In vitro and in vivoimmunomodulatory effects of cobalt protoporphyrin administered in combinationwith immunosuppressive drugs.

BACKGROUND: Immunosuppressive strategies are designed to take advantage of potential synergies between drugs to possibly decrease the risk of side-effects. In the present study, the ability of Cobalt protoporphyrin (CoPP) to potentiate the effect of the immunosuppressive drugs mycophenolate sodium (MPS) or cyclosporin A (CsA) was explored in vitro and in vivo. METHODS: In vitro analyses of proliferation and apoptosis were performed on primate T cell cultures, following incubation with the immunosuppressive drugs MPS or CsA, alone or in combination with CoPP. In vivo the effect of CoPP and CsA combination therapy was assessed in a rat heterotopic cardiac allotransplantation model. RESULTS: In vitro results suggest that co-administration of CoPP with CsA or MPS increases immunosuppressive effects of these drugs when combined with CoPP. In particular, the co-administration of CoPP with CsA resulted in the synergistic induction of lymphocyte apoptosis. In vivo, animals immunosuppressed with CsA (1.5 mg/kg) or CoPP (20 mg/kg) alone, had a median survival of 7 or 8 days, respectively. In contrast, animals immunosuppressed with CsA (1.5 mg/kg) combined with CoPP (20 mg/kg) had significantly prolonged median survival (12 days), compared to recipients treated with CsA or CoPP alone (p<0.05). CONCLUSION: Our in vitro and in vivo studies demonstrate that CoPP can potentiate the immunomodulatory effects of CsA, ultimately extending allograft survival

Life supporting pig-to-primate xenotransplantation model using transgenic GalT KO pigs: The Padua experience

Background: Survival of pig organs transplanted into primates is limited due to the early occurrence of acute humoral xenograft rejection (AHXR). Generation of a1,3-galactosyltransferase gene-knockout (GTKO) and/or human complement regulatory protein (hCRP) transgenic pigs avoided occurrence of hyperacute rejection in pig-to-primate xenotransplantation. The aim of this study was to investigate the added value deriving from further genetic modifications, including the addition of coagulation regulating molecules, in a life supporting pig to primate renal xenotransplantation model. Methods: Twelve bilaterally nephrectomized cynomolgus monkeys received a kidney from GTKO pigs. Animals in Group 1 (n = 4) received a kidney from GTKO, CD55 donor pigs. Animals in Group 2 (n = 6) were transplanted with a kidney from GTKO pigs transgenic for human CD39, CD55, CD59 and fucosyltransferase (TF) proteins. Animals in Group 3 (n = 2) received a kidney from GTKO pigs transgenic for human CD55, endothelial protein C receptor (EPCR) and thrombomodulin (TM). All recipients received cyclophosphamide (up to 4 doses perioperatively), cyclosporin A (35 mg/kg twice a day), mycophenolate sodium (40\u201360 mg/kg daily) and steroids. Complete hematological and biochemical analyses were routinely performed in all xenotransplanted animals. Pre- and post-transplantation sera were tested for binding of IgM and IgG to donor porcine aortic endothelial cells (PAEC) by flow cytometry. Tissue sections from all explanted kidneys were stained with hematoxylin eosin. The xenografts were also examined for the presence of IgM, IgG, C5b-9 and fibrin by immunohystochemistry. In addition, cellular infiltrates were characterized. Results: Median survival of the animals in the study was 8, 16 and 29 days for Group 1, 2 and 3, respectively. Except for one animal euthanized due to abdominal bleeding, all primates were euthanized in the presence of kidney failure. In all cases a humoral immune response against donor PAEC was observed. Indeed, anti PAEC antibodies IgM and IgG increased as early as on day 4 and 7 days, respectively. Antibodies persisted throughout the post-operative period. Higher antibody titers were observed in Group 2 animals. At euthanasia all xenografted kidneys presented grade II and III AHXR with considerable deposition of IgM, IgG, C5b9 and fibrin. Interestingly, failure in animals from Group 3 occurred in the presence of a more pronounced deposition of IgG. Conclusion: Preliminary data suggest that, in our model, the use of Gal deficient donor kidneys multitransgenic pig with endothelial specific human EPCR and TM proteins extend recipient's survival. This in vivo data confirms that control of coagulation derangements is beneficial in solid organ xenotransplantation

Implementation of the three R's Tenet to primate research: application of a new device as a means of long-term enteral administration of drugs or nutritional supplementation

In experimental research entailing the use of animals, the application of the three R's Tenet (reduction, refinement, replacement) is now diligently adhered to by the scientific community and incorporated in an increasing number of legal frameworks. In particular, the refinement of procedures, by theidentification of strategies that enable the non-stressful administration of food or drugs to nonhuman primates exposed to long-term studies, is strongly recommended. Neuron pig-to-primate xenotransplantation is a complex animal model, requiring oral treatments (and, occasionally, nutritional supplementation) for several months. Sixteen 4–8 year-old purpose-bred cynomolgus monkeys (Macaca fascicularis), weighing between 2.6 and 4.4 kg were used as recipients in xenotransplantation studies and underwent surgical positioning of a surgical placed gastrostomy (SPG). The device consists of an injection port placed subcutaneously on the anterior chest wall and of a silicon valved catheter inserted into the gastric lumen. The device was left in place for a period ranging between 1 and 12 months. In six cases, the SPG was successfully utilized for several months, until the experimental end-point was reached, avoiding the forced handling of nonhuman primates postoperatively. In six cases, the SPG had to be removed prior to reaching the end-point due to local infection at the site of implant that promptly regressed with SPG removal and antibiotic treatment. In two cases, the SPG-implanted primates were euthanized for reasons unrelated to the SPG or the xenotransplantation procedure. Finally, in two ongoing animals the SPG implanted prior to xenotransplantation is currently utilized after more than 3 months in the absence of any complication. The SPG device described minimizes the forced handling of nonhuman primates, otherwise needed to ensure the oral administration of substances by gavage. In this light, the device represents an effective refinement that fully complies with the three R's tenet, that should be considered by Primate Centres exposing nonhuman primates to long-term daily oral administration

Clinicopathological findings in non-human primate recipients of porcine renal xenografts: quantitative and qualitative evaluation of proteinuria

BackgroundImmunological and histopathological features in pig-to-primate renal xenotransplantation are widely studied. Only limited data have been reported about clinicopathological findings in primate recipients of life-supporting renal xenografts. In human medicine, proteinuria represents a common complication in kidney transplantation and is associated with impaired graft survival. The detection of low molecular weight proteins of tubular origin is considered an early method for predicting potential graft rejection. In this study, the presence and the significance of quantitative and qualitative proteinuria were evaluated in xenotransplanted non-human primates in which kidney function was supported only by the transplanted organ.MethodsEight bilaterally nephrectomized cynomolgus monkeys (Macaca fascicularis) were transplanted with a single kidney from α1,3-galactosyltransferase gene-knockout (GTKO) pigs transgenic for human CD39, CD55, CD59, and α1,2-fucosyltransferase. In addition to hematological and biochemical analyses, quantitative and qualitative analysis of proteinuria was evaluated by urinary protein-to-creatinine ratio (UPC ratio) and sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE), respectively.ResultsThe main hematological and biochemical changes recorded after transplantation were a progressive anemia and a severe and progressive decrease in total proteins. In urine samples, the UPC ratio was low before transplantation and increased after transplantation. Similarly, SDS-AGE was negative before transplantation, but bands consistent with mixed (i.e., tubular and glomerular) proteinuria were observed in all samples collected post-transplantation.ConclusionsThe study of clinicopathological changes in cynomolgus monkey renal xenograft recipients provides a valid help in monitoring the health conditions in the post-transplant period. Moreover, the evaluation of UPC ratio and the use of SDS-AGE technique in urine samples of cynomolgus monkey renal xenograft recipients may be considered a valid, inexpensive, and less time-consuming method than more sophisticated techniques in monitoring proteinuria. Proteinuria and presence of low molecular weight (LMW) proteins were consistently found in urine after transplantation, independent of fluctuations in renal function.Laura Pintore, Saverio Paltrinieri, Marta Vadori, Federica Besenzon, Laura Cavicchioli, Giulia Maria De Benedictis, Fiorella Calabrese, Emanuele Cozzi, Mark B. Nottle, Simon C. Robson, Peter J. Cowan and Massimo Castagnar