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.     

Blood group A determinants with mono- and difucosyl type 1 chain in human erythrocyte membranes

Application of a monoclonal antibody defining monofucosyl type 1 chain A (AH21) revealed the presence of a glycolipid having the same thin-layer chromatography mobility as Aa but showing a clear reactivity with AH21. This glycolipid was detectable in Lea-b- erythrocytes but not in Lea+b- or Lea-b+ erythrocytes. Another monoclonal antibody defining difucosyl type 1 chain A (HH3) detected the presence of a glycolipid component reacting with this antibody in Lea-b+ erythrocytes but not in Lea+b- or Lea-b- erythrocytes. The component defined by monoclonal antibody AH21 and that defined by HH3 were isolated and characterized by 1H NMR spectrometry and methylation analysis as having the structures (Formula: see text) The 1H NMR spectra of these glycolipid antigens were characterized by resonances for anomeric protons that are identical with those of glycolipids with type 1 chain previously isolated but distinctively different from those of type 2 chain analogues. Resonances reflecting ceramide composition are characteristic for these antigens from human erythrocytes and are distinguishable from those of the same antigen from other sources.     

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+).     

Structures of mucin-type sugar chains of the galactosyltransferase purified from human milk. Occurrence of the ABO and Lewis blood group determinants

The mucin-type sugar chains of human milk galactosyltransferase samples purified from two donors with different blood types were released by alkaline borohydride treatment and quantitatively labeled by N-[3H]acetylation. The radioactive oligosaccharides thus obtained were fractionated by high performance liquid chromatography and immobilized lectin chromatography, and their structures were studied by sequential digestion with endo- or exoglycosidases, methylation analysis, and periodate oxidation. It was revealed that the structures of the mucin-type sugar chains of galactosyltransferase are extremely various, and many blood group determinants are expressed on more than 13 different backbone sugar chains. The characteristic features of the sugar chains could be summarized as follows. 1) The sugar chains of both samples are composed of core 1, Gal beta 1----3GalNAc, and core 2, GlcNAc beta 1----6(Gal beta 1----3)GalNAc. 2) One or two N-acetyllactosamine repeating units extend from the core through GlcNAc beta 1----6Gal and GlcNAc beta 1----3 Gal linkages. 3) Blood group determinants are expressed in accord with the blood types of the donors: sample 1 from a donor of blood type O, Lea+b- contains oligosaccharides with Lea and X determinants, and sample 2 from a donor of B, Lea-b- contains those with H, X, Y, and B determinants.     

Monoclonal antibody-defined antigen of human uterine endometrial carcinomas is Leb

A monoclonal antibody, MSN-1, generated by immunizing a mouse with a human uterine endometrial adenocarcinoma cell line, SNG-II, was strongly and specifically reactive with neutral glycosphingolipids from cancer tissues of patients with uterine endometrial adenocarcinomas. The glycosphingolipid antigen was purified from pooled human meconia, which contained the antigen at the concentration of 1.95 mumol/g dry weight. Its structure was determined by NMR, negative ion FABMS, permethylation analysis, and TLC-immunostaining with monoclonal anti-Lc4Cer antibody, and was concluded to be the III4IV2Fuc2Lc4Cer,Leb antigen of the human Lewis blood system. On ELISA, the monoclonal antibody was found to be strongly reactive with Leb, slightly with Lea and not at all with A, B, H, or IV2FucGg4Cer. The amount of Leb in cancerous regions in the patients with the Lea-b+ blood group was significantly increased compared to that in normal regions in the same patients, and it was a newly appearing antigen in the cancerous tissue of a patient with the Lea+b- blood group.     

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.     

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.     

Detection of Lewis(a) antigen in sera of ovarian carcinoma patients by MOv2-MOv8 double-determinant radioimmunoassay

The monoclonal antibodies MOv2 and MOv8, raised against ovarian carcinoma, were found to be directed against two non-crossreacting epitopes expressed on the same molecule. Immunochemical analysis of the MOv8 recognized epitope showed that the Le(a) oligosaccharide, or commercial anti-Le(a) MAb, but not the anti-Le(b) MAb, prevented Ov8 binding to the reference target cell line (SW626), indicating that it is carried by the Le(a) antigen. Since we previously reported that MOv2 also recognises the Le(a) antigen, these data suggest that Mov8 and Mov2 were directed against different epitopes on the same oligosaccharide chain. Bearing in mind the knowledge of the biochemical nature of the monoclonal antibody recognized epitopes (CaMOv2 and CaMOv8), the presence of the circulating molecules recognized by them was analyzed by double determinant immunoradiometric assay (DDIRMA) in 103 sera from ovarian carcinoma patients. Patients with clinical evidence of the disease (ED) with MOv2 and MOv8 reactive and negative tumors had sera reactivity in 67% and 19% respectively. Also, 26% of the patients with no clinical evidence of disease (NED) had positive sera. When we investigated the relationship between MOv2-MOv8 DDIRMA sera positivity and red blood cells (RBC) Lewis phenotype, a strong correlation was found between the Le(a)+ phenotype and DDIRMA sera reactivity in healthy donors (6/6) and in ovarian carcinoma patients (9/10) whatever their clinical condition. No Le(a)- healthy donors gave evidence of MOv2-MOv8 reactive sera. In contrast, 33% and 57% of the sera from ED carcinoma patients with respectively Lea-b+ and Lea-b- phenotype were positive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetics of plasma concentration of von Willebrand factor.

The plasma concentration of von Willebrand factor (vWf) shows a very wide range in individuals without bleeding disorders. In a twin study we found that 60% of the variance of the plasma concentration of vWf is due to genetic factors. Individuals with AB0 blood group 0 have a lower concentration of vWf than individuals with blood group A, B or AB. Thirty percent of the genetic variance was due to an effect of the AB0 locus. Since the Lewis substances show great structural similarity to the ABH blood group substances we compared the vWf concentration in individuals with and without the Lea antigen on the red cell surface. Individuals lacking the Lea antigen had a lower vWf concentration than individuals who had this antigen. Le(a+b-) people are nonsecretors and Le(a-b+) people are secretors of ABH substance. The lowest vWf concentration was found in blood group 0 secretors. Both the AB0 locus and the Secretor locus may be major loci for the determination of the plasma concentration of vWf.     

Characterization of monoclonal antibodies specific for the Lewis a human blood group determinant

Four hybridoma cell lines were derived from the spleen cells of mice immunized with the neutral glycolipids of human meconium. The antibodies secreted by these lines were specific for the Lewis a antigen of the human Lewis blood group system as determined by solid phase immunoassay using synthetic carbohydrate antigens and by plate binding assay and thin layer chromatography-autoradiography using natural glycolipid antigens. Coating protein A-bearing Staphylococcus aureus with one of the antibodies yielded a stable reagent that produced rapid agglutination of Lewis a positive human erythrocytes. The fine structural specificity of these antibodies was assessed by competition radioimmunoassay using synthetic structural analogs of Lewis a conjugated to bovine serum albumin. One antibody was specific for the Lewis a trisaccharide (Gal beta 1 leads to 3(Fuc alpha 1 leads to 4) beta GlcNAc), while a second recognized the entire Lea (1 leads to 3) beta Gal tetrasaccharide. The third and fourth were directed at topography largely provided by only the alpha Fuc and beta GlcNAc units. These monoclonal antibodies not only represent potentially useful reagents for detecting the Lewis a antigen but also provide a system for studying precise relationships between anticarbohydrate antibody structure and binding specificity.     

IgG antibodies to Lewis type 2 antigens in serum of H. pylori-infected and noninfected blood donors of different Lewis(a,b) blood-group phenotype

Individuals of the Le(b+)/secretor phenotype revealed a stronger natural immune response to Le(x) and Le(y) epitopes irrespective of Helicobacter pylori serologic status. In contrast, H. pylori-infected Le(b-) type individuals showed a significantly higher proportion of strong responders to Le(x) antigen compared with the H. pylori-uninfected subgroup. The data suggest that the immune response to Lewis type 2 determinants is related to both the H. pylori serologic status and the Le(a,b) phenotype of the host.     

Molecular mechanisms of expression of Lewis b antigen and other type I Lewis antigens in human colorectal cancer.

Lewis b (Leb) antigens are gradiently expressed from the proximal to the distal colon, i.e., they are abundantly expressed in the proximal colon, but only faintly in the distal colon. In the distal colon, they begin to increase at the adenoma stage of cancer development and then increase with cancer progression. We aimed to clarify the molecular basis of Leb antigen expression in correlation with the expression of other type I Lewis antigens, such as Lewis a (Lea) and sialylated Lewis a (sLea), in colon cancer cells. Considering the Se genotype and the relative activities of the H and Se enzymes, the amounts of Leb antigens were proved to be determined by both the H and Se enzymes in noncancerous and cancerous colon tissues. But the Se enzyme made a much greater contribution to determining the Lebamounts than the H enzyme. In noncancerous colons, the Se enzyme were gradiently expressed in good correlation with the Leb expression, while the H enzyme was constantly expressed throughout the whole colon. In distal colon cancers, the H and Se enzymes were both significantly upregulated in comparison with in adjacent noncancerous tissues. In proximal colon cancers, expression of the H enzyme alone was highly augmented. The augmented expression of Leb antigens in distal colon cancers is caused mainly by upregulation of the Se enzyme and partly by the H enzymes, while it is caused by upregulation of the H enzyme alone in proximal colon cancers. The Se gene dosage profoundly influences the amounts of the Leb, Lea, and sLea antigens in whole colon tissues, regardless of whether they are noncancerous or cancerous tissues. It suggests that the Se enzyme competes with alpha2,3 sialyltransferase(s) and the Le enzyme for the type I acceptor substrates.     

Solution structure of the Lewis x oligosaccharide determined by NMR spectroscopy and molecular dynamics simulations.

The Lewis x (Lex) determinant is a trisaccharide fragment that has been implicated as a specific differentiation antigen, as a tumor antigen, and as a key component of the ligand for the endothelial leukocyte adhesion molecule, ELAM-1. High-resolution nuclear magnetic resonance spectroscopy shows it to have a relatively rigid structure. Only a small range of glycosidic dihedral angles in the trisaccharide produce simulated nuclear Overhauser effect spectra agreeing with data measured for the human milk pentasaccharide, lacto-N-fucopentaose-3, which contains the Lex determinant. Independently, the same average structure for the Lex determinant arises from in vacuo molecular dynamics simulations. The proposed conformation of the Lex trisaccharide is very similar to that recently determined for the closely related Lea trisaccharide. In agreement with the recent finding that both sialylated Lea and Lex react with ELAM-1, the results presented here show that the Lea and Lex determinants contain very similar carbohydrate domains.     

Functional Role for Piezo1 in Stretch-evoked Ca2+ Influx and ATP Release in Urothelial Cell Cultures

The urothelium is a sensory structure that contributes to mechanosensation in the urinary bladder. Here, we provide evidence for a critical role for the Piezo1 channel, a newly identified mechanosensory molecule, in the mouse bladder urothelium. We performed a systematic analysis of the molecular and functional expression of Piezo1 channels in the urothelium. Immunofluorescence examination demonstrated abundant expression of Piezo1 in the mouse and human urothelium. Urothelial cells isolated from mice exhibited a Piezo1-dependent increase in cytosolic Ca²⁺ concentrations in response to mechanical stretch stimuli, leading to potent ATP release; this response was suppressed in Piezo1-knockdown cells. In addition, Piezo1 and TRPV4 distinguished different intensities of mechanical stimulus. Moreover, GsMTx4, an inhibitor of stretch-activated channels, attenuated the Ca²⁺ influx into urothelial cells and decreased ATP release from them upon stretch stimulation. These results suggest that Piezo1 senses extension of the bladder urothelium, leading to production of an ATP signal. Thus, inhibition of Piezo1 might provide a promising means of treating bladder dysfunction.     

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.     

Temporal and spatial dimensions of postnatal growth of the mouse urinary bladder urothelium

Postnatal growth and renewal of mouse urothelium start on the day of birth. In the present study, temporal and spatial dimensions of urothelial growth were studied during the first two postnatal weeks. Quantitative analysis showed that the rate of urothelial cell proliferation is significantly higher during all 14 postnatal days than in adult mice. Three peaks of proliferative and mitotic activity were revealed: on the day of birth and postnatal day 1, on days 6 and 7, and on day 14. The high proliferation rate around the day of birth and at postnatal days 6 and 7 coincides with cell death in the urothelium. Semiquantitative analysis showed that during all 14 postnatal days, the urothelial proliferative response is mostly confined to the basal cell layer. Urothelial cells divide predominantly in parallel to the plain of the urothelium on all chosen postnatal days. Increased portions of urothelial cells, dividing perpendicularly to the urothelium were observed only on the day of birth and on postnatal day 7. Our results suggest that postnatal growth of mouse urothelium is particularly the result of an increasing number of cells in individual cell layers and not the result of an increasing number of cell layers.     

Urothelial umbrella cells of human ureter are heterogeneous with respect to their uroplakin composition: different degrees of urothelial maturity in ureter and bladder?

Urothelial umbrella cells are characterized by apical, rigid membrane plaques, which contain four major uroplakin proteins (UP Ia, Ib, II and III) forming UPIa/UPII and UPIb/UPIII pairs. These integral membrane proteins are thought to play an important role in maintaining the physical integrity and the permeability barrier function of the urothelium. We asked whether the four uroplakins always coexpress in the entire human lower urinary tract. We stained immunohistochemically (ABC-peroxidase method) paraffin sections of normal human ureter (n = 18) and urinary bladder (n = 10) using rabbit antibodies against UPIa, UPIb, UPII and UPIII; a recently raised mouse monoclonal antibody (MAb), AU1, and two new MAbs, AU2 and AU3, all against UPIII; and mouse MAbs against umbrella cell-associated cytokeratins CK18 and CK20. Immunoblotting showed that AU1, AU2 and AU3 antibodies all recognized the N-terminal extracellular domain of bovine UPIII. By immunohistochemistry, we found that in 15/18 cases of human ureter, but in only 2/10 cases of bladder, groups of normal-looking, CK18-positive umbrella cells lacked both UPIII and UPIb immunostaining. The UPIb/UPIII-negative cells showed either normal or reduced amounts of UPIa and UPII staining. These data were confirmed by double immunofluorescence microscopy. The distribution of the UPIb/UPIII-negative umbrella cells was not correlated with localized urothelial proliferation (Ki-67 staining) or with the distribution pattern of CK20. Similar heterogeneities were observed in bovine but not in mouse ureter. We provide the first evidence that urothelial umbrella cells are heterogeneous as some normal-looking umbrella cells can possess only one, instead of two, uroplakin pairs. This heterogeneity seems more prominent in the urothelium of human ureter than that of bladder. This finding may indicate that ureter urothelium is intrinsically different from bladder urothelium. Alternatively, a single lineage of urothelium may exhibit different phenotypes resulting from extrinsic modulations due to distinct mesenchymal influence and different degrees of pressure and stretch in bladder versus ureter. Additional studies are needed to distinguish these two possibilities and to elucidate the physiological and pathological significance of the observed urothelial and uroplakin heterogeneity.     

Detection of non-papillary, non-invasive transitional cell G1 carcinoma as revealed by increased DNA instability and other cancer markers

The method to reveal DNA-instability as demonstrated by immunohistochemical staining with anti-cytidine antibody after acid hydrolysis (DNA-instability test) was used as a marker of malignancy. The test was applied to paraffin-embedded sections taken from 15 urinary bladders, renal pelvic cavities, and ureters bearing multiple carcinoma in situ (CIS) and totally 31 papillary urothelial cancers. The serial sections of the same tissues were also subjected to immunohistochemical staining for PCNA, p53, DFF45, and VEGF. The DNA-instability test was positive in 100% cancer lesions irrespective of the grades, and apparently normal urothelium, and hyperplastic and dysplastic urothelial lesions also showed the areas with clones positively stained with DNA-instability testing, and the percent numbers of positive areas in them were 28.3%, 37.7%, and 61.5%, respectively. These clones, which were present in apparently normal urothelium and in hyperplastic and dysplastic urothelial lesions, showed higher percent values of PCNA-positive-cells, in comparison to the values estimated in the areas with negatively stained DNA-instability testing, and the former values were statistically not different from those in carcinoma lesions. Furthermore, the percent numbers of areas positive for p53, DFF45, and VEGF, with positive DNA-instability testing were also much higher than those with negative DNA-instability testing in apparently normal urothelium, and hyperplastic and dysplastic urothelial lesions, and the former values were again comparable to those in cancer lesions with no statistical differences. These clones were regarded as already being malignant and should be the direct precursors of progressed cancer lesions. They will make progression through two different pathways, one to papillary non-invasive G1 cancers by neovascularization induced by paracrine secretion of VEGF, and another to flat CIS G2 without secretion of VEGF; thus the clones should be regarded as non-papillary, non-invasive Gl TCC, or CIS G1. It should be always taken into account that the probability for apparently normal urothelium, and hyperplastic and dysplastic urothelial lesions to contain cancer clones, will be high already, especially in tumor-bearing bladders.     

Expansion and long-term culture of differentiated normal rat urothelial cells in vitro

The objective of this study is to establish a reliable cell culture system for the long-term culture of rat urothelial cells (RUC), in which the cells multiply in vitro and form stratified polarized urothelium. Urothelial cells were harvested by the enzymatic digestion of the urothelium exposed by the eversion of resected rat bladders. Primary cultures were initiated in keratinocyte serum-free medium (KSFM) for selective proliferation of urothelial cells. Subsequently, the cells were propagated in a mixture of conditioned medium (CM) derived from Swiss 3T3 cell culture supernatant and KSFM (CM-KSFM). Mean population doubling time was 13.8 +/- 0.9 h. RUC were successfully maintained for 18 passages over a period of 4-5 mo. Detailed investigations of culture conditions showed that CM-KSFM yielded a differentiated multilayer structure. The stratified urothelial sheets measuring 4 x 6 cm2 could be formed and then detached using dispase. Cytokeratin pattern in both the cultured urothelial monolayer and engineered stratified layers was similar to those seen in vivo, as assessed with monoclonal antibody against cytokeratin 17. Ultrastructural morphology showed microvilli, basal cell layer, and desmosomes between adjacent cells in the stratified urothelium.