Mucin glycoarray in gastric and gallbladder epithelia

<p>Abstract</p> <p>Background</p> <p>Mucins are critical cytoprotective glycoproteins and alterations of epithelial gastric mucins have been described in different pathological conditions. The purpose of the present study was to evaluate the putative usefulness of mucins in understanding the progression of gastric cancer and gallstone formation in a better perspective.</p> <p>Methods</p> <p>Formalin-fixed paraffin-embedded gastric biopsy specimens and surgically resected gallbladder tissue samples were sectioned. Alcian Blue (AB) staining was performed to identify sialomucins (staining blue at pH 2.5) and sulfomucins (staining brown at pH 1.0) and then Periodic acid-Schiff's (PAS) staining to visualize the neutral mucins (staining magenta).</p> <p>Results</p> <p>In normal gastric and gallbladder mucosae, we found that neutral mucins were predominant, whereas in intestinal metaplasia, gastric carcinoma and stone-containing gallbladder, a significant increase of acidic mucins was found.</p> <p>Conclusion</p> <p>We suggest that the sulfomucins have a greater role in gallstone formation than the neutral mucins and also that the sialomucins and sulfomucins play an important role in cancer progression and metastasis. Our results challenge the glycobiologists to delve deeper in elucidating the role of mucins in gastric malignancy and in gallstone formation.</p

Colonic phenotype of the ileum in Crohn's disease: A prospective study before and after ileocolonic resection

Background: Colonic metaplasia has been described in pouchitis. In a prospective study, we investigated whether colonic phenotype may develop in Crohn's disease (CD) ileum. The expression of sulfomucins (colonic mucin), sialomucins, and CD10 (small intestine mucin and phenotype) was evaluated before and after ileocolonic resection for CD. Methods: From February 2007 to March 2010, 22 patients with CD undergoing surgery were enrolled. Clinical (Crohn's Disease Activity Index &gt;150) and endoscopic recurrence (Rutgeerts score ≥1) rates were assessed at 6 and 12 months. Ileal samples were taken at surgery (T0), at 6 (T1), and 12 months (T2) for histology, histochemistry (High Iron Diamine-Alcian Blue), and immunohistochemistry (anti-CD10). Results: In 22 patients, recurrence was assessed at 6 and 12 months (clinical recurrence 9% and 18%; endoscopic recurrence 73% and 77%). In all 22 patients, ileal samples were taken at 6 and 12 months (involved area in patients with recurrence). In 19 of 22 (86.3%) patients, the involved ileum was also studied at surgery. At T0, T1, and T2, the expression of sialomucins and CD10 (small intestine mucin and phenotype) was comparable and higher (P &lt; 0.0001) than the expression of sulfomucins (colonic mucin) (mean [range], T0:82 [35-100] versus 75 [0-100] versus 16 [0-50]; T1:96 [60-100] versus 94.7 [50-100] versus 3.89 [0-40]; T2:93.3 [60-100] versus 88.1 [25-100] versus 6.6 [0-40]). The expression of small-intestine mucin and phenotype was higher at T1 (P = 0.025) versus T0 (P = 0.026). Differently, the expression of colonic mucin was lower at T1 versus T0 (P = 0.027). Conclusions: In CD, the ileum involved by severe/established lesions develops a "metaplastic" colonic mucosa phenotype. Differently, CD ileum with no lesions or with early recurrence maintains the "native" small intestine type mucin secretion and phenotype

Characterisation of aberrant crypt foci in carcinogen-treated rats: association with intestinal carcinogenesis.

Carcinogen-treated rats develop foci of aberrant crypts in the colon (ACFs) that have been interpreted as preneoplastic lesions. To characterise ACFs further, we studied in the unsectioned colon of rats the number, multiplicity, some morphological characteristics and the type of mucin production in ACFs. In ACFs observed 115 days after the administration of 50 mg kg-1 1,2-dimethylhydrazine (DMH), crypt multiplicity [number of aberrant crypts (AC) per focus] was positively correlated (P < 0.0001) with the reduction of goblet cells, and with luminal and nuclear alterations in the cells surrounding the lumen of the ACs. We studied mucin production in the unsectioned colon, demonstrating that ACFs producing sulphomucins (like the normal distal rat colon) were progressively reduced when ACF multiplicity increased, whereas ACFs containing sialomucins (correlated with an increased risk of colon cancer) or both sulphomucins and sialomucins increased with crypt multiplicity. We also studied ACFs in the colon and the occurrence of intestinal tumours in rats treated with azoxymethane (AOM; 64 mg kg-1). A significant association was found (P = 0.04) between tumours and the presence of 'large' ACFs (AC/ACF > 14 crypts) and a borderline significant association (P = 0.057) between the presence of tumours and sialomucin-producing ACFs. We found no association between the number of ACFs, ACF multiplicity and the presence of tumours

In vivo and in vitro effects of retinoids on the histological changes in colorectal tissue

The purpose of this study was to determine the histological changes induced by high physiological levels of dietary retinoids on rat colorectal tissue in vivo and on human colorectal adenomas and adenocarcinomas in vitro. For the in vivo study, young male rats were fed stock diets supplemented with 6, 30, or 60 Retinol Equivalents (RE) of retinol (Retinol A, B, and C, respectively) or $-carotene (0-carotene A, B, and C, respectively) for two weeks. The animals were sacrificed arid a segment of colorectal tissue was fixed at the end of the experiment. The High Iron Diamine-Alcian Blue and Periodic Acid Thionin/Potassium Hydroxide/Periodic Acid Schiff stains were used to determine the numbers of goblet cells and mast cells and amount, type, and location of mucus produced by goblet cells and connective tissue. The in vitro study was performed on human tumors exposed to retinol (75 or 250 ug/100 ml medium, Retinol 1 and 2, respectively) and B-carotene (250 or 500 ug/100 ml medium, B-carotene 1 and 2, respectively), for six days. Histological stains and dependent variables were the same as; the in vivo study. Number of plasma cells and lymphocytes were also determined

Tissue sulfomucin and sialomucin content in colon mucosa without intestinal transit subjected to intervention with curcuma longa (curcumin)

To measure the tissue sulfomucin and sialomucin content of the colon mucosa without fecal flow, subjected to intervention with curcumin, and the influence of the concentration used and the intervention time. Methods: Thirty-six rats were subjected to proximal right colostomy and distal mucous fistula. They were divided into two groups according to whether sacrifice was performed two or four weeks after the intervention. Each group was divided into three subgroups according to the enema applied daily: saline alone; curcumin at 50 mg/kg/day or curcumin at 200 mg/kg/day. Acid mucins were diagnosed using the Alcian blue technique. The mucin content was quantified by means of computer-assisted image analysis. The significance level of 5% was used throughout (p < 0.05). Results: There were dose-related increases in the quantities of sulfomucins in the animals subjected to interventions with curcumin, both after two weeks (p < 0.00001) and after four weeks (p < 0.00001). There were increases in sialomucin quantity that were concentration-related (p < 0.00001) and time-related (p < 0.00001). Conclusion: Curcumin enemas increase the quantity of acid mucins in the intestinal flow in the excluded colon, with dose and time dependency323182193FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAUL

On the Relationship between Sialomucin and Sulfomucin Expression and Hydrogenotrophic Microbes in the Human Colonic Mucosa

The colonic mucus layer is comprised primarily of acidomucins, which provide viscous properties and can be broadly classified into sialomucins or sulfomucins based on the presence of terminating sialic acid or sulfate groups. Differences in acidomucin chemotypes have been observed in diseases such as colorectal cancer and inflammatory bowel disease, and variation in sialo- and sulfomucin content may influence microbial colonization. For example, sulfate derived from sulfomucin degradation may promote the colonization of sulfate-reducing bacteria (SRB), which through sulfate respiration generate the genotoxic gas hydrogen sulfide. Here, paired biopsies from right colon, left colon, and rectum of 20 subjects undergoing routine screening colonoscopies were collected to enable parallel histochemical and microbiological studies. Goblet cell sialo- and sulfomucins in each biopsy were distinguished histochemically and quantified. Quantitative PCR and multivariate analyses were used to examine the abundance of hydrogenotrophic microbial groups and SRB genera relative to acidomucin profiles. Regional variation was observed in sialomucins and sulfomucins with the greatest abundance of each found in the rectum. Mucin composition did not appear to influence the abundance of SRB or other hydrogenotrophic microbiota but correlated with the composition of different SRB genera. A higher sulfomucin proportion correlated with higher quantities of Desulfobacter, Desulfobulbus and Desulfotomaculum, relative to the predominant Desulfovibrio genus. Thus, acidomucin composition may influence bacterial sulfate respiration in the human colon, which may in turn impact mucosal homeostasis. These results stress the need to consider mucus characteristics in the context of studies of the microbiome that target intestinal diseases

Glycosaminoglycans in the tongue of birds

We examined the literature to verify whether adaptations and modifications in the structure and glandular secretions of birds' tongues are related to habitat and can be ascribed to evolutionary processes. The data are discussed in relation to species taxonomy, following the Sibley and Ahlquist classification [C. G. Sibley and J. E. Alquist, Philogeny and Classification of Birds. A Study in Molecular Evolution (Yale University Press, New Haven, 1990)]. The following conclusions are drawn: gustatory papillae and taste buds are present in varying numbers in most species. The composition of gland secretions is also found to be variable. Proteic secretion is documented in Larus modestus, Sula variegata, Fulica atra only. Acid proteoglycans both with sulfomucins and carboxymucins, and also glycoproteins, are consistently found. Sialic and hyaluronic acids are found in many species. Our overview indicates that the presence or absence of gustatory papillae is related to adaptation processes that these structures undergo in response to environmental factors, and that the absence of front tongue glands can be ascribed to habitat and feeding habits. Referring to the Sibley and Ahlquist classification, proteins are present in the glandular secretion of less evolved species, whereas more evolved species exhibit a gradual decrease in proteins with the exception of hyaluronic acid, which is absent, and a progressive increase in glycoproteins and acid proteoglycans

Nepmucin, a novel HEV sialomucin, mediates L-selectin–dependent lymphocyte rolling and promotes lymphocyte adhesion under flow

Lymphocyte trafficking to lymph nodes (LNs) is initiated by the interaction between lymphocyte L-selectin and certain sialomucins, collectively termed peripheral node addressin (PNAd), carrying specific carbohydrates expressed by LN high endothelial venules (HEVs). Here, we identified a novel HEV-associated sialomucin, nepmucin (mucin not expressed in Peyer's patches [PPs]), that is expressed in LN HEVs but not detectable in PP HEVs at the protein level. Unlike conventional sialomucins, nepmucin contains a single V-type immunoglobulin (Ig) domain and a mucin-like domain. Using materials affinity-purified from LN lysates with soluble L-selectin, we found that two higher molecular weight species of nepmucin (75 and 95 kD) were decorated with oligosaccharides that bind L-selectin as well as an HEV-specific MECA-79 monoclonal antibody. Electron microscopic analysis showed that nepmucin accumulates in the extended luminal microvillus processes of LN HEVs. Upon appropriate glycosylation, nepmucin supported lymphocyte rolling via its mucin-like domain under physiological flow conditions. Furthermore, unlike most other sialomucins, nepmucin bound lymphocytes via its Ig domain, apparently independently of lymphocyte function–associated antigen 1 and very late antigen 4, and promoted shear-resistant lymphocyte binding in combination with intercellular adhesion molecule 1. Collectively, these results suggest that nepmucin may serve as a dual-functioning PNAd in LN HEVs, mediating both lymphocyte rolling and binding via different functional domains

Common Bile-Duct Mucosa in Choledochoduodenostomy Patients — Histological and Histochemical Study

We describe the histological and histochemical changes of the common bile-duct mucosa in specimens obtained by means of peroral cholangioscopy, 1–12 years after choledochoduodenal anastomosis. Our findings — hyperplasia of the superficial epithelium, metaplastic goblet cells containing predominantly acid sialomucins, and pyloric-like gland formation containing neutral mucins — express a morphological and functional differentiation of the common bile-duct mucosa that probably facilitates its survival in a different environment. We consider that these adaptive changes may explain the uneventful long-term postoperative period of choledochoduodenostomized patients

Clinical and Molecular Analyses of Intestinal Goblet Cell Acidomucins and Cysteine Metabolism

Goblet cells are key contributors to intestinal mucosal barrier function through their role in production of mucins and other important secretory products, which include trefoil factor 3 (TFF3) and resistin like molecule ?? (RETNLB). Goblet cell mucins show great structural heterogeneity but can be broadly classified into neutral and acidic subtypes (chemotypes), which can be further classified into sulfomucins or sialomucins based on the presence of terminal sulfate or sialic acid groups on the oligosaccharide chains. Sulfomucins and sialomucins vary regionally throughout the gastrointestinal tract and changes in their expression have been observed in diseases such as inflammatory bowel disease and colorectal cancer. It is likely that there is variation in these chemotypes among individuals and that both regional and interindividual variation in sulfo- and sialomucins may influence microbial colonization. However, quantitative data and a description of interindividual variation in sulfo- and sialomucin content in the human colon are lacking. There is an absence of data describing the relationship between microbial species and these mucin chemotypes in the human colon. In Chapter 2, we begin to fill these gaps in the literature via a pilot study examining sulfo- and sialomucins in the healthy human colon and their relationship with sulfate reducing bacteria (SRB). Quantitative differences in mucin abundance among specific regions of the colon and individuals are described. In addition, we observe that the relationship between these mucin chemotypes and SRB is not influenced by location in the colon, but rather by the host. In Chapter 3, we begin to identify factors that may contribute to changes in sulfomucin in disease and possibly interindividual variation, using human adenocarcinoma-derived LS174T cells, which have a goblet cell-like phenotype and produce both sulfo- and sialomucins. We specifically focused on the effects of bacterial flagellin, IL-13, and TNF?? on the expression of genes encoding the major secretory mucin, MUC2, and Golgi sulfotransfereases, CHST5 and GAL3ST2. In addition, expression of sulfomucin and Sulfo Lea antigen, which is synthesized in part by GAL3ST2, was examined. Overall, the results indicated that both host and microbial factors influence sulfomucin expression, possibly via modulation of CHST5 and GAL3ST2 expression. Because goblet cells synthesize and secrete cysteine-rich products, including mucins, TFF3, and RETNLB, they may have a high cysteine requirement. Furthermore, they may require additional cysteine, which can be catabolized to sulfate, for mucin sulfation. Cysteine is also used in the production of other proteins as well as essential molecules including glutathione, taurine, and pyruvate. However, high cysteine levels are cytotoxic. Mammals regulate cysteine metabolism to maintain levels within a range that is sufficient for synthesis of essential molecules but below the level of cytotoxicity through the use of cysteine dioxygenase (CDO), which catalyzes the first step in cysteine catabolism. In Chapter 4, CDO expression and localization in mouse small and large intestine is examined using immunohistochemical and immunofluorescence staining techniques. We observed that CDO is expressed in goblet cells as well as Paneth and enteroendocrine cells, which are also members of the secretory lineage, while expression was absent in absorptive enterocytes. We postulate that this striking difference in CDO expression between the absorptive and secretory cell lineages is due to higher cysteine catabolism requirements in goblet, Paneth, and enteroendocrine cells relative to absorptive epithelial cells, either to synthesize additional taurine and sulfate or to metabolize excess cysteine when cells are not actively synthesizing cysteine-rich secretory products. In Chapter 5, we examine the possibility that goblet cells are using CDO to synthesize taurine and determine whether inflammatory stimuli alter taurine biosynthesis, using the LS174T cell line as a model. There are two pathways for taurine synthesis. One pathway involves the oxidation of cysteine via CDO to cysteinesulfinate, which is decarboxylated by cysteinesulfinic acid decarboxylase (CSAD) to hypotaurine. The other pathway involves the conversion of cysteine to Coenzyme A, which releases cysteamine during turnover. Cysteamine is then oxidized to hypotaurine via cysteamine dioxygenase (ADO). In both pathways, hypotaurine is then oxidized to taurine. We confirm that LS174T cells synthesize taurine via the ADO pathway and possibly the CDO/CSAD pathway. We also demonstrate that certain inflammatory triggers can enhance taurine biosynthesis and/or export, which may provide taurine for other cell types in an in vivo setting. The anti-inflammatory effects of taurine in the intestine have been described, so these results may represent a novel mechanism by which goblet cells attenuate inflammation. Overall, the findings described in this dissertation provide novel information regarding the role of intestinal goblet cells in mucosal barrier function