ABSTRACT
BACKGROUND/AIMS: To bridge acute liver failure (ALF) patients to orthotopic liver transplantation, several bioartificial liver (BAL) systems have been developed. The bio-component of most BAL systems consists mainly of porcine hepatocytes. Plasma or blood of ALF patients is perfused through the BAL thereby contacting porcine hepatocytes. Xenogeneic BAL systems may suffer from hyperacute rejection similar to whole-organ xenotransplants. Hyperacute rejection is mediated by antibodies directed against Galalpha(1-3)Gal, a carbohydrate structure present on most mammalian cells. Galalpha(1-3)Gal is produced by the enzyme alpha1,3-galactosyltansferase (alphaGal-T). Conflicting data have been published concerning Galalpha(1-3)Gal expression on hepatocytes in intact porcine liver. We investigated whether isolated porcine hepatocytes express Galalpha(1-3)Gal. METHODS: Immunofluorescence, flow cytometry, RT-PCR and enzyme activity assays were performed on freshly isolated and cultured porcine hepatocytes and liver biopsies. Anti-Galalpha(1-3)Gal antibodies were measured in plasma from patients treated with BAL by ELISA. RESULTS: Isolated porcine hepatocytes express (alphaGal-T) at low levels and Galalpha(1-3)Gal is present in low quantities on these cells, in contrast to hepatocytes in situ. Furthermore, IgG and IgM anti-Galalpha(1-3)Gal are depleted from the plasma of ALF patients during BAL treatment. CONCLUSIONS: Isolation and culture of porcine hepatocytes induce Galalpha(1-3)Gal expression, which may elicit immunological responses potentially compromising BAL functionality.
Subject(s)
Disaccharides/metabolism , Hepatocytes/physiology , Hepatocytes/transplantation , Liver Failure, Acute/therapy , Liver, Artificial , Animals , Antibodies , Bioreactors , Cells, Cultured , Disaccharides/immunology , Flow Cytometry , Fluorescent Antibody Technique , Galactosyltransferases/genetics , Galactosyltransferases/metabolism , Graft Rejection/immunology , Graft Rejection/metabolism , Graft Rejection/physiopathology , Graft Survival , Hepatocytes/cytology , Humans , Liver Failure, Acute/metabolism , Liver Failure, Acute/pathology , RNA, Messenger/analysis , Sus scrofa , Transplantation, HeterologousABSTRACT
The synthesis of sufficient amounts of oligosaccharides is the bottleneck for the study of their biological function and their possible use as drug. As an alternative for chemical synthesis, we propose to use Escherichia coli as a "living factory." We have addressed the production of the Galp alpha(1-3)Galp beta(1-4)GlcNAc epitope, the major porcine antigen responsible for xenograft rejection. An E. coli strain was generated which simultaneously expresses NodC (to provide the chitin-pentaose acceptor), beta(1-4) galactosyltransferase LgtB, and bovine alpha(1-3) galactosyltransferase GstA. This strain produced 0.68 g/L of the heptasaccharide Galp alpha(1-3)Galp beta(1-4)(GlcNAc)(5), which harbours the xenoantigen at its non-reducing end, establishing the feasibility of this approach.
Subject(s)
Antigens/metabolism , Bacterial Proteins , Escherichia coli Proteins/metabolism , Escherichia coli/immunology , Escherichia coli/metabolism , Glycosyltransferases/genetics , N-Acetyllactosamine Synthase/metabolism , Transplantation, Heterologous/immunology , Animals , Antigens/chemistry , Carrier Proteins/metabolism , Cattle , Chromatography , Epitopes , Escherichia coli Proteins/immunology , Gene Transfer Techniques , Intracellular Signaling Peptides and Proteins , Models, Biological , N-Acetylglucosaminyltransferases/metabolism , N-Acetyllactosamine Synthase/immunology , Plasmids/metabolism , Polysaccharides/biosynthesis , Recombinant Proteins/chemistry , Swine , Temperature , Time Factors , TrisaccharidesABSTRACT
The disaccharide galactose(alpha)1,3 galactose (the alphaGal epitope) is the major xenoantigen responsible for the hyperacute vascular rejection occurring in pig-to-primates organ transplantation. The synthesis of the alphaGal epitope is catalyzed by the enzyme alpha1,3-galactosyltransferase (alpha1,3GalT). To be able to control porcine alpha1,3GalT gene expression specifically, we have analyzed the upstream portion of the alpha1,3GalT gene, and identified the regulatory sequences. Porcine alpha1,3GalT transcripts were detected by 5' RACE analysis, and the corresponding genomic sequences were isolated from a phage library. The porcine alpha1,3GalT gene consists of at least 10 different exons, four of which contain 5' untranslated sequence. Four distinct promoters, termed A-D, drive alpha1,3GalT gene transcription in porcine cells. A combination of alternative promoter usage and alternative splicing produces a series of transcripts that differ in their 5' portion, but encode the same protein. Promoters A-C have been isolated, and functionally characterized using luciferase reporter gene assays in transfected porcine endothelial cells (PEC-A). Promoter preference in porcine endothelial cells was estimated on the basis of relative transcript levels as determined by real-time quantitative PCR. More than 90% of the alpha1,3GalT transcripts in PEC-A cells originate from promoter B, which has characteristics of a housekeeping gene promoter. While promoter preference remains unchanged, alpha1,3GalT mRNA levels increase by 50% in 12 h upon tumour necrosis factor alpha-activation of PEC-A cells. However, the magnitude of this change induced by inflammatory conditions could be insufficient to affect cell surface alpha1,3-galactosylation.