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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 h FUT2 ×h GLA 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 h FUT2 ×h GLA 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 - h FUT2 ×h GLA cells as compared to the TSA - nTG group. Interestingly, the levels of Galα1→3Gal expression in TSA-treated h FUT2 ×h GLA 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 h FUT2 and h GLA 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.

Background: Animal tissues and tissue-derived biomaterials are widely used in the field of xenotransplantation and regenerative medicine. A potential immunogenic risk that affects the safety and effectiveness of xenografts is the presence of remnant α -Gal antigen (synthesized by GGTA1 or/and iGb3S ). GGTA1 knockout mice have been developed as a suitable model for the analysis of anti-Gal antibody-mediated immunogenicity. However, we are yet to establish whether GGTA1/iGb3S double knockout (G/i DKO) mice are sensitive to Gal antigen-positive xenoimplants., Methods: α -Gal antigen expression in the main organs of G/i DKO mice or bovine bone substitutes was detected via a standardized ELISA inhibition assay. Serum anti- α -Gal antibody titers of G/i DKO mice after immunization with rabbit red blood cells (RRBC) and implantation of raw lyophilized bone substitutes (Gal antigen content was 8.14 ± 3.17 × 10 12 /mg) or Guanhao Biotech bone substitutes (50% decrease in Gal antigen relative to the raw material) were assessed. The evaluation of total serum antibody, inflammatory cytokine, and splenic lymphocyte subtype populations and the histological analysis of implants and thymus were performed to systematically assess the immune response caused by bovine bone substitutes and bone substitute grafts in G/i DKO mice., Results: α -Gal epitope expression was reduced by 100% in the main organs of G/i DKO mice, compared with their wild-type counterparts. Following immunization with RRBC, serum anti-Gal antibody titers of G/i DKO mice increased from 80- to 180-fold. After subcutaneous implantation of raw lyophilized bone substitutes and Guanhao Biotech bone substitutes into G/i DKO mice, specific anti- α -Gal IgG, anti- α -Gal IgM, and related inflammatory factors (IFN- γ and IL-6) were significantly increased in the raw lyophilized bone substitute group but showed limited changes in the Guanhao Biotech bone substitute group, compared with the control., Conclusion: G/i DKO mice are sensitive to Gal antigen-positive xenogeneic grafts and can be effectively utilized for evaluating the α -Gal-mediated immunogenic risk of xenogeneic grafts., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2020 Anliang Shao et al.)

Background: Xenografts are an attractive alternative to traditional bone grafts because of the large supply from donors with predictable morphology and biology as well as minimal risk of human disease transmission. Clinical series involving xenograft bone transplantation, most commonly from bovine sources, have reported poor results with frequent graft rejection and failure to integrate with host tissue. Failures have been attributed to residual alpha-Gal epitope in the xenograft which humans produce natural antibody against. To the authors' knowledge, there is currently no xenograft-derived bone graft substitute that has been adopted by orthopedic surgeons for routine clinical use., Methods: In the current study, a bone scaffold intended to serve as a bone graft substitute was derived from porcine cancellous bone using a tissue decellularization and chemical oxidation protocol. In vitro cytocompatibility, pathogen clearance, and alpha-Gal quantification tests were used to assess the safety of the bone scaffold intended for human use., Results: In vitro studies showed the scaffold was free of processing chemicals and biocompatible with mouse and human cell lines. When bacterial and viral pathogens were purposefully added to porcine donor tissue, processing successfully removed these pathogens to comply with sterility assurance levels established by allograft tissue providers. Critically, 98.5% of the alpha-Gal epitope was removed from donor tissue after decellularization as shown by ELISA inhibition assay and immunohistochemical staining., Conclusions: The current investigation supports the biologic safety of bone scaffolds derived from porcine donors using a decellularization protocol that meets current sterility assurance standards. The majority of the highly immunogenic xenograft carbohydrate was removed from donor tissue, and these findings support further in vivo investigation of xenograft-derived bone tissue for orthopedic clinical application., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Hyperacute rejection (HAR) depends on the response of xenoreactive antibodies principally against porcine α-Gal epitope. Methods eliminating HAR include GGTA1 inactivation, regulation of the complement system and modification of the oligosaccharide structure of surface proteins in donor's cells. Transgenic animals designed for the purpose of xenotransplantation with single modification do not display full reduction of the α-Gal epitope level, which means that a accumulation of several modifications in one transgenic individual is needed. The aim of the study was to create a molecular and cytogenetic profile of a double transgenic animal with α1,2-fucosyltransferase and α-galactosidase expression. As a result of interbreeding of an individual with α1,2-fucosyltransferase expression with an individual with α-galactosidase expression 12 living piglets were obtained. PCR revealed the pCMVFUT gene construct was present in four individuals and pGAL-GFPBsd in three, including one with a confirmed integration of both the gene constructs. Fluorescence in situ hybridization confirmed the site of transgene integration, which corresponded to the mapping site of the transgenes which occurred in the parental generations. Karyotype analysis did not show any changes in the structure or the number of chromosomes (2n = 38, XX). As for the results pertaining to the single transgenic individuals, expression analysis demonstrated a high extent of α-Gal epitope level reduction on the surface of cells, whereas human serum cytotoxicity tests revealed the smallest decrease in longevity of cells in the obtained double transgenic individual (4.35 %). The tests suggest that the co-expression of both the transgenes leads to a considerable reduction of the α-Gal antigen level on the surface of cells and a decrease of xenotransplant immunogenicity.

Background: Among the bone graft sources used currently, the availability of autografts is limited and allografts are expensive. Therefore, xenobone grafts have drawn attention as a new source of bone grafts, although immunologic rejection issues are unresolved. This study used a GalT knockout mouse model to investigate the effects of reducing the alpha-gal epitope using alpha-galactosidase on the union of porcine xenobone grafts., Methods: Sixty-eight alpha-gal knockout C57/BL6 mice and eight wild-type mice were used. The mice were divided into five groups: In group 1 (26 alpha-gal knockout mice), an alpha-galactosidase-treated porcine xenograft was transplanted into the mouse femur to reduce antigenicity, and intramedullary fixation was performed. In group 2 (26 alpha-gal knockout mice), a non-treated porcine xenobone graft was performed. In group 3 (eight alpha-gal knockout C57/BL6 mice), syngenic bone grafts were performed. In group 4 (eight wild-type C57/BL6 mice), syngenic bone grafts were performed. In group 5 (eight C57/BL6 alpha-gal knockout mice), a bone defect model was obtained by maintaining the gap of the osteotomy site. Groups 3, 4, and 5 were used for positive and negative control groups. Qualitative immunohistochemical analysis of the porcine bone was performed to detect the presence of the alpha-gal epitope in groups 1 and 2. The concentration of the anti-alpha-gal antibody was evaluated using a quantitative enzyme-linked immunosorbent assay (ELISA) at the time of sacrifice (3, 4, and 5 weeks after the operation). Histologic and radiologic results (Goldberg method) for the bone union were compared., Results: The qualitative immunohistochemical analysis showed that the alpha-gal epitope was reduced when xenobone grafts were treated with alpha-galactosidase. Compared with group 2, group 1 showed a low anti-alpha-gal antibody concentration in the ELISA results. In group 2, the anti-alpha-gal antibody concentration increased with time. Group 1 showed significantly better histologic union than group 2, but the amount of radiologic union was similar in the two groups., Conclusions: Alpha-galactosidase treatment of a porcine xenobone graft can reduce the alpha-gal epitope. This reduction in the antigen could significantly reduce the humoral immune response to the alpha-gal antigen in C57/BL6 alpha-gal knockout mice, leading to significant improvements in histologic union. This study provides a relevant GalT knockout mouse model for detecting the effects of alpha-gal epitope reduction by alpha-galactosidase on the union of porcine xenobone grafts., (© 2014 John Wiley & Sons A/S.)

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.

PURPOSE. To solve the shortage of donor corneas, a decellularizing method based on hypertonic saline treatment was introduced, and a favorable outcome was observed in pig-to-rabbit lamellar corneal transplantation. This study was an investigation of the efficacy of pig-to-nonhuman primate lamellar corneal transplantation, using both decellularized and fresh porcine corneas to assess feasibility as a substitute for human corneas. METHODS. Nine Chinese rhesus macaques underwent lamellar corneal transplantation using both decellularized (n = 5) and fresh (n = 4) porcine corneas. Clinically acceptable graft size (7.5 mm in diameter) and minimal immunosuppression based on topical and systemic corticosteroids were applied. Rejection signs, histology of porcine grafts, and serial changes in recipients' blood profile, including memory T-cell subset, anti-α-Gal and donor pig-specific antibodies, and complement were evaluated. Changes in aqueous complement concentration were also assessed at 4 weeks after transplantation. RESULTS. Of the decellularized porcine lamellar grafts, 80% remained transparent for more than 6 months, whereas half of the fresh porcine lamellar grafts developed chronic rejection. Rejected grafts showed extensive cellular infiltration, predominantly CD8(+) T lymphocytes and macrophages. Immunologic profiles of the recipients with rejected grafts showed a significant increase in the concentration of aqueous complement, an enhancement of memory T cells, and an abrupt increase in donor pig-specific antibodies. CONCLUSIONS. The findings suggested that decellularized porcine cornea could be a promising substitute for human corneal allograft. Fresh porcine cornea may be a feasible option for a substitute if combined with more potent immunosuppression or if obtained from transgenic pigs with complement-regulatory proteins.

Anti-graft antibodies are often associated with graft rejection. Under special conditions, grafts continue to function normally even in the presence of anti-graft antibodies and complement. This condition is termed accommodation. We developed a xenograft accommodation model in which baby Lewis rat hearts are transplanted into Rag/GT-deficient mice, and accommodation is induced by repeated i.v. injections of low-dose anti-alpha-Gal IgG(1). The accommodated grafts survived a bolus dose of anti-alpha-Gal IgG(1), while freshly transplanted second grafts were rejected. To study the mechanism of anti-alpha-Gal IgG(1)-mediated accommodation, both real-time PCR and immunohistochemical staining revealed elevated expression of DAF, Crry and CD59 in the accommodated grafts. In vitro exposure of rat endothelial cells to anti-alpha-Gal IgG(1) also induced the up-regulation of DAF, Crry and CD59, as revealed by Western blot analyses, and was associated with an acquired resistance to antibody and complement-mediated lysis in vitro. Collectively, these studies suggest that the up-regulation of complement regulatory proteins may abrogate complement-mediated rejection and permit the development of xenograft accommodation.

Purpose: To investigate the antigenicity of porcine corneal stroma as xenograft to man., Methods: The localization of alpha-gal epitope in the porcine eye was determined using biotinylated Griffonia simplicifolia 1 isolectin B4. Porcine corneal stromal was inserted into corneal stromal pockets of cynomolgus monkeys. Immunohistochemistry was performed to analyze the immunological reaction in the monkey., Results: Immunohistochemistry showed no alpha-gal epitope in the porcine cornea except for several keratocytes in the anterior-most part. Haze and keratic precipitates developed in two corneas out of three corneas that were followed up until 6 months after the surgery. In these two corneas, infiltrating cells included CD4+, CD8+, or HAM56+ cells, suggesting that haze and keratic precipitates were induced by cellular rejection to porcine corneal stroma., Conclusions: Porcine corneal stroma induces no hyperacute rejection but mild cellular rejection when transplanted in the cornea of cynomolgus monkeys.

Aim: To provide information on the specificity of induced anti-pig antibodies (Abs) in baboons after exposure and sensitization to pig antigens., Materials and Methods: Baboons (n=7) received either porcine mobilized peripheral blood progenitor cells (n=3), kidney (n=3) or heart (n=1) transplants. After rejection of these cells or organs, pre- and post-rejection sera were analyzed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry to detect and measure anti-Galactosealpha1,3Galactose (Gal) and anti-non-Gal Abs. To study the anti-non-Gal carbohydrate response, the sera were incubated with pig red blood cells pretreated with alpha-galactosidase (to remove Gal) and three other exoglycosidases to remove other potential oligosaccharide epitopes, and studied by flow cytometry. To study the anti-swine leukocyte antigen (SLA) response, non-Gal Abs from two baboons sensitized with kidneys from inbred miniature swine of dd or aa haplotype, respectively, were adsorbed on cells of aa, cc, or dd haplotypes, and binding to aa, cc or dd cells was measured by flow cytometry. Cytotoxicity of anti-non-Gal Abs was tested in vitro by a complement-mediated cytotoxicity assay, using pig cells as targets., Results: In pre-transplant and pre-rejection sera, anti-Gal Abs were detected, but anti-non-Gal Abs were either absent or at minimal levels. After exposure to pig antigens, baboons developed induced anti-Gal and anti-non-Gal Abs. No anti-non-Gal Abs directed to the tested carbohydrate epitopes could be detected. Anti-non-Gal Abs showed minor evidence of specific SLA haplotype reactivity, suggesting that the major Ab response was to pan-pig determinants. Anti-non-Gal Abs showed a low level of complement-mediated lysis of pig cells in vitro., Conclusions: In this limited study, no Ab response to non-Gal carbohydrates was observed, and anti-SLA specificity was minor, indicating that most induced anti-non-Gal Ab was directed against non-specific pig proteins, including SLA-epitopes.