The production of homozygous pigs with a disruption in the GGTA1 gene, which encodes α1,3galactosyltransferase (α1,3GT), represented a critical step toward the clinical reality of xenotransplantation. Unexpectedly, the predicted complete elimination of the immunogenic Galα(1,3)Gal carbohydrate epitope was not observed as Galα(1,3)Gal staining was still present in tissues from GGTA1−/− animals. This shows that, contrary to previous dogma, α1,3GT is not the only enzyme able to synthesize Galα(1,3)Gal. As iGb3 synthase (iGb3S) is a candidate glycosyltransferase, we cloned iGb3S cDNA from GGTA1−/− mouse thymus and confirmed mRNA expression in both mouse and pig tissues. The mouse iGb3S gene exhibits alternative splicing of exons that results in a markedly different cytoplasmic tail compared with the rat gene. Transfection of iGb3S cDNA resulted in high levels of cell surface Galα(1,3)Gal synthesized via the isoglobo series pathway, thus demonstrating that mouse iGb3S is an additional enzyme capable of synthesizing the xenoreactive Galα(1,3)Gal epitope. Galα(1,3)Gal synthesized by iGb3S, in contrast to α1,3GT, was resistant to down-regulation by competition with α1,2fucosyltransferase. Moreover, Galα(1,3)Gal synthesized by iGb3S was immunogenic and elicited Abs in GGTA1 −/− mice. Galα(1,3)Gal synthesized by iGb3S may affect survival of pig transplants in humans, and deletion of this gene, or modification of its product, warrants consideration.

The glycosphingolipid isoglobotrihexosylceramide, or isogloboside 3 (iGb3), is believed to be critical for natural killer T (NKT) cell development and self-recognition in mice and humans. Furthermore, iGb3 may represent an important obstacle in xenotransplantation, in which this lipid represents the only other form of the major xenoepitope Galα(1,3)Gal. The role of iGb3 in NKT cell development is controversial, particularly with one study that suggested that NKT cell development is normal in mice that were rendered deficient for the enzyme iGb3 synthase (iGb3S). We demonstrate that spliced iGb3S mRNA was not detected after extensive analysis of human tissues, and furthermore, the iGb3S gene contains several mutations that render this product nonfunctional. We directly tested the potential functional activity of human iGb3S by expressing chimeric molecules containing the catalytic domain of human iGb3S. These hybrid molecules were unable to synthesize iGb3, due to at least one amino acid substitution. We also demonstrate that purified normal human anti-Gal immunoglobulin G can bind iGb3 lipid and mediate complement lysis of transfected human cells expressing iGb3. Collectively, our data suggest that iGb3S is not expressed in humans, and even if it were expressed, this enzyme would be inactive. Consequently, iGb3 is unlikely to represent a primary natural ligand for NKT cells in humans. Furthermore, the absence of iGb3 in humans implies that it is another source of foreign Galα(1,3)Gal xenoantigen, with obvious significance in the field of xenotransplantation.

Human membrane cofactor protein (CD46) controls complement activation and when expressed sufficiently as a transgene protects xenografts against complement‐mediated rejection, as shown here using non‐immunosuppressed baboons and heterotopic CD46 transgenic pig kidney xenografts. This report is of a carefully engineered transgene that enables high‐level CD46 expression. A novel CD46 minigene was validated by transfection and production of a transgenic pig line. Pig lymphocytes were tested for resistance to antibody and complement‐mediated lysis, transgenic tissues were characterized for CD46 expression, and kidneys were transplanted to baboons without immunosuppression. Absorption of anti‐Galα(1,3)Gal epitope (anti‐GAL) serum antibodies was measured. Transgenic pigs expressed high levels of CD46 in all tissues, especially vascular endothelium, with stable expression through three generations that was readily monitored by flow cytometry of transgenic peripheral blood mononuclear cells (PBMC). Transgenic PBMC pre‐sensitized with antibody were highly resistant to human complement‐mediated lysis which readily lysed normal pig PBMC. Normal pig kidneys transplanted without cold ischemia into non‐immunosuppressed adult baboons survived a median of 3.5 h (n = 7) whereas transgenic grafts (n = 9), harvested at ∼24‐h intervals, were either macroscopically normal (at 29, 48 and 68 h) or showed limited macroscopic damage (median > 50 h). Microscopic assessment of transplanted transgenic kidneys showed only focal tubular infarcts with viable renal tissue elsewhere, no endothelial swelling or polymorph adherence and infiltration by lymphocytes beginning at 3 days. Coagulopathy was not a feature of the histology in four kidneys not rejected and assessed at 48 h or later after transplantation. Baboon anti‐GAL serum antibody titers were high before transplantation and, in one extensively analyzed recipient, reduced ∼8‐fold within 5.5 h. The data demonstrate that a single CD46 transgene controls hyperacute kidney graft rejection in untreated baboons despite the presence of antibody and complement deposition. The expression levels, tissue distribution and in vitro functional tests indicate highly efficient CD46 function, controlling both classical and alternative pathway complement activation, which suggests it might be the complement regulator of choice to protect xenografts.

The integration of growth and the acute-phase response is investigated by comparing the mRNA levels in rat liver during acute inflammation with those after partial hepatectomy. Northern analysis is carried out for the mRNAs for thiostatin, alpha 2-macroglobulin, alpha 1-antitrypsin, inter-alpha-trypsin inhibitor subunit 1, haptoglobin, ceruloplasmin, transferrin, vitamin D-binding protein, alpha 1-acid glycoprotein, beta-fibrinogen, apolipoproteins A-IV and E, albumin, transthyretin, alpha 2-HS-glycoprotein, retinol-binding protein, beta-tubulin, c-myc protooncogene, glyceraldehyde-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, ornithine transcarbamylase, and alcohol dehydrogenase. The acute-phase response dominates during the first 18 h. Changes in mRNA levels related to growth of the liver become important thereafter, and the capacity for an acute-phase response of plasma protein synthesis becomes greatly reduced. The early increase in the level of ceruloplasmin mRNA observed during inflammation is abolished during regeneration, and that of vitamin D-binding protein mRNA is converted into a decrease. The mRNAs levels of glyceraldehyde-3-phosphate dehydrogenase increase, and those for phosphoenolpyruvate carboxykinase decrease during regeneration. Ornithine transcarbamylase mRNA levels are found to exhibit negative acute-phase regulation. The pattern of transcriptional regulation is similar during inflammation and regeneration.

CD46 is a ubiquitous human cell surface receptor for the complement components C3b and C4b and for various pathogens, including the measles virus and human herpes virus 6. Ligand binding to CD46 affects (i) protection of autologous cells from complement attack by breakdown of complement components, (ii) intracellular signals that affect the regulation of immune cell function, (iii) antigen presentation, and (iv) down-regulation of cell surface CD46. Recent evidence indicates that CD46 signaling can link innate and acquired immune function. The molecular mechanisms for these processes and the importance of intracellular trafficking of the receptor have not yet been elucidated. We demonstrate here that, in nonlymphoid cells, CD46 is constitutively internalized via clathrin-coated pits, traffics to multivesicular bodies, and is recycled to the cell surface. However, cross-linking of CD46 at the cell surface, by either multivalent antibody or by measles virus, induces pseudopodia that engulf the ligand in a process similar to macropinocytosis, and leads to the degradation of cell surface CD46. Thus, we have elucidated two pathways for CD46 internalization, which are regulated by the valence of cross-linking of CD46 and which utilize either clathrin-coated pits or pseudopodial extension. This has important implications for CD46 signaling, antigen presentation, CD46 down-regulation, and engulfment of pathogens.

The current success rate of transplant surgery and immunosuppression has led to a demand for organs that has outstripped the supply. This has required investigation of alternate strategies. Therefore, allotransplantation across the ABO blood group barrier has commenced, and pig-to-human xenotransplantation is under consideration. The first immunological barrier to both these types of transplantation is the prevention of the antibody-mediated rejection. This rejection is a result of natural preformed antibodies circulating in the serum of the recipient binding to either ABO (for allo) or alpha-galactose (alpha-Gal) (for xeno) antigens expressed on the donor tissue. These antibodies recognise antigens that are, in both cases, carbohydrate molecules with the characteristic feature that the nonreducing terminal carbohydrate is either a Gal or N-acetlygalactosamine residue in an alpha1,3 linkage. These epitopes are synthesised by closely related members of a single family of glycosyltransferases. This review discusses the carbohydrate antigens, the enzymes involved in their synthesis and the consequences of natural antibodies binding these antigens.

SummaryIn the early 1990s, the Gal α (1,3)Gal carbohydrate linkage was found to be the major xenoepitope causing hyperacute rejection. This carbohydrate, the antibodies that bind to it, and the enzyme that produces it ( α 1,3-galactosyltransferase) were the foci of research by many groups. Nearly a decade later, α 1,3-galactosyltransferase knockout pigs were finally produced; hyperacute rejection could be avoided in these pigs. Having achieved this goal, enthusiasm declined for the study of glycosyltransferases and their carbohydrate products. To examine whether this decline was premature, we evaluate whether gene deletion has indeed solved the initial rejection problem or, in fact, created new problems. This review addresses this by examining the impact of the gene deletion on cell surface carbohydrate. Surprisingly, Gal α (1,3)Gal is still present in α 1,3-galactosyltransferase knockout animals: it is possibly synthesized on lipid by iGb3 synthase. Furthermore, removal of α Gal resulted in the exposure of the N-acetyllactosamine epitope. This exposed epitope can bind natural antibodies and perhaps should be capped by transgenic expression of another transferase. We believe the continued study of glycosyltransferases is essential to examine the new issues raised by the deletion of α 1,3-galactosyltransferase.

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