Carbohydrate antigens and xenotransplantationAspects of preparation, analysis and pharmaceutical administration of glyco-sphingolipids. Mechanisms of hyperacute xenorejection in rodent models

Abstract

Cell surface carbohydrates are important antigens in transfusion and transplantation. In pig to human xenotransplantation, carbohydrate antigens contribute to hyperacute rejection of the graft. The role of carbohydrate antigens in small animal models for xenotransplantation, previously poorly researched, was investigated. Glycolipid based carbohydrate antigens of donor tissues were prepared and characterised by mass spectrometry, 1H-NMR spectroscopy and thin layer chromatography immuno- and chemical staining. The anti-carbohydrate antibody response was studied using well characterised donor and other glycolipids. Pig kidney glycolipids were comprehensively isolated by HPLC. A new sensitive electrospray ionisation mass spectrometry technique was specially developed for glycolipid analysis.The organ of biosynthesis for the Lewis glycolipid antigens in human blood is disputed. To study the possible reabsorption of these antigens from degraded shed intestinal epithelial cells, Lewis negative rats were fed large amounts of pure Leb glycolipids. There was a transient absorption of the glycolipids to the rat intestinal lining, but no transfer to the systemic circulation could be detected. Most of the Leb glycolipid was excreted intact. Uptake of Leb glycolipids onto rat erythrocytes was studied by serology.Serum components inducing hyperacute rejection in the vascularised concordant mouse heart to rat xenotransplantation system was studied by decomplementation and passive immunoglobulin transfer. Hyperacute rejection was induced by intravenous injections of immunoglobulin preparations, and abrogated by complement depletion. The ability to induce hyperacute rejection was carried by the IgG, and probably not the IgM, fraction of rat serum. Two antigens, a possibly proteinaceous erythrocyte antigen and an unidentified lymphocyte antigen, were identified as potential causes of induced hyperacute rejection.IgG and IgM antibodies towards the Forssman glycolipid, present in mouse vascular tissue, were found in serum from grafted rats, contrasting with only low titre IgM anti-Forssman in naive rats. Attempts were made to neutralise anti-Forssman antibodies with soluble and solid phase saccharides. The Forssman antigen, as shown by immunoelectron microscopy and mouse endothelial primary cell culture, was not expressed on mouse endothelial cells, but on a perivascular macrophage-like cell with dendritic morphology. Anti-Forssman antibodies themselves did not induce hyperacute rejection.Rat natural IgM antibodies also bound guinea pig Forssman glycolipid.