Blood group antigens in differentiated thyroid neoplasms

Alteration of cell-surface blood group antigens during malignant transformation is a well-known phenomenon that has not yet been sufficiently investigated in thyroid gland neoplasms. We evaluated 50 normal thyroid glands and 141 differentiated thyroid neoplasms (29 follicular adenomas, 30 follicular carcinomas, 56 papillary carcinomas, and 26 medullary carcinomas) both by the immunoperoxidase technique, using monoclonal antibodies against blood group antigens (A, B, H, Le(a), Le(b), Le(x), and Le(y)) and precursor substances (T, Tn, and sTn), and by affinity to the lectin from Arachis hypogea, to determine the usefulness of these antigens as tumor markers and prognostic factors. Neoplastic tissues showed immunostaining with concordant and nonconcordant expression of ABH antigens. There were statistically significant differences between normal and neoplastic tissues but not among the different neoplasms. Statistically significant differences in Lewis antigen expression were noted between normal and neoplastic tissues and between benign and malignant tumors. Tn and sTn antigen expression showed statistically significant differences between normal and neoplastic tissues. In conclusion, blood group antigens are tumor markers that are expressed more frequently in malignant than in benign neoplasms. The presence of metastases was correlated with enhanced peanut lectin receptors and a loss of A or B antigens.     

ABH blood group antigen significance as markers of endothelial differentiation of mesenchymal cells

The expression pattern of A, B and H blood group antigens was evaluated by staining frozen sections with specific monoclonal antibodies developed by us and using the indirect immunoperoxidase method. The expression of blood group antigens was ubiquitously upregulated in the endothelial cells of fetal organs. In the process of their differentiation to endothelial naive embryonic mesenchymal cells expressed cytoplasmic ABH antigens. They were assumed as products of the activation of the respective genes. ABH antigen expression was considered as suggestive evidence for the assumption that blood group antigens could serve as early immunomorphologic markers of endothelial differentiation of mesenchymal cells, thus specifying the location of future blood vessels. Extending the conceptual framework of blood group antigens' significance we consider them as being possibly involved in the process of fetal morphogenesis.     

Effect of O-glycosylated mucin on invasion and metastasis of HM7 human colon cancer cells

Mucinous colorectal cancers have a poorer prognosis than colorectal cancers which produce a low amount of mucin, but the exact mechanism is not well understood. The present study was undertaken to elucidate the possible mechanisms of invasion and metastasis of colon cancer cells producing high levels of mucin using mucin glycosylation inhibitor, benzyl-alpha-N-acetylgalactosamine. The binding activity of treated HM7 cells to endothelial leukocyte adhesion molecule (ELAM-1) was significantly decreased and fixed cell binding of MoAb SNH-3 and 19-9 (specific for sialyl Le(x) and sialyl Le(a), respectively) was also significantly decreased. Metalloproteinase activity in conditioned medium and invasion of matrigel-coated porous filters by treated HM7 cells were decreased. However, there was no difference between control and treated HM7 cells in terms of matrix protein binding. These results suggest that O-glycosylated mucin is important in the invasive and metastatic properties of HM7 human colon cancer cells.     

Staphylococcus aureus binding to human nasal mucin

Colonization of human nasal mucosa with Staphylococcus aureus sets the stage for subsequent systemic infection. This study characterizes S. aureus adhesion to nasal mucosa in vitro and investigates the interaction of S. aureus with human nasal mucin. S. aureus binding to cell-associated and cell-free mucus was greater than to nonmucin-coated epithelial cells. Scanning electron microscopy of S. aureus incubated with human nasal mucosal tissue showed minimal binding to ciliated respiratory epithelium. In a solid-phase assay, S. aureus bound to purified human nasal mucin-coated wells significantly more than to bovine serum albumin-coated microtiter wells. Binding to mucin was saturable in a dose- and time-dependent fashion. Staphylococcal adherence to human nasal mucin was inhibited by bovine submaxillary mucin but not by fibrinogen. Pretreatment of mucin with periodate but not with pronase reduced adherence. Trypsin treatment of the bacteria significantly reduced adherence to mucin. 125I-labelled nasal mucin bound to two surface proteins (138 and 127 kDa) of lysostaphin-solubilized S. aureus. Binding to human nasal mucin occurs in part via specific adhesin-receptor interactions involving bacterial proteins and the carbohydrate moiety in mucin. These experiments suggest that S. aureus binding to mucin may be critical for colonization of the nasopharyngeal mucosa.     

Functional heterogeneity of colonic adenocarcinoma mucins for inhibition of Entamoeba histolytica adherence to target cells

Mucins secreted from the gastrointestinal epithelium from the basis of the adherent mucus layer which is the host's first line of defense against invasion by Entamoeba histolytica. Galactose and N-acetyl-D-galactosamine residues of mucins specifically inhibit binding of the amebic 170 kDa heavy subunit Gal-lectin to target cells, an absolute prerequisite for pathogenesis. Herein we characterized the secretory mucins isolated from the human colon and from three human colonic adenocarcinoma cell lines: two with goblet cell-like (LS174T and T84) and one with absorptive cell-like morphology (Caco-2). By Northern blot analysis the intestinal mucin genes MUC2 and MUC3 were constitutively expressed by confluent LS174T and Caco-2 cells, whereas T84 cells only transcribed MUC2 and not MUC3 mRNA. 3H-glucosamine and 3H-threonine metabolically labeled proteins separated as high M, mucins in the void (Vo > 10(6) Da) of Sepharose-4B column chromatography and remained in the stacking gel of SDS-PAGE as depicted by fluorography. All mucin preparations contained high amounts of N-acetyl-glucosamine, galactose, N-acetyl-galactosamine, fucose and sialic acid, saccharides typical of the O-linked carbohydrate side chains. Mucin samples from the human colon and from LS174T and Caco-2 cells inhibited E. histolytica adherence to chinese hamster ovary cells, whereas mucins from T84 cells did not. These results suggest that genetic heterogeneity and/or posttranslational modification in glycosylation of colonic mucins can affect specific epithelial barrier function against intestinal pathogens.     

p95vav associates with tyrosine-phosphorylated SLP-76 in antigen-stimulated T cells

Proliferation, migration-associated differentiation, and cell death occur continuously and in a spatially well-organized fashion along the crypt-villus axis of the mouse small intestine, making it an attractive system for studying how these processes are regulated and interrelated. A pathway for producing glycoconjugates was engineered in adult FVB/N transgenic mice by expressing a human alpha 1,3/4-fucosyltransferase (alpha 1,3/4-FT; EC 2.4.1.65) along the length of this crypt-villus axis. The alpha 1,3/4-FT can use lacto-N-tetraose or lacto-neo-N-tetraose core chains to generate Lewis (Le) blood group antigens Le(a) or Le(x), respectively, and H type 1 or H type 2 core chains to produce Leb and Le(y). Single- and multilabel immunohistochemical studies revealed that expression of the alpha 1,3/4-FT results in production of Le(a) and Leb antigens in both undifferentiated proliferated crypt cells and in differentiated postmitotic villus-associated epithelial cells. In contrast, Le(x) antigens were restricted to crypt cells. Villus enterocytes can be induced to reenter the cell cycle by expression of simian virus 40 tumor antigen under the control of a promoter that only functions in differentiated members of this lineage. Bitransgenic animals, generated from a cross of FVB/N alpha 1,3/4-FT with FVB/N simian virus 40 tumor antigen mice, expand the range of Le(x) expression to include villus-associated enterocytes that have reentered the cell cycle. Thus, the fucosylations unveil a proliferation-dependent switch in oligosaccharide production, as defined by a monoclonal antibody specific for the Le(x) epitope. These findings show that genetic engineering of oligosaccharide biosynthetic pathways can be used to define markers for entry into, or progression through, the cell cycle and to identify changes in endogenous carbohydrate metabolism that occur when proliferative status is altered in a manner that is not deleterious to the system under study.     

Tactile salience influences extinction

The Kidd (JK) blood group system is clinically important in transfusion medicine. Alloantibodies to antigens in this system may be produced following blood transfusion or during pregnancy and can result in serious haemolytic transfusion reactions and haemolytic disease of the newborn (HDN). JK antigens on erythrocytes are carried by glycoproteins with the capacity to transport urea through cell membranes. cDNA complementary to mRNA transcribed at the JK locus was cloned in 1994. The molecular basis of the Jk(a)/Jk(b) blood group polymorphism was recently shown to be a single nucleotide substitution predicting an amino acid change (Asp280Asn) in an extracellular loop of the JK glycoprotein. After confirmation of the JK gene polymorphism we developed a rapid and robust technique for JK genotyping with allele-specific primers in a single-tube PCR. In addition, a 217 bp intron located at nucleotides 811-812 in the JK gene was found and sequenced. The genotyping test was validated with samples from 106 Caucasian Swedish and 13 Black South African random blood donors. Complete phenotype-genotype correlations were obtained. However, four Jk(a-b-) samples of Polynesian and Finnish origin typed as Jk(b)Jk(b). Potential use of the presented method can be predicted in clinical transfusion medicine including prenatal determination of the JK genotype in a fetus at risk for HDN caused by JK antibodies.     

Diurnal variations in tear glycoproteins: evidence for an epithelial origin for the major non-reducible > or = 450 kDa sialoglycoprotein(s)

PURPOSE: To characterize the nature and origin of changes in tear glycoproteins accompanying eye closure. METHODS: Reflex (R) and overnight closed (C) eye tears collected by capillary tubes were centrifuged with the resulting R pellets (primarily desquamated epithelial cells) and C pellets (primarily PMN and some epithelial cells) extracted in acidic PBS. Extracts and supernatants were separated by size-exclusion HPLC and/or SDS-PAGE. Gels were stained or blotted and immune- or lectin-probed. An HPLC glycoprotein fraction of > or = 450 kDa isolated from all four sources was characterized before and after partial deglycosylation, using antibodies specific to known mucin and carbohydrate epitopes. Immunofluorescence microscopy was carried out on human conjunctiva, using as probe a MAb to salivary mucin specific for a sialyl Lea epitope, which was found to cross-react specifically with the major non-reducible high molecular weight sialoglycoproteins (SGs) in tears. These SGs were immunoprecipitated and blot-probed along with tissue extracts. RESULTS: R fluid contained minor amounts of numerous glycoproteins, including probably several of inducible lacrimal secretory origin. Results confirmed sIgA as the principal source of the intense reducible glycoprotein bands common to C fluid. Smaller amounts of free secretory component and serum glycoproteins were also visualized. The HPLC fraction (> or = 450 kDa) consisted of four major non-reducible glycoproteins. In R fluid, this fraction (< 1% total protein) consisted primarily of two entities: a 450-500 kDa SG and a larger asialoglycoprotein. The SG accounts for as much as 85% of the total protein in the R pellet extract. C fluid was associated with a selective increase in SGs and a shift in distribution to two SGs > 500 kDa. All SGs exhibited a common antigenicity reacting specifically with the MAb for the sialyl Lea epitope. SGs indistinguishable in size and antigenicity were recovered in epithelial extracts. Immunofluorescence microscopy revealed that reactivity was localized to the epithelial plasma membrane, increasing in intensity from basal to apical cells. Although these SGs exhibited some properties in common with MUC1, immunological and other data suggest a unique SG. CONCLUSIONS: Tear glycoproteins are derived from four principal sources. In R fluid, an inducible lacrimal secretion predominates. In C fluid, a constitutive sIgA secretion predominates, augmented by a serum exudate and SGs derived at least in part from the epithelium. In R fluid and pellet extracts, the SGs consist primarily of a 450-500 kDa species that is most probably derived from the plasma membrane. Larger antigenically related SGs are prevalent in C fluid.     

Differential expression of human high-molecular-weight salivary mucin (MG1) and low-molecular-weight salivary mucin (MG2)

Two distinct mucin components of saliva, MG1 and MG2, have been identified based on chemical composition and molecular weights (high and low, respectively) in saliva. With the aim of characterizing the expression pattern of salivary mucins, we have prepared monoclonal antibodies (MAbs) directed against the peptide core of MG1 and against a synthetic peptide derived from the MG2 (MUC7) sequence. MAb PANH2 raised against partially deglycosylated MG1 stained a high-molecular-weight smear in Western blots of partially purified MG1. PANH2 binding was increased by deglycosylation with trifluoromethanesulfonic acid as well as with subsequent periodate treatment, and was eliminated by pronase treatment, strongly suggesting that MAb PANH2 was directed to a peptide epitope of MG1. MAb PANH3 raised against a synthetic peptide derived from the MG2 (MUC7) sequence reacted with the native molecule and stained a narrow smear of ca. 200,000 to 210,000 in Western blots of concentrated saliva and a lower-molecular-weight smear of trifluoromethanesulfonic-acid-treated MG2. Immunohistology on frozen sections of human salivary glands showed that MAb PANH2 selectively labeled mucous cells, whereas MAb PANH3 labeled subpopulations of serous cells. Double-direct immunofluorescence staining with PANH2 and PANH3 demonstrated that the staining patterns were non-overlapping. The development of these antibody probes will facilitate studies of mucin expression in diseases of salivary glands.     

Histo-blood group Lewis genotyping from human hairs and blood

The expression of histo-blood group Lewis antigens is determined by the Lewis-type alpha 1-->3/4fucosyltransferase (Le enzyme) encoded by Fuc-TIII gene (Le gene). The genotyping of Le genes by the PCR-RFLP methods established recently and partly modified in this study was found to be useful not only for determining the genuine Lewis blood types of samples such as human hairs and blood stains but also for distinguishing non-genuine Lewis-negative phenotypes frequently observed in pregnant women from genuine ones. The availability of the present PCR-RFLP methods for the paternity tests was also discussed.     

Expression of carbohydrate antigen sialyl Le(a): a new functional prognostic factor in gastric cancer

PURPOSE: The prognostic value of the altered expression of carbohydrate antigens sialyl Le(a) (sLe(a)) and sialyl Le(x) (sLe(x)), which have been implicated as functional ligands in heterotypic-cell-adhesion systems in the multistep process of tumor metastasis, were evaluated. PATIENTS AND METHODS: The level of expression of sLe(a) and sLe(x) antigens was examined immunohistochemically in paraffin-embedded tumor samples from 137 patients who underwent resection for gastric cancer. Correlation between the antigens' expression, various established clinicopathologic factors, and prognosis were studied by univariate and multivariate analysis. RESULTS: Tumors that were positive for the sLe(a) antigen were significantly more likely to be large (P = .035), to be localized at the proximal third of the stomach (P = .018), to have an infiltrate appearance (P = .013), to have an invasive mode both in depth of invasion (P = .028) and in lymphatic invasion (P = .002), and to be classified as late stage (P = .011) than those that were negative for sLe(a), whereas the sLe(x) antigen status was not correlated with any clinicopathologic factors. The overall survival of patients with an sLe(a)-antigen-positive tumor was significantly poorer than that of those with an sLe(a)-antigen-negative tumor (P = .0001). Survival within each pathologic stage differed also (stage I, P = .030; stage II, P = .046; stage III, P = .026, respectively). A Cox regression analysis with multiple covariates showed that positive sLe(a) antigen status was an independent prognostic factor for a worse outcome in patients with gastric cancer. According to the mode of recurrence, increased sLe(a) antigen expression significantly affected both peritoneal dissemination and liver metastasis. CONCLUSION: Increased expression of the sLe(a) antigen may serve as a potent prognostic indicator for recurrence in patients with gastric cancer. Careful follow-up and intensive therapy are required for patients with an sLe(a)-antigen-positive gastric cancer.     

Tissue antigens in the saliva of rats. Demonstration of salivary gland specific and nonspecific antigens

Antisera were raised in rabbits against the half-saturated ammonium sulfate soluble fraction of rat saliva. With these antisera, at least four antigens were demonstrated by immunodiffusion in the saliva and in a saline extract of the parotid gland; three of these antigens were also found in a saline extract of the submaxillary gland. Two of the four antigens were salivary gland specific, the other two being shared by the liver,pancreas and testis. The immunofluorescent pattern indicates that these antigens originate from the acinar epithelium of the salivary glands and probably constitute products of secretion and/or cellular turnover.     

Heterogeneity of carbohydrate chains of acidic bronchial mucin isolated from the spatum of two subjects with chronic bronchitis

Acidic glycoproteins having blood group H activity were isolated from the sputum of two patients suffering from chronic bronchitis by reduction of the fibrillar mucus, chromatography on ECTEOLA-cellulose and gel filtration on Sepharose 4-B. These glycoproteins were degraded with alkaline borohydride and the degradation products were fractionated by chromatography on ionexchange resins and by gel filtration. The carbohydrate chains have a wide heterogeneity with regard to acidity and molecular size. Therefore carbohydrate chain heterogeneity which was already observed for bronchial glycoproteins isolated from a patient suffering from cystic fibrosis is not specific for cystic fibrosis.     

Detection of four human milk groups with respect to Lewis blood group dependent oligosaccharides

Neutral oligosaccharides in human milk samples from approximately 50 women were analysed applying a recently developed high-pH anion-exchange chromatographic method. Three different oligosaccharide patterns could be detected in accordance with milk groups that had been already described. These oligosaccharide groups correspond to the Lewis blood types Le(a-b+), Le(a+b-) and Le(a-b-). In addition to these oligosaccharide patterns, a new carbohydrate pattern was detected in a milk sample from a Le(a-b-) individual. Here, only nonfucosylated oligosaccharides and compounds bearing alpha1,3 linked fucosyl residues were found, whereas structures with alpha1,2 and alpha1,4 fucosyl linkages were missing. This finding led to the hypothesis that there are four different oligosaccharide milk groups that fit well to the genetic basis of the Lewis blood group system.     

Synthesis of N-acetylneuraminyl-alpha 2,3(6)lactose-malate dehydrogenase conjugate for detecting sialic acid terminal groups on glycoproteins via homogeneous lectin-based enzyme-linked binding assay

An N-acetylneuraminyl-alpha 2,3(6)lactose-malate dehydrogenase (MDH-Lac-Neu5Ac) conjugate is prepared via an isothiocyanate conjugation method using a p-aminophenethylamino derivative of sialyllactose. The newly synthesized conjugate can be utilized as a reagent in a novel homogeneous lectin-based, enzyme-linked, competitive binding assay (1-3) for probing the specific carbohydrate structure and content of intact glycoproteins. The enzymatic activity of the MDH-Lac-Neu5Ac conjugate is shown to be significantly inhibited (35%) by sialic acid-binding lectin, Limax flavus agglutinin (LFA), and this inhibition is reversed by mucin, a glycoprotein possessing sialic acid terminals. The asialo form of mucin, however, binds weakly to LFA, yielding no substantial increase in the MDH-Lac-Neu5Ac activity at comparable glycoprotein concentrations. Use of the newly synthesized conjugate in conjunction with LFA or other lectins capable of binding sialic acid may provide a rapid and convenient way to detect the presence and relative amount of sialic acid terminal groups within intact glycoprotein structures.     

Characterization of placentation-specific binucleate cell glycoproteins possessing a novel carbohydrate. Evidence for a new family of pregnancy-associated molecules

The ovine binucleate cell-specific glycoproteins recognized by the monoclonal antibody SBU-3 first appear at the initiation of placentation, and their expression continues throughout gestation. These placenta-specific proteins have not been detected in any other adult or fetal sheep tissues and are specific to the materno-fetal interface. The SBU-3 monoclonal antibody recognizes the carbohydrate epitope common to a group of proteins ranging in molecular mass from 30 to 200 kDa whose function during pregnancy remains undefined. The biochemical properties of these uniquely expressed glycoproteins were investigated by analyzing both the carbohydrate and protein portion of the molecules. Analysis of phytohemagglutinin and concanavalin A binding to electrophoretically separated SBU-3 proteins revealed that the major proteins between 40 and 70 kDa bind phytohemagglutinin. In contrast, concanavalin A bound only to minor proteins in the SBU-3 glycoprotein preparation. Analysis of the carbohydrate conjugated to the SBU-3 glycoproteins revealed that the major chains are sialylated O-linked and complex partially sialylated multiple antennary N-linked chains. The presence of N-glycolylneuraminic acid in an N-linked structure indicates the unique nature of this carbohydrate epitope. The differential binding to phytohemagglutinin and concanavalin A provided a method for further purification and characterization of the major protein components with monoclonal antibody immunoaffinity-purified SBU-3 proteins being further separated by concanavalin A-Sepharose chromatography. Microsequence analysis of the major non-concanavalin A-binding proteins (69, 62, and 57 kDa) revealed partial homology to ovine and bovine pregnancy-associated glycoprotein and rabbit pepsinogen F. Immunoblot analysis of the SBU-3 proteins showed cross-reactivity with polyclonal antisera directed against ovine placental-associated glycoprotein and pregnancy-specific glycoprotein B. These results suggest that together these glycoproteins represent members of a binucleate cell-derived family of pregnancy-associated molecules in the ruminant placenta.     

Is ABH nonsecretor status a risk factor for obstructive lung disease?

In a multidisciplinary study of risk factors for chronic obstructive pulmonary disease (COPD), a significantly more impairment of forced expiration was observed in ABH nonsecretors than in ABH secretors among 1017 white adults. (ABH refers to the "A" and "B" antigens of the ABO blood group system and "H", the heterogenetic substance which is found in persons of all ABO types including type "O".) Nonsecretors had significantly lower mean values of forced expiratory volume in one second as a percentage of forced vital capacity (FEV1/FVC%) and a significantly larger proportion of them had aberrant values, defined as FEV1/FVC% less than 68. These differences remained when mean values or rates of aberrancy were adjusted for other factors reported to alter risk of airway obstruction. In view of the known COPD-peptic ulcer and nonsecretor-duodenal ulcer associations, these findings suggest that the ability to secrete ABH antigens into secretions of the respiratory and gastrointestinal tract may have a protective effect on epithelialized organs in general, or on the lung and portions of the gut specifically.     

Immunoelectrophoretic analysis of the porcine zona pellucida

Antigens of the porcine zona pellucida were evaluated by 2-dimensional and line immunoelectrophoretic techniques. Zona antigen preparations studied included heat-solubilized isolated zonae pellucidae (SIZP), a purified 60 000 Mr glycoprotein (PPZA, purified pig zona antigen), and two fractions of this 60 000 Mr zona component which had been exposed to SDS (ZP3-E1C and ZP3-E2C). Antisera were raised to intact zonae pellucidae (IZP), SIZP and PPZA. Collectively, electrophoretic data revealed that the porcine zona system is antigenically complex with each zona antiserum tested detecting numerous antigens in the various zona preparations. These antigens, however, all had similar electrophoretic mobilities, and this limited the resolution of these techniques. The 60 000 Mr pig zona macromolecule (ZP3) appeared to be the most immunogenic of the three major pig zona glycoproteins since antisera prepared against IZP or SIZP reacted primarily with this component. However, the 60 000 Mr component does share antigenic determinants with the other major zona glycoproteins as revealed by cross-reactions of the antisera with the various zona preparations. Electrophoretic studies also suggested that the various zona antisera could distinguish, with different degrees of sensitivity, multiple antigenic determinants on the individual zona macromolecules. These studies also indicated that SDS treatment of zona glycoproteins does alter the antigenicity of the macromolecule, both with respect to the total number and individual identity of antigens detected.     

A new method for the determination of the ABO blood type of semen by immunoblotting using anti-ABH antibodies following immunoprecipitation

A new method that enables the ABO blood type of semen to be determined by identifying the molecular weight of a blood group substance (BGS) has been developed. In order to produce an antibody against a BGS (p84) detected on the sperm plasma membrand (SPM), p84 was purified from seminal plasma, injected into a rabbit. When seminal plasma was immunoprecipitated with the anti-p84 antibody, SDS-PAGE analysis of the immune complex yielded three specific polypeptides with molecular masses of 84, 51 and 25 kDa. The first of these polypeptides, p84, showed ABH antigenic activity when subjected to immunoblotting. The 51 and 25 kDa proteins corresponded to rabbit IgG heavy and light chains, respectively. Using this method, ABH antigenic activity of a band with a molecular mass of 84 kDa was detected in 25 seminal plasma samples by immunoblotting. When serial two-fold dilutions of seminal plasma were analyzed using this method, clear immunoreactive 84 kDa bands were observed up to 60-fold dilution. When seminal plasma was mixed with vaginal fluid at varying ratios, ABH antigenic activity of 84 kDa immunoreactive bands could be detected until the seminal plasma: vaginal fluid mixture reached a ratio of 1:20. These results suggest that this method will be applicable to the analysis of forensic samples of semen contaminated by vaginal fluid, such as those obtained from victims of sexual assault.