Structural characterization of blood group A glycolipids in blood group A liver tissue in situ perfused with O blood: the dominating presence of type 1 core chain A antigens.

BACKGROUND: Biochemical studies of organ blood group antigen expression show a mixed pattern originating from both the organ tissue and remaining blood cells trapped in the organ despite in vitro perfusion of the vascular tree. The blood group A glycolipid expression was studied in a unique case in which a human liver had been in situ perfused by recipient blood. CASE HISTORY: A blood group O recipient was re-transplanted with an ABO incompatible A1Le (a - b +) liver. Because of discrepancy in size, liver segments II and III were removed 2 h after re-vascularization. Thereafter, the removed A1 liver segment was physiologically in situ perfused with O blood, eliminating a major part of the donor blood cells/plasma. EXPERIMENTAL: Total neutral glycolipids were isolated from the liver tissue and separated by high-performance liquid chromatography. Purified glycolipid fractions were stained with anti-A monoclonal antibodies (mAbs) and structurally characterized by mass spectrometry and proton nuclear magnetic resonance (NMR) spectroscopy. RESULTS: Two blood group A reactive glycolipid compounds were isolated. One component had a thin-layer chromatography (TLC) mobility as a six-sugar glycolipid and reacted with mAbs specific for A type 1 mono-fucosyl structures. The second glycolipid fraction migrated as seven-sugar components and reacted with mAbs specific for type 1 difucosyl (ALeb) as well as Leb determinants. Mass spectrometry of the six-sugar component showed a structure similar to a blood group A hexaglycosylceramide with one fucose. Mass spectrometry and proton NMR spectroscopy of the seven-sugar fraction revealed a mixture of blood group Leb hexa- and ALeb hepta-glycosylceramides, respectively. All fractions were non-reactive with antibodies specific for A antigens based on types 3 and 4 core chain structures. In addition, TLC immunostaining of glycolipids isolated from blood group A livers, harvested for organ transplantation but discarded for various reasons, revealed trace amounts of several A glycolipids with a complex pattern. CONCLUSION: The in situ perfused liver tissue contains blood group A glycolipids based exclusively on type 1 core chains. The secretor gene (Se) codes for a fucosyltransferase acting on all core chain precursors while the H-gene fucosyltransferase only utilizes the type 2 chain precursor. Whether this explains that only A type 1 chain compounds were found has to be established.

Expression of carbohydrate xenoantigens on porcine peripheral nerve.

BACKGROUND: The use of thin easily revascularized cutaneous nerve autografts, which has been the gold standard, or the alternative use of nerve allografts or artificial grafts for nerve reconstructing have all their pros and cons. Nerve xenotransplantation may offer a potential alternative. In a potential pig to human nerve xenograft transplantation set-up several porcine antigen barriers have to be considered such as carbohydrate antigens system like the blood group A/O, the Galalpha1-3Gal (alphaGal) and the Hanganutziu-Deicher (HD) antigens. The swine leukocyte protein antigens system may also have to bee considered. The knowledge of the antigen expression on pig peripheral nerves is today limited. The present study describes the distribution of glycolipid based carbohydrate xenoantigens in ischiadicus nerve from blood group A and O pigs. METHODS: Glycolipid fractions were separated on thin layer chromatography plates and immunostained with human AB sera, biotinylated Griffonia simplicifolia isolectin B4, monoclonal antibodies reacting with the HD antigen and with blood group A antigens based on different core saccharide structures. In addition, the subcellular distribution of alphaGal and HD antigens were studied by light- and electron-microscopical immunohistochemistry. The total amount of neutral glycolipids was 15 mg/g tissue for both blood group A and O nerves with mono-glycosylceramides as the dominating component. RESULTS AND CONCLUSIONS: The total amount of acidic glycolipids (gangliosides and sulpholipids) was 9 mg/g tissue for both the blood group O and A nerves with sulphatides as the dominating components. Analyses of the glycolipid fractions showed strong expression of both the alphaGal and the HD antigens in nerves from both blood group A and O pigs. In addition, small amounts of blood group A antigens were expressed in nerves from blood group A pigs. Staining of neutral glycolipids from blood group A pigs using monoclonal antibodies reacting with A antigen having different core structures suggested that the A epitope expressed on pig ischiadicus nerves is based on the type 1 core chain structure. Light and electron microscopical studies on the alphaGal and HD-antigen distribution revealed that the neural cells were alphaGal antigen negative. Endothelial cells of blood vessels, and lymphatic and perineural cells expressed alphaGal antigen. Both endothelial cells and myelinized axons revealed positively labelled for the HD antigen.

Release of pig leukocytes during pig kidney perfusion and characterization of pig lymphocyte carbohydrate xenoantigens.

The Galalpha1-3Gal (alphaGal) antigen is considered the main xenoantigen in the pig to human species combination but other porcine antigens have to be considered such as the swine lymphocyte antigen (SLA), the blood group A/O and the Hanganutziu-Deicher (H-D) antigens. The H-D antigens are N-glycolyl-neuraminic acid (NeuGc) terminated gangliosides that are widely distributed in mammalian species but absent in humans. Upon exposure to a vascularized pig organ, the human recipient can be immunized by direct interaction with the pig tissue or/and by transfer of tissue/cells from the organ into the recipient. In the present work, we describe the release of cells from porcine kidneys upon perfusion and the expression of glycolipid based alphaGal, blood group A/O and H-D antigens in pig lymphocytes. Pig kidneys were flushed with 20 ml of NaCl or Lidocain containing 5000 U heparin, and thereafter perfused with 3000-ml perfusion solution and the cells released were counted and examined microscopically. Neutral glycolipid and ganglioside fractions were extracted from purified pig lymphocytes. The extracted components were characterized by thin layer chromatography, degradation and mass spectrometry. The expression of alphaGal and H-D epitopes on cells released from pig kidneys and purified pig lymphocytes were studied by immune electron microscopy. A total amount of about 300 x 106 leukocytes, mainly lymphocytes were released in the perfusate from the kidneys, of which about 100 x 106 cells were eluated in the 600 to 2400 ml perfusate fraction. Immunelectron microscopical analysis with Griffonia simplicifolia isolectin B4 showed staining of pig leukocytes and other cells, morphologically similar to endothelial cells, released in the perfusate. The purified porcine lymphocytes contained 930 microg neutral glycolipid (4.2 microg/mg cell protein) of which 95% was glycolipids with one to four sugar residues. Immunostaining of the neutral glycolipid fractions revealed alphaGal terminated compounds migrating in the five and 10 to 12 sugar regions and blood group A compounds in the six and eight sugar regions. Two major gangliosides NeuGc-GM3 and NeuGc-GD3 were found in the pig lymphocytes. In a patient extracorporeally xenoperfused with a pig kidney, an increased staining of both alphaGal terminated structures as well as the H-D reactive gangliosides were found in the post-perfusion serum samples. In summary, leukocytes, mainly lymphocytes are released from pig kidneys during perfusion which may contribute to immunization of human xenograft recipients.