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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 need.sup.1-3. Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative.sup.4. Genetically multi-modified pig hearts that lack galactose-[alpha]1,3-galactose epitopes ([alpha]1,3-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 baboons.sup.5. 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 once.sup.6. Here we show that [alpha]1,3-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 xenotransplantation.sup.7. [alpha]1,3-galactosyltransferase-knockout pig hearts that express human CD46 and human thrombomodulin require non-ischaemic preservation with continuous perfusion and post-transplantation growth control to ensure long-term orthotopic function of the xenograft in baboons., Author(s): Matthias Längin [sup.1] [sup.2] , Tanja Mayr [sup.1] [sup.2] , Bruno Reichart [sup.2] , Sebastian Michel [sup.3] , Stefan Buchholz [sup.3] , Sonja Guethoff [sup.2] [sup.3] , Alexey Dashkevich [...]

Introduction: Orthotopic cardiac xenotransplantation has seen notable improvement, leading to the first compassionate use in 2022. However, it remains challenging to define the clinical application of cardiac xenotransplantation, including the back‐up strategy in case of xenograft failure. In this regard, the heterotopic thoracic technique could be an alternative to the orthotopic procedure. We present hemodynamic data of heterotopic thoracic pig‐to‐baboon transplantation experiments, focusing on perioperative xenograft dysfunction and xenograft overgrowth. Methods: We used 17 genetically modified piglets as donors for heterotopic thoracic xenogeneic cardiac transplantation into captive‐bred baboons. In all animals, pressure probes were implanted in the graft's left ventricle and the recipient's ascending aorta and hemodynamic data (graft pressure, aortic pressure and recipient's heart rate) were recorded continuously. Results: Aortic pressures and heart rates of the recipients' hearts were postoperatively stable in all experiments. After reperfusion, three grafts presented with low left ventricular pressure indicating perioperative cardiac dysfunction (PCXD). These animals recovered from PCXD within 48 h under support of the recipient's heart and there was no difference in survival compared to the other 14 ones. After 48 h, graft pressure increased up to 200 mmHg in all 17 animals with two different time‐patterns. This led to a progressive gradient between graft and aortic pressure. With increasing gradient, the grafts stopped contributing to cardiac output. Grafts showed a marked weight increase from implantation to explantation. Conclusion: The heterotopic thoracic cardiac xenotransplantation technique is a possible method to overcome PCXD in early clinical trials and an experimental tool to get a better understanding of PCXD. The peculiar hemodynamic situation of increasing graft pressure but missing graft's output indicates outflow tract obstruction due to cardiac overgrowth. The heterotopic thoracic technique should be successful when using current strategies of immunosuppression, organ preservation and donor pigs with smaller body and organ size. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 <.01). MAP and SVRI were higher in baboons than piglets (MAP: 99 ± 22 mm Hg vs 62 ± 11 mm Hg, P <.01; SVRI: 1823 ± 581 dyn*s/cm5*m2 vs 827 ± 204 dyn*s/cm5*m2, P <.01). GEDI and ELWI did differ significantly between both species, but measurements were within similar ranges (GEDI: 523 ± 103 mL/m2 vs 433 ± 78 mL/m2, P <.01; ELWI: 10 ± 3 mL/kg vs 11 ± 2 mL/kg, P <.01). Regarding adult human reference values, CI was similar to both baboons and piglets, but all other parameters were different. Conclusions: Parameters of preload, afterload, and contractility differ between baboons and piglets. In particular, baboons have a much higher afterload than piglets, which might be instrumental in causing perioperative xenograft dysfunction and post‐operative myocardial hypertrophy after orthotopic pig‐to‐baboon cardiac xenotransplantation. Most transpulmonary thermodilution‐derived parameters obtained from healthy piglets and baboons lie outside the reference ranges for humans, so human normal values should not be used to guide treatment in those animals. Our data provide reference values as a basis for developing algorithms for perioperative hemodynamic management in pig‐to‐baboon cardiac xenotransplantation. [ABSTRACT FROM AUTHOR]

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 constantpressure coronary perfusion was achieved. During workingmode 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 < 0.01), but remained stable throughout the following study period (3 h WM 517 ± 103 mm Hg·mL/g·min; p ¼ 0.63). Along with the elevated workload during WM, myocardial metabolism and oxygen consumption increased compared with LM (0.021 ± 0.08 vs. 0.06 ± 0.01 mL/min/g; 1 h after reperfusion). Histologic examination of the myocardium revealed no structural damage. 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. [ABSTRACT FROM AUTHOR]

Abstract: Background: Regulatory T cells (Treg) play an important role in maintenance of homeostasis in vivo. Treg application to alleviate allo‐organ rejection is being studied extensively. However, natural Treg (nTreg) expansion in vitro is laborious and expensive. Antigen‐specific Treg are more effective and require lower cell numbers than use of nTreg for immune control. The baboon, as a non‐human primate experimental animal model, is widely used in xenotransplantation research. An effective method to generate baboon xeno‐specific Treg would benefit research on immune tolerance in xenotransplantation using this model system. Method: Baboon tolerogenic dendritic cells (tolDC) were generated in 3 days from monocytes isolated from baboon peripheral blood mononuclear cells in medium supplemented with anti‐inflammatory cytokines. After loading with porcine‐specific (PS) in vitro‐transcribed RNA (ivtRNA), tolDC were used to induce CD4+ T cells to become porcine‐specific Treg (PSTreg) in cocultures supplemented with IL‐2 and rapamycin for 10 days. Anti‐inflammatory and inflammatory cytokine expression was evaluated at the mRNA and protein levels in both baboon tolDC and PSTreg. Functional assays, suppression of activation markers on porcine‐specific effector T cells (PSTeff) and inhibition of PSTeff proliferation, were used to test PSTreg specificity. Results: TolDC generated with this method exhibited a tolerogenic phenotype, expressed CCR7 and produced high levels of IL‐10 and TGF‐β1, whereas IL‐12p40 and IFN‐γ were not expressed. PSTreg were successfully generated in cocultures of CD4+ T cells and PS ivtRNA‐loaded tolDC. They exhibited a CD3+ CD4+ CD25+ CD127low/− CD45RAlow Foxp3+ phenotype and were characterized by high expression of IL‐10 and TGF‐β1 mRNA and protein. They showed upregulated expression of EBI3 and GARP mRNA. PSTreg exhibited highly suppressive effects toward PSTeff, secreting high amounts of IL‐10 and TGF‐β1 cytokine upon interaction with PSTeff and suppressing IFN‐γ expression on PSTeff. Conclusion: In this study, a fast 3‐day method to generate baboon‐derived tolDC is provided that allows subsequent induction of PSTreg displaying high porcine‐antigen specificity and expression of IL‐10 and TGF‐β1. Porcine‐specific baboon Treg can be used in porcine solid organ or cell xenotransplantation studies through adoptive cell transfer into host baboons. [ABSTRACT FROM AUTHOR]

Background The complement system plays a crucial role in acute xenogeneic reactions after cardiac transplantation. We used an ex vivo perfusion model to investigate the effect of Cp40, a compstatin analog and potent inhibitor of complement at the level of C3. Methods Fifteen wild-type pig hearts were explanted, cardiopleged, and reperfused ex vivo after 150 minutes of cold ischemia. Hearts were challenged in a biventricular working heart mode to evaluate cardiac perfusion and function. In the treatment group (n=5), the complement cascade was blocked at the level of C3 using Cp40, using diluted human blood. Untreated human and porcine blood was used for controls. Results Throughout the perfusion, C3 activation was inhibited when Cp40 was used (mean of all time points: 1.11 ± 0.34% vs 3.12 ± 0.48% control activation; P<.01). Compared to xenoperfused controls, the cardiac index improved significantly in the treated group (6.5 ± 4.2 vs 3.5 ± 4.8 mL/min/g; P=.03, 180 minutes perfusion), while the concentration of lactate dehydrogenase as a maker for cell degradation was reduced in the perfusate (583 ± 187 U/mL vs 2108 ± 1145 U/mL, P=.02). Histological examination revealed less hemorrhage and edema, and immunohistochemistry confirmed less complement fragment deposition than in untreated xenoperfused controls. Conclusions Cp40 efficiently prevents C3 activation of the complement system, resulting in reduced cell damage and preserved function in wild-type porcine hearts xenoperfused ex vivo. We suggest that this compstatin analog, which blocks all main pathways of complement activation, could be a beneficial perioperative treatment in preclinical and in future clinical xenotransplantation. [ABSTRACT FROM AUTHOR]

The authors reflect on the guidelines offered by the Xenotransplantation International Society For heart and Lung (ISHLT) Advisory Board. They inform that initial clinic xenogeneic heart transplantation has provided a solution for heart failure patients suffering from severe right ventricular disfunction and coagulation disorders. They mention that xenotransplantation in non-human primates related to their health is time consuming and costly.

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 × 10 7 vs 8.8 ± 2.9 × 10 6 ; P < .01), IgM (4.4 ± 3.7 × 10 6 vs 1.7 ± 1.2 × 10 6 ; P < .01), and the complement activation product C4b/c (3.5 ± 1.3 × 10 6 vs 2.3 × 10 6  ± 9.4 × 10 5 ; 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 × 10 4 /mm 3 vs 1.3 ± 1.4 × 10 5 /mm 3 ; 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., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)