Results of Two Cases of Pig-to-Human Kidney Xenotransplantation.

BACKGROUND: Xenografts from genetically modified pigs have become one of the most promising solutions to the dearth of human organs available for transplantation. The challenge in this model has been hyperacute rejection. To avoid this, pigs have been bred with a knockout of the alpha-1,3-galactosyltransferase gene and with subcapsular autologous thymic tissue. METHODS: We transplanted kidneys from these genetically modified pigs into two brain-dead human recipients whose circulatory and respiratory activity was maintained on ventilators for the duration of the study. We performed serial biopsies and monitored the urine output and kinetic estimated glomerular filtration rate (eGFR) to assess renal function and xenograft rejection. RESULTS: The xenograft in both recipients began to make urine within moments after reperfusion. Over the 54-hour study, the kinetic eGFR increased from 23 ml per minute per 1.73 m2 of body-surface area before transplantation to 62 ml per minute per 1.73 m2 after transplantation in Recipient 1 and from 55 to 109 ml per minute per 1.73 m2 in Recipient 2. In both recipients, the creatinine level, which had been at a steady state, decreased after implantation of the xenograft, from 1.97 to 0.82 mg per deciliter in Recipient 1 and from 1.10 to 0.57 mg per deciliter in Recipient 2. The transplanted kidneys remained pink and well-perfused, continuing to make urine throughout the study. Biopsies that were performed at 6, 24, 48, and 54 hours revealed no signs of hyperacute or antibody-mediated rejection. Hourly urine output with the xenograft was more than double the output with the native kidneys. CONCLUSIONS: Genetically modified kidney xenografts from pigs remained viable and functioning in brain-dead human recipients for 54 hours, without signs of hyperacute rejection. (Funded by Lung Biotechnology.).

Infectious Diseases and Clinical Xenotransplantation.

Xenotransplantation, transplantation into humans of vascularized organs or viable cells from nonhuman species, is a potential solution to shortages of transplantable human organs. Among challenges to application of clinical xenotransplantation are unknown risks of transmission of animal microbes to immunosuppressed recipients or the community. Experience in allotransplantation and in preclinical models suggests that viral infections are the greatest concern. Worldwide, the distribution of swine pathogens is heterogeneous and cannot be fully controlled by international agricultural regulations. It is possible to screen source animals for potential human pathogens before procuring organs in a manner not possible within the time available for surveillance testing in allotransplantation. Infection control measures require microbiological assays for surveillance of source animals and xenograft recipients and research into zoonotic potential of porcine organisms. Available data suggest that infectious risks of xenotransplantation are manageable and that clinical trials can advance with appropriate protocols for microbiological monitoring of source animals and recipients.

Porcine endogenous retroviruses in xenotransplantation.

Xenotransplantation using pig cells, tissues or organs is under development to alleviate the shortage of human donor organs. Meanwhile, remarkably long survival times of pig organs in non-human primates have been reported, as well as the functionality of pig kidneys and hearts in brain-dead humans. Most importantly, two transplantations of pig hearts in patients were performed with survival times of the patients of 8 and 6 weeks. Xenotransplantation may be associated with the transmission of porcine microorganisms including viruses to the recipient. Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs and cannot be eliminated like other viruses can. PERVs are able to infect certain human cells and therefore pose a risk for xenotransplantation. It is well known that retroviruses are able to induce tumors and immunodeficiencies. However, until now, PERVs were not transmitted in all infection experiments using small animals and non-human primates, in all preclinical xenotransplantation trials in non-human primates and in all clinical trials in humans. In addition, several strategies including antiretrovirals, PERV-specific small interfering RNA, vaccines and genome editing using CRISPR/Cas have been developed to prevent PERV transmission.

Close contacts of xenograft recipients: Ethical considerations due to risk of xenozoonosis.

With decades of pre-clinical studies culminating in the recent clinical application of xenotransplantation, it would appear timely to provide recommendations for operationalizing oversight of xenotransplantation clinical trials. Ethical issues with clinical xenotransplantation have been described for decades, largely centering on animal welfare, the risks posed to the recipient, and public health risks posed by potential spread of xenozoonosis. Much less attention has been given to considerations relating to potentially elevated risks faced by those who may care for or otherwise have close contact with xenograft recipients. This paper examines the ethical and logistical issues raised by the potential exposure to xenozoonotic disease faced by close contacts of xenotransplant recipients-defined herein as including but not limited to caregivers, household contacts, and sexual partners-which warrants special attention given their increased risk of exposure to infection compared to the general public. We discuss implications of assent or consent by these close contacts to potentially undergo, along with the recipient, procedures for infection screening and possible quarantine. We then propose several options and recommendations for operationalizing oversight of xenotransplantation clinical trials that could account for and address close contacts' education on and agency regarding the risk of xenozoonosis.

Novel factors potentially initiating acute antibody-mediated rejection in pig kidney xenografts despite an efficient immunosuppressive regimen.

Antibody-mediated rejection (AMR) is a common cause of graft failure after pig-to-nonhuman primate organ transplantation, even when the graft is from a pig with multiple genetic modifications. The specific factors that initiate AMR are often uncertain. We report two cases of pig kidney transplantation into immunosuppressed baboons in which we identify novel factors associated with the initiation of AMR. In the first, membranous nephropathy was the initiating factor that was then associated with the apparent loss of the therapeutic anti-CD154 monoclonal antibody in the urine when severe proteinuria was present. This observation suggests that proteinuria may be associated with the loss of any therapeutic monoclonal antibody, for example, anti-CD154 or eculizumab, in the urine, resulting in xenograft rejection. In the second case, the sequence of events and histopathology tentatively suggested that pyelonephritis may have initiated acute-onset AMR. The association of a urinary infection with graft rejection has been well-documented in ABO-incompatible kidney allotransplantation based on the expression of an antigen on the invading microorganism shared with the kidney graft, generating an immune response to the graft. To our knowledge, these potential initiating factors of AMR in pig xenografts have not been highlighted previously.

Monitoring for PERV Following Xenotransplantation.

Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs. PERV-A, PERV-B and PERV-C can be released as infectious virus particles and PERV-A and PERV-B can infect human cells in culture. PERV-C does not infect human cells, but high-titer recombinant PERV-A/C can infect them. Retroviruses are able to induce immunosuppression and/or tumors in the infected host. Numerous methods have been developed to study PERV in donor pigs. No PERV infections were observed in infection experiments as well as in preclinical and clinical xenotransplantation trials. Despite this, several strategies have been developed to prevent PERV infection of the recipient. PCR-based and immunological methods are required to screen xenotransplant recipients. Since the proviruses are integrated into the pig genome, PERV infection has to be distinguished from microchimerism, e.g., the presence of pig cells in the recipient, which is common in xenotransplantation. Sensitive PCR methods using pig short interspersed nuclear elements (SINE) sequences allow to detect pig cells easily. Virus infection can also be detected by an increase of viral genomic or mRNA in human cells. The method of choice, however, is to screen for specific antibodies against PERV using different recombinant PERV proteins, purified viruses or peptides.

Heart of the matter-infection and xenotransplantation.

In this clinicopathological conference, invited experts discussed a previously published case of a patient with nonischemic cardiomyopathy who underwent heart transplantation from a genetically modified pig source animal. His complex course included detection of porcine cytomegalovirus by plasma microbial cell-free DNA and eventual xenograft failure. The objectives of the session included discussion of selection of immunosuppressive regimens and prophylactic antimicrobials for human xenograft recipients, description of infectious disease risk assessment and mitigation in potential xenograft donors and understanding of screening and therapeutic strategies for potential xenograft-related infections.

Porcine endogenous retrovirus: classification, molecular structure, regulation, function, and potential risk in xenotransplantation.

Xenotransplantation with porcine organs has been recognized as a promising solution to alleviate the shortage of organs for human transplantation. Porcine endogenous retrovirus (PERV), whose proviral DNAs are integrated in the genome of all pig breeds, is a main microbiological risk for xenotransplantation. Over the last decades, some advances on PERVs' studies have been achieved. Here, we reviewed the current progress of PERVs including the classification, molecular structure, regulation, function in immune system, and potential risk in xenotransplantation. We also discussed the problem of insufficient study on PERVs as well as the questions need to be answered in the future work.

The prevention strategies of swine viruses related to xenotransplantation.

Xenotransplantation is considered a solution for the shortage of organs, and pigs play an indispensable role as donors in xenotransplantation. The biosecurity of pigs, especially the zoonotic viruses carried by pigs, has attracted attention. This review introduces several viruses, including porcine endogenous retroviruses that are integrated into the pig genome in a DNA form, herpesviruses that have been proven to clearly affect recipient survival time in previous xenotransplant surgeries, the zoonotic hepatitis E virus, and the widely distributed porcine circoviruses. The detail virus information, such as structure, caused diseases, transmission pathways, and epidemiology was introduced in the current review. Diagnostic and control measures for these viruses, including detection sites and methods, vaccines, RNA interference, antiviral pigs, farm biosecurity, and drugs, are discussed. The challenges faced, including those posed by other viruses and newly emerged viruses, and the challenges brought by the modes of transmission of the viruses are also summarized.

Mechanisms and strategies to promote cardiac xenotransplantation.

End stage heart failure is a terminal disease, and the only curative therapy is orthotopic heart transplantation. Due to limited organ availability, alternative strategies have received intense interest for treatment of patients with advanced heart failure. Recent studies using gene-edited porcine organs suggest that cardiac xenotransplantation may provide a future source of organs. In this review, we highlight the historical milestones for cardiac xenotransplantation and the gene editing strategies designed to overcome immunological barriers, which have culminated in a recent cardiac pig-to-human xenotransplant. We also discuss recent results of studies on the engineering of human-porcine chimeric organs that may provide an alternative and complementary strategy to overcome some of the major immunological barriers to producing a new source of transplantable organs.

Measuring success in pig to non-human-primate renal xenotransplantation: Systematic review and comparative outcomes analysis of 1051 life-sustaining NHP renal allo- and xeno-transplants.

Facile gene editing has accelerated progress in pig to non-human-primate (NHP) renal xenotransplantation, however, outcomes are considered inferior to NHP-allotransplantation. This systematic review and outcomes analysis of life-sustaining NHP-renal transplantation aimed to benchmark "preclinical success" and aggregated 1051 NHP-to-NHP or pig-to-NHP transplants across 88 articles. Although protocols varied, NHP-allotransplantation survival (1, 3, 12months, 67.5%, 37.1%, 13.2%) was significantly greater than NHP-xenotransplantation (1, 3, 12 months, 38.8%, 14.0%, 4.4%; p < .001); a difference partially mitigated by gene-edited donors containing at least knockout of alpha-1,3-galactosyltransferase (1, 3, 12 months, 47.1%, 24.2%, 7.6%; p < .001). Pathological analysis demonstrated more cellular rejection in allotransplantation (62.8% vs. 3.1%, p < .001) and more antibody-mediated rejection in xenotransplantation (6.8% vs. 45.5%, p < .001). Nonrejection causes of graft loss between allotransplants and xenotransplants differed; infection and animal welfare (1.7% vs. 11.2% and 3.9% vs. 17.0%, respectively, p < .001 for both). Importantly, even among a subgroup of unsensitized rhesus macaques under long-term immunosuppression, NHP-allotransplant survival was significantly inferior to clinical allotransplantation (6 months, 36.1% vs. 94.0%; p < .001), which suggests clinical outcomes with renal xenografts may be better than predicted by current preclinical data.

What is the clinical relevance of deviant serum calcium and phosphate levels after pig-to-primate kidney xenotransplantation?

Experience from human renal allotransplantation informs us that disturbances in serum calcium and phosphate levels are relatively common. Post-transplant hypercalcemia is associated with an increased risk of recipient mortality, but not of graft loss or nephropathy, and post-transplant hyperphosphatemia with an increased risk of both recipient mortality and death-censored graft failure, but neither post-transplant hypocalcemia nor hypophosphatemia is associated with adverse outcome. Studies after pig-to-nonhuman primate kidney xenotransplantation have demonstrated consistent supranormal serum calcium and subnormal serum phosphate levels. If these trends in serum electrolyte levels were to occur following pig-to-human kidney xenotransplantation, the data from allotransplant studies would indicate an increased risk of recipient mortality (associated with hypercalcemia) but not of graft loss or nephropathy, and no adverse outcome from hypophosphatemia. Furthermore, some nonhuman primates are now surviving in a healthy state for longer than a year after life-supporting pig kidney transplantation, suggesting that chronic hypercalcemia and/or hypophosphatemia are not detrimental to long-term survival, and should not prevent clinical trials of pig kidney transplantation from being undertaken.

Moving xenotransplantation from bench to bedside: Managing infectious risk.

Xenotransplantation of organs from swine in immunosuppressed human recipients poses many of the same challenges of allotransplantation relative to the risk for infection, malignancy, or graft rejection in proportion to the degree of immunosuppression and epidemiologic exposures. The unique features of xenotransplantation from pigs relative to infectious risk center on the potential for unusual organisms derived from swine causing productive infection, "xenosis" or "xenozoonosis," in the host. Based on experience in allotransplantation, the greatest hazard is due to viruses, due to the relative lack of information regarding the behavior of these potential pathogens in humans, the absence of validated serologic and molecular assays for swine-derived pathogens, and uncertainty regarding the efficacy of therapeutic agents for these organisms. Other known, potential pathogens (i.e., bacteria, fungi, parasites) tend to be comparable to those of humans. Concerns remain for unknown organisms in swine that may replicate in immunosuppressed humans. Clinical trials of genetically modified organs sourced from swine in immunosuppressed humans with organ failure are under development. Such trials require informed consent regarding potential infectious risks to the recipient, determination of breeding characteristics of swine, assessments of potential risks to the public and healthcare providers, consideration of ethical issues posed by this novel therapy, and defined strategies to monitor and address infectious episodes that may be encountered by healthcare teams. Clinical trials in xenotransplantation will allow improved definition of potential infectious risks.

Early T Cell Activation Metrics Predict Graft-versus-Host Disease in a Humanized Mouse Model of Hematopoietic Stem Cell Transplantation.

Acute graft-versus-host disease (GVHD) is a frequent complication of hematopoietic transplantation, yet patient risk stratification remains difficult, and prognostic biomarkers to guide early clinical interventions are lacking. We developed an approach to evaluate the potential of human T cells from hematopoietic grafts to produce GVHD. Nonconditioned NBSGW mice transplanted with titrated doses of human bone marrow developed GVHD that was characterized by widespread lymphocyte infiltration and organ pathology. Interestingly, GVHD was not an inevitable outcome in our system and was influenced by transplant dose, inflammatory status of the host, and type of graft. Mice that went on to develop GVHD showed signs of rapid proliferation in the human T cell population during the first 1-3 wk posttransplant and had elevated human IFN-γ in plasma that correlated negatively with the expansion of the human hematopoietic compartment. Furthermore, these early T cell activation metrics were predictive of GVHD onset 3-6 wk before phenotypic pathology. These results reveal an early window of susceptibility for pathological T cell activation following hematopoietic transplantation that is not simply determined by transient inflammation resulting from conditioning-associated damage and show that T cell parameters during this window can serve as prognostic biomarkers for risk of later GVHD development.

Initial experimental experience of triple-knockout pig red blood cells as potential sources for transfusion in alloimmunized patients with sickle cell disease.

BACKGROUND: Blood transfusion remains important in the treatment of patients with sickle cell disease (SCD). However, alloimmunization after blood transfusion is associated with patient morbidity and mortality. Triple-knockout (TKO) pigs (i.e., pigs in which the three known xenoantigens to which humans have anti-pig antibodies have been deleted) may be an alternative source of RBCs for these patients because many humans have no preformed antibodies to TKO pig RBCs (pRBCs). METHODS AND MATERIALS: In an in vitro study, plasma from alloimmunized (n = 12) or non-alloimmunized (n = 12) SCD patients was used to determine IgM/IgG binding to, and CDC of, TKO pRBCs. In an in vivo study, after an estimated 25% of blood volume was withdrawn from two capuchin monkeys, CFSE-labeled TKO pRBCs were transfused. Loss of TKO pRBCs was monitored by flow cytometry, and 7 weeks later, 25% of blood was withdrawn, and CFSE-labeled monkey RBCs were transfused. RESULTS: The in vitro study demonstrated that plasma from neither alloimmunized nor non-alloimmunized SCD patients bound IgM/IgG to, or induced CDC of, TKO pRBCs. In the in vivo study, survival of TKO pRBCs in the two capuchin monkeys was of 5 and 7 days, respectively, whereas after allotransfusion, survival was >28 days. CONCLUSIONS: In conclusion, (1) in the present limited study, no antibodies were detected that cross-reacted with TKO pRBCs, and (2) TKO pigs may possibly be an alternate source of RBCs in an emergency if no human RBCs are available.

Post-stapedotomy reparative granuloma following use of acellular porcine small intestinal submucosa.

PURPOSE: There have been multiple proposed etiologies of reparative granuloma following stapes surgery. In this report, we present the first case of post-stapedectomy reparative granuloma following the use of Biodesign (Cook Medical, Bloomington, IN) otologic graft material, an acellular matrix derived from porcine small intestinal submucosa, and review the literature of post-stapes surgery reparative granuloma. PATIENT: 50-year-old woman who developed a reparative granuloma following stapedotomy with acellular porcine intestinal submucosa presenting with profound hearing loss and vertigo. INTERVENTION: Middle ear exploration with excision of granuloma and revision stapedotomy. MAIN OUTCOME MEASURES: Audiologic outcomes as measured by pure-tone air and bone conduction thresholds and word recognition scores. Improvement in vertigo. MAIN FINDINGS: Surgical excision of the reparative granuloma with revision stapedotomy resolved vertigo. Hearing has improved progressively postoperatively. CONCLUSIONS: We report the first case of post-stapedotomy reparative granuloma following the use of acellular porcine intestinal submucosa. Although exact etiology cannot be determined from a single case report, this illustrates the need for careful use of novel foreign graft material. This case additionally confirms that removal of granuloma and inciting materials can salvage serviceable hearing.

Comprehensive Analysis of Cardiac Xeno-Graft Unveils Rejection Mechanisms.

Porcine heart xenotransplantation is a potential treatment for patients with end-stage heart failure. To understand molecular mechanisms of graft rejection after heart transplantation, we transplanted a 31-day-old alpha-1,3-galactosyltransferase knockout (GTKO) porcine heart to a five-year-old cynomolgus monkey. Histological and transcriptome analyses were conducted on xenografted cardiac tissue at rejection (nine days after transplantation). The recipient monkey's blood parameters were analyzed on days -7, -3, 1, 4, and 7. Validation was conducted by quantitative real-time PCR (qPCR) with selected genes. A non-transplanted GTKO porcine heart from an age-matched litter was used as a control. The recipient monkey showed systemic inflammatory responses, and the rejected cardiac graft indicated myocardial infarction and cardiac fibrosis. The transplanted heart exhibited a total of 3748 differentially expressed genes compared to the non-transplanted heart transcriptome, with 2443 upregulated and 1305 downregulated genes. Key biological pathways involved at the terminal stage of graft rejection were cardiomyopathies, extracellular interactions, and ion channel activities. The results of qPCR evaluation were in agreement with the transcriptome data. Transcriptome analysis of porcine cardiac tissue at graft rejection reveals dysregulation of the key molecules and signaling pathways, which play relevant roles on structural and functional integrities of the heart.

Trichostatin A-Assisted Epigenomic Modulation Affects the Expression Profiles of Not Only Recombinant Human α1,2-Fucosyltransferase and α-Galactosidase A Enzymes But Also Galα1→3Gal Epitopes in Porcine Bi-Transgenic Adult Cutaneous Fibroblast Cells.

This study was conducted to explore whether trichostatin A-assisted epigenomic modulation (TSA-EM) can affect the expression of not only recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) immune system enzymes but also Galα1→3Gal epitopes in ex vivo proliferating adult cutaneous fibroblast cells (ACFCs) derived from hFUT2×hGLA bi-transgenic pigs that had been produced for the needs of future xenotransplantation efforts. The ACFC lines were treated with 50 nM TSA for 24 h and then the expression profiles of rhα1,2-FT and rhα-Gal A enzymes were analyzed by Western blot and immunofluorescence. The expression profiles of the Galα1→3Gal epitope were determined by lectin blotting and lectin fluorescence. The ACFCs derived from non-transgenic (nTG) pigs were served as the negative (TSA-) and positive (TSA+) control groups. For both hFUT2×hGLA and nTG samples, the expression levels of α1,2-FT and α-Gal A proteins in TSA+ cells were more than twofold higher in comparison to TSA- cells. Moreover, a much lower expression of the Galα1→3Gal epitopes was shown in TSA- hFUT2×hGLA cells as compared to the TSA- nTG group. Interestingly, the levels of Galα1→3Gal expression in TSA-treated hFUT2×hGLA and nTG ACFCs were significantly higher than those noticed for their TSA-untreated counterparts. Summing up, ex vivo protection of effectively selected bi-transgenic ACFC lines, in which TSA-dependent epigenetic transformation triggered the enhancements in reprogrammability and subsequent expression of hFUT2 and hGLA transgenes and their corresponding transcripts, allows for cryopreservation of nuclear donor cells, nuclear-transferred female gametes, and resultant porcine cloned embryos. The latter can be used as a cryogenically conserved genetic resource of biological materials suitable for generation of bi-transgenic cloned offspring in pigs that is targeted at biomedical research in the field of cell/tissue xenotransplantation.