Mitsuhiro Sekijima

Porcine cytomegalovirus infection is associated with early rejection of kidney grafts in a pig to baboon xenotransplantation model.

Background Recent survivals of our pig-to-baboon kidney xenotransplants have been markedly shorter than the graft survivals we previously reported. The discovery of high levels of porcine cytomegalovirus (pCMV) in one of the rejected xenografts led us to evaluate whether this reduction in graft survival might be because of the inadvertent introduction of pCMV into our &agr;1,3-galactosyltransferase gene knockout swine herd. Methods Archived frozen sections of xeno-kidney grafts over the past 10 years were analyzed for the presence of pCMV, using real-time polymerase chain reaction. Three prospective pig-to-baboon renal transplants using kidneys from swine delivered by cesarean section (C-section) and raised in isolation were likewise analyzed. Results Kidney grafts, from which 8 of the 18 archived samples were derived were found to be pCMV-negative, showed a mean graft survival of 48.3 days and were from transplants performed before 2008. None showed signs of disseminated intravascular coagulopathy and were lost because of proteinuria or infectious complications. In contrast, 10 of the archived samples were pCMV positive, were from kidney transplants with a mean graft survival of 14.1 days, had been performed after 2008, and demonstrated early vascular changes and decreased platelet counts. Three prospective xenografts from swine delivered by C-section were pCMV negative and survived an average of 53.0 days. Conclusions Decreased survivals of &agr;1,3-galactosyltransferase gene knockout renal xenografts in this laboratory correlate temporally with latent pCMV in the donor animals and pCMV in the rejected xeno-kidneys. Transmission of pCMV to swine offspring may be avoided by C-section delivery and scrupulous isolation of donor animals.

Results of life-supporting galactosyltransferase knockout kidneys in cynomolgus monkeys using two different sources of galactosyltransferase knockout Swine.

Background Various durations of survival have been observed in the xenotransplantation of life-supporting &agr;-1,3-galactosyltransferase knockout (GalT-KO) porcine kidneys into nonhuman primates. Although others have demonstrated loss of GalT-KO—transplanted kidneys within 2 weeks, we have reported an average survival of 51 days with the cotransplantation of the kidney and vascularized thymus and an average of 29 days with the kidney alone. 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 Massachusetts General Hospital (MGH)-Nippon Institute for Biological Science (NIBS) GalT-KO pigs and five GalT-KO grafts were from MEIJI GalT-KO swine. All cynomolgus recipients were treated identically. Results Recipients of kidneys from the MGH GalT-KO kidneys swine, produced by nuclear transfer in Japan, survived an average of 28.7 days, whereas recipients of MEIJI GalT-KO kidneys 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 cytomegalovirus, whereas the MGH-derived swine were negative. Conclusion This is the first study comparing renal xenotransplantation from two different sources of GalT-KO swine into nonhuman primates at a single center. The results demonstrate that porcine cytomegalovirus may be responsible for early loss of GalT-KO swine kidney xenografts.

High Incidence of Xenogenic Bone Marrow Engraftment in Pig-to-Baboon Intra-Bone Bone Marrow Transplantation

Previous attempts of α‐1,3‐galactocyltransferase knockout (GalTKO) pig bone marrow (BM) transplantation (Tx) into baboons have demonstrated a loss of macro‐chimerism within 24 h in most cases. In order to achieve improved engraftment with persistence of peripheral chimerism, we have developed a new strategy of intra‐bone BM (IBBM) Tx. Six baboons received GalTKO BM cells, with one‐half of the cells transplanted into the bilateral tibiae directly and the remaining cells injected intravenously (IBBM/BM‐Tx) with a conditioning immunosuppressive regimen. In order to assess immune responses induced by the combined IBBM/BM‐Tx, three recipients received donor SLA‐matched GalTKO kidneys in the peri‐operative period of IBBM/BM‐Tx (Group 1), and the others received kidneys 2 months after IBBM/BM‐Tx (Group 2). Peripheral macro‐chimerism was continuously detectable for up to 13 days (mean 7.7 days; range 3–13) post‐IBBM/BM‐Tx and in three animals, macro‐chimerism reappeared at days 10, 14 and 21. Pig CFUs, indicating porcine progenitor cell engraftment, were detected in the host BM in four of six recipients on days 14, 15, 19 and 28. In addition, anti‐pig unresponsiveness was observed by in vitro assays. GalTKO/pCMV‐kidneys survived for extended periods (47 and 60 days). This strategy may provide a potent adjunct for inducing xenogeneic tolerance through BM‐Tx.

Protective effect of neutralization of the extracellular high-mobility group box 1 on renal ischemia-reperfusion injury in miniature swine.

Background Strategies that reduce ischemia-reperfusion injury (IRI) have the potential to expand the numbers of available organs for transplantation. Recent reports in rodent models have demonstrated that high-mobility group box 1 (HMGB1) acts as an alarm in initiating the inflammatory response resulting from ischemic injury. The aim of this study was to evaluate the cytoprotective effects of anti-HMGB1 antibodies on renal IRI in preclinical large animals. Methods One hundred twenty minutes of warm and 60 min of cold renal ischemia were induced in 8 CLAWN miniature swine. Three of eight animals received intravenous anti-HMGB1 antibody at 1 mg/kg just before the reperfusion of renal blood flow. Renal function was assessed by serum creatinine and renal biopsy. Serum levels of interleukin (IL)-1&bgr;, IL-6, and HMGB1 were measured. Results The concentration of HMGB1 increased as early as 30 min after reperfusion and before the elevation of IL-1&bgr; and IL-6. Serum creatinine levels were markedly elevated, peaking at a median of 5 days (peak creatinine levels: 11.6±1.6 mg/dL) and recovering by day 14. Anti-HMGB1 antibody injection dramatically decreased renal damage as well as serum levels of HMGB1 associated with IRI. Renal function returned to near normal by day 9, and peak creatinine levels were markedly lower (7.4±0.2 mg/dL), and biopsies possessed fewer pathologic changes when compared to the control group. Conclusion 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.

Kidney-induced cardiac allograft tolerance in miniature swine is dependent on MHC-matching of donor cardiac and renal parenchyma.

Kidney allografts possess the ability to enable a short course of immunosuppression to induce tolerance of themselves and of cardiac allografts across a full‐MHC barrier in miniature swine. However, the renal element(s) responsible for kidney‐induced cardiac allograft tolerance (KICAT) are unknown. Here we investigated whether MHC disparities between parenchyma versus hematopoietic‐derived “passenger” cells of the heart and kidney allografts affected KICAT. Heart and kidney allografts were co‐transplanted into MHC‐mismatched recipients treated with high‐dose tacrolimus for 12 days. Group 1 animals (n = 3) received kidney and heart allografts fully MHC‐mismatched to each other and to the recipient. Group 2 animals (n = 3) received kidney and heart allografts MHC‐matched to each other but MHC‐mismatched to the recipient. Group 3 animals (n = 3) received chimeric kidney allografts whose parenchyma was MHC‐mismatched to the donor heart. Group 4 animals (n = 3) received chimeric kidney allografts whose passenger leukocytes were MHC‐mismatched to the donor heart. Five of six heart allografts in Groups 1 and 3 rejected <40 days. In contrast, heart allografts in Groups 2 and 4 survived >150 days without rejection (p < 0.05). These data demonstrate that KICAT requires MHC‐matching between kidney allograft parenchyma and heart allografts, suggesting that cells intrinsic to the kidney enable cardiac allograft tolerance.

Adoptive Transfer of Renal Allograft Tolerance in a Large Animal Model

Our recent studies in an inbred swine model demonstrated that both peripheral and intra‐graft regulatory cells were required for the adoptive transfer of tolerance to a second, naïve donor‐matched kidney. Here, we have asked whether both peripheral and intra‐graft regulatory elements are required for adoptive transfer of tolerance when only a long‐term tolerant (LTT) kidney is transplanted. Nine highly‐inbred swine underwent a tolerance‐inducing regimen to prepare LTT kidney grafts which were then transplanted to histocompatible recipients, with or without the peripheral cell populations required for adoptive transfer of tolerance to a naïve kidney. In contrast to our previous studies, tolerance of the LTT kidney transplants alone was achieved without transfer of additional peripheral cells and without strategies to increase the number/potency of regulatory T cells in the donor. This tolerance was systemic, since most subsequent, donor‐matched challenge kidney grafts were accepted. These results confirm the presence of a potent tolerance‐inducing and/or tolerance‐maintaining cell population within LTT renal allografts. They suggest further that additional peripheral tolerance mechanisms, required for adoptive transfer of tolerance to a naïve donor‐matched kidney, depend on peripheral cells that, if not transferred with the LTT kidney, require time to develop in the adoptive host.

Role of bone marrow maturity, insulin‐like growth factor 1 receptor and forkhead box protein N1 in thymic involution and rejuvenation

Thymic involution is associated with age‐related changes of the immune system. Utilizing our innovative technique of transplantation of a thymus as an isolated vascularized graft in MHC‐inbred miniature swine, we have previously demonstrated that aged thymi are rejuvenated after transplantation into juvenile swine. Here we have studied the role of insulin‐like growth factor (IGF) and forkhead‐box protein‐N1 (FOXN1) as well as bone marrow (BM) in thymic rejuvenation and involution. We examined thymic rejuvenation and involution by means of histology and flow cytometry. Thymic function was assessed by the ability to induce tolerance of allogeneic kidneys. Aged thymi were rejuvenated in a juvenile environment, and successfully induced organ tolerance, while juvenile thymi in aged recipients involuted and had a limited ability to induce tolerance. However, juvenile BM inhibited the involution process of juvenile thymi in aged recipients. An elevated expression of both FOXN1 and IGF1 receptors (IGF‐1R) was observed in juvenile thymi and rejuvenated thymi. Juvenile BM plays a role in promoting the local thymic milieu as indicated by its ability to inhibit thymic involution in aged animals. The expression of FOXN1 and IGF‐1R was noted to increase under conditions that stimulated rejuvenation, suggesting that these factors are involved in thymic recovery.

Transgenic Expression of hCD47 Minimizes the Development of Proteinuria Following Pig-to-Baboon Kidney Transplantation and Reduces Phagocytosis of Porcine Endothelial Cells and Podocytes

Background The early development of proteinuria following pig renal xenotransplantation (XKTx) was a major obstacle to prolonged life-supporting GalTKO XKTx in baboons. We have recently shown that CTLA-4Ig and Rituximab reduce proteinuria in association with downregulatation of the inflammatory response of CD80/SMPDL-3 in porcine glomeruli, allowing GalTKO thymokidneys (TK) without other transgenes (Tg) to function for >6 months. In this study, we addressed the hypothesis that failure of porcine CD47 to provide inhibitory signals to baboon macrophages via SIRP&agr; triggers an inflammatory cascade that promotes proteinuria. We addressed this hypothesis by using hCD47 Tg porcine GalTKO animals as the source of TK. Methods Study 1 (in vivo study to assess the effect of hCD47 Tg on GalTKO TK donor on post-XKTx proteinuria): Two baboons received hCD47+/hCD55+ GalTKO kidneys (Group 1). One received hCD55+ GalTKO kidney (Group 2). As a control (Group 3), 6 baboons received GalTKO kidneys without additional Tg. All animals received an anti-CD154 mAb-based immunosuppressive regimen without CTLA4-Ig. Expression of hCD47 or hCD55 by donor kidneys was assessed histologically. All recipients had anti-non-Gal NAb antibody-complement mediated cytotoxicity lower than 35%. Study 2 (in vitro study): Expression of SIRP&agr; and hCD47 in porcine glomeruli following XKTx was investigated. Phagocytosis of porcine endothelial cells (EC) as well as podocytes with/without hCD47 was assessed in co-culture assays. Results Study 1: No elicited anti-pig ab developed following hCD47/CD55 Tg XKTx in any recipients. All of the recipients in Groups 2 and 3 had to be euthanized before POD70 in association with the development of massive proteinuria (Fig 1A-2, A-1). In contrast, of the two baboons in Group 1, the animal that expressed hCD47 highly in glomeruli (Fig. 2 left) developed only minimal proteinuria (>120 days). The other animal, which expressed hCD47 at a lower level (Fig 2 right) developed only 1+ and occasionally 2+ proteinuria and was euthanized at POD 69 due to graft growth (Fig.1A-3). Study 2: Expression of SIRP&agr; on glomerular cells markedly decreased in GalTKO kidneys following XKTx, while no change in SIRP&agr; was seen in hCD47+ GalTKO kidneys. Statistically significant reductions of phagocytosis of both porcine EC and podocytes were observed when hCD47 was expressed on porcine ECs (top in Fig 3) or podocytes (bottom in Fig 3). Figure. No caption available. Figure. No caption available. Figure. No caption available.Summary and ConclusionsThese results demonstrate that (1) high expression of hCD47 on porcine glomerular cells in xenografts may prevent the development of proteinuria; and (2) the expression of hCD55 alone in kidneys does not prevent early post-XKTx proteinuria in these models. Taken together, these results suggest that interspecies incompatibilities between CD47 and SIRP&agr; induce glomerular endothelial damage that penetrates the glomerular network and ultimately leads to disruption and damage of porcine podocytes.NIH grant P01AI45897.

Inhalation of Carbon Monoxide Protects Liver Against Warm Ischemia-Reperfusion Injury in CLAWN Miniature Swine

Background Carbon monoxide (CO) is produced endogenously as a by-product of heme catalysis, and has been shown to reduce hepatic ischemia-reperfusion injury (IRI) in murine models. We previously reported the beneficial effects of perioperative low-dose inhalation of CO on lung IRI in CLAWN miniature swine. Since CO is well known to be a toxic gas, translational research to evaluate the safety and effectiveness of CO-therapy in various organs using large animals is essential prior to its use in the clinic. The aims of this research were to establish a large animal model of hepatic warm IRI and to evaluate the cytoprotective effects of inhaled CO on ameliorating IRI in this model. Methods Hepatic warm ischemia was induced for 45 minutes by clamping portal vein and proper hepatic artery under the extracorporeal portojugular bypass to prevent visceral congestion. Six CLAWN miniature swine were divided evenly into 2 groups: a CO-treated group and a control group. In the CO-treated group, animals were continuously inhaled with CO from the time of laparotomy until 2 hours after reperfusion (345 minutes in total). CO concentration was adjusted based on COHb levels in the blood (⩽15%). Liver injury was evaluated by serum AST/ALT levels as well as histologies of liver biopsies. Serum cytokine levels (TNF-&agr;, IL-6, HMGB1) were measured to characterize the inflammatory response to hepatic IRI and evaluate the effect of CO on that response. Results Serum AST/ALT levels were markedly increased following 45-minutes hepatic warm ischemia and recovering by day 4. Peak AST/ALT levels of the CO-treated group were significantly lower than those of the control group (control vs CO-treated; AST: 2224 ± 326 vs 456 ± 81 U/L; ALT: 98 ± 50 vs 23 ± 5 U/L; p<0.05). Liver biopsies at 2 hours after reperfusion from the control animals demonstrated hemorrhage, necrosis, congestion and extensive vacuolization of the hepatocytes associated with neutrophil infiltrates. TUNEL staining revealed diffuse apoptotic cells among hepatocytes as well. In sharp contrast, biopsies at 2 hours showed limited areas of congestion and vacuolization or apoptotic cells of the hepatocytes in the CO-treated group. Liver biopsies at 4 days after reperfusion in the control group demonstrated numerous positive PCNA cells indicating active liver regeneration following IRI, while minimal positive PCNA cells in the CO-treated group. Furthermore, CO-therapy successfully inhibited to increase in serum levels of proinflammatory cytokines especially for HMGB1 (control vs CO-treated: 163 ± 56 vs 21 ± 7 ng/mL; 1-hour reperfusion; p<0.05, 164 ± 43 vs 14 ± 2 ng/mL; 2-hour reperfusion; p<0.05). Conclusion To our knowledge, this is the first demonstration that the perioperative administration of low-dose inhaled CO decreases warm IRI in livers in a clinically relevant, large animal model. This protective effect is mediated in part by inhibiting proinflammatory responses and apoptosis of the hepatocytes.

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.