Semiquantitative immunohistochemical studies of blood group antigen A,B,H, Lea, Leb structures and Ii backbone chains in the normal human cervix and in cervical adenocarcinoma

Summary Epithelia frequently express blood group antigens and these are often perturbed in neoplasia. This study has characterized the range of expression of ABH and Lewis terminal structures and the Ii backbone chains in the normal human cervix by semiquantitative immunohistochemistry. Effects of the secretor gene were defined by determination of salivary secretor status. Modifications of blood group antigen expression in cervical adenocarcinoma were also addressed.Normal cervical squamous and glandular epithelia showed a range of expression of the antigens studied. Lewis-gene-negative cases showed no expression of Lewis antigens. Secretor status had no effect on ABH expression in squamous epithelium, but it did have a marked effect on ABH expression in glands and on Le^b expression in both squamous and glandular epithelia. Patterns of expression of i chains in squamous epithelium suggest that these may be the carriers of ABH and Lewis antigens in a proportion of cases. Distinct patterns of expression were seen in glandular tubal metaplasia and in endothelium.Adenocarcinomas showed topographical rather than quantitative changes in blood group antigen expression with more extensive luminal expression of ABH, Lewis and Ii structures than that seen in normal glands. This change is distinct from those usually associated with malignancy.     

Insights into the expression of ABH and Lewis antigens through human bone marrow transplantation.

Twelve information bone marrow transplants, with at least one difference in ABO and/or Lewis types between donor and recipient, were retrospectively studied. ABH and Lewis antigens were determined in plasma, erythrocytes, and lymphocytes. Donor lymphocytes acquired the ABH and Lewis antigens from the recipient's plasma in the same way that donor erythrocytes acquired the Lewis antigens from it. Lymphocytotoxicity detected type 1 ABH and Lewis antigens only, providing evidence for the existence of combined ABH and Lewis antigens on lymphocytes. This was in contrast with the ABH antigens on type 2 chains of red cells, which are devoid of Lewis specificities. The differences in genetic control, probable chemical structure, and cellular origin of these two types of ABH antigens are presented in a theoretical model that accounts for most of the known data.     

Norwalk virus-like particles bind specifically to A,H and difucosylated Lewis but not to B histo-blood group active glycosphingolipids

Noroviruses and norovirus virus-like particles (VLPs) exhibit strain specific patterns in their binding to ABH and Lewis histo-blood group antigens. In this study we demonstrate for the first time specific binding of Norwalk virus VLPs to type 1 and type 2 chain glycosphingolipids (GSLs) carrying ABH and Lewis antigens. N-succinimidyl-3-tributylstannyl benzoate (ATE) was precursor labeled with ¹²⁵I and then conjugated to VLPs. The ¹²⁵I-VLPs were used in GSL thin-layer chromatogram binding assays and displayed binding to H type 1, Lewis b, A type 1, A Lewis b GSLs but no binding to B type 1 or B Lewis b GSLs. For the type 2 chain GSLs the Norwalk VLPs bound to H type 2, Lewis y, A type 2 and A Lewis y. In addition, the VLPs bound to several complex GSLs from blood group O and A, but not from blood group B red blood cells.     

Frequency of Lewis blood groups and ABH and Lewis substance secretion in Schleswig-Holstein]

According to the hypothesis of Ceppellini and Morgan the Lewis blood groups are formed by the secondary attachment of the Lewis substances to the red blood cells and this process is genetically controlled by the genes of the ABH und Lewis(a)-substance secretion (SE, se, L and 1). The correctness of this hypothesis is demonstrated by determination of the Lewis blood groups and the ABH and Lewis secretor status with different suitable antisera and by estimation of the gene frequencies Se, se, L and 1 in a sample of 382 blood donors from Schleswig-Holstein and by determination of the same groups in 73 pairs of parents with 156 children. There are no significant differences between observation and expectation in the sample as well as in the family investigation and there are no critical pairs of parents having children with "impossible" Lewis blood group. The results suggest to make more use of the Lewis blood groups.     

Blood group antigens on human erythrocytes-distribution,structure and possible functions

Human erythrocyte blood group antigens can be broadly divided into carbohydrates and proteins. The carbohydrate-dependent antigens (e.g.,ABH, Lewies, Ii, P₁, P-related, T and Tn) are covalently attached to proteins and/or sphingolipids, which are also widely distributed in body fluids, normal tissues and tumors. Blood group gene-specific glycosyltransferases regulate the syntheses of these antigens. Protein-dependent blood group antigens (e.g., MNSs, Gerbich, Rh, Kell, Duffy and Cromer-related) are carried on proteins, glycoproteins and proteins with glycosylphosphatidylinositol anchor. The functions of these molecules on human erythrocytes remain unknown; some of them may be involved in maintaining the erythrocyte shape. This review describes the distribution, structures and probable biological functions of some of these antigens in normal and pathological conditions.     

Relationship between lectin binding properties and the expression of blood group ABH antigens in vascular endothelia and red blood cells from 18 primate species

Summary The reactivity was examined of horseradish peroxidase labelledUlex europaeus agglutinin-I (UEA-I) andGriffonia simplicifolia agglutinin I-B₄ (GSAI-B₄) with red blood cells and vascular endothelium in formalin-fixed, paraffin embedded tissues from 18 primate species. The expression of blood group ABH antigens in these cells as well as secretions from other tissues was also examined by the indirect immunoperoxidase method using monoclonal anti-ABH antibodies as primary antibodies. In Prosimians and New World monkeys which lack ABH antigens on both red blood cells and endothelial cells, but produce these antigens in other tissue secretions, GSAI-B₄ always reacted with both red blood cells and endothelial cells. In Old World monkeys, which express blood group antigens on endothelial cells but not on red blood cells, neither GSAI-B₄ nor UEA-I reactivity were observed, except the endothelial cells from blood group B or O individuals occasionally reacted with GSAI-B₄ or UEA-I, respectively. Although UEA-I reactivity was not observed in the endothelial cells of gibbon, it reacted with these cells from chimpanzees. In these two anthropoid apes, both endothelial cells and red blood cells expressed ABH antigens as in humans. These results suggest the close evolutionary relationship between the expression of blood group ABH antigens and lectin binding properties of red blood cells and endothelial cells in primate species.     

Red cell H-deficient,salivary ABH secretor phenotype of Reunion island. Genetic control of the expression of H antigen in the skin

Based on the genetic model proposing thatH andSe are two structural genes, we predicted that the red cell H-deficient, salivary ABH secretor phenotype should be found on Reunion island, where a large series of H-deficient non-secretor families have been previously described. Two such Reunion individuals are now reported. POU [A_h, Le(a–b+), secretor of A, H, Le^a and Le^b in saliva] and SOU [O_h, Le(a–b+), secretor of H, Le^a and Le^b in saliva]. Both are devoid of H -2-fucosyltransferase activity in serum. In addition, the preparation of total non-acid glycosphingolipids from plasma and red cells of POU revealed the type 1ALe^b heptaglycosylceramide and small amounts of the monofucosylated type 1 A hexaglycosylceramide. Both glycolipids possess an H structure probably synthesised by the product of theSe gene. No other blood group A glycolipids, with types 2, 3 or 4 chains, normally present in the presence of the product of theH gene, were found on red cells or plasma of POU.TheH,Se andLe genetic control of the expression of ABH and related antigens in different tissue structures of the skin is described in 54 H-normal individuals of known ABO, secretor and Lewis phenotypes; in one red cell H-deficient salivary secretor (SOU); and in one H-deficient non-secretor (FRA). Sweat glands express ABH under the control of theSe gene. Sweat ducts express ABH under the control of bothH andSe genes and Lewis antigens under the control ofLe and bothH andSe genes. Epidermis, vascular endothelium and red cells express ABH under the control of theH gene. The products ofH andSe genes are usually expressed in different cells. However, the results illustrate that in some structures, like the epithelial cells of sweat ducts, both the products ofH andSe genes can contribute to the synthesis of the same Le^b structure.     

Lewis phenotypes and secretor character in the "Castilla la Nueva"-region (Spain). ABH and Lewis antigen levels in salivary secretion

In 1980 blood and saliva samples were taken from Spanish students of the University of Madrid. Red cells were analysed for A1B2BO and Lewis blood groups. Saliva samples were tested to detect the specific group substances ABH, Lea and Leb. A slightly higher frequency of the "le" gene (0.419) was found in our sample as compared to other Spanish samples. The phenotype frequencies of ABH secretors (77.2%) and non-secretors (22.8%) are in the range of other European populations. The levels of A and B antigens of individuals belonging to these blood groups were similar, whereas the average titration of the H substance showed the relation O greater than A2 greater than A1 greater than A1B greater than B. Analysis of variance proved this heterogeneity to be statistically significant. The amount of Lea substance in non-secretors was higher than in secretors. This shows again that the ABH secretor status has some influence on the quantity of this antigen. The average titration of the Leb substance in secretors was higher than that of Lea in individuals belonging to O, A and AB blood groups, but not in those with blood group B.     

Lewis histo-blood group α1,3/α1,4 fucose residues may both mediate binding to GII.4 noroviruses

Human noroviruses cause recurrent epidemics of gastroenteritis known to be dominated by the clinically important GII.4 genotype which recognizes human Secretor gene-dependent ABH histo-blood group antigens (HBGAs) as attachment factors. There is increasing evidence that GII.4 noroviruses have undergone evolutionary changes to recognize Lewis antigens and non-Secretor saliva. In this study, we have investigated the possibilities of the Lewis α1,3/α1,4 fucoses as mediators of binding of GII.4 noroviruses to Lewis antigens. The study was carried out using molecular dynamics simulations of Lewis type-1 and type-2 chain HBGAs in complex with VA387 P domain dimers in explicit water. Based on the computer simulations, we suggest the possibility of two receptor binding modes for Lewis HBGAs: the "Secretor pose" with the Secretor Fucα1,2 in the binding site and the "Lewis pose" with the Lewis Fucα1,3/α1,4 residues in the binding site. This was further supported by an extensive GlyVicinity analysis of the Protein Data Bank with respect to the occurrence of the Lewis and Secretor poses in complexes of Lewis antigens with lectins and antibodies as well as GII norovirus strains. The Lewis pose can also explain the interactions of GII.4 norovirus strains with Le(x) and SLe(x) structures. Moreover, the present model suggests binding of complex branched polysaccharides, with the Lewis antigens at the nonreducing end, to P domain dimers of GII.4 strains. Our results are relevant for understanding the evolution of norovirus binding specificities and for in silico design of future antiviral therapeutics.     

Blood group antigen expression is involved in <Emphasis Type="Italic">C. albicans</Emphasis> interaction with buccal epithelial cells

Human blood group polymorphisms are known to be determined by the expression of A, B or H antigens and the Lewis antigens. Protection against microbial infections has been associated with inheritance of polymorphisms in genes encoding and regulating the expression of ABH and Lewis antigens in bodily secretions and epithelial tissue surfaces, subsequently resulting in the presentation of different glycosylated terminal antigens on the cell surface. We investigated the role of blood group antigens in diversifying the glycosylation of buccal epithelial cells (BEC) that line the oral cavity. Specifically, we characterized and statistically evaluated the expression of histo-blood group (A, B, O) antigens on N-and O-linked glycans from BEC membrane proteins of various individuals that represented different blood group type and secretor status using a porous graphitic carbon liquid chromatography electrospray ionization mass spectrometry (PGC-LC-ESI-MS) based glycomics approach. From these BEC membrane proteins a total of 77 N-glycan and 96 O-glycan structures were structurally characterized from 19 individuals and relatively quantitated. The N-glycans from the secretor individuals did not express any A/B blood group determinants, but contained several terminal H-antigens. Apart from the non-secretors, the N-glycan profiles of BEC from all blood groups displayed similar glycan types, while varying in their relative intensities between individuals. However, multivariate analysis of the O-glycans from individuals displayed segregation patterns clearly associated with their blood group type and secretor status. In adhesion assays the oral pathogen Candida albicans showed a significantly higher interaction to blood group O type BECs relative to other blood groups.     

Structural basis for the recognition of lewis antigens by genogroup I norovirus

Noroviruses (NoVs) bind to histo-blood group antigens, namely, ABH antigens and Lewis antigens. We previously showed the NoVs GI/2, GI/3, GI/4, and GI/8 were able to strongly bind to Lewis a (Le^a) antigen, which is expressed by individuals who are nonsecretors. In this study, to investigate how Lewis antigens interact with GI NoV virion protein 1 (VP1), we determined the crystal structures of the P domain of the VP1 protein from the Funabashi 258 (FUV258) strain (GI/2) in complexes with Le^a, Le^b, H type 1, or A type 1 antigens. The structures were compared with those of the NV/68 strain (GI/1), which does not bind to the Le^a antigen. The four loop structures, loop P, loop S, loop A, and loop B, continuously deviated by more than 2 Å in length between the Cα atoms of the corresponding residues of the FUV258 and NV/68 P domains. The most pronounced differences between the two VP1 proteins were observed in the structures of loop P. In the FUV258 P domain, loop P protruded toward the next protomer, forming a Le^a antigen-binding site. The Gln389 residue make a significant contribution to the binding of the Le^a antigen through the stabilization of loop P as well as through direct interactions with the α4-fucosyl residue (α4Fuc) of the Le^a antigen. Mutation of the Gln389 residue dramatically affected the degree of binding of the Lewis antigens. Collectively, these results suggest that loop P and the amino acid residue at position 389 affect Lewis antigen binding.     

Role of ABO secretor status in mucosal innate immunity and H. pylori infection

The fucosylated ABH antigens, which constitute the molecular basis for the ABO blood group system, are also expressed in salivary secretions and gastrointestinal epithelia in individuals of positive secretor status; however, the biological function of the ABO blood group system is unknown. Gastric mucosa biopsies of 41 Rhesus monkeys originating from Southern Asia were analyzed by immunohistochemistry. A majority of these animals were found to be of blood group B and weak-secretor phenotype (i.e., expressing both Lewis a and Lewis b antigens), which are also common in South Asian human populations. A selected group of ten monkeys was inoculated with Helicobacter pylori and studied for changes in gastric mucosal glycosylation during a 10-month period. We observed a loss in mucosal fucosylation and concurrent induction and time-dependent dynamics in gastric mucosal sialylation (carbohydrate marker of inflammation), which affect H. pylori adhesion targets and thus modulate host-bacterial interactions. Of particular relevance, gastric mucosal density of H. pylori, gastritis, and sialylation were all higher in secretor individuals compared to weak-secretors, the latter being apparently "protected." These results demonstrate that the secretor status plays an intrinsic role in resistance to H. pylori infection and suggest that the fucosylated secretor ABH antigens constitute interactive members of the human and primate mucosal innate immune system.     

Identification of Le antigens in meconium of a phenotypically a Le(ab) non-secretor individual

Blood group active fucolipids of human meconium have been shown to correlate to the ABH and Lewis blood groups and to the secretor status of the corresponding children. Using a monoclonal anti-Le^b antibody and an antibody to chromatogram binding assayy the presence of Le^b antigens in meconium of a phenotypically A Le(a⁺b⁻) non-secretor indivual is here demonstrated. Phenotype was determined on cord blood and saliva obtained 2 years after birth.     

Identification of Leb antigens in meconium of a phenotypically a Le(a+b−) non-secretor individual

Blood group active fucolipids of human meconium have been shown to correlate to the ABH and Lewis blood groups and to the secretor status of the corresponding children. Using a monoclonal anti-Le^b antibody and an antibody to chromatogram binding assayy the presence of Le^b antigens in meconium of a phenotypically A Le(a⁺b^?) non-secretor indivual is here demonstrated. Phenotype was determined on cord blood and saliva obtained 2 years after birth.     

Conformational studies on the ABH and Lewis blood group oligosaccharides

The possible conformations for the ABH and Lewis blood group oligosaccharides have been studied by an energy-minimisation procedure using empirical potential functions. It has been found that the conformation of the core structure is not altered significantly by the addition of l-fucose, galactose or N-acetyl galactosamine residues at the non-reducing end. Correlation of the preferred conformations with their known binding properties suggests that the differences between type 1 and type 2 structures become significant only when a large enough fragment of the determinant is considered. It is suggested that non-specific reagents may have small binding sites while the reagents that are specific for type 1 or type 2 structures may have larger binding sites. A two-pocket model has been proposed for antibodies and lectins which can distinguish the A1 and A2 antigens.     

The significance of Epstein Barr Virus (EBV) & DNA Topoisomerase II alpha (DNA-Topo II alpha) immunoreactivity in normal oral mucosa, Oral Epithelial Dysplasia (OED) and Oral Squamous Cell Carcinoma (OSCC)

Background

The glycosylation of a great number of molecules, glyco-protein or glycolipids, has been of interest for decades.

Objective

To compare the expressive patterns of the isoantigenic determinants of histo-blood groups ABH and Lewis in squamous and simple epithelium and in precursors and cancers of the cervix.

Methods

A total of 36 lesions and neoplasms (10 LG-SIL, 16 HG-SIL and 10 invasive carcinomas) have been studied with immunohistochemical techniques, using monoclonal antibodies (MoAb BG1 to BG8) for precursor chains, blood-group ABH and Lewis group Le^a, Le^b, Le^x, and Le^y, and four types of lectins. In addition, we have studied the expression of p53 protein and PCNA, establishing the rate of proliferation of each lesion. Using PCR techniques, we have also detected part of the intron of the E6 gene of HPV-16.

Results

In the invasive cervical carcinomas, we observed a loss of expression of the Le^x antigen (p < 0.01). With regard to the progression of the different lesions studied, we found alterations in the patterns of expression of the antigens of the ABH and Lewis blood groups. There was a tendency towards a loss of expression and heterogeneous patterns in the more advanced lesions, as well as over-expression of the Le^y antigens. With PCNA, we established a proliferative rate which tended to be greater in relation to the progression of the cervix neoplasms.

Conclusion

These results indicate that there is a relation between the losses of histo-blood groups and the progression of the squamous intraepithelial lesions.     

Carbohydrate recognition factors of a Talpha (Galbeta1-->3GalNAcalpha1-->Ser/Thr) and Tn (GalNAcalpha1-->Ser/Thr) specific lectin isolated from the seeds of Artocarpus lakoocha

Artocarpus lakoocha agglutinin (ALA), isolated from the seeds of A. lakoocha fruit, is a galactose-binding lectin and a potent mitogen of T and B cells. Knowledge obtained from previous studies on the affinity of ALA was limited to molecular and submolecular levels of Galbeta1-->3GalNAc (T) and its derivatives. In the present study, the carbohydrate specificity of ALA was characterized at the macromolecular level according to the mammalian Gal/GalNAc structural units and corresponding glycoconjugates by an enzyme-linked lectinosorbent (ELLSA) and inhibition assays. The results indicate that ALA binds specifically to tumor-associated carbohydrate antigens GalNAcalpha1-->Ser/Thr (Tn) and Galbeta1-->3 GalNAcalpha1-->Ser/Thr (Talpha). It barely cross-reacts with other common glycotopes on glycoproteins, including ABH blood group antigens, Galbeta1-->3/4GlcNAc (I/II) determinants, T/Tn covered by sialic acids, and N-linked plasma glycoproteins. Dense clustering structure of Tn/Talpha-containing glycoproteins tested resulted in 2.4 x 10(5)-6.7 x 10(5)-fold higher affinities to ALA than the respective GalNAc and Gal monomer. According to our results, the overall affinity of ALA for glycans can be ranked respectively: polyvalent Tn/Talpha glycotopes > monomeric Talpha and simple clustered Tn > monomeric Tn > GalNAc > Gal; while other glycotopes: Galalpha1-->3/4Gal (B/E), Galbeta1-->3/4GlcNAc (I/II), GalNAcalpha1-->3Gal/GalNAc (A/F), and GalNAcbeta1-->3/4Gal (P/S) were inactive. The strong specificity of ALA for Tn/Talpha cluster suggests the importance of glycotope polyvalency during carbohydrate-receptor interactions and emphasizes its value as an anti-Tn/T lectin for analysis of glycoconjugate mixtures or transformed carbohydrates.     

Expression of Lewis antigenic determinants in colorectal adenocarcinomas

Expression of type 1 and type 2 chain Lewis antigens was studied in 32 rectal adenocarcinoma specimens; the results were correlated with the patients' Lewis phenotype and secretor status. In addition, the pattern of expression of these antigens was analyzed in adjacent and distant normal mucosa. We used an indirect immunofluorescence technique with p-phenylenediamine counterstaining (Oriol technique) and a panel of monoclonal antibodies directed against the different antigenic specificities. Normal distal colonic mucosa only expresses monofucosylated structures (Lea and X) arising from activity of the alpha 1-3,4-fucosyltransferase coded by the Le gene. Rectal adenocarcinomas also show Lea and X, but also reexpress blood group antigens ABH and exhibit difucosylated determinants (Leb and Y). The accumulation of mono- and difucosylated type 2 chain in neoplastic processes, independently of the Le and Se genes, could be due to the enzymes coded by reactivation of the H and X genes. Blood group antigens form a complex signal code, genetically regulated, which intervenes in differentiation, growth and cellular recognition processes, and which may undergo important modifications during malignant transformation. These alterations could be useful in the diagnosis and prognosis of some types of carcinoma.     

The ABO, Lewis and related blood group antigens; a review of structure and biosynthesis

Numerous studies have shown that the antigenic determinants of the ABO blood group system are closely related in biochemical terms to the antigenic determinants of the Hh, P, Lewis and Ii blood group systems. The blood group antigens of each of these systems are formed by the addition of specific sugars to an oligosaccharide precursor chain which may be bound through sphingosine to fatty acids (glycolipid) or through serine or threonine to a peptide chain (glycoproteins). The direct gene products of each of these blood group systems are the glycosyltransferase enzymes which catalyse the addition of the specific sugar thus conferring the specified blood group activity to the glycolipid or glycoprotein molecule. The antigenic determinants of the ABO and Lewis systems in addition to red cells also exist in the body secretions in soluble form when the relevant genes are expressed in the phenotype. The antigens expressed on both the red cells and in the secretions are determined by the interaction of Hh, Sese, ABO and Lele genes.     

The ABO, Lewis and related blood group antigens; a review of structure and biosynthesis

Abstract Numerous studies have shown that the antigenic determinants of the ABO blood group system are closely related in biochemical terms to the antigenic determinants of the Hh, P, Lewis and Ii blood group systems. The blood group antigens of each of these systems are formed by the addition of specific sugars to an oligosaccharide precursor chain which may be bound through sphingosine to fatty acids (glycolipid) or through serine or threonine to a peptide chain (glycoproteins). The direct gene products of each of these blood group systems are the glycosyltransferase enzymes which catalyse the addition of the specific sugar thus conferring the specified blood group activity to the glycolipid or glycoprotein molecule. The antigenic determinants of the ABO and Lewis systems in addition to red cells also exist in the body secretions in soluble form when the relevant genes are expressed in the phenotype. The antigens expressed on both the red cells and in the secretions are determined by the interaction of Hh, Sese, ABO and Lele genes.