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International audience; DMBT1 (deleted in malignant brain tumor 1), a human mucin-like glycoprotein, belonging to the scavenger receptor cystein-rich (SRCR) superfamily, is mainly secreted from mucosal epithelia. It has been shown previously that interaction of hensin, the rabbit ortholog of DMBT1, with galectin 3, a β-galactoside-binding lectin, induces a terminal differentiation of epithelial cells. In this paper, we have used surface plasmon resonance (SPR), to analyse the binding of galectin 3 to two purified samples of human DMBT1:recombinant DMBT1 produced in CHO cells and DMBT1 isolated from intestinal tissues. Characterization of their glycosylation profile by nano-ESI-Q-TOF tandem mass spectrometry showed significant differences in O-glycans between the two DMBT1 samples. Results obtained by SPR demonstrated that the oligosaccharide side chains of DMBT1 are recognized by the carbohydrate-recognition domain (CRD) of galectin 3 and modification in the pattern of oligosaccharides modulates the binding parameters of DMBT1 with galectin 3. Moreover, using immunohistochemistry on paraffin-embedded colonic tissue sections, we could show a co-localisation of DMBT1 and galectin 3 in human intestine, suggesting a potential physiological interaction.

International audience; Intestinal mucins are very high molecular weight glycoproteins secreted by goblet cells lining the crypt and the surface of the colonic mucosa. Profound alterations of mucin O-glycans are observed in diseases such as cancer and inflammation, modifying the function of the cell and its antigenic and adhesive properties. Based on immunohistochemical studies, certain cancer- and inflammation- associated glycans have been defined as oncofetal antigens. However, little or no chemical analysis has allowed the structural elucidation of O-glycans expressed on human fetal mucins. In this paper, mucins were isolated from different regions of the normal human intestine (ileum, right, transverse and left colon) of eight fetuses with A, B or O blood group. After alkaline borohydride treatment, the released oligosaccharides were investigated by nanoESI Q-TOF MS/MS (electrospray ionization quadrupole time-of-flight tandem mass spectrometry). More than 117 different glycans were identified, mainly based on core 2 structures. Some core 1, 3 and 4 oligosaccharides were also found. Most of the structures were acidic with NeuAc residues mainly α2-6 linked to the N-acetylgalactosaminitol and sulphate residues 3-linked to galactose or 6-linked to GlcNAc. In contrast to adult human intestinal mucins, Sda/Cad determinants were not expressed on fetal mucin O-glycans and the presence of an acidic gradient along the intestinal tract was not observed. Similar patterns of glycosylation were found in each part of the intestine and the level of expression of the major oligosaccharides was in the same order of magnitude. This study could help determining new oncofetal antigens, which can be exploited for the diagnosis or the treatment of intestinal diseases.

Transposition of intestinal segments is frequently used for bladder reconstruction. Following transposition, bowel segments continue to produce mucus and a correlation between excessive mucus production and complications such as urinary tract infection or catheter blockage has been observed for a long time. However, no information is currently available on the change of mucin expression and glycosylation under these abnormal conditions. In this study, the variable number tandem repeat region and the irregular repeat domain of human MUC2 were isolated as two glycopeptide populations after reduction and trypsin digestion followed by gel chromatography from urine of patients transposed with urinary bladders. After alkaline borohydride treatment, the oligosaccharides released from the whole MUC2 mucin and the two glycosylated domains were investigated by nanoESI Q-TOF MS/MS (electrospray ionization quadrupole time-of-flight tandem mass spectrometry). More than 60 different glycans were identified, mainly based on sialylated core 3 structures. Some core 1, 2 and 4 oligosaccharides were also found. Most of the structures were acidic with NeuAc residues mainly alpha2-6 linked to the N-acetylgalactosaminitol and sulphate residues exclusively 3-linked to galactose. No expression of blood group A and B or Sda/Cad determinants was observed. Similar patterns of glycosylation were found in the tandem repeat region and the irregular repeat domain and the level of expression of the major oligosaccharides were in the same order of magnitude. The most interesting feature of this study was that sialyl-Tn antigen, which is considered as a tumour antigen, was the oligosaccharide most highly expressed. This result suggests that mucins from intestinal transposed segments are abnormally glycosylated.

Background Micro-array analysis of promoter hypermethylation provides insight into the role and extent of DNA methylation in colorectal cancer (CRC) development, and allows direct comparison with DNA mutations. Methods Colonic biopsy samples were obtained endoscopically from 10 normal, 23 adenoma (17 low grade (LGD) and 6 high grade dysplasia (HGD)), and 8 ulcerative colitis patients. CRC samples were obtained from 24 patients (17 primary, 7 metastatic (mCRC)). Field effects were analyzed in tissues 1cm(n=5) and 10cm(n= 5) from CRC margins. Tissue materials were analyzed for DNA methylation status using a 96 gene panel and expression levels were assayed using whole genomie mRNA arrays. SFRP1 was examined by immunohistochemistry. 5-aza treatment was performed on HT29 cells to analyze reversal of DNA methylation. Results 10 genes (including SFRP1, MAL, SLIT2, SEPTIN9) showed hypermethylation in more than 85% of tumor samples. The number of methylated gene alterations were: HGD(56) > LGD(40) > CRC(37) > mCRC(12). 2 genes (MAL and SFRP1) distinguished precancerous and cancerous lesions from inflammed and healthy tissue (p

Background and Aim: Several studies have looked at the effects of different types of fiber (i.e., prebiotics) on gut microbiota and symptoms in patients with IBS; however there is lack of consistent response to prebiotics among patients. In order to determine the reason for lack of predictable responses in patients, here we investigate the effect of a specific dietary change on gut microbial composition using humanized mice. Methods: Germ free (GF) mice were colonized with human feces (humanized) by gavage of 200μl of frozen feces mixed with an equal volume of pre-reduced PBS. Microbial composition was assessed by 16S rRNA based pyrosequencing using the 454-titanium platform. Non-targeted fecal metabolomics was performed using UPLC-MS. Data analysis was performed using QIIME and MetaboAnalyst. Results: GF mice were humanized using feces from three healthy human donors: two males and one female (n=4-8/donor). Four weeks following humanization, fecal samples were collected from each mouse. Un-weighted UniFrac based PCoA plots of the 16S rRNA microbial composition data demonstrate reproducible reconstitution of the corresponding human fecal inoculum in humanized mice. The metabolomic profile of each sample was used as a read-out of the microbiota functional status. PCoA of these metabolomicsbased "fingerprints" revealed that reproducible aspects of microbiota functional status are dictated by the human fecal inoculum. Together these data show that individual aspects of the human gut microbiota's composition and metabolomic activity can be reconstructed within humanized mice. The presence of most fecal metabolites was independent of the fecal source (human or humanized mouse) suggesting that much of the microbiota functional status is resilient to modification by the host species and largely shared between individuals. In order to determine if dietary intervention impacts all three groups of humanized mice similarly, diet was switched from standard diet to FOS-enriched diet (fructo-oligosaccharide, commonly used prebiotic; 10%w/v). Fecal samples were collected before and after dietary intervention. Un-weighted UniFrac based PCoA plots of the 16S rRNA microbial composition data show distinct diet-induced changes in microbial composition between the three groups of humanized mice, with only a small effect seen in one group. Conclusion: Gut microbial composition and function can be reliably reconstructed in humanized mice making them a good model to study effects of environmental factors on the human gut microbiota. The effect of prebiotics/dietary intervention on gut microbial composition depends on the initial microbial community composition. This study may therefore provide new insight into the observed differences in patient responses to prebiotics in previous studies.

The intestinal mucus layer plays a key role in themaintenance of host-microbiota homeostasis. The production of goblet cells, which secrete mucus, is modulated by microbiota, but neither the species nor the mechanisms involved in this process are still unknown. We studied how two prominent commensal bacteria may influence the mucus production by goblet cells and the profile of mucin glycosylation in gnotobiotic rats. We have chosen Bacteroides thetaiotaomicron, which is characterized by its high mucus-polysaccharides degrading potential, and Faecalibacterium prausnitzii, which is a sensor of intestinal health. Germ free rats (GF) were orally inoculated with B. thetaiotaomicron either alone or with a mix of B. thetaiotaomicron and F. prausnitzii leading respectively to mono-associated (Bt-rats) and di-associated rats (Bt+Fp-rats). A panel of goblet cells markers was analyzed by histological staining, immunohistochemistry, quantitative PCR and Western blot in colon epithelium. The mucin O-glycosylation was determined by MALDI TOF mass spectrometry. In Bt-rats, the goblet cells number and the expression of mucus-related genes (muc2, muc4, klf4, c1galt1 and b4galt4 mRNAs) were increased compared to GF ones. KLF4 protein, a transcription factor involved in goblet cell terminal differentiation, was also increased in Bt-rats, whereas a decrease in Chromogranin A protein, a marker of enteroendocrine cells was observed. We propose that B. thetaiotaomicron provokes an imbalance inside the secretory lineage by favoring mucus production at the expense of enteroendocrine cells. When B. thetaiotaomicron was associated to F. prausnitzii, the effects on goblet cells were reduced/ decreased/diminished. Indeed, the number of goblet cells per crypt and the amount of KLF4 protein were lower in Bt+Fp-rats than in Bt-rats. We then analyzed the mucus quality by studying the profile of mucin O-glycosylation. In Bt-rats, a decrease in the production of sulfated (4.5% of total oligosaccharides instead of 12.9%) and neutral (40.1% instead of 52.8%) oligosaccharides was observed and was correlated to an increased proportion of sialylated O-glycans carrying NeuAc (24.2% instead of 18.9%) or NeuGc (31.2% instead of 15.4%) residues compared to GF rats. Thus, B. thetaiotaomicron impacts the composition of mucin O-glycans, with a decrease in sulfated and neutral oligosaccharides in favor of sialylated ones. Furthermore, glycosylation of mucins from Bt+Fp-rats resembled to those of GF rats. As previously observed for goblet cells, F. prausnitzii seemed to decrease the effect of B. thetaiotaomicron on mucus. Using a novel gnotobiotic model, which is the first described with F. prausnitzii, we showed how the balance between B. thetaiotaomicron and F. prausnitzii plays a key role in protecting epithelium via their respective effects on mucus.