Hongji Yang

The role of anti-non-Gal antibodies in the development of acute humoral xenograft rejection of hDAF transgenic porcine kidneys in baboons receiving anti-Gal antibody neutralization therapy.

Background. The present study was undertaken to determine the role of preformed and induced anti-non-Gal antibodies in the rejection of hDAF pig-to-baboon kidney xenotransplants after anti-Gal antibody neutralization therapy. Methods. Seven baboons received life-supporting kidney transplants from hDAF transgenic pigs. Anti-Gal antibodies were neutralized by GAS914 or TPC (a Gal PEG glycoconjugate polymer). Group 1 (n=5) underwent a conventional immunosuppressive therapy with FK506, rabbit anti-thymocyte serum/immunoglobulin, mycophenolate mofetil, and steroids. Group 2 (n=2) received an anti-humoral immunity regimen with LF15-0195, Rituxan and cobra venom factor in addition to ATG, FK506 and steroids. Levels of anti-non-Gal antibodies and their mediated complement-dependent cytotoxic activities (CDC) were detected by flow cytometry using Gal knockout (k/o) pig lymphocytes (LC) or endothelial cells (EC) as targets. Results. Continuous infusion of GAS914/TPC significantly reduced anti-Gal antibodies. In Group 1, four of five baboons developed severe acute humoral xenograft rejection (AHXR) and the rejection was associated with either a high level of preformed anti-non-Gal IgG or a marked elevation in induced anti-non-Gal IgG and IgM. Sera collected at the time of AHXR had a high level of CDC to porcine LC/EC from Gal k/o animals. The intensive anti-humoral therapy in Group 2 completely inhibited both anti-Gal and non-Gal antibody production and prevented AHXR. However, this therapy was not well tolerated by the baboons. Conclusion. In a pig-to-baboon kidney transplant model, both preformed and induced anti-non-Gal antibodies are strongly associated with the pathogenesis of AHXR when anti-Gal antibodies are neutralized.

Monotherapy with LF 15-0195, an analogue of 15-deoxyspergualin, significantly prolongs renal allograft survival in monkeys

Background. LF 15-0195 is a novel, more potent, and less toxic analogue of 15-deoxyspergualin, an antibiotic used as an immunosuppressive agent to prevent rejection of organ transplants. This study was undertaken to determine whether LF 15-0195 monotherapy would prevent renal allograft rejection in a nonhuman primate model. Methods. In the study groups, recipients received LF 15-0195 monotherapy at doses of 0.065 mg/kg per day (group 2, n=4), 0.13 mg/kg per day (group 3, n=4), or 0.2 mg/kg per day (group 4, n=4), administered subcutaneously, on postoperative days 0 to 14. Results. Group 1 consisted of untreated control recipients, all of which developed advanced graft rejection after surviving for an average of 6.5±0.6 days. LF 15-0195 treatment significantly prolonged graft survival in groups 2, 3, and 4, to 20±20 days, 49±5 days, and 39±4 days, respectively. Animals in groups 3 and 4 demonstrated no evidence of rejection during LF 15-0195 treatments. The animals maintained stable renal function for 2 weeks after LF 15-0195 withdrawal but gradually developed rejection at 5 to 6 weeks. Pathologic studies demonstrated that vascular graft rejection was attenuated in LF 15-0195-treated allografts, compared with control specimens. These groups also demonstrated transient reductions in lymphocyte counts during treatment, which returned to normal levels 2 weeks after LF 15-0195 withdrawal. Total serum concentrations of IgM and IgG decreased by a mean of 20.4% and a mean of 31.4%, respectively, at the end of LF 15-0195 treatment (postoperative day 14). LF 15-0195 did not significantly alter thrombocyte counts or hemoglobin levels. Necropsy studies showed no evidence of drug toxicity in the heart, liver, spleen, intestines, stomach, or colon. Conclusions. LF 15-0195 monotherapy significantly prolonged renal allograft survival in monkeys. These encouraging data suggest that this novel agent may be of future value in clinical transplantation.

Improvement in rejection of human decay accelerating factor transgenic pig-to-primate renal xenografts with administration of rabbit antithymocyte serum.

Background. Survival in pig-to-baboon kidney xenotransplantation is currently limited by acute humoral xenograft rejection (AHXR). We hypothesized that the administration of rabbit antithymocyte serum (RATS) would delay or prevent AHXR as compared with a cyclophosphamide (CyP)-based immunosuppressive regimen. Methods. Nine baboons received life-supporting heterotopic single-kidney transplants from human decay accelerating factor transgenic pigs. Immunosuppression consisted of GAS (a galactosyl &agr;-1,3-galactose analog), cyclosporine, and steroids. Group 1 (n=2) was also treated with CyP and a rapamycin derivative (RAD), group 2 (n=4) received RATS and RAD, and group 3 (n=3) received only RATS. Animals were maintained until death or sacrifice because of uncontrollable rejection or other complications. Graft histopathology was assessed at the study endpoint. Results. Mean survival was 28±11.3 days, 23±2.5 days, and 20±2.5 days for groups 1, 2, and 3 (not significant). Graft rejection was the cause of death in both CyP-treated animals. One RATS-treated animal died of rejection; the others died of infections or bleeding. Two RATS-treated animals developed posttransplant lymphoproliferative disorder, and one died of cytomegalovirus pneumonitis. Histopathology revealed severe AHXR in group 1 kidneys, involving 100±0% of the tissue examined. In contrast, AHXR was reduced in groups 2 and 3, involving 21±14% and 18±28%, respectively, of the tissue examined (P <0.01). Conclusions. Substitution of RATS for CyP was well tolerated and resulted in reduced severity of AHXR in this model. Complications seen in RATS-treated animals may be preventable through the use of standard prophylaxis for infections. Our data suggest that further studies are warranted to explore the use of antilymphocyte agents in xenotransplantation.

The influence of baseline expression of human decay accelerating factor transgene on graft survival and acute humoral xenograft rejection.

Background. Transgenic pigs expressing human decay accelerating factor (hDAF) have been widely used as donors in various non-human primate transplant models. Despite the use of similar immunosuppressive protocols, there is marked variation in graft survival among centres. The present study was undertaken to determine whether the level of hDAF expression in the pig kidney correlates with the degree of rejection and duration of graft survival. Methods. hDAF transgenic pigs were provided from two suppliers: Guelph Imutran Centre (G) and Harlan Sprague Dawley (H). Following a bilateral nephrectomy, a single hDAF pig kidney was implanted in the baboon, which was subsequently treated with conventional immunosuppressive protocols. The pig’s contralateral kidney was collected to provide baseline data. The severity of acute humoral xenograft rejection (AHXR) was graded as stage I-III. hDAF expression was measured using morphologic analysis comparing the contralateral and grafted kidneys at the endpoint. Results. Baseline hDAF expression in kidneys from pigs provided by supplier G was significantly higher than that from supplier H (P<0.01). Furthermore, the survival of baboons receiving grafts from G pigs was significantly longer than those receiving grafts from H pigs (P<0.05). In addition, reduction of hDAF expression at the endpoint was associated with a higher degree of AHXR. Severe apoptosis or necrosis was found in grafts with AHXR II-III. Conclusions. Pig kidneys from different suppliers have variable baseline hDAF expression, which may have an influence on graft survival. Reduced expression of hDAF in the terminal graft was associated with the severity of rejection.

Ex vivo and extracorporeal perfusion with hDAF pig kidneys

The present study was undertaken to determine whether human decay accelerating factor (hDAF) transgene would prevent hyperacute rejection (HAR) while perfused with human blood or extracorporeally in baboons. Four hDAF pig kidneys and three non‐hDAF pig kidneys were perfused ex vivo with fresh human blood for 6 h. Additionally four hDAF pig kidneys and four non‐hDAF pig kidneys were extracorporeally perfused in baboons and pigs, respectively, for 3 h. In ex vivo perfusion, the color of hDAF pig kidneys remained pink at the end of 6‐h perfusion and they had normal histology, while non‐hDAF kidneys developed HAR. HDAF pig kidneys had superior function over non‐transgenic pig kidneys. Urine output was 17.31 ± 3.70 ml/h for hDAF pig kidneys, and only 5.81 ± 0.26 ml/h for non‐hDAF kidneys (P < 0.05). Creatinine clearance was 1.16 ± 1.24 ml/min for hDAF kidneys and 0.22 ± 0.15 ml/min for non‐hDAF kidneys (P < 0.05). Other functional data including potassium, urine specific density, and osmolality were normal in the hDAF kidneys, while in non‐hDAF kidneys, serum potassium was elevated to over 9 mmol/l by the end of perfusion (P < 0.01). Non‐hDAF kidneys also lost more sodium through urine than hDAF kidneys (173.67 ± 14.05 mmol/l vs. 109 ± 31 mmol/l, P < 0.05). In the extracorporeal perfusion, all the baboons tolerated the procedure well with normal hemodynamic and hemotologic profiles. These baboons were well until killed 42 to 56 days after perfusion, although their antiporcine antibodies were greatly elevated. We conclude that hDAF transgene protects against HAR, allowing the pig kidney to function normally while perfused with human blood, and that extracorporeal perfusion using hDAF pig kidneys is a safe procedure in baboons.

(1) The role of anti‐non‐Gal antibodies in xenograft rejection in a pig‐to‐baboon kidney transplantation model

It has been well documented that a specific oligosaccharide (Gala1–3Galb1–4GlcNAc; Gal) on the pig cell surface not only causes hyperacute rejection (HAR), but also initiates acute humoral xenograft rejection (AHXR) when pig organs were transplanted into high-order primates, including humans, apes and old-world monkeys [1–3]. Recently, a1,3-galactosyltransferase gene-knockout (Gal-KO) pigs were developed by cloning technology [4–6]. It is expected that Gal-KO pig organs will continue to stimulate anti-graft response against epitopes other than Gal antigens (non-Gal antigens). These antigens could be numerous carbohydrates and xenoproteins on the pig cell surface. However, the role of anti-non-Gal antibodies remains elusive. Based on our presentation at the IXA Symposium of World Transplant Congress in Boston, this review paper summarizes the results of our recent study of pig to baboon kidney transplantation. Group 1: baboons received human decay accelerating factor (hDAF) transgenic pig kidneys and neutralizing Gal Abs therapy. These therapies included GAS914 (a polymeric form of Gal trisaccharide) and TPC (a Gal polyethylene glycol conjugate), and both can neutralize Gal antibodies [7]. Group 2: baboons received Gal K/O pig kidney grafts [8]. Group 3: baboons received hDAF transgenic pig kidneys, which were Gal positive and no neutralizing Gal Ab therapy was given [9]. Baseline immunosuppression for the three groups included either CyP or ATG for induction, either CsA or FK506, either rapamycin or mycophenolate mofetil and steroids for maintenance. In group 4, four non-immunosuppressed baboons received extracorporeal perfusion with hDAF pig kidneys and then were followed up to 6–7 weeks [10].