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.     

The high-molecular-weight human mucin is the primary salivary carrier of ABH, Le(a), and Le(b) blood group antigens

Because many bacteria interact with the carbohydrate portions of receptor molecules, factors controlling glycosylation probably influence the ability of salivary components to mediate bacterial adherence/clearance. Important sources of diversity in glycosylation are the ABO, secretor, and Lewis genes, which code for glycosyltransferases that add specific sugar sequences to the termini of carbohydrate chains of glycolipids and glycoproteins. We identified, by Western blotting, salivary glycoproteins carrying the ABH and Le(a) or Le(b) antigens. Samples of whole, unstimulated saliva were obtained from 19 subjects whose blood group was determined by agglutination of red blood cells with specific antisera. After centrifugation, the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Glycoproteins carrying blood group antigens were identified by staining the blot with monoclonal antisera specific for the A, B, H, Le(a), or Le(b) antigens. The most intensely staining component from all the samples migrated at the same position as the high-molecular-weight mucin. Saliva samples from the nonsecretors contained only the Le(a) antigen. Samples from the secretors contained one or more of the ABH antigens and, variably, the Le(b) antigen. In all cases, the salivary blood group antigens corresponded to those found on the red blood cells of the same subject. The functional consequences of the expression of blood group antigens on the high-molecular-weight mucin are not known, but their presence could modulate the adherence of certain oral microorganisms that interact preferentially with this molecule.     

Effects of troglitazone and metformin on glucose and lipid metabolism: alterations of two distinct molecular pathways

Using an experimental model of rat colon adenocarcinoma, we have recently shown that the presence of H blood-group antigen on variants of the CD44 adhesion molecule carrying amino acids encoded by exon v6 (CD44v6), increased the cells' tumorigenicity. In the present study, colon adenocarcinomas were induced by 1,2-dimethylhydrazine treatment in rats. Using immunohistochemistry, biopsies of normal, precancerous and carcinomatous colon mucosa were evaluated for expression A and H blood group antigens and CD44s and CD44v6 antigens. Normal rat colon showed strong and homogeneous expression of blood-group antigen A, but weak expression of H antigen. Several weeks before the appearance of tumours, dysplastic glands were strongly stained with anti-H reagents, while their A antigen was lost. Expression of CD44v6 was weak and restricted to some cells at the bottom of normal crypts. No obvious change was observed before appearance of severe dysplasia. In carcinomas, a strong but irregular expression of A, H and CD44v6 antigens was observed. In moderately differentiated carcinomas, A and H antigens were present at the apical surface of cells, whereas CD44v6 was found at the basolateral side. Only carcinomatous cells with loss of polarity, found in poorly differentiated cancers or infiltrated in the muscularis mucosae, were found to coexpress blood-group H or A and CD44v6 antigens at their surface.     

Relationship between ABO blood group and levels of Gal alpha,3Galactose-reactive human immunoglobulin G

BACKGROUND: The terminal Gal alpha1,3Galactose (alphaGal) determinant is present on all porcine glycoproteins and glycolipids, but is not expressed by human cells. Consequently human sera contain anti-alphaGal natural antibodies. The human blood group B antigen [Gal alpha1,3(Fuc1,2)Galactose] is differentiated from the alphaGal epitope by the presence of a fucosyl group. METHODS: To determine whether the expression of the B antigen has any effect on the level of alphaGal-reactive natural antibodies, equal numbers (n=12) of A, B, AB, and O serum samples were evaluated by ELISA and flow cytometry. RESULTS: A significant reduction in IgG alphaGal reactivity was observed with serum samples from B antigen-expressing donors (B, AB) relative to non-B antigen-expressing donors (A, O). CONCLUSIONS: These results are consistent with the possibility that anti-alphaGal antibodies in non-B antigen-expressing individuals include a subset that is reactive with the structurally related B antigen and that this subset is absent in B and AB individuals.     

Dendritic cells of the oral mucosa and the induction of oral tolerance. A local affair

The oral mucosa is an important site to induce immunological tolerance to protein antigens. Previously we have established that oral contacts to allergen can lead to systemic tolerance in both humans and experimental animals. Because of the importance of tolerance induction as a possible way to modulate allergic reactivity, we wished to study the mechanisms involved in efficient tolerance induction via the oral mucosa. Dendritic Langerhans' cells in both skin and oral epithelium are the first cells to encounter antigen. Therefore, possible functional differences between Langerhans' cells from skin and oral mucosa were studied by migration and transfer experiments. It was found that dendritic cells derived from the oral mucosa were not able to transfer tolerance, but that they acted as antigen-presenting cells in sensu stricto irrespective of the source and route of antigen administration.     

Intestinal metaplasia with adherent Helicobacter pylori: a hybrid epithelium with both gastric and intestinal features

Helicobacter pylori seem to avoid areas of intestinal metaplasia in the gastric mucosa, but attachment of these bacteria to epithelium with the appearance of incomplete intestinal metaplasia has been documented. To characterize the nature of the epithelium to which H pylori was attached, we carried out an immunohistochemical study using monoclonal antibodies against gastric surface mucous cell mucins (M1), blood group-related carbohydrates antigens (Le(a), sialyl Le(a), Le(b), type 1H, and type 2H) and sialyl Tn antigen. The results of this study suggest that these areas of H pylori attachment represent a hybrid epithelium whose cells share characteristics of both gastric surface mucous cells and intestinal metaplastic cells. Whether all areas of incomplete intestinal metaplasia represent an intermediate stage between the normal gastric epithelium and the fully developed complete type of metaplasia remains to be determined.     

Increased synovial endothelium binding and transendothelial migration of mononuclear cells during Salmonella infection

OBJECTIVE: To investigate the adhesion and extravasation capacity of peripheral blood mononuclear cells (PBMC) and the transport of bacterial antigens within these cells during Salmonella infection. METHODS: Thirteen patients who were part of 2 outbreaks of Salmonella enteritidis infection were included in this study. The capacity of PBMC from these patients to bind to vascular endothelium in inflamed synovium was tested using a Stamper-Woodruff-type frozen-section assay. The same cells were studied for the presence of Salmonella antigens by immunofluorescence staining. The transendothelial migration of mononuclear cells containing Salmonella or its components through unstimulated endothelial cell layer was quantified. RESULTS: The capacity of PBMC to adhere to synovial vessels was significantly increased during Salmonella infection (P=0.0003). Monocytes had a transiently high adhesive state between 2 and 5 weeks after the patients had eaten the contaminated food. The cells containing Salmonella antigens were concentrated in the transmigrated population. CONCLUSION: During acute Salmonella infection the increased binding of PBMC to vascular endothelium in inflamed synovium and enhanced transmigration of PBMC containing Salmonella may be the key factors leading to transport of bacterial antigens through the endothelial barrier and initiation of arthritis in susceptible individuals.     

Some characteristics of the ridge mucosa before and after implant installation. A prospective study in humans

The aim of the present investigation was to study some tissue characteristics of the ridge mucosa before and after implant installation. 9 partially edentulous patients were included in the study. At the time of fixture installation, 1 recipient site in each patient was selected for soft tissue biopsy. Abutment connection and restorative therapy were performed after 3-6 months. When the implants had been in function for about 6 months, a soft tissue sample was obtained from the keratinized peri-implant mucosa at the 1 implant site from which the first biopsy was obtained. Each biopsy was divided into 1 mesial and 1 distal portion. The mesial tissue portion was fixed in a buffered fixative and embedded in EPON. Sections were produced, stained in PAS and toluidine blue and used for histometric and morphometric analyses. The distal portion of the biopsies were embedded, snap frozen in liquid nitrogen and stored in a freezer at -70 degrees C. From each tissue portion, 15 sections were prepared in a cryostat and exposed to immunohistochemical staining. A panel of monoclonal antibodies was used and the avidin-biotin method for staining was applied. The sections were examined morphometrically. Both tissues harbored a well keratinized oral epithelium and a connective tissue, the composition of which was close to identical in terms of collagen, cells and vascular structures. The peri-implant mucosa, however, also included a junctional epithelium which evidently allowed the penetration of products from the oral cavity. As a consequence, the periimplant mucosa in comparison to the masticatory mucosa was found to contain significantly enhanced numbers of different inflammatory cells.     

Cutaneous lymphocyte associated antigen (CLA) and alpha e beta 7 integrins are expressed by mononuclear cells in skin and oral lichen planus

The cutaneous lymphocyte associated- (CLA-) positive subset of lymphocytes appears to migrate preferentially into skin by interacting with E-selectin on vascular endothelium; lymphocytes expressing the alpha e beta 7 integrin accumulate preferentially in the epithelium of the gastrointestinal tract. To determine whether the mononuclear cell population of the oral mucosa resembles that of the skin or intestine, and using lichen planus as a model, the proportions of CLA- and alpha e beta 7-positive cells in the epithelium, lamina propria and peripheral blood were compared by immunostaining and flow cytometry. In both skin and oral lichen planus, selective accumulation of CLA-positive cells was seen in the epithelium but not in the lamina propria. In contrast, large numbers of alpha e beta 7-positive intraepithelial cells were found in oral but not in skin lichen planus. These results show that in terms of CLA and alpha e beta 7 expression there are important differences in the mononuclear cell population of oral mucosa and skin.     

Immunohistochemical and virological study of skin in the papular-purpuric gloves and socks syndrome

The pathogenesis of viral exanthems remains unclear. We have undertaken an immunohistochemical study of lesional skin biopsies in patients with the papular-purpuric gloves and socks syndrome (PPGSS) secondary to parvovirus B19 infection. Intracytoplasmic staining of the dermal endothelial cells, keratinocytes, and sweat glands was shown with an antibody to parvovirus B19. There were perivascular dermal infiltrates with T cells, sometimes with exocytosis. By polymerase chain reaction, virus DNA was detected in all skin biopsies and in one serum sample. The cutaneous manifestations of parvovirus infection seem secondary to infection of the endothelium and epidermis.     

Isolation of myosin heavy chain from small skeletal muscle samples by preparative continuous elution gel electrophoresis: application to measurement of synthesis rate in human and animal tissue

We studied seven patients aged 14 to 40 years who received living-related kidney transplants and had allograft survivals of 26 to 29 years. The blood urea and creatinine were either within normal limits or marginally elevated. Histopathologic examination showed only mild mesangial expansion, interstitial fibrosis, and arteriosclerosis. Immunoperoxidase staining with anti-HLA antibodies or in situ hybridization with a Y chromosome probe showed persistence of donor tubular epithelium and vascular endothelium within the graft. Recipient-derived glomerular cells were seen in one case, and interstitial lymphocytic infiltrates were seen in all cases. A review of the clinicopathologic data available for these cases indicated that both central and peripheral immunologic mechanisms contributed to the maintenance of prolonged graft survival. This extended survival was independent of six antigen matching, down-regulation of donor HLA antigen expression, and ingrowth of host epithelium/endothelium into the allograft.     

Lipoprotein profile, diet and body composition in athletes practicing mixed an anaerobic activities

To investigate the prevalence and possible role of anti-endothelial cell antibodies (AECA) in the pathogenesis of systemic lupus erythematosus (SLE), cell membrane antigen was prepared from cultured human umbilical vein endothelial cells and immunoblotting performed to detect AECA in SLE sera. IgG-AECA could be detected in 41 (86%) of 47 SLE patients. They were highly specific and failed to react with membrane antigens of human peripheral blood mononuclear cells or granulocytes. IgG-AECA reacted with endothelial membrane antigens which ranged from 15 to 200 kDa in molecular size. Further analysis of the antigens reacting with IgG-AECA revealed some interesting correlations between specific species of antibodies with certain clinical manifestations. Thus, patients having lupus nephritis, vasculitis, and hypocomplementemia had IgG-AECA against a 66-kDa membrane antigen; those with thrombocytopenia had IgG-AECA against a 55-kDa antigen; those with pleuritis had IgG-AECA against an 18-kDa antigen. These results indicate that IgG-AECA in the sera of SLE patients consist of heterogenous species.     

Sialosyl-Le(a) and Sialosyl-Le(x) antigens in adhesion of cancer cells and tumor progression

Sialosyl-Le(a) and sialosyl-Le(x) are tumor-associated carbohydrate antigens present in different types of human tumors. They are commonly found on the cell surface of a variety of adenocarcinomas such as lung cancer, gastric cancer, pancreatic cancer and colorectal cancer, and in serum of cancer patients. Both antigens have been proposed as important diagnostic markers and they are used in detecting and monitoring of these diseases. Recently, it has been shown that sialosyl-Le(a) and sialosyl-Le(x) carbohydrate structures are ligands for selectins, newly described family of adhesion molecules. Selectins function as lymphocyte-homing and leukocyte enrollment receptors, or as activation dependent cell surface receptors of platelets and endothelial cells. Several lines of evidence suggest that sialosyl-Le(a) and sialosyl-Le(x) are responsible for adhesion of human cancer cells to endothelium. It has been shown that E-selectin and P-selectin present on endothelial cells mediate these interactions. The mentioned facts suggest that selectins and their carbohydrate ligands can play an important role in a selective homing of tumor cells during metastasis.     

Oral tolerance is determined at the level of draining lymph nodes

In the skin and in the epithelium of the oral mucosa a comparable network of Langerhans cells can be found. Antigen application on either epithelium leads to rapid emigration of Langerhans cells to the draining lymph nodes. Application on the oral mucosa leads to tolerance induction while application on the skin results in sensitization of the animal. Here we show that there are no differences in the antigen presentation capacity of oral mucosa- and skin-derived dendritic cells. However, measurement of IFN-gamma and IL-5 production, as representatives of Th1 and Th2 cytokines respectively, in total lymph node suspensions after sensitization via the skin or oral mucosa demonstrated a skewing of the response towards Th2 after antigen application on the oral mucosa. Together with our previous studies, in which it was shown that oral tolerance induction is not inherent to oral mucosa-derived dendritic cells, we postulate that oral tolerance is determined at the level of draining lymph nodes influenced by local cytokine profiles.     

Biochemical characterization and molecular cloning of a novel endothelial-specific sialomucin

We have generated rat monoclonal antibodies (MoAbs) against cell surface antigens of the mouse endothelioma cell line bEND.3. Three antibodies (V.1A7, V.5C7, and V.7C7) were selected, all of which recognize a 75-kD antigen on bEND.3 cells and bind selectively to endothelial cells in cryostat sections of mouse tissues. A cDNA for the antigen was isolated from a bEND.3 pCDM8 expression library by using transient expression in COS-7 cells and immunoselection with the three MoAbs. This cDNA coded for a novel, type I membrane protein of 248 amino acids with an extracellular domain rich in threonine and serine residues (35%). The protein is sensitive to O-sialoglycoprotein endopeptidase, indicating that it belongs to the class of sialomucin-like proteins. Therefore, we suggest the name endomucin. Treatment of isolated endomucin by sialidase and O-glycosidase reduced the apparent molecular weight to 45 kD and abolished binding of all three antibodies, indicating that carbohydrates are directly or indirectly involved in the formation of the antibody epitopes. Immunohistological analysis of all examined mouse tissues showed that endomucin is an endothelial antigen found in venous endothelium as well as in capillaries, but not on arterial endothelium. Interestingly, high endothelial venules of peripheral and mesenteric lymph nodes as well as of Peyers's patches were negative for staining with the three MoAbs.     

Clostridium difficile toxin A binding to human intestinal epithelial cells

Clostridium difficile radiolabelled toxin A ([3H]-toxin A) bound to human duodenal and colonic epithelial cells isolated from endoscopic biopsies. Binding was greater at 4 degrees C than 37 degrees C, consistent with the thermal binding characteristic of toxin A to a carbohydrate moiety. At 37 degrees C colonic cells bound significantly more [3H]-toxin A than duodenal cells. The amount of [3H]-toxin A binding varied considerably between individuals. [3H]-toxin A was displaced by unlabelled toxin A by 50% for duodenal cells and 70% for colonic cells with 94.3 nM unlabelled toxin A. Low non-displacable binding was observed in some samples at 4 degrees C and 37 degrees C, suggesting that these cells came from individuals incapable of specifically binding toxin. Pre-treating cells with alpha- or beta-galactosidases to cleave terminal alpha- and beta-galactose residues reduced [3H]-toxin A binding. There was also a reduction in [3H]-toxin A binding after heat treating cells, which is suggestive of protein binding. The reduction in binding varied between individuals. The reduction of [3H]-toxin A binding, after the removal of beta-linked galactose units, implicates these as components of the receptor and adds credence to the idea that the Lewis X, Y and I antigens may be involved in toxin A binding to human intestinal epithelial cells. However, because the Lewis antigens do not possess terminal alpha-galactose units, the reduction in binding after alpha-galactosidase treatment suggests that other receptors may be involved in toxin A binding to some human intestinal cells. These data are the first demonstration of direct toxin A binding to human intestinal epithelial cells.     

Dietary fructooligosaccharides prevent postgastrectomy anemia and osteopenia in rats

We evaluated possible modes of epithelial cell destruction and restoration in minor salivary gland biopsies from patients with SS. Minor salivary gland biopsies from 10 primary Sj?gren's syndrome (pSS) patients and eight control individuals were evaluated by immunohistochemical staining for the expression of apoptosis-related molecules, substances released by activated cytotoxic T cells, as well as proteins involved in epithelial cell repair. The results were analysed by computer screen analysis and they were expressed as average percentages. Apoptosis-promoting molecules, Fas antigen and Fas ligand were observed in ductal and acinar epithelial cells as well as in infiltrating mononuclear cells of minor salivary glands from SS patients in comparison with control biopsies. Bax protein, which acts as a death-promoter message, was expressed in the ductal and acinar epithelial cells and in mononuclear infiltrating cells of SS patients compared with control individuals, while Bcl-2, an inhibitor of apoptosis, was primarily found in the lymphocytic infiltrates. In situ DNA fragmentation assay (TUNEL) revealed that epithelial cells were apoptotic in patients with SS compared with control subjects. Immunohistochemical staining for perforin and granzyme B, released from granules of activated cytotoxic lymphocytes, revealed their presence in lymphocytic infiltrates of patients with SS compared with control biopsies. pS2, a member of the trefoil protein family which functions as promoter of epithelial cell repair and cell proliferation, was expressed in epithelial cells in biopsies from SS patients. These studies suggest that the functional epithelium of minor salivary glands in patients with SS appears to be influenced by both intrinsic and extrinsic mechanisms of destruction, while a defensive mechanism of epithelial restoration seems to be active.     

Potential role of molecular mimicry between Helicobacter pylori lipopolysaccharide and host Lewis blood group antigens in autoimmunity

Helicobacter pylori is involved in gastritis, gastric and duodenal ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. Earlier studies already suggested a role for autoimmune phenomena in H. pylori-linked disease. We now report that lipopolysaccharides (LPS) of H. pylori express Lewis y, Lewis x, and H type I blood group structures similar to those commonly occurring in gastric mucosa. Immunization of mice and rabbits with H. pylori cells or purified LPS induced an anti-Lewis x or y or anti-H type I response, yielding antibodies that bound human and murine gastric glandular tissue, granulocytes, adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma cells. Experimental oral infections in mice or natural infection in humans yielded anti-Lewis antibodies also. The beta chain of gastric (H+,K+)-ATPase, the parietal cell proton pump involved in acid secretion, contained Lewis y epitopes; gastric mucin contained Lewis x and y antigenic determinants. Growth in mice of a hybridoma that secretes H. pylori-induced anti-Lewis y monoclonal antibodies resulted in histopathological evidence of gastritis, which indicates a direct pathogenic role for anti-Lewis antibodies. In conclusion, our observations demonstrate that molecular mimicry between H. pylori LPS and the host, based on Lewis antigens, and provide understanding of an autoimmune mechanism for H. pylori-associated type B gastritis.