ABH Blood Group Research Articles

Galectin-3 Induces Endothelial Cell Morphogenesis and Angiogenesis

Increasing evidence suggests that carbohydrate-binding proteins play an essential role in tumor growth and metastasis. However, conflicting results on their function in the regulation of cell proliferation and differentiation during angiogenesis have been reported. We have examined the role of galectin-3 in the regulation of human umbilical vein endothelial cell proliferation, differentiation, migration, and neovascularization. Galectin-3, a carbohydrate-binding protein, with specificity for type 1 and 11 ABH blood group epitopes and polylactosamine glycan containing cell surface glycoproteins, is the major nonintegrin cellular laminin-binding protein. Because galectin-3 expression was shown to be associated in some tumor systems with metastasis, we questioned whether it induces endothelial cell morphogenesis. Here we show that galectin-3 affects chemotaxis and morphology and stimulates capillary tube formation of HUV-EC-C in vitro and angiogenesis in vivo. Endothelial cell morphogenesis is a carbohydrate-dependent process, as it is neutralized by specific sugars and antibodies. These findings demonstrate that endothelial cell surface carbohydrate recognition event(s) can induce a signaling cascade leading to the differentiation and angiogenesis of endothelial cells.

Pathogenesis and management of recurrent urinary tract infection in women.

A number of pathogenic factors for the development of recurrent urinary tract infection, such as prolonged vaginal colonization with uropathogenic Escherichia coli, nonsecretion of ABH blood-group antigens, impaired local immune response, oestrogen deficiency in postmenopausal women and altered vaginal milieu caused by the use of contraceptives, are involved. Long-term use of antimicrobial agents is the cornerstone of prevention of recurrent urinary tract infection. Other approaches currently used involve self-start (on demand) therapy, oestrogen replacement in postmenopausal women, behavioural changes and alternative therapies, such as acupuncture.

Interaction of cholera toxin and Escherichia coli heat-labile enterotoxin with glycoconjugates from rabbit intestinal brush border membranes: relationship with ABH blood group determinants.

The capacity of cholera toxin (CT) and type I heat-labile enterotoxin produced by Escherichia coli isolated from human intestine (LTh) to interact with glycoconjugates bearing ABH blood group determinants from rabbit intestinal brush border membranes (BBM) was studied. On the basis of the type of intestinal compounds related to the human ABH blood group antigens, rabbits were classified as AB or H. Toxin binding to the intestinal glycolipids and glycoproteins depends on the blood group determinant borne by the glycoconjugate and on the analyzed toxin. LTh was capable of interacting preferentially with several blood group A- and B-active BBM glycolipids compared to those isolated from animals lacking these antigens (H rabbits). Also, LTh preferably bound to several BBM glycoproteins from AB rabbit intestines compared to those from H ones. One of these glycoproteins, the sucrase-isomaltase complex (EC 3.2.1.48-10) isolated from AB and H rabbits showed the same differential LTh binding. Conversely, CT practically did not recognize either blood group A-, B-, or H-active glycolipids and glycoproteins. These results may be relevant for carrying out in vivo experiments in rabbits in order to disclose the role of ABH active-glycoconjugates in the secretory response induced by LTh in rabbit intestine.

Tissue-specific expression of Le(Y) antigen in high endothelial venules of human lymphoid tissues.

In this study, we demonstrated that the anti-Le(Y) antibody (BM-1) especially reacted with high endothelial venules (HEVs) in peripheral lymph nodes of blood group O individuals. The Le(Y) expression on HEVs showed a unique tissue-specific pattern, i.e., a large amount of the Le(Y) expression in peripheral lymph nodes and no or small amounts in mesenteric lymph node. Statistical analysis showed that there was the significant difference between the percentage of Le(Y)-positive HEVs in peripheral lymph nodes and mesenteric lymph nodes. No expression of Le(Y) was observed in vessels of Payer's patch, thymus, spleen and other non-lymphoid organs. In blood group A or B individuals, the reactivity between HEVs and anti-Le(Y) antibody increased after enzyme digestion with alpha-N-acetylgalactosaminidase or alpha-galactosidase. These findings show that the expression of difucosylated blood group ABH antigens are especially expressed on HEVs in peripheral lymph nodes. Furthermore, the tissue-specific pattern suggests that these antigens may be related to intercellular adhesion between lymphocytes and HEVs.

The metabolism of 6-deoxyhexoses in bacterial and animal cells

L-fucose and L-rhamnose are two 6-deoxyhexoses naturally occurring in several complex carbohydrates. In prokaryotes both of them are found in polysaccharides of the cell wall, while in animals only L-fucose has been described, which mainly participates to the structure of glycoconjugates, either in the cell membrane or secreted in biological fluids, such as ABH blood groups and Lewis system antigens. L-fucose and L-rhamnose are synthesized by two de novo biosynthetic pathways starting from GDP-D-mannose and dTDP-D-glucose, respectively, which share several common features. The first step for both pathways is a dehydration reaction catalyzed by specific nucleotide-sugar dehydratases. This leads to the formation of unstable 4-keto-6-deoxy intermediates, which undergo a subsequent epimerization reaction responsible for the change from D- to L-conformation, and then a NADPH-dependent reduction of the 4-keto group, with the consequent formation of either GDP-L-fucose or dTDP-L-rhamnose. These compounds are then the substrates of specific glycosyltransferases which are responsible for insertion of either L-fucose or L-rhamnose in the corresponding glycoconjugates. The enzyme involved in the first step of GDP-L-fucose biosynthesis in E. coli, i.e., GDP-D-mannose 4,6 dehydratase, has been recently expressed as recombinant protein and characterized in our laboratory. We have also cloned and fully characterized a human protein, formerly named FX, and an E. coli protein, WcaG, which display both the epimerase and the reductase activities, thus indicating that only two enzymes are required for GDP-L-fucose production. Fucosylated complex glycoconjugates at the cell surface can then be recognized by specific counter-receptors in interacting cells, these mechanisms initiating important processes including inflammation and metastasis. The second pathway starting from dTDP-D-glucose leads to the synthesis of antibiotic glycosides or, alternatively, to the production of dTDP-L-rhamnose. While sets of data are available on the first enzyme of the pathway, i.e., dTDP-D-glucose dehydratase, the enzymes involved in the following steps still need to be identified and characterized.

GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from Escherichia coli, a key enzyme in the biosynthesis of GDP-L-fucose, displays the structural characteristics of the RED protein homology superfamily.

The process of guanosine 5'-diphosphate L-fucose (GDP-L-fucose) biosynthesis is conserved throughout evolution from prokaryotes to man. In animals, GDP-L-fucose is the substrate of fucosyltransferases that participate in the biosynthesis and remodeling of glycoconjugates, including ABH blood group and Lewis-system antigens. The 'de novo' pathway of GDP-L-fucose biosynthesis from GDP-D-mannose involves a GDP-D-mannose 4,6 dehydratase (GMD) and a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GMER). Neither of the catalytic mechanisms nor the three-dimensional structures of the two enzymes has been elucidated yet. The severe leukocyte adhesion deficiency (LAD) type II genetic syndrome is known to result from deficiencies in this de novo pathway. The crystal structures of apo- and holo-GMER have been determined at 2.1 A and 2.2 A resolution, respectively. Each subunit of the homodimeric (2 x 34 kDa) enzyme is composed of two domains. The N-terminal domain, a six-stranded Rossmann fold, binds NADP+; the C-terminal domain (about 100 residues) displays an alpha/beta topology. NADP+ interacts with residues Arg12 and Arg36 at the adenylic ribose phosphate; moreover, a protein loop based on the Gly-X-X-Gly-X-X-Gly motif (where X is any amino acid) stabilizes binding of the coenzyme diphosphate bridge. The nicotinamide and the connected ribose ring are located close to residues Ser107, Tyr136 and Lys140, the putative GMER active-site center. The GMER fold is reminiscent of that observed for UDP-galactose epimerase (UGE) from Escherichia coli. Consideration of the enzyme fold and of its main structural features allows assignment of GMER to the reductase-epimerase-dehydrogenase (RED) enzyme homology superfamily, to which short-chain dehydrogenase/reductases (SDRs) also belong. The location of the NADP+ nicotinamide ring at an interdomain cleft is compatible with substrate binding in the C-terminal domain.

The molecular and supramolecular chemistry of carbohydrates: chemical introduction to the glycosciences

1: Configuration of monosaccharides. 2: Conformation of monosaccharides and their derivatives. 3: Alkyl and aryl glycosides and glycosamines. 4: Nomenclature. 5: Reactions of hydroxyl groups. 6: Reactions of carbonyl groups and hemiacetals. 7: Changes of configuration, unsaturated and branched-chain sugars. 8: Sugars in chiral synthesis. 9: Oligosaccharides : configuration and analysis. 10: Chemical transformations and synthesis of oligosaccharides. 11: Associations with anions, cations, and inorganic molecules. 12: Sialic and sialylated oligosaccharides. 13: Glycoconjugates. 14: Structure of some crystallized sugar-protein complexes. 15: Antigens and antibodies. Lectins. 16: ABH and related blood group antigens. 17: Important recognition events involving oligosaccharides in the living world. 18: Oligosaccharides as ligands to DNA

Distribution of ABH blood group epitopes on inner surface of dental hard tissue: serological, immunohistochemical and ultrastructural study on odontoblasts.

This study was designed to investigate localization of ABH antigens on the inner surface of human tooth hard tissues. The tooth samples, extracted therapeutically, were cut and dried at room temperature for a few weeks. Scanning electron microscopic observation disclosed that the inner surface was exclusively covered with the odontoblastic zone and virtually no blood vessels remained in the samples. Blood group activities of the tooth fragments, which were detected with absorption-elution test, were markedly decreased when the odontoblastic zone was scraped off. To visualize the activity, the avidin-biotin-peroxidase complex immunostaining was performed on the odontoblasts of fresh teeth followed by embedding. Decalcification process was omitted to preserve the antigenicity. Group specific colorization was successfully developed on the cell surface of the odontoblasts. Immunoreactivity of the cell membrane of the odontoblast including the odontoblastic process was also confirmed by immunotransmission microscopic observation. On the other hand, localization of epitopes on intrinsic dentine without cell components could not be visualized. The results of these experiments indicate that the odontoblasts are one of potent sources of blood group antigenicity for blood grouping of the human teeth.

Blood groups Lewis(b) and ABH expression in gastric mucosa: lack of inter-relation with Helicobacter pylori colonisation and occurrence of gastric MALT lymphoma.

BACKGROUND: Blood group Lewis(b) antigens mediate Helicobacter pylori attachment to gastric mucosa with attachment being particularly strong in subjects with ABH blood group O. AIMS: To determine whether H pylori...

Further refinement of the description of the ligand-binding characteristics for the galactoside-binding mistletoe lectin, a plant agglutinin with immunomodulatory potency.

The galactoside-binding lectin from mistletoe (Viscum album L.) is a biological response modifier, eliciting e.g. enhanced secretion of cytokines. This immunological activity warrants the further analysis of its ligand-binding properties with special attention paid to blood group epitopes. To avoid the microheterogeneity and complexity of naturally occurring glycoproteins, chemically strictly defined neoglycoconjugates and a panel of synthetic oligosaccharides were employed in solid-phase assays for direct binding and assessment of the relative inhibitory capacity. Since label incorporation into the lectin, although performed under protective conditions, or surface immobilization by adsorption to plastic may affect its affinity characteristics, the extent of neoglycoconjugate binding in the absence of any interfering substance and in the presence of oligosaccharides was determined comparatively with labeled and with immobilized lectin. In principle, these two factors could be excluded to markedly alter binding features. In addition to lactose, the blood group determinants H and B were strongly reactive. A fucose residue can thus especially be accommodated to the binding site when linked to the non-reducing unit. N-Acetyllactosamine was nearly as potent as an inhibitor as lactose. Lec and the A determinant were notably inferior to the other ABH blood group epitopes. Le(a) and Le(x) and their sialylated derivatives displayed only very weak binding capacity. Among the two natural isomers of sialyllactose, the alpha 2,6-form displayed a higher level of inhibitory capacity than the alpha 2,3-derivative. Isomeric variants of the Thomsen-Friedenreich antigen, too, reduced lectin binding to the lactose-carrying polymer. Their capacities were surpassed by those of the H and the B determinants and a related form of the latter, the P1 epitope. An overlap of specificity with the immunomodulatory human galectin-3 is thus measurable for H/B-like structures. The documented differential reactivity of the mistletoe lectin to blood group oligosaccharides may have a bearing on the responsiveness of blood group-positive cell populations.

Tyramine-containing poly(4-nitrophenylacrylate) as iodinatable ligand carrier in biodistribution analysis.

Targeted label or drug delivery requires access to convenient carrier systems and methods for efficient ligand conjugation. The main purpose of this study is to design an iodinatable synthetic polymer, whose application in vivo in tumor-bearing mice is tested with several related carbohydrate ligands, namely ABH and Lewis blood group epitopes. Tyramine and aminopropyl derivatives of the synthetic oligosaccharides were attached to poly(4-nitrophenylacrylate). Following iodination, the biodistribution of the sugar-free and the substituted polymers was determined in tumor-bearing mice. Flow cytofluorimetric analysis assessed tumor cell binding of further ligand types to human tumor cells in vitro. Quantitative ligand incorporation was achieved under mild conditions. Whereas the ligand-free poly[N-(2-hydroxyethyl)acrylamide] (MW 30 kDa) showed preferential accumulation in kidney, neoglycopolymers were found in substantial amounts in liver, kidney or spleen. The nature of the carbohydrate structure quantitatively influenced the distribution pattern. Tumor cell binding of blood group determinants and three further ligand types revealed non-uniform intensity in labeling and percentage of positive cells even in comparison between lines with identical histogenetic origin. Carbohydrate-exposing poly[N-(2-hydroxyethyl)acrylamide] polymers with tyramine as an iodine acceptor distribute in mice with a profile which is quantitatively influenced by small structural variations of the ligand part. Further refinement of the ligand structure may increase the level of selectivity for organ and tumor accumulation.

Hemagglutinins in fungus extracts and their blood group specificity

A total of 833 fungi harvested from 1977 to 1994 were tested and 422 extracts (47.8%) produced hemagglutination of human red cells. The lectins in fungus extracts which showed blood-group-specific or related reactions were partially purified by ammonium sulfate precipitation and gel filtration on a Sephadex G-100 column. Anti-H-like agglutinins were found in extracts of Pleurocybella porrigens, Naematoloma sublateritium and Pholiota squarrosa. These extracts agglutinated strongly with human group O red cells and rather weakly with A and B cells. Anti-A agglutinins were found in extracts of Hohenbuehelia serotina, Paxillus panuoides, Melanoleuca melaleuca and Hygrophorus capreolarius. The extract of Clavulinopsis fusiformis contained anti-B agglutinin. The ABH reactivities of the extracts were cofirmed by an agglutination inhibition test with ABH secretor saliva and blood group substances from human gastric linings and by the destruction of inhibiting activity using blood-group-specific decomposing enzymes. L-Fucose was the most active inhibiting monosacharide of anti-H-like agglutinins. The reaction of anti-A agglutinins was strongly inhibited by N-acetyl-D-galactosamine. D-Galactose and raffinose and melibiose which contain alpha-galactosyl residues were potent inhibitors of C. fusiformis agglutinin.

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.

Simultaneous expression of keratan sulphate epitope (a sulphated poly-N-acetyllactosamine) and blood group ABH antigens in papillary carcinomas of the human thyroid gland

The monoclonal antibody 5-D-4 recognizes heavily sulphated forms of keratan sulphate epitope. It reacted strongly with the cell surfaces of most thyroid papillary carcinomas from all the individuals examined, independently of the blood group of the patients. Cells of follicular variants of papillary carcinomas were also labelled by 5-D-4. In contrast, no labelling with this antibody was observed in other types of thyroid neoplasms, or in normal tissues. The reactivity of 5-D-4 with papillary carcinomas was markedly reduced or abolished by prior digestion with endo-beta-galactosidase, keratanase II, or N-glycosidase F. Although keratanase digestion had no effect on 5-D-4 labelling, it revealed the binding sites of Griffonia simplicifolia agglutinin II (GSA-II), which recognizes terminal N-acetylglucosamine in a limited number of carcinoma cells from some individuals. Blood group ABH antigens, which are simultaneously expressed together with keratan sulphate epitope in cancer cells, were eliminated by digestion with endo-beta-galactosidase and N-glycosidase F, but were resistant to keratanase and keratanase II treatment. These results indicate that keratan sulphate oligosaccharides are cancer-associated and are probably oncofoetal antigens, as are the blood group antigens in human thyroid glands. The results suggests that poly-N-acetyllactosamine, which is ubiquitously and consistently produced in papillary carcinomas, is modified in two different ways: sulphation on the 6-position of at least some units of either galactose or N-acetylglucosamine or both, and decoration of non-reducing termini with the blood group antigens. Along with the endo-beta-galactosidase-GSA-II labelling procedure, labelling with 5-D-4 may be a useful diagnostic means for distinguishing papillary carcinoma from other types of thyroid neoplasms.

Expression of Gal beta 1-4GlcNAc sequences by human gastrointestinal neoplasms and their precursors as detected by Erythrina cristagalli and Erythrina corallodendron lectins

Lectins from Erythrina cristagalli (EGA) and Erythrina corallodendron (ECorA) are well-known to detect type 2 chain oligosaccharides (Gal beta 1-4GlcNAc). These carbohydrate moieties are the biosynthetic precursors of various ABH and Lewis blood group antigens and are therefore also related to tumor-associated carbohydrate antigens. For this reason, we investigated the expression of ECA and ECorA binding sites in a series of gastric, colorectal and pancreatic carcinomas as well as corresponding normal tissues. Additionally, a series of hyperplastic and adenomatous colorectal polyps was analyzed. According to our results, both lectins exhibited a strong reactivity with the great majority of gastrointestinal carcinomas. Regarding gastric carcinomas, a stronger reactivity with intestinal-type compared to the diffuse-type species could be observed. Some poorly differentiated tumors were not or only very faintly stained. In the case of colorectal carcinomas, liver metastases which were investigated comparatively, exhibited the same binding pattern as the primary tumors. Colorectal adenomas were stained in about half of the cases without significant relation to the grade of cellular atypia. Positivity observed in normal epithelia (i.e, gastric superficial epithelia, fundus neck cells and deep pyloric mucous glands, pancreatic acini and ductal structures) is in keeping with histogenetic relations between these normal histological structures and corresponding neoplasms. In areas exhibiting intestinal metaplasia, various portions showed cytoplasmic staining of columnar cells and/or of goblet cell vacuoles. On the other hand, columnar and goblet cells in normal colorectal tissue were only weakly stained in a number of specimens. Therefore, it can be concluded that Gal beta 1-4GlcNAc is overexpressed in neoplastic colorectal tissues. Summarized, ECA and ECorA are suitable tools to analyze the expression of Gal beta 1-4GlcNAc, the common precursor substance of various tumor-associated type 2 chain antigens in neoplastic tissues.

Effects of the carbohydrate-binding protein galectin-3 on the invasiveness of human breast carcinoma cells.

Galectin-3 is a Mr 30,000 protein with carbohydrate-binding specificity for type I and II ABH blood group epitopes and polylactosamine glycans expressed on cell surface and extracellular matrix glycoproteins such as laminin. Cell lines propagated from human normal mammary epithelia and from benign or infiltrating components of primary breast tumours express low levels of galectin-3 in the cytoplasm. However, galectin-3 when added exogenously in solution or when bound within a three-dimensional matrix markedly enhanced the migration of the primary tumour cell lines through a Matrigel barrier. Galectin-3 expression in the cytoplasm and intercellularly on surface membranes was greatly increased in cell lines propagated from malignant ascites and pleural effusions of late stage breast cancer. These cell lines were non-invasive in the Matrigel assay and exogenous galectin-3 had no enhancing effect on invasiveness. These results suggest that galectin-3 could play multiple roles in cell metastasis at an early invasive stage by acting in a paracrine manner to stimulate cell migration through an extracellular matrix, and in later stage cancers in synergy with other mediators of cell-cell aggregation. However, endogenous galectin-3 expression in human breast cancers is not correlated directly with their invasive potential in vitro.

Immunocytochemical evidence for a modulation of galectin 3 (Mac-2), a carbohydrate binding protein, in pulmonary fibrosis.

Galectin 3 is endogenous mammalian carbohydrate-binding protein with affinity for terminal beta-galactose residues, polylactosamine glycans, and ABH-blood group carbohydrate epitopes. To determine the distribution and regulation of galectin 3 during pulmonary injury, which is known to be accompanied by profound changes in the carbohydrate moieties of cell surface glycoproteins of alveolar cells, a rat model of irradiation-induced lung inflammation and repair was used. Immunocytochemistry showed that in normal rat lungs, galectin 3 was localized to alveolar macrophages, with weaker staining of bronchial epithelial cells. Shortly after irradiation-induced lung injury, when there is active proliferation of type II alveolar epithelial cells and re-epithelialization of alveolar basement membranes by type I cells, the total galectin concentration in the lung increased dramatically. This increase was due in part to an increased population of galectin 3-positive interstitial and alveolar macrophages. In addition, galectin 3 was expressed prominently at the surface of the newly formed type I alveolar epithelium and to lesser extent at the apical surface of type II cells. These findings suggest that the increased synthesis and secretion of galectin 3 during irradiation-induced lung injury, together with ligation of secreted lectin at the surface of alveolar epithelial cells, may play roles in pulmonary alveolar epithelial expansion and differentiation during injury and repair.

Blood group A antigen expression in platelets is prominently associated with glycoprotein Ib and IIb. Evidence for an A1/A2 difference.

Blood group ABH antigens are associated with platelets as intrinsic determinants and extrinsically adsorbed antigens, and exist both on glycosphingolipids and on glycoproteins (GPs). We now provide direct evidence that the blood group ABH antigens are prominently associated with platelet GPIb and GPIIb. By immunoprecipitation, a murine monoclonal anti-A antibody precipitated surface-biotin-labelled blood group A1 platelet membrane proteins with electrophoretic characteristics identical to those of GPIb/IX and GPIIb/IIIa. By immunoblotting of SDS-PAGE separated blood group A1 platelet proteins the monoclonal anti-A antibody bound to proteins with electrophoretic characteristics identical to those of GPIb and GPIIb. When immunoaffinity purified GPIb/IX and GPIIb/IIIa, derived from blood group O, A1 and A2 platelets, were employed for immunoblotting, GPIb and GPIIb only from A1 platelets bound the monoclonal anti-A antibody. By ELISA, wherein monoclonal antibodies specific for GPIb (APl) and the GPIIb/IIIa complex (AP2) were used to capture and hold antigens from platelet lysate, human anti-A antibodies reacted with these proteins derived from blood group A1 platelets; proteins from blood group A2, O and B platelets showed no reactivity. These results indicate that blood group A antigen is associated with GPIb and GPIIb derived from blood group A1 but not A2 platelets.

Loss of ABH antigen expression in bladder cancer is not caused by loss of heterozygosity of the ABO locus.

The significance of loss of heterozygosity (LOH) of chromosome 9 as an early event in human bladder tumors has been demonstrated by several studies. We have studied LOH of the ABO gene locus at 9q34.2 by means of polymerase chain reaction (PCR) genotyping of tumor preparations and leukocytes. Twenty-two tumors, all of which were immunohistochemically negative for ABH antigens, were examined. Eleven tumors were from blood-group O individuals, and were examined by denaturing gradient gel electrophoresis (DGGE). They showed normal band patterns with no sign of single base mutations or LOH. All II A and B tumors were sorted on a fluorescence-activated cell sorter (FACS) according to DNA content, thereby making it possible to study differences in genotype between subpopulations of cells in the same tumor. In 2 genotypically AO cases, we found 2 aneuploid subpopulations. In both cases, the most abnormal, with the highest DNA content, showed complete loss of the O allele, leaving the A allele intact. As all tumors were ABH antigen-negative, this study demonstrates that LOH of the ABO locus on chromosome 9q34 is not the cause of loss of blood-group ABH expression in human bladder cancer.

Minimally differentiated erythroleukaemia (AML M6 'variant'): a rare subset of AML distinct from AML M6. Groupe Français d'Hématologie Cellulaire.

We describe eight cases of erythroleukaemia distinct from FAB-AML M6, which demonstrate minimal erythroid differentiation not associated with a myeloblastic component. Three infants (including a Down's syndrome) and two adults presented with a de novo leukaemia. One case was preceded by an untreated refractory anaemia with excess of blasts and one by polycythaemia vera. One case presented with an inaugural blast crisis of chronic myeloid leukaemia. In four patients the leukaemic cells showed a proerythroblast-like morphology. The four other were initially classified as undifferentiated AL (two cases) or AML MO (two cases) because of the immature aspect of the cells, their lack of myeloperoxidase activity and the absence of B, T lymphoid and myeloid (My) marker expressions apart from the CD33 antigen. Immunophenotyping in three cases showed an immature erythroblast profile (glycophorins A and B+, spectrin+). In the five others the erythroid nature was recognized by the expression of ABH blood group system on fresh cells (four cases) and glycophorin A on cells after 3 d in vitro culture with erythropoietin (EPO) + IL3 (two cases). Moreover, an erythroid colony growth of leukaemic origin was observed in three patients. In conclusion, the study of erythroid marker expression is of particular importance when immunophenotyping leukaemic cells with a proerythroblast-like morphology or an undifferentiated aspect and a HLA DR-, CD36++, B-, T-, My- (CD33 +/-) phenotype. We propose the term AML M6 'variant' for this rare type of AML.