Porcine pancreata may be considered a potential source of islets for transplantation into diabetic recipients; however, whether porcine islet grafts will be susceptible to damage by natural antibody‐mediated hyperacute rejection remains unknown. In this study, we performed Western blots to determine whether membrane proteins present on porcine neonatal islet cells (NIC) are recognized by xenoreactive antibodies present in human sera. Western blots of freshly isolated porcine NICs with AB sera detected the presence of 14 antigens (MW 24–164 kDa) and 4 antigens (MW 101–150 kDa) to which antiserum against human IgM and IgG bound, respectively. The most prominent antigens with IgM reactivity had MWs of 36, 63, and 120 kDa, whereas for IgG, the most intensely reactive antigen had a MW of 120 kDa. When membrane fractions prepared from purified porcine aortic endothelial cells and LLC‐PK1 cells were analyzed, the major antigens had molecular weights comparable to those seen for NICs. After culturing the NICs for 5 days, the number of detected xenoreactive antigens binding IgM or IgG decreased and the antigens present at 36, 63, and 120 kDa with IgM reactivity were shown to have a decreased intensity of binding. Incubation of cultured porcine NICs for 18 hr in the presence of human AB serum containing complement resulted in a 55% loss of cellular insulin content (P < 0.0001), a 45% reduction in recoverable DNA (P < 0.0001), and a marked reduction in insulin secretory response to an in vitro glucose challenge. Recovery and viability of porcine NICs was not affected when incubated with AB serum depleted of anti‐Gal antibodies with Synsorb 90. These results demonstrate that natural human antibodies of both IgM and IgG subtypes bind to antigens present on Department of Laboratory Medicine and complement reduces islet cell survival and functional viability. Adsorbing serum with the αGal(1–3)βGal(1–4)βGlc carbohydrate removes natural human antibody‐mediated destruction of porcine neonatal islet cell grafts.
Pig organs express aGal antigen and thus are hyperacutely rejected if perfused by human blood. Human B/A antigens are similar to pig aGal antigen, suggesting that the corresponding antibodies may crossreact. Our purpose was to determine if there is a human ABO blood-group difference in porcine±human xenotransplantation. Plasma from six A, five B, seven AB, and six O individuals pooled by blood group were tested in an ex-vivo porcine working heart model. Blood-group A plasma-perfused hearts survived 20 14 min (n = 5), B 241 9 min (n = 3), AB 151 37 min (n = 5), and O 9 1 min (n = 8). A and O were different (p < 0.001) from B and AB. Function was significantly better in group B. Edema accumulation and creatine kinase change was highest in A and O. All groups had comparable levels of anti-aGal antibody, as well as comparable perfusion and operative conditions. Multivariate linear regression analysis showed the anti-B antibody levels to be predictive of survival (p < 0.001). At higher plasma concentrations, hearts perfused with B plasma survived longer (p =0.01) than AB (218 45 min, n = 4 vs. 6 0 min, n = 3). These results suggest a human ABO blood-group difference in porcine-to-human xenotransplantation, which may be mediated by the anti-A and anti-B antibodies.
The identification of xenoantigens on the surface of endothelial cells is important for understanding the mechanism of hyperacute rejection and development of abrogating methods. The objective of our study was to identify the porcine antigens that, when bound by xenoreactive antibodies in human serum, result in cytotoxicity of porcine cells. Human AB and O sera were adsorbed with porcine aortae, erythrocytes, platelets, and a broad spectrum of immobilized carbohydrate moieties (Synsorbs). Aortae and erythrocytes were able to adsorb the xenoreactive antibodies that were cytotoxic to porcine cells (LLC‐PK1), determined using an MTT cytotoxicity assay. Only carbohydrates having the αGal(1–3)βGal(1–4) moieties (Synsorbs 90, 115) were able to significantly reduce cytotoxicity with both types of sera. Western blots of porcine aortic endothelial cells (PAEC) and LLC‐PK1 cell membrane extracts probed with unadsorbed sera indicate the binding of xenoreactive IgM to approximately 17 and 11 antigen bands, respectively, having molecular weights ranging from 20–133 kDa. Anti‐IgG development showed 8 and 11 antigen bands on PAEC and LLC‐PK1 membrane preparations, respectively. When blots were performed using adsorbed AB sera, the binding to all antigens was still observed. When Synsorb 90 bound antibodies were used to probe the blots, the majority of antigens were still detected. This suggests that the binding of xenoantibodies to the most prominent antigens, as detected by Western blot procedures may not be the ones to which cytotoxic xenoreactive antibodies bind. Alternative approaches are required to identify such antigens.
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