BACKGROUND: The primary limitation to kidney transplantation is organ shortage. Recent progress in gene editing and immunosuppressive regimens has made xenotransplantation with porcine organs a possibility. However, evidence in pig-to-human xenotransplantation remains scarce, and antibody-mediated rejection (AMR) is a major obstacle to clinical applications of xenotransplantation. METHODS: We conducted a kidney xenotransplantation in a brain-dead human recipient using a porcine kidney with five gene edits (5GE) on March 25, 2024 at Xijing Hospital, China. Clinical-grade immunosuppressive regimens were employed, and the observation period lasted 22 days. We collected and analyzed the xenograft function, ultrasound findings, sequential protocol biopsies, and immune surveillance of the recipient during the observation. RESULTS: The combination of 5GE in the porcine kidney and clinical-grade immunosuppressive regimens prevented hyperacute rejection. The xenograft kidney underwent delayed graft function in the first week, but urine output increased later and the single xenograft kidney maintained electrolyte and pH homeostasis from postoperative day (POD) 12 to 19. We observed AMR at 24 h post-transplantation, due to the presence of pre-existing anti-porcine antibodies and cytotoxicity before transplantation; this AMR persisted throughout the observation period. Plasma exchange and intravenous immunoglobulin treatment mitigated the AMR. We observed activation of latent porcine cytomegalovirus toward the end of the study, which might have contributed to coagulation disorder in the recipient. CONCLUSIONS 5GE and clinical-grade immunosuppressive regimens were sufficient to prevent hyperacute rejection during pig-to-human kidney xenotransplantation. Pre-existing anti-porcine antibodies predisposed the xenograft to AMR. Plasma exchange and intravenous immunoglobulin were safe and effective in the treatment of AMR after kidney xenotransplantation.
Transplantation, Heterologous/methods* ; Kidney Transplantation/methods* ; Heterografts/pathology* ; Immunoglobulins, Intravenous/administration & dosage* ; Graft Survival/immunology* ; Humans ; Animals ; Sus scrofa ; Graft Rejection/prevention & control* ; Kidney/pathology* ; Gene Editing ; Species Specificity ; Immunosuppression Therapy/methods* ; Plasma Exchange ; Brain Death ; Biopsy ; Male ; Aged

OBJECTIVE: To prepare decellularized nerve grafts from alpha-1, 3-galactosyltransferase (GGTA1) gene-edited pigs and explore their biocompatibility for xenotransplantation. METHODS: The sciatic nerves from wild-type pigs and GGTA1 gene-edited pigs were obtained and underwent decellularization. The alpha-galactosidase (α-gal) content in the sciatic nerves of GGTA1 gene-edited pigs was detected by using IB4 fluorescence staining and ELISA method to verify the knockout status of the GGTA1 gene, and using human sciatic nerve as a control. HE staining and scanning electron microscopy observation were used to observe the structure of the nerve samples. Immunofluorescence staining and DNA content determination were used to evaluate the degree of decellularization of the nerve samples. Fourteen nude mice were taken, and subcutaneous capsules were prepared on both sides of the spine. Decellularized nerve samples of wild-type pigs ( n=7) and GGTA1 gene-edited pigs ( n=7) were randomly implanted in the subcutaneous capsules. Blood was drawn at 1, 3, 5, and 7 days after implantation to detect neutrophil counting. RESULTS: IB4 fluorescence staining and ELISA detection showed that GGTA1 gene was successfully knocked out in the nerves of GGTA1 gene-edited pigs. HE staining showed that the structure of the decellularized nerve from GGTA1 gene-edited pigs was well preserved; the nerve basement membrane tube structure was visible under scanning electron microscopy; no cell nuclei was observed, and the extracellular matrix components was retained in the nerve grafts by immunofluorescence staining; and the DNA content was significantly reduced when compared with the normal nerves ( P<0.05). In vivo experiments showed that the number of neutrophils in the two groups were similar at 1, 3, and 7 days after implantation, with no significant difference ( P>0.05); only at 5 days, the number of neutrophils was significantly lower in the GGTA1 gene-edited pigs than in the wild-type pigs ( P<0.05). CONCLUSION The decellularized nerve grafts from GGTA1 gene-edited pigs have well-preserved nerve structure, complete decellularization, retain the natural nerve basement membrane tube structure and components, and low immune response after xenotransplantation through in vitro experiments.
Animals ; Transplantation, Heterologous ; Galactosyltransferases/genetics* ; Sciatic Nerve/immunology* ; Swine ; Tissue Engineering/methods* ; Humans ; Graft Rejection/prevention & control* ; Gene Editing ; Mice ; Mice, Nude ; Heterografts/immunology* ; Animals, Genetically Modified ; Tissue Scaffolds ; Decellularized Extracellular Matrix

BACKGROUND: Anti-Gal is a major antibody induced in non-human primates (NHPs) after xenotransplantation. To understand the mechanism of graft rejection, we investigated the association between anti-Gal responses and graft failure in NHP recipients of porcine islet transplantation (PITx). METHODS: Intraportal PITx was performed in 35 diabetic NHPs, and graft function was monitored. Early graft failure (EGF) was defined as loss of graft function within a month after PITx. Seven, 19, nine NHPs received immunosuppression (IS) without CD40 pathway blockade (Group I), with anti-CD154 (Group II), and with anti-CD40 (Group III), respectively. The anti-Gal levels on day 0 and day 7 of PITx were measured by ELISA. RESULTS: The frequency of EGF was significantly lower in Group II (26.3%) than in Group I (100%, P=0.0012) and Group III (77.8%, P=0.0166). While levels of anti-Gal IgG in Group I and anti-Gal IgM in Group III increased on day 7 compared with day 0 (P=0.0156 and 0.0273), there was no increase in either on day 7 in Group II. The ratio of anti-Gal IgM or IgG level on day 7 to that on day 0 (Ratio7/0) was significantly higher in recipients with EGF than without EGF (P=0.0009 and 0.0027). ROC curve analysis of anti-Gal IgM Ratio7/0 revealed an area under the curve of 0.789 (P=0.0003). CONCLUSIONS: IS with anti-CD154 suppressed anti-Gal responses and prevented EGF in PITx. Anti-Gal IgM Ratio7/0, being associated with EGF, is a predictive marker for EGF.
Animals ; Antibodies/blood/immunology ; Antigens, CD40/immunology ; Area Under Curve ; CD40 Ligand/immunology ; Disaccharides/*immunology ; Epidermal Growth Factor/blood ; Graft Rejection/*immunology ; Immunoglobulin G/blood ; Immunoglobulin M/*blood ; Immunosuppressive Agents/therapeutic use ; *Islets of Langerhans Transplantation ; Macaca mulatta ; ROC Curve ; Swine ; Transplantation, Heterologous

α-Gal, the main xenotransplantation antigen, can lead to hyperacute rejection (HAR) in xenotransplantation. This study was purposed to investigate the effect of recombinant α-galactosidase (α-Gal antigen) on the Holstein-Friesian(H-F) red blood cells (RBC). The enzymelysis method was used to digest the α-Gal antigen on H-F RBC; the saline and anti-human globulin methods were used to perform the agglutination test of H-F RBC and human plasma; the flow cytometry was used to detect the α-Gal antigen on surface of H-F RBC, fluorescence intensity of FITC-IB4 and FITC-IgG labeled RBC. The results indicated that the saline and anti-human globulin method showed α-galactosidase-treated H-F RBC fail to agglutinate with human pooled plasma; the flow cytometry showed the fluorescence intensity of FITC-IB4 and FITC-IgG labeled RBC decrease 99.0% and 87.8%, respectively. It is concluded that the novel α-galactosidase can be used to cleared the α-Gal antigen on the surface of H-F RBC and α-galactosidase-treated H-F RBC may be considered as human blood substitute.
Animals ; Blood Substitutes ; Cattle ; Erythrocytes ; immunology ; Female ; Humans ; Transplantation, Heterologous

The study was aimed to investigate the effect of anti-mouse CD122 antibody on the hematopoietic repopulating capacity of cord blood CD34⁺ cells in a humanized murine model-non obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After sublethal irradiation with γ-ray, NOD/SCID mice were intraperitoneally injected with 200 µg mouse isotype control antibody or anti-mouse CD122 antibody. Human cord blood CD34⁺ cells or phosphate-buffered saline (PBS) were injected via the tail vein at 6-8 hours later. Cohort of the mice injected with anti-mice CD122 antibody or control antibody alone were sacrificed at different time point (at week 2, 3, and 4 weeks) after the injection, and the percentage of NK cells in the peripheral blood was analyzed by flow cytometry. To evaluate the effect of anti-mouse CD122 antibody on the repopulating capacity of cord blood CD34⁺ cells in the recipient mice, phenotype analysis was performed in the bone marrow at 6 and 8 weeks after the transplantation. The results showed that the proportion of NK cells in the peripheral blood were (4.6 ± 0.6)% and (5.7 ± 1.7)% at week 2 and 3 after anti-CD122 antibody injection respectively,which decreased by 60%, compared with the mice injected with isotype control antibody. After 6 and 8 weeks of cord blood CD34⁺ cell transplantation,the percentage of human CD45⁺ in the bone marrow of the recipient mice treated with anti-mice CD122 antibody was (63.0 ± 12.2)% and (53.2 ± 16.3)%,respectively,which were dramatically higher than that in the mice treated with isotype control antibody (7.7 ± 3.6)% and (6.1 ± 2.4)%. Moreover,at 8 weeks after transplantation,human CD34⁺ cells appeared significantly in the recipients treated with anti-CD122 antibody. It is concluded that the anti-mouse CD122 antibody enhances the hematopoietic repopulating capacity of cord blood CD34⁺ cells in the NOD/SCID mice through decreasing the proportion of NK cells.
Animals ; Antibodies ; immunology ; Antigens, CD34 ; Bone Marrow ; Cord Blood Stem Cell Transplantation ; Fetal Blood ; immunology ; Hematopoietic Stem Cell Transplantation ; Hematopoietic System ; cytology ; immunology ; Humans ; Interleukin-2 Receptor beta Subunit ; immunology ; Killer Cells, Natural ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Transplantation, Heterologous

OBJECTIVETo review the current status and progress on pig islet xenotransplantation.

DATA SOURCESData used in this review were mainly from English literature of Pubmed database. The search terms were "pig islet" and "xenotransplantation".

STUDY SELECTIONThe original articles and critical reviews selected were relevant to this review's theme.

RESULTSPigs are suggested to be an ideal candidate for obtaining available islet cells for transplantation. However, the potential clinical application of pig islet is still facing challenges including inadequate yield of high-quality functional islets and xenorejection of the transplants. The former can be overcome mainly by selection of a suitable pathogen-free source herd and the development of isolation and purification technology. While the feasibility of successful preclinical pig islet xenotranplantation provides insights in the possible mechanisms of xenogeneic immune recognition and rejection to overwhelm the latter. In addition, the achievement of long-term insulin independence in diabetic models by means of distinct islet products and novel immunotherapeutic strategies is promising.

CONCLUSIONSPig islet xenotransplantation is one of the prospective treatments to bridge the gap between the needs of transplantation in patients with diabetes and available islet cells. Nonetheless, further studies and efforts are needed to translate obtained findings into tangible applications.

Animals ; Graft Rejection ; immunology ; prevention & control ; Islets of Langerhans Transplantation ; immunology ; methods ; Swine ; Transplantation, Heterologous ; methods

Non-human primate studies must be conducted prior to the clinical trial of xenotransplantation. In order to develop clinically applicable immune-modulatory regimen through non-human primate studies, close monitoring of xenogeneic immune responses is required. We adopted multiplex cytokine analysis in assessment of the immune status during the course of pig-to-non-human primate islet transplantation. This study aimed to assess the feasibility of this multiplex cytokine assay in the development of immune-modulatory regimen. Using this assay, we were able to detect different cytokines with a minimal usage of blood samples, and this allowed us to detect various immunological situations in the recipients. Detection of TNF-alpha surge (347.8 pg/mL) guided us to block TNF-alpha in the early phase of transplantation. Supportive information for in vivo efficacy of cytokine neutralizing antibody could be speculated by in vitro neutralization assay (1,250 pg/mL --> 0 pg/mL). In addition, periodic monitoring of cytokines in peripheral blood allowed the detection of the infection episode prior to other routine assays. These benefits of multiplex cytokine assay may be generally applied to other pre-clinical research, which is a prerequisite for clinical trials.
Animals ; Antibodies, Neutralizing/immunology ; Blood Cell Count ; Cytokines/*blood/immunology ; Immunoassay/*methods ; Interleukin-6/blood ; Islets of Langerhans Transplantation/*immunology ; Macaca mulatta ; Swine ; Transplantation, Heterologous ; Tumor Necrosis Factor-alpha/blood/immunology

αGal, a xenotransplantations antigen (XTA), can lead to hyper acute reaction (HAR) in xenotransplantation. α-Galactosidase from B. fragilis is a novel galactosidase belong to CAZy GH110 which can clear the terminal αGal from branched and linear oligosaccharides. This study was purposed to investigate the removal effect of a novel α-galactosidase on α-Gal XTA on surface of red blood cells. The αGal XTA from the red blood cells of cattle, pig, dog and rabbit was digested by using recombinant α-galactosidase; the α-Gal antigens on surface of cells was detected by flow cytometry. The results showed that the XTA was disappeared completely or mainly. It is concluded that the novel α-galactosidase is a potential enzyme to remove the XTA on the surface of xenotransplants and can be used to overcome the HAR in xenotransplantation.
Animals ; Antigens, Heterophile ; immunology ; Cattle ; Dogs ; Epitopes ; Erythrocytes ; immunology ; Macaca mulatta ; Mice ; Mice, Inbred BALB C ; Rabbits ; Swine ; Transplantation, Heterologous ; alpha-Galactosidase ; immunology

BACKGROUNDCostimulatory signals play a vital role in T cell activation. Blockade of costimulatory pathway by CTLA4Ig or CD40LIg have enhanced graft survival in experimental transplantation models yet mechanisms remain undetermined. We investigated the effects of CTLA4Ig and CD40LIg gene transfer on islet xenografts rejection in rats.

METHODSHuman islets were infected with recombinant adenoviruses containing CTLA4Ig and CD40LIg genes and implanted beneath the kidney capsule of diabetic rats. Levels of blood sugar, morphological changes, and survival of grafts were recorded. Expressions of CTLA4Ig, CD40LIg and insulin were detected by immunohistochemical staining and cytokines levels were quantified by enzyme-linked immunosorbent assay (ELISA).

RESULTSBlood glucose levels in transplant rats decreased to normal level on the 2nd day post transplantation. The mean blood glucose in the control group, CTLA4Ig transfected group, CD40LIg transfected group and CTLA4Ig + CD40LIg cotransfected group increased on days 8, 24, 21, 68, post transplantation respectively. The grafts in control group, CTLA4Ig transfected group, CD40LIg transfected group and CTLA4Ig + CD40LIg cotransfected group survived for (8 ± 1), (29 ± 4), (27 ± 3), and (74 ± 10) days, respectively. Survival in CTLA4Ig + CD40LIg cotransfected group was significantly longer. Survivals of CTLA4Ig transfected group and CD40LIg transfected group were significantly longer than control group. In control animals, serum interleukin-2 and tumor necrosis factor α concentration significantly increased within seven days post transplantation. Haematoxylin eosin staining of grafts showed live islets in situ of transplant rats without inflammatory cell infiltration. Immunohistochemical staining confirmed the expression of insulin at islets in all experimental groups.

CONCLUSIONSTransfer of CTLA4Ig and CD40LIg genes, especially the cotransfer of both, inhibits rejection of murine islet xenografts. Downregulated expressions of Th1 cells related cytokines might be related to the beneficial effects.

Abatacept ; Animals ; Enzyme-Linked Immunosorbent Assay ; Graft Rejection ; therapy ; Graft Survival ; genetics ; physiology ; Humans ; Immunoconjugates ; genetics ; metabolism ; Immunohistochemistry ; Insulin ; metabolism ; Islets of Langerhans Transplantation ; immunology ; methods ; Rats ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transplantation, Heterologous ; immunology ; methods

It has been reported that the immune response due to alpha-Gal epitopes is an important factor in tissue valve failure. The elimination of the interaction between the natural anti-Gal antibodies and alpha-gal epitopes on the xenografts is a prerequisite to the success of xenografts in humans. Previously, we reported that the green coffee bean alpha-galactosidase could remove all alpha-Gal epitopes from cell surface of porcine aortic valve and pericardial tissue, but it has limitations on cost effectiveness. In this study we wanted to know whether the recently produced recombinant human alpha-galactosidase A has the same effective enzymatic activity as green coffee bean alpha-galactosidase in removing alpha-Gal epitopes from the same tissues. After treating fresh porcine aortic valve and pericardial tissue with recombinant alpha-galactosidase A, each sample was stained with Griffonia simplicifolia type I isolectin B4 indirect immunoperoxidase avidin-biotin technique. We then examined whether the alpha-Gal epitopes were reduced or abolished in each consecutive concentration of recombinant alpha-galactosidase A by comparing the degree of the Griffonia simplicifolia isolectin B4 staining. As a result, the recombinant alpha-galactosidase A could remove cell surface alpha-Gals on porcine aortic valve and pericardial tissue as effectively as green coffee bean alpha-galactosidase.
Adolescent ; Animals ; Aortic Valve/chemistry/cytology/*immunology ; Child ; Coffea/enzymology ; Epitopes/*immunology ; Heart Valve Prosthesis Implantation ; Humans ; Pericardium/chemistry/cytology/*immunology ; Recombinant Proteins/genetics/*immunology ; Swine ; Transplantation, Heterologous/immunology ; alpha-Galactosidase/genetics/*immunology