Upregulated Tß4 expression in inflammatory bowel disease impairs the intestinal mucus barrier by inhibiting autophagy in mice.

Disrupted intestinal barrier homeostasis is fundamental to inflammatory bowel disease. Thymosin ß4 (Tß4) improves inflammation and has beneficial effects in dry-eye diseases, but its effects on the intestinal mucus barrier remain unknown. Therefore, this study evaluated the underlying regulatory mechanisms and effects of Tß4 by examining Tß4 expression in a mouse model with dextran sodium sulfate (DSS)-induced colitis and colonic barrier damage. Additionally, we intraperitoneally injected C57BL/6 mice with Tß4 to assess barrier function, microtubule-associated protein 1 light chain 3 (LC3II) protein expression, and autophagy. Finally, normal human colon tissue and colon carcinoma cells (Caco2) were cultured to verify Tß4-induced barrier function and autophagy changes. Mucin2 levels decreased, microbial infiltration increased, and Tß4 expression increased in the colitis mouse model versus the control mice, indicating mucus barrier damage. Moreover, Tß4-treated C57BL/6 mice had damaged intestinal mucus barriers and decreased LC3II levels. Tß4 also inhibited colonic mucin2 production, disrupted tight junctions, and downregulated autophagy; these results were confirmed in Caco2 cells and normal human colon tissue. In summary, Tß4 may be implicated in colitis by compromising the integrity of the intestinal mucus barrier and inhibiting autophagy. Thus, Tß4 could be a new diagnostic marker for intestinal barrier defects.

Colonic mucin-2 attenuates acute necrotizing pancreatitis in rats by modulating intestinal homeostasis.

Mucin-2 (MUC2) secreted by goblet cells participates in the intestinal barrier, but its mechanism in acute necrotizing pancreatitis (ANP) remains unclear. In acute pancreatitis (AP) patients, the functions of goblet cells (MUC2, FCGBP, CLCA1, and TFF3) decreased, and MUC2 was negatively correlated with AP severity. ANP rats treated with pilocarpine (PILO) (PILO+ANP rats) to deplete MUC2 showed more serious pancreatic and colonic injuries, goblet cell dysfunction, gut dysbiosis, and bacterial translocation than those of ANP rats. GC-MS analysis of feces showed that PILO+ANP rats had lower levels of butyric acid, isobutyric acid, isovaleric acid, and hexanoic acid than those of ANP rats. The expression of MUC2 was associated with colonic injury and gut dysbiosis. All these phenomena could be relieved, and goblet cell functions were also partially reversed by MUC2 supplementation in ANP rats. TNF-α-treated colonoids had exacerbated goblet cell dysfunction. MUC2 expression was negatively correlated with the levels of pro-inflammatory cytokines (IL-1ß and IL-6) (p < .05) and positively related to the expression of tight junction proteins (Claudin 1, Occludin, and ZO1) (p < .05). Downregulating MUC2 by siRNA increased the levels of the pro-inflammatory cytokines in colonoids. MUC2 might maintain intestinal homeostasis to alleviate ANP.

Protective effects of Bifidobacterium bifidum FL-228.1 on dextran sulfate sodium-induced intestinal damage in mice.

PURPOSE: Numerous studies have found that probiotics benefit the intestinal barrier. However, the prophylactic effects of probiotics on the intestinal barrier, i.e., if probiotics exert protective effects in healthy individuals to defend them against harmful elements, have seldomly been reported. The present study aimed to investigate the possible mechanisms of potential strains with the function of preventing intestinal barrier damage. METHODS: This study investigated nine potential probiotic strains using in vitro and in vivo models on their intestinal barrier-protecting properties. Transcriptomic was then employed to decipher the underlying mechanisms of action of the strains. RESULTS: The results showed that the strains, to varying degrees, regulated the ratio of interleukin (IL)-10 and IL-12 in peripheral blood mononuclear cells (PBMCs), increased the transepithelial electrical resistance (TEER) values, and decreased Caco-2 cell monolayers permeability. Correspondingly, the strains showed different prophylactic efficacies in protecting mice from dextran sulfate sodium (DSS)-induced intestinal barrier damage. Remarkably, Bifidobacterium bifidum FL-228.1 (FL-228.1) showed the best prophylactic efficacies in protecting mice from DSS-induced intestinal barrier damage. Further research suggested that FL-228.1 exerted its prophylactic effects by enhancing mucin 2 (Muc2) production and Claudin (Cldn)-4 in the colon. Furthermore, the transcriptomic and protein-protein interactions (PPI) analyses indicated that the inhibition of NLRP3 and the activation of PPARγ and TLR2 could be involved in protecting the intestinal barrier by FL-228.1. CONCLUSION: Bifidobacterium bifidum FL-228.1 may be developed as a promising probiotic for the prevention of intestinal barrier damage via PPARγ/NLRP3/ TLR2 pathways by enhancing Muc2 and Cldn-4.

An Increase in Mucin2 Expression Is Required for Colon Cancer Progression Mediated by L1.

An induction in the expression of the cell adhesion receptor L1, a Wnt target gene, is a characteristic feature of Wnt/ß-catenin activation in colon cancer cells at later stages of the disease. We investigated the proteins secreted following L1 expression in colon cancer cells and identified Mucin2 among the most abundant secreted proteins. We found that suppressing Mucin2 expression in L1-expressing colon cancer cells inhibits cell proliferation, motility, tumorigenesis, and liver metastasis. We detected several signaling pathways involved in Mucin2 induction in L1-expressing cells. In human colon cancer tissue, Mucin2 expression was significantly reduced or lost in the adenocarcinoma tissue, while in the mucinous subtype of colon cancer tissue, Mucin2 expression was increased. An increased signature of L1/Mucin2 expression reduced the survival rate of human colon cancer patients. Thus, induction of Mucin2 expression by L1 is required during mucinous colon cancer progression and can serve as a marker for diagnosis and a target for therapy.

Oxidized phospholipids cause changes in jejunum mucus that induce dysbiosis and systemic inflammation.

We previously reported that adding a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to a Western diet (WD) ameliorated systemic inflammation. To determine the mechanism(s) responsible for these observations, Ldlr-/- mice were fed chow, a WD, or WD plus Tg6F. We found that a WD altered the taxonomic composition of bacteria in jejunum mucus. For example, Akkermansia muciniphila virtually disappeared, while overall bacteria numbers and lipopolysaccharide (LPS) levels increased. In addition, gut permeability increased, as did the content of reactive oxygen species and oxidized phospholipids in jejunum mucus in WD-fed mice. Moreover, gene expression in the jejunum decreased for multiple peptides and proteins that are secreted into the mucous layer of the jejunum that act to limit bacteria numbers and their interaction with enterocytes including regenerating islet-derived proteins, defensins, mucin 2, surfactant A, and apoA-I. Following WD, gene expression also decreased for Il36γ, Il23, and Il22, cytokines critical for antimicrobial activity. WD decreased expression of both Atoh1 and Gfi1, genes required for the formation of goblet and Paneth cells, and immunohistochemistry revealed decreased numbers of goblet and Paneth cells. Adding Tg6F ameliorated these WD-mediated changes. Adding oxidized phospholipids ex vivo to the jejunum from mice fed a chow diet reproduced the changes in gene expression in vivo that occurred when the mice were fed WD and were prevented with addition of 6F peptide. We conclude that Tg6F ameliorates the WD-mediated increase in oxidized phospholipids that cause changes in jejunum mucus, which induce dysbiosis and systemic inflammation.

Role of Mucin 2 Glycoprotein and L-Fucose in Interaction of Immunity and Microbiome within the Experimental Model of Inflammatory Bowel Disease.

Many factors underlie the development of inflammatory bowel disease (IBD) in humans. In particular, imbalance of microbiota and thinning of the mucosal layer in the large intestine play a huge role. Pathogenic microorganisms also exacerbate the course of diseases. In this research the role of mucin 2 deficiency in the formation of intestinal microflora in the experimental model using the Muc2 gene knockout mice in the presence of Helicobacter spp. was investigated. Also, restorative and anti-inflammatory effect of the dietary L-fucose in the Muc2-/- mice on microflora and immunity was evaluated. For this purpose, bacterial diversity in feces was studied in the animals before and after antibiotic therapy and role of the dietary L-fucose in their recovery was assessed. To determine the effect of bacterial imbalance and fucose on the immune system, mRNA levels of the genes encoding pro-inflammatory cytokines (Tnf, Il1a, Il1b, Il6) and transcription factors of T cells (Foxp3 - Treg, Rorc - Th17, Tbx21 - Th1) were determined in the colon tissue of the Muc2-/- mice. Significant elimination of bacteria due to antibiotic therapy caused decrease of the fucose levels in the intestine and facilitated reduction of the regulatory T cell transcription factor (Foxp3). When the dietary L-fucose was added to antibiotics, the level of bacterial DNA of Bacteroides spp. in the feces of the Muc2-/- mice was partially restored. T regulatory cells are involved in the regulation of inflammation in the Muc2-/- mice. Antibiotics reduced the number of regulatory T cell but did not decrease the inflammatory response to infection. Fucose, as a component of mucin 2, helped to maintain the level of Bacteroides spp. during antibiotic therapy of the Muc2-/- mice and restored biochemical parameters, but did not affect the inflammatory response.

Fenretinide Reduces Intestinal Mucin-2-Positive Goblet Cells in Chronic Alcohol Abuse.

INTRODUCTION: Alcohol-induced thickening of the gut mucosal layer and increased expression of goblet cell gel-forming mucins, such as mucin-2 (MUC2) are associated with disruptions to the gut barrier in alcoholic liver disease (ALD). Interest in drugs that can target gut mucins in ALD has grown; however to date, no studies have examined the properties of drugs on expression of gut mucins in models of ALD. We previously demonstrated that at 10 mg/kg/day, the drug fenretinide (N-[4-hydroxyphenyl] retinamide [Fen]), a synthetic retinoid, mitigates alcohol-associated damage to the gut barrier and liver injury in a murine model of ALD. METHODS: In this study, we specifically sought to examine the effects of Fen on gut goblet cells, and expression of mucins, including MUC2 using a 25-day Lieber-DeCarli model of chronic alcohol intake. RESULTS: Our results show that chronic alcohol intake increased gut-mucosal thickening, goblet cell numbers, and mRNA and protein expression of MUC2 in both the ileum and colon. Alcohol intake was associated with marked decreases in ileal and colonic Notch signaling, levels of Notch ligands Dll1 and Dll4, and increases in the expression of Notch-associated genes indispensable for goblet cell specification, including Math1 and Spdef. Interestingly, ileal and colonic expression of KLF4, which is involved in terminal differentiation of goblet cells, was reduced in mice chronically fed alcohol. Coadministration of alcohol with Fen at 10 mg/kg/day significantly reduced alcohol-associated increases in ileal and colonic mucosal thickening, ileal Muc2, colonic Muc2, Muc5ac and Muc6 mRNAs, and goblet cell numbers. We also found that Fen strongly prevented alcohol-mediated suppression of the Notch ligand Dll1, Notch signaling, and alcohol-induced increases in expression of Notch-associated goblet cell specification genes in both the ileum and colon. In the absence of alcohol, Fen treatments alone at 10 mg/kg/day had no effects on any of the goblet cell-related endpoints. CONCLUSION: These data show for the first time that the drug Fen possesses mucosal layer-modulating properties in response to chronic alcohol abuse. These data warrant further preclinical examination of Fen given the need for anti-ALD drugs and emerging evidence of a role for intestinal goblet cell mucins in the progression of ALD.

Mechanism of endometrial MUC2 in reproductive performance in mice through PI3K/AKT signaling pathway after lipopolysaccharide treatment.

The objective of this study was to investigate the effects of exposure to endotoxin on the reproductive performance of humans and animals in pregnancy and delivery period. Mucin is considered to play a critical role in protecting the tissue epithelium. At pregnancy period, the MUC2 expression of uterus in the High LPS group was significantly higher than that in the Control group. The glycosaminoglycans of gland cells were secreted into the uterine cavity to protect the uterus. Then, the MUC2 layer became thinner, and LPS entered the lamina propria of the uterus. The mRNA expression of tight junction proteins showed a marked drop, and morphological damage of the uterus occurred. Subsequently, the glycosaminoglycans of gland cells in the High LPS and Low LPS groups increased with the increasing LPS dose, and the damage to the endometrial epithelium was repaired in female mice at Day 5 postdelivery. A low dose of LPS activated the PI3K/AKT signaling pathways to increase the glycosaminoglycans particles, while a high dose of LPS inhibited the PI3K/AKT signaling pathway to decrease the glycosaminoglycans particles. Taken together, our results suggest that gland cells secreted glycosaminoglycans particles into the uterine cavity by exocytosis to increase the thickness of the mucus layer to protect the uterus and that this process was regulated by PI3K/AKT signaling pathways.

Emerging Themes in the Molecular Pathogenesis of Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) are ubiquitous diarrheal pathogens that thrive in areas lacking basic human needs of clean water and sanitation. These genetically plastic organisms cause tremendous morbidity among disadvantaged young children, in the form of both acute diarrheal illness and sequelae of malnutrition and growth impairment. The recent discovery of additional plasmid-encoded virulence factors and elucidation of their critical role in the molecular pathogenesis of ETEC may inform new approaches to the development of broadly protective vaccines. Although the pathogens have been closely linked epidemiologically with nondiarrheal sequelae, these conditions remain very poorly understood. Similarly, while canonical effects of ETEC toxins on cellular signaling promoting diarrhea are clear, emerging data suggest that these toxins may also drive changes in intestinal architecture and associated sequelae. Elucidation of molecular events underlying these changes could inform optimal approaches to vaccines that prevent acute diarrhea and ETEC-associated sequelae.

Deletion of mucin 2 induces colitis with concomitant metabolic abnormalities in mice.

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-/-) was used to investigate the impact of intestinal disease on metabolic dysfunction. Before the onset of severe colitis, such as rectal prolapse, Muc2-/- mice exhibited impaired glucose clearance. Defects were noted in the insulin signaling pathway corresponding with upregulated genes in lipid utilization pathways, increased mitochondrial number, and peroxisome proliferator-activated coactivator 1α (PGC-1α), a transcription factor central to energy metabolism regulation. Parallel to these metabolic alterations, Muc2-/- mice exhibited systemic inflammation and bacteremia. We further characterized the dysbiotic microbiome's predicted functional categories given its contributing role to the colitic phenotype in the Muc2-/- mice. In addition to less butyrate levels, we show an increased predisposition to lipid metabolism and lipid biosynthesis pathways in the microbiome associated with the host's altered metabolic state. This study establishes the Muc2-/- mouse model that develops spontaneous colitis, as an ideal model for studying early comorbid metabolic dysfunction. Clarification of the underlying etiology of two phenotypes in this model could unravel important clues regarding the treatment of metabolic comorbidities during colitis.NEW & NOTEWORTHY This study discloses the impaired systemic energy metabolism in a classic colitis murine model (Muc2-/- knock-out model). Investigating the interaction between colitis and metabolic disorders helps to extend our knowledge on deciphering inflammatory bowel disease-associated comorbidities and provides new insight into clinical treatment.

Mechanical stimulation activates Piezo1 to promote mucin2 expression in goblet cells.

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.

Chronic stress promotes colitis by disturbing the gut microbiota and triggering immune system response.

Chronic stress is known to promote inflammatory bowel disease (IBD), but the underlying mechanism remains largely unresolved. Here, we found chronic stress to sensitize mice to dextran sulfate sodium (DSS)-induced colitis; to increase the infiltration of B cells, neutrophils, and proinflammatory ly6Chi macrophages in colonic lamina propria; and to present with decreased thymus and mesenteric lymph node (MLN) coefficients. Circulating total white blood cells were significantly increased after stress, and the proportion of MLN-associated immune cells were largely changed. Results showed a marked activation of IL-6/STAT3 signaling by stress. The detrimental action of stress was not terminated in IL-6-/- mice. Interestingly, the composition of gut microbiota was dramatically changed after stress, with expansion of inflammation-promoting bacteria. Furthermore, results showed stress-induced deficient expression of mucin-2 and lysozyme, which may contribute to the disorder of gut microbiota. Of note is that, in the case of cohousing, the stress-induced immune reaction and decreased body weight were abrogated, and transferred gut microbiota from stressed mice to control mice was sufficient to facilitate DSS-induced colitis. The important role of gut microbiota was further reinforced by broad-spectrum antibiotic treatment. Taken together, our results reveal that chronic stress disturbs gut microbiota, triggering immune system response and facilitating DSS-induced colitis.

Fucose Ameliorates Tritrichomonas sp.-Associated Illness in Antibiotic-Treated Muc2-/- Mice.

The mucus layer in the intestine plays a critical role in regulation of host-microbe interactions and maintaining homeostasis. Disruptions of the mucus layer due to genetic, environmental, or immune factors may lead to inflammatory bowel diseases (IBD). IBD frequently are accompanied with infections, and therefore are treated with antibiotics. Hence, it is important to evaluate risks of antibiotic treatment in individuals with vulnerable gut barrier and chronic inflammation. Mice with a knockout of the Muc2 gene, encoding the main glycoprotein component of the mucus, demonstrate a close contact of the microbes with the gut epithelium which leads to chronic inflammation resembling IBD. Here we demonstrate that the Muc2-/- mice harboring a gut protozoan infection Tritrichomonas sp. are susceptible to an antibiotic-induced depletion of the bacterial microbiota. Suppression of the protozoan infection with efficient metronidazole dosage or L-fucose administration resulted in amelioration of an illness observed in antibiotic-treated Muc2-/- mice. Fucose is a monosaccharide presented abundantly in gut glycoproteins, including Mucin2, and is known to be involved in host-microbe interactions, in particular in microbe adhesion. We suppose that further investigation of the role of fucose in protozoan adhesion to host cells may be of great value.

Calcium-activated chloride channel regulator 1 (CLCA1) forms non-covalent oligomers in colonic mucus and has mucin 2-processing properties.

Calcium-activated chloride channel regulator 1 (CLCA1) is one of the major nonmucin proteins found in intestinal mucus. It is part of a larger family of CLCA proteins that share highly conserved features and domain architectures. The CLCA domain arrangement is similar to proteins belonging to the ADAM (a disintegrin and metalloproteinase) family, known to process extracellular matrix proteins. Therefore, CLCA1 is an interesting candidate in the search for proteases that process intestinal mucus. Here, we investigated CLCA1's biochemical properties both in vitro and in mucus from mouse and human colon biopsy samples. Using immunoblotting with CLCA1-specific antibodies and recombinant proteins, we observed that the CLCA1 C-terminal self-cleavage product forms a disulfide-linked dimer that noncovalently interacts with the N-terminal part of CLCA1, which further interacts to form oligomers. We also characterized a second, more catalytically active, N-terminal product of CLCA1, encompassing the catalytic domain together with its von Willebrand domain type A (VWA). This fragment was unstable but could be identified in freshly prepared mucus. Furthermore, we found that CLCA1 can cleave the N-terminal part of the mucus structural component MUC2. We propose that CLCA1 regulates the structural arrangement of the mucus and thereby takes part in the regulation of mucus processing.

Human intestinal enteroids as a model of Clostridioides difficile-induced enteritis.

Clostridioides difficile is an important nosocomial pathogen that produces toxins to cause life-threatening diarrhea and colitis. Toxins bind to epithelial receptors and promote the collapse of the actin cytoskeleton. C. difficile toxin activity is commonly studied in cancer-derived and immortalized cell lines. However, the biological relevance of these models is limited. Moreover, no model is available for examining C. difficile-induced enteritis, an understudied health problem. We hypothesized that human intestinal enteroids (HIEs) express toxin receptors and provide a new model to dissect C. difficile cytotoxicity in the small intestine. We generated biopsy-derived jejunal HIE and Vero cells, which stably express LifeAct-Ruby, a fluorescent label of F-actin, to monitor actin cytoskeleton rearrangement by live-cell microscopy. Imaging analysis revealed that toxins from pathogenic C. difficile strains elicited cell rounding in a strain-dependent manner, and HIEs were tenfold more sensitive to toxin A (TcdA) than toxin B (TcdB). By quantitative PCR, we paradoxically found that HIEs expressed greater quantities of toxin receptor mRNA and yet exhibited decreased sensitivity to toxins when compared with traditionally used cell lines. We reasoned that these differences may be explained by components, such as mucins, that are present in HIEs cultures, that are absent in immortalized cell lines. Addition of human-derived mucin 2 (MUC2) to Vero cells delayed cell rounding, indicating that mucus serves as a barrier to toxin-receptor binding. This work highlights that investigation of C. difficile infection in that HIEs can provide important insights into the intricate interactions between toxins and the human intestinal epithelium.NEW & NOTEWORTHY In this article, we developed a novel model of Clostridioides difficile-induced enteritis using jejunal-derived human intestinal enteroids (HIEs) transduced with fluorescently tagged F-actin. Using live-imaging, we identified that jejunal HIEs express high levels of TcdA and CDT receptors, are more sensitive to TcdA than TcdB, and secrete mucus, which delays toxin-epithelial interactions. This work also optimizes optically clear C. difficile-conditioned media suitable for live-cell imaging.

Acute high-dose titanium dioxide nanoparticle exposure alters gastrointestinal homeostasis in mice.

Human exposure to a wide variety of engineered nanoparticles (NPs) is on the rise and use in common food additives increases gastrointestinal (GI) exposure. Host health is intricately linked to the GI microbiome and immune response. Perturbations in the microbiota can affect energy harvest, trigger inflammation and alter the mucosal barrier leading to various disease states such as obesity and inflammatory bowel diseases. We hypothesized that single high-dose titanium dioxide (TiO2 ) NP exposure in mice would lead to dysbiosis and stimulate mucus production and local immune populations. Juvenile mice (9-10 weeks) were gavaged with 1 g/kg TiO2 NPs and examined for changes in mucosa-associated bacteria abundance, inflammatory cytokines, mucin expression and body mass. Our data provide support that TiO2 NP ingestion alters the GI microbiota and host defenses promoting metabolic disruption and subsequently weight gain in mice.

Mucin 2 (MUC2) modulates the aggressiveness of breast cancer.

PURPOSE: Tumors that secrete large volumes of mucus are chemotherapy resistant, however, mechanisms underlying this resistance are unknown. One protein highly expressed in mucin secreting breast cancers is the secreted mucin, Mucin 2 (MUC2). While MUC2 is expressed in some breast cancers it is absent in normal breast tissue, implicating it in breast cancer. However, the effects of MUC2 on breast cancer are largely unknown. This study examined the role of MUC2 in modulating breast cancer proliferation, response to chemotherapy and metastasis. METHODS: Using patient derived xenografts we developed two novel cell lines, called BCK4 and PT12, which express high levels of MUC2. To modulate MUC2 levels, BCK4 and PT12 cells were engineered to express shRNA targeted to MUC2 (shMUC2, low MUC2) or a non-targeting control (shCONT, high MUC2) and proliferation and apoptosis were measured in vitro and in vivo. BCK4 cells with shCONT or shMUC2 were labeled with GFP-luciferase and examined in an experimental metastasis model; disease burden and site specific dissemination were monitored by intravital imaging and fluorescence guided dissection, respectively. RESULTS: Proliferation decreased in BCK4 and PT12 shMUC2 cells versus control cells both in vitro and in vivo. Chemotherapy induced minimal apoptosis in control cells expressing high MUC2 but increased apoptosis in shMUC2 cells containing low MUC2. An experimental metastasis model showed disease burden decreased when breast cancer cells contained low versus high MUC2. Treatment with Epidermal Growth Factor (EGF) increased MUC2 expression in BCK4 cells; this induction was abolished by the EGF-receptor inhibitor, Erlotinib. CONCLUSIONS: MUC2 plays an important role in mediating proliferation, apoptosis and metastasis of breast cancer cells. MUC2 may be important in guiding treatment and predicting outcomes in breast cancer patients.

Consumption of non-digestible oligosaccharides elevates colonic alkaline phosphatase activity by up-regulating the expression of IAP-I, with increased mucins and microbial fermentation in rats fed a high-fat diet.

We have recently reported that soluble dietary fibre, glucomannan, increased colonic alkaline phosphatase (ALP) activity and the gene expression without affecting the small-intestinal activity and that colonic ALP was correlated with gut mucins (index of intestinal barrier function). We speculated that dietary fermentable carbohydrates including oligosaccharides commonly elevate colonic ALP and gene expression as well as increase mucin secretion and microbial fermentation. To test this hypothesis, male Sprague-Dawley rats were fed a diet containing 30 % lard with or without 4 % fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), raffinose (RAF) and lactulose (LAC), which are non-digestible oligosaccharides or isomalto-oligosaccharides (IMOS; some digestible oligosaccharides) for 2 weeks. Colon ALP activity, the gene expression and gut luminal variables including mucins, organic acids and microbiota were measured. Colonic ALP was significantly elevated in the FOS, RAF and LAC groups, and a similar trend was observed in the GOS group. Colonic expression of intestinal alkaline phosphatase (IAP -I), an ALP gene, was significantly elevated in the FOS, GOS and RAF groups and tended to be increased in the LAC group. Dietary FOS, GOS, RAF and LAC significantly elevated faecal mucins, caecal n-butyrate and faecal ratio of Bifidobacterium spp. Dietary IMOS had no effect on colonic ALP, mucins, organic acids and microbiota. Colon ALP was correlated with mucins, caecal n-butyrate and faecal Bifidobacterium spp. This study demonstrated that non-digestible and fermentable oligosaccharides commonly elevate colonic ALP activity and the expression of IAP-I, with increasing mucins and microbial fermentation, which might be important for protection of gut epithelial homoeostasis.

EXPRESSION OF MUC2 GLYCOPROTEIN ANTIBODY AND VASCULAR ENDOTHELIAL GROWTH FACTOR IN BARRETT'S MUCOSA.

Higher expression of the mucin 2 (MUC2) glycoprotein and vascular endothelial growth factor (VEGF) in Barrett's mucosa may be associated with a higher risk of esophageal adenocarcinoma development. Thirty-six patients diagnosed with Barrett's esophagus (BE), short-segment, were included in the study due to unsuccessful treatment with proton pump inhibitors. The diagnosis was confirmed by histopathologic analysis of the tissue obtained by esophagogastric junction biopsy. Expression of MUC2 and VEGF was determined by immunohistochemistry. We found four patients in early stage of adenocarcinoma and 32 patients with BE; five of them had indication for argon plasma coagulation treatment, one for radiofrequency ablation and one for endoscopic mucosal resection; 25 patients were treated with proton pump inhibitors. Regression of BE occurred in 25 (69.44%) patients. MUC2 positivity is unique for goblet cells in patients with BE, but it is not the only marker. VEGF is an indicator of angiogenesis in the mucosa of patients with BE and adenocarcinoma.

The Muc2 mucin coats murine Paneth cell granules and facilitates their content release and dispersion.

Paneth cells are a key subset of secretory epithelial cells found at the base of small intestinal crypts. Unlike intestinal goblet cells, which secrete the mucin Muc2, Paneth cells are best known for producing an array of antimicrobial factors. We unexpectedly identified Muc2 staining localized around Paneth cell granules. Electron microscopy (EM) confirmed an electron lucent halo around these granules, which was lost in Paneth cells from Muc2-deficient (-/-) mice. EM and immunostaining for lysozyme revealed that Muc2-/- Paneth cells contained larger, more densely packed granules within their cytoplasm, and we detected defects in the transcription of key antimicrobial genes in the ileal tissues of Muc2-/- mice. Enteroids derived from the small intestine of wild-type and Muc2-/- mice revealed phenotypic differences in Paneth cells similar to those seen in vivo. Moreover, lysozyme-containing granule release from Muc2-/- enteroid Paneth cells was shown to be impaired. Surprisingly, Paneth cells within human ileal and duodenal tissues were found to be Muc2 negative. Thus Muc2 plays an important role in murine Paneth cells, suggesting links in function with goblet cells; however human Paneth cells lack Muc2, highlighting that caution should be applied when linking murine to human Paneth cell functions. NEW & NOTEWORTHY We demonstrate for the first time that murine Paneth cell granules possess a halo comprised of the mucin Muc2. The presence of Muc2 exerts an impact on Paneth cell granule size and number and facilitates the release and dispersal of antimicrobials into the mucus layer. Interestingly, despite the importance of Muc2 in murine Paneth cell function, our analysis of Muc2 in human intestinal tissues revealed no trace of Muc2 expression by human Paneth cells.