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Aflatoxin M1 (AFM1), ochratoxin A (OTA), and zearalenone (ZEA) are mycotoxins commonly found in milk. Mycotoxin contamination has caused food safety concerns worldwide since most of the toxic effects in humans are serious. The combined toxic effects of these mycotoxins on intestinal epithelial cells have not been reported. Herein, we investigated the combined effects of AFM1, OTA, and ZEA on intestinal integrity and define the underlying mechanisms(s) of their effects in Caco-2/HT29-MTX co-cultures. Our results showed that the mixtures of AFM1 + OTA, AFM1 + ZEA, and AFM1 + ZEA + OTA significantly increased epithelial permeability. Immunofluorescence analysis and transmission electron microscopy revealed that mycotoxins altered TJ proteins morphology and disrupted their structures. Also, the present study showed that mixtures of mycotoxins significantly modulated MUC5AC and MUC5B mRNA levels and protein secretion. This study demonstrated that the effects of mixtures of mycotoxins on intestinal barrier function were more significant than AFM1 alone. More importantly, the damage of intestinal integrity caused by mycotoxins was correlated to the change of the TJ proteins location and the decrease of mucin secretion. Mixtures of AFM1, OTA, and ZEA in food might pose a health risk to consumers, particularly in children, and toxin risks should be considered.

BACKGROUND A body of evidence has suggested that mucins play an important role in adhesion, invasion, and cancer metastasis. However, this evidence is scarce and sometimes confusing. OBJECTIVE We performed a systematic review and meta-analysis of available studies to better define the role of mucins in the behavior of colorectal cancer (CRC). METHODS Medical literature was searched through November 30, 2017, using suitable keywords. Pooled estimates, that is, odd ratios (ORs), were obtained using fixed or random-effects models, as appropriate. Heterogeneity between studies was evaluated with the Cochran Q test and I values, whereas the likelihood of publication bias was assessed by constructing funnel plots. Their symmetry was estimated by the Begg and Mazumdar adjusted rank correlation test and by the Egger regression test. RESULTS A total of 2234 CRC patients were included in 12 studies, eligible for meta-analysis. There was a significant difference concerning total mucin expression between CRC patients and controls [pooled ORs (95% confidence interval)=8.156 (2.624-25.354), test for overall effect Z=3.627, P

Chitosans have attracted the interest of the medicinal chemists as mucous adhesive excipients capable of increasing the residence period of drugs inside mucous membranes. Their interactions with the oligomeric mucus gel-forming glycoprotein mucin 2 throughout the intestine determine the level of mucus adhesion, which can be potentiated by the insertion of thiolated substituents on its structure. In this work, we studied the interactions between the mucin 2 and thiolated chitosans, ranking them based on the free energy of receptor-ligand interaction. Results show that when non-bonded interactions were considered, the chitosan

Intraductal papillary mucinous neoplasm (IPMN) of the pancreas is characterized by cystic dilation of the pancreatic duct, caused by mucin hypersecretion, with slow progression via the adenoma-carcinoma sequence mechanism. Mutation of GNAS at codon 201 is found exclusively in IPMNs, occurring at a rate of 41-75%. Recent advances in molecular biological techniques have demonstrated that GNAS mutation might play a role in the transformation of IPMNs after the appearance of neoplastic cells, rather than in the tumorigenesis of IPMNs. GNAS mutation is observed frequently in the intestinal subtype of IPMNs with MUC2 expression, and less frequently in IPMNs with concomitant pancreatic ductal adenocarcinoma (PDAC). Research has focused on assessing GNAS mutation status in clinical practice using various samples. In this review, we discuss the clinical application of GNAS mutation assessment to differentiate invasive IPMNs from concomitant PDAC, examine the clonality of recurrent IPMNs in the remnant pancreas using resected specimens, and differentiate pancreatic cystic lesions using cystic fluid collected by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA), duodenal fluid, and serum liquid biopsy samples.

ALK-positive (ALK+) lung adenocarcinoma usually shows a more advanced-staged disease with frequent nodal metastasis and highly aggressive outcomes compared with EGFR-mutated lung cancers. The aim of this study was to investigate the expression profiles of several mucins in ALK + lung cancers to gain insight into the relationship between the more aggressive biological nature of ALK + lung cancers and the role of mucins. We examined the immunohistochemical profiles of mucins MUC1, MUC2, MUC5AC, and MUC6 in 19 ALK + lung cancers compared with 42 EGFR-mutated lung cancers. ALK + cancers were found to occur in younger patients and were characterized by a solid-predominant histologic subtype with frequent signet ring cells and peritumoral muciphages. By contrast, EGFR-mutated cancers lacked ALK-specific histological patterns. Although all MUC1 and MUC5AC were expressed in both subtypes, MUC1 expression in ALK + cancers was visualized exclusively through cytoplasmic staining, whereas those in EGFR-mutated cancers were predominantly membranous staining in apical area (92.9%) and focally in cytoplasmic staining (7.1%). MUC5AC expression in ALK + cancers was exclusively visualized through cytoplasmic staining (100%), whereas EGFR-mutated cancers showed predominantly perinuclear dot-like patterns (90.5%) and focal cytoplasmic staining (9.5%). MUC2 and MUC6 expression was not detected in either type of lung cancer. Conclusions The high frequency of both MUC1 and MUC5AC cytoplasmic expression, coupled with a lack of MUC2 and MUC6 expression in ALK + lung cancer may contribute to the biologically aggressive behavior of ALK + cancer. Inhibitors to these types of mucins may thus act as a barrier to cancerous extension reducing their aggressive behavior.

Constipation is an acute or chronic illness attributed to various causes, ranging from lifestyle habits to side effects of a disease. To improve the laxative effects of some traditional medicines, herbal mixtures of Liriope platyphylla, Glycyrrhiza uralensis, and Cinnamomum cassia (LGC) were evaluated for their mechanism of action and therapeutic effects in loperamide (Lop)-induced constipated Sprague Dawley rats by examining alterations in excretion parameters, histological structure, mucin secretion, and related protein levels. Food intake and water consumption were constant for all animals. We observed that the Lop+LGC-treated group had significantly greater excretion of stool and urine than was observed in the Lop+Vehicle-treated group. Administration of LGC in the constipation model restored the intestinal transit ratio to normal levels, and increased the number of goblet cells, mucosal layer, and muscle thickness. Mucin secretion was greater in the Lop+LGC-treated group than in the Lop+Vehicle-treated group, and the expression of MUC2 and AQP8 genes were also increased. In addition, reverse transcription polymerase chain reaction and Western blot revealed an increase in the muscarinic acetylcholine receptors (mAChRs) in the Lop+LGC-treated group compared to the Lop+Vehicle-treated group. Furthermore, compared with the Lop+Vehicle-treated group, treatment with LGC reduced the phosphorylation of PKC and PI3K, and expression of Gα protein, but increased levels of IP3. Our results suggest that the traditional herbal mixture of LGC induces a potent laxative effect in Lop-induced constipation through mucosal tissue changes and mucin production. We also demonstrated that the laxative effect of LGC is closely related to the expression of mAChR and its downstream signals, suggesting the possibility of developing a constipation-laxative agent using LGC.

Aims Bisphenol A (BPA) can induce intestinal epithelial cell barrier dysfunction; however, its effects on the intestinal mucus barrier remain unclear. We used LS174T cells as a model to investigate the effects of BPA on the functions of intestinal goblet cells. Main methods This study used CCK-8, flow cytometry, ELISA and real-time PCR to investigate the effects of BPA on mitochondrial dynamics, oxidative stress and apoptosis in goblet cells. In addition, mucin synthesis and secretion were evaluated using PAS staining and a PAS assay, respectively. Key findings Our results indicate that BPA reduced cell viability in a time- and concentration-dependent manner. BPA induced mitochondrial dysfunction, as indicated by the depolarization of the mitochondrial membrane potential, inhibition of mitochondrial respiratory chain complex enzyme activity and reduction of ATP production. Moreover, BPA caused oxidative stress by significantly increasing the accumulation of ROS, as well as oxidative stress products, and reducing the antioxidant capacity. Furthermore, BPA induced intestinal goblet cell apoptosis, accompanied by increased DNA fragmentation, caspase-3, -8, -9,-10 gene expression and enzyme activity. Additionally, BPA inhibited the synthesis and secretion of mucin 2. Significance Our data suggest that BPA affected the secretory function of intestinal goblet cells by inducing mitochondrial dysfunction, oxidative stress, and apoptosis.

Background Ampullary adenocarcinoma (AA) originates from either intestinal (INT) or pancreaticobiliary (PB) epithelium. Different prognostic factors of recurrence have been identified in previous studies. Methods In 91 AA patients of the AGEO retrospective multicentre cohort, we evaluated the centrally reviewed morphological classification, panel markers of Ang et al. including CK7, CK20, MUC1, MUC2 and CDX2, the 50-gene panel mutational analysis, and the clinicopathological AGEO prognostic score. Results Forty-three (47%) of the 91 tumours were Ang-INT, 29 (32%) were Ang-PB, 18 (20%) were ambiguous (Ang-AMB) and one could not be classified. Among these 90 tumours, 68.7% of INT tumours were Ang-INT and 78.2% of PB tumours were Ang-PB. MUC5AC expression was detected in 32.5% of the 86 evaluable cases. Among 71 tumours, KRAS, TP53, APC and PIK3CA were the most frequently mutated genes. The KRAS mutation was significantly more frequent in the PB subtype. In multivariate analysis, only AGEO prognostic score and tumour subtype were associated with relapse-free survival. Only AGEO prognostic score was associated with overall survival. Conclusions Mutational analysis and MUC5AC expression provide no additional value in the prognostic evaluation of AA patients. Ang et al. classification and the AGEO prognostic score were confirmed as a strong prognosticator for disease recurrence.

The in vitro study of the pathogenesis of inflammatory bowel disease (IBD) requires a cell model which closely reflects the characteristics of the in vivo intestinal epithelium. This study aimed to investigate the application of L-pNIPAM hydrogel as a scaffold to develop a long-term 3D co-culture model of Caco-2 and HT29-MTX cells under conditions analogous to inflammation, to determine its potential use in studying IBD. Monocultures and co-cultures were layered on L-pNIPAM hydrogel scaffolds and maintained under dynamic culture conditions for up to 12 weeks. Treatments with IL-1β, TNFα, and hypoxia for 1 week were used to create an inflammatory environment. Following prolonged culture, the metabolic activity of Caco-2 monoculture and 90% Caco-2/10% HT29-MTX co-cultures on L-pNIPAM hydrogels were increased, and finger-like structures, similar in appearance to villi were observed. Following treatment with IL-1β, TNFα and hypoxia, ALP and ZO-1 were decreased, MUC2 increased, and MUC5AC remained unchanged. ADAMTS1 was increased in response to hypoxia. Caspase 3 expression was increased in response to TNFα and hypoxic conditions. In conclusion, L-pNIPAM hydrogel supported long-term co-culture within a 3D model. Furthermore, stimulation with factors seen during inflammation recapitulated features seen during IBD.

Background/Aim: The objective of this study was to investigate the relationship between MUC2 immunostaining and clinicopathological characteristics in a subset of colorectal carcinomas (CRCs). Materials and Methods: A total of 128 CRCs, 50 local nodal metastases, and 42 normal colonic mucosae were retrieved from the archives at the Department of Pathology at King Abdulaziz University, Jeddah, Saudi Arabia. Immunohistochemistry was performed using anti-MUC2 antibody. A cut-off of 25% of positive immunostaining was used to define low and high immunostaining. Statistical tests were used to determine the association of MUC2 with clinicopathological characteristics and survival. Results: MUC2 immunostaining was observed in 66.7% in normal colonic mucosa. Low MUC2 immunostaining was higher in primary CRC (P = 0.003) and nodal metastasis (80%) (P < 0.001). There was significant association of low MUC2 immunostaining in CRC with age group below 60 years (P = 0.05) and occurrence of lymphovascular invasion (P = 0.034). Other clinicopathological parameters were not correlated with MUC2 immunostaining. Regression analysis revealed that low MUC2 immunostaining was an independent predictor of lymphovascular invasion (P = 0.041). In the Kaplan–Meier survival analysis, there was a significant longer disease-free survival in patients with low MUC2 immunostaining (P = 0.045). However, there was no association between MUC2 immunostaining and overall survival (P = 0.601). Conclusion: MUC2 immunostaining may have distinct clinical significance and provide valuable information and could be considered as an important independent prognostic factor while planning the adjuvant therapy in CRC. In future perspective, characterization of MUC2 immunostaining on a large number of cases and molecular studies may be needed.

Short-chain fatty acids contribute to normal bowel function and prevent bacterial infections. In particular, butyrate is a promising candidate that plays an important role in regulating the functional integrity of the gastrointestinal tract by stimulating mucin secretion. We investigated whether butyrate treatment modulates mucin secretion and bacterial adherence in LoVo cells. In addition, the possible signaling pathways were also examined in connection with the upregulation of mucin secretion. The results showed that butyrate induced mucin secretion in LoVo cells, resulting in the inhibition of Escherichia coli adhesion by increasing the adherence of Lactobacillus acidophilus and Bifidobacterium longum. The gene expression analysis suggests that mitogen-activated protein kinase (MAPK) signaling pathways including Cdc42-PAK pathway appears to be involved in stimulating mucin secretion. More importantly, butyrate induced the increased actin expression and polymerization in LoVo cells, which could be attributable to the Cdc42-PAK signaling pathway, implicated in actin cytoskeleton and mucin secretion. Our results provide a molecular basis in modulating bacterial adherence and the MAPK signaling pathway for the improved homeostasis of colonic epithelial cells.

Changes in the lining of the small intestine following chemotherapy have been extensively studied, although also occurs in the large intestine. The aim of this study was to assess the consequences of Epirubicin-based therapy on goblet cells (GCs) and mucus production on colonic mucosa, immediately and after short-time of chemotherapy administration to oncohematological patients, by clinical and histopathological analysis. We assessed the mucus production, composition, and distribution by Alcian Blue (pH 2.5)-Periodic Acid-Schiff (PAS) staining, alongside with the immunoexpression of mucin (MUC)2, MUC4 and inflammatory markers in a series of oncohematological patients, immediately and after short-time of Epirubicin-based chemotherapy cumulative therapy cessation. We showed that GCs number decrease slightly at 48 hours, while mucous secretion became mixed (with a few neutral) after three weeks. Overall, the secretion was increased immediately after the Epirubicin administration, due to the activation of inflammatory pathways, assessed by increased immunostaining of tumor necrosis factor-alpha (TNF-α) at 48 hours. The MUC2 and MUC4 showed a decreased immunoexpression at 48 hours after the Epirubicin administration compared to controls and partially restored three weeks after the cessation. Overall, it is highly plausible that all these key players revolve around the chemotherapy-induced mucositis in oncohematological patients and highlights the morphofunctional particularities of the GCs, which further modulates the clinical outcome of the patient.

This meta-analysis aimed to assess the association of MUC-2 expression with clinicopathological parameters in gastric carcinoma (GC) patients.Clinical databases based on the study aim were searched in detail. The relative risk ratios (RRs) and associated 95% confidence intervals (95% CIs) were computed after eligible trials were included in the study.Nineteen trials involving 2,363 GC patients were included in this meta-analysis. The expression of MUC-2 showed correlation with clinical stage (I/II vs. III/IV) (RR = 1.09, 95% CI: 1.00-1.18, I2 = 24%, p = 0.194), and lymphatic invasion (present vs. absent) (RR = 0.83, 95% CI: 0.72-0.95, I2 = 22.3%, p = 0.252). However, no significant association was identified between the MUC-2 expression and other clinicopathological parameters, including gender (male vs. female), tumor size (5 vs. ≤5 cm), Lauren's classification (intestinal vs. diffuse), tumor differentiation (poorly vs. well and moderately), lymph node metastasis (present vs. absent), vascular invasion (present vs. absent), and 5-year survival (yes vs. no) of GC patients.Our meta-analysis findings suggested that MUC-2 positive cases were correlated with lower tumor stage and lower rate of lymphatic invasion. Further clinical studies are warranted to confirm the role of MUC-2 in clinical practice.

Endocrine mucin-producing sweat gland carcinoma (EMPSGC) is a rare low-grade adnexal malignancy with a predilection for the eyelids of elderly White women, which is associated with invasive mucinous carcinoma with endocrine features in one-third of cases. EMPSGC is characterized by the presence of neuroendocrine differentiation and mucin production. However, EMPSGC displays a variety of architectural patterns including solid, cribriform, papillary, and cystic growth. In addition, EMPSGC may also display nonendocrine cytologic features, such as apocrine change. Because of their variable appearance, EMPSGC can show significant morphologic overlap with certain histologic mimics, namely basal cell carcinoma, hidrocystoma, apocrine hidradenoma, and tubular adenoma. In addition, the often limited sampling of this anatomically delicate area can make the diagnosis of EMPSGC challenging. EMPSGC expresses neuroendocrine markers, including synaptophysin and chromogranin, often in a focal distribution. However, insulinoma-associated protein 1 (INSM1) has been found to be a more sensitive marker for EMPSGC. Recent studies have also demonstrated the expression of the gel-forming mucin 2 (MUC2) in EMPSGC, possibly signifying a lacrimal or conjunctival origin of these neoplasms. In this article, we discuss EMPSGC in the context of its histologic mimics (BCC, hidrocystoma, apocrine hidradenoma, and tubular adenoma) and we investigate the utility of the immunohistochemical expression of INSM1 and MUC2 in the distinction of EMPSGC from them. We demonstrate that INSM1 and MUC2 can reliably distinguish EMPSGC from these histologic mimics.

Mucus forms an important protective barrier that minimizes bacterial contact with the colonic epithelium. Intestinal mucus is organized in a complex network with several specific proteins, including the mucin-2 (MUC2) and the abundant IgGFc-binding protein, FCGBP. FCGBP is expressed in all intestinal goblet cells and is secreted into the mucus. It is comprised of repeated von Willebrand D (vWD) domain assemblies, most of which have a GDPH amino acid sequence that can be autocatalytically cleaved, as previously observed in the mucins MUC2 and mucin-5AC. However, the functions of FCGBP in the mucus are not understood. We show that all vWD domains of FCGBP with a GDPH sequence are cleaved and that these cleavages occur early during biosynthesis in the endoplasmic reticulum. All cleaved fragments, however, remain connected via a disulfide bond within each vWD domain. This cleavage generates a C-terminal-reactive Asp-anhydride that could react with other molecules, such as MUC2, but this was not observed. Quantitative analyses by MS showed that FCGBP was mainly soluble in chaotropic solutions, whereas MUC2 was insoluble, and most of the secreted FCGBP was not covalently bound to MUC2. Although FCGBP has been suggested to bind immunoglobulin G, we were unable to reproduce this binding in vitro using purified proteins. In conclusion, while the function of FCGBP is still unknown, our results suggest that it does not contribute to covalent crosslinking in the mucus, nor incorporate immunoglobulin G into mucus, instead the single disulfide bond linking each fragment could mediate controlled dissociation.

Specific diets regulate neuroimmune responses and modify risk of inflammatory bowel diseases, including ulcerative colitis. A link between gut and brain inflammation is also emerging. We hypothesized that adjusting dietary fatty acid composition modulates the neuroimmune responses in the mucin 2 knock out mice model of spontaneous colitis. Mice were randomly divided into three groups and fed isocaloric diets that only differed in their fatty acid composition. Diets enriched with anhydrous milk fat, corn oil, or Mediterranean diet fats were used. After nine weeks, brain and serum concentrations of ten inflammatory cytokines were measured. Three of these cytokines, including interleukin (IL)-2, IL-12 p70 and interferon-γ, were differentially expressed in the brains of animals from the three diet groups while there were no differences in the serum concentrations of these cytokines. Since only limited information is available about the functions of IL-2 in the central nervous system, in vitro experiments were performed to assess its effects on microglia. IL-2 had no effect on the secretion of neurotoxins and nitric oxide by microglia-like cells, but it selectively regulated phagocytic activity and reactive oxygen species production by stimulated microglia-like cells. Modulation of microglial reactive oxygen species through altered brain IL-2 concentrations could be one of the mechanisms linking diets with modified risk of neuroimmune disorders including Parkinson's disease.

Resistant starch (RS) has caught much attention for its potential to exert a beneficial impact on intestine and certain members of its resident microbiota. In this study, we examined how dietary RS promotes intestinal barrier in meat ducks by microbiome-metabolomics analysis. Ducklings were fed corn-soybean basal diet or RS diet. Dietary RS improved intestinal morphology and enhanced barrier function in ileum, evidenced by lower permeability and upregulated tight junction proteins and Mucin-2 gene expression. Microbiome analysis showed that RS administration elevated the proportion of Firmicutes and butyrate-producing bacteria, and increased butyrate contents in cecum. Furthermore, significant alterations in metabolic profiles were observed, with most of these were associated with the amino acid metabolism (especially tryptophan), lipid metabolism, and intestinal inflammation. Together, diet with RS improved gut integrity and caused corresponding alterations in gut metabolome and microbiome, yielding better insights of the mechanism by RS improved the gut system of ducks.

BACKGROUND: Mucinous pancreatic cystic lesions (PCLs) express different mucin (MUC) types according to their histomorphologic types. High cystic fluid viscosity may help in the detection of mucinous PCLs. We hypothesized that high cystic fluid viscosity may be suggestive of a certain MUC type in mucinous PCLs. METHODS: Prespecified MUC types (MUC1, MUC2, MUC4, MUC5AC, and MUC6) were evaluated in 18 definitively diagnosed mucinous PCLs with sufficient tissue material and prediagnostic cyst fluid viscosity evaluation—string sign (SS)—test. We evaluated the agreement of MUC expression with positive SS test results. Later, we compared cystic fluid carcinoembryonic antigen (CEA) between the prespecified MUC expressing and nonexpressing cyst types. RESULTS: A total of 18 mucinous PCL patients, 11 females, with mean age ± SD (59.7 ± 13.3) were included. Almost all malignant mucinous PCLs expressed MUC1 (71.4%) (P = .023). We found no significant agreement between the prespecified MUC types and positive SS, except MUC4 which had mild agreement. Also, no significant relation was found between cystic fluid CEA levels and MUC expression (P = .584). CONCLUSION: We did not detect a significantly moderate or good agreement between the prespecified MUC types and SS test. MUC1 was highly expressed in malignant mucinous cysts; however, it was incompatible with the SS test. MUC4 expression showed mild agreement with the SS test in a small number of patients.

BACKGROUND AND AIM Studies on the regulation of mucin2 expression in intestinal goblet cells by the endocrine system and the immune system have been comprehensive, but the effects of abundant mechanical factors in the intestinal microenvironment on goblet cells are not clear. METHODS We constructed mechanical stimulation models in vivo and in vitro to explore the effect of mechanical stimulation on intestinal goblet cells. Piezo1 expression and function were regulated through model mouse and drugs to explored whether Piezo1 mediated mechanical stimulation. RESULTS The results showed that hydrostatic pressure could promote mucus secretion in the mouse colon, and both traction force and shear force could promote the expression of mucin2 in the LS174T cell line. We further found that the Piezo1 protein, which was abundantly expressed in goblet cells, acted as a mechanoreceptor. Knockout of Piezo1 in the intestinal epithelial cells of mice could reduce the promotion of mucus secretion by pressure stimulation, and the specific downregulation of Piezo1 protein in LS174T cells or Piezo1 inhibitor treatment could significantly reduce the promotion of mucin2 expression in goblet cells by mechanical stimulation; however, treatment with a Piezo1 agonist had the opposite effect. Moreover, we found that Piezo1 regulated mucin2 expression through the downstream Erk/Ca2+ pathway. CONCLUSION In short, our study confirmed for the first time that goblet cells are mechanoreceptive cells that can directly sense mechanical stimulation in the intestinal tract and respond back through the Piezo1-Erk/Ca2+ -mucin2 pathway.

Most athletes continually endure mental and physical stress from intense exercise. Fructo-oligosaccharide (FOS) can reduce physical exhaustion, but the concrete mechanism behind it still needs further research. In this study, the effect of FOS on colonic mucosal barriers was investigated using an exercise-induced stress mouse model. Except for control individuals, mice were subject to cycles of 2-day exercise (at 20 rpm) interleaved by 5-day rest. The mice experienced a total of 6 days of exercise during the feeding period. FOS improved common indicators of exhaustion, such as glycogen storage in muscle. 16S rRNA data supported that changes in the gut microbiome were also closely related to stress status. Notably, Anaerotruncus was increased in mice under stress, while FOS facilitated the growth of Dorea, which is negatively associated with exhaustion. The RNA-seq analysis revealed that FOS could maintain the integrity of colonic epithelial barriers. For example, FOS significantly restored the expression of tight junctions (Occludin and Zonula occludens-1) in the colon, which was impaired under a stress state. Besides, the NOD-like receptor family pyrin domain containing 6 (NLRP6) inflammasome might contribute to the protection of the colonic mucosa by promoting the secretion of IL-18, Mucin2 (Muc2) and intestine lectin 1 (Itln1) in FOS-treated individuals. In short, FOS administration attenuated the damage of colonic mucosal barriers in exercise-induced stressed mice.

Intestinal barrier dysfunction is an essential pathological change in inflammatory bowel disease (IBD). The mucus layer and the intestinal epithelial tight junction act together to maintain barrier integrity. Studies showed that chitosan oligosaccharide (COS) had a positive effect on gut health, effectively protecting the intestinal barrier in IBD. However, these studies usually focused on its impact on the intestinal epithelial tight junction. The influence of COS on the intestinal mucus layer is still poorly understood. In this study, we explored the effect of COS on intestinal mucus in vitro using human colonic mucus-secreted HT-29 cells. COS relieved DSS (dextran sulfate sodium)-induced mucus defects. Additionally, the structural characteristics of COS greatly influenced this activity. Finally, we evaluated the protective effect of COS on intestinal barrier function in mice with DSS-induced colitis. The results indicated that COS could manipulate intestinal mucus production, which likely contributed to its intestinal protective effects.

Patients with inflammatory bowel disease (IBD) are at increased risk of under-recognized metabolic comorbidities. Chronic intestinal inflammation in IBD along with changes to the gut microbiome leads to broader systemic effects. Despite the existence of multiple animal models to study colitis, limited studies have examined the metabolic abnormalities associated with these models. In this study, a spontaneous model of colitis (mucin 2 knock-out mouse, Muc2

Intestinal mucus protects epithelial and immune cells from the gut resident microorganisms, and provides growth-promoting factors as mucus-derived O-glycans for beneficial bacteria. A lack of intestinal protective mucus results in changes in the commensal microflora composition, mucosal immune system reprogramming, and inflammation. Previous work has shown that fucose, the terminal glycan chain component of the intestinal glycoprotein Mucin2, and fucoidan polysaccharides have an anti-inflammatory effect in some mouse models of colitis. This study evaluates the effect of fucose on reproductive performance in heterozygous mutant Muc2 female mice. We found that even though Muc2+/− females are physiologically indistinguishable from C57Bl/6 mice, they have a significantly reduced reproductive performance upon dietary fucose supplementation. Metagenomic analysis reveals that the otherwise healthy wild-type siblings of Muc2−/− animals have reduced numbers of some of the intestinal commensal bacterial species, compared to C57BL/6 mice. We propose that the changes in beneficial microflora affect the immune status in Muc2+/− mice, which causes implantation impairment. In accordance with this hypothesis, we find that macrophage polarization during pregnancy is impaired in Muc2+/− females upon addition of fucose. Metabolic profiling of peritoneal macrophages from Muc2+/− females reveals their predisposition towards anaerobic glycolysis in favor of oxidative phosphorylation, compared to C57BL/6-derived cells. In vitro experiments on phagocytosis activity and mitochondrial respiration suggest that fucose affects oxidative phosphorylation in a genotype-specific manner, which might interfere with implantation depending on the initial status of macrophages. This hypothesis is further confirmed in BALB/c female mice, where fucose caused pregnancy loss and opposed implantation-associated M2 macrophage polarization. Taken together, these data suggest that intestinal microflora affects host immunity and pregnancy outcome. At the same time, dietary fucose might act as a differential regulator of macrophage polarization during implantation, depending on the immune status of the host.

The authors wish to make the following corrections to this paper [...]

Mucus produced by goblet cells in the gastrointestinal tract forms a biological barrier that protects the intestine from invasion by commensals and pathogens. However, the host-derived regulatory network that controls mucus secretion and thereby changes gut microbiota has not been well studied. Here, we identify that Forkhead box protein O1 (Foxo1) regulates mucus secretion by goblet cells and determines intestinal homeostasis. Loss of Foxo1 in intestinal epithelial cells (IECs) results in defects in goblet cell autophagy and mucus secretion, leading to an impaired gut microenvironment and dysbiosis. Subsequently, due to changes in microbiota and disruption in microbiome metabolites of short-chain fatty acids, Foxo1 deficiency results in altered organization of tight junction proteins and enhanced susceptibility to intestinal inflammation. Our study demonstrates that Foxo1 is crucial for IECs to establish commensalism and maintain intestinal barrier integrity by regulating goblet cell function.

Cancer cells in intraoperative peritoneal washings (PW) indicate increased peritoneal recurrence. Detection of CEA or CK20 genes indicates poor prognosis. We assessed long-term prognosis of patients with amplification of cancer-related genes in PW obtained intraoperatively during curative gastric cancer surgery.PW was collected before and immediately after curative gastrectomy. CEA, CK20, TFF1, MUC2, and FABP1-mRNA were selected as marker genes for reverse transcription polymerase chain reaction. Peritoneal recurrence-free survival (PRFS) and overall survival (OS) after7-year follow-up were examined using the Kaplan-Meier method.Of 138 patients who underwent gastrectomy with negative cytological findings at laparotomy, 80 patients showed negative cancer-related gene amplification in preoperative PW. Fifty-eight patients were excluded due to positive gene amplification, which suggested presence of preoperative peritoneal cancer cells. The 80 patients had mRNA amplification in PW after surgery. Amplification of multiple and single cancer-related marker genes was observed in 38 and 21 patients; 21 cases had marker-negative results. Five-year PRFS was 69.1%, 95.2%, and 100% in multi-marker-positive, single marker-positive, and marker-negative cases, respectively. Multi-marker-positive patients had significantly worse PRFS than the other groups (p 0.05). Multivariate analysis in the Cox proportional hazards model identified multi-marker-positivity as an independent prognostic factor for PRFS (hazard ratio, 7.6; 95% confidence interval, 1.07-62.63; p = 0.046), and multi-marker-positive patients had significantly worse OS than other groups (p 0.01).Multi-marker cancer-related gene amplification in PW is associated with worse prognosis in PRFS and OS even after a long follow-up; PRFS can be stratified by the number of genes amplified.

The intestinal mucosa is in continuous contact with milliard of microorganisms, thus intestinal epithelial barrier is a critical component in the arsenal of defense mechanisms required to prevent infection and inflammation. Mucin 2 (MUC2), which is produced by the goblet cells, forms the skeleton of the intestinal mucus and protects the intestinal tract from self-digestion and numerous microorganisms. Dedicator of cytokinesis 4 (DOCK4) is a member of the DOCK-B subfamily of the DOCK family of guanine nucleotide exchange factors. It is reported that DOCK4 plays a critical role in the repair of the barrier function of the intestinal epithelium after chemical damage. In this study, the role of DOCK4 in the goblet cell differentiation and MUC2 production is explored. Disordered intestinal epithelium and shortage of goblet cells were observed in DOCK4 gene knockout mice. Furthermore, DOCK4 deletion contributed to the low expression of MUC2 and the goblet cell differentiation/maturation factors including growth factor independent 1 (Gfi1) and SAM pointed domain epithelial-specific transcription factor (Spdef) in mouse ileums and colons. Overexpression of DOCK4 caused a marked increase in Gfi1, Spdef, and MUC2, while siRNA knockdown of endogenous DOCK4 significantly decreased Gfi1, Spdef, and MUC2 in HT-29 cells. In addition, MUC2, DOCK4, and the goblet cell differentiation/maturation factors mRNA levels were decreased in colorectal cancer samples compared with normal colons. A significant positive correlation was found between MUC2 and DOCK4. In conclusion, DOCK4 may serve as a critical regulator of goblet cell differentiation and MUC2 production in the intestine.

Supplemental Digital Content is available in the text., BACKGROUND: Lymph node metastasis is the single most important prognostic risk factor for recurrence in patients with colon cancer who have undergone curative surgery. The routine method for detecting disseminated tumor cells in lymph nodes is microscopic examination of one or a few hematoxylin and eosin-stained tissue sections by a trained pathologist. This method, however, is insensitive mainly because less than 1% of the lymph node volume is examined, leading to misclassification. OBJECTIVE: This study aimed to investigate whether analysis of a selected group of biomarker mRNAs improves detection and characterization of lymph node metastases/micrometastases compared with the routine method. DESIGN: This study is a side-by-side comparison of biomarker mRNA analysis and histopathology of 185 lymph nodes from patients with colon cancer representing stages I to IV, and an investigation of the importance of lymph node tissue volume for tumor cell detection. SETTINGS: This is a collaborative study between a high-volume central hospital and a preclinical university institution. PATIENTS: Fifty-seven patients who had undergone tumor resection for colon cancer were included. MAIN OUTCOME MEASURES: The primary outcomes measured were mRNA copies per 18S rRNA copy of CEACAM5, KLK6, SLC35D3, POSTN, and MUC2 by multiplex assay and metastases/micrometastases detected by histopathology. RESULTS: The number of tumor cell-positive lymph nodes was 1.33-fold higher based on CEACAM5 mRNA levels compared with histopathological examination. Increasing the tissue volume analyzed for CEACAM5 levels from an 80-µm section to half a lymph node increased the number of positive nodes from 34 of 107 to 80 of 107 (p < 0.0001). Similarly, the number of positive nodes for the aggressiveness marker KLK6 increased from 9 of 107 to 24 of 107. LIMITATIONS: Only a limited number of individual lymph nodes per patient was available for analysis. CONCLUSIONS: mRNA analysis of CEACAM5, KLK6, and SLC35D3 improves the detection of tumor cells in lymph nodes from patients surgically treated for colon cancer, and, together with POSTN and MUC2, it further allows characterization of the tumor cells with respect to aggressiveness and the tumor cell environment. See Video Abstract at http://links.lww.com/DCR/B650. EL ANÁLISIS DE ARNM DE CEACAM5, KLK6, SLC35D3, POSTN Y MUC2 MEJORA LA DETECCIÓN Y PERMITE LA CARACTERIZACIÓN DE CÉLULAS TUMORALES EN LOS GANGLIOS LINFÁTICOS DE PACIENTES CON CÁNCER DE COLON ANTECEDENTES: Las metástasis en los ganglios linfáticos son el factor de riesgo pronóstico más importante de recurrencia en pacientes con cáncer de colon que se han sometido a cirugía curativa. El método de rutina para detectar células tumorales diseminadas en los ganglios linfáticos es el examen microscópico de una o algunas secciones de tejido teñidas con hematoxilina-eosina por un patólogo capacitado. Sin embargo, este método es insensible principalmente porque se examina menos del 1% del volumen de los ganglios linfáticos, lo que conduce a una clasificación errónea. OBJETIVO: Investigar si el análisis de un grupo seleccionado de ARNm de biomarcadores mejora la detección y caracterización de metástasis / micrometástasis en los ganglios linfáticos en comparación con el método de rutina. DISEÑO: Una comparación en paralelo del análisis de ARNm de biomarcadores y la histopatología de 185 ganglios linfáticos de pacientes con cáncer de colon que representan las etapas I-IV, e investigación de la importancia del volumen de tejido de los ganglios linfáticos para la detección de células tumorales. ENTORNO CLINICO: Estudio colaborativo entre un hospital central de alto volumen y una institución universitaria preclínica. PACIENTES: Cincuenta y siete pacientes que han sido sometidos a resección tumoral por cáncer de colon. PRINCIPALES MEDIDAS DE VALORACION: copias de ARNm / copia de ARNr 18S de CEACAM5, KLK6, SLC35D3, POSTN y MUC2 mediante análisis múltiple y metástasis / micrometástasis detectadas por histopatología. RESULTADOS: El número de ganglios linfáticos con células tumorales positivas fue 1,33 veces mayor según los niveles de ARNm de CEACAM5 en comparación con el examen histopatológico. El aumento del volumen de tejido analizado para los niveles de CEACAM5 de una sección de 80 µm a la mitad de un ganglio linfático aumentó el número de ganglios positivos de 34/107 a 80/107 (p

Background Compound sophorae decoction (CSD), a Chinese Herbal decoction, is frequently clinically prescribed for patients suffered from ulcerative colitis (UC) characterized by bloody diarrhea. Yet, the underlying mechanism about how this formulae works is remain elusive. Methods In the present study, the experimental colitis in C57BL/6 J mice was induced by oral administration of standard diets containing 3% dextran sodium sulfate (DSS), and CSD was given orally for treatment at the same time. The clinical symptoms including stool and body weight were recorded each day, and colon length and its histopathological changes were observed. Apoptosis of colonic epithelium was studied by detecting protein expression of cleaved caspase-3, and cell proliferation by Ki-67 immunohistochemistry. Tight junction complex like ZO-1 and occludin were also determined by transmission electron microscope and immunofluorescence. The concentration of FITC-dextran 4000 was measured to evaluate intestinal barrier permeability and possible signaling pathway was investigated. Mucin2 (MUC2) and notch pathway were tested through western blot. The M1/M2 ratio in spleen and mesenteric lymph nodes were detected by flow cytometry. And the mRNA levels of iNOS and Arg1 were examined by qRT-PCR. Results CSD could significantly alleviate the clinical manifestations and pathological damage. Body weight loss and DAI score of mice with colitis were improved and shortening of colon was inhibited. The administration of CSD was able to reduce apoptotic epithelial cells and facilitate epithelial cell regeneration. Increased intestinal permeability was reduced in DSS-induced colitis mice. In addition, CSD treatment obviously up-regulated the expression of ZO-1 and occludin and the secretion of MUC2, regulated notch signaling, and decreased the ratio of M1/M2. Conclusions These data together suggest that CSD can effectively mitigate intestinal inflammation, promote phenotypic change in macrophage phenotype and enhance colonic mucosal barrier function by, at least in part, regulating notch signaling in mice affected by DSS-induced colitis.

Intestinal epithelial cell endoplasmic reticulum (ER) stress has been implicated in intestinal inflammation. It remains unclear whether ER stress is an initiator of or a response to inflammation. Winnie mice, carrying a Muc2 gene mutation resulting in intestinal goblet cell ER stress, develop spontaneous colitis with a depleted mucus barrier and increased bacterial translocation. This study aims to determine whether the microbiota was required for the development of Winnie colitis, and whether protein misfolding itself can initiate inflammation directly in absence of the microbiota. To assess the role of microbiota in driving Winnie colitis, WT and Winnie mice on the same background were rederived into the germ-free facility and housed in the Trexler-type soft-sided isolators. The colitis phenotype of these mice was assessed and compared to WT and Winnie mice housed within a specific pathogen-free facility. We found that Winnie colitis was substantially reduced but not abolished under germ-free conditions. Expression of inflammatory cytokine genes was reduced but several chemokines remained elevated in absence of microbiota. Concomitantly, ER stress was also diminished, although mucin misfolding persisted. RNA-Seq revealed that Winnie differentiated colon organoids have decreased expression of the negative regulators of the inflammatory response compared to WT. This data along with the increase in Mip2a chemokine expression, suggests that the epithelial cells in the Winnie mice are more responsive to stimuli. Moreover, the data demonstrate that intestinal epithelial intrinsic protein misfolding can prime an inflammatory response without initiating the unfolded protein response in the absence of the microbiota. However, the microbiota is necessary for the amplification of colitis in Winnie mice. Genetic predisposition to mucin misfolding in secretory cells initiates mild inflammatory signals. However, the inflammatory signal sets a forward-feeding cycle establishing progressive inflammation in the presence of microbiota. Abbreviations: Endoplasmic Reticulum: ER; Mucin-2: Muc-2; GF: Germ-Free; Inflammatory Bowel Disease: IBD

AHCC® is a standardized extract of cultured mushroom (Lentinula edodes) mycelia with a wide variety of therapeutic effects including anti-inflammatory, antitumor and antiviral effects. Trichinellosis, a food-borne parasitic zoonosis is caused by the nematode Trichinella spp. Infection with Trichinella is characterized by the induction of a Th1-type response at the beginning of the intestinal phase, followed by a dominant Th2-type response which is essential for parasite expulsion. The aim of this study was to evaluate the immunomodulatory effect of AHCC® in a murine model of Trichinella spiralis infection. Swiss CD1 mice were infected with T. spiralis larvae and treated with AHCC®. Standard treatment with albendazole (ABZ) was used as control in the assessment of parasite burden. The small intestine was taken out and the proximal segment was evaluated for several parameters: gene expression of immune and stress-reticulum mediators, histological damage score, goblet cell count and Mucin 2 (Muc2) gene expression. AHCC® modulated expression levels of both Th1 and Th2 cytokines and reduced histological damage score. In addition, AHCC® diminished the number of adults of T. spiralis in treated animals. AHCC® treatment anticipates T. spiralis expulsion and increases goblet cell number and Muc2 gene expression.

The disruption of the protective intestinal barrier—the ‘leaky gut’—is a common complication of the inflammatory bowel disease. There is limited data on the mechanisms of the intestinal barrier disruption upon low-grade inflammation characteristic of patients with inflammatory bowel disease in clinical remission. Thus, animal models that recapitulate the complexity of chronic intestinal inflammation in vivo are of particular interest. In this study, we used Mucin-2 (Muc2) knockout mice predisposed to colitis to study intestinal barrier upon chronic inflammation. We used 4-kDa FITC-Dextran assay and transmission electron microscopy to demonstrate the increased intestinal permeability and morphological defects in intercellular junctions in Muc2 knockout mice. Confocal microscopy revealed the disruption of the apical F-actin cytoskeleton and delocalization of tight junction protein Claudin-3 from the membrane. We further demonstrate mitochondrial damage, impaired oxygen consumption and the reduction of the intestinal ATP content in Muc2 knockout mice. Finally, we show that chemically induced mitochondrial uncoupling in the wild type mice mimics the intestinal barrier disruption in vivo and causes partial loss of F-actin and membrane localization of Claudin-3. We propose that mitochondrial damage and metabolic shifts during chronic inflammation contribute to the leaky gut syndrome in Muc2 knockout animal model of colitis.

Di-2-ethylhexyl phosphate (DEHP) and its main toxic metabolite mono-2-ethylhexyl phthalate (MEHP) are the typical endocrine disrupting chemicals (EDCs) and widely affect human health. Our previous research reported that synthetic nonionic dietary emulsifier polysorbate 80 (P80, E433) had the promotional effect on the oral absorption of DEHP in rats. The aim of this study was to explore its mechanism of promoting oral absorption, focusing on the mucus barrier and mucosal barrier of the small intestine. A small molecule fluorescent probe 5-aminofluorescein-MEHP (MEHP-AF) was used as a tracker of MEHP in vivo and in vitro. First of all, we verified that P80 promoted the bioavailability of MEHP-AF in the long-term and low-dose exposure of MEHP-AF with P80 as a result of increasing the intestinal absorption of MEHP-AF. Afterwards, experimental results from Western blot, qPCR, immunohistochemistry, and immunofluorescence showed that P80 decreased the expression of proteins (mucus protein mucin-2, tight junction proteins claudin-1 and occludin) related to mucus barrier and mucosal barrier in the intestine, changed the integrity of intestinal epithelial cell, and increased the permeability of intestinal epithelial mucosa. These results indicated that P80 promoted the oral absorption of MEHP-AF by altering the intestinal mucus barrier and mucosal barrier. These findings are of great importance for assessing the safety risks of some food emulsifiers and clarifying the absorption mechanism of chemical pollutants in food, especially for EDCs.

Broilers are often deprived of feed and water for up to 48 h after hatch. This delayed access to feed (DAF) can inhibit small intestine development. The objective of this study was to determine the effects of DAF on small intestinal morphology, mRNA abundance of the goblet cell marker Muc2 and absorptive cell marker PepT1, and the distribution of goblet cells in young broilers. Cobb 500 chicks, hatching within a 12-h window, were randomly allocated into 3 groups: control with no feed delay (ND), 24-h feed delay (DAF24), and 36-h feed delay (DAF36). Morphology, gene expression, and in situ hybridization analyses were conducted on the duodenum, jejunum, and ileum at 0, 24, 36, 72, 120, and 168 h after hatch. Statistical analysis was performed using a t test for ND and DAF24 at 24 h. A 2-way ANOVA and Tukey's HSD test (P < 0.05) were used for ND, DAF24, and DAF36 from 36 h. At 24 to 36 h, DAF decreased the ratio of villus height/crypt depth (VH/CD) in the duodenum but increased VH/CD in the ileum due to changes in CD, whereas at 72 h, DAF decreased VH/CD due to a decrease in VH. The mRNA abundance of PepT1 was upregulated, while Muc2 mRNA was downregulated in DAF chicks. Cells expressing Muc2 mRNA were present along the villi and in the crypts. The ratio of the number of goblet cells found in the upper half to the lower half of the villus was greater in DAF chicks than in ND chicks, suggesting that DAF affected the appearance of new goblet cells. The number of Muc2 mRNA-expressing cells in the crypt, however, was generally not affected by DAF. In conclusion, DAF transiently affected small intestinal morphology, upregulated PepT1 mRNA, downregulated Muc2 mRNA, and changed the distribution of goblet cells in the villi. By 168 h, however, these parameters were not different between ND, DAF24, and DAF36 chicks. Virginia Agricultural Experiment Station; Hatch Program of the National Institute of Food and Agriculture, U.S. Department of Agriculture (NIFA-USDA); NIFA-USDAUnited States Department of Agriculture (USDA) [2017-67015-26588] This project was funded in part by the Virginia Agricultural Experiment Station, the Hatch Program of the National Institute of Food and Agriculture, U.S. Department of Agriculture (NIFA-USDA), and NIFA-USDA grant 2017-67015-26588.

A hallmark of enteroaggregative Escherichia coli (EAEC) infection is the formation of an intestinal biofilm, which comprises a mucus layer with immersed bacteria. Pic is an autotransporter secreted by EAEC, and other E. coli pathotypes, and has been involved in two apparently contradictory phenotypes, as a mucus secretagogue and as a mucinase. Here, we investigated this Pic dual activity, mucus secretagogue capability and mucinolytic activity, in human goblet cells that secrete MUC2 and MUC5AC. Pic induced mucus hypersecretion directly in the goblet cells, without other intestinal cell types involved. At the same time, Pic exhibited strong proteolytic activity on the secreted mucins. These activities were independent since a mutation in the serine protease motif (PicS258I) abolished mucin degradation while maintaining the mucus secretagogue activity intact. Furthermore, deoxycholic acid (DCA)-induced mucins were proteolytically degraded when goblet cells were co-incubated with DCA/Pic, while co-incubation with DCA/PicS258I induced a synergistic effect on mucus hypersecretion. Pic was more efficient degrading MUC5AC than MUC2, but no degradation was detected with Pic inactivated at the active site by mutation or pharmacological inhibition. Remarkably, Pic cleaved MUC2 and MUC5AC in the C-terminal domain, leaving N-terminal subproducts, impacting the feature of gel-forming mucins and allowing mucus layer penetration by EAEC. Astonishingly, Pic stimulated rapid mucin secretion in goblet-like cells by activating the intracellular calcium pathway resulting from the PLC signal transduction pathway, leading to the production of DAG and releasing IP3, a second messenger of calcium signaling. Therefore, the dual activity of Pic, as a mucus secretagogue and a mucinase, is relevant in the context of carbon source generation and mucus layer penetration, allowing EAEC to live within the layer of mucus but also access epithelial cells.

Goblet cells (GCs) are specialized cells of the intestinal epithelium contributing critically to mucosal homeostasis. One of the functions of GCs is to produce and secrete MUC2, the mucin that forms the scaffold of the intestinal mucus layer coating the epithelium and separates the luminal pathogens and commensal microbiota from the host tissues. Although a variety of ion channels and transporters are thought to impact on MUC2 secretion, the specific cellular mechanisms that regulate GC function remain incompletely understood. Previously, we demonstrated that leucine-rich-repeat-containing protein 26 (LRRC26), a known regulatory subunit of the Ca2+-and voltage-activated K+channel (BK channel), localizes specifically to secretory cells within the intestinal tract. Here, utilizing a mouse model in which MUC2 is fluorescently tagged allowing visualization of single GCs in intact colonic crypts, we show that murine colonic GCs have functional LRRC26-associated BK channels. In the absence of LRRC26, BK channels are present in GCs, but are not activated at physiological conditions. In contrast, all tested MUC2-negative cells completely lacked BK channels. Moreover, LRRC26-associated BK channels underlie the BK channel contribution to the resting transepithelial current across mouse distal colonic mucosa. Genetic ablation of either LRRC26 or BK-pore forming α-subunit in mice results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate (DSS). These results demonstrate that normal potassium flux through LRRC26-associated BK channels in GCs has protective effects against colitis in mice.SignificanceA primary function of goblet cells (GCs) of the intestinal epithelium is to generate a protective mucus layer lining the intestinal lumen. GC dysfunction is linked to Inflammatory Bowel Disease (IBD). GC mucus secretion is thought to be dependent on contributions of an ensemble of anion and cation fluxes, although understanding remains limited. Here, it is shown in mouse colon that the Ca2+- and voltage-dependent BK-type K+channel, specifically in association with the LRRC26 regulatory subunit, plays a critical role in normal GC function, protecting mice against chemically-induced colitis. The results demonstrate that normal K+fluxes mediated by LRRC26-containing BK channels are required for normal GC function, potentially providing insights into the potential role of BK channels in IBD.

The mucin Muc2 is a major constituent of the mucus layer that covers the intestinal epithelium and creates a barrier between epithelial cells and luminal commensal or pathogenic microorganisms. The Gram-positive foodborne pathogen Listeria monocytogenes can cause enteritis and also disseminate from the intestine to give rise to systemic disease. L. monocytogenes can bind to intestinal Muc2, but the influence of the Muc2 mucin barrier on L. monocytogenes intestinal colonization and systemic dissemination has not been explored. Here, we used an orogastric L. monocytogenes infection model to investigate the role of Muc2 in host defense against L. monocytogenes. Compared to wild-type mice, we found that Muc2−/− mice exhibited heightened susceptibility to orogastric challenge with L. monocytogenes, with higher mortality, elevated colonic pathology, and increased pathogen burdens in both the intestinal tract and distal organs. In contrast, L. monocytogenes burdens were equivalent in wild-type and Muc2−/− animals when the pathogen was administered intraperitoneally, suggesting that systemic immune defects related to Muc2 deficiency do not explain the heightened pathogen dissemination observed in oral infections. Using a barcoded L. monocytogenes library to measure intrahost pathogen population dynamics, we found that Muc2−/− animals had larger pathogen founding population sizes in the intestine and distal sites than observed in wild-type animals. Comparisons of barcode frequencies suggested that the colon becomes the major source for seeding the internal organs in Muc2−/− animals. Together, our findings reveal that Muc2 mucin plays a key role in controlling L. monocytogenes colonization, dissemination, and population dynamics.

Dietary protein sources have profound effects on children and young animals, and are important for the gut barrier function and immune resilience. Milk and soy are the main sources of protein for children and young animals after weaning. The objective of this study was to compare the effects of dairy and soy proteins on the intestinal barrier in early development. Weanling C57BL/6 mice were fed AIN-93G diets prepared with casein or soy protein isolate (SPI) for 21 days. Compared with those fed with the casein diet, mice fed with the SPI diet did not change their body weight and organ coefficients, but increased their feed intake and ratio of feed to gain. SPI lowered the level of luminal secretory immunoglobulin A (SIgA) and downregulated the levels of IL-4, IL-13, polymeric immunoglobulin receptor (Pigr), Janus kinase 1 (Jak1), signal transducer and activator of transcription 6 (Stat6), and transforming growth factor-&beta, (Tgfb) in the mouse ileum. Western blotting of ileal proteins confirmed that SPI suppressed the activation of the JAK1/STAT6 signaling pathway. Furthermore, SPI attenuated intestinal mucin production, as demonstrated by the decreased numbers of intestinal goblet cells and the reduced relative expression levels of mucin 1 (Muc1), mucin 2 (Muc2), trefoil factor 3 (Tff3), glucose-regulated protein 94 (Grp94), and anterior gradient homolog 2 (Agr2). The results indicated that the SPI diet could attenuate mouse intestinal immunity, as demonstrated by decreased SIgA and mucin production in the intestine. Therefore, we suggest that our findings should be of consideration when SPI or casein are used as dietary protein sources.

Background Colorectal cancer (CRC) remains a serious health concern worldwide. Despite advances in diagnosis and treatment, about 15 to 30% of stage II CRC patients subjected to tumor resection with curative intent, develop disease relapse. Moreover, the therapeutic strategy adopted after surgery is not consensual for these patients. This supports the imperative need to find new prognostic and predictive biomarkers for stage II CRC. Methods For this purpose, we used a one-hospital series of 227 stage II CRC patient samples to assess the biomarker potential of the immunohistochemical expression of MUC2 mucin and CDX2 and SOX2 transcription factors. The Kaplan-Meier method was used to generate disease-free survival curves that were compared using the log-rank test, in order to determine prognosis of cases with different expression of these proteins, different mismatch repair (MMR) status and administration or not of adjuvant chemotherapy. Results In this stage II CRC series, none of the studied biomarkers showed prognostic value for patient outcome. However low expression of MUC2, in cases with high expression of CDX2, absence of SOX2 or MMR-proficiency, conferred a significantly worst prognosis. Moreover, cases with low expression of MUC2 showed a significantly clear benefit from treatment with adjuvant chemotherapy. Conclusion In conclusion, we observe that patients with stage II CRC with low expression of MUC2 in the tumor respond better when treated with adjuvant chemotherapy. This observation supports that MUC2 is involved in resistance to fluorouracil-based adjuvant chemotherapy and might be a promising future predictive biomarker in stage II CRC patients.

Throughout the intestinal epithelium surface there is an intricate polymer network composed by gel-forming mucins, which plays a protective role due to the formation of a physical, chemical and immunological barrier between the organism and the environment. Mucin 2 (MUC2) is the main mucin in the small and large intestine, and it is expressed specifically in the gastrointestinal tract (GIT), which makes its promoter region an important candidate for expression of heterologous genes of biotechnological interest in the GIT of bovine and other ruminants. In order to characterize the bovine MUC2 promoter we designed primers to amplify and isolate a candidate region for this promoter. The amplified sequence was confirmed by sequencing and cloned into a plasmid vector containing the luciferase (LUC) reporter gene. The regulatory sites of the MUC2 promoter already described in the literature were used to find the putative regulatory sites in the bovine MUC2 promoter region. With these data, some deletions were performed in order to find the promoter sequence with greatest expression capacity and specificity. The constructions were tested by transient transfection assays in LoVo cells (human colorectal adenocarcinoma) and bovine fibroblasts. The quantification of the relative expression of the promoter was measured using dual-luciferase assays. Real-time PCR was performed to analyze the expression of endogenous MUC2. The results presented herein prove that the isolated sequence corresponds to the promoter of bovine MUC2 gene, since it was able to induce expression of a reporter gene in an in vitro cell culture experimental platform.

The potential for ingestion of copper oxide nanomaterials (CuO NMs) is increasing due to their increased exploitation. Investigation of changes in gene expression allows toxicity to be detected at an early stage of NM exposure and can enable investigation of the mechanism of toxicity. Here, undifferentiated Caco-2 cells, differentiated Caco-2 cells, Caco-2/HT29-MTX (mucus secreting) and Caco-2/Raji B (M cell model) co-cultures were exposed to CuO NMs andcopper sulphate(CuSO4) in order to determine their impacts. Cellular responses were measured in terms of production ofreactive oxygen species(ROS), the gene expression of an antioxidant (haem oxygenase 1 (HMOX1)), the pro-inflammatory cytokine (interleukin 8 (IL8)), the metal binding (metallothionein 1A and 2A (MT1AandMT2A)) and the mucus secreting (mucin 2 (MUC2)), as well as HMOX-1 protein level. While CuSO4induced ROS production in cells, no such effect was observed for CuO NMs. However, these particles did induce an increase in the level of HMOX-1 protein and upregulation ofHMOX1,MT2A,IL8andMUC2genes in all cell models. In conclusion, the expression ofHMOX1,IL8and MT2A were responsive to CuO NMs at 4 to 12h post exposure when investigating the toxicity of NMs using intestinalin vitromodels. These findings can inform the selection of endpoints, timepoints and models when investigating NM toxicity to the intestinein vitroin the future.

Approximately 1 billion people worldwide suffer from zinc deficiency, with severe consequences for their well-being, such as critically impaired intestinal health. In addition to an extreme degeneration of the intestinal epithelium, the intestinal mucus is seriously disturbed in zinc-deficient (ZD) animals. The underlying cellular processes as well as the relevance of zinc for the mucin-producing goblet cells, however, remain unknown. To this end, this study examines the impact of zinc deficiency on the synthesis, production, and secretion of intestinal mucins as well as on the zinc homeostasis of goblet cells using the in vitro goblet cell model HT-29-MTX. Zinc deprivation reduced their cellular zinc content, changed expression of the intestinal zinc transporters ZIP-4, ZIP-5, and ZnT1 and increased their zinc absorption ability, outlining the regulatory mechanisms of zinc homeostasis in goblet cells. Synthesis and secretion of mucins were severely disturbed during zinc deficiency, affecting both MUC2 and MUC5AC mRNA expression with ongoing cell differentiation. A lack of zinc perturbed mucin synthesis predominantly on the post-translational level, as ZD cells produced shorter O-glycans and the main O-glycan pattern was shifted in favor of core-3-based mucins. The expression of glycosyltransferases that determine the formation of core 1-4 O-glycans was altered in zinc deficiency. In particular, B3GNT6 mRNA catalyzing core 3 formation was elevated and C2GNT1 and C2GNT3 elongating core 1 were downregulated in ZD cells. These novel insights into the molecular mechanisms impairing intestinal mucus stability during zinc deficiency demonstrate the essentiality of zinc for the formation and maintenance of this physical barrier.

New findings What is the central question of this study? What is the localization and distribution pattern of adenomatous polyposis coli (APC) in intestinal epithelial cells? Does this distribution change in different regions of the colon or in the condition of inflammation? What is the main finding and its importance? Colonic epithelia from mice and humans contain a subset of goblet cells displaying high APC levels. The number of APChigh goblet cells increases in inflamed tissue, which also displays increased GRP78, indicating potential stress from mucin production. In cultured human colon cells, expression of interleukin 1 pathway components (inducers of MUC2 expression) is reduced upon APC depletion raising the potential for APC participation in an inflammatory response. Abstract Adenomatous polyposis coli (APC) serves as a gatekeeper of intestinal homeostasis by promoting cellular differentiation and maintaining crypt architecture. Although appreciated as a critical colon tumour suppressor, roles for APC in disease states such as inflammation have yet to be fully delineated. This study aimed to characterize the localization of APC protein in gastrointestinal tissues from human patients with active inflammatory bowel disease and mice with dextran sodium sulfate (DSS)-induced colitis. Fluorescence immunohistochemistry revealed a subset of goblet cells with elevated Apc staining intensity in the small intestines and proximal/medial colons of mice. Upon induction of colitis with DSS, these 'APChigh ' goblet cells remained in the proximal and medial colon, but now were also observed in the distal colon. This phenotype was recapitulated in humans, with APChigh goblet cells observed only in the descending colons of patients with active ulcerative colitis. In cultured human colon cells derived from normal tissue, APC depletion reduced expression of mRNAs encoding the interleukin 1 (IL1) signalling pathway components IL1β and interleukin-1 receptor (IL1R), known regulators of Muc2 expression. Treating cancer cells lacking wild-type APC with IL1β, or induction of full-length APC in these cells led to increases in IL1R and MUC2 expression. Combining IL1β treatment with APC induction led to an increase of MUC2 expression greater than expected for additive affects, suggesting that APC sensitizes cells to IL1 signalling. These findings suggest that APC has novel roles in maintaining proper goblet cell function, thus providing further evidence for APC as an important factor in intestinal tissue homeostasis and disease.

Publisher's version (útgefin grein), The Colonic Mucosal Barrier (CMB) is the site of interaction between the human body and the colonic microbiota. The mucus is the outer part of the CMB and is considered as the front-line defense of the colon. It separates the host epithelial lining from the colonic content, and it has previously been linked to health and diseases. In this study, we assessed the relationship between red meat and whole-grain intake and (1) the thickness of the colonic mucus (2) the expression of the predominant mucin gene in the human colon (MUC2). Patients referred to colonoscopy at the University Hospital of Southern Denmark-Sonderjylland were enrolled between June 2017 and December 2018, and lifestyle data was collected in a cross-sectional study design. Colonic biopsies, blood, urine, and fecal samples were collected. The colonic mucus and bacteria were visualized by immunostaining and fluorescence in situ hybridization techniques. We found a thinner mucus was associated with high red meat intake. Similarly, the results suggested a thinner mucus was associated with high whole-grain intake, albeit to a lesser extent than red meat. This is the first study assessing the association between red meat and whole-grain intake and the colonic mucus in humans. This study is approved by the Danish Ethics Committee (S-20160124) and the Danish Data Protecting Agency (2008-58-035). A study protocol was registered at clinical trials.gov under NCT04235348., This study is a part of SysCID project, which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 733100, The Region of Southern Denmark”, “The University Hospital of Southern Denmark-Sonderjylland”, “The University of Southern Denmark”, and “Knud og Edith Eriksens mindefond”.

To investigate the effect of electroacupuncture (EA) on irritable bowel syndrome (IBS) in mice through regulating nucleotide-binding oligomerization domain protein-like receptor family pyrin domain containing 6 (NLRP6).Water-avoidance stress (WAS) mice model was used to investigate the effects and the mechanism of EA. Abdominal withdrawal reflex test, open field test, and intestinal motility test were used to evaluate visceral sensitivity, anxiety, and intestinal motility in mice. The expressions of NLRP6, Mucin-2 (MUC2) and E-cadherin were determined using immunofluorescence and Western blotting assays.EA significantly upregulated the expression of NLRP6 in the intestine of mice. Moreover, EA increased the expressions of MUC2 and E-cadherin in WAS mice.Our study found that the relief of IBS symptoms by EA may involve the increase in the expression of NLRP6 in WAS mice.

With the ulcerative colitis (UC) incidence increasing worldwide, it is of great importance to prevent and treat UC. However, efficient treatment options for UC are relatively limited. Due to the potentially serious adverse effects of existing drugs, there is an increasing demand for alternative candidate resources derived from natural and functional foods. Astragalin (AG) is a type of anti-inflammatory flavonoid, with Moringa oleifera and Cassia alata being its main sources. In this study, we investigated the therapeutic effects of AG on mice with dextran sulfate sodium (DSS)-induced colitis. Our results suggested that AG treatment reduced weight loss and the disease activity index (DAI), prevented colon shortening and alleviated colonic tissue damage. AG treatment reduced the expression of pro-inflammatory cytokines and related mRNAs (such as TNF-α, IL-6, and IL-1β), inhibited colonic infiltration by macrophages and neutrophils, ameliorated metabolic endotoxemia, and improved intestinal mucosal barrier function (increased expression levels of mRNAs such as ZO-1, occludin, and Muc2). Western blot analysis revealed that AG downregulated the NF-κB signaling pathway. Moreover, AG treatment partially reversed the alterations in the gut microbiota in colitis mice, mainly by increasing the abundance of potentially beneficial bacteria (such as Ruminococcaceae) and decreasing the abundance of potentially harmful bacteria (such as Escherichia-Shigella). Ruminococcaceae and Enterobacteriaceae (Escherichia-Shigella) were thought to be the key groups affected by AG to improve UC. Therefore, AG might exert a good anti-UC effect through microbiota/LPS/TLR4/NF-kB-related pathways in mice. The results of this study reveal the anti-inflammatory effect and mechanism of AG and provide an important reference for studying the mechanisms of natural flavonoids involved in preventing inflammation-driven diseases.