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M S Sandrin

Publications and source records attributed to M S Sandrin.

At least 19 recordsLinked to original sources
PubMed1998

Peptide mimics of a tumor antigen induce functional cytotoxic T cells.

The ability to mimic peptide/peptide and/or peptide/carbohydrate structures may be important in generating cross-reactive antibodies for autoimmune and other diseases. We show that the peptide sequence DAHWESWL can mimic the conformation of the unrelated MUC1 peptide SAPDTRPAP(G). Mice immunized with mannan-MUC1-peptides make cytotoxic T lymphocytes (CTLs) and are protected from MUC1+ tumors. We show that the same specific anti-MUC1 responses can be produced by immunizing with the DAHWESWL peptide; furthermore, specific tumor protection is obtained in a manner similar to that with MUC1 immunization. The DAHWESWL peptide immunization leads to CTLs that recognize H2Dd and H2Ld but not H2b or human leukocyte antigens-group A (HLA-A) *0201 presented MUC1 peptides. However, mutation of the DAHWESWL peptide to a more HLA-A*0201-compatible structure with appropriate anchors (DLHWASWV), leads to the production of CTLs in HLA-A*0201 mice.

Amino Acid Sequence↗
PubMed1997

Combined transgenic expression of alpha-galactosidase and alpha1,2-fucosyltransferase leads to optimal reduction in the major xenoepitope Galalpha(1,3)Gal.

Hyperacute rejection of pig organs by humans involves the interaction of Galalpha(1,3)Gal with antibodies and complement. Strategies to reduce the amount of xenoantigen Galalpha(1,3)Gal were investigated by overexpression of human lysosomal alpha-galactosidase in cultured porcine cells and transgenic mice. The overexpression of human alpha-galactosidase in cultured porcine endothelial cells and COS cells resulted in a 30-fold reduction of cell surface Galalpha(1,3)Gal and a 10-fold reduction in cell reactivity with natural human antibodies. Splenocytes from transgenic mice overexpressing human alpha-galactosidase showed only a 15-25% reduction in binding to natural human anti-Galalpha(1,3)Gal antibodies; however, this decrease was functionally significant as demonstrated by reduced susceptibility to human antibody-mediated lysis. However, because there is residual Galalpha(1,3)Gal and degalactosylation results in the exposure of N-acetyllactosamine residues and potential new xenoepitopes, using alpha-galactosidase alone is unlikely to overcome hyperacute rejection. We previously reported that mice overexpressing human alpha1,2-fucosyltransferase as a transgene had approximately 90% reduced Galalpha(1,3)Gal levels due to masking of the xenoantigen by fucosylation; we evaluated the effect of overexpressing alpha-galactosidase and alpha1,2-fucosyltransferase on Galalpha(1,3)Gal levels. Galalpha(1, 3)Gal-positive COS cells expressing alpha1,3-galactosyltransferase, alpha1,2-fucosyltransferase, and alpha-galactosidase showed negligible cell surface staining and were not susceptible to lysis by human serum containing antibody and complement. Thus, alpha1, 2-fucosyltransferase and alpha-galactosidase effectively reduced the expression of Galalpha(1,3)Gal on the cell surface and could be used to produce transgenic pigs with negligible levels of cell surface Galalpha(1,3)Gal, thereby having no reactivity with human serum and improving graft survival.

Animals↗
PubMed1997

Down-regulation of Gal alpha(1,3)Gal expression by alpha1,2-fucosyltransferase: further characterization of alpha1,2-fucosyltransferase transgenic mice.

BACKGROUND: In pig-to-primate transplantation, antibody-mediated hyperacute rejection is the consequence of binding of natural antibodies to Gal alpha(1,3)Gal on pig endothelium. The elimination of the Gal alpha(1,3)Gal antigen from pig cells should prevent hyperacute rejection. Using in vitro techniques, we have previously reported that using the alpha1,2-fucosyltransferase gene induces the preferential expression of H substance with a concomitant reduction in the expression of Gal alpha(1,3)Gal. The aim of the present study was to examine the effect of expressing the alpha1,2-fucosyltransferase gene in vivo on Gal alpha(1,3)Gal. METHODS: Three alpha1,2-fucosyltransferase transgenic lines of mice were produced and characterized serologically and histologically. RESULTS: Immunohistological studies showed heavy staining for H substance in liver, spleen, kidney, and heart, with a reduction in staining for Gal alpha(1,3)Gal. In addition, there was a reduction in the binding of human anti-Gal alpha(1,3)Gal antibody to lymphocytes from alpha1,2-fucosyltransferase transgenic mice and a substantial decrease in complement-mediated cytolysis of alpha1,2-fucosyltransferase transgenic lymphocytes when compared with that obtained with normal mice. CONCLUSIONS: The findings have important implications, in that alpha1,2-fucosyltransferase transgenic pigs could be produced as a source for humans. Such pigs should have a reduced expression of Gal alpha(1,3)Gal.

ABO Blood-Group System↗
PubMed1997

Human natural killer lymphocytes directly recognize evolutionarily conserved oligosaccharide ligands expressed by xenogeneic tissues.

BACKGROUND: In discordant xenogeneic species combinations, vascularized transplants are hyperacutely rejected, due to binding of xenoreactive natural antibodies (XNA) to selected tissues of the graft, followed by activation of the complement and coagulation cascades. A major epitope recognized by human XNA is the terminal disaccharide Gal alpha(1,3)Gal. Poorly defined, early cell-mediated events also contribute to recognition and rejection of discordant xenografts, and we have suggested a role of natural killer (NK) lymphocytes in this process. METHODS: Human NK cells were used as effectors in functional assays of adhesion to and lysis of xenogeneic discordant endothelial cells in vitro. Adhesion and lysis inhibition experiments were performed using a large panel of carbohydrates, as well as F(ab')2 fragments of human XNA. COS cells transduced with the porcine alpha-galactosyltransferase were also used as targets for NK cell adhesion. RESULTS: We demonstrate that XNA-reactive carbohydrate epitopes expressed by xenogeneic cells, including Gal alpha(1,3)Gal, are also directly recognized by human NK cells. First, selected carbohydrates in solution displace with comparable efficiency both XNA and NK cell binding to xenogeneic endothelium; second, XNA F(ab')2 fragments selectively inhibit human NK cell adhesion to porcine endothelium, but not to human endothelium; third, unstimulated NK lymphocytes adhere selectively to COS-7 cells expressing the porcine glycosyltransferase that encodes the Gal alpha(1,3)Gal epitope. CONCLUSIONS: Collectively, our findings suggest that humoral and cellular components of the natural immune response against heterologous species independently evolved recognition patterns directed against overlapping carbohydrate determinants.

3T3 Cells↗
PubMed1997

Transgenic approaches for the reduction of Galalpha(1,3)Gal for xenotransplantation.

The major barrier to clinically successful xenotransplantation is the lack of effective therapies aimed at eliminating antibody and complement -dependent hyperacute rejection. This review examines transgenic strategies to eliminate or reduce expression of the major pig to human xenoantigen Galalpha(1,3)Gal such that the epitope is no longer recognized by natural human antibodies, by the use of glycosidases and/or glycosyltransferases that can competitively and effectively inhibit the activity of the alpha1,3galactosyltransferase gene and thereby eliminate the xenoantigen Galalpha(1,3)Gal.

Animals↗
PubMed1997

Natural human anti-Gal alpha(1,3)Gal antibodies react with human mucin peptides.

We have recently demonstrated that both antibodies to Gal alpha(1,3)Gal, and the Gal alpha(1,3)Gal binding lectin (IB4), bind a synthetic peptide (DAHWESWL), there being a similar recognition of carbohydrate and peptide structures. We now report that the anti-Gal alpha(1,3)Gal antibodies and IB4 lectin also react with peptides encoded by mucin genes (MUC 1, 3, 4)-sequences known to be rich in serine, threonine and proline. This activity was demonstrated (1) by the ability of mucin derived peptides to block the reaction of anti-Gal alpha(1,3)Gal antibodies and IB4 lectin with a Gal alpha(1,3)Gal+ pig endothelial cell line; the reactions were specific and did not occur with a random peptide containing the same sequences or with other mucin peptides; (2) by the fact that anti-mucin1 antibodies could react with the Gal alpha(1,3)Gal expressed after transfection of COS cells (Gal alpha(1,3)Gal-,Muc1-) with cDNA encoding the pig alpha, 3galactosyltransferase; and (3) that the IB4 lectin and anti-Gal alpha(1,3)Gal antibodies could react with mucin 1 found on the surface of human breast cancer cells. Thus natural occurring anti-Gal alpha(1,3)Gal antibodies found in all human serum can react with self (Muc1) peptides expressed in large amounts on the surface of tumour cells but not on normal cells. The findings are of interest and serve to explain the previously reported findings that human cells can, at times, express Gal alpha(1,3)Gal; such expression is an artefact, the reaction is due to the phenomenon described herein, i.e. that anti-Gal alpha(1,3)Gal antibodies react with mucin peptides.

Amino Acid Sequence↗
PubMed1996

Switching amino-terminal cytoplasmic domains of alpha(1,2)fucosyltransferase and alpha(1,3)galactosyltransferase alters the expression of H substance and Galalpha(1,3)Gal.

When alpha(1,2)fucosyltransferase cDNA is expressed in cells that normally express large amounts of the terminal carbohydrate Galalpha(1,3)Gal, and therefore the alpha(1,3)galactosyltransferase (GT), the Galalpha(1,3)Gal almost disappears, indicating that the presence of the alpha(1,2)fucosyltransferase (HT) gene/enzyme alters the synthesis of Galalpha(1,3)Gal. A possible mechanism to account for these findings is enzyme location within the Golgi apparatus. We examined the effect of Golgi localization by exchanging the cytoplasmic tails of HT and GT; if Golgi targeting signals are contained within the cytoplasmic tail sequences of these enzymes then a "tail switch" would permit GT first access to the substrate and thereby reverse the observed dominance of HT. Two chimeric glycosyltransferase proteins were constructed and compared with the normal glycosyltransferases after transfection into COS cells. The chimeric enzymes showed Km values and cell surface carbohydrate expression comparable with normal glycosyltransferases. Co-expression of the two chimeric glycosyltransferases resulted in cell surface expression of Galalpha(1,3)Gal, and virtually no HT product was expressed. Thus the cytoplasmic tail of HT determines the temporal order of action, and therefore dominance, of these two enzymes.

Animals↗
PubMed1996

Isolation and characterization of cDNA clones for Humly9: the human homologue of mouse Ly9.

Ly9 is a mouse cell membrane antigen found on all lymphocytes and coded for by a gene that maps to chromosome 1. We previously described the isolation and characterization of a full-length cDNA clone for mouse Ly9. Using cross-species hybridization we isolated cDNA clones encoding the human homologue Humly9. Analysis of the predicted protein sequence suggests that the extra-cellular portion of the Humly9 molecules is composed of four Ig-like domains: a V domain (V) without disulphide bonds and a truncated C2 domain (tC2) with two disulphide bonds, a second V domain without disulphide bonds and a second tC2 with two disulphide bonds, i.e., as V-tC2-V-tC2. The gene encoding Humly9 was mapped to chromosome 1 by analysis of human/hamster hybrids, and more specifically to the 1q22 region by in situ hybridization. The protein sequence data support the view that Humly9 belongs to the immunoglobulin-superfamily subgroup which includes CD48, CD2, and LFA-3.

Amino Acid Sequence↗
PubMed1995

A novel mechanism of retrovirus inactivation in human serum mediated by anti-alpha-galactosyl natural antibody.

Type C retroviruses endogenous to various nonprimate species can infect human cells in vitro, yet the transmission of these viruses to humans is restricted. This has been attributed to direct binding of the complement component C1q to the viral envelope protein p15E, which leads to classical pathway-mediated virolysis in human serum. Here we report a novel mechanism of complement-mediated type C retrovirus inactivation that is initiated by the binding of "natural antibody" [Ab] (anti-alpha-galactosyl Ab) to the carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R expressed on the retroviral envelope. Complement-mediated inactivation of amphotropic retroviral particles was found to be restricted to human and other Old World primate sera, which parallels the presence of anti-alpha-galactosyl natural Ab. Blockade or depletion of anti-alpha-galactosyl Ab in human serum prevented inactivation of both amphotropic and ecotropic murine retroviruses. Similarly, retrovirus was not killed by New World primate serum except in the presence of exogenous anti-alpha-galactosyl Ab. Enzyme-linked immunosorbent assays revealed that the alpha-galactosyl epitope was expressed on the surface of amphotropic and ecotropic retroviruses, and Western blot analysis further localized this epitope to the retroviral envelope glycoprotein gp70. Finally, down-regulation of this epitope on the surface of murine retroviral particle producer cells rendered them, as well as the particles liberated from these cells, resistant to inactivation by human serum complement. Our data suggest that anti-alpha-galactosyl Ab may provide a barrier for the horizontal transmission of retrovirus from species that express the alpha-galactosyl epitope to humans and to other Old World primates. Further, these data provide a mechanism for the generation of complement-resistant retroviral vectors for in vivo gene therapy applications where exposure to human complement is unavoidable.

3T3 Cells↗
PubMed1995

Oxidative/reductive conjugation of mannan to antigen selects for T1 or T2 immune responses.

The induction of CD8+ cytotoxic T lymphocytes (CTLs) is desirable for immunization against many diseases, and recombinant-synthetic peptide antigens are now favored agents to use. However, a major problem is how to induce CTLs, which requires a T1-type response to such synthetic antigens. We report that T1-type (generating high CTL, low antibody) or T2-type (the reciprocal) responses can be induced by conjugation of the antigen to the carbohydrate polymer mannan: T1 responses are selected by using oxidizing conditions; T2 responses are selected by using reducing conditions for the conjugation. Using human MUC1 as a model antigen in mice, immunization with oxidized mannan-MUC1 fusion protein (ox-M-FP) led to complete tumor protection (challenge up to 5 x 10(7) MUC1+ tumor cells), CTLs, and a high CTL precursor (CTLp) frequency (1/6900), whereas immunization with reduced mannan-MUC1 FP (red-M-FP) led to poor protection after challenge with only 10(6) MUC1+ tumor cells, no CTLs, and a low CTLp frequency (1/87,800). Ox-M-FP selects for a T1 response (mediated here by CD8+ cells) with high interferon gamma (IFN-gamma) secretion, no interleukin 4 (IL-4), and a predominant IgG2a antibody response; red-M-FP selects for a T2-type response with IL-4 production and a high predominant IgG1 antibody response but no IFN-gamma.

3T3 Cells↗
PubMed1995

Biochemical studies of pig xenoantigens detected by naturally occurring human antibodies and the galactose alpha(1-3)galactose reactive lectin.

The xenotransplantation of pig organs to humans is now receiving serious consideration because of the shortage of human donors for organ transplants. However, such xenografts would be hyperacutely rejected due to naturally occurring antibodies, present in all human sera, that react with pig antigens on the surface of endothelial cells, leading to complement fixation and the rapid onset of intravascular coagulation. A major target of these human natural antibodies is the terminal nonreducing disaccharide Gal alpha (1,3)Gal, and we now report on the array of molecules that are galactosylated by the alpha 1,3-galactosyltransferase. Pig lymphocytes and endothelial cells (both of which bear Gal alpha(1,3)Gal epitopes) were surface iodinated and the 125I-labeled molecules were precipitated with either human antibodies or the lectin from Griffonia simplicifolia (IB4, which binds to Gal alpha(1,3)Gal epitopes). The precipitated molecules were analyzed by gel electrophoresis and autoradiography. Five major groups of molecules were identified by one-dimensional SDS/PAGE (alpha 220 kDa, beta 160-180 kDa, gamma 120 kDa, delta 64 kDa, epsilon 40 kDa); the beta molecule was different in the 2 cell types (beta 1 of lymphocytes and beta 2 of endothelial cells). Two-dimensional SDS/PAGE analysis revealed that each of these groups of molecules resolved into further species of different charge (presumably due to different glycosylation) and also different molecular mass to give at least 20 different Gal alpha(1,3)Gal+ surface molecules. None of these molecules appeared to be present as disulfide-associated dimers. It is clear that there are many galactosylated molecules on the cell surface; indeed, using longer exposures of the autoradiographs, at least 40 different Gal alpha (1,3)Gal+ molecules could be identified. Several of these molecules are likely to have been identified by others, e.g., the 115-kDa, 125-kDa, and 135-kDa triad identified by Platt. Strategies to overcome hyperacute rejection could include modification or deletion of the alpha 1,3-galactosyltransferase gene, which would simultaneously delete all the Gal alpha(1,3)Gal epitopes on these molecules.

Animals↗
PubMed1995

Tissue expression, structure and function of the murine Ly-6 family of molecules.

Murine Ly-6 molecules are a family of cell surface glycoproteins which have interesting patterns of tissue expression during haematopoiesis from multipotential stem cells to lineage committed precursor cells, and on specific leucocyte subpopulations in the peripheral lymphoid tissues. These interesting patterns of tissue expression suggest an intimate association between the regulation of Ly-6 expression and the development and homeostasis of the immune system. Ly-6 molecules are low molecular weight phosphatidyl inositol anchored glycoproteins with remarkable amino acid homology throughout a distinctive cysteine rich protein domain that is associated predominantly with O-linked carbohydrate. These molecules are encoded by multiple tightly linked genes located on Chr. 15 which have conserved geneomic organization. The in vivo functions of Ly-6 molecules are not known although in vitro studies suggest a role in cellular activation. This review will summarize our understanding of Ly-6 with regard to tissue expression, molecular structure, gene organization and function.

Amino Acid Sequence↗
PubMed1995

Enzymatic remodelling of the carbohydrate surface of a xenogenic cell substantially reduces human antibody binding and complement-mediated cytolysis.

The major obstacle to successful discordant xenotransplantation is the phenomenon of hyperacute rejection (HAR). In the pig-to-primate discordant transplant setting, HAR results from the deposition of high-titre anti-alpha-galactosyl antibodies and complement activation leading to endothelial cell destruction and rapid graft failure. To overcome HAR, we developed an enzymatic carbohydrate remodelling strategy designed to replace expression of the Gal alpha-1,3-Gal xenoepitope on the surface of porcine cells with the non-antigenic universal donor human blood group O antigen, the alpha-1,2-fucosyl lactosamine moiety (H-epitope). Xenogenic cells expressing the human alpha-1,2-fucosyltransferase expressed high levels of the H-epitope and significantly reduced Gal alpha-1,3-Gal expression. As a result, these cells were shown to be resistant to human natural antibody binding and complement-mediated cytolysis.

Animals↗
PubMed1994

Gal alpha(1,3)Gal is the major xenoepitope expressed on pig endothelial cells recognized by naturally occurring cytotoxic human antibodies.

Hyperacute rejection, mediated by natural antibody, is the major barrier to xenotransplantation. The studies reported herein were aimed at evaluating antibody-mediated cytotoxicity and the role of the Gal alpha(1,3)Gal epitope, which we had previously demonstrated was the major epitope of pig cells detected by naturally occurring human antibodies. Also, we had shown that this epitope could be induced in non-expressing cells by the transfection of a cDNA clone encoding alpha(1,3)galactosyl transferase, the enzyme that produces this epitope. The importance of the Gal alpha(1,3)Gal epitope was supported by (1) sugar inhibition studies; (2) complete absorption of cytotoxic antibodies by melibiose-sepharose columns; and (3) the ability of normal human serum to lyse COS cells after transfection with a cDNA clone encoding alpha(1,3)galactosyl transferase. These findings strongly suggest that the majority of cytotoxic human antibodies that would recognize a xenogeneic graft are directed to the Gal alpha(1,3)Gal epitope.

Animals↗