In our studies of life-supporting GalT-KO pig-to-baboon kidneys, we have found that some recipients developed increased serum creatinine with growth of the grafts, without histologic or immunologic evidence of rejection. We hypothesized that the rapid growth of orthotopic pig grafts in smaller baboon recipients may have led to deterioration of organ function. To test this hypothesis for both kidneys and lungs, we have assessed whether the growth of outbred (Yorkshire) organ transplants in miniature swine is regulated by intrinsic (graft) or extrinsic (host environment) factors. Yorkshire kidneys exhibited persistent growth in miniature swine, reaching 3.7x their initial volume over 3 months vs. 1.2x for miniature swine kidneys over the same time period. Similar rapid early growth of lung allografts was observed and, in this case, led to organ dysfunction. For xenograft kidneys, a review of our results suggests that there is a threshold of 25cm3/kg for kidney graft volume/recipient body weight that induces cortical ischemia in transplanted GalT-KO kidneys in baboons. These results suggest that intrinsic factors are responsible, at least in part, for growth of donor organs and that this property should be taken into consideration for growth-curve mismatched transplants, especially for life-supporting organs transplanted into a limited recipient space.

Background Various durations of survival have been observed in the xenotransplantation of life-supporting alpha-1,3-galactosyltransferase knockout (GalT-KO) porcine kidneys into nonhuman primates (NHPs). While others have demonstrated loss of GalT-KO transplanted kidneys within two weeks, we have reported an average survival of 51 days with the co-transplantation of the kidney and vascularized thymus and an average of 29 days with the kidney alone. In order to determine the factors responsible for this difference in survival time, we performed xenogeneic kidney transplantations into cynomolgus monkeys with an anti-CD40L-based regimen using two different strains of GalT-KO swine, one derived from MGH-Miniature swine and the other obtained from Meji University. Materials and Methods Eight cynomolgus moneys received GalT-KO kidneys. Three kidney grafts were from MGH/NIBS GalT-KO pigs and 5 GalT-KO grafts were from MEIJI GalT-KO swine. All cynomolgus recipients were treated identically. Results Recipients of kidneys from the MGH GalT-KO swine, produced by nuclear transfer in Japan, survived an average of 28.7 days, while recipients of MEIJI GalT-KO swine survived an average of 9.2 days. Among the differences between these two groups, one potentially revealing disparity was that the MEIJI swine were positive for porcine-CMV, while the MGH-derived swine were negative. Conclusions This is the first study comparing renal xenotransplantation from two different sources of GalT-KO swine into NHPs at a single center. The results demonstrate that porcine-CMV may be responsible for early loss of GalTKO swine kidney xenografts.

To our knowledge, this is the first evidence of histologically viable porcine lung grafts beyond 7 days in baboons. Our results indicate that GalT-KO pig lungs are highly susceptible to acute humoral rejection and that this may be mitigated by transgenic expression of hCD47.

Background We have recently demonstrated that human‐CD47 (hCD47) expressed on endothelial cells of porcine lung xenografts extended median graft survival from 3.5 days to 8.7 days in baboons. Intra‐bone bone marrow transplantation (IBBMTx) in a pig‐to‐baboon model was previously shown to markedly prolong the duration of macrochimerism up to 21 days from 1 to 4 days by intravenous BMTx. We now examined whether the use of hCD47 transgenic (Tg) BM further prolonged the duration of chimerism following IBBMTx. We then tested if lung xenograft survival was prolonged following IBBMTx. Methods Baboons received GalTKO‐hCD47/hCD55Tg (n = 5) or ‐hCD55Tg (n = 1) or ‐hCD46/HLA‐E Tg (n = 1) pig IBBMTx. Macrochimerism, anti‐pig T cells and antibody responses were assessed. Animals received lung xenografts from either hCD47+ or hCD47‐ porcine lungs 1‐3 months later. Results All baboons that received hCD47Tg porcine IBBM maintained durable macrochimerism >30 days, and two maintained chimerism for >8 weeks. Notably, anti‐pig antibody levels decreased over time and anti‐pig cellular unresponsiveness developed following IBBMTx. Lungs from hCD47Tg IBBMTx matched pigs were transplanted at day 33 or day 49 after IBBMTx. These animals showed extended survival up to 13 and 14 days, while animals that received lungs from hCD47 negative pigs displayed no prolonged survival (1‐4 days). Conclusion This is the first report demonstrating durable macrochimerism beyond 8 weeks, as well as evidence for B cell tolerance in large animal xenotransplantation. Using hCD47Tg pigs as both IBBMTx and lung donors prolongs lung xenograft survival. However, additional strategies are required to control the acute loss of lung xenografts.

Fully MHC-mismatched lungs in CLAWN miniature swine were consistently rejected within 63 days, suggesting that this is a robust large animal model ideal for investigating mechanisms and treatment of lung rejection. Perioperative low-dose CO inhalation prolonged graft survival and inhibited antidonor antibody production and was associated with decreased proinflammatory mediators in this model.

Introduction Lung transplant recipients with documented gastroesophageal reflux disease (GERD) are at increased risk for graft dysfunction. Here, we present the first large-animal model of gastric aspiration following allogeneic lung transplantation and some preliminary data demonstrating the effect of chronic aspiration on the direct and indirect pathways of allorecognition. Methods Left orthotopic lung transplants (n=3) were performed in miniature swine across an MHC class I disparity, followed by 12 days of high-dose cyclosporine (CyA). At the time of transplantation, a trans-tracheal catheter was placed at the carina, above the bronchial anastomosis. A gastrostomy tube was placed for daily aspiration of gastric contents. Subsequently, graft lungs were instilled with gastric aspirate daily (3cc/hr × 8hr/day) for 50 days. Recipients were followed with daily CBC, scheduled chest radiographs, and biopsies. In vitro studies, including cell-mediated lympholysis (CML), mixed lymphocyte reactions (MLR), and peptide proliferation assays (PPA), were performed. Results from these three recipients were compared to those of historical controls (n=6) who were treated identically, except for the tracheal cannulation and simulated gastric aspiration. Results Two of the experimental animals were sacrificed with non-viable lungs soon after the POD 50 biopsy. In both cases the native lung was normal. The third animal survived to 180 days without evidence of chronic rejection. After immunosuppressive treatment, all animals demonstrated donor-specific hyporesponsiveness by assays of direct alloresponse (CML, MLR). A significant response to synthetic donor-derived class I peptide, however, was seen in all animals. A more pronounced and diffuse response was seen in the animals rejecting their grafts. The historical controls showed medium-term graft survival with evidence of chronic rejection in the majority of animals, as previously reported. Conclusion In a model of GERD after lung transplantation, a spectrum of clinical outcomes was observed. The in vitro data suggest that acid reflux enhances the indirect alloresponse to processed donor class I antigen, giving mechanistic insight into the manner in which GERD may be deleterious to the transplanted lung.

In this study, we demonstrated the beneficial effects of perioperative administration of anti-HMGB1 antibody in reducing renal IRI in a clinically relevant, large animal model.

Background Nuclear transfer (NT) technologies offer a means for producing the genetically modified pigs necessary to develop swine models for mechanistic studies of disease processes as well as to serve as organ donors for xenotransplantation. Most previous studies have used commercial pigs as surrogates. Method and Results In this study, we established a cloning technique for miniature pigs by somatic cell nuclear transfer (SCNT) using Nippon Institute for Biological Science (NIBS) miniature pigs as surrogates. Moreover, utilizing this technique, we have successfully produced an α-1, 3-galactosyltransferase knockout (GalT-KO) miniature swine. Fibroblasts procured from a NIBS miniature pig fetus were injected into 1312 enucleated oocytes. The cloned embryos were transferred to 11 surrogates of which five successfully delivered 13 cloned offspring; the production efficiency was 1.0% (13/1312). In a second experiment, lung fibroblasts obtained from neonatal GalT-KO MGH miniature swine were used as donor cells and 1953 cloned embryos were transferred to 12 surrogates. Six cloned offspring were born from five surrogates, a production efficiency of 0.3% (6/1953). Conclusions These results demonstrate successful establishment of a miniature pig cloning technique by SCNT using NIBS miniature pigs as surrogates. To our knowledge, this is the first demonstration of successful production of GalT-KO miniature swine using miniature swine surrogates. This technique could help to ensure a stable supply of the cloned pigs through the use of miniature pig surrogates and could expand production in countries with limited space or in facilities with special regulations such as specific pathogen-free or good laboratory practice.