Intra-bone Bone Marrow Transplantation in Pig-to-Nonhuman Primates for the Induction of Tolerance Across Xenogeneic Barriers.

Mixed chimerism and thymic tissue transplantation strategies have achieved xenogeneic tolerance in pig-to-mouse models, and both have been extended to pig-to-baboon models. A mixed chimerism strategy has shown promise toward inducing tolerance in allogeneic models in mice, pigs, nonhuman primates (NHP), humans, and a rat-to-mouse small animal xeno-model. However, even though α-1,3-galactosyltransferase gene knockout (GalTKO) pigs have been used as bone marrow (BM) donors, direct intravenous injection of porcine BM cells was detected for only up to 4 days (peripheral macro-chimerism) in one case, and the rest lost chimerism within 2 days.Recent data in allogeneic models demonstrated that direct injection of donor BM cells into recipient BM spaces (intra-bone bone marrow transplantation: IBBMTx) produces rapid reconstitution and a higher survival rate compared to i.v. injection. In order to minimize the loss of injected porcine BM peripherally before reaching the BM space, Yamada developed a xeno-specific regimen including IBBMTx coated with a collagen gel matrix in a preclinical pig-to-baboon model (Yamada IBBMTx). This strategy aims to achieve improved, persistent macro-chimerism as well as engraftment of BM across a xenogeneic barrier. The initial study published in 2015 demonstrated that this IBBMTx strategy leads to markedly prolonged peripheral macro-chimerism detectable for up to 23 days. Furthermore, a more recent study using human CD47-transgenic (Tg) GalTKO pigs as xeno-donors achieved long-lasting macro-chimerism >60 days with evidence of reduction of anti-pig natural antibodies (nAb). This is the longest macro-chimerism that has ever been achieved in a preclinical large animal xenotransplant model to date. In this chapter, we introduce a brief summary of our achievements in regard to successful tolerance induction by utilizing our novel strategy of IBBMTx as well as describe the step-by-step methodology of surgical and in vitro procedures that are required for this project.

Transgenic expression of human CD47 reduces phagocytosis of porcine endothelial cells and podocytes by baboon and human macrophages.

BACKGROUND: Our initial studies utilizing a galactosyl-α1-3-galactosyltransferase gene knockout (GalTKO) pig-to-baboon renal transplant model demonstrated that the early development of nephrotic syndrome has been a significant obstacle to the long-term survival of baboon recipients. We have recently documented that sphingomyelin phosphodiesterase-3 (SMPDL3b) and CD80 expressed on podocytes in porcine kidney grafts contribute to this complication. We have hypothesized that one regulator of immune function is CD47 and that incompatibilities in CD47 between pig and baboon could potentially affect macrophage function, increasing the susceptibility of the kidney grafts to immunologically induced injury. METHODS: In order to address this hypothesis in vitro, we isolated and cultured porcine podocytes and ECs from GalTKO alone, human CD47 (hCD47)/hCD55 expressing transgenic (Tg) GalTKO swine, and GalTKO hCD46/hCD55 Tg swine along with baboon or human macrophages. RESULTS: We found that baboon macrophages phagocytosed porcine ECs in a similar manner to human macrophages, and this response was significantly reduced when porcine ECs and podocytes expressed hCD47/hCD55 but not hCD46/hCD55 without hCD47. Furthermore, masking hCD47 by anti-hCD47 antibody on hCD47/hCD55Tg ECs restored phagocytosis. These results are consistent with the hypothesis that CD47 incompatibility plays an important role in promoting macrophage phagocytosis of endogenous cells from the transplanted kidney. CONCLUSIONS: The similar levels of phagocytosis of porcine cells by baboon and human macrophages suggest that the expression of hCD47Tg on glomerular cells in donor porcine kidneys may prove to be a key strategy for preventing proteinuria following kidney xenotransplantation in a pig-to-human as well as a pig-to-baboon model.

Intra-bone bone marrow transplantation from hCD47 transgenic pigs to baboons prolongs chimerism to >60 days and promotes increased porcine lung transplant survival.

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.

The Protective Effects of Carbon Monoxide Against Hepatic Warm Ischemia-Reperfusion Injury in MHC-Inbred Miniature Swine.

BACKGROUND: The development of treatment strategies to protect against ischemia-reperfusion injury (IRI) to livers is important not only for liver surgeries but also in regard to increasing the utilization of livers from marginal donors. In this study, we examined whether inhalational carbon monoxide (CO) therapy reduced IRI after a 45-min (min) warm ischemia (WI) in a miniature swine model. MATERIALS AND METHODS: Six CLAWN miniature swine underwent a 45-min hepatic WI induced by clamping the portal vein and proper hepatic artery. Three animals were subjected to control conditions while the remaining three were treated with CO inhalation for a total of 345-min, including 120-min after reperfusion to maintain a concentration of CO-Hb under 15% (CO-treated group). IRI of the livers was evaluated by liver function tests, serum pro-inflammatory cytokines, and liver biopsies. RESULTS: All controls had statistically significant increased levels of liver enzymes compared to the CO-treated group (p < 0.05). In controls, liver biopsies at 2 h after reperfusion showed marked histological changes including diffuse hemorrhage, congestion, necrosis, vacuolization, and neutrophil infiltration with apoptosis. In contrast, the CO-treated group showed less obvious or only minimal histological changes. Furthermore, increases in high-mobility group box 1, TNF-α, and IL-6 in sera that were induced by IRI in controls were markedly inhibited by the CO treatment. CONCLUSION: We demonstrated that low-dose CO inhalation reduces hepatic warm IRI, potentially through downregulation of pro-inflammatory mediators and activation of anti-apoptotic pathways. To our knowledge, this is the first report demonstrating CO inhalation attenuated hepatic IRI following WI in a large animal model.

Preparation of hybrid porcine thymus containing non-human primate thymic epithelial cells in miniature swine.

BACKGROUND: We have achieved greater than a 6-month survival of a life-supporting kidney co-transplanted with a vascularized thymic graft into non-human primates (NHPs). Although we have achieved pig-specific unresponsiveness in vitro, immunosuppression was not able to be fully weaned. Studies in mice and humanized mice suggest that a hybrid pig thymus (Hyb-thy)-containing host thymic epithelial cells (TECs) can optimize intra-thymic selection, achieving xenograft tolerance with improved reconstitution of T-cell function. METHODS: We have tested the feasibility of the preparation of a Hyb-thy that contains NHP TECs in the donor thymic grafts. We first prepared the Hyb-thy in the donor pigs 2-3 weeks before xeno-Tx. We performed six cases of Hyb-thy preparation in six juvenile miniature swine. Two pigs received non-manipulated cynomolgus monkey thymic cells that were isolated from an excised atrophic thymus via injection into their thymic lobes (Group 1). The remaining four received thymic cells that were isolated from non-atrophic thymic glands (Groups 2 and 3). Pigs in Group 2 received unmanipulated thymic cells in one thymic lobe, as well as CD2-positive cell-depleted TEC-enriched cells in the contralateral lobe. Pigs in Group 3 received TEC-enriched cells alone. RESULTS: All thymus-injected pigs received tacrolimus and rapamycin until endpoint (POD16). We detected cynomolgus monkey TEC networks in pig thymus from Groups 1 and 3, while pigs in Group 2 rejected the thymic cells. We demonstrated the preparation of Hyb-thy in pigs using tacrolimus plus rapamycin therapy. CONCLUSIONS: Our results suggest that the enrichment of TEC from the excised NHP thymus facilitated NHP TEC engraftment in pig thymus.

Upregulation of CD80 on glomerular podocytes plays an important role in development of proteinuria following pig-to-baboon xeno-renal transplantation - an experimental study.

We have previously reported that co-transplantation of the kidney with vascularized donor thymus from α-1,3-galactosyltransferase gene knockout pigs with an anti-CD154 with rituximab-based regimen led to improved xenograft survival in baboons with donor-specific unresponsiveness. However, nephrotic syndrome emerged as a complication in which the glomeruli showed mild mesangial expansion with similarities to minimal change disease (MCD) in humans. Since MCD is associated with CD80 expression in glomeruli and elevated urinary excretion, we evaluated a potential role for CD80 in xenograft nephropathy. Study 1 confirmed high urinary CD80 excretion in nephrotic animals with renal xenografts showing CD80 expression in glomeruli. In Study 2, baboons receiving xenografts received CTLA4-Ig once a week from the second postoperative week or no CTLA4-Ig. The non-CTLA4-Ig group developed severe proteinuria with modest mesangial expansion with high urinary excretion of CD80 and documented CD80 expression in glomerular podocytes. All of the recipients in non-CTLA4-Ig groups had to be euthanized before POD 60. In contrast, CTLA4-Ig group showed a marked reduction in proteinuria and survived significantly longer, up to 193 days. These results demonstrate that anti-CD80 targeted therapy represents a promising strategy for reduction of proteinuria following renal xeno-transplantation with improved survival.

Estimation of mortality risk for vascular operations by trainees vs attending surgeons.

OBJECTIVE: The development of the ability to estimate patient mortality preoperatively in surgical trainees has not been well studied. DESIGN: Prospective comparative study in which the expected perioperative mortality risk and the maximum tolerable mortality at which operation would still be offered were estimated by the operating surgeons immediately before planned vascular procedures. SETTING: University vascular surgery teaching service. PARTICIPANTS: Predicted and maximum allowable mortality risks were compared between trainees and attending surgeons, with the mortality calculated using the Veterans Administration Surgical Quality Improvement Program (VASQIP) as a reference. RESULTS: Surveys were performed before 379 procedures over a 10-month period. The median expected mortality risk predicted by trainees (2%; interquartile range [IQR]: 1%-5%) was higher than the risk predicted by attending surgeons (1%; IQR: 0.8%-3%) (p < 0.01). The median expected mortality risk calculated by VASQIP (0.8%; IQR: 0.4%-1.7%) was less than that estimated by trainees by a median of 0.3% (IQR: 0.2%-3.2%) or and that by attending surgeons by 0.3% (IQR: 0.2-1.3%) (p < 0.01). The median maximum tolerable mortality risk predicted by trainees (10%; IQR: 5%-27.5%) was equal to the risk predicted by attending surgeons (10%; IQR: 5%-17.5%). The perioperative mortality calculated by VASQIP exceeded the maximum tolerable mortality offered by trainees or attending surgeons in 1% of cases each. Discrepancies between expected mortality and maximum tolerable mortality for trainees and attending surgeons were greater for younger (postgraduate year 1 or 2) trainees (0.8%; IQR: 0-3.0%) than for more senior (postgraduate year 4 or 5) trainees (0.4%; IQR: 0.1%-2.0%). CONCLUSION: Surgeons in training overestimated the perioperative mortality risk of operations and were willing to tolerate a greater mortality risk compared with attending surgeons. Both trainee and attending surgeons tended to overestimate the perioperative mortality risk compared with that calculated by VASQIP.