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.     

The Lewis blood group system among Chinese in Taiwan.

The nonsecretor gene se is absent (or very rare) among Chinese in Taiwan and the previously reported Le(a+b-) phenotype in this population is in fact Le(a+b+) as proven by the presence of small amounts of Leb antigen on red blood cells. Salivary ABH substances in this phenotype are usually (although not always) markedly reduced. The Chinese Le(a+b+) phenotype is postulated to be the result of a weak secretor gene Se omega. Although the Le(a+b+) phenotype is very rare in Caucasians, it has a frequency of 25% in Chinese. All Le(a-b-) Chinese are ABH secretors and have varying amounts of Lea and/or Leb substances in saliva.     

侗族九个红细胞血型系统和ABH分泌型的分布

报道了广西侗族的ABO、MNSs、Rhesus、Duffy、Kidd、P、Diego、Lewis和Xg等九种红细胞血型系统和ABH唾液分泌型的分布。共调查了201名父母均系侗族而彼此无血缘的学生,其中男116名,女85名。结果表明,广西侗族中ABO系统的r基因(0.6286)、MN系统的m基因(0.6294)、Duffy系统的Fy~a基因(0.9651)、Kidd系统的JK~a基因(0.4628)和Rhesus系统的CDe染色体(0.7532)等频率都很高,ABO系统的q基因(0.1672)、P系统的P_1基因(0.1333)和Lewis系统的Le~a基因(0.3232)等频率较低。MNSs系统的S基因(0.0124)频率很低,而MS染色体连锁率却为零。Xg系统的Xg~a基因频率(0.3746)与汉族和维吾尔族一样,处于低水平。Lewis系统的Le(a )表型者中发现八例是ABH唾液分泌型,但分泌的物质不是A便是B,而分泌H物质的唾液分泌型者全部都是Le(a-)型。六个民族间遗传距离分析表明,侗族与壮族在血缘上最近,其次是与朝鲜族、蒙古族、汉族相近,而与维吾尔族最远。     

Genetic and nongenetic influences on the ABH and Lea antigen levels of saliva and milk.

The saliva and milk of 250 parturient women were studied in relation to ABH antigen levels; part of the sample was also investigated for the Lewis (Lea) substance. The levels of A and B are higher in saliva, and those of H and Lea higher in milk. The H average salivary titers presented the relationship O greater than A2 greater than A1 greater than B greater than AB, but these differences were not present in milk. In addition, the salivary levels of A and B are similar in individuals of these groups but B greater than A in AB persons, and A1 greater than A2; while in milk A greater than B in A, B and AB subjects, and A1 approximately equal to A2. The amount of Lea substance depends of the ABH secretor status in both secretions; but independently of this difference, the average titers were always higher in milk. Correlation coefficients between the levels observed in the two secretions are statistically significant for the A substance in A persons (0.46), H in B (0.58) and Lea in all subjects tested (0.47). A stepwise multiple regression analysis performed to verify the influence of four genetic and six nongenetic variables in the ABH levels of both fluids indicated only one consistent modifying factor: ABO type.     

Factor VIII and factor IX in a twin population. Evidence for a major effect of ABO locus on factor VIII level.

In order to establish the relative importance of genetic factors on the variation in plasma concentration of coagulation factors VIII and IX, these parameters were determined in 74 monozygotic and 84 like-sexed dizygotic twin pairs. The twins belonged to two age groups: 33-39 years and 57-62 years. Factor VIII was determined as factor VIII coagulant antigen (VIIICAg) and as factor VIII-related antigen (VIIIRAg). Factor IX was determined as factor IX antigen (IXAg). A higher value for each coagulation factor was found in the older-age group compared to the younger group, whereas no difference was found between the sexes. A significant correlation was found between values for VIIIRAg and VIIICAg (r = .56). For VIIICAg, it could be demonstrated that the age effect was secondary to the age effect on VIIIRAg. The concentration of VIIICAg and VIIIRAg varied among ABO blood types, being lowest in type O individuals, higher in A2 individuals, and highest in A1 and B individuals. The effect of the ABO locus on VIIICAg was secondary to an effect on VIIIRAg. Analysis of variance revealed a significant genetic influence on the variance of VIIICAg and VIIIRAg with a heritability estimate of .57 for VIIICAg and .66 for VIIIRAg. This is in agreement with a previous hypothesis of an effect of several autosomal genes on factor VIII concentration. Thirty percent of the genetic variance of VIIIRAg was due to the effect of ABO blood type. The ABO locus is therefore a major locus for the determination of factor VIII concentration. No significant genetic effect on the variation in plasma concentration of IXAg could be detected.     

Methodologic and genetic influence on immunohistochemical demonstration and semiquantitation of blood group antigen A in human ureter urothelium

In order to improve the accuracy and prognostic value of ABH blood group antigen loss in urothelial tumors, the effect of Lewis blood type and methodologic factors on detectability and distribution of blood group antigen A in human formalin-fixed, paraplast-embedded urothelium and endothelium was investigated by means of the Tween 20-modified indirect immunoperoxidase staining technique. Urothelium of Lea-b+ and Lea-b- individuals expressed significant higher amounts of blood group antigen A compared to urothelium of Lea+b- individuals. The expression on endothelial cells was related to vessel type and size, but not related to Lewis types. Compared to human anti-A, monoclonal anti-A demonstrated blood group antigen A with higher sensitivity and, due to reduced background staining, higher specificity. Consequently monoclonal anti-A detected blood group antigen A in the urothelium of Lea+b- individuals where human anti-A failed to stain, and different staining patterns became apparent. Both a two- to fourfold variation in the proportion between tissue section area and volume, and the volume of anti-A applied induced minor changes in sensitivity and specificity. The monoclonal anti-A method and knowledge about erythrocyte Lewis types might prove valuable in evaluating changes in blood group antigen-A expression in urothelial tumors.     

Histochemical localization and analysis of blood group-related antigens in human pancreas using immunostaining with monoclonal antibodies and exoglycosidase digestion

We examined the distribution of blood group-related antigens using an indirect immunoperoxidase method with monoclonal antibodies (MAb) directed to A, B, H, Lewis a (Lea), Lewis b (Leb), Lewis x (Lex), and Lewis y (Ley) antigens and Type 1 precursor chain in human pancreas. Effects of prior digestion with exoglycosidases on MAb stainings were simultaneously investigated. A, B, H, Leb, and Ley antigens were detected in acinar cells and interlobular duct cells but not in centroacinar cells, intercalated duct cells, and islet of Langerhans cells. The expression of these antigens in acinar cells was not dependent on Lewis type and secretor status of the tissue donors, whereas that in interlobular duct cells was strictly dependent on secretor status. The distribution pattern of these antigens in acinar cells was not homogeneous, i.e., cells producing H antigens expressed both Leb and Ley antigens but not A or B antigens, whereas those producing A or B antigens did not secrete Leb and Ley as well as H antigens. Digestion with alpha-N-acetylgalactosaminidase or alpha-galactosidase resulted in the appearance of Leb and Ley antigens as well as H antigen in acinar cells producing A and/or B antigens. Type 1 precursor chain was not detected in pancreatic tissues from secretors but appeared in acinar cells producing H antigen after alpha-L-fucosidase digestion, which also disclosed Lex but not Lea antigen in acinar cells expressing both Leb and Ley. In some non-secretors, MAb against Type 1 precursor chain reacted with acinar cells without enzyme digestion. Although Lea antigen was not detected in acinar cells, it was found in centroacinar cells, intercalated duct cells, and interlobular duct cells from all individuals examined except two Le(a-b-) secretors. After sialidase digestion, Lex antigen appeared in centroacinar and intercalated duct cells from some individuals. Sialidase digestion also elicited reactivity with MAb against Type 1 precursor chain in islet of Langerhans cells from some individuals. These results demonstrate the complexity in the pattern of expression and regulation of blood group-related antigens in different cell types of human pancreas. Such complexity may largely be ascribed to differences in individual genotypes and in gene expression patterns of different cell types.     

Histochemical evaluation of secretory glycoproteins in human salivary glands with lectin-horseradish peroxidase conjugates

Paraffin sections of submandibular, sublingual, minor salivary, and parotid glands from ten human autopsy cases were stained with a battery of ten lectins conjugated to horseradish peroxidase. Variable affinity for one or another lectin between mucous cells in a gland evidenced cellular heterogeneity in mucin production. Mucous cells of a given type of gland varied among individuals, but for a single individual appeared markedly but not completely similar from one type of salivary gland to another. The individual variation related, in part, to the ABO blood group and secretor status of the individual. For mucous cells in secretors of blood group A and B all antigens stained strongly for the presence of terminal alpha-N-acetylgalactosamine or alpha-galactose, respectively. Mucous cells in AB secretors contained both antigens, whereas those of O (H) secretors lacked both. Mucous cells of three presumed nonsecretors, two of whom were immature infants and possibly too young to produce ABO antigen, failed to stain. Mucous cells in glands from the presumed nonsecretors, however, revealed a staining pattern consistent with the presence of Lea antigen. Mucous cells of nonsecretors stained with Lotus tetragonolobus agglutinin but not with Ulex europeus I agglutinin, whereas mucous cells of ABO secretors stained with both lectins. This difference in lectin binding indicated that sites reactive only with Lotus tetragonolobus agglutinin contain 1----4 linked fucosyl residues and sites stained by both lectins contain fucose linked 1----2 to the oligosaccharide. Staining of mucous cells of nonsecretors with Pisum sativum agglutinin indicate that either the lectin binds to internal N-acetylglucosamine of Lea substance or the mucous cells contain an N-glycosidic glycoprotein of the type thought to bind this lectin. Serous cells stained less strongly than mucous cells and differed in lectin affinities from one type of gland to another in an individual. Staining of serous cells of a given gland varied markedly among different subjects. This individual variability did not relate to blood group as terminal sugars demonstrative of A or B blood group antigens were not detected in any serous cells. Serous cells in the submandibular glands from the two immature infants were unreactive with all lectin conjugates. Secretions in parotid and submandibular serous cells generally contained a higher content of fucose than those in sublingual serous cells, which contained higher levels of a terminal galactose-sialic acid dimer. Some but not other cells of striated and interlobular ducts of submandibular glands of one subject stained for alpha-N-acetylgalactosamine.     

A serum test for cystic fibrosis using monoclonal antibody 19-9

Monoclonal antibody 19-9 detects a sialosylated Lea antigen with the following sugar sequence: NeuNAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal. . . . This antigen is detected as a mucin in the sera of many patients with gastrointestinal and pancreatic cancer. Elevated levels of sialosylated Lea antigen are also detected in serum from 14 of 16 patients with cystic fibrosis (87%). One of the two negative patients belongs to the Le(a-b-) blood group and so is unable to synthesize the sialosylated Lea antigen. The high percentage of cystic fibrosis patients with elevated sialosylated Lea antigen suggests that the 19-9 antibody may be useful for diagnosis of cystic fibrosis. Antibodies to other sialosylated carbohydrates in mucins may also be useful for detection of cystic fibrosis and may allow diagnosis of patients belonging to the Le(a-b-) blood group.     

Expression of Lewis histo-blood group glycolipids in the plasma of individuals of Le(a+b+) and partial secretor phenotypes

Red cell Lewis antigens are carried by glycosphingolipids passively absorbed from plasma. Plasma was collected from a spectrum of individuals with normal and unusual Lewis/secretor phenotypes in order to investigate the glycolipid basis for the unusual phenotypes. Samples were obtained from: a Le(a+b–) ABH nonsecretor who secreted Lewis substances; a Le(a+b–) partial secretor; Le(a+b+) partial secretors; Le(a+b+) secretors; and a full range of normal Lewis/secretor phenotypes as controls. The Le(a+b+) samples represented Polynesian, Asian and Réunion Island ethnic backgrounds. Nonacid glycolipids were prepared, separated by thin-layer chromatography, and then immunostained with potent monoclonal antibodies of known specificity. Despite different serological profiles of the Le(a+b–) and Le(a+b+) Polynesian samples, their plasma glycolipid expressions were very similar, with both Le^a and Le^b co-expressed. The copresence of Le^a and Le^b in Le(a+b+) samples is in marked contrast to Caucasians with normal Lewis phenotypes, who have predominantly either Le^a or Le^b. These results suggest that there is a range of the secretor transferases in different individuals, possibly due to different penetrance or to several weak variants. We also show that Lewis epitopes on longer and/or more complex core chains appear to be predominant in the Polynesian Le(a+b+) samples. The formation of these extended glycolipids is compatible with the concept that in the presence of reduced secretor fucosyltransferase activity, increased elongation of the precursor chain occurs, which supports the postulate that fucosylation of the precursor prevents or at least markedly reduces chain elongation.Abbreviations CBA chromatogram binding assay - TLC thin-layer chromatography     

Two familial cases of dissociation of saliva Lea levels and erythrocyte Lewis types

Two familial cases in which the saliva Lea levels were seen to dissociate with the donors' red blood cell (RBC) Lewis types are reported. In case 1, the saliva from a donor with an RBC type of Le(a+b-) contained a low level of Lea antigen. A low Lea level was also observed in the saliva from this proband's father who has an RBC type of Le(a+b-). In case 2, the saliva from a donor with an RBC type of Le(a-b+) contained a high level of Lea antigen. High Lea levels were also present in the saliva of this proband's father and brothers with an RBC type of Le(a-b+).     

Histochemical demonstration of O-glycosidically linked, type 3 based ABH antigens in human pancreas using lectin staining and glycosidase digestion procedures

Histochemical analyses of the chemical structures of sugar sequences with or without blood group specificity were carried out by combined stepwise digestion of tissue sections with exo- and endoglycosidases and subsequent lectin stainings in formalin-fixed, paraffin-embedded human pancreas. In acinar cells from blood group A or AB secretor individuals, sequential digestion with alpha-N-acetylgalactosaminidase and alpha-L-fucosidase imparted reactivity with peanut agglutinin (PNA) in cells reactive with Dolichos biflorus agglutinin as well as those with Ulex europaeus agglutinin I(UEA-I). Simple fucosidase digestion imparted the PNA reactivity only in UEA-I reactive cells. Sequential digestion with alpha-galactosidase and fucosidase likewise liberated the PNA binding sites in Griffonia simplicifolia agglutinin I-B4 reactive cells from blood group B and AB secretors. Sialidase digestion liberated the PNA binding sites not only in acinar cells but also intercalated duct cells, islet cells of Langerhans and endothelial cells. The PNA reactivity obtained by these enzyme digestions was eliminted by endo-alpha-N-acetylgalactosaminidase (endo-GalNAcdase) digestion. Preexisting PNA affinity in acinar cells from non-secretors was also susceptible to endo-GalNAcdase treatment. Following the endo-GalNAcdase digestion, fucosidase or sialidase digestion recovered the PNA reactivity in acinar cells from nonsecretors. These results show that ABH determinants carried on O-glycosidically linked type 3 chain (D-galactose-(beta 1-3)-N-acetyl-D-galactosamine alpha 1-serine or threonine) are secreted in pancreatic acinar cells and suggest that product coded by the secretor gene is required for the complete conversion of type 3 precursor chains into H determinants.     

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.     

Blood group A glycosphingolipid accumulation in the hair of patients with alpha-N-acetylgalactosaminidase deficiency

In the hair of individuals with blood group AB, the level of blood group A glycosphingolipids is much lower than that of blood group B. We hypothesized that in hair, blood group A determinants are converted by alpha-N-acetylgalactosaminidase (alpha-NAGA, E.C.3.2.1.49) to H determinants. To address our hypothesis, the relative amount of ABH glycosphingolipids in hairs and nails of normal subjects, patients with Kanzaki disease, and heterozygous carriers of alpha-NAGA deficiency were analyzed by dot-blotting and enzyme-linked immunosorbent assay. In hair from normal subjects with blood group B, ABH glycosphingolipids consisted of 88% blood group B- and 12% blood group H glycosphingolipids. In blood group A subjects, 14% were group A- and 86% were group H glycosphingolipids. In Kanzaki patients, 81% were blood group A- and 19% were blood group H glycosphingolipids. In 2 alpha-NAGA deficiency carriers, the ABH glycosphingolipids consisted of 67% blood group A- and 33% blood group H glycosphingolipids. These results indicate that blood group A glycosphingolipids are catabolized to H glycosphingolipids by alpha-NAGA, resulting in lower levels of blood group A glycosphingolipids in the hair of normal subjects, and alpha-NAGA deficiency causes accumulation of blood group A glycosphingolipids in the hair of Kanzaki patients. This finding is of clinical relevance because it suggests that hair may be used to diagnose and assess the alpha-NAGA status of individuals.     

Aberrant secretors in donors and patients with duodenal ulcer

Results of the screening of aberrant (paradoxal) secretors in 14372 healthy donors and 614 patients with ulcus duodeni are presented. An extremely high frequency of aberrant secretors was established (12.6%) in patients with ulcus duodeni in comparison to donors (0.10%). A new type of aberrant secretors was registered in patients of A1 and A1B blood groups. Their salivas normally inhibited anti-A sera, but the expected inhibition of anti-A1 reagents (dolichos biflorus) could not be observed. The data of comparative quantitative investigations of the inhibiting strength of the ABH antigen in salivas of normal secretors, nonsecretors and aberrant secretors are presented. Various theoretical explanations of aberrant secretors are discussed.     

Isolation, composition and reactivity of the neutral glycoproteins from human meconiums with specificities of the ABO and Lewis systems.

Blood-group-specific A1, B, AB, H and Lea neutral glycoproteins were isolated from suitable pools of human normal meconiums by a preliminary fractionation with a cationic detergent at pH5 and 9 (borate), followed by ion-exchange and gel chromatography. The ABH materials have sedimentation coefficients of about 10S-11S, whereas the Lea preparation, not strictly homogeneous, shows a coefficient of 7S. From the detailed analytical data collected, the following relations are deduced between these various substances; they all possess a common peptide core; there are stable ratios of N-acetylglucosamine/N-acetylgalactosamine in the B, H and Lea materials and of N-acetylglucosamine/galactose in A, H and Lea materials, from which the numbers of A and B determinants are estimated. In the ABH substances, the ratio of glucosamine to the sum of threonine and serine is stable. Presumably because of genetic factors, the amount of fucose varies among the different glycoproteins, but it is always definitely lower than in the average cyst substances. Various serological tests and precipitin methods were used to measure the potency, purity and integrity of the preparations, including comparisons between A1 and A2 substances from this source. The Leb activity did not appear as high as it is in glycoproteins from adults and a possible interpretation would be the immature Lewis system as observed on erythrocytes; this could explain their very strong inhibiting power towards iso-agglutinins. This family of substances with various specificities has common features with that prepared from ovarian cysts, but differs clearly on some points.     

Expression of blood group A and B antigens in nuclear heterochromatin in mucous cells of human cervical glands.

Using a post-embedding immunogold labeling procedure, we found that monoclonal antibody against A (MAb-A) or B antigen (MAb-B) reacted with nuclear heterochromatin regions, as well as secretory granules, in mucous cells of human cervical glands. Systematic and critical observation of specimens from 24 individuals of different blood groups revealed that the labeling pattern with MAb with strictly dependent on the blood group (A,B, or O) of the donors, i.e., MAb-A reacted with the heterochromatin from blood group A and AB but not with B and O individuals. Labeling with MAb-B was also specific for the heterochromatin from blood group B donors. On the other hand, MAb against H antigen did not react with the heterochromatin from any individuals examined, despite the fact that H antigens were detected by the MAb in secretory granules. Such specific reactions provide evidence that certain types of blood group-related antigens exist in the nuclear heterochromatin in mucous cells of human cervical glands. In contrast to the secretory granules in which ABH antigens were recognized by blood group-specific lectin, heterochromatin regions had little or no affinity for these lectins. Furthermore, the secretory status of individuals affected the staining intensity with MAb in secretory granules but not in the heterochromatin. These results suggest that the blood group substances found in the heterochromatin may have different molecular properties from those in the secretory granules, although both have the same determinant structures of ABH antigens.     

Individuals with Le(a+b-) blood group have increased susceptibility to symptomatic vibrio cholerae O1 infection

Background

Human genetic factors such as blood group antigens may affect the severity of infectious diseases. Presence of specific ABO and Lewis blood group antigens has been shown previously to be associated with the risk of different enteric infections. The aim of this study was to determine the relationship of the Lewis blood group antigens with susceptibility to cholera, as well as severity of disease and immune responses to infection.

Methodology

We determined Lewis and ABO blood groups of a cohort of patients infected by Vibrio cholerae O1, their household contacts, and healthy controls, and analyzed the risk of symptomatic infection, severity of disease if infected and immune response following infection.

Principal Findings

We found that more individuals with cholera expressed the Le(a+b−) phenotype than the asymptomatic household contacts (OR 1.91, 95% CI 1.03–3.56) or healthy controls (OR 1.90, 95% CI 1.13–3.21), as has been seen previously for the risk of symptomatic ETEC infection. Le(a–b+) individuals were less susceptible to cholera and if infected, required less intravenous fluid replacement in hospital, suggesting that this blood group may be associated with protection against V. cholerae O1. Individuals with Le(a–b−) blood group phenotype who had symptomatic cholera had a longer duration of diarrhea and required higher volumes of intravenous fluid replacement. In addition, individuals with Le(a–b−) phenotype also had lessened plasma IgA responses to V. cholerae O1 lipopolysaccharide on day 7 after infection compared to individuals in the other two Lewis blood group phenotypes.

Conclusion

Individuals with Lewis blood type Le(a+b−) are more susceptible and Le(a–b+) are less susceptible to V. cholerae O1 associated symptomatic disease. Presence of this histo-blood group antigen may be included in evaluating the risk for cholera in a population, as well as in vaccine efficacy studies, as is currently being done for the ABO blood group antigens.     

Subcellular localization of blood group substances ABH in human salivary glands

We investigated the subcellular localization of ABH antigens in human submandibular, sublingual, and buccal glands by applying a post-embedding immunogold method using monoclonal antibodies specific for A, B, and H antigens. In most glands the immunoreactivity was usually restricted to mucous cells, in which only secretory granules and sometimes Golgi cisternae were specifically labeled. A and B antigens were demonstrated only in the glands of type A, B, and AB subjects, while H antigen was visualized in glands from individuals of all blood types. Moreover, differences were observed in the relative distribution of ABH antigens, depending on the type of gland.