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ABSTRACT: Infection of gastric mucosa by Helicobacter pylori induces an inflammatory response with increased levels of proinflammatory cytokines. Among them, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 induce the activation of signaling pathways that regulate genes expression, such as MUC2 and MUC4 intestinal mucins ectopically detected in gastric tumors. This study evaluated if the predominant inflammatory cell type correlates with MUC2 and MUC4 expression in human intestinal gastric tumors (n=78). In addition, we analyzed the regulatory effects of the associated inflammatory signaling pathways on their expression in gastric cancer cell lines, and in a mouse model with hyperactivated STAT3 signaling pathway. Tumors with predominant lymphoplasmocytic infiltrate (chronic inflammation), presented higher levels of MUC2 and were more differentiated than tumors with predominant polymorphonuclear infiltrate (acute inflammation). These differences can be attributed to specific cytokines, because TNF-alpha and IL-1beta induced MUC2 but no MUC4 expression in gastric cancer cell lines. The two groups of tumors expressed similar levels of MUC4 that correlated with the expression of STAT3 transcription factor, implicated in the activation of genes through the IL-6 pathway. In gastric tissues from gp130(+/+), gp130(Y757F/Y757F) and gp130(Y757F/Y757F) Stat3(-/+) mice, Muc2 was not detected, whereas Muc4 was found in the gastric tumors developed in the gp130(Y757F/Y757F) mice, with hyperactivated STAT3. These data indicate that the signaling pathways associated with the inflammatory response can modulate the expression of MUC2 and MUC4 intestinal mucin genes, in human and mouse gastric tumors.
Oncogene 03/2010; 29(12):1753-62. · 6.37 Impact Factor
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ABSTRACT: Mucus hypersecretion is a hallmark of nasal polyposis (NP). Corticosteroids (CS) are first-line treatment for NP, decreasing their size and inflammatory component. However, their effect on mucin production is not well-understood. The aim of this (pilot) study was to investigate CS effect on mucin expression in NP.
Patients were randomized in control (n = 9) and treatment (oral prednisone for 2 weeks and intranasal budesonide for 12 weeks; n = 23) groups. Nasal polyposis from nonasthmatic (NP; n = 13), aspirin-tolerant (NP-ATA; n = 11) and aspirin-intolerant (NP-AIA; n = 8) asthmatics were studied. Nasal polyposis biopsies were obtained before (w0) and after 2 (w2) and 12 (w12) weeks of CS treatment. Secreted (MUC5AC, MUC5B and MUC8) and membrane-tethered (MUC1, MUC4) mucins (immunohistochemistry) and goblet cells (Alcian blue-periodic acid Schiff) were quantified in both epithelium and glands. Rhinorrea and nasal obstruction were also assessed.
At w2, steroids increased MUC1 (from 70 to 97.5) and MUC4 (from 80 to 100) in NP-ATA patients' epithelium compared with baseline (w0). At w12, steroids decreased MUC5AC (from 40 to 5) and MUC5B (from 45 to 2.5) in NP-ATA patients' epithelium and glands, respectively, compared with baseline. No mucin presented significant changes in NP-AIA patients. MUC5AC and MUC5B expression correlated with goblet and mucous cell numbers, respectively, and MUC5AC also with rhinorrea score.
These results suggest: (i) CS up-regulate membrane (MUC1, MUC4) while down-regulate secreted (MUC5AC, MUC5B) mucins; (ii) there exists a link between secreted mucin expression and goblet cell hyperplasia; and (iii) NP from AIA may develop resistance to CS treatment.
Allergy 07/2008; 63(10):1368-76. · 6.27 Impact Factor
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ABSTRACT: Erythropoietin (EPO) is a hormone that regulates red blood cell production. Recombinant human EPO (rHuEPO) and NESP (novel erythropoiesis stimulating protein) have been produced for therapeutic purposes and also to improve sports performance. The primary sequences of rHuEPO and NESP differ by just five amino acids. Due to the high homology, no antibodies that are able to discriminate between both molecules have been obtained until now. The aim of the present work was to design synthetic peptides corresponding to the sequence that differs between EPO and NESP (87-90aa), that can then be used as immunogens to develop specific rabbit polyclonal antibodies for selectively detecting EPO and NESP. Three peptides were synthesized: EPO (81-95), NESP (81-95), and NESP (86-104), and these were coupled to KLH and OVA for immunization and screening purposes, respectively. The sera obtained were tested by ELISA on synthetic peptide-OVA conjugates and purified by immunoaffinity chromatography against the corresponding synthetic peptide. The specific purified antibodies were characterized by ELISA, SDS-PAGE, and isoelectric focusing, followed by western blot. Antisera raised against EPO (81-95) recognized rHuEPO but not NESP. In contrast, anti-NESP (84-106) sera gave a specific anti-NESP response only after immunoaffinity purification on a NESP (86-91) column. An efficient strategy for generating specific antibodies against EPO and NESP can be achieved by selecting suitable synthetic peptides. The antibodies obtained are able to differentiate between rHuEPO and NESP, and may be particularly useful for screening purposes in both therapeutic and antidoping contexts.
Analytical and Bioanalytical Chemistry 09/2007; 388(7):1531-8. · 3.78 Impact Factor
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ABSTRACT: The female uterine cervix has 2 characteristic populations of epithelial cells: the endocervix is composed by mucus-secreting cells that express several mucin genes, and the exocervix has a typical stratified squamous epithelium and does not express secreted mucins. Among human mucin genes, the MUC4 sequence has a transmembrane domain, and its molecular structure suggests that it has a protective role and also may be implicated in intracellular signalling. The aim of this study is to analyze whether changes in the expression of MUC4 can be detected associated with the squamous dysplastic transformation of exocervical epithelium. MUC4 expression has been analyzed by immunohistochemistry, Western blotting, and in situ hybridization. Using immunohistochemical techniques, MUC4 is found in normal endocervix (n = 11) and is absent or only focally detected in the normal stratified cervical epithelium (n = 18). In samples from squamous metaplasia (n = 9), MUC4 is variably expressed (10% to 50% positive cells), whereas MUC4 is strongly detected in dysplastic cervical epithelia. The greatest number of positive cells is found in samples with moderate and severe dysplasia in which MUC4 is detected in 100% of the analyzed samples (n = 16). These results have been confirmed by Western blotting and by detection of MUC4 transcripts using in situ hybridization. The present data suggest that MUC4 is activated during the process of squamous dysplastic transformation and may be used as a marker for this pathologic process.
Human Pathlogy 12/2001; 32(11):1197-202. · 2.88 Impact Factor
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ABSTRACT: In normal stomach, MUC5AC and MUC6 apomucins are associated with Lewis types 1 and 2, respectively, and this association is lost during gastric carcinogenesis. The expression of gastric (MUC5AC, MUC6) and intestinal (MUC2, MUC4) apomucins and Lewis antigens during gastric development, using single and double labeling immunohistochemistry on fetal tissues (15-41 weeks), was analyzed and related to the tumor expression patterns. Apomucin expression in other fetal tissues was also analyzed. In gastric samples, MUC2 is detected in 14 of 19 showing no correlation with fetal age, and MUC4 is not detected. MUC5AC and MUC6 are always highly detected and are coexpressed and associated with both types of Lewis antigens. These patterns change progressively with the development of the adult gastric morphology. MUC2 is detected in the small intestine, colon, and pancreas; MUC4 is expressed in the colon; MUC5AC is detected in the small intestine; and MUC6 is found in the duodenum and pancreas. The patterns of apomucin expression and association with Lewis antigens during development are complex, but there is a trend toward the establishment of the adult pattern, with the exception of MUC4, which is not detected. These patterns found in fetal stomach indicate that alterations reported in gastric tumors do not fully recapitulate a developmental phenotype.
Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry 04/2001; 9(1):42-8. · 1.63 Impact Factor
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ABSTRACT: In normal gastric epithelium, MUC5AC is detected in superficial epithelium associated with Lewis type 1 antigens and MUC6 is detected in antral glands with Lewis type 2. Therefore, the stomach constitutes an excellent model to examine the role of glycosyltransferases in determining the specificity of apomucin glycosylation.
To determine the molecular basis of this association and to examine changes in expression of gastric and intestinal apomucins and their association with Lewis antigens during the gastric carcinogenesis process.
Fucosyltransferase (FUT1, FUT2, FUT3) and mucin (MUC5AC, MUC6) transcripts were detected using reverse transcription-polymerase chain reaction. Apomucin (MUC2, MUC4, MUC5AC, MUC6) and Lewis antigen (types 1 and 2) expression were analysed using single and double immunohistochemistry and in situ hybridisation.
In the normal stomach, FUT1 is exclusively detected associated with MUC6; FUT2 is only detected when MUC5AC is present. This co-regulation is lost in gastric tumours, as is differential expression of MUC5AC and MUC6 in normal gastric epithelial cells. In gastric tumours, especially those with the intestinal phenotype, MUC2 and MUC4 genes are upregulated, and gastric-type and intestinal-type mucins are coexpressed. These changes are early events in the gastric carcinogenesis process, as they are detected in intestinal metaplasia.
The glycosylation pattern found in normal gastric epithelium is dictated by the specific set of fucosyltranferases expressed by the cells rather than by the apomucin sequence. The development of intestinal metaplasia and gastric cancer is associated with the appearance of cellular phenotypes that are absent from normal epithelium.
Gut 10/2000; 47(3):349-56. · 10.11 Impact Factor
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ABSTRACT: To investigate the expression of MUC6 mucin in gastric carcinomas, we generated a novel monoclonal antibody (MAb CLH5) using an MUC6 synthetic peptide. MAb CLH5 reacted exclusively with the MUC6 peptide and with native and deglycosylated mucin extracts from gastric tissues. MAb CLH5 immunoreactivity was observed in normal gastric mucosa restricted to pyloric glands of the antrum and mucopeptic cells of the neck zone of the body region. In a series of 104 gastric carcinomas, 31 (29.8%) were immunoreactive for MUC6. The expression of MUC6 was not associated with histomorphological type or with clinicopathological features of the carcinomas. Analysis of the co-expression of MUC6 with other secreted mucins (MUC5AC and MUC2) in 20 gastric carcinomas revealed that different mucin core proteins are co-expressed in 55% of the cases. MUC6 was co-expressed and co-localized with MUC5AC in 45% and with MUC2 in 5% of the cases. Expression of MUC2 alone was observed in 25% of the cases. All carcinomas expressing MUC2 mucin in more than 50% of the cells were of the mucinous type according to the WHO classification. The co-expression of mucins was independent of the histomorphological type and stage of the tumors. In conclusion, we observed, using a novel well-characterized MAb, that MUC6 is a good marker of mucopeptic cell differentiation and is expressed in 30% of gastric carcinomas, independent of the clinicopathological features of the cases. Furthermore, we found that co-expression and co-localization of mucins in gastric carcinomas is independent of histomorphology and staging. Finally, we observed that intestinal mucin MUC2 is expressed as the most prominent mucin of the mucins tested in mucinous-type gastric carcinomas.
Journal of Histochemistry and Cytochemistry 04/2000; 48(3):377-88. · 2.72 Impact Factor
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Methods in molecular biology (Clifton, N.J.) 02/2000; 125:353-68.
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ABSTRACT: Intestinal metaplasia is a well-established premalignant condition of the stomach that is characterized by mucin carbohydrate modifications defined by histochemical methods. The purpose of the present study was to see whether the expression of mucin core proteins was modified in the different types of intestinal metaplasia and to evaluate the putative usefulness of mucins as "molecular markers" in this setting. We used a panel of monoclonal antibodies with well-defined specificities to MUC1, MUC2, MUC5AC, and MUC6 to characterize the expression pattern of mucins. In contrast to normal gastric mucosa, the complete form or type I intestinal metaplasia (n = 20) displayed little or no expression of MUC1, MUC5AC, or MUC6 in the metaplastic cells and strong expression of the intestinal mucin MUC2 in the goblet cells of all cases. The incomplete forms of intestinal metaplasia, type II (n = 25) and type III (n = 16), expressed MUC1 and MUC5AC in every case, both in goblet and in columnar cells. MUC6 was also expressed in 16 cases of type II intestinal metaplasia and in 11 cases of type III intestinal metaplasia. The intestinal mucin MUC2 was expressed in every case of incomplete intestinal metaplasia, mostly in goblet cells. The mucin expression profile in the different types of intestinal metaplasia allows the identification of two patterns: one defined by decreased levels of expression of "gastric" mucins (MUC1, MUC5AC, and MUC6) and expression of MUC2 intestinal mucin, which corresponds to type I intestinal metaplasia, and the other defined by coexpression of "gastric mucins" (MUC1, MUC5AC, and MUC6) together with the MUC2 mucin, encompassing types II and III intestinal metaplasia. Our results challenge the classical sequential pathway of intestinal metaplasia (from type I to type III via a type II intermediate step).
Cancer Research 04/1999; 59(5):1003-7. · 7.86 Impact Factor
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ABSTRACT: Among the human mucin complementary DNAs thus far identified, two (MUC5AC and MUC6) were cloned from stomach libraries. This study examines the distribution of MUC6 in normal tissues and compares it with that of MUC5AC as well as with the expression of Lewis blood group antigens.
Affinity-purified rabbit antibodies detecting epitopes within the repetitive sequence of MUC5AC and MUC6 were used in enzyme-linked immunosorbent assays and immunohistochemical assays. RNA expression was analyzed by in situ hybridization. Double-labeling immunofluorescence was used to study apomucin and Lewis antigen coexpression.
MUC6 is detected in the stomach, colon, gallbladder, and endocervix. Two patterns of staining are observed, perinuclear and diffuse cytoplasmic, possibly reflecting differences in MUC6 glycosylation. Using both immunohistochemical assays and in situ hybridization on stomach tissue sections, MUC6 is expressed mainly in antral mucous cells, whereas MUC5AC is detected mainly in the superficial epithelium and neck glands. In antral mucosa, MUC6+ cells express Lewis(y), whereas MUC5AC+ cells express Lewis(b) and sialyl-Lewis(a).
It was concluded that MUC6 has a distinct tissue distribution pattern, different from that of MUC1-MUC5; MUC5AC and MUC6 are expressed by different cellular populations in normal stomach; and in this tissue, MUC5AC+ cells and MUC6+ cells show different patterns of Lewis antigen expression.
Gastroenterology 10/1995; 109(3):723-34. · 11.68 Impact Factor
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ABSTRACT: Sialyl-Tn, defined by monoclonal antibody (MAb) B72.3, shows restricted normal-tissue distribution but is expressed in a wide variety of carcinomas. To analyze the immunogenicity of sialyl-Tn, mice were immunized with ovine submaxillary mucin (OSM) in combination with monophosphoryl lipid A (MPLA), liposomes, or adjuvants that activate macrophages (rIL-1, rIFN-gamma, rM-CSF, IL-1-derived peptides) or T cells (rIL-2). The level and specificity of the immune response were analyzed by ELISA. rIL-1 and rIFN-gamma induced a very high and specific antibody response, whereas the effect of rM-CSF was dose-dependent: at a low dose it induced a high-level specific antibody response and at the high dose level it induced a polyclonal non-specific response. These results indicate that cytokines are powerful adjuvants which modulate both the magnitude and specificity of the immune response. More studies are necessary to determine the optimal doses in animal models and in active specific immunotherapy of patients with cancer.
International Journal of Cancer 09/1993; 55(1):148-52. · 5.44 Impact Factor
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ABSTRACT: The MUC2 intestinal mucin gene contains tandem repeats of 23 amino acid length that are rich in threonine.
Mouse monoclonal antibody LDQ10 was raised against chemically deglycosylated mucin isolated from LS174T colon cancer nude mouse xenografts.
LDQ10 reacts with deglycosylated colon cancer mucin and with a synthetic peptide encompassing the MUC2 tandem repeat sequence. In immunohistochemical assays, strong reactivity with goblet cells in colon, small bowel, and stomach is observed; weaker reactivity with mucin-producing cells in other epithelial tissues is shown. The epitope recognized by LDQ10 is localized in the rough endoplasmic reticulum of normal colonic goblet cells. LDQ10 also shows strong reactivity with colorectal and stomach cancers and weaker reactivity with pancreas, breast, and bladder cancers.
Antibody LDQ10 detects a peptide epitope of MUC2 that becomes cryptic on glycosylation. Altered synthesis of the MUC2 apomucin takes place in a variety of epithelial cancers.
Gastroenterology 02/1993; 104(1):93-102. · 11.68 Impact Factor
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ABSTRACT: The brush border of normal small-intestine epithelial cells is rich in enzymes that are involved in the digestive process. Such molecules can be used as markers to analyze cell lineages and differentiation properties of colorectal cancers. Monoclonal antibodies detecting dipeptidyl peptidase-IV, aminopeptidase N, endopeptidase F, sucrase-isomaltase, alkaline phosphatase, maltase-glucoamylase and lactase have been used to analyze the phenotype of colorectal cancers, adjacent mucosa and histologically normal distant mucosa. The avidin-biotin peroxidase complex method was used. Expression of dipeptidyl peptidase-IV, aminopeptidase N, sucrase-isomaltase and alkaline phosphatase was common in non-neoplastic mucosa adjacent to, and distant from, the tumor; in contrast, endopeptidase F, maltase-glucoamylase and lactase were rarely expressed in normal distant mucosa and more frequently expressed in mucosa adjacent to the tumor. Dipeptidyl peptidase-IV, aminopeptidase N, endopeptidase F, sucrase-isomaltase and alkaline phosphatase were frequently expressed in colorectal cancers, whereas maltase-glucoamylase and lactase were rarely expressed. Two general patterns of antibody reactivity were observed: diffuse cytoplasmic and apical; apical reactivity was generally associated with more differentiated tumors. A logistic predictive regression model indicated that enzyme expression in colorectal cancers followed a coordinate pattern, but was unrelated to the location of the tumor, Dukes stage or differentiation grade. In conclusion, expression of brush-border-associated enzymes occurs frequently in colorectal cancers and is regulated in a co-ordinated manner. These markers can be used for the phenotypic sub-classification of colorectal cancers.
International Journal of Cancer 06/1992; 51(2):173-81. · 5.44 Impact Factor
