ABH Antigens Research Articles

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.

Prognostic value of an assessment of ABH(0) isoantigens and Thomsen-Friedenreich (TF) antigen expression in patients with urinary bladder tumours.

The discriminating and prognostic value of the expression of ABH(0) and TF antigens in tumour cells of 76 patients with urinary bladder tumours was evaluated. In patients with superficial tumours the most frequently defined phenotype was ABH(-) TF(+) and in those with deep tumours ABH(-) TF(-). The lowest frequency of recurrences was seen in patients with TFCr(+) antigenic phenotype and no patient died in this group. The mean lifespan of patients with TF(-) tumours was shorter (21 months) than of those with TF(+) tumours. For detection of tumours with higher risk of recurrences an analysis of the combination of markers revealed to be more predictive than analysis of a single marker.

Computer simulation of histo-blood group oligosaccharides: energy maps of all constituting disaccharides and potential energy surfaces of 14 ABH and Lewis carbohydrate antigens.

The three-dimensional structures of fourteen histo-blood groups carbohydrate antigens have been established through a combination of molecular mechanics and conformational searching methods. The conformational space available for each disaccharide, constituents of these determinants, has been throroughly characterized. The results have been organized in a data bank fashion. Larger relatives, i.e. 14 tri- and tetrasaccharides of histo-blood group antigens, have been modelled using a different method for exploring the complex potential energy surface. This approach is aimed at establishing all the possible families of conformations, along with the conformational pathways. Different conformational behaviours are exhibited by these oligosaccharides. Some of them, i.e. Le(x) and Le(y) tri and tetrasaccharides, are very rigid; 99% of their populations belong to the same conformational family. Others, like H type 1, H type 2 or H type 6 oligosaccharides, are essentially rigid, but a secondary conformational family, corresponding to 3-4% of the total population, can arise. Finally, the H types 3 and 4 trisaccharides, and the A type 1 and A type 2 tetrasaccharides are predicted to behave rather flexibly. The information gathered in the present investigation has been used to analyse the body of experimental evidence, either physical or biological, available for this series of carbohydrate antigens. Of special interest are the several different alignments that can be proposed for these molecules. They yield a realistic definition of the three-dimensional features of the epitopes thereby providing essential information about how carbohydrate antigens are recognized by proteins.

Flow cytometry analysis of dual red blood cell populations after bone marrow transplantation.

Flow cytometry represents an alternative method to agglutination assays for the accurate quantification of mixed field populations of erythrocytes observed after bone marrow transplantation. Murine monoclonal antibodies directed against the blood group ABH antigens were selected and processed in order to prepare ready-to-use fluorescent reagents. Anti-A (NaM87-1F6; IgG3), anti-B (NaM9-2E11; IgG3) and anti-H (NaM19-7E11; IgM) were purified, labelled with fluorescein isothiocyanate, and used in a direct flow cytometry assay. Anti-A1 (NaM1-1C9; IgG3) was no longer active after FITC-labelling and then was used in an indirect assay. The agglutination was prevented by formaldehyde pretreatment of erythrocytes. Using artificially-made double populations of erythrocytes, measured values with mixtures of 1-100% of cells were very closely related to expected values, showing both the sensitivity and the accuracy of the method. From careful investigation of a series of bone-marrow transplanted patients, we conclude that engraftments could be demonstrated earlier by flow cytometry than by agglutination, because minor populations (1-10%) of cells could be determined accurately only with labelled reagents. In addition, the disappearance of the donor cells on a long-term follow-up of patients enabled an earlier detection of graft failure in one case. The proposed method provides appreciable help to follow engraftment in patients and may have more general applications for the study of other haemopoietic chimaeras.

Histochemical differences of the lectin affinities of backbone polylactosamine structures carrying the ABO blood group antigens in papillary carcinoma and other types of thyroid neoplasm.

Endo-beta-galactosidase from Escherichia freundii cleaves linear polylactosamine structure as follows: R-GlcNAc-beta 1-3Gal-beta 1-4GlcNAc-beta 1-R' + H2O-->R-GlcNAc-beta 1-3Gal + GlcNAc-beta 1-R'. Staining with Griffonia simplicifolia agglutinin II (GSA-II) following enzyme digestion reveals the distribution of R-GlcNac-beta 1-3Gal-beta 1-4GlcNAc-beta 1-R' structures in tissue sections. In this study, the procedure was applied to formalin-fixed, paraffin-embedded tissue sections from 26 cases of papillary carcinomas including 2 follicular variants, 8 follicular carcinomas, 7 adenomas, 1 anaplastic carcinoma and 1 medullary carcinoma in order to investigate whether different types of polyactosamine-containing structure are produced in these thyroid neoplasms. Simultaneously, the susceptibility of the ABH antigens expressed in these neoplastic cells to endo-beta-galactosidase digestion was examined. Most of the papillary carcinoma cells from all the individuals examined were strongly stained by GSA-II following enzyme digestion. Without enzyme digestion, little or no reactivity with GSA-II was observed. Among other types of neoplasms, only one case of follicular carcinoma exhibited reactivity with GSA-II following enzyme digestion. ABH antigens were expressed in 22 cases of papillary carcinomas, 2 adenomas, 5 follicular carcinomas and 1 anaplastic carcinoma, and their expression was dependent on the ABO blood group of the patients. Endo-beta-galactosidase digestion resulted in the elimination of these antigens not only in papillary carcinomas but also in other neoplasms.(ABSTRACT TRUNCATED AT 250 WORDS)

ABH and Lewis immunohistochemistry of the human eye

ABH and Lewis antigens are detected by immunohistochemistry exclusively in the anterior cornea epithelium and in the conjunctiva bulbi, but not in the ciliary body, in the retina, or in the vitreous body of the eye. The ABH antigens found in the vitreous humor by the absorption elution technique (Rordorf et al., Forensic Sci. Int. 41, 1989, 111–116) are probably glycosphingolipids, which are not detected by immunohistochemistry. They may come from the plasma of uveal blood vessels, or be produced by local cells.

ABH and Colton blood group antigens on Aquaporin-1, the human red cell water channel protein

The recent identification of the red cell water transporter (AQP1) has led to the identification of the "aquaporins", a new class of membrane proteins which function as water-selective transport proteins and are involved in many physiological processes. Identification of the chromosomal localization of the corresponding gene led to the recognition that AQP1 is the structural basis of the Colton blood group antigens. Analysis of individuals with the Colton null phenotype led to the recognition that homozygosity for knockout mutations in the corresponding gene is exceedingly rare but is without a significant clinical phenotype, predicting a redundancy in expression of other aquaporin homologs. These studies demonstrate the importance which molecular studies in red cell blood group antigens may play in diverse areas of biomedical research. Moreover, they provide another example that blood group antigens may be polymorphisms in functionally important proteins on the red cell surface.

Biological functions of carbohydrate ABH blood group determinants

The ABH blood group determinants are carbohydrates found in different human cells and tissues. The modulation in their expression in the course of ontogenesis, as well as the studies on their subcellular localization, impose a discussion on the role of ABH antigens as molecules participating in cell interactions, contact inhibition, adhesion and metastases. It is supposed that they are also involved in cellular differentiation during human embryogenesis. The described association between blood groups and disease and the de novo acquisition of blood group determinants support the idea that ABH antigens are related to pathological processes. The modulation in blood group antigen expression observed in several tumors demands a clarification of their diagnostic and prognostic value as tumor-associated markers. Biomedical Reviews 1994; 10: 55-63.

Subcellular distribution of blood group ABH and related antigens

Blood group ABH and related antigens are expressed not only in red blood cells but also abundantly in many epithelial cells of human and other vertebrate species. Recent electron microscope observations have revealed the precise distribution of these antigens in various cellular constituents such as the Golgi complex, cell membrane, mucous granules and nuclear heterochromatin. These findings may contribute to elucidate the biosynthetic mechanism of these substances as well as their physiological and pathological function.

Demonstration of HLA and ABH Antigens in Fresh and Frozen Human Menisci by Immunohistochemistry

The expression of HLA and ABH antigens in fresh, frozen, and twice-frozen normal human meniscal tissue was evaluated with use of immunohistochemistry. A generalized expression of Class-I and Class-II HLA antigens was found in the endothelial and synovial cells of all three forms of tissue. Fibrochondrocytes were Class-I positive and Class-II negative. ABH antigens were expressed by endothelial cells in relationship to the blood group of the patient. Freezing preserved most of the HLA and ABH molecular structure. Although the normal meniscus consists of a relatively few chondrocytes embedded in an extracellular matrix, it also contains Class-II and ABH-positive endothelial cells and Class-II-positive synovial cells. These antigens are present at the moment of transplantation and could evoke an immune response in the host that would modulate the results of meniscal allografting.

Variation of Blood Group Antigen Expression on Vaginal Cells and Mucus in Secretor and Nonsecretor Women

The expression of blood group antigens on the surface of urothelial cells and in mucus is controlled partly by the blood type and secretor status of the individual. To our knowledge, the possibility that the levels of these antigens vary with time has not been previously assessed. We determine if the pattern and/or intensity of blood group antigen expression on vaginal epithelial cells and mucus changed with time. Cell and mucus specimens were collected weekly for 3 months from 10 women: 5 (2 secretors and 3 nonsecretors) with and 5 (3 secretors and 2 nonsecretors) without a history of urinary tract infections. In addition, samples were collected on 5 consecutive days from 5 of these individuals. The cell and mucus samples were assayed for ABH and Lewis blood group antigens using monoclonal antibodies in cell concentration immunofluorescence and enzyme-linked fluorogenic assays, respectively. Although the pattern of antigen expression in the vaginal cell and mucus samples was consistent with the blood type and secretor status of an individual, in all women the level of antigen expression changed significantly and rapidly during the 3-month and 5-day periods. The results show a previously unrecognized phenomenon and demonstrate that the expression of blood group antigens on vaginal cells and in mucus is a dynamic process.

Blood Group Antigen Expression on Vaginal Cells and Mucus in Women with and without A History of Urinary Tract Infections

Adherence of bacteria to carbohydrate receptors on the surface of vaginal epithelial cells is a critical event that precedes bacterial colonization of the vaginal mucosa and subsequent urinary tract infection. Blood group antigens, carbohydrate structures bound to lipids or proteins, constitute an important component of the uroepithelial cell membrane and may affect susceptibility to urinary tract infections. To determine if the ABH and Lewis antigen expression in women with a history of urinary tract infections differed from that of women without such a history, vaginal specimens from 87 women (42 patients and 45 controls) were analyzed for the detection of these antigenic determinants using monoclonal antibodies in quantitative immunoassays. The profile of ABH antigen expression was generally consistent with the ABO phenotype of the individual and appeared to be influenced by the secretor status. Secretors expressed higher levels of A, B and H determinants than nonsecretors. In addition, Lewis antigens were detected on vaginal cells and in mucus. Samples from nonsecretors expressed higher levels of Lea and Lex antigens, whereas secretors expressed higher levels of Leb and Ley antigens. The levels of antigen expression varied widely among individuals with the same blood type and secretor status. Comparisons between patient and control groups showed no significant differences in ABH or Lewis antigen expression overall, or when controlling for ABO or secretor phenotypes, respectively. These findings confirm our previous observations on healthy women, and document the heterogeneity of blood group antigen expression on vaginal epithelial cells and mucus from women with a history of urinary tract infections.

Profiles of expression of carbohydrate chain structures during human intrahepatic bile duct development and maturation: A lectin-histochemical and immunohistochemical study

We investigated expression of carbohydrate chain structures during human intrahepatic bile duct development and maturation. Results of staining for ABH antigens were negative in the ductal plate and biliary cells migrating into the mesenchyma but were positive in newly formed bile ducts in the fetal liver and in maturing bile ducts in the postnatal liver. Staining for Lewis(a), sialyl Lewis(a) and Lewisb antigens was negative in the ductal plate but positive in migrating biliary cells, newly formed ducts and maturing ducts. Staining for sialyl Lewisx antigen was positive in the ductal plate, migrating biliary cells and newly formed ducts but negative in the postnatal liver. Lewis(y) antigen staining was negative in the ductal plate and migrating biliary cells but positive in newly formed ducts and maturing ducts. Staining for T and Tn antigens was negative in the ductal plate, migrating biliary cells and newly formed ducts but positive in maturing ducts. Staining for Lewisx, sialyl Tn and carcinoembryonic antigen was negative in all tissues. Receptors of Ulex europaeus agglutinin I and soybean agglutinin were absent in the ductal plate, migrating biliary cells and newly formed ducts but present in maturing large ducts. Staining for receptors of concanavalin A and Ricnus communis agglutinin I was negative in the ductal plate but positive in migrating biliary cells, newly formed ducts and maturing ducts. Staining for the receptor of succinylated wheat germ agglutinin was always positive. These findings suggest that carbohydrate chain structures change during intrahepatic bile duct development and maturation and that new carbohydrate chain structures gradually emerge as intrahepatic bile ducts develop and mature.

Expression of type-2 histo-blood group carbohydrate antigens (Le(x), Le(y), and H) in normal and malignant human endometrium.

Changes in expression of histo-blood group ABH and Lewis antigens are common alterations in carcinomas. Using immunohistochemistry, we have evaluated the expression of type-2 histo-blood group antigens in normal and malignant endometrial tissues in relation to genetic and hormonal factors. The Le(x), sialosyl-Le(x), and Le(y) antigens were inconstantly expressed in the normal endometrium. The expression was uninfluenced by the secretor status but was related to the ABO blood group status in Oestradiol (E2) stimulated endometria. Le(y) was expressed most frequently in proliferating endometria from blood group 0 individuals. Le(x) and Le(y) were maximally expressed in atrophic endometria, and Le(x) and Le(y) staining scores correlated inversely with serum levels of E2 in normal, non-secretory endometria. No correlation was found in adenomatous hyperplasias and endometrial carcinomas, which when compared with atrophic endometria, showed a loss of Le(x) and Le(y) and an increased H-carbohydrate expression at apical membranes. Carcinomas from non-secretors showed lower expression of Le(y) and H-antigens than carcinomas from secretors. Our findings suggest that the genetic and hormonal influence on glycosylation based on type-2 chain carbohydrates differ between normal and malignant endometrium. This difference is probably related to specific tumour-associated qualitative and quantitative changes in the fucosyltransferases.

Changes in ABH antigen expression on red cells during in vivo aging: a flow cytometric analysis.

During aging of red cells they undergo a multitude of physical and chemical changes. Age-dependent changes in the expression of ABH major blood group antigens on red cells were studied in a two-variable flow cytometric analysis. The expression of the antigens was measured after staining with fluorescence antibodies (for A and B) or lectin (for H). Cell age was related to size as measured by forward light scattering. Aging senescent cells are smaller and have low forward light scattering properties. The results indicated that, within a given population, the A and/or B and H antigens decreased with the decrease in forward light scattering (i.e., increase in age), but A and B antigen expression decreased to a greater extent than did H. During aging, red cells lose the antigens of the major blood groups, but the degree of this loss is related to the fact that these antigens are located on the cell surface.

Fucosylated Thomsen-Friedenreich antigen in alpha-anomeric configuration in human gastric surface epithelia: an allogeneic carbohydrate antigen possibly controlled by the Se gene.

Human gastric surface epithelial cells display the ABH blood group antigens with the core structure of N-acetyllactosamine (NAcLc). Their expression is under the control of the secretor gene Se. The Thomsen-Friedenreich (T)-antigen (Gal beta 1-3GalNAc) is another core structure of the ABH antigens. We examined the gastric surface epithelial expression of T- and alpha 1-2 fucosylated T (FucT) histochemically with peanut agglutinin (PNA) and monoclonal antibody (MAb) MBr1, respectively. Eight of 24 individuals exhibited the PNA-reactive antigen (i.e., T-expressers) and others the MBr1-reactive antigen (i.e., FucT-expressers). alpha-L-fucosidase digestion of the FucT-positive tissues and beta-galactosidase digestion of the T-positive tissues, respectively, made them reactive with PNA and the antibody specific for GalNAc alpha-O-Ser/Thr. There was a remarkable correlation among reactivities with MBr1, Ulex europaeus lectin 1 (UEA1), and anti-Leb MAb CO-431. ABH blood group status had no correlation with this expression. We conclude that human gastric surface epithelial cells constitutionally synthesize T in alpha configuration (i.e., Gal beta 1-3GalNAc alpha-O-Ser/Thr) and that it was alpha 1-2 fucosylated only in the FucT-expressers. alpha 1-2 fucosylation of T is suggested to be regulated by the Se gene.

Immunocytochemical Localization of Blood Group ABH Antigens in the Human Parotid Gland

The aim of this study was to determine whether the parotid gland, like other salivary glands, produces blood group antigens. A post-embedding immunogold staining (IGS) method for ABH antigens was applied to osmicated and nonosmicated, routinely prepared specimens of normal human parotid glands. A and B antigens were not detected, whereas H antigen was found in acini and ducts of glands from A, B, and O subjects. In osmicated samples, the only sites of H labeling were secretory granules of a few acinar cells and those of most intercalated duct cells. In both cell types, only the pale matrix of the granules was stained. In non-osmicated samples, secretory granules showed the same distribution pattern of H, although gold particles were more abundant, and basolateral membranes of acinar and striated duct cells and the apical vesicles of striated duct cells were also labeled. Our results suggest that small amounts of H antigen are secreted by the parotid gland.

Histochemical Demonstration of Endo-.BETA.-Galactosidase Susceptible Poly-N-Acetyllactosamine with the Blood Group Specificities in Papillary Carcinomas of the Human Thyroid Glands.

Blood group ABH and related antigens are not expressed in normal thyroid glands. However, cells of thyroid papillary carcinoma, especially their luminal surfaces expressed ABH antigens in a manner compatible with the ABO blood group of patients. Sialyl Lewis a (CA 19-9) and sialyl precursor type 1 chain were likewise intensely and frequently expressed in these cells from most of the individuals examined. On the other hand, Lewis x, sialyl Lewis x and sialyl precursor type 2 chain were minor components of the carbohydrate antigens expressed in these cells. In contrast, type 2H, type 2A and presumably type 2B antigens were preferentially expressed in these cancer cells. Prior digestion with endo-β-galactosidase from Escherichia freundii eliminated or reduced the reactivity of monoclonal antibodies and lectins specific for these ABH and related antigens with carcinoma cells. Along with the elimination or reduction of staining with these reagents, binding sites with Griffonia simplicifolia agglutinin-II appeared in the corresponding tissue sites indicating the presence of poly-N-acetyllactosamine structures. These results suggested that poly-N-acetyllactosamine structure containing linear domain susceptible to endo-β-galactosidase digestion is a common and ubiquitous precursor of the blood group-related antigens and its synthesis is prerequisite for the oncofetal expression of these antigens in papillary carcinoma of the thyroids.

Human prostate acid phosphatase is carrier protein for ABH blood group antigens of semen.

Prostate acid phosphatase (PAcP) was characterized as one of carrier proteins for ABH blood group antigens in semen. The diluted semen was sensitized with anti-PAcP. The resulting immunocomplex was subjected to SDS-PAGE after being washed with saline. SDS-PAGE patterns of the immunocomplex consisted of four bands. After electroblotting from gel to nitrocellulose filter, immunostaining using anti-PAcP or monoclonal anti-blood group antibodies was performed. The result showed that the protein of 50 kD was the subunit of PAcP and the band of 94 kD was identified with blood group antigens respectively. Other two bands were rabbit IgG against PAcP and its fragment. When semen was treated with the chemicals such as papain, SDS, 2-mercaptoethanol and dithiothreitol, the ABH of antigen which was captured by anti-PAcP were considerably reduced. The reduction of ABH activity by these treatments indicated that ABH antigens were located on PAcP in semen which was inactivated by the treatment. Therefore, PAcP was one of proteins carrying ABH epitopes in semen in addition to alpha 2-seminoglycoprotein.

ABH antigens as recognition sites for the activation of red blood cell anion exchange by the lectin Ulex europaeus agglutinin I

The blood group antigen H (blood group O) and fucose-specific lectin Ulex europaeus agglutinin I (UEA1) (10 micrograms/ml) was found to increase the rate constant of Cl- efflux into 100 mM Na+ oxalate media by about 40% in erythrocytes taken from antigen H donors. In 100 mM K+ oxalate, 150 mM Na+ pyruvate and in 150 mM Na+ acetate media the lectin elevated the rate constant of Cl- efflux by 20-50%. The acceleration of Cl- efflux by UEA1 was completely blocked by 10 microM 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) indicating that the effect of the lectin is mediated by the anion exchanger of human erythrocytes (band 3 protein). In antigen A1 erythrocytes no significant stimulation of anion exchange by UEA1 was seen. The activation of Cl- efflux was completely prevented by addition of 1 mM fucose to the medium. These results suggest that the effect of UEA1 is mediated through interaction with the fucose residues of H antigens. Increasing extracellular Ca++ from 0.5 to 5 mM in Na+ pyruvate or Na+ acetate media slightly reduced the acceleration of anion exchange by the lectin. On the other hand, replacing part of extracellular chloride by bicarbonate did not considerably alter the (previously reported) stimulatory effect of UEA1 on red blood cell Ca++ uptake. This suggests that the acceleration of anion exchange and of Ca++ uptake by UEA1, respectively, are mediated by different mechanisms. It is concluded that UEA1 activates anion exchange of human erythrocytes most probably by a direct interaction with H antigens present on extracellular domains of the band 3 protein.