Search

The blockade of the CD40/CD40L immune checkpoint is considered essential for cardiac xenotransplantation. However, it is still unclear which single antibody directed against CD40 or CD40L (CD154), or which combination of antibodies, is better at preventing organ rejection. For example, the high doses of antibody administered in previous experiments might not be feasible for the treatment of humans, while thrombotic side effects were described for first-generation anti-CD40L antibodies. To address these issues, we conducted six orthotopic pig-to-baboon cardiac xenotransplantation experiments, combining a chimeric anti-CD40 antibody with an investigational long-acting PASylated anti-CD40L Fab fragment. The combination therapy effectively resulted in animal survival with a rate comparable to a previous study that utilized anti-CD40 monotherapy. Importantly, no incidence of thromboembolic events associated with the administration of the anti-CD40L PAS-Fab was observed. Two experiments failed early because of technical reasons, two were terminated deliberately after 90 days with the baboons in excellent condition and two were extended to 120 and 170 days, respectively. Unexpectedly, and despite the absence of any clinical signs, histopathology revealed fungal infections in all four recipients. This study provides, for the first time, insights into a combination therapy with anti-CD40/anti-CD40L antibodies to block this immune checkpoint. [ABSTRACT FROM AUTHOR]

Introduction: Inflammatory responses and coagulation disorders are a relevant challenge for successful cardiac xenotransplantation on its way to the clinic. To cope with this, an effective and clinically practicable anti‐inflammatory and anti‐coagulatory regimen is needed. The inflammatory and coagulatory response can be reduced by genetic engineering of the organ‐source pigs. Furthermore, there are several therapeutic strategies to prevent or reduce inflammatory responses and coagulation disorders following xenotransplantation. However, it is still unclear, which combination of drugs should be used in the clinical setting. To elucidate this, we present data from pig‐to‐baboon orthotopic cardiac xenotransplantation experiments using a combination of several anti‐inflammatory drugs. Methods: Genetically modified piglets (GGTA1‐KO, hCD46/hTBM transgenic) were used for orthotopic cardiac xenotransplantation into captive‐bred baboons (n = 14). All animals received an anti‐inflammatory drug therapy including a C1 esterase inhibitor, an IL‐6 receptor antagonist, a TNF‐α inhibitor, and an IL‐1 receptor antagonist. As an additive medication, acetylsalicylic acid and unfractionated heparin were administered. The immunosuppressive regimen was based on CD40/CD40L co‐stimulation blockade. During the experiments, leukocyte counts, levels of C‐reactive protein (CRP) as well as systemic cytokine and chemokine levels and coagulation parameters were assessed at multiple timepoints. Four animals were excluded from further data analyses due to porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) infections (n = 2) or technical failures (n = 2). Results: Leukocyte counts showed a relevant perioperative decrease, CRP levels an increase. In the postoperative period, leukocyte counts remained consistently within normal ranges, CRP levels showed three further peaks after about 35, 50, and 80 postoperative days. Analyses of cytokines and chemokines revealed different patterns. Some cytokines, like IL‐8, increased about 2‐fold in the perioperative period, but then decreased to levels comparable to the preoperative values or even lower. Other cytokines, such as IL‐12/IL‐23, decreased in the perioperative period and stayed at these levels. Besides perioperative decreases, there were no relevant alterations observed in coagulation parameters. In summary, all parameters showed an unremarkable course with regard to inflammatory responses and coagulation disorders following cardiac xenotransplantation and thus showed the effectiveness of our approach. Conclusion: Our preclinical experience with the anti‐inflammatory drug therapy proved that controlling of inflammation and coagulation disorders in xenotransplantation is possible and well‐practicable under the condition that transmission of pathogens, especially of PCMV/PRV to the recipient is prevented because PCMV/PRV also induces inflammation and coagulation disorders. Our anti‐inflammatory regimen should also be applicable and effective in the clinical setting of cardiac xenotransplantation. [ABSTRACT FROM AUTHOR]

Cardiac xenotransplantation has seen remarkable success in recent years and is emerging as the most promising alternative to human cardiac allotransplantation. Despite these achievements, acute vascular rejection still presents a challenge for long-term xenograft acceptance and new insights into innate and adaptive immune responses as well as detailed characterizations of signaling pathways are necessary. In allotransplantation, endothelial cells and their sugar-rich surface—the endothelial glycocalyx—are known to influence organ rejection. In xenotransplantation, however, only in vitro data exist on the role of the endothelial glycocalyx so far. Thus, in the current study, we analyzed the changes of the endothelial glycocalyx components hyaluronan, heparan sulfate and syndecan-1 after pig-to-baboon cardiac xenotransplantations in the perioperative (n = 4) and postoperative (n = 5) periods. These analyses provide first insights into changes of the endothelial glycocalyx after pig-to-baboon cardiac xenotransplantation and show that damage to the endothelial glycocalyx seems to be comparable or even less pronounced than in similar human settings when current strategies of cardiac xenotransplantation are applied. At the same time, data from the experiments where current strategies, like non-ischemic preservation, growth inhibition or porcine cytomegalovirus (a porcine roseolovirus (PCMV/PRV)) elimination could not be applied indicate that damage of the endothelial glycocalyx also plays an important role in cardiac xenotransplantation. [ABSTRACT FROM AUTHOR]

Background: Preoperative size matching is essential for both allogeneic and xenogeneic heart transplantation. In preclinical pig‐to‐baboon xenotransplantation experiments, porcine donor organs are usually matched to recipients by using indirect parameters, such as age and total body weight. For clinical use of xenotransplantation, a more precise method of size measurement would be desirable to guarantee a "perfect match." Here, we investigated the use of transthoracic echocardiography (TTE) and described a new method to estimate organ size prior to xenotransplantation. Methods: Hearts from n = 17 genetically modified piglets were analyzed by TTE and total heart weight (THW) was measured prior to xenotransplantation into baboons between March 2018 and April 2022. Left ventricular (LV) mass was calculated according to the previously published method by Devereux et al. and a newly adapted formula. Hearts from n = 5 sibling piglets served as controls for the determination of relative LV and right ventricular (RV) mass. After explantation, THW and LV and RV mass were measured. Results: THW correlated significantly with donor age and total body weight. The strongest correlation was found between THW and LV mass calculated by TTE. Compared to necropsy data of the control piglets, the Devereux formula underestimated both absolute and relative LV mass, whereas the adapted formula yielded better results. Combining the adapted formula and the relative LV mass data, THW can be predicted with TTE. Conclusions: We demonstrate reliable LV mass estimation by TTE for size matching prior to xenotransplantation. An adapted formula provides more accurate results of LV mass estimation than the generally used Devereux formula in the xenotransplantation setting. TTE measurement of LV mass is superior for the prediction of porcine heart sizes compared to conventional parameters such as age and total body weight. [ABSTRACT FROM AUTHOR]

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]

Xenotransplantation is a promising approach to reduce organ shortage, while genetic modification of donor pigs has significantly decreased the immunogenic burden of xenotransplants, organ rejection is still a hurdle. Genetically modified pig organs are used in xenotransplantation research, and the first clinical pig‐to‐human heart transplantation was performed in 2022. However, the impact of genetic modification has not been investigated on a cellular level yet. Endothelial cells (EC) and their sugar‐rich surface known as the glycocalyx are the first barrier encountering the recipient's immune system, making them a target for rejection. We have previously shown that wild type venous but not arterial EC were protected against heparan sulfate (HS) shedding after activation with human serum or human tumor necrosis factor alpha (TNF훼). Using a 2D microfluidic system we investigated the glycocalyx dynamics of genetically modified porcine arterial and venous EC (Gal훼1,3 Gal knock‐out, transgenic for human CD46 and thrombomodulin, GTKO/hCD46/hTM) after activation with human serum or human TNF훼. Interestingly, we observed that GTKO/hCD46/hTM arterial cells, additionally to venous cells, do not shed HS. Unscathed HS on GTKO/hCD46/hTM EC correlated with reduced complement deposition, suggesting that protection against complement activation contributes to maintaining an intact glycocalyx layer on arterial EC. This protection was lost on GTKO/hCD46/hTM cells after simultaneous perfusion with human serum and human TNF훼. HS shedding on arterial cells and increased complement deposition on both arterial and venous cells was observed. These findings suggest that GTKO/hCD46/hTM EC revert to a proinflammatory phenotype in an inflammatory xenotransplantation setting, potentially favoring transplant rejection. [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]