DOES ISOLATION PROCEDURE MODIFY THE EXPRESSION OF GAL ALPHA 1,3GAL ON PORCINE PANCREATIC ISLETS?

Abstract

P1081 Aims: Gal alpha 1,3Gal (Gal) is the first target for antibody-mediated rejection in pig-to-non-human primate xenograft. The Galactosyl expression on porcine islets remains controversial but is usually reported on vascular endothelial cells in pig pancreas. Although the Gal expression in adult pigs has been reported as low or absent, it seems important to assess whether the Gal expression changed on pig islets from adult pigs prior to and after isolation procedure. In fact, the isolation procedure might damage the vascular structure in pig islets thereby eliminating the expression of Gal. Methods: This work was performed using adult pig (Landrace, 2 years old, n=9) pancreases. Biopsies, prior and after islets isolation were realized. The immunoperoxydase technique using the alpha Gal-specific biotinylated BS-1 isolectin B4 (1:25 dilution; Rayat, 1998) and von Willebrand’s Factor (vWF) were performed respectively for Gal and vasculature structure analysis. Transmission electron microscopy (TEM) was performed to analyse the endothelial ultrastructure prior to and after isolation. Image analysis was undertaken with Scion Image, Beta 4.02, acquisition and analysis software (SCION CORPORATION, Maryland, USA) to determine the percentage of Gal positive proportion and locate the vascular structure in pig islets. Islets were isolated by Liberase PI digestion following a static digestion method. Results: Vascular structure analysis of pig islets demonstrated that capillaries of large islets (>100 μm), essentially found in adult pig donors (mean diameter:169±10μm), were mainly located within the islets between the pancreatic beta cells. After isolation, immunohistochemistry in fact showed that vWF positive staining cells were located in the middle of freshly isolated large islets. Gal staining confirmed that islets endothelial cells essentially expressed Gal epitope and represented a proportion of 16±2% of total islets area in native pig pancreases. After isolation, however, a significant decrease of Gal staining (- 49%) was observed in pig islets but the expression was still positive even in adult pig islets. The decrease of Gal expression could be the consequence of enzymatic pancreatic digestion process at the level of peripheral capillaries (in common with exocrine tissue). Although vWF staining cells were found within islets after isolation, TEM has demonstrated that continuous capillaries structure was lost during isolation process and endothelial cells appeared as single spindle shapes cells. Conclusions: Adult pig islets still expressed the Gal epitope after isolation procedure. The availability of pig deficient in Gal expression as a source of islets may be beneficial for extending islets graft survival in non-human primate models and in humans.