Gene transfer of the Bcl-2 gene confers cytoprotection to isolated adult porcine pancreatic islets exposed to xenoreactive antibodies and complement

Background. Exposing adult porcine pancreatic islets (PI) to xenoreactive n atural antibodies (XNA) induces brisk inflammatory injury that involves act ivation of the complement system. Gene transfer of Bcl-2 has been shown to protect PI from apoptosis and necrosis in several models. In this study, we investigated the effect of Bcl-2 gene transfer on Protection of PI from Pr imate XNA and complement-mediated injury. Methods. The PI were isolated from adult female sows. Only islet preparatio ns that exhibited >90% viability and purity were used. Fresh rhesus monkey serum served as the XNA source. Gene transfer of Bcl-2 was achieved with an adenoviral vector (AdBcl-2) at 500 particle forming units (pfu)/cell. The Bcl-2 expression was confirmed by Western blot technique. Untransfected and transfected PI were incubated in 50% fresh complete serum (CS) or heat-ina ctivated (HI) rhesus serum for 24 hours. The PI viability was analyzed with acridine orange and ethidium bromide staining. Antibody and complement-med iated cytotoxicity were tested by intracellular lactate dehydrogenase (LDH) release. The PI function was assessed in vitro by static incubation studie s and in vivo after intraportal transplantation in diabetic severe combined immunodeficiency (SCID) mice. Results. The AdBcl-2 gene transfer resulted in Bcl-2 gene expression in >90 % of PI cells,. Following exposure to MVA, <15% of the untransfected cells were viable. Similar results were obtained in PI transfected with a similar recombinant adenovirus encoding the reporter gene E coli <beta>-galactosid ase (AdLacZ), an irrelevant gene. A significant increase in LDH release was observed in control PI after, exposure to CS compared with PI that overexp ressed Bcl-2 (82.89%+/-7.78% vs 34.31%+/-5.4%, P<.005). Higher insulin rele ase was observed in vitro in PI transfected with Bcl-2 compared with untran sfected PI or islets transfected with AdLacZ (stimulation index of 0.9<plus /minus>0.31, 0.9 +/-0.3 vs 2.67 +/-0.4, respectively). Only PI heated with AdBcl-2 were able to achieve euglycemia after exposure to XNA and complemen t after transplantation Conclusions. Transfer of the antiapoptotic and antinecrotic Bcl-2 gene into PI can reduce primate XNA and complement-mediated lysis. Cytoprotection of PI with Bcl-2 has potential to improve survival of PI xenotransplants.