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M E Breimer

Publications and source records attributed to M E Breimer.

At least 19 recordsLinked to original sources
PubMed1998

Biotinylated platelets have an impaired response to agonists as evidenced by in vitro platelet aggregation tests.

Biotinylation of platelets, using a water soluble biotin analogue which reacts with primary amines, has been proposed to be a reliable technique for study of in vivo survival of platelets and their subpopulations. The information about the influence of this technique on platelet function has been limited. In the present work we studied the effect of in vitro biotinylation on platelet function and activation. Washed human platelets, at a concentration of 1 x 10(9)/L, were biotinylated with five different concentrations of sulfo-NHS-biotin or NHS-LC-biotin, ranging from 0 to 5 mM. The degree of platelet activation during and after biotinylation was monitored by measuring the externalization of P-selectin, and the platelet function was evaluated by aggregometry. It was observed that biotinylated platelets, in a dose dependent manner, displayed an impaired aggregation response. A slight increase in platelet membrane P-selectin occurred during the labelling procedure.

Adenosine Diphosphate↗
PubMed1998

Electrospray ionization and collision-induced dissociation time-of-flight mass spectrometry of neutral glycosphingolipids.

A series of native naturally occurring neutral glycosphingolipids has been analysed by electrospray ionization tandem mass spectrometry using a hybrid magnetic sector-TOF instrument. The collision-induced dissociation products of precursor ions were detected by an orthogonal acceleration time-of-flight mass spectrometer as the second analyser. Glycosphingolipids, with mono- to hexa-saccharide chain lengths with different ceramide constituents, were studied. The result of electrospray ionization in the positive ion mode generally showed singly charged molecular ions with Na+ as adduct, [M + Na]+. The sensitivity of the electrospray ionization was greatly enhanced by addition of NaCl, LiCl (forming [M + Li]+) or KCl (yielding [M + K]+) to the sample. A comparison between the collision-induced dissociation of precursor molecular ions of monoglycosylceramides, using Na+, Li+ and K+ as adducting species, showed that the intensity of the fragment ions and the extent of the daughter ion fragmentation of the molecular ions, are dependent on the type of adduct used. The daughter ion spectra of Li+ adduct ions showed intense sequence fragment ions, both of the saccharide chain and the ceramide moiety, and were superior to those obtained using Na+ or K+. The collision-induced dissociation spectra of the [M + Li]+ ions, of glycosphingolipids containing di- to hexasaccharides, are also presented. Proposed possible fragments, resulting from the CID of the molecular ions [M + Li]+ of monoglycosylceramides, are shown.

Animals↗
PubMed1998

Characterization of a mouse monoclonal IgG3 antibody to the tumor-associated globo H structure produced by immunization with a synthetic glycoconjugate.

Globo H (Fuc alpha1 --> 2Galbeta1 --> 3GalNAcbeta1 --> 3Gal alpha1 --> 4Galbeta1 --> 4Glc) is a carbohydrate structure that shows enhanced expression in many human carcinomas. From mice immunized with a globo H-KLH (keyhole limpet hemocyanin) synthetic conjugate an IgG3 monoclonal antibody (mAb VK-9) was derived that recognizes the globo H structure. Serological analysis showed that the minimal structure recognized by this mAb was the tetrasaccharide sequence Fuc alpha1 --> 2Galbeta1 --> 3GalNAcbeta1 --> 3Gal. An isomeric structure with an internal alphaGalNAc linkage was also recognized but less efficiently. mAb VK-9 did not react with many related structures, such as galactosylgloboside, globoside, H type 1, H type 2 blood group structures or fucosyl-gangliotetraosyl ceramide, but did react weakly with globo A ceramide. Not only did mAb VK-9 react with carbohydrate-protein conjugates but it could also recognize globo H-ceramide and human tumor cells expressing globo H. These results suggest that globo H-KLH could be explored as a vaccine in the treatment of carcinoma patients.

Animals↗
PubMed1998

An ELISA technique for quantitation of human xenoantibodies binding to pig cells: application in patients with pig kidneys extracorporeally connected to the circulation.

A quantitative ELISA technique for determination of human anti-pig xenoantibody number in serum samples has been established using pig lymphocytes and pig/rabbit erythrocytes as target cells and a pool of serum from human blood group AB donors. The number of low affinity antibodies binding to the cells was determined by quantitation following the use of aqueous washing of the cells and separation of bound and unbound antibodies with the phthalate oil method. The efficiency of different soluble Gal(alpha)1-3Gal-terminating di- and tri-saccharides to inhibit antibody binding was tested and found to vary between 70-90% at a saccharide concentration of 10 mg/ml. The assay was used to evaluate the antibody changes in two patients who, after plasmapheresis treatments, had pig kidneys extracorporeally connected to their blood circulation. The number of anti-pig IgM/IgG antibodies bound to each pig lymphocyte were reduced from 5,600/13,200 to 1,300/3,100 in patient 1 and from 1,200/6,500 to 500/2,100 in patient 2 by three consecutive daily plasmapheresis treatments. Although the lymphocytotoxic titers were reduced to very low levels, the antibody numbers still present in the blood of patient 1 caused a hyperacute rejection of the pig kidney. However, the antibody levels in patient 2 did not cause rejection of this kidney during 15 min perfusion time. A strong anti-pig antibody response 3 weeks after the perfusion experiment was found in patient 1 as shown by 27,600/245,300 IgM/IgG molecules bound to pig lymphocytes corresponding to an increase of lymphocytotoxic titer from 8 to 512. The second patient showed a much weaker immune response with 1,400/19,800 IgM/IgG antibodies corresponding to a lymphocytotoxic titer increase from 8 to 32. The use of this quantitation technique enables more accurate investigation of antibody binding to xenogenic target cells than conventional titration techniques.

Animals↗
PubMed1998

Characterisation of human natural anti-sheep xenoantibodies.

Currently, the pig species is regarded as the most likely organ donor for human xenotransplantation in the future. However, it cannot be granted that the pig will be the optimal species of choice. We have studied human anti-sheep antibodies in comparison with anti-pig antibodies. The anti-sheep lymphocytotoxic and hemagglutination titers were in the range 8 to 128 and 2 to 32, respectively, in single individuals, which were considerably lower than the anti-pig titers of these individuals. Perfusion of sheep kidneys with human blood reduced the anti-sheep xenoantibody titers to zero as measured by lymphocytotoxic, hemagglutination, and sheep aortic endothelial cell antibody binding assays. The perfused kidneys showed generalised depositions of human IgM and C3c in the vascular tree and focal depositions of C1q and fibrin. Obliteration of capillaries by human platelets and polymorphonuclear cells were observed. Total neutral glycolipid fractions were isolated from sheep intestinal, pancreatic, and kidney tissues. By using a chromatogram binding assay, a monoclonal anti-Forssman antibody identified a single compound with five sugar residues in all organs. Several glycolipid bands were stained in all organs by the Gal(alpha)1-specific lectin I-B4 from Griffonia (Bandeiraea) Simplicifolia. A human AB serum pool showed staining by both IgG and IgM antibodies of the Forssman and Gal(alpha)1-terminating components as well as some other, not structurally identified, components. The Forssman and Gal(alpha)1-reactivity in human sera could be eliminated by immunoadsorption using Forssman and Gal(alpha)1-3Gal-immunoadsorbent columns, respectively. Immunostaining of sheep kidney tissue sections showed the presence of Gal(alpha)1-terminating epitopes by immunoperoxidase and immunogold silver staining techniques. Proximal convoluted tubules showed a strong staining, while thin loops of Henle, collecting ducts, urothelium, and vessels showed a weaker staining. Distal convoluted tubules and thick loops of Henle were completely negative. In summary, human serum contains anti-sheep xenoantibodies reacting mainly with the Forssman and Gal(alpha)1-determinants in sheep tissues and the anti-sheep antibody titers are lower than the corresponding anti-pig titers.

Animals↗
PubMed1997

Biochemical and enzymatic characterization of blood group ABH and related histo-blood group glycosphingolipids in the epithelial cells of porcine small intestine.

Non-acid glycosphingolipids were isolated from small intestinal epithelial cells of a single blood group A pig. One very predominant blood group compound was obtained chemically pure upon HPLC fractionation. It was characterized by mass spectrometry and 1H NMR spectroscopy to be the type 1 chain blood group A hexaglycosylceramide. Support for the presence of minute amounts of additional A glycolipids was obtained by mass spectrometry and immunostaining of TLC plates with anti-A antibodies specific for A type 2 chain, A type 3 and 4 chain, and the ALe(b) determinant. Among precursor chains, globoside (type 4) and lactotetraosylceramide (type 1) were immunologically identified, whereas no neolactotetraosylceramide (type 2) and gangliotetraosylceramide reactivities were detected. We addressed the question whether the predominant expression of type 1 chain based A glycolipids reflects a restricted glycolipid precursor chain specificity of the alpha 1-2 fucosyl- and/or the alpha 1-3 N-acetylgalactosaminyltransferases, or if the biosynthesis of the precursor chains themselves is regulated. All precursor core saccharides, lacto- (type 1), neolacto-(type 2), and gangliotetraosylceramide as well as globopentaosylceramide (type 4), could serve as acceptors for fucose in vitro when a crude microsomal fraction obtained from mechanically released, porcine intestinal epithelial cells was used as an enzyme source. Under the same conditions an N-acetylgalactosamine residue could be transferred to the blood group H structures based on these core saccharide chains. Lactotriaosylceramide, but not gangliotriaosylceramide, could serve as an acceptor for UDP-galactose. When the product was digested with beta-galactosidase (EC 3.2.1.23) from S.pneumoniae, under conditions where it specifically cleaves Gal beta 1-4 residues, approximately 40% of the radioactivity was cleaved off, indicating that a substantial amount of neolactotetraosylceramide was made in vitro, as opposed to the predominance of lactotetraosylceramide-based structures found in vivo.

ABO Blood-Group System↗
PubMed1996

Blood group glycosphingolipid expression in kidney of an individual with the rare blood group A1 Le(a-b+) p phenotype: absence of blood group structures based on the globoseries.

Total neutral glycolipid fractions were isolated from kidney and ureter tissue obtained at autopsy of an individual of the rare blood group A1 Le(a-b+) p. The amount of glycolipids isolated were 3.7 and 2.5 mg g-1 dry tissue weight for the kidney and ureter tissue, which is in the range of reference blood group P kidneys. Part of the kidney glycolipid fraction was subfractionated by HPLC. Glycolipid compounds were structurally characterized by thin-layer chromatography (chemical detection and immunostaining with monoclonal antibodies), proton NMR spectroscopy and mass spectrometry. Globotriaosyl- and globotetraosyl-ceramides, which are the major compounds in kidneys of P individuals, were absent in the p kidney, and a comparatively increased amount of monoglycosyl- and lactosylceramides was found. A shift to longer fatty acyl chains in the ceramide part of lactosylceramides was noted. Elongated globoseries compounds with five to seven sugar residues, including the blood group A type 4 chain structure, were lacking. A slight increase in neolactotetraosyl- and blood group X pentaglycosyl-ceramides was noticed. The study confirms an enzymatic block in the conversion of lactosylceramide to elongated globoseries compounds in the kidney tissue similar to that of erythrocytes of p individuals.

Animals↗
PubMed1996

Physiological and histological characterisation of a pig kidney in vitro perfusion model for xenotransplantation studies.

A pig kidney perfusion model aimed for use in immunological and physiological xenotransplantation research has been developed. Organ viability was characterised by clearance studies, functional response to hormones/diureticum and by light microscopical examination. The pig kidney was perfused in a specially designed plexiglass chamber, using a roller pump and a small membrane oxygenator (O2/CO2, 95/5). The recirculating perfusate used was autologous pig blood diluted by Tyrodes solution to a hematocrit of 30%, at a total starting volume of 600-650 ml. The temperature was 37 degrees C. It was crucial for good organ function that the nephrectomy operating time, as well as the warm (1-2 min) and cold ischemia (average 43 min) times were minimized. The average total perfusion time was 151 minutes. Physiological parameters were measured during 10-15 minute periods at average times of 40, 63, 88 and 142 minutes. The clearance values of inulin in these periods were 54 +/- 13, 59 +/- 15, 48 +/- 23, 27 +/- 5 and for PAH; 103 +/- 14, 121 +/- 14, 106 +/- 30, 114 +/- 34 ml/min/100 g tissue weight. The plasma flows were 123 +/- 12, 155 +/- 17, 136 +/- 36 and 206 +/- 57 ml/min/100 g. The injection of 0.5 micrograms of alpha ANP to the perfusate resulted in a significant decrease in vascular resistance, and increase in urine production (+107%), as well as sodium (+112%) and potassium (+46%) excretion. Ten mg furosemide doubled the urine production and sodium excretion, while potassium excretion increased marginally. The number of leucocytes decreased by 39% during the perfusion, while the platelet count was unaffected. Light microscopy of the renal tissue after termination of the experiments revealed endothelial damage to variable extent. Loss of endothelial cells was most obvious at the level of arcuate and interlobular arteries, while the endothelium was intact in larger arteries and veins. Accumulation of polymorphonuclear granulocytes was found predominantly in the peritubular vessels, and to a lesser degree in the cortical venules. In the tubular cells, only minimal epithelial swelling and irregular cytoplasmic vacuolisation was found. Thus, a good functional viability can be maintained during 2 hours in vitro perfusion, although a decline in function as well as structural damage can be seen at the end of the experiment.

Animals↗
PubMed1995

The expression of carbohydrate blood group antigens correlates with heat resistance.

Recent data indicate that cells may resist heat shock via more than one route: heat shock protein synthesis and other still ill-defined mechanisms. We investigated this phenomenon using four types of cells derived from a single rat colon carcinoma: clones REGb and PROb; PRO A+, a glycosylation variant of PROb selected for its high expression of blood group A antigen; and Ph8, a thermoresistant variant of PROb selected by repeated sublethal heat treatments. Basal heat resistance was clearly associated with the level of cell surface expression of blood group H and A antigens. Biosynthesis of these carbohydrate structures requires two glycosyltransferases, H and A enzymes, whose activities are also correlated with basal heat resistance. In addition, heat sensitive REGb cells were rendered more resistant by transfection with the gene encoding for H enzyme, allowing expression of H antigen. Thus, these terminal glycosylations could play a role as cellular protectors against heat treatment. Blood group carbohydrate antigens were mainly located on O-linked carbohydrate chains of a major glycoprotein of 200 kDa and to a lesser extent on N-linked chains. Only trace amounts were present as glycolipids.

ABO Blood-Group System↗
PubMed1993

Expression of glycolipid blood group antigens in single human kidneys: change in antigen expression of rejected ABO incompatible kidney grafts.

Total neutral glycolipid fractions were separated into molecular species on thin-layer chromatography plates and detected by immunostaining with monoclonal anti-blood group antibodies. Blood group A antigens based on type 1, 2, 3 and 4 carbohydrate core saccharides were present in kidneys of A1 and A1B individuals. Blood group A2 individuals expressed only small amounts of A antigen compared to A1 individuals especially of the type 3 and 4 compounds. Kidneys from non-secretor individuals contained less A antigen compared to secretor individuals, and in both groups a variation in the antigen expression between single individuals was noted. Blood group A type 2 and 3 (which is an extension of A type 2) antigens were present both as basic 6 and 9 sugar structures as well as extended saccharide chains migrating in the 8 to 11 sugar interval. In contrast, the type 1 chain based A and Lewis antigens were only present as their basic 5 to 7 sugar chains, and no elongated structures were found. Four cases of A2 kidneys initially transplanted into O recipients and removed after 5, 12, 21 days and 4 years, respectively, were also analyzed. Two of these kidneys, originating from the same donor, showed a difference in A antigen expression. The kidney functioning for four years (lost due to chronic rejection) completely lacked X antigen with five sugar residues (present in all other individuals) and contained a large amount of A antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

ABO Blood-Group System↗
PubMed1992

The blood group B type-4 heptaglycosylceramide is a minor blood group B structure in human B kidneys in contrast to the corresponding A type-4 compound in A kidneys. Structural and in vitro biosynthetic studies.

Blood group A glycolipid antigens have been found based upon at least four different core saccharides (types 1 to 4). The biological significance of this structural polymorphism is not known, although the successful outcome of transplantations of blood group A2 kidneys to blood group O individuals have been partly explained by the low expression of A type-3 and -4 chain glycolipid antigens in A2 kidneys. If graft rejection due to ABO incompatibility is, in any way, correlated to the expression of type-3 and -4 chain blood group glycolipids, it is of interest to identify possible blood group B structures based on these core saccharides. In a non-acid glycosphingolipid fraction isolated from human blood group B kidneys, mass spectrometry, high-temperature gas chromatography-mass spectrometry and probing of thin-layer chromatograms with Gal alpha 1-4Gal-specific Escherichia coli and monoclonal anti-B antibodies provided evidence for minute amounts of a Gal alpha 1-3(Fuc alpha 1-2)Gal beta-HexNAc-Gal alpha 1-4Gal beta-Hex-Ceramide structure consistent with a B type-4 chain heptaglycosylceramide. In contrast, blood group A kidneys have the corresponding A type-4 chain heptaglycosylceramide as the predominant blood group A glycolipid. No, or very low activity of the blood group B gene enzyme on the type-4 chain blood group H hexaglycosylceramide precursor was found by biosynthetic experiments in vitro, which might explain the low expression of type-4 chain blood group B heptaglycosylceramides in human blood group B kidneys.

ABO Blood-Group System↗
PubMed1992

Glycosphingolipid expression in spontaneously aborted fetuses and placenta from blood group p women. Evidence for placenta being the primary target for anti-Tja-antibodies.

A 12-week-old fetus and one 17-week-old fetus + placenta were obtained after spontaneous abortions from two women of blood group p. The 17-week-old fetus was dissected into intestine, liver, brain and residual tissue. Nonacid glycosphingolipid fractions were prepared from the tissues. Glycolipid characterization was carried out using thin layer chromatography immunostained with monoclonal antibodies and bacteria and by 1H NMR spectroscopy and mass spectrometry. In the placental fraction substantial amounts of globotetraosylceramide (P-antigen) and globotriaosylceramide (Pk-antigen) were identified. In contrast, the fetuses contained only trace amounts of these structures, as revealed by immunostaining. These results indicate that the primary target for the antibodies of the anti-Tja serum is the placenta tissue, resulting in termination of the pregnancy.

Abortion, Spontaneous↗
PubMed1992

Characterisation of the anti-A antibody response following an ABO incompatible (A2 to O) kidney transplantation.

Anti-A,B antibodies produced in a blood group OLe(a-b-) recipient receiving a kidney graft from a blood group A2Le(a-b+) donor have been analysed for their ability to bind to different glycosphingolipid antigens. Solid-phase RIA using pure glycosphingolipid antigens and a chromatogram binding assay using total nonacid glycosphingolipid fractions from erythrocytes of different human blood group phenotypes together with pure glycolipid antigens were used as assay systems. Serum antibodies were shown to bind equally well to A (types 1, 2, 3 and 4) and B (types 1 and 2) antigenic structures but no binding to H antigens (types 1, 2 and 4) was detected. After adsorption of serum antibodies on A1 Le(a-b+) erythrocytes there was a residual anti-A antibody activity which could not be adsorbed by synthetic A-trisaccharides coupled to crystalline silica (Synsorb-A). These residual antibodies, which are not present in a pretransplant serum sample, had a specificity for the A antigen with type 1 core saccharide chain and the binding epitope obviously included both the N-acetylgalactosamine and the N-acetylglucosamine. The fucose residue was apparently not obligate for binding. The conformation of the sugar units involved in the binding epitope was determined.

ABO Blood-Group System↗
PubMed1992

Serological and immunochemical characterization of anti-PP1Pk (anti-Tja) antibodies in blood group little p individuals. Blood group A type 4 recognition due to internal binding.

Serum samples from 13 blood group little p individuals were tested by radioimmunoassay for their IgG antibody subclass distribution against the P, P1 and Pk antigens. There was no uniform subclass distribution pattern, although all but one had IgG3 antibodies against all the P system antigens tested. Studies were performed adsorbing anti-Tja serum sequentially to columns with synthetic carbohydrate antigenic determinants within the P system coupled to silica beads (SynsorbsR). The effect on agglutinin and indirect antiglobulin titers was determined after adsorption to SynsorbsR with different P-system antigens (P1, Pk, P). Adsorption to all the three SynsorbsR was needed to eliminate or strongly reduce antibody titers. The effect on IgM, IgG, IgA as well as IgG subclass antibody binding to P, P1 and Pk antigens was also determined by radioimmunoassay and chromatogram binding assay. Anti-PP1Pk antibodies from a little p woman with repeated abortions were shown to bind to glycosphingolipid antigens prepared from one of the aborted placentae using a chromatogram binding assay. This binding was eliminated by serum adsorption to SynsorbsR with P1, Pk and P carbohydrates. Anti-PP1Pk antibodies were also shown to bind to extended structures in the globoseries, i.e. globopentaosylceramide, globohexaosylceramide (globo-H) and globoheptaosylceramide (globo-A). This binding is most probably due to antibodies recognizing internal sequences in the carbohydrate chain. Attempts were made to visualize the binding epitope of the antibodies by computer molecular modelling.

Adult↗
PubMed1992

Non-acid glycosphingolipid expression in plasma of an A1 Le(a-b+) secretor human individual: identification of an ALeb heptaglycosylceramide as major blood group component.

Total non-acid glycosphingolipids were isolated from plasma of an A1 Le(a-b+) secretor individual with Refsum's disease (phytanic acid storage disease). The glycolipids were separated into 11 fractions by open column chromatography and by HPLC. The fractions were analyzed by thin-layer chromatography and tested for different blood group A activities as well as blood group Le(a )and Leb activity. The fractions were structurally characterized by proton NMR spectroscopy and FAB mass spectrometry and in selected cases by EI mass spectrometry of the permethylated and permethylated-reduced derivatives. Degradation analysis was performed on partially permethylated or permethylated-reduced alditol acetates. The dominating blood group compound was found to be a blood group A active type 1 chain difucosylheptaglycosylceramide. Other blood group compounds were identified as a blood group A active type 1 chain monofucosylhexaglycosylceramide, a blood group Leb hexaglycosylceramide, a blood group H active type 1 chain pentaglycosylceramide, and a globotetraosylceramide (the P-antigen). The presence of a Le(a) glycosphingolipid and blood group A type 3/4 chain structures were also found by immunostaining. Glucosyl-, lactosyl-, and globotriaosylceramides were the dominating short chain compounds. The amount of phytanic acid incorporated into the monoglycosylceramide fraction was found to be less than 5% of the fatty acids.

Animals↗
PubMed1992

Basic biochemistry of cell surface carbohydrates and aspects of the tissue distribution of histo-blood group ABH and related glycosphingolipids.

Cell surface carbohydrates may be protein- or lipid-linked. The structural polymorphism of the oligosaccharide chains is extensive due to variations in monosaccharide composition, carbohydrate sequence, branching, linkage position and linkage anomericity. Blood group ABH and related glycosphingolipids show a remarkable tissue-specific expression with possible implications in areas such as transfusion medicine, transplantation surgery and oncology. This communication gives a condensed description of the present knowledge of the tissue-specific distribution of histo-blood glycolipids in humans.

ABO Blood-Group System↗