1-generation animals was determined at regular intervals over a period of 14 months by a sensitive real-time PCR-based detection method testing blood, nasal swabs and cultured peripheral blood mononuclear cells (PBMCs). F1 sows were used as recipients of genetically modified embryos to generate a xenotransplant donor herd. Offspring were tested for PCMV accordingly. All offspring have remained PCMV negative over the whole observation period of 14 months. A completely PCMV-negative pig herd for xenotransplantation has thus been successfully established.

Heart transplantation is the only cure for patients with terminal cardiac failure, but the supply of allogeneic donor organs falls far short of the clinical need1–3. Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative4. Genetically multi-modified pig hearts that lack galactose-α1,3-galactose epitopes (α1,3-galactosyltransferase knockout) and express a human membrane cofactor protein (CD46) and human thrombomodulin have survived for up to 945 days after heterotopic abdominal transplantation in baboons5. This model demonstrated long-term acceptance of discordant xenografts with safe immunosuppression but did not predict their life-supporting function. Despite 25 years of extensive research, the maximum survival of a baboon after heart replacement with a porcine xenograft was only 57 days and this was achieved, to our knowledge, only once6. Here we show that α1,3-galactosyltransferase-knockout pig hearts that express human CD46 and thrombomodulin require non-ischaemic preservation with continuous perfusion and control of post-transplantation growth to ensure long-term orthotopic function of the xenograft in baboons, the most stringent preclinical xenotransplantation model. Consistent life-supporting function of xenografted hearts for up to 195 days is a milestone on the way to clinical cardiac xenotransplantation7.

The porcine cytomegalovirus (PCMV) is a herpesvirus that may pose a risk for xenotransplantation using pig cells, tissues, or organs. Here, three orthotopic pig heart transplantations into baboons were studied. To detect PCMV, a real-time PCR and a Western blot assay based on four PCMV protein sequences, including two tegument proteins, were used. The transmission of PCMV from the donor pig to the recipient baboon was found in two cases, despite PCMV not being detected in the blood of the donor pigs by real-time PCR. Although it was not in the blood, PCMV was detected in different organs of the donor pigs, and in sibling animals. Immunohistochemistry using an antiserum that is specific for PCMV detected virus protein-expressing cells in all of the organs of the recipient baboon, most likely representing disseminated pig cells. Therefore, for the first time, the distribution of PCMV in organs of the donor pigs and the recipient baboons was described. In addition, baboon cytomegalovirus (BaCMV) was found activated in the recipient, and a screening for hepatitis E virus (HEV) and porcine lymphotropic herpesviruses (PLHV) was performed. For the first time, a cross-reactivity between antibodies directed against PCMV and BaCMV was found.

Background In pig‐to‐human xenotransplantation, early cellular rejection reactions are mediated by natural killer cells (NK cells). Human NK cells are inhibited by HLA‐E via CD94/NKG2A receptors. To protect porcine grafts against human NK cell responses, transgenic GTKO pigs expressing hCD46 and HLA‐E have been generated. The aim of this study was to test the effect of this genetic modification on xenogeneic, and in particular human NK cell response, using an ex vivo perfusion model of pig hearts with human blood. Methods Cardiopleged and explanted genetically modified (gm) pig hearts (GTKO/hCD46/HLA‐E/hβ2‐microglobulin) and wild‐type (wt) controls (n = 6 each) were reperfused and tested in an 8 hours ex vivo perfusion system using freshly drawn human blood. Cardiac function was evaluated during a 165‐minute period in working heart mode. Myocardial damage, antibody deposition, complement activation, and coagulation parameters were evaluated histologically at the end of perfusion. The number of NK cells in the perfusate was determined by flow cytometry at baseline and at 8 hours; tissue infiltration by NK cells was quantified by immunofluorescence microscopy using NKp46 staining of frozen sections. Results Deposition of IgG (1.2 ± 1 × 107 vs 8.8 ± 2.9 × 106; P < .01), IgM (4.4 ± 3.7 × 106 vs 1.7 ± 1.2 × 106; P < .01), and the complement activation product C4b/c (3.5 ± 1.3 × 106 vs 2.3 × 106 ± 9.4 × 105; P > .01) was lower in gm than wt hearts. NK cell percentages of leukocytes in the perfusate decreased from 0.94 ± 0.77% to 0.21 ± 0.25% (P = .04) during xenoperfusion of wt hearts. In contrast, the ratio of NK cells did not decrease significantly in the gm hearts. In this group, NK cell myocardial infiltration after 480 minutes of perfusion was lower than in wt organs (2.5 ± 3.7 × 104/mm3 vs 1.3 ± 1.4 × 105/mm3; P = .0001). The function of gm hearts was better preserved compared to wt organs, as demonstrated by higher cardiac index during the first 2 hours of ex vivo perfusion. Conclusion GTKO, hCD46, and HLA‐E expression in porcine hearts reduced complement deposition, complement dependent injury, and myocardial NK cell infiltration during perfusion with human blood. This tested combination of genetic modifications may minimize damage from acute human‐anti‐pig rejection reactions and improve myocardial function after xenotransplantation.

Xenotransplantation using porcine cells, tissue or organs may be associated with transmission of various pathogens that can induce zoonosis. Highly specific and sensitive methods detecting porcine endogenous retroviruses (PERVs), hepatitis E virus (HEV), porcine cytomegalovirus (PCMV) were developed and used to screen (i) wild-type Gottingen Minipigs, a breed produced under specified pathogen-free (spf) conditions, (ii) wild-type Aachen Minipigs, a newly created pig breed in Germany, and (iii) pigs produced carrying various transgenic modifications to prevent transplant rejection were analysed. Moreover, some animals were tested for the presence of >49 other microorganisms, incl. porcine lymphotropic herpes viruses (PLHV). Human-tropic PERV-A and PERV-B were found in all pigs und PERV-C was detected in all tested Gottingen Minipigs and in more than 90% of the other pigs. Although PERV-C does not infect human cells, it may represent a special risk when it recombines with PERV-A, as high-titre PERV-A/C were able to infect human cells in vitro. The expression level of PERV in all examined animals, as analysed in a mitogen stimulation assay, was found to same low range as previously reported for German landrace pigs. HEV was found in 27% of the Gottingen Minipigs, the prevalence of the herpes viruses was below 10%. Immunological methods based on recombinant viral proteins were developed to screen for antibodies against PERV, HEV, PCMV and PLHV. Antibodies against HEV were found only in few animals, whereby in piglets they were of maternal origin. The presence of HEV in sera of three sows six days after parturition and in their offspring in a well characterised SPF facility suggests vertical transmission of the virus. In contrast to PERVs, which are integrated in the porcine genome, HEV and herpes viruses can be eliminated using Caesarean section and eventually antiviral treatment. Most importantly, when islet cells from non-transgenic and transgenic pigs expressing immunomodulatory molecules were transplanted into marmosets, no transmission of PERV, HEV, PCMV and PLHV was observed, although four out of 14 donor pigs were PCMV positive. No transmission of PERV, HEV, PCMV and PLHV was observed after hearts from triple transgenic pigs were transplanted into baboons. These data indicate that sensitive and reliable methods for the detection of pathogens are available and that screening of putative donor pigs of porcine xenotransplants is critical to ensure microbiological safety in clinical xenotransplantation. Supported by the German Research Foundation (DFG) Transregio Collaborative Research Centre 127.

Background: Heme oxygenase-1 (HO-1) is an inducible stress-responsive enzyme converting heme to bilirubin, carbon monoxide, and free iron, which exerts anti-inflammatory and antiapoptotic e...

Although acute cellular rejection after heart transplantation (HTX) can be controlled by full-dose calcineurin inhibitor (CNI)-based immunosuppressive regimens, cardiac allograft vasculopathy (CAV), nephrotoxicity, and malignancy remain ongoing problems. To evaluate the potential beneficial effects of sirolimus and CNI reduction, we compared de novo low-dose tacrolimus and sirolimus with standard tacrolimus and mycophenolate mofetil (MMF)-based immunosuppression after HTX.We analyzed a long-term follow-up cohort of 126 patients who underwent HTX during the period 1998-2005 and received either de novo low-dose tacrolimus/sirolimus (lowTAC/SIR; n = 61) or full-dose tacrolimus/MMF (TAC/MMF; n = 64).Freedom from treatment switch was less in the low TAC/SIR group than in the TAC/MMF group (51.7% vs 73.0%, p = 0.038) 8 years after HTX. Freedom from acute rejection was 90.6% in the low TAC/SIR group vs 80.3% in the TAC/MMF group (p = 0.100). There was no difference in freedom from International Society for Heart and Lung Transplantation CAV grade ≥ 1 (55.4% vs 60.0%, p = 0.922), time until CAV diagnosis (4.2 ± 2.0 years vs 3.2 ± 2.4 years, p = 0.087), and CAV severity (p = 0.618). The benefit of reduced early maximum creatinine for low TAC/SIR treatment (1.8 ± 0.9 mg/dl vs 2.4 ± 1.1 mg/dl in TAC/MMF group, p0.001) did not continue 5 years and 8 years after HTX (1.4 ± 0.4 mg/dl vs 1.7 ± 1.2 mg/dl, p = 0.333, and 1.6 ± 1.1 mg/dl vs 1.6 ± 0.8 mg/dl, p = 0.957). The trend for superior survival at 5 years with low TAC/SIR treatment (93.1% vs 81.3% in TAC/MMF group, p = 0.051) could not be confirmed after 8 years (84.7% vs 75.0%, p = 0.138). Multivariate analysis at 8 years did not reveal any benefit of low TAC/SIR treatment.Reduction of de novo CNI did not result in superior long-term renal function. Low-dose mechanistic target of rapamycin inhibition did not achieve any benefit in CAV prevention compared with full-dose TAC/MMF after HTX.