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

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

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

Glycocalyx dynamics and the inflammatory response of genetically modified porcine endothelial cells.

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 (TN

Impact of porcine cytomegalovirus on long-term orthotopic cardiac xenotransplant survival

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 TNF alpha 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 clinical and genetic spectrum of autosomal-recessive TOR1A-related disorders.

In the field of rare diseases, progress in molecular diagnostics led to the recognition that variants linked to autosomal-dominant neurodegenerative diseases of later onset can, in the context of biallelic inheritance, cause devastating neurodevelopmental disorders and infantile or childhood-onset neurodegeneration. TOR1A-associated arthrogryposis multiplex congenita 5 (AMC5) is a rare neurodevelopmental disorder arising from biallelic variants in TOR1A, a gene that in the heterozygous state is associated to torsion dystonia-1 (DYT1 or DYT-TOR1A), an early-onset dystonia with reduced penetrance. While 15 individuals with TOR1A-AMC5 have been reported (less than 10 in detail), a systematic investigation of the full disease-associated spectrum has not been conducted. Here, we assess the clinical, radiological and molecular characteristics of 57 individuals from 40 families with biallelic variants in TOR1A. Median age at last follow-up was 3 years (0-24 years). Most individuals presented with severe congenital flexion contractures (95%) and variable developmental delay (79%). Motor symptoms were reported in 79% and included lower limb spasticity and pyramidal signs, as well as gait disturbances. Facial dysmorphism was an integral part of the phenotype, with key features being a broad/full nasal tip, narrowing of the forehead and full cheeks. Analysis of disease-associated manifestations delineated a phenotypic spectrum ranging from normal cognition and mild gait disturbance to congenital arthrogryposis, global developmental delay, intellectual disability, absent speech and inability to walk. In a subset, the presentation was consistent with fetal akinesia deformation sequence with severe intrauterine abnormalities. Survival was 71% with higher mortality in males. Death occurred at a median age of 1.2 months (1 week - 9 years) due to respiratory failure, cardiac arrest, or sepsis. Analysis of brain MRI studies identified non-specific neuroimaging features, including a hypoplastic corpus callosum (72%), foci of signal abnormality in the subcortical and periventricular white matter (55%), diffuse white matter volume loss (45%), mega cisterna magna (36%) and arachnoid cysts (27%). The molecular spectrum included 22 distinct variants, defining a mutational hotspot in the C-terminal domain of the Torsin-1A protein. Genotype-phenotype analysis revealed an association of missense variants in the 3-helix bundle domain to an attenuated phenotype, while missense variants near the Walker A/B motif as well as biallelic truncating variants were linked to early death. In summary, this systematic cross-sectional analysis of a large cohort of individuals with biallelic TOR1A variants across a wide age-range delineates the clinical and genetic spectrum of TOR1A-related autosomal-recessive disease and highlights potential predictors for disease severity and survival

Active Replication of Porcine Cytomegalovirus (PCMV) Following Transplantation of a Pig Heart into a Baboon despite Undetected Virus in the Donor Pig

Viral zoonoses may represent a serious problem when xenotransplantation using pig cells, tissues, and organs will be performed. Recently we reported multiorgan failure, mainly liver failure, in a baboon (Papio anubis) after orthotopic transplantation of a heart from a genetically modified pig and showed that the baboon was not infected with hepatitis E virus (HEV). Since pigs are frequently infected with porcine cytomegalovirus (PCMV), and the infection is latent and therefore difficult to detect, screening for PCMV was performed, using a highly sensitive real-time PCR and a nested PCR. In addition, to confirm the absence of HEV, more specific immunological methods and a real-time PCR were used. Only two out of six available pigs, including the selected donor animal, were found negative for both viruses. Nevertheless, PCMV was detected in the blood of the baboon. The titre of PCMV in the blood was above 3x104/ ml and analysis of its sequence confirmed that it was PCMV, but not baboon CMV. The data highlight a considerable risk of PCMV infection and call for regular screening of donor pigs applying the most sensitive detection methods

Myofibrillar myopathy: a rare but important differential diagnosis of camptocormia in a patient with Parkinson’s Disease

Abstract Here we report on a patient with Parkinson's Disease and camptocormia due to Myofibrillar Myopathy Type 3. By leading the reader through the clinical reasoning process and highlighting the respective red flags we aim to increase the readers’ awareness for the differential diagnosis of camptocormia