Xenotransplantation最新文献

Background: Significant progress has been made in the long-term survival of non-human primates after orthotopic gene-edited pig cardiac xenotransplantation. However, to our knowledge, there are no reports of the successful reversal of an acute rejection episode in such an experiment. We present evidence suggesting that rejection can be reversed with corticosteroids and complement inhibition.

Methods: Orthotopic transplantation of a pig heart (with 69 gene-edits) was carried out in a baboon. The immunosuppressive regimen was based on CD40/CD154 T cell co-stimulation pathway blockade and rapamycin. Cardiac function remained excellent until Day 162, when there were increases in heart rate, ventricular septal wall thickness, left ventricular end-diastolic pressures (LVEDP), and troponin level, which were associated with a low serum level of rapamycin (<4 ng/mL). Anti-rejection treatment was begun with an increase in rapamycin dosage, steroid bolus therapy, two doses of a C1-esterase inhibitor, and an extra dose of the anti-CD154mAb.

Results: There was a rapid correction of all hemodynamic parameters, and the troponin T level (which had risen to 139 ng/L) returned to pre-rejection levels. Ventricular septal thickness and LVEDP returned to pre-rejection levels after treatment. The baboon remains well with normal graft function. Baseline heart rate remains faster than before the rejection episode.

Conclusions: As we transition to the clinical application of gene-edited pig cardiac xenotransplantation, the ability to treat rejection is of vital importance. The optimal treatment for rejection remains uncertain but we suggest that systemic complement inhibition is important.

The molecular barriers that cause acute xenograft rejection have been identified and addressed by generating genetically modified (GM) animals, knocked out for specific xenoantigens (xenoAgs), and expressing regulatory molecules for both complement and coagulation pathways among others. The focus of xenotransplantation research now lies in delayed xenograft rejection. Dendritic cells (DC) are a specific subpopulation of professional antigen-presenting cells (APC) that play a crucial role in the context of organ transplantation. DCs, originating from both the xenograft and the recipient, have the capacity to present xenoAgs to the recipient's immune system via their respective major histocompatibility complex (MHC) molecules leading to rejection. These processes are known as direct and indirect presentation, respectively. However, under certain microenvironmental conditions, DC develops into anti-inflammatory regulatory cells that can induce immunological tolerance. The purpose of this review is to summarize current knowledge on the general characteristics and functions of DC from species relevant to xenotransplantation, specifically humans, non-human primates (NHP), and pigs. It will also cover the process of xenoAg presentation, different methods for generating DC with regulatory properties in vitro, and finally, discuss the current strategies for using regulatory DC to improve xenograft acceptance by inducing tolerance.

Xenotransplantation, the transplantation of organs from pigs to humans, presents significant challenges due to immune rejection, which is driven by molecular incompatibilities between species. This study investigates the compatibility between human thrombin and porcine protease-activated receptor-1 (PAR-1), a key regulator of both coagulation and inflammatory responses. Human thrombin activates PAR-1 in human vascular endothelial cells, but our results demonstrate that human thrombin does not effectively activate PAR-1 in porcine vascular endothelial cells due to differences in amino acid sequences, particularly at the thrombin cleavage site and the Hir domain. Protein-protein docking analysis further reveals that porcine PAR-1 forms less stable interactions with human thrombin compared to human PAR-1, resulting in reduced activation. This molecular incompatibility likely contributes to impaired nitric oxide (NO) production, endothelial dysfunction, and increased inflammation, which are critical for the survival of transplanted organs. Additionally, experiments using the PAR-1 inhibitor vorapaxar (Vor) show that inhibiting PAR-1 signaling can suppress inflammatory cytokine and chemokine expression in co-cultures of human macrophages and porcine endothelial cells. These findings suggest that selective PAR-1 inhibitors or targeted therapies regulating thrombin-PAR-1 signaling may improve the success rate of xenotransplantation. However, further in vivo studies are needed to validate these findings and explore therapeutic interventions targeting thrombin-PAR-1 interactions to enhance xenograft survival.

Renal transplantation is the optimal treatment for end-stage kidney disease, but the rising demand for organs necessitates exploring alternative graft sources, including xenotransplantation. This study evaluated attitudes toward xenotransplantation among different social groups in a Muslim country. A structured survey assessing demographics, religiosity levels, attitudes toward xenotransplantation, and the influence of religious authorities was completed by 988 participants: 376 clergy members (38.1%), 206 physicians (20.9%), 162 dialysis patients and their relatives (16.4%), and 244 individuals from other groups (24.7%). Significant sociodemographic differences were observed in gender, marital status, and education (p < 0.001). Physicians and dialysis patients/relatives were likelier to support living donor transplantation (48.5%, 67.3%), whereas clergy members often reported no opinion (44.9%). Favorable attitudes toward xenotransplantation were the highest among physicians (66%) and dialysis patients/relatives (67.3%). When the animal was specified as a pig, 73.1% of the Clergy group exhibited unfavorable attitudes. However, this group significantly changed their opinion after the favorable fatwa issued by the religious authority. Logistic regression identified male gender, lower religiosity, and higher education as predictors of favorable attitudes, with physicians and dialysis patients/relatives demonstrating the strongest associations compared to clergy. To infer, collaboration between religious and scientific authorities is essential to address concerns and emphasize the potential benefits of xenotransplantation.

Soft tissue repair patches of decellularized extracellular matrices (ECM) with inherently preserved structural components and biomacromolecules are desirable in regenerative applications. This study characterizes three detergent-based decellularization methods to fabricate acellular porcine liver matrices to remove antigenic determinants without compromising the structural integrity, glycosaminoglycans (GAG) content, and bound growth factors within the resulting ECM. Three detergents chosen for decellularization were sodium dodecyl sulfate (SDS), SDS with sodium deoxycholate (SDS+SDC-combinatorial method), and triton X-100 followed by SDS. Combinatorial detergent decellularization effectively removed cellular components and retained intact collagenous structure with minimal residual DNA and protein. It also preserved significantly higher amounts of GAG, HGF, and bFGF. TX100 decellularization was highly destructive with the least preservation of GAG and GFs. The SDS method showed an intermediate level of preservation of biomolecules. The correlation obtained between GAG and GFs revealed quantification of GAG to be an indirect way of estimating the bound GFs preserved within the ECM. In vitro experiments revealed the non-cytotoxic nature of the scaffolds. The study revealed that, among the three methods of decellularization, the ECM scaffold fabricated by combinatorial detergent decellularization is extremely promising to be used as a soft tissue repair patch with inherent bioactive molecules for scaffold-based regenerative therapies.

Clinical xenotransplantation has the potential to address shortages of human organs for patients with end-stage organ failure. Advances in genetic engineering, immunosuppressive regimens, and infectious disease diagnostics have improved prospects for clinical xenotransplantation. Management of the infectious risks posed by clinical xenotransplantation requires biosecure breeding and validated methods for microbiological surveillance of source animals and recipients. Novel infection control protocols may complement biosafety requirements. Infectious risks in xenotransplantation include both known human pathogens common to immunosuppressed organ recipients and from porcine organisms or xenozoonoses for which the clinical manifestations are less well defined and for which microbial assays and therapies are more limited. Some pig-specific organisms do not infect human cells but have systemic manifestations when active within the xenograft. The human risk posed by porcine endogenous retroviruses (PERV) is uncertain. There are no documented transmissions of PERV in humans and swine are available with inactivated genomic PERV loci. Metagenomic sequencing will complement more traditional diagnostic tools in the detection of any unknown pathogens in xenotransplantation recipients. Such data are required for the development of protocols for donor and recipient microbiological surveillance, infection control, and antimicrobial therapies that will enhance the safety of clinical xenotransplantation.

Introduction: "Delayed" antibody-mediated xenograft rejection is one of the most important obstacles to clinical application of pig organ xenografts. The aim of this study was to assess the impact of a structured desensitization regimen including proteasome inhibition and next-generation costimulation blockade on xenoreactive antibodies.

Methods: Rhesus macaques with moderate-high pre-treatment xenoreactive antibody titers (N = 2) were selected. Recipients received twice-weekly carfilzomib (20 mg/m²), anti-CD154 (20 mg/kg) every other week, and CD4 and CD20 lymphocyte cell depletion. Bone marrow was acquired to assess plasma cell depletion in response to proteasome inhibition. A flow cytometry-based xenoreactive crossmatch assay was performed to assess levels of circulating xenoreactive antibodies.

Results: The desensitization regimen resulted in a >50% depletion of CD38+CD27+ bone marrow plasma cells; these changes were progressive over the duration of the desensitization treatment period. The desensitization strategy and plasma cell depletion resulted in a progressive reduction in anti-pig IgG antibodies. Following xenotransplantation, both desensitized recipients demonstrated superior graft survival to a highly xenoreactive recipient (MST 30 days vs. 6 days), but neither desensitized recipient experienced prolonged graft survival.

Conclusions: A structured desensitization regimen including proteasome inhibition and costimulation blockade results in plasma cell depletion and resultant reduction in circulating xenoreactive anti-pig IgG antibodies, with a modest improvement in xenograft survival. This desensitization regimen has promise for pig-to-NHP xenotransplant models.

Porcine kidney xenotransplantation for end-stage renal disease (ESRD) has reached the stage of clinical testing following major advances in donor pig genetic modifications and effective immunosuppressive strategies through decades of rigorous translational research. Reports of pig kidney xenograft survival beyond 1 year posttranplant in nonhuman primate (NHP) models justify optimism for its potential as an alternative to allotransplantation. In the United States, experimental transplantations of genetically engineered (GE) porcine kidneys into brain-dead subjects and a small number of ESRD patients have shown no evidence of hyperacute rejection and adequate pig kidney function for up to several months. Here we discuss pre-clinical/clinical results, infectious disease, ethical, and regulatory considerations, and propose evidence-based recommendations. For initial clinical trials in kidney xenotransplantation, we make the following recommendations: (i) transplantation with organs from a triple knockout (TKO) donor pig, preferably with added human transgenes, (ii) an immunosuppressive regimen with induction therapy to deplete T (and possibly B) cells, and maintenance therapy based on a cluster of differentiation (CD)40/CD154 co-stimulation pathway blockade, (iii) the patient should be fully acceptable as a candidate for allotransplantation but should be unlikely ever to receive an allograft. Patients aged 60-69 years (extendable to 40-75 years, if one of the criteria mentioned below is present), of blood group B or O, and with diabetes are most at risk in this regard. Other patients who could be considered are (i) those who have lost two or more previous kidney allografts from recurrent disease in the graft, (ii) those with broad human leukocyte antigen (HLA)-reactivity but no evidence of anti-pig antibodies, including swine leukocyte antigen (SLA), and (iii) those with failing vascular access. Clinical pilot studies in carefully and highly selected patients with no alternative therapy will provide the foundation upon which to base subsequent formal expanded clinical trials.

Recent landmark clinical translation of xenotransplantation depended upon multiple innovations by the xenotransplant community, including the introduction of a variety of source pig genetic modifications, technical innovations, and novel immunosuppressive strategies, as well as the development of ethical and regulatory frameworks to support translation to the clinic. Each organ, tissue, or cell type intended for xenotransplantation will require application-specific preclinical milestones to be met in order to predict "success", as measured by ethical, safe, and efficacious translation to the clinic. Based on successful pre-clinical results and emerging evidence from decedent studies and initial clinical cases, evidence-based infectious disease, ethical, and regulatory considerations are emerging, and will be the foundations for the application-specific position papers that are currently under development. Here, we describe significant landmark events focusing upon safe and efficacious results underpinned by appropriate guidance documents developed over the past three decades that enabled recent translation to the clinic for heart and kidney xenografts. These steps have been undertaken over the past three decades by the xenotransplant community specifically led by the International Xenotransplantation Association (IXA) in consultation with the Transplantation Society (TTS) and the World Health Organization (WHO) to usher xenotransplantation to the clinic.

Porcine endogenous retroviruses (PERVs) are integrated into the genome of all pigs and can infect human cells in culture. However, no PERV infections have been reported in recipients following preclinical or clinical xenotransplantation or deliberate infection experiments. Detection of PERV infection in transplanted recipients is challenging due to microchimerism, such as the presence of pig cells containing PERV proviruses in the recipient. Based on our previous publications on PERV detection in xenotransplant recipients, particularly from the first clinical trials, we developed a comprehensive strategy to screen for PERV infections. Recipients can be monitored for increasing levels of viral genomic RNA and mRNA using real-time reverse transcriptase (RT)-PCR, which can indicate PERV expression and replication. To test this strategy, explanted pig hearts and organs from baboons after pig heart transplantation were analyzed. No PERV genomic RNA or mRNA was detected in these tissues, although both were found in PERV-producing human control cells. Screening for antibodies against PERV as indirect evidence of infection is the method of choice. Recombinant viral proteins were prepared for use in Western blot assays. Animal antisera generated through immunization with recombinant PERV proteins served as positive controls. No antibodies against PERV were detected in transplanted baboons, even though microchimerism was observed in many of the animals' organs. For effective antibody screening, at least two PERV proteins should be used as antigens.