Rapid Destruction of Porcine Islets of Langerhans by Fresh Human Blood

Abstract

484 Previous studies, by us and others have demonstrated only minor effects of human complement active serum on porcine islets in vitro. In this study, adult porcine islets were exposed to fresh non-heparinized human blood in a device originally devised by us for studies on complement activation.Method: Adult porcine islets (API) were isolated, using standard procedures and cultured in RPMI containing 10 % porcine serum for 1-4 days. One, 2, or 5 µl of API (1µl=1000 IEQ) suspended in 100 µl medium, was placed in a circular device consisting of a surface-heparinized cardiovascular plastic tube. Subsequently, 7 ml of nonheparinized fresh human blood was added and the device placed in an incubator at 37°C and rocked back and forth for 5, 15, 30 or 60 minutes. Blood supplemented with 100µl culture medium served as controls. Thrombocytes (TRC), differential leukocyte count, C3a, soluble Terminal Complement Complex (TCC), Thrombin-Anti-Thrombin (TAT) and insulin was measured. Results: The table below summarizes the findings after 60 minutes: When API were placed in the device, there was a rapid consumption of TRC during the first 5-15 minutes and an extensive, visible coagulation could be observed. In parallel, there was a significant increase in C3a, TCC and TAT compared to the controls. By 15-30 minutes there was a selective loss of neutrophils and monocytes with little or no effect on lymphocytes. The changes where equally pronounced whether 1, 2 or 5 µl of islets had been added. Islet cell destruction was manifest within 30 minutes, as evidenced by the release of large amounts of insulin.Conclusion: When mixed with fresh non-heparinized human blood adult porcine islets caused a marked activation of the complement and coagulation systems, and induced consumption of neutrophils and monocytes. During these events the islets were destroyed. The events mimic those seen in hyperacute xenograft rejection. These findings are in marked contrast to those seen in previous in vitro studies, with human serum and may have important implications for clinical islet xenotransplantation with regards to the protective measures required.