The introduction of h-DAF transgenic porcine organs into pre-clinical pig-to-primate discordant xenotransplantation has led to complete and reliable abrogation of hyperacute xenograft rejection (HAR). Despite additional heavy immunosuppression however, most xenografts are still lost due to acute vascular rejection (AVR), with current treatment protocols being of only limited value. In a life-supporting model of pig-to-primate kidney transplantation, unmodified (n=8) or h-DAF-transgenic (n=9) porcine kidneys were transplanted into cynomolgus monkeys under cyclophosphamide (CyP), cyclosporine and low-dose steroid immunosuppression. Longest recipient survival was 11 days in the control group and 68 days in the h-DAF transgenic group. Stable initial graft function with recipient survival >4 days was generated in eight animals (two controls and six transgenics). In these animals, plasma complement levels were analyzed during ongoing AVR. Compared with baseline levels, a two-fold increase in C3a levels and a four-fold increase in sC5b-9 levels were measured. In parallel to systemic complement activation, increased deposition of C3 and C5b-9 along with massive staining for recipient IgM immunoglobulins was detected in the xenografts on immunohistochemistry. We conclude that acute vascular xenograft rejection of porcine kidneys in cynomolgus monkeys is associated with classical pathway complement activation following binding of induced recipient anti-porcine antibodies. This complement activation can be observed despite membrane bound expression of human complement regulators in the porcine xenografts. Therefore, additional short-term fluid phase complement inhibition seems necessary for the future development of protocols designed for treatment of AVR in the pig-to-primate combination.

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At present, the major barrier to successful discordant xenotransplantation of unmodified or complement regulator transgenic porcine xenografts is acute vascular xenograft rejection (AVR). AVR is associated with the intragraft deposition of induced recipient xenoreactive antibodies and subsequent complement activation. In a life-supporting pig to primate kidney xenotransplantation setting using h-DAF transgenic donor organs and postoperative immunosuppression, episodes of AVR were either treated with boluses of cyclophosphamide and steroids or with the same regimen supplemented by a three-day course of C1-Inhibitor, a multifunctional complement regulator. In 8 out of 10 animals stable initial graft function was achieved; in all animals one or more episodes of AVR were observed. When, in 4 animals, C1-Inhibitor was added to the standard anti-rejection treatment regimen, AVR was successfully reversed in 6 out of 7 episodes, while in another group of 4 animals receiving the standard anti-rejection treatment 0 out of 4 episodes of AVR responded to treatment. Response to anti-rejection treatment was associated with a significant increase in recipient survival time. We conclude that AVR of h-DAF transgenic porcine kidneys can be successfully treated by additional short-term fluid phase complement inhibition.

Organs transplanted between phylogenetically disparate species, such as from the pig into the primate, are subject to hyperacute rejection (HAR). This form of xenograft rejection is mediated by preformed natural antibodies and is believed to occur invariably in discordant xenografts thus leading to rapid destruction and complete thrombosis of the graft. Recent data, however, have shown that in the porcine to cynomolgus monkey setting, HAR is not inevitably seen after porcine kidney transplantation. The influence of preoperative antiporcine antibody levels in the recipient, cold ischemia time, and donor organ weight on the onset of HAR was investigated by using unmodified large white pigs (aged 3-12 weeks) as organ donors and adult cynomolgus monkeys (aged 1.5-3.5 years) as recipients. Porcine kidney xenotransplantation was performed in either a non-life-supporting model (n=7) or in a life-supporting model (n=8). In both models, no correlation was found between cold ischemia time and HAR. When preoperative anti-porcine antibody levels were investigated, a significant increase in incidence of HAR was observed in animals with elevated anti-porcine IgM (P<0.05) but not IgG levels (P=NS). Interestingly, although 5 of 12 grafts with an organ weight of less than 50 g underwent HAR, none of three grafts with a donor organ weight of more than 70 g showed signs of HAR. In addition, all three larger grafts showed intraoperative and postoperative urine production, although only in 1 (48 g) of the 12 grafts weighing less than 50 g primary graft function was observed. In one animal, a second porcine kidney (23 g) was successfully transplanted (without HAR) immediately after HAR and subsequent removal of a first porcine kidney (20 g). These results indicate that in the porcine to cynomolgus monkey setting anti-porcine IgM rather than IgG anti-porcine antibody levels seem to be of predominant importance for the induction of HAR. By increasing the donor organ size and weight the frequency of the onset of HAR can be at least reduced. This is most likely due to immunoabsorption of the recipients preformed antibodies in the porcine kidney without lethal damage for the graft.

Cold rolling has detrimental effect on the formability of sheet metals. It is, however, inevitable in producing sheet high quality surfaces. The effects of cold rolling on the forming limits of stretch sheets are not investigated comprehensively in the literature. In this study, a through experimental study is conducted to observe the effect of different cold rolling thickness reduction on the formability of sheet metals. Since the experimental procedure of such tests are costly, an artificial intelligence is also adopted to predict effects of cold thickness reduction on the formability of the sheet metals. In this regard, St14 sheets are examined using tensile, metallography, cold rolling and Nakazima’s hemi-sphere punch experiments. The obtained data are further utilized to train and test an adaptive neural network fuzzy inference system (ANFIS) model. The results indicate that cold rolling reduces the formability of the sheet metals under stretch loading condition. Moreover, the tensile behavior of the sheet alters considerably due to cold thickness reduction of the same sheet metal. The trained ANFIS model also successfully trained and tested in prediction of forming limits diagrams. This model could be used to determine forming limit strains in other thickness reduction conditions. It is discussed that determination of forming limit diagrams is not an intrinsic property of a chemical composition of the sheet metals and many other factors must be taken into account.