Novel SLA class I alleles of Chinese pig strains and their significance in xenotransplantation

To lay background for studying rejection mechanisms in xenotransplantation and developing the strategies for intervention, class I genes of swine leukocyte antigens (SLA) of three Chinese pig strains Bm, Gz and Yn were cloned and sequenced. The cDNA of the class I loci P1 and P14 were amplified by RT-PCR and subjected to insert into sequencing vectors. All six allelic sequences we examined, each two for one Chinese strain, are not identical to those reported, which allows these novel sequences receiving their accession numbers AY102467- AY102472 from GenBank. This study further reveals that the homologies of MHC class I genes in their primary structures and the deduced amino acids between Chinese pigs (SLA) and human (HLA-A*0201) are better than those between pigs and mice (H-2Db/H-2Kb). The comparison also indicates that the amino acid residues critical for recognition by human KIRs are altered in the swine class I molecules. The amino acids responsible for binding human CD8 coreceptor are largely conserved although there are two critical residues substituted. A functional test indicated that the human T cells specific for the prokaryotically expressed SLA Plprotein could respond quite well in vitro to the class I-positive swine chon-drocytes and PBMCs in presence of human APCs. This implies that, due to the substitution of two critical residues, the inaccessibility of human CD8 coreceptor to swine class I molecule might be contributable to the indirect pathway that the human T cells have to use for recognizing the SLA class I xenogeneic antigens.

The role of donor hepatocytes and/or splenocytes pre-injection in reducing islet xenotransplantation rejection

OBJECTIVE: To observe the effect of tail vein injection with donor hepatocyte and/or splenocyte on islets xenotransplantation rejection. METHODS: New-born male pigs and BALB/C mice were selected as donors and recipients respectively. Islets xenotransplantation was performed in recipients just after the third time of tail vein injection with donor hepatocytes and/or splenocytes. Macrophage phagocytosis, NK killing activity, T lymphocyte transforming function of spleen cells, antibody forming function of B lymphocytes, and T lymphocyte subsets were taken to monitor transplantation rejection. The effects of this kind of transplantation were indicated as variation of blood glucose and survival days of recipients. RESULTS: Streptozotocin (STZ) succeeded in inducing diabetes mellitus models of mice. Pre-injection of donor hepatocytes, and splenocytes or their mixture via tail vein was effective in preventing donor islets transplantation from rejection, which was demonstrated by the mentioned immunological marks. And each group of transplantation could decrease the blood glucose of recipients and prolong the survival days. Pre-injection of mixture of donor hepatocytes and splenocytes was more effective in preventing rejection than pre-injection of donor hepatocytes or splenocytes separately. CONCLUSION: We propose that pre-injection of donor hepatocytes, splenocytes separately or their mixture before donor islets transplantation is a good way to prevent rejection.

Establishment of Xenotransplantation Model of Human CN-AML with FLT3-ITD^(mut)/NPM1 in NOD/SCID Mice

Summary： Patients with FLT3-ITD^mmutt/NPM1- cytogenetically normal acute myeloid leukemia （CN-AML）, as high-risk molecular group in CN-AML, are associated with a worse prognosis than other CN-AML patients. It is beneficial to generate xenotransplantation model of FLT3-ITD^mut/NPM1- CN-AML to better understand the pathogenesis and therapeutic strategies of such AML subtype. The purpose of present study was to establish the xenotransplantation model in NOD/SCID mice with FLT3-ITD^mut/NPM1- CN-AML primary cells. The FLT3-ITD^mut/NPM1- CN-AML primary cells from 3 of 7 cases were successfully transplanted into NOD/SCID mice, and human CD45 positive cells were detected in the peripheral blood, spleen and bone marrow of mice by using flow cytometry. Infiltration of human leukemia cells in various organs of mice was observed by using immunohistochemistry. Gene analysis confirmed sustained FLT3/ITD mutation without NPM1 mutation in mice. By performing serial transplantation, it was found that characteristics of the leukemia cells in secondary and tertiary genera- tion models remained unchanged. Moreover, in vivo cytarabine administration could extend survival of NOD/SCID mice, which was consistent with clinical observation. In conclusion, we successfully estab- lished xenotransplantation model of human FLT3-ITD^mut/NPM1- CN-AML in NOD/SCID mice. The model was able to present primary disease and suitable to evaluate the curative effects of new drugs or therapy strategies.

Establishment of Reproducible Xenotransplantation Model of T Cell Acute Lymphoblastic Leukemia in NOD/SCID Mice

T cell acute lymphoblastic leukemia(T-ALL) is an aggressive leukemia.However the poor prognosis and low morbidity restrict further analysis of the disease.Therefore there is an increasing demand to develop animal models for identifying novel therapeutic approaches.In this study,we inoculated the anti-mouse CD122 monoclonal antibody conditioned NOD/SCID mice with the leukemia cells from 9 T-ALL patients and 1 cell line via the tail vein.Four of the 9 patients and the cell line were successfully engrafted.Flow cytometry detected high percentage of human CD45 + cells in recipient mice.Immunohistochemistry showed infiltration of human CD45 + cells in different organs.Serial transplantation was also achieved.In vivo drug treatment showed that dexamethasone could extend survival,which was consistent with clinical observation.These results demonstrated that we successfully established 5 xenotransplantation models of T-ALL in anti-mCD122 mAb conditioned NOD/SCID mice,which recapitulated the characteristics of original disease.

Intracerebral xenotransplantation of semipermeable membrane- encapsuled pancreatic islets

AIM： To identify the decreasing effect of xenotransplantion in combination with privileged sites on rejection and death of biological semipermeable membrane-（BSM） encapsulated implanted islets. METHODS： After the BSM experiment in vitro, BSM- encapsulated SD rat＇s islet-like cell clusters （ICCs） were xenotransplanted into normal dog＇s brain. Morphological changes were observed under light and transmission electron microscope. The islets and apoptosis of implanted B cells were identified by insulin-TONEL double staining. RESULTS： The BSM used in our study had a favorable permeability, some degree of rigidity, lighter foreign body reaction and toxicity. The grafts consisted of epithelioid cells and loose connective tissue. Severe infiltration of inflammatory cells was not observed. The implanted ICCs were identified 2 mo later and showed typical apoptosis. CONCLUSION： BSM xenotransplantation in combination with the privileged site can inhibit the rejection of implanted heterogeneous ICCs, and death of implanted heterogeneous B cells is associated with apoptosis. 2005 The WJG Press and Elsevier Inc. All rights reserved.

Expression of L amino acid transport system 1 and analysis of iodine-123-methyltyrosine tumor uptake in a pancreatic xenotransplantation model using fused high-resolution-micro-SPECT-MRI

BACKGROUND:The specificity in discriminating pancreatitis is limited in the positron emission tomography(PET)using Fluorine-18-fluorodeoxyglucose.Furthermore,PET is not widely available compared to the single photon emission computed tomography(SPECT).Since amino acids play a minor role in metabolism of inflammatory cells,the potential of the SPECT tracer,3-[ 123 I]iodo-L-α-methyltyrosine(123I-IMT),for detecting pancreatic cancer was examined in xenotransplantation models of human pancreatic carcinoma in mice. METHODS: 123 I-IMT was injected to eight mice inoculated with subcutaneous or orthotopic pancreatic tumors.Fused high-resolution-micro-SPECT(Hi-SPECT)and magnetic resonance imaging were performed.The gene expression level of L amino acid transport-system 1(LAT1)was analyzed and correlated with tumor uptake of 123 I-IMT. RESULTS:A high uptake of 123 I-IMT was detected in all tumor-bearing mice.The median tumor-to-background ratio (T/B)was 12.1(2.0-13.2)for orthotopic and 8.4(1.8-11.1)for subcutaneous xenotransplantation,respectively.Accordingly, the LAT1 expression in transplanted Colo357 cells was increased compared to non-malignant controls.CONCLUSIONS:Our mouse model could show a high 123 I-IMT uptake in pancreatic cancer.Fused MRI scans facilitate precise evaluation of uptake in the specific regions of interest.Further studies are required to confirm these findings in tumors derived from other human pancreatic cancer cells.Since amino acids play a minor role in the metabolism of inflammatory cells,the potential for application of 123 I-IMT to distinguish pancreatic tumor from inflammatory pancreatitis warrants further investigation.

Xenotransplantation of embryonic pig pancreas for treatment of diabetes mellitus in non-human primates

Transplantation therapy for diabetes in humans is limited by the low availability of human donor whole pancreas or islets. Outcomes are complicated by immunosuppressive drug toxicity. Xenotransplantation is a strategy to overcome supply problems. Implantation of tissue obtained early during embryogenesis is a way to reduce transplant immunogenicity. Pig insulin is biologically active in humans. In that regard the pig is an appropriate xenogeneic organ donor. Insulin-producing cells originating from embryonic pig pancreas obtained very early following pancreatic primordium formation [embryonic day 28 (E28)] engraft long-term in rhesus macaques. Endocrine cells originating from embryonic pig pancreas transplanted in host mesentery migrate to mesenteric lymph nodes, engraft, differentiate and improve glucose tolerance in rhesus macaques without the need for immune suppression. Transplantation of embryonic pig pancreas is a novel approach towards beta cell replacement therapy that could be applicable to humans.

Response of the xenograft endothelium in the concordant xenotransplantation

To investigate the response of the xenograft endothelium in the concordant hamster to rat cardiac xenotransplantation and the mechanism of acute vascular rejection. Methods： The animals were divided into 5 groups randomly： control group, CsA group, splenectomy group, DO splenectomy＋CsA group and D3 splenectomy＋CsA group. Hamster heart was heterotopicaly transplanted to rat abdominal cavity. The graft survival was monitored by palpation of the rat abdominal wall. The histological and ultrastructural changes of the xenogafts were investigated. NF-κB and P-selectin expression in the xenograft were detected. Heme Oxigenase-1 and Bcl-2 expression were also detected in the xenografts of different groups. Results： The mean survival time of the xenografts in control group, CsA group, splenectomy group, DO splenectomy＋CsA group and D3 splenectomy＋CsA group was 3.4±0.55, 3.8±0,45, 6.4± 1.52, 30 and 7.4 ±1.14 days. The rejected graft showed typical acute vascular rejection in control group, CsA group,splenectomy group and D3 splenectomy＋CsA group. Endothelial cells of the rejected xenograft showed dramatic assembly of ribosomes and expansion of the rough endoplasmic reticulum. However, the endothelium of the long-term survived grafts in DO splenectomy＋CsA group showed normal architecture. NF-κB and P-selectin expression were detected in the rejected xenografts. HO-1 expression was observed in the long-term survived xenografts in DO splenectomy＋CsA group. Conclusion： The endothelial cells of the xenograft might be activated during the acute vascular rejection. Expression of HO-1 might inhibit the upregulation of NF-κB and adhesion molecular which decreases the activation of the endotheliuna of the graft.

Current advances in xenotransplantation

BACKGROUND: Transplantation of organs is a well-known and accepted life-saving procedure for end-stage kidney, liver, heart and lung diseases. The insufficient number of donor organs limits the application of this technique and leads to unnecessary loss of life. Experimental techniques such as xenotransplantation are extremely important to determine new methods of creating organ availability. DATA SOURCES; A literature search of Pubmed database was conducted and research articles reviewed. RESULTS: Xenotransplantation is a progressive field of research. Human complement regulatory protein ( hDAF) transgenic pigs and new immunosuppressive strategies that reduce xenoreactive αgal antibodies, have decreased rates of acute vascular rejection. Transplantation of α-1, 3-galac-tosyltransferase knock-out pig organs into baboons has resulted in the longest graft survival to date. Coagulation pathways have been identified as having a role in graft rejection. In vitro studies of porcine endogenous retroviruses (PERVs) show encouraging results that zoonosis will be less hindering to xenotransplantation than once thought. CONCLUSIONS: Several recent advances in xenotransplantation research have brought this technique closer to clinical application. The Ethics Committee of the International Xenotransplantation Association has made recommendations to ensure maintenance of ethical standards. Advancement will depend on the development of pig models, novel immunosuppressive strategies to target the innate immune system, and new ways to create donor specific tolerance. Prevention of rejection and transmission of infectious agents remain unresolved issues. In the future, it is feasible that xenotransplantation will be used to resolve this medical dilemma.

A humanized mouse model to study human immune response in xenotransplantation

BACKGROUND: A major barrier to the clinical application of xenotransplantation as a treatment option for patients is T cell-mediated rejection. Studies based on experimental rodent models of xenograft tolerance or rejection in vivo have provided useful information about the role of T cell immune response in xenotransplantation. However not all observations seen in rodents faithfully recapitulate the human situation This study aimed to establish a humanized mouse model of xenotransplantation, which mimics xenograft rejection in the context of the human immune system. METHODS: NOD-SCID IL2rγ -/- mice were transplanted with neonatal porcine islet cell clusters (NICC) followed by reconstitution of human peripheral blood mononuclear cells (PBMC). Human leukocyte engraftment and islet xenograft rejection were confirmed by flow cytometric and histological analyses. RESULTS: In the absence of human PBMC, porcine NICC transplanted into NOD-SCID IL2rγ -/- mice revealed excellent graft integrity and endocrine function. Human PBMC demonstrated a high level of engraftment in NOD-SCID IL2rγ -/- mice. Reconstitution of NICC recipient NOD-SCID IL2rγ -/- mice with human PBMC led to the rapid destruction of NICC xenografts in a PBMC number-dependent manner. CONCLUSIONS: Human PBMC-reconstituted NOD-SCID IL2rγ -/- mice provide an ideal model to study human immune responses in xenotransplantation. Studies based on this humanized mouse model will provide insight for improving the outcomes of clinical xenotransplantation.

An improved model of heterotopic cardiac xenotransplantation from guinea－pig to rat

Shortage of allogenic donor heart remains a serious problem all over the world leading to an upsurge in the study of xenografic cardiac transplantation. This study, based on the cuff technique, modified the grafting procedure in some aspects and successfully established a model of cervical cardiac xenotransplantation from guinea-pig to rat. Of 112 xenografting procedures performed in this group, the success rate of operation was 98. 2%, with only 2 cases of operative failure. Mean survival of the xenografts was 14±5. 5 min. The result showed that the modified model is more available and reproducible with much less bleeding than the original model, thus providing a more practical and stable experimental model for studying the mechanisms of hyperacute vascular rejection and the prevention of rejection in discordant cardiac xenotransplantation.

Use of zebrafish embryos as avatar of patients with pancreatic cancer:A new xenotransplantation model towards personalized medicine

BACKGROUND The response to chemotherapy treatment of patients with pancreatic ductal adenocarcinoma(PDAC)is difficult to predict and the identification of patients who most likely will benefit from aggressive chemotherapy approaches is crucial.The concept of personalized medicine has emerged in the last years with the objective to tailor the medical treatment to the individual characteristics of each patient,and particularly to the tumor biology of each patient.The need for invivo xenotransplantation models for cancer patients has increased exponentially,and for this reason zebrafish avatars have gained popularity.Preliminary studies were conducted also with PDAC tissue.AIM To develop a simple,not expensive,diffusible zebrafish embryo model as avatar for patients affected by PDAC.METHODS Tumor tissue was taken from the surgical specimen by the histopathologist.After its fragmentation into small pieces,they are stained with CM-Dil.Small pieces of stained tissue were transplanted into the yolk of wt AB zebrafish embryos with a glass capillary needle.Embryos were incubated at 35°C in E3 medium supplemented with 1%Pen/Strep in the presence or absence of drugs for the following days in respect of the treatment plan(Gemcitabine;Gemcitabine and Oxaliplatin;Gemcitabine and nab-Paclitaxel;5-Fluorouracil and Folinic acid and Oxaliplatin and Irinotecan).The response of zebrafish xenografts to the chemotherapy options has been analyzed by monitoring the fluorescent stained area at 2 h post injection(hpi),1 d and 2 d post injection(dpi).In each time point,the mean size of the stained area was measured by ImageJ and it was normalized with respect to the 1 dpi time point mean relative tumor area(RTA).We evaluated the effect of the chemotherapy exposition comparing the mean RTA of each treated subgroup and the control group and evaluating the percentage reduction of the mean RTA by comparing each treated subgroup with the control group.RESULTS Between July 2018 and October 2019,a total of 15 patients with pancreatic cancer were prospectively enrolled.In all cases,it was possible to take a fragment of the tumor from the surgical specimen for the xenotransplantation in the zebrafish embryos.The histological examination confirmed the presence of a PDAC in all cases.In absence of chemotherapy(control group),over time the Dil-stained area showed a statistically significant increase in all cases.A statistically significant reduction of the mean RTA in the treated subgroups for at least one chemotherapy scheme was reported in 6/15(40%)cases.The analysis of the percentage reduction of the RTA in treated subgroups in comparison to the control group revealed the presence of a linear relationship in each subgroup between the percentage reduction of the RTA and the number of cases reporting each percentage threshold considered for the analysis.CONCLUSION Our model seems to be effective for the xenotransplantation of PDAC tissue and evaluation of the effect of each chemotherapy scheme on the xenotransplanted tumor tissue.

Is the near coming xenotransplantation era relieving us from needing to look for more non-living organ donors?

Despite organ transplantation being the most successful treatment for end-stage organ dysfunction,the number of annual solid organ transplantations is much lower than that required to satisfy the demand of patients on waiting lists.The explanation for this phenomenon is the relative scarcity of non-living organ donors due to several factors,such as:(1)Late arrival of patients with a neurocritical condition to an emergency service;(2)lack of detection of those patients as possible organ donors by health professionals dedicated to procurement or by clinicians at emergency and intensive care units,for instance;(3)late transfer of the patient to an intensive care unit to try to recover their health and to provide hemodynamic,ventilatory,and metabolic support;(4)lack of confirmation of the physiological status of the possible donor;(5)late or incorrect positive diagnosis of the subject’s death,either due to brain or cardiac death;(6)difficulty in obtaining legal authorization,either by direct relatives or by the authority,for the extraction of organs;and(7)deficient retrieval surgery of the organs actually donated.The recent reports of relatively successful xenotransplants from genetically modified pigs open the possibility to fix this mismatch between supply and demand,but some technical(organ rejection and opportunistic infections),and economic issues,still remain before accepting a progressive replacement of the organ sources for transplantation.An approximate economic cost analysis suggests that the hypothetical acquisition cost of any genetically modified pig derived organ is high and would not even satisfy the solid organ demand of the wealthiest countries.

Microbiosis in lung allotransplantation and xenotransplantation:State of the artandfuture perspective

The respiratory tract is known to harbor a microbial community including bacteria,viruses,and fungi.New techniques contribute enormously to the identification of unknown or culture-independent species and reveal the interaction of the community with the host immune system.The existing respiratory microbiome and substantial equilibrium of the transplanted microbiome from donor lung grafts provide an extreme bloom of dynamic changes in the microenvironment in lung transplantation(LT)recipients.Dysbiosis in grafts are not only related to the modified microbial components but also involve the kinetics of the host-graft“talk,”which signifies the destination of graft allograft injury,acute rejection,infection,and chronic allograft dysfunction development in short-and long-term survival.Microbiome-derived factors may contribute to lung xenograft survival when using genetically multimodified pig-derived organs.Here,we review the most advanced knowledge of the dynamics and resilience of microbial communities in transplanted lungs with various pretransplant indications.Conceptual and analytical points of view have been illustrated along the time series,gaining insight into the microbiome and lung grafts.Future endeavors on precise tools,sophisticated models,and novel targeted regimens are needed to improve the long-term survival in these patients.

The importance of public engagement in clinical xenotransplantation

Over the past several decades,significant scientific progress in xenotransplantation has brought the field to the threshold of clinical trials.In the past 3 years in the United States,experimental pig kidney and heart xenotransplantation have been performed on human subjects recently declared dead by neurological criteria(decedents).In addition,two pig heart transplants have been carried out in living patients under the United States Food and Drug Administration's expanded access guidelines.However,though there has been a flurry of activity there remain unanswered questions regarding how the public views xenotransplantation,what concerns may exist,and how to address these concerns in a meaningful way.This paper aims to underscore the importance of public engagement in xenotransplantation,emphasizing the ongoing need for studies to assess public opinions.The current evidence on public engagement studies is reviewed and gaps in our understanding are identified.We propose practical steps to advance this field.Additional studies to determine the extent of racial/ethnic differences in attitudes to xenotransplantation should be conducted.Empirical and descriptive analysis of certain religious viewpoints—especially minority faiths—would be valuable.As public engagement is an important aspect of public acceptance of novel research that is accompanied by risk,we suggest that xenotransplantation biotechnology companies might consider leading the way in funding this research.

Establishment of a donor pig for xenotransplantation clinical trials based on the principle of Changsha Communiqué

Background:Xenotransplantation is a potential way to reduce the shortage of the needed organ grafts for the end-stage disease.Immune rejection,physiological incompatibility and bio-safety are the most critical issues.Methods:To ensure the safety and efficacy of gene editing,second-and thirdgeneration sequencing technologies have allowed us to obtain a clearer genetic background of donor pigs for xenotransplantation.Based on the Changsha Communiqué,the local DPF-excluded lists and DPF donor facility were established in Changsha,China.A pig-to-human islet clinical trial was conducted and overseen by the respective Chinese governmental agency.Results:The DPF standards for pig husbandry eliminated specific pathogens in donor pigs.We have established a PERV-C free,genetic information clean,DPF donor for xenotransplantation.A clinical trial of ten adult patients(9M:1F)with type 1 diabetes who received DPF porcine islet xenotransplantation via the portal vein were performed.Clinical accepted immunosuppressant drugs and autologous Treg were used for controlling immune rejection.No cross-species infection events occurred in this trial,and importantly,no cross-species transmission of PERV was found.Conclusions:Xenotransplantation is a pioneer study and safety is the most important issue.The fundamental principles for establishing xenotransplantation donor pigs should follow the Changsha Communiqué(2008),the second WHO consultation,and the 2018 Changsha Communiquéwhich would finally help reducing the risks of xenotransplantation.

Porcine cytomegalovirus in xenotransplantation:The new frontier in human transplantation?

The first gene-edited pig heart transplantation taking place in March this year in Maryland,USA,was hailed as a step into a new world[1,2],a promise to finally serve those who are waiting for organs on the heart transplant waitlist.Much effort had been put into this endeavor in the past decades,first to deal with the issue of complement activation and hyperacute humoral rejection[3,4],second to deal with PERV by inactivating the main enzyme reverse transcriptase,thus cells still produce virus particles which are able to infect new human cells,but they cannot integrate in the genome of the new target cell[5–7]and third to control unwanted growth of the organ after transplantation using e.g.mTOR inhibitors[8–10].

Xenotransplantation--reflections on the bioethics

Background:Similar to most countries in the world,China has a severe shortage of human organs,and this is one of the main issues restricting the application of organ transplantation technology.In 2019 alone,only 19,454(23.90%)of the 81,410 people waiting were able to receive organ transplants.There is an increasing focus from both the medical profession and society on how to fill the gap between supply and demand.Methods:Xenotransplantation using animal organs is being considered as one option to make up for the shortage of human organs for transplantation.For some years now,the international medical community has been examining the possibility of using animal organs for human transplant.However,the research has faced two important types of challenges:scientific and ethical issues.Results:In January 2021,the first clinical trial of transgenic pig heart transplantation into a human recipient was completed by the Medical Center of the University of Maryland in the United States.This has stimulated enthusiasm and interest in xenotransplantation.Conclusions:The trend towards xenotransplantation has highlighted global problems such as the severe shortage of organ transplant donors and the high cost of organ transplantation.China needs to consider how to cope with the scientific,public health,and social ethics challenges of xenotransplantation clinical trials.

Expert consensus on clinical trials of human xenotransplantation in China

The history of xenotransplantation started in the 19th century.After a few decades of investigation,significant breakthroughs and preclinical milestones have been achieved worldwide.With the recent transplantation of genetically modified porcine kidneys and heart into humans,these ground-breaking achievements have attracted great attention worldwide,in the hope that xenotransplantation might alleviate or even solve the problem of organ shortage.On January 20,2022,the China Organ Transplantation Development Foundation convened a symposium on“The History,Current Situation and Future of Human Xenotransplantation Clinical Trials,”where ways to promote the ethical and sustainable development of xenotransplantation in China were discussed among the participating experts.A formal consensus was reached as the product of the symposium,outlining the expert opinions on scientific,regulatory,and ethical issues of clinical trials of xenotransplantation in China.