Xenotransplantation最新文献

Advancements in xenotransplantation intersecting with modern machine perfusion technology offer promising solutions to patients with liver failure providing a valuable bridge to transplantation and extending graft viability beyond current limitations. Patients facing acute or acute chronic liver failure, post-hepatectomy liver failure, or fulminant hepatic failure often require urgent liver transplants which are severely limited by organ shortage, emphasizing the importance of effective bridging approaches. Machine perfusion is now increasingly used to test and use genetically engineered porcine livers in translational studies, addressing the limitations and costs of non-human primate models. Current reports about artificial and bioartificial liver support combined with xenografts showcase the potential in ex vivo xenogeneic perfusion. Breakthroughs, such as the perfusion of genetically modified porcine liver with FDA-approved machine perfusion systems connected to human blood circulation, underscore the interest and potential feasibility of a "liver dialysis" bridge to allotransplantation or recovery. This review provides an overview of the past and current research in the field of ex vivo pig liver xenoperfusion.

Background: Gene-edited pigs for xenotransplantation usually contain one or more transgenes encoding human complement regulatory proteins (CRPs). Because of species differences, human CRP(s) expressed in gene-edited pigs may have difficulty inhibiting the activation of exogenous rabbit complement added to a complement-dependent cytotoxicity (CDC) assay. The use of human complement instead of rabbit complement in CDC experiments may more accurately reflect the actual regulatory activity of human CRP(s).

Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from one GTKO pig and two GTKO/hCD55 pigs with a high or low level of hCD55 expression. After incubation of heat-inactivated normal human sera (HINHS) with porcine PBMCs, CDC levels were measured after the addition of commercial rabbit complement or human complement. In addition, a modified one-step CDC method was established using pooled normal human sera (NHS) without the addition of exogenous complement.

Results: There was no significant difference in the binding of IgM/IgG to PBMCs from the three pigs. Both rabbit and human complement mediated a significant cytotoxic effect on GTKO pig PBMCs (98.97% vs. 82.73%). Even the high expression of hCD55 only had a very limited inhibitory effect on rabbit complement-mediated cytotoxicity (81.70% vs. 98.97%). However, regardless of whether the expression level was high or low, hCD55 had a very remarkable inhibitory effect on human complement-mediated cytotoxicity (2.94% and 23.83% vs. 82.73%; p < 0.01). Similar results were obtained using the modified one-step CDC method. In addition, the inhibitory effect of hCD55 on C3c and C5b-9 deposition on pig PBMCs was positively correlated with the expression level of hCD55.

Conclusion: The use of human complement instead of rabbit complement in CDC assays can better reflect the actual cytotoxic effect of human xenoantibodies against pig PBMCs expressing human CRP(s), and thus may have potential application to gene-edited pig-to-human xenotransplantation.

Background: The removal of preformed antibodies with cleaving enzyme like IdeS (Imlifidase) has demonstrated therapeutic potential in organ transplantation for sensitized recipients. However, preformed xenoreactive antibodies (XAbs) against porcine glycans are predominantly IgM and considered detrimental in pig-to-human xenotransplantation.

Methods: Recombinant IceM, an endopeptidase cleaving IgM, was generated in Escherichia coli. Four maximally MHC-mismatched rhesus macaques underwent two serial skin transplantations to model allosensitized patients awaiting xenotransplantation. IceM was administered IV in allosensitized animals at 28 and 56 days after the first skin transplantation to assess in vivo IgM cleavage. Total IgG and IgM were quantified with western blot, and anti-pig (xenoreactive) IgG/IgM were evaluated using flowcrossmatch. B cell and its subpopulations were assessed using flow cytometry.

Results: IceM selectively cleaved human IgM, while showing no cleavage activity toward other isotypes including IgG, IgA, IgD, and IgE. Additionally, IceM cleaves only human and non-human primate IgM in vitro, but not in sera from other species. At a dose of 0.5 mg/kg, IceM reduced xenoreactive IgM levels to 13.76% ± 4.98% of baseline (B cell flow crossmatch) at 24 h post-administration, with baseline levels restored approximately 2 weeks after treatment. Additionally, animals showed similar kinetics of xenoreative IgM degradation with the repeated dose of IceM.

Conclusion: In this study, we report a recombinant bacterial enzyme that selectively cleaves IgM in human and non-human primate sera. Repeat administration of IceM in macaques enables selective, robust clearance of circulating xenoreactive IgM. This approach will be useful in treating preformed natural and rebound IgM in xenotransplantation.

Organ transplants are used to treat many end-stage diseases, but a shortage of donors means many patients cannot be treated. Xenogeneic organs have become an important part of filling the donor gap. Many current studies of kidney, heart, and liver xenotransplantation have used gene-edited pig organs on brain-dead recipients. However, the endocrine system, immune system, and nervous system of brain-dead people are changed, which are different from that of real patients transplanted, and the current research results of brain death (BD) recipients are also different. So there are drawbacks to using brain-dead people for xenotransplantation. In addition, although the policy requires the use of non-human primate (NHP) experiments as the research standard for xenotransplantation, there are still differences between NHP and humans in terms of immunity. Therefore, to better study xenotransplantation, new models may be needed in addition to NHP and brain-dead individuals. Humanized animal models or organoids may be able to fill this gap.

Introduction: There is no standard protocol for management of organ preservation for orthotopic, life-sustaining cardiac xenotransplantation, particularly for hearts from pediatric sized donors. Standard techniques and solutions successful in human allotransplantation are not viable. We theorized that a solution commonly used in reparative cardiac surgery in human children would suffice by exploiting the advantages inherent to xenotransplantation, namely the ability to reduce organ ischemic times by co-locating the donor and recipient.

Methods: Orthotopic cardiac xenotransplantation was performed from genetically engineered pigs to size matched baboons. A dose of modified Del Nido cardioplegia initiated donor heart arrest and was followed by a second dose mixed with recipient blood prior to implant. Hemodynamics and cardiac function were tracked with a combination of invasive and non-invasive measures.

Results: Mean ischemic time and cardiopulmonary bypass times were 54.1 ± 14.6 and 84.1 ± 14 min respectively. The ejection fraction following chest closure was preserved at >50% for all animals. This finding persisted at 48hours. Mean inotropic score at 24 h post-implant was 9.7 ± 3.

Conclusion: Del Nido cardioplegia solution when combined with short graft ischemic times demonstrates promising outcomes to avoid primary graft dysfunction for cardiac xenografts in a small animal model of life-sustaining orthotopic cardiac xenotransplantation. Ex vivo perfusion systems may be unnecessary for successful clinical implementation of this evolving technology.

Introduction: Although there is a plethora of literature on electrocardiographic changes following cardiac allotransplantation, there is little in the field of cardiac xenotransplantation. The only published literature to date is that of the first pig-to-human cardiac xenotransplantation. Here we take a close look at the electrocardiographic parameters in four non-human primate recipients of orthotopic cardiac xenotransplantation to develop baseline metrics for comparison.

Methods: Orthotopic cardiac xenotransplantion was carried out in four non-human primate recipients. Electrocardiographic parameters were followed at various intervals using an internal hemodynamic monitoring system (DSI) as well as a standard 12-lead electrocardiogram (ECG). ECG intervals were then compared to published literature on porcine ECG intervals and pig-to-human cardiac xenotransplantation.

Results: There were no significant differences observed between timepoints for HR, PR, QRS, QT, or QTc after cardiac xenotransplantation for each animal subject. ECG parameters were statistically similar to those of in situ mini-pig hearts in the literature. ECG parameters from the DSI on average were shorter than those from a traditional ECG, however, DSI parameters were consistent over time.

Conclusion: These results demonstrate the possibility of conduction health for genetically engineered porcine donor hearts following cardiac xenotransplantation. Ongoing work to compare the results of an ECG in a porcine donor heart before and after implantation into a NHP is necessary to better characterize variables that may be at play in the function of the conduction system.

Background: The Federal Ethics Committee on Non-Human Biotechnology (ECNH) of Switzerland is an independent expert committee appointed by the Federal Council and mandated to advise the federal authorities from an ethical perspective in the field of nonhuman biotechnology and gene technology. Due to recent developments in the field of xenotransplantation after the introduction of genome editing technologies, the ECNH has commissioned an expert report on the ethical questions of xenotransplantation with a focus on animal ethics. The subject of the inquiry is, in particular, if current developments in the field of xenotransplantation raise new questions regarding ethics in the nonhuman realm or if existing questions have to be re-examined and answered anew.

Methods: An interdisciplinary approach was applied to answer this question. Based on the latest empirical results from medicine and biotechnology, xenotransplantation is analyzed and evaluated with reference to the dignity of the creature (Würde der Kreatur)-which is defined in the Swiss Federal Constitution-and the dignity of animals (Tierwürde) that is stipulated in the Swiss Animal Welfare Act and the Federal Act on Non-Human Gene Technology, as well as contemporary positions in the ethics of the human-animal relationship.

Results: The report concludes that genome editing for xenotransplantation does not generate any qualitatively new ethical issues concerning ethics in the nonhuman realm. However, contemporary biotechnological developments must be taken as an opportunity to discuss existing ethical issues in an urgent and intensified manner, particularly regarding the significance of animals' moral standing. The lack of consideration of animal-related aspects and the neglect of current developments and the state of the art of animal ethics in the recent discussion about xenotransplantation is a scientific, ethical, and political issue because animals are most negatively affected by xenotransplantation. This is especially relevant because the contemporary state of the art in animal ethics tends to consider and protect animals more strongly than in the past.

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 (H_max). 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 H_max 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.

Conventional T cell-directed immunosuppression is the mainstay of standard-of-care therapy to prevent graft rejection in clinical organ transplantation. However, it remains ineffective in preventing experimental and clinical organ xenograft rejection. Here, we explored the impact of allogeneic versus xenogeneic antigen stimulation on human T cell responses and gene profile. A comparable proliferative human T cell response was observed in vitro following stimulation with either human or pig cells. Yet, elevated High mobility group box-1 (HMGB1) levels were following xenogeneic but not allogeneic stimulation, suggesting a pro-inflammatory response. Next, human peripheral blood mononuclear cells (PBMC) were cultured with allogeneic human, "concordant" xenogeneic monkey, or "discordant" xenogeneic pig, intact cells, or cell lysates. Flow-sorted CD3⁺T cells were analyzed for gene expression using NanoString. A distinct pro-inflammatory gene profile was observed in human CD3⁺T cells following co-culture with discordant xenogeneic pig cells, but not concordant xenogeneic monkey cells or allogeneic human cells. Uniquely, stimulation with pig cells induced the expression of the transcription factor NCF4, which promotes inflammasome activation. Pig cell lysate, but not intact pig cells, induced high expression of the DNA-binding cytokine interleukin-26 gene. Collectively, these observations highlight the impact of xenogeneic stimulation of human T cells in pig xenograft recipients and concomitant inflammatory responses, which may contribute to immunosuppression-resistant xenograft rejection. Finally, the impact of genetic engineering of donor pigs on human T cell transcriptomic gene profile is yet to be determined.