Untersuchungen zur perioperativen kardialen Xenograft-Dysfunktion im ex-vivo Modell sowie nach heterotop thorakaler und orthotoper Xenotransplantation

Aufgrund des bestehenden Spenderorganmangels gewinnt das Forschungsfeld der Xenotransplantation zunehmend an Bedeutung. Eine von mehreren Hürden für die orthotope kardiale Xenotransplantation ist eine primäre Insuffizienz des Spenderorgans, die nicht in direktem Zusammenhang mit einer antikörpervermittelten hyperakuten Abstoßung steht. Diese wird als perioperative kardiale Xenograft-Dysfunktion (PCXD) bezeichnet und ist aus herzchirurgischer aber insbesondere auch anästhesiologischer und intensivmedizinischer Sicht von Bedeutung. Als Modell für die initiale Phase nach xenogener kardialer Transplantation wurde ein ex-vivo Perfusionssystem entwickelt, mit dem Konzepte zur Therapie der PCXD untersucht wurden: Mittels Inhibition des Komplements C3 konnten der Myokardschaden reduziert und die Herzfunktion während xenogener Perfusion verbessert werden. Durch die Kombination der genetischen Modifikationen GGTA1-KO, hCD46 und HLA-E des Spenderorgans reduzierte sich die initiale xenogene Reaktion, insbesondere jene der NK-Zellen. Präklinische Untersuchungen wurden in zwei Großtiermodellen durchgeführt: Im heterotop thorakalen Transplantationsmodell trat eine PCXD ebenfalls auf. In keinem Fall führte diese jedoch zum Abbruch des Versuchs, da das Empfängerherz das Transplant hämodynamisch unterstützen konnte. Es gelang der Nachweis, dass die primäre Dysfunktion des Grafts bei diesen Versuchen vollständig reversibel war. Im orthotopen Transplantationsmodell war die PCXD maßgeblich für Versuchsabbrüche innerhalb der ersten 48 Stunden verantwortlich. Das Auftreten dieser war jedoch abhängig von der kardioplegischen Lösung, die zur Präservation der Herzen während der Transplantation verwendet wurde. Ein Zusammenhang mit einer viralen Infektion des Spenderherzens, insbesondere durch das Hepatitis E Virus, konnte nicht nachgewiesen werden. Aus den durch diese Arbeit gewonnenen Erkenntnissen können Strategien zur Verbesserung der primären Graftfunktion abgeleitet werden, die ein Langzeitüberleben auch im orthotopen Transplantationsmodell möglich erscheinen lassen

Porcine Lymphotropic Herpesvirus (PLHV) Was Not Transmitted During Transplantation of Genetically Modified Pig Hearts into Baboons

Porcine lymphotropic herpesviruses -1, -2, and -3 (PLHV-1, PLHV-2, and PLHV-3) are gammaherpesviruses that are widespread in pigs. These viruses are closely related to the human pathogens Epstein–Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV), both of which are known to cause severe diseases in humans. To date, however, no definitive association has been established between PLHVs and any disease in pigs. With the growing interest in xenotransplantation as a means to address the shortage of human organs for transplantation, the safety of using pig-derived cells, tissues, and organs is under intense investigation. In preclinical trials involving pig-to-nonhuman primate xenotransplantation, another porcine herpesvirus—porcine cytomegalovirus, a porcine roseolovirus (PCMV/PRV)—was shown to be transmissible and significantly reduced the survival time of the xenotransplants. In the present study, we examined donor pigs and their respective baboon recipients, all of which were part of preclinical pig heart xenotransplantation studies, for the presence of PLHV. PLHV-1, PLHV-2, and PLHV-3 were detected in nearly all donor pigs; however, no evidence of PLHV transmission to the baboon recipients was observed

The Coronary Microcirculation in Hamster-to-Rat Cardiac Xenografts

BACKGROUND The aim of this study was to establish a new experimental model to directly analyse the coronary microcirculation in cardiac xenografts. METHODS Intravital fluorescence microscopy (IVM) of the subepicardial microcirculation in heterotopically transplanted hamster-to-rat cardiac xenografts was performed at 30 and 90 min of reperfusion. We quantitatively assessed the microcirculatory perfusion characteristics as well as the interactions of leukocytes and platelets with the endothelium of postcapillary coronary venules in non-sensitised as well as sensitised recipients. RESULTS In this first experimental IVM study of cardiac xenografts, we successfully visualised the subepicardial microcirculation, i.e. feeding arterioles, nutritive capillaries and draining postcapillary venules, during reperfusion. Leukocyte-endothelial and platelet-endothelial cell interactions could be quantified. In the non-sensitised group, the myocardial microcirculation remained stable during the observation period of 90 min, whereas in the sensitised group, xenografts were rejected immediately. CONCLUSIONS We established a model for the assessment of the microcirculatory dysfunction and inflammation during ischaemia/reperfusion injury in hamster-to-rat cardiac xenografts

Evidence for Microchimerism in Baboon Recipients of Pig Hearts

Xenotransplantation, like allotransplantation, is usually associated with microchimerism, i.e., the presence of cells from the donor in the recipient. Microchimerism was reported in first xenotransplantation trials in humans, as well as in most preclinical trials in nonhuman primates (for review, see Denner, Viruses 2023, 15, 190). When using pigs as xenotransplantation donors, their cells contain porcine endogenous retroviruses (PERVs) in their genome. This makes it difficult to discriminate between microchimerism and PERV infection of the recipient. Here, we demonstrate the appropriate virological methods to be used for the identification of microchimerism, first by screening for porcine cellular genes, and then how to detect infection of the host. Using porcine short interspersed nuclear sequences (SINEs), which have hundreds of thousands of copies in the pig genome, significantly increased the sensitivity of the screening for pig cells. Second, absence of PERV RNA demonstrated an absence of viral genomic RNA or expression as mRNA. Lastly, absence of antibodies against PERV proteins conclusively demonstrated an absence of a PERV infection. When applying these methods for analyzing baboons after pig heart transplantation, microchimerism could be demonstrated and infection excluded in all animals. These methods can be used in future clinical trials

Transthoracic echocardiography is a simple tool for size matching in cardiac xenotransplantation

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

Current Status of Cardiac Xenotransplantation: Report of a Workshop of the German Heart Transplant Centers, Martinsried, March 3, 2023

This report comprises the contents of the presentations and following discussions of a workshop of the German Heart Transplant Centers in Martinsried, Germany on cardiac xenotransplantation. The production and current availability of genetically modified donor pigs, preservation techniques during organ harvesting, and immunosuppressive regimens in the recipient are described. Selection criteria for suitable patients and possible solutions to the problem of overgrowth of the xenotransplant are discussed. Obviously microbiological safety for the recipient and close contacts is essential, and ethical considerations to gain public acceptance for clinical applications are addressed. The first clinical trial will be regulated and supervised by the Paul-Ehrlich-Institute as the National Competent Authority for Germany, and the German Heart Transplant Centers agreed to cooperatively select the first patients for cardiac xenotransplantation

Hemodynamic evaluation of anesthetized baboons and piglets by transpulmonary thermodilution: Normal values and interspecies differences with respect to xenotransplantation

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/m(2) vs 5.3 +/- 1.0/min/m(2), 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/cm(5)*m(2) vs 827 +/- 204 dyn*s/cm(5)*m(2), P < .01). GEDI and ELWI did differ significantly between both species, but measurements were within similar ranges (GEDI: 523 +/- 103 mL/m(2) vs 433 +/- 78 mL/m(2), 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

AChR deficiency due to ε-subunit mutations: two common mutations in the Netherlands

Congenital myasthenic syndromes are a clinically and genetically heterogeneous group of hereditary disorders affecting neuromuscular transmission. We have identified mutations within the acetylcholine receptor (AChR) ε-subunit gene underlying congenital myasthenic syndromes in nine patients (seven kinships) of Dutch origin. Previously reported mutations ε1369delG and εR311Q were found to be common; ε1369delG was present on at least one allele in seven of the nine patients, and εR311Q in six. Phenotypes ranged from relatively mild ptosis and external ophthalmoplegia to generalized myasthenia. The common occurrence of εR311Q and ε1369delG suggests a possible founder for each of these mutations originating in North Western Europe, possibly in Holland. Knowledge of the ethnic or geographic origin within Europe of AChR deficiency patients can help in targeting genetic screening and it may be possible to provide a rapid genetic diagnosis for patients of Dutch origin by screening first for εR311Q and ε1369delG

Ultrasound Assessment of Pleural Effusions After Orthotopic Pig‐to‐Baboon Cardiac Xenotransplantation

Background: Pleural effusions develop frequently after cardiac surgery in humans. Lung ultrasound is an essential non-invasive tool in the diagnosis and treatment of these effusions. Pleural effusions also develop regularly after preclinical cardiac xenotransplantation experiments. Unlike in the human setting, modern ultrasound devices lack pre-installed tools for calculating the volume of pleural effusions in baboons. The aim of this study was to analyze ultrasound examinations of pleural effusions after orthotopic pig-to-baboon cardiac xenotransplantation experiments in order to develop a formula for calculating the effusion volume based on ultrasound measurements. Methods: Hearts from seven genetically modified (GGTA1-KO, hCD46/hTBM transgenic) juvenile pigs were orthotopically transplanted into male baboons. Postoperatively, the baboons were tested regularly for the development of pleural effusions using ultrasound. When thoracocentesis was required, the drained effusion volume (EV) was compared to ultrasound-derived calculations using various formulas. These calculations were based on measuring the distance between lung and diaphragm at the effusions’ maximum height (Hmax). Subsequently, the most promising formula was used to describe the interobserver variability between trained and untrained staff members to predict effusion volumes based on ultrasound measurements. Results: Ultrasound measurement correlated very strongly with the absolute EV (r = 0.9156, p < 0.0001), with EV indexed to total body weight (r = 0.9344, p < 0.0001) and with EV indexed to body surface area (BSA) (r = 0.9394, p < 0.0001). The ratio between Hmax and EV increased with total body weight and BSA and also depended on the baboon species. The sonographic measurements taken by an experienced and an inexperienced observer showed only low interobserver variability. A Bland–Altman plot of both observers’ measurements showed an overall bias of –2.39%. Conclusion: Ultrasound imaging provides a simple and non-invasive tool for measuring pleural effusion quantity in baboons. This facilitates simple and efficient monitoring even in the hands of untrained personnel and may guide the decision-making to perform thoracocentesis