Antibody responses to glycolipid-borne carbohydrates require CD4+ T cells but not CD1 or NKT cells.

Naturally occurring anti-carbohydrate antibodies play a major role in both the innate and adaptive immune responses. To elicit an anti-carbohydrate immune response, glycoproteins can be processed to glycopeptides and presented by the classical antigen-presenting molecules, major histocompatibility complex (MHC) Class I and II. In contrast, much less is known about the mechanism(s) for anti-carbohydrate responses to glycolipids, although it is generally considered that the CD1 family of cell surface proteins presents glycolipids to T cells or natural killer T (NKT) cells.

The cytoplasmic and transmembrane domains of secretor type alpha1,2fucosyltransferase confer atypical cellular localisation.

Carbohydrate structures influence many aspects of cell biology. Manipulating the glycosyltransferase enzymes, that sequentially add carbohydrate moieties to proteins and lipids as they pass through the Golgi and secretory pathway, can alter these carbohydrate epitopes. We previously demonstrated that the eight amino acid cytoplasmic tail of alpha1,2fucosyltransferase (FT) contained a sequence for Golgi localisation.

Humans lack iGb3 due to the absence of functional iGb3-synthase: implications for NKT cell development and transplantation.

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 Galalpha(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).

Carbohydrate residues downstream of the terminal Galalpha(1,3)Gal epitope modulate the specificity of xenoreactive antibodies.

Carbohydrates are involved in many immunological responses including the rejection of incompatible blood, tissues and organs. Carbohydrate antigens with Galalpha(1,3)Gal epitopes are recognized by natural antibodies in humans and pose a major barrier for pig-to-human xenotransplantation. Genetically modified pigs have been established that have no functional alpha1,3-galactosyltransferase (alpha1,3GT), which transfers alphaGal to N-acetyllactosamine (LacNAc) type oligosaccharides.

ABO blood group and related antigens, natural antibodies and transplantation.

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.

Recognition of a carbohydrate xenoepitope by human NKRP1A (CD161).

Background: Many immunologically important interactions are mediated by leukocyte recognition of carbohydrates via cell surface receptors. Uncharacterized receptors on human natural killer (NK) cells interact with ligands containing the terminal Galalpha(1,3)Gal xenoepitope. The aim of this work was to isolate and characterize carbohydrate binding proteins from NK cells that bind alphaGal or other potential xenoepitopes, such as N-acetyllactosamine (NAcLac), created by the deletion of alpha1,3galactosyltransferase (GT) in animals.

Lentiviral expression of CTLA4Ig inhibits primed xenogeneic lymphocyte proliferation and cytokine responses.

Background: Co-stimulatory blockade is known to inhibit lymphocyte responses and to prolong allograft and xenograft survival. The present study examines the effect of transgenic expression of cytotoxic T lymphocyte-associated molecule-4 immunoglobulin (CTLA4Ig) by a porcine endothelial cell line (PIEC) transduced by a lentiviral vector, on primed xenogeneic T-cell proliferative and cytokine responses.

Methods: Splenocytes from mice primed with PIEC were used as responder cells in a secondary proliferative assay.

The molecular basis for galalpha(1,3)gal expression in animals with a deletion of the alpha1,3galactosyltransferase gene.

The production of homozygous pigs with a disruption in the GGTA1 gene, which encodes alpha1,3galactosyltransferase (alpha1,3GT), represented a critical step toward the clinical reality of xenotransplantation. Unexpectedly, the predicted complete elimination of the immunogenic Galalpha(1,3)Gal carbohydrate epitope was not observed as Galalpha(1,3)Gal staining was still present in tissues from GGTA1(-/-) animals. This shows that, contrary to previous dogma, alpha1,3GT is not the only enzyme able to synthesize Galalpha(1,3)Gal.

Alpha1,3-galactosyltransferase knockout pigs are available for xenotransplantation: are glycosyltransferases still relevant?

In the early 1990s, the Galalpha(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 (alpha1,3-galactosyltransferase) were the foci of research by many groups. Nearly a decade later, alpha1,3-galactosyltransferase knockout pigs were finally produced; hyperacute rejection could be avoided in these pigs.

Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non-immunosuppressed baboons.

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.

Ligand binding determines whether CD46 is internalized by clathrin-coated pits or macropinocytosis.

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.

Histidine 271 has a functional role in pig alpha-1,3galactosyltransferase enzyme activity.

Alpha(1,3)Galactosyltransferase (GT) is a Golgi-localized enzyme that catalyzes the transfer of a terminal galactose to N-acetyllactosamine to create Galalpha(1,3)Gal. This glycosyltransferase has been studied extensively because the Galalpha(1,3)Gal epitope is involved in hyperacute rejection of pig-to-human xenotransplants. The original crystal structure of bovine GT defines the amino acids forming the catalytic pocket; however, those directly involved in the interaction with the donor nucleotide sugars were not characterized.

The cytoplasmic tail of alpha 1,3-galactosyltransferase inhibits Golgi localization of the full-length enzyme.

It is currently under debate whether the mechanism of Golgi retention of different glycosyltransferases is determined by sequences in the transmembrane, luminal, or cytoplasmic domains or a combination of these domains. We have shown that the cytoplasmic domains of alpha1,3-galactosyltransferase (GT) and alpha1,2-fucosyltransferase (FT) are involved in Golgi localization. Here we show that the cytoplasmic tails of GT and FT are sufficient to confer specific Golgi localization.

The cytoplasmic tail of alpha 1,2-fucosyltransferase contains a sequence for golgi localization.

The Golgi apparatus has a central role in the glycosylation of proteins and lipids. There is a sequential addition of carbohydrates by glycosyltransferases that are distributed within the Golgi in the order in which the glycosylation occurs. The mechanism of glycosyltransferase retention is considered to involve their transmembrane domains and flanking regions, although we have shown that the cytoplasmic tail of alpha1,2-fucosyltransferase is important for its Golgi localization.

Transgenic expression of a CD46 (membrane cofactor protein) minigene: studies of xenotransplantation and measles virus infection.

CD46 (membrane cofactor protein) is a human cell-surface regulator of activated complement and a receptor for the measles virus. A CD46 transgenic mouse line with an expression pattern similar to that of human tissues has been produced, to develop an animal model of (i) the control of complement activation by complement regulators in hyperacute rejection of xenografts, and (ii) measles virus infection. The mouse line was made using a CD46 minigene that includes promoter sequence and the first two introns of genomic CD46, which was coinjected into mouse ova with chicken lysozyme matrix attachment region DNA.

Interactions between the ectodomains of haemagglutinin and CD46 as a primary step in measles virus entry.

Recombinant soluble forms of the ectodomains of measles virus haemagglutinin (sH) and of its receptor CD46 (sCD46) were obtained as a purified disulphide-bonded sH homodimer with an apparent molecular mass of 160 kDa and a purified sCD46 monomer with an apparent molecular mass of 60 kDa, without detectable contamination with moesin. Purified sH bound to purified and immobilized sCD46 and this binding was specifically inhibited by sCD46 in solution. sCD46 bound to wild-type H expressed on the cell surface and inhibited measles virus binding to CD46-expressing cells.

Translation is enhanced after silent nucleotide substitutions in A+T- rich sequences of the coding region of CD46 cDNA.

Specific sequences in the coding region of CD46 (membrane cofactor protein) transcripts have been shown to have a marked effect on translation. Two A+T-rich regions of CD46 cDNA were altered by mutation without changing the CD46 amino acid sequence (silent nucleotide substitution). In one region, the A+T content was reduced from 78% to 55% and in the other a putative polyadenylation addition sequence was disrupted.

Engineering of recombinant soluble CD46: an inhibitor of complement activation.

Human CD46 (membrane cofactor protein) is a type 1 glycoprotein that functions to protect autologous cells from complement-mediated damage by binding C3b and C4b for their factor I-mediated cleavage. We now describe the production and function of recombinant soluble CD46 (rsCD46), which was produced as a truncated form by mutagenesis using the splice overlap extension polymerase chain reaction, by inserting a translational stop codon into the CD46 cDNA at the junction of the transmembrane and extracellular domains. After transfection of an expression construct into 293-EBNA (Epstein-Barr nuclear antigen)-transformed cells, secretion of rsCD46 protein was detected by immunoradiometric assay using monoclonal antibodies.

A functional analysis of recombinant soluble CD46 in vivo and a comparison with recombinant soluble forms of CD55 and CD35 in vitro.

The human cell surface complement regulatory proteins CD46 (MCP), CD55 (DAF) and CD35 (CR1) protect autologous cells from complement-mediated damage by inhibiting C3 and C5 convertases. This regulatory potential has previously been exploited in the treatment of some models of inflammatory injury by the generation of recombinant soluble (rs) proteins, such as rsCD55 and rsCD35 . More recently, we have shown that rsCD46 inhibits complement activation in the fluid phase.

Transcriptional activity of the phosphoenolpyruvate carboxykinase gene decreases in regenerating rat liver.

Rat cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene expression and enzyme activity in liver were studied in rats fasted for 12 hours before and after partial hepatectomy, sham operation or no operation. Transcriptional activity and mRNA levels decreased in regenerating liver compared to sham-operated and unoperated controls. In contrast, PEPCK enzyme activity in regenerating liver was similar to that in the livers of sham-operated and unoperated controls.

Gene expression in regenerating and acute-phase rat liver.

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.