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Diabetes leads to intestinal barrier dysfunction. 5-Hydroxytryptamine 4 receptor (5-HT

Endocrine mucin-producing sweat gland carcinoma is a low-grade eyelid tumor. Small biopsies and insensitive immunohistochemistry predispose to misdiagnosis. We aimed to identify clarifying immunohistochemical markers, molecular markers, or both. Clinicopathologic data (22 cases) were reviewed. Immunohistochemistry (insulinoma-associated protein 1, BCL-2, mucin 2 [MUC2], mucin 4, androgen receptor, β-catenin, and Merkel cell polyomavirus) and next-generation sequencing (Memorial Sloan Kettering integrated mutation profiling of actionable cancer targets, 468 genes) were performed (3 cases). Female patients (n = 15) and male patients (n = 7) (mean age 71.8 years; range 53–88 years) had eyelid or periorbital tumors (>90%) with mucin-containing solid or cystic neuroendocrine pathology. Immunohistochemistry (insulinoma-associated protein 1, BCL2, androgen receptor, retinoblastoma-associated protein 1, and β-catenin) was diffusely positive (5/5), MUC2 partial, mucin 4 focal, and Merkel cell polyomavirus negative. Memorial Sloan Kettering integrated mutation profiling of actionable cancer targets identified 12 single-nucleotide variants and 1 in-frame deletion in 3 cases, each with DNA damage response or repair (BRD4, PPP4R2, and RTEL1) and tumor-suppressor pathway (BRD4, TP53, TSC1, and LATS2) mutations. Microsatellite instability, copy number alterations, and structural alterations were absent. Insulinoma-associated protein 1 and MUC2 are positive in endocrine mucin-producing sweat gland carcinoma. MUC2 positivity suggests conjunctival origin. Multistep pathogenesis involving DNA damage repair and tumor-suppressor pathways may be implicated.

The backbone of all colorectal cancer classifications including the consensus molecular subtypes (CMS) highlights microsatellite instability (MSI) as a key molecular pathway. Although mucinous histology (generally defined as >50% extracellular mucin-to-tumor area) is a “typical” feature of MSI, it is not limited to this subgroup. Here, we investigate the association of CMS classification and mucin-to-tumor area quantified using a deep learning algorithm, and the expression of specific mucins in predicting CMS groups and clinical outcome. A weakly supervised segmentation method was developed to quantify extracellular mucin-to-tumor area in H&E images. Performance was compared to two pathologists’ scores, then applied to two cohorts: (1) TCGA (n = 871 slides/412 patients) used for mucin-CMS group correlation and (2) Bern (n = 775 slides/517 patients) for histopathological correlations and next-generation Tissue Microarray construction. TCGA and CPTAC (n = 85 patients) were used to further validate mucin detection and CMS classification by gene and protein expression analysis for MUC2, MUC4, MUC5AC and MUC5B. An excellent inter-observer agreement between pathologists’ scores and the algorithm was obtained (ICC = 0.92). In TCGA, mucinous tumors were predominantly CMS1 (25.7%), CMS3 (24.6%) and CMS4 (16.2%). Average mucin in CMS2 was 1.8%, indicating negligible amounts. RNA and protein expression of MUC2, MUC4, MUC5AC and MUC5B were low-to-absent in CMS2. MUC5AC protein expression correlated with aggressive tumor features (e.g., distant metastases (p = 0.0334), BRAF mutation (p

Background LncRNA-DANCR is involved in inflammation and acts as a major contributor to colon cancer. The effects and mechanism of LncRNA-DANCR were first investigated in a DSS-induced colitis model in vivo and vitro. Material and methods Sprague-Dawley rats were given DSS to induce the colitis model. TNF-α, IL-1β, IL-6 levels and expression of intestinal adhesion proteins ZO-1 and MUC2 in colon tissues and DSS-induced NCM460 cells were measured using corresponding kits. A hematoxylin and eosin (H&E) staining assay was performed to evaluate colon tissue pathology conditions. Protein expression levels in DSS-induced NCM460 cells were evaluated by Western blotting, and cell apoptosis was detected using a TUNEL assay. Gene levels in DSS-induced NCM460 cells were evaluated by PCR. The StarBase online tool was used to predict the LncRNA-DANCR target. The LncRNA-DANCR target was verified using a luciferase reporter assay. Results LncRNA-DANCR was up-regulated in DSS-induced groups of rats. TNF-α, IL-1β and IL-6 expression was significantly increased in DSS-induced groups of rats and cells. Zo-1 and MUC2 expression levels were decreased in DSS-induced groups of rats. Silencing LncRNA-DANCR reduced inflammation, cell apoptosis and up-regulated ZO-1, MUC2 and Claudin-1 in DSS-induced cells. MiR-125b-5p was the downstream LncRNA-DANCR target. All LncRNA-DANCR effects in the colitis model were reversed by the miR-125b-5p inhibitor. Conclusion LncRNA-DANCR/miR-125b-5p, which may act as a regulatory axis in inflammation, apoptosis and barrier function dysregulation, can provide an essential reference for the development of new drugs in colitis treatment.

Background & Aims Alterations in intestinal MUC2 mucin and microbial diversity are closely linked with important intestinal pathologies; however, their impact on each other and on intestinal pathogenesis has been vaguely characterized. Therefore, it was of interest in this study to delineate distinct and cooperative function of commensal microbiota and the Muc2 mucus barrier in maintaining intestinal epithelial barrier function. Methods Muc2 mucin deficient (Muc2–/–) and sufficient (Muc2+/+) littermates were used as a model for assessing the role of Muc2. To quantify the role of the microbiota in disease pathogenesis, Muc2+/+ and Muc2–/– littermates were treated with a cocktail of antibiotics that reduced indigenous bacteria, and then fecal transplanted with littermate stool and susceptibility to dextran sulphate sodium (DSS) quantified. Results Although, Muc2+/+ and Muc2–/– littermates share similar phyla distribution as evidenced by 16S sequencing they maintain their distinctive gastrointestinal phenotypes. Basally, Muc2–/– showed low-grade colonic inflammation with high populations of inflammatory and tolerogenic immune cells that became comparable to Muc2+/+ littermates following antibiotic treatment. Antibiotics treatment rendered Muc2+/+ but not Muc2–/– littermates highly susceptibility to DSS-induced colitis that was ILC3 dependent. Muc2–/– microbiota was colitogenic to Muc2+/+ as it worsened DSS-induced colitis. Microbiota dependent inflammation was confirmed by bone-marrow chimera studies, as Muc2–/– receiving Muc2+/+ bone marrow showed no difference in their susceptibility toward DSS induced colitis. Muc2–/– microbiota exhibited presence of characteristic OTUs of specific bacterial populations that were transferrable to Muc2+/+ littermates. Conclusions These results highlight a distinct role for Muc2 mucin in maintenance of healthy microbiota critical in shaping innate host defenses to promote intestinal homeostasis., Graphical abstract

It is conceivable that pathological conditions can cause intestinal barrier disruption and innate immune dysfunction. However, very limited information has been reported on the effect of seasonal variance on intestinal barriers and innate immunity. The present study was designed to investigate the seasonal variance in intestinal epithelial barriers and the associated innate immune response of turtle intestines during hibernation and nonhibernation periods. Goblet cells (GCs) demonstrated dynamic actions of the mucosal barrier with strong Muc2 protein expression during hibernation. However, weak Muc2 expression during nonhibernation was confirmed by immunohistochemistry, immunofluorescence and immunoblotting. Furthermore, light and transmission electron microscopy revealed that the hypertrophy of GCs resulted in the hypersecretion of mucus granules (MGs) and created a well-developed mucosal layer during hibernation. The absorptive cells (ACs), forming a physical barrier of tight junctions, and desmosomes were firmly anchored during hibernation. Conversely, during nonhibernation, the integrity of tight junctions, adherence junctions and desmosomes was noticeable expanded, causing increased paracellular permeability. As further confirmation, there was strong zonula occluden-1 (ZO-1) and connexins 43 (Cx43) protein expression during hibernation and weak ZO-1 and Cx43 expression during nonhibernation. Moreover, the expression level of the innate immune response proteins Toll-like receptors 2 and 4 (TLR2 and 4) were enhanced during hibernation and were reduced during nonhibernation. These results provide rich information about the seasonal fluctuations that interrupt intestinal epithelial barriers and innate immune response, which might be essential for protection and intestinal homeostasis.

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.

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.

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.

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.

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.

Purpose: Early application of protease inhibitors through the intestinal lumen could increase survival following experimental shock by blocking the pancreatic digestive enzymes. Hence, it was hypothesized that two-route injection (intraintestinal + intravenous) of ulinastatin (UTI), a broad-spectrum protease inhibitor, could better alleviate intestinal injury than single-route injection (either intravenous or intraintestinal). Methods: A sepsis model induced by lipopolysaccharide on rats was established. The rats were randomly divided into five groups: sham, sepsis, UTI intravenous injection (Uiv), UTI intraintestinal injection (Uii), and UTI intraintestinal + intravenous injection (Uii + Uiv) groups. The mucosal barrier function, enzyme-blocking effect, levels of systemic inflammatory cytokines, and 5-day survival rate were compared among groups. The small intestinal villus height (VH), crypt depth (CD), and two components of mucosal barrier (E-cadherin and mucin-2) were measured to evaluate the mucosal barrier function. The levels of trypsin and neutrophil elastase (NE) in the intestine, serum, and vital organs were measured to determine the enzyme-blocking effect. Results: Compared with the single-route injection group (Uiv or Uii), the two-route injection (Uii + Uiv) group displayed: (1) significantly higher levels of VH, VH/CD, E-cadherin, and mucin-2; (2) decreased trypsin and NE levels in intestine, plasma, and vital organs; (3) reduced systemic inflammatory cytokine levels; and (4) improved survival of septic rats. Conclusion: Two-route UTI injection was superior to single-route injection in terms of alleviating intestinal injury, which might be explained by extensive blockade of proteases through different ways. Keywords: Sepsis, Ulinastatin, Intestines, Trypsin, Elastase

Intestinal epithelial cell wound healing involves cell migration, proliferation, and differentiation. Although numerous studies have analyzed the migration of absorptive epithelial cells during wound healing, it remains unclear how goblet cells restitute and how MUC2 mucin production affects this process. In this study, we examined the role of high MUC2 production in goblet cell migration during wound healing and demonstrated that during high MUC2 output, goblet cells migrated slower because of impaired production of wound healing factors and endoplasmic reticulum (ER) stress. Two goblet cell lines, HT29-H and HT29-L, that produced high and low MUC2 mucin, respectively, were used. HT29-L healed wounds faster than HT29-H cells by producing significantly higher amounts of fibroblast growth factor (FGF) 1, FGF2, vascular endothelial growth factor-C, and matrix metallopeptidase 1. Predictably, treatment of HT29-H cells with recombinant FGF2 significantly enhanced migration and wound healing. High MUC2 biosynthesis in HT29-H cells induced ER stress and delayed migration that was abrogated by inhibiting ER stress with tauroursodeoxycholic acid and IL-22. FGF2- and IL-22–induced wound repair was dependent on STAT1 and STAT3 signaling. During wound healing after dextran sulfate sodium–induced colitis, restitution of Math1M1GFP+ goblet cells occurred earlier in the proximal colon, followed by the middle and then distal colon, where ulceration was severe. We conclude that high MUC2 output during colitis impairs goblet cell migration and wound healing by reducing production of growth factors critical in wound repair.

Purpose Barrett's esophagus is one of the main risk factors for increased incidence of esophageal adenocarcinoma. In this study, we studied protein expression levels and cellular localizations of MUC-1, MUC-2, MUC-5AC, CK7, and cytoplasmic p27 to assess the relationship between the expression of each of these proteins and the disease progression on endoscopic biopsies. Materials and methods Immunohistochemical analyses were performed using antibodies produced against MUC-1, MUC-2, MUC-5AC, CK7, and p27. Endoscopic specimens of esophageal mucosa were obtained from 72 patients who underwent esophagectomy for Barrett’s esophagus, metaplasia, dysplasia, or esophageal adenocarcinoma developed from Barrett’s esophagus. Results Multilayer squamous epithelium showed only MUC-1 positivity in the EAC group while MUC-2 and MUC-5AC staining could not be detected in this group. Strong and diffused membranous or cytoplasmic staining of CK7 was observed at squamous, ductal, surface columnar and/or glandular epithelium. c-p27 staining was diffused and moderate in the cellular membranes observed in all groups except for esophageal epithelial metaplasia without intestinal metaplasia. Additionally, weakly focal cytoplasmic staining in squamous epithelium of p27 in EAC was detected. Conclusions Barrett's esophagus, which has a heterogeneous epithelium, might yield different diagnosis based on endoscopic evaluation and immunohistological investigation. Thus, the use of MUC1, p27, and CK7 might strengthen the truthful diagnosis. MUC-1, CK7, and c-p27 immunostaining can be used as the predictive markers for esophageal cancer progression from Barrett’s esophagus.

G Protein Coupled Receptor 109A (GPR109A), which belongs to the G protein coupled receptor family, can be activated by niacin, butyrate, and β-hydroxybutyric acid. Here, we assessed the anti-inflammatory activity of sodium butyrate (SB) on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis mice, an experimental model that resembles Crohn's disease, and explored the potential mechanism of SB in inflammatory bowel disease (IBD). In vivo, experimental GPR109a−/− and wild-type (WT) mice were administered SB (5 g/L) in their drinking water for 6 weeks. The mice were then administered TNBS via rectal perfusion to imitate colitis. In vitro, RAW246.7 macrophages, Caco-2 cells, and primary peritoneal macrophages were used to investigate the protective roles of SB on lipopolysaccharide (LPS)-induced inflammatory response and epithelium barrier dysfunction. In vivo, SB significantly ameliorated the inflammatory response and intestinal epithelium barrier dysfunction in TNBS-induced WT mice, but failed to provide a protective effect in TNBS-induced GPR109a−/− mice. In vitro, pre-treatment with SB dramatically inhibited the expression of TNF-α and IL-6 in LPS-induced RAW246.7 macrophages. SB inhibited the LPS-induced phosphorylation of the NF-κB p65 and AKT signaling pathways, but failed to inhibit the phosphorylation of the MAPK signaling pathway. Our data indicated that SB ameliorated the TNBS-induced inflammatory response and intestinal epithelium barrier dysfunction through activating GPR109A and inhibiting the AKT and NF-κB p65 signaling pathways. These findings therefore extend the understanding of GPR109A receptor function and provide a new theoretical basis for treatment of IBD., Highlights • Sodium butyrate maintains the gut epithelium barrier and protects against inflammation in TNBS-induced colitis-model mice • Targeting GPR109A anti-inflammatory and pro-intestinal epithelium barrier functions is a new strategy for IBD treatment Butyrate, produced by microbial fermentation in the bowel, has a protective effect toward maintaining the integrity of the gut epithelium barrier, which can decrease inflammatory responses in inflammatory bowel disease (IBD) such as Crohn's disease, although the underlying mechanism remains unknown. Here, we used cell and animal models of colitis to demonstrate that butyrate-mediated activation of the GPR109A receptor, which is known to suppress the inflammatory effects in various diseases, underlies its protective effects on colon health. Our results demonstrate that activating GPR109A thus may represent a novel approach to treat IBD, for which effective treatments are urgently required.

An experiment was conducted to investigate the effects of different dietary threonine (Thr) levels and immune stress on Pekin ducklings' growth performance, carcass traits, serum immune parameters, and intestinal mucin 2 (MUC2) and nuclear factor kB (NF-κB) gene expressions. A total of 320 Pekin ducklings was randomly assigned to a 5 × 2 factorial arrangement of treatments. Each treatment group consisted of 4 replicate pens with 8 ducks per pen. Ducklings were fed 5 graded levels of Thr: 0.49, 0.56, 0.60, 0.65, and 0.76% from hatch to 21 d of age. At 11 d of age, ducks in the stressed groups were challenged with bovine serum albumin (BSA), and ducks in the unstressed groups were injected with normal saline water. The results showed that increasing Thr supplementation from 0.49 to 0.56% in the diet can improve BWG; feed consumption; weight and relative weight of breast and leg; weight of liver, bursa of Fabricius, spleen, and thymus; serum natural immune globulin A (IgA) concentration; and MUC2 gene expression in the ileum of 21-day-old Pekin ducks, significantly (P < 0.05). Immune stress with BSA had a significant effect on 21-day-old Pekin ducklings' BWG, feed consumption, and weight and relative weight of breast and thymus (P < 0.05), but no interaction between BSA and dietary Thr content was noticed in our experiment in 21-day-old Pekin ducks (P < 0.05). Dietary Thr requirements of the unstressed groups and stressed groups based on broken-line model analyses for ducks' BWG were 0.705 and 0.603%, respectively, and for ducks' feed consumption were 0.724 and 0.705%, respectively.

Colorectal cancer (CRC) exhibits complex pathogenesis via compromised intestinal mucosal barrier. It is accepted that goblet cells secrete mucin which line the intestinal mucosal barrier and offer wide range protection and maintain the gut integrity. The principal mucin in the small and large intestine which is Mucin2 (MUC2) is predominantly expressed in the goblet cells which play a pivotal role in intestinal homeostasis. Its disruption is associated with diverse diseases and carcinomas. MUC2 has lately been identified as a principal marker in various mechanisms and secretory cell lineage. While MUC2 expression is regulated by various modulators, alterations in its expression are associated with immunomodulation, differences in tumor immunity and also regulation of microbiota. In the light of current literature, the present review explicates the regulation, functional mechanisms and essential role of MUC2 in colorectal cancer and aids in providing deep understanding of pathogenesis of the disease and also specifies the importance of the MUC2 in gaining more insights about the subtypes of colorectal cancer and how it can succour in approximating the prognosis and survival of the patients.

The purpose of this study was to evaluate the relieving effect of tilapia head glycolipids (TH-GLs) on dextran sulfate sodium (DSS)-induced colitis in mice and to further explore its mechanism. Mice were orally administered 3% (w/v) DSS to establish a model of ulcerative colitis (UC), and subsequently treated with TH-GLs or sulfasalazine. In addition, the expression of key targets in the intestinal mucosal barrier and the inflammatory signal pathway were studied by combining immunochemical analysis techniques. The results showed that varying doses of TH-GLs can significantly improve colon lesions caused by DSS, reduce histological scores, increase mucus secretion, extend colon length, increase weight, and inhibit the occurrence of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), Interleukin-1β (IL-1β), and Interleukin- 6 (IL-6). Further, studies have shown that TH-GLs increase the secretion of MUC2 and up-regulate the expression of tight junction related proteins, such as ZO-1 and Occludin. In addition, TH-GLs significantly down-regulated the protein expression levels of TNF-α, IKK-β, and nuclear factor-κB (NF-κB). Here, we have elucidated the potential mechanism of TH-GLs in protecting mice with colitis. In general, this study shows that TH-GLs could improve the symptoms of UC by improving the gut barrier and inhibiting inflammatory signals, which provides a scientific basis for future clinical applications.

The effects of supplemental dietary threonine (Thr) on laying performance, expression of intestinal mucin 2 (MUC2) and secretory IgA (sIgA), and intestinal microbiota of laying hens fed a low CP diet were investigated. A total of 240 Lohmann Brown laying hens, 28 wk of age, was allocated to 3 dietary treatments, each of which included 5 replicates of 16 hens. Hens were fed a control diet (16% CP), a low CP diet (14% CP), or a low CP diet supplemented with 0.3% L-Thr for 12 weeks. Chemical analyses of the diets for Thr are 0.49, 0.45, and 0.69%, respectively. Lowering dietary CP impaired egg production and egg mass of laying hens. Dietary Thr supplementation to the low CP diet increased (P 0.05) egg production and egg mass. In addition, ileal sIgA contents and MUC2 and sIgA mRNA expression were increased (P 0.05) by dietary Thr addition. Dietary CP reduction reduced (P 0.05) intestinal bacterial diversity, whereas dietary Thr supplementation to the low CP diet recovered the bacteria diversity and significantly increased the abundance of potential beneficial bacteria. In conclusion, dietary Thr supplementation to a low CP diet could affect intestinal health and hence productivity via regulating intestinal mucin and sIgA expression, and microbial population of laying hens.

Secretory IgA (sIgA) and its transcytosis receptor, polymeric immunoglobulin receptor (pIgR), along with mucus, form the first lines of intestinal defense. Threonine (Thr) is a major component of intestinal mucins and IgA, which are highly secreted under lipopolysaccharide (LPS) induced inflammation. In the current study, the effect of Thr on the secretory immune system was determined in an ex vivo chicken ileal explant model. Results showed that a 2-hour Thr-deprivation of culture medium induced a compensatory increase in the mRNA expression of interleukin-8 (IL-8), mucin 2 (MUC2), and IgA during LPS challenge, and this increase was suppressed with Thr addition to the media (P ≤ 0.05), suggesting that Thr was required for mucin and IgA production after exposure to LPS. Similarly, a 2-hour culture of explants from birds fed a Thr adequate diet showed an increase in the mRNA abundance of IL-8, MUC2, and IgA with LPS treatment (P ≤ 0.003), which had a trend to be attenuated with Thr supplementation in the media (P ≤ 0.10). In contrast, explants from birds fed a Thr deficient diet had no response to LPS treatment. These results indicated that in vivo Thr deficiency induced impaired inflammatory and secretory immune responses in broiler chicks. Furthermore, our results revealed that induction of MUC2 and pIgR gene expression required nuclear factor-κB (NF-κB) activation. Additionally, IgA transcytosis may be dependent on extracellular-regulated protein kinase (ERK) activation, which may indirectly impact pIgR gene expression.

Background Neurenteric cysts are rare central nervous system lesions derived from an endodermal origin. There is no consensus concerning pathogenesis because of the paucity of occurrences. We report an immunohistochemical study of 10 cases with neurenteric cysts and postulate its pathogenesis. Methods Ten patients underwent surgical treatment for neurenteric cysts from 1995 to 2015. We retrospectively reviewed clinical, radiologic, operative, and pathologic findings for these patients. Immunohistochemical stains were completed in all cases to distinguish cell type and origin. Results Three cell types were identified: pseudostratified-ciliated, goblet-columnar, and simple cuboidal cells. All cases were positive for cytokeratin 7, and negative for cytokeratin 20, caudal-type homeobox 2, mucin 2, thyroid transcription factor 1, human chorionic gonadotropin, placental alkaline phosphatase, and cluster of differentiation 31. Four of them had positive staining for mucin 5AC, with expression only in goblet-columnar cells. According to the immunohistochemical results, the cells resembled the respiratory tract (pseudostratified-ciliated), stomach (goblet-columnar), and respiratory bronchioles (simple cuboidal). Seventy-five percent of cases with recurrence had a goblet-columnar component, emphasizing the importance of total resection of the cyst and complete pathologic examination. Conclusions We postulate that the cystic tumor was derived from multipotent endodermal cells that migrated and traveled along the neuroectoderm, with incomplete differentiation into various cell types as a result of an unsuitable microenvironment. Because the neurenteric canal was only the channel of migration rather than a component of the cysts, the term neuroendodermal cysts is more precise in presenting the embryopathogenesis.

The main structural component of the mucus in the gastrointestinal tract is the MUC2 mucin. It forms large networks that in colon build the loose outer mucous layer that provides the habitat for the commensal flora and the inner mucous layer that protects the epithelial cells by being impenetrable to bacteria. The epithelial cells in mice lacking MUC2 are not adequately protected from bacteria, resulting in inflammation and the development of colon cancer as found in human ulcerative colitis. Correct processing of the MUC2 mucin is the basis for the building of these protective networks. During the biosynthesis of the MUC2 mucin, post-translational modifications are formed resulting in reduction-insensitive bonds between MUC2 monomers. By the use of γ-glutamyltranspeptidase and isopeptidase activity in leech saliva, we could show that the molecular nature of these reduction-insensitive bonds is isopeptide bonds formed between side chains of lysine and glutamine. Transglutaminase 2 has an affinity to the MUC2 CysD2 domain in the nanomolar range and can catalyze its cross-linking. By using mass spectrometry, we identified MUC2 residues involved in this cross-linking. This shows for the first time that transamidation is not only stabilizing the skin and the fibrin clot, but is also important for the correct intracellular processing of MUC2 to generate protective mucus.

Crosstalk between the Notch signaling pathway and Caudal-related homeobox 2 (Cdx2) has important roles in the development of Barrett's esophagus (BE). We investigated the expression and function of the Notch signaling ligand Delta-like 1 (Dll1) during the development of BE. We determined the expression levels of Dll1 and intracellular signaling molecules related to Notch signaling ((Notch1, Hairy/enhancer of split 1 (Hes1), and Atonal homolog 1 (ATOH1)) in human esophageal squamous and Barrett's epithelium samples. Next, those expression levels in esophageal squamous cells (Het-1A) and Barrett's esophageal cells (CP-A and BAR-T) following stimulation with either bile acids or gamma-secretase inhibitor were investigated. Finally, changes in those expression levels following transfection of a Cdx2 or Dll1 expression vector into Het-1A cells were examined. In addition, changes in those expression levels following knockdown of Cdx2 or Dll1 in CP-A cells were also examined. Dll1 was found to be upregulated and localized in the cell membrane and cytoplasm in BE. Bile acids enhanced cytoplasmic expression of Dll1 in CP-A cells, while cleaved Notch1 expression did not change, suggesting lack of a Dll1 agonistic effect on Notch signaling. Cells transfected with Cdx2 revealed significantly enhanced Dll1, while forced expression of Dll1 enhanced ATOH1, Cdx2, and MUC2 expression levels. Nevertheless, enhanced Dll1 did not induce Hes1 expression, suggesting that Dll1 may primarily function as an intracellular signaling molecule and not a Notch agonistic ligand in the canonical pathway. In addition, knockdown of Cdx2 completely abrogated any increase in Dll1 expression upon treatment with bile acids. Our results revealed a novel function of Dll1: facilitation of intestinal metaplasia in conjunction with Cdx2 expression. Furthermore, they suggest that intracellular induction of Dll1 expression in esophageal epithelial cells due to Cdx2 induction in response to bile acids has important roles in BE development.

Secreted mucins constitute a crucial part of the gel that protects respiratory and digestive epithelia, being MUC2/Muc2 the predominant gel-forming mucin of the intestine while MUC5AC/Muc5ac is one of the gel-forming mucins most expressed at the airways. In this study, we have analyzed Muc2 and Muc5ac during rat development by using immunohistochemistry, Western blotting and RT-PCR. We demonstrated that rat Muc2 was expressed in fetal intestinal goblet cells of surface epithelium of villi and developing Lieberkühn crypts. In neonates and adults, Muc2 was expressed at luminal goblet cells of small and large intestine and at gastric mucous and glandular cells. Muc5ac protein was observed in embryonic gastric and lung samples; expression increased during development and postnatal and adult life. After birth, a low reaction was detected at the tracheal surface epithelium and glands, which increased in adults. Fil: Ferretti, Valeria. Universidad Nacional de La Plata. Facultad de Ciencias Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Segal Eiras, Amada. Universidad Nacional de La Plata. Facultad de Ciencias Médicas; Argentina Fil: Barbeito, Claudio Gustavo. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Croce, María Virginia. Universidad Nacional de La Plata. Facultad de Ciencias Médicas; Argentina

In this study we identified mechanisms at the colonic mucosa by which MUC2 mucin regulated the production of β-defensin in a proinflammatory milieu but functionally protected susceptible bacteria from its antimicrobial effects. The regulator role of MUC2 on production of β-defensin 2 in combination with the proinflammatory cytokine interleukin-1β (IL-1β) was confirmed using purified human colonic MUC2 mucin and colonic goblet cells short hairpin RNA (shRNA) silenced for MUC2. In vivo, Muc2(-/-) mice showed impaired β-defensin mRNA expression and peptide localization in the colon as compared with Muc2(+/-) and Muc2(+/+) littermates. Importantly, purified MUC2 mucin abrogated the antimicrobial activity of β-defensin 2 against nonpathogenic and enteropathogenic Escherichia coli. Sodium metaperiodate oxidation of MUC2 removed the capacity of MUC2 to stimulate β-defensin production and MUC2's inhibition of defensin antimicrobial activity. This study highlights that a defective MUC2 mucin barrier, typical in inflammatory bowel diseases, may lead to deficient stimulation of β-defensin 2 and an unbalanced microbiota that favor the growth of β-defensin-resistant microbes such as Clostridium difficile.

Mucus provides a barrier to bacteria and toxins while allowing nutrient absorption and waste transport. Unlike colonic mucus, small intestinal mucus structure is poorly understood. This study aimed to provide evidence for a continuous, structured mucus layer and assess the diffusion of different sized particles through it. Mucus structure was assessed by histology and immunohistochemistry. Ultra-structure was assessed by scanning electron microscopy. Tracking of 100 nm and 500 nm latex beads was conducted using ex vivo porcine mucus. The porcine jejunum and ileum were filled with mucus. Layered MUC2 staining was visible throughout the small intestine, covering villus tips. Scanning electron microscopy showed net-like mucin sheets covering villi (211 ± 7 nm pore diameter). Particle tracking of 100 nm latex beads, showed no inhibition of diffusion through mucus while 500 nm beads displayed limited diffusion. These results suggest a continuous mucus layer exists throughout the small intestine, which is highly stratified adjacent to the epithelium. The network observed is consistent with previous observations and correlates with stratified MUC2 staining. Mucin pore size is consistent with free diffusion of 100 nm and limited diffusion of 500 nm particles. Small Intestinal mucus structure has important implications for drug delivery systems and prevention and treatment of conditions like mucositis and inflammatory bowel disease.

The expression patterns of secreted (MUC2 and MUC5AC) and membrane-tethered (MUC1, MUC4, MUC12 and MUC13) mucins were monitored in healthy pigs and pigs challenged orally with Lawsonia intracellularis. These results showed that the regulation of mucin gene expression is distinctive along the GI tract of the healthy pig, and may reflect an association between the function of the mucin subtypes and different physiological demands at various sites. We identified a specific depletion of secreted MUC2 from goblet cells in infected pigs that correlated with the increased level of intracellular bacteria in crypt cells. We concluded that L. intracellularis may influence MUC2 production, thereby altering the mucus barrier and enabling cellular invasion.

In order to demonstrate transglutaminase activity in biological samples immunological as well as glutamine- and amine-donor based assays are commonly used. However, the identification of the transglutaminase reaction product, i. e. the isopeptide cross-linked peptides/proteins or the deamidated protein/peptide are often neglected. This article describes a workflow for the detection of the products of transglutaminase-catalyzed reactions. In particular, possible pitfalls and traps that can arise during the mass spectrometry-based identification of isopeptide cross-links are addressed and characterised on actual samples.

Late gestation is a key period for intestinal development. Maternal heat exposure may induce intestinal dysfunction of offspring. To investigate the responses of intestinal morphology and function of offspring to the maternal heat stress (HS), twelve first-parity Landrace × Large White sows were assigned to thermoneutral (TN) (18-22 °C; n = 6) or HS (28-32 °C; n = 6) treatment groups at 85 d of gestation until natural farrowing. Twenty-four newborn piglets (two piglets at medium body weight from each litter) were randomly selected and divided into in utero thermoneutral (IUTN, n = 12) and heat-stressed (IUHS, n = 12) groups according to the sow’s treatment. Blood and intestinal samples were harvested to evaluate stress hormone levels, intestinal morphology, integrity and barrier function in the newborn piglets. Our results showed that maternal HS piglets exhibited increased serum adrenocorticotropic hormone (ACTH) concentration compared with that observed in the IUTN group. IUHS piglets showed lower lactase activities in the jejunum and ileum, whereas no significant differences were found between the two groups in the length of intestine, villus length or crypt depth. Serum diamine oxidase (DAO) activity was increased in IUHS piglets. IUHS piglets also exhibited decreased ZO-1, ZO-2 and MUC2 mRNA expression in the jejunum, while the protein levels were not affected. Additionally, IUHS piglets had a lower apoptotic percentage and FAS mRNA expression in the jejunum than those in the IUTN group. Taken together, these results demonstrate that high ambient temperature during late gestation of primiparous sows causes stress response in neonatal piglets, compromising intestinal permeability and mucosal barrier function, which may be partly mediated by inducing intestinal apoptosis.

That an alteration of the intestinal permeability is associated with gut barrier function has been increasingly evident, which plays an important role in human and animal health. Bisphenol A (BPA), an industrial compound used worldwide, has recently been classified as an environmental pollutant. One of our earlier studies has demonstrated that BPA disrupts the intestinal barrier function by inducing apoptosis and inhibiting cell proliferation in the human colonic epithelial cells line. In this study, we investigated the effects of dietary BPA uptake on the colonic barrier function in mice, as well as the intestinal permeability. Dietary BPA uptake was observed to destroy the morphology of the colonic epithelium and increase the pathology score. The levels of endotoxin, diamine peroxidase, D-lactate, and zonulin were found to have been significantly elevated in both plasma and colonic mucosa. A decline in the number of intestinal goblet cells and in mucin 2 gene expression was observed in the mice belonging to the BPA group. The results of immunohistochemistry revealed that the expression of tight junction proteins (ZO-1, occludin, and claudin-1) in colonic epithelium of BPA mice decreased significantly, and their gene abundance was also inhibited. Moreover, dietary BPA uptake was also found to have significantly reduced colonic microbial diversity and altered microbial structural composition. The functional profiles of colonic bacterial community exhibited adverse effects of dietary BPA intake on the endocrine and digestive systems, as well as the transport and catabolism functions. Collectively, our study highlighted that dietary BPA increased the colonic permeability, and this effect was closely related to the disruption of intestinal chemistry and physical and biological barrier functions.

Colorectal primary signet ring cell carcinoma (SRCC) is a rare entity accounting for nearly 1% of all colorectal carcinomas. It is an independent prognostic factor associated with less favorable outcome. This aggressiveness is mainly due to the intrinsic biology of these tumors. Here is an overview of the literature related to clinicopathological features, molecular biology, and management of SRCC of the colon and the rectum.

MUC2 is the major gel-forming mucin of the colon forming a protective gel barrier organized into an inner stratified and an outer loose layer. The MUC2 N-terminus (D1-D2-D′D3 domains) has a dual function in building a net-like structure by disulfide-bonded trimerization and packing the MUC2 polymer into an N-terminal concatenated polygonal platform with the C-termini extending perpendicularly by pH- and calcium-dependent interactions. We studied the N-terminal D′D3 domain by producing three recombinant variants, with or without Myc tag and GFP (green fluorescent protein), and analyzed these by gel filtration, electron microscopy and single particle image processing. The three variants were all trimers when analyzed upon denaturing conditions but eluted as hexamers upon gel filtration under native conditions. Studies by electron microscopy and three-dimensional maps revealed cage-like structures with 2- and 3-fold symmetries. The structure of the MUC2 D3 domain confirms that the MUC2 mucin forms branched net-like structures. This suggests that the MUC2 mucin is stored with two N-terminal concatenated ring platforms turned by 180° against each other, implicating that every second unfolded MUC2 net in mature mucus is turned upside down.

The mucus layer coating the gastrointestinal tract serves as the first line of intestinal defense against infection and injury. Probiotics promote mucin production by goblet cells in the intestine. p40, a Lactobacillus rhamnosus GG-derived soluble protein, has been shown to transactivate the EGF receptor (EGFR) in intestinal epithelial cells, which is required for inhibition of apoptosis and preservation of barrier function in the colon, thereby ameliorating intestinal injury and colitis. Because activation of EGFR has been shown to up-regulate mucin production in goblet cells, the purpose of this study was to investigate the effects and mechanisms of p40 regulation of mucin production. p40 activated EGFR and its downstream target, Akt, in a concentration-dependent manner in LS174T cells. p40 stimulated Muc2 gene expression and mucin production in LS174T cells, which were abolished by inhibition of EGFR kinase activity, down-regulation of EGFR expression by EGFR siRNA transfection, or suppression of Akt activation. Treatment with p40 increased mucin production in the colonic epithelium, thus thickening the mucus layer in the colon of wild type, but not of Egfr(wa5) mice, which have a dominant negative mutation in the EGFR kinase domain. Furthermore, inhibition of mucin-type O-linked glycosylation suppressed the effect of p40 on increasing mucin production and protecting intestinal epithelial cells from TNF-induced apoptosis in colon organ culture. Thus, these results suggest that p40-stimulated activation of EGFR mediates up-regulation of mucin production, which may contribute to the mechanisms by which p40 protects the intestinal epithelium from injury.

Mucinous differentiation is associated with both CpG island methylator phenotype and microsatellite instability in colorectal cancer. The mucinous phenotype derives from abundant expression of the colonic goblet cell mucin, MUC2, and de novo expression of gastric foveolar mucin, MUC5AC. We, therefore, investigated the protein expression levels of MUC2 and MUC5AC, as well as MUC5B and MUC6, in molecular subtypes of colorectal cancer. Seven-hundred and twenty-two incident colorectal carcinomas occurring in 702 participants of the Melbourne Collaborative Cohort Study were characterized for methylator status, MLH1 methylation, somatic BRAF and KRAS mutations, microsatellite-instability status, MLH1, MSH2, MSH6, and PMS2 mismatch repair, and p53 protein expression, and their histopathology was reviewed. Protein expression levels of MUC2, MUC5AC, MUC5B, MUC6, and the putative mucin regulator CDX2 were compared with molecular and clinicopathological features of colorectal cancers using odds ratios and corresponding 95% confidence intervals. MUC2 overexpression (>25% positive tumor cells) was observed in 33% colorectal cancers, MUC5B expression in 53%, and de novo MUC5AC and MUC6 expression in 50% and 39%, respectively. Co-expression of two or more of the mucins was commonly observed. Expression of MUC2, MUC5AC and MUC6 was strongly associated with features associated with tumorigenesis via the serrated neoplasia pathway, including methylator positivity, somatic BRAF p.V600E mutation, and mismatch repair deficiency, as well as proximal location, poor differentiation, lymphocytic response, and increased T stage (all P

We report 3 patients all men between 45 and 64 years of age with unilocular or multilocular mucinous cystadenomas of the kidney. One tumor arose from the renal pelvis, and 2 involved the entire pyelocaliceal system. The tumors measured between 2.4 and 37 cm in greatest dimension. Two patients were asymptomatic, and 1 had recurrent attack of acute pyelonephritis. Microscopically, the morphology and immunophenotype (CK20, MUC2, and CDX2 positive) of the tumors were similar to the colonic adenomas. Two patients were asymptomatic 24 and 64 months after surgery, including the patient with mucinous cystadenoma and intramucosal carcinoma. One patient died of acute myocardial infarction, and his tumor was an autopsy finding. Only 17 cases of mucinous cystadenomas and 5 cases of mucinous cystadenocarcinomas have been reported. Of the 17 mucinous cystadenomas, 2 arose in horseshoe kidneys. The mean size of these neoplasms was 15 cm (2.4-37 cm). Despite their large size, some patients with mucinous cystadenomas were asymptomatic. Sixty percent were associated with renal lithiasis. Thirty percent progressed to mucinous adenocarcinomas, and only 2 cases showed areas of intramucosal carcinomas. Two cases were associated with carcinoid tumors, similar to those reported in the appendix. Most patients were asymptomatic after surgery, and only 1 patient died by abdominal sepsis related to adenomucinosis. The 3 examples of mucinous cystadenomas of the pyelocaliceal system reported here, and those previously published indicate that they are very uncommon neoplasms with morphology and intestinal immunophenotype similar to the colonic adenomas.

Human mucin-2 (MUC-2) is the first line of innate host defense in preventing pathogen-induced epithelial injury. Entamoeba histolytica (Eh) colonizes the mucus layer by binding of the parasite's surface galactose lectin to galactose and N-acetyl-d-galactosamine residues on colonic MUC-2, preventing parasite contact-dependent cytolysis of epithelial cells. We quantified early innate responses to Eh in wild-type and MUC-2-deficient mice (Muc2(-/-)) using closed colonic loops. Eh infection in wild-type but not Muc2(-/-) mice induced a time-dependent increase in (3)H-labeled mucin and nonmucin glycoprotein secretions. Immunohistochemical staining revealed intense MUC-2 secretion, which formed a thick, protective mucus plug overlying the surface epithelium, entrapping Eh. In Muc2(-/-) mice, Eh induced a pronounced time-dependent secretory exudate with increased gross pathology scores and serum albumin leakage. Colonic pathology, secretory responses, and increased proinflammatory cytokine secretions of TNF-α, IFN-γ, and IL-13 correlated with altered expression of the tight junction proteins claudin-2, occludin, and ZO-1. We identified the putative Eh virulence factor that elicits the proinflammatory responses and alters tight junction permeability as Eh cysteine protease A5 (EhCP-A5). The present findings demonstrate that colonic mucins confer both luminal and epithelial barrier functions and that, in the absence of MUC-2, mice are more susceptible to Eh-induced secretory and proinflammatory responses mediated by EhCP-A5.

Several studies demonstrated that fermented milks may provide a large number of bioactive peptides into the gastrointestinal tract. We previously showed that beta-casomorphin-7, an opioid-like peptide produced from bovine β-casein, strongly stimulates intestinal mucin production in ex vivo and in vitro models, suggesting the potential benefit of milk bioactive peptides on intestinal protection. In the present study, we tested the hypothesis that the total peptide pool (TPP) from a fermented milk (yoghurt) may act on human intestinal mucus-producing cells (HT29-MTX) to induce mucin expression. Our aim was then to identify the peptide(s) carrying the biological activity and to study its impact in vivo on factors involved in gut protection after oral administration to rat pups (once a day, 9 consecutive days). TPP stimulated MUC2 and MUC4 gene expression as well as mucin secretion in HT29-MTX cells. Among the four peptide fractions that were separated by preparative reversed-phase high-performance liquid chromatography, only the C2 fraction was able to mimic the in vitro effect of TPP. Interestingly, the sequence [94-123] of β-casein, present only in C2 fraction, also regulated mucin production in HT29-MTX cells. Oral administration of this peptide to rat pups enhanced the number of goblet cells and Paneth cells along the small intestine. These effects were associated with a higher expression of intestinal mucins (Muc2 and Muc4) and of antibacterial factors (lysozyme, rdefa5). We conclude that the peptide β-CN(94-123) present in yoghurts may maintain or restore intestinal homeostasis and could play an important role in protection against damaging agents of the intestinal lumen.

As a result of chronic inflammation of their colon, patients with ulcerative colitis or Crohn's disease are at risk of developing colon cancer. In this paper, we consider the progression of colitis-associated colon cancer. Unlike normal colon mucosa, the inflammed colon mucosa undergoes genetic mutations, affecting, in particular, tumor suppressors TP53 and adenomatous polyposis coli (APC) gene. We develop a mathematical model that involves these genes, under chronic inflammation, as well as NF-κB, β-catenin, MUC1 and MUC2. The model demonstrates that increased level of cells with TP53 mutations results in abnormal growth and proliferation of the epithelium; further increase in the epithelium proliferation results from additional APC mutations. The model may serve as a conceptual framework for further data-based study of the early stage of colon cancer.

The aim of this study was to investigate the effect of dietary probiotic inclusion level on mucin composition (monosaccharide ratio), mucosal morphometry, mucus layer staining intensity, and mucus layer thickness along the broiler intestinal tract. One-day-old male Cobb broilers were administered maize-soybean meal basal (BD) diets for 42 d and depending on the feed additive used, broilers were allocated into the following 5 experimental treatments: control C (BD, no additive), treatment P1 (10(8) colony forming units of probiotic/kg of BD), treatment P2 (10(9) cfu of probiotic/kg of BD), treatment P3 (10(10) cfu of probiotic/kg of BD), and treatment A (2.5 mg avilamycin/kg of BD). Intestinal samples from duodenum, ileum, and cecum of 14- and 42-d-old broilers were collected and analyzed. Mannose (Man) decreased linearly with increasing probiotic level in duodenum (P=0.015) and ileum (P=0.042) of 14-d-old broilers. N-Acetyl-glucosamine and galactose decreased linearly (P=0.012 and P=0.001, respectively), while fucose increased linearly (P

TMF/ARA160 is a Golgi-associated protein with several cellular functions, among them direction of the NF-κB subunit, p65 RelA, to ubiquitination and proteasomal degradation in stressed cells. We sought to investigate the role of TMF/ARA160 under imposed stress conditions in vivo. TMF(-/-) and wild-type (WT) mice were treated with the ulcerative agent dextran sulfate sodium (DSS), and the severity of the inflicted acute colitis was determined. TMF(-/-) mice were found to be significantly less susceptible to DSS-induced colitis, with profoundly less bacterial penetration into the colonic epithelia. Surprisingly, unlike in WT mice, no bacterial colonies were visualized in colons of healthy untreated TMF(-/-) mice, indicating the constitutive resistance of TMF(-/-) colonic mucus to bacterial retention and penetration. Gene expression analysis of colon tissues from unchallenged TMF(-/-) mice revealed 5-fold elevated transcription of the muc2 gene, which encodes the major component of the colonic mucus gel, the MUC2 mucin. Accordingly, the morphology of the colonic mucus in TMF(-/-) mice was found to differ from the mucus structure in WT colons. The NF-κB subunit, p65, a well known transcription inducer of muc2, was up-regulated significantly in TMF(-/-) intestinal epithelial cells. However, this did not cause spontaneous inflammation or increased colonic crypt cell proliferation. Collectively, our findings demonstrate that absence of TMF/ARA160 renders the colonic mucus refractory to bacterial colonization and the large intestine less susceptible to the onset of colitis.

Cdx2 expression in esophageal stem cells induced by reflux bile acids may be an important factor for development of Barrett's esophagus, whereas Notch signaling is a molecular signaling pathway that plays an important role in the determination of cell differentiation. ATOH1 (a factor associated with Notch signaling) plays an important role in differentiation of stem cells into goblet cells. However, the relationship between the Notch signaling pathway and Cdx2 expression in the development of Barrett's esophagus has not been explored. The aim of this study was to investigate the interrelationship between Notch signaling and Cdx2 in esophageal epithelial cells. The expressions of Cdx2, MUC2, and intracellular signaling molecules related to Notch signaling (Notch1, Hes1, and ATOH1) were examined using real-time polymerase chain reaction (PCR) and immunohistochemical staining with biopsy specimens obtained from esophageal intestinal metaplasia (IM) with goblet cells (IM⁺) and columnar epithelium not accompanied by goblet cells (IM⁻). For in vitro experiments, we employed human esophageal epithelial cell lines (OE33, OE19, and Het-1A). After forced Cdx2 expression by applying a Cdx2 expression vector to the cells, changes in the expressions of Notch1, Hes1, ATOH1, Cdx2, and MUC2 were analyzed by real-time PCR and western blot analysis. Changes in expressions of Notch1, Hes1, ATOH1, Cdx2, and MUC2 in cells were analyzed following stimulation with bile acids in the presence or absence of Cdx2 blocking with Cdx2-siRNA. Suppressed Hes1 and enhanced ATOH1 and MUC2 expressions were identified in IM⁺ specimens. Forced expression of Cdx2 in cells suppressed Hes1, and enhanced ATOH1 and MUC2 expressions, whereas bile acids suppressed Hes1, and enhanced ATOH1, Cdx2, and MUC2 expressions. On the other hand, these effects were blocked by siRNA-based Cdx2 downregulation. Enhanced expression of Cdx2 by stimulation with bile acids may induce intestinal differentiation of esophageal columnar cells by interaction with the Notch signaling pathway.

SummaryThe intestinal mucus barrier prevents pathogen invasion and maintains host-microbiota homeostasis. We show that fatty acid synthase (FAS), an insulin-responsive enzyme essential for de novo lipogenesis, helps maintain the mucus barrier by regulating Mucin 2, the dominant mucin in the colon and a central component of mucus. Inducible Cre recombinase-directed inactivation of the FAS gene in the colonic epithelium of mice is associated with disruptions in the intestinal mucus barrier as well as increased intestinal permeability, colitis, systemic inflammation, and changes in gut microbial ecology. FAS deficiency blocked the generation of palmitoylated Mucin 2, which must be S-palmitoylated at its N terminus for proper secretion and function. Furthermore, a diabetic mouse model exhibited lower FAS levels and a decreased mucus layer, which could be restored with insulin treatment. Thus, the role of FAS in maintaining intestinal barrier function may explain the pathogenesis of intestinal inflammation in diabetes and other disorders.

Mucins represent major components of the mucous layer in the stomach, protecting the underlying epithelium from acid, mechanical trauma, proteases and pathogenic bacteria. Previous studies have shown an association of neoplastic transformation in the stomach with aberrant mucin levels, suggesting a potential role of genetic variation in mucin genes in the development of gastric cancer (GC). We assessed the association of genetic variation in candidate single nucleotide polymorphisms (SNPs) in mucin genes with the risk of chronic atrophic gastritis (CAG), a well-established precursor of GC in the German population-based ESTHER study. We genotyped MUC1 T31T, MUC2 L58P, MUC2 V116M, MUC5B E34G, MUC5B R51W, MUC5B rs2014486 (intronic) and MUC6 V619M for 533 serologically defined CAG cases and 1054 age- and sex-matched controls. None of the analysed SNPs was associated with CAG. However, large studies are needed to disclose or exclude potential weak associations of these SNPs with CAG risk.

The present study investigated the alteration of intestinal microbiota, innate immunity-related genes, and bacterial translocation in rats with cirrhosis and liver transplantation.Specific pathogen-free Sprague-Dawley rats were randomized into 4 groups: (1) normal controls (N); (2) liver cirrhosis (LC); (3) normal control groups with liver transplantation (LTN); and (4) liver cirrhosis with liver transplantation (LTC). We examined plasma endotoxin, bacterial tacslocation, denaturing gradient gel electrophoresis (DGGE) profile of intestinal mucosa-associated bacteria, abundance of key bacterial populations, and expression of innate immunity-related gene.The LTC and LC group, showed higher endotoxin levels (1.08±0.73 EU/mL and 0.74±0.70 EU/mL, respectively) than the N group (0.27±0.13 EU/mL; P.05). the incidence of bacterial translocation (BT) to liver and mesenteric lymph nodes (MLN), and the number of total bacteria were increased significantly in the LTC and LC groups compared with the N group (P.05). The counts of Lactobacilli and Bacteroides were lower, whereas Enterobacteria were higher in the LC than the N group (P.05). Mucins (MUC2, MUC3) and Toll-like receptors (TLR2, TLR4) messenger RNA (mRNA) expression were significantly higher in the LC and LTC groups than the N group (P.05). The marked difference between the groups in the overall structure of the bacterial community was also generated by DGGE profiles.Liver cirrhosis disturbs intestinal microbiota and innate immunity-related genes, which contributes to endotoxemia and bacterial translocation. These had not completely recovered in cirrhotic rats until 1 month after orthotopic liver transplantation.

The aim of this study was to characterize the physiologic expression of "intestinal" features in gallbladders of infants and children. The study group consisted of 56 pediatric (age, 2 weeks to 7 years) and 15 adult (15-25 years) patients who underwent incidental cholecystectomy during surgery for other lesions. All gallbladders examined were histologically unremarkable without inflammation, gallstones, or neoplasia. The presence of goblet cells and the expression of cytokeratin 7, cytokeratin 20, mucin core protein 2, and caudal-related homeobox protein 2 were examined. Intestinal features were frequently detected in the pediatric gallbladders: goblet cells in 34 cases (61%), cytokeratin 20 expression in 25 (45%), mucin core protein 2 expression in 32 (57%), and caudal-related homeobox protein 2 expression in 16 (29%). In contrast, none of these features was identified in adult gallbladders. The expression of mucin core protein 2 was mostly restricted to goblet cells in pediatric gallbladders, whereas cytokeratin 20 and caudal-related homeobox protein 2 were expressed in both goblet and nongoblet cells. Cytokeratin 7 was diffusely and consistently expressed in both pediatric and adult gallbladder epithelium including goblet cells. Intestinal features became less frequent with age and were scarce in children aged 6 to 7 years. Thus, goblet cells were identified in 14 (93%) of 15 children aged

Composite tumors are rare neoplasms containing a mixture of 2 different cellular components present in roughly equal proportions. It is hypothesized that composite tumors arise from a multipotential stem cell with subsequent bidirectional differentiation. We present an unusual composite tumor of the stomach composed equally of signet ring cell carcinoma and low-grade neuroendocrine carcinoma. Twenty-one additional patients with signet ring cell carcinomas of the stomach were studied to determine the prevalence of neuroendocrine differentiation by morphology and immunohistochemistry for synaptophysin and chromogranin A. Immunohistochemistry for mucins 5AC and 2 was performed to assess for divergent differentiation toward foveolar and intestinal mucin phenotypes, respectively, and to evaluate for any potential relationship with neuroendocrine differentiation. We found morphologic evidence of neuroendocrine carcinoma in 4 (19%) of 21 consecutive signet ring carcinomas. E-cadherin immunostaining was subsequently performed on these 4 tumors plus the index case. All 5 tumors demonstrated concordance between the signet ring and neuroendocrine components. There was no distinct relationship to mucin 5AC/mucin 2 profiles, with the exception that all 11 intramucosal signet ring cell carcinomas from 4 patients with germ line cadherin 1 gene mutations were composed exclusively of mucin 5AC+ signet ring cells that lacked intestinal mucin and neuroendocrine differentiation. The concordant E-cadherin status in the neuroendocrine and signet ring cell tumor components and the frequent admixture of mucin 5AC+ cells with foveolar differentiation and mucin 2+ cells with intestinal differentiation may support the hypothesis that composite tumors arise from a common stem cell with bilineage or multilineage differentiation.

Population genetic analyses of bacterial genes whose products interact with host tissues can give new understanding of infection and disease processes. Here we show that strains of the genetically diverse gastric pathogen Helicobacter pylori from Amerindians from the remote Peruvian Amazon contain novel alleles of cagA, a major virulence gene, and reveal distinctive properties of their encoded CagA proteins. CagA is injected into the gastric epithelium where it hijacks pleiotropic signaling pathways, helps Hp exploit its special gastric mucosal niche, and affects the risk that infection will result in overt gastroduodenal diseases including gastric cancer. The Amerindian CagA proteins contain unusual but functional tyrosine phosphorylation motifs and attenuated CRPIA motifs, which affect gastric epithelial proliferation, inflammation, and bacterial pathogenesis. Amerindian CagA proteins induced less production of IL-8 and cancer-associated Mucin 2 than did those of prototype Western or East Asian strains and behaved as dominant negative inhibitors of action of prototype CagA during mixed infection of Mongolian gerbils. We suggest that Amerindian cagA is of relatively low virulence, that this may have been selected in ancestral strains during infection of the people who migrated from Asia into the Americas many thousands of years ago, and that such attenuated CagA proteins could be useful therapeutically.

Threonine is an essential amino acid necessary for synthesis of intestinal (glyco)proteins such as mucin MUC2 to maintain adequate gut barrier function. In premature infants, reduced barrier function may contribute to the development of necrotizing enterocolitis (NEC). Human milk protects against NEC compared with infant formula. Therefore, we hypothesized that formula feeding decreases the MUC2 synthesis rate concomitant with a decrease in intestinal first-pass threonine utilization, predisposing the preterm neonate to NEC. Preterm pigs were delivered by caesarian section and received enteral feeding with formula (FORM; n = 13) or bovine colostrum (COL; n = 6) for 2 d following 48 h of total parenteral nutrition. Pigs received a dual stable isotope tracer infusion of threonine to determine intestinal threonine kinetics. NEC developed in 38% of the FORM pigs, whereas none of the COL pigs were affected (P = 0.13). Intestinal fractional first-pass threonine utilization was lower in FORM pigs (49 6 2%) than in COL pigs (60 6 4%) (P = 0.02). In FORM pigs compared with COL pigs, protein synthesis (369 6 31 mg·kg 21 ·d 21 vs. 615 6 54 mg·kg 21 ·d 21 ; P = 0.003) and MUC2 synthesis (121 6 17%/d vs. 184 6 15%/d; P = 0.02) were lower in the distal small intestine (SI). Our results suggest that formula feeding compared with colostrum feeding in preterm piglets reduces mucosal growth with a concomitant decrease in first-pass splanchnic threonine utilization, protein synthesis, and MUC2 synthesis in the distal SI. Hence, decreased intestinal threonine metabolism and subsequently impaired gut barrier function may predispose the formula-fed infant to developing NEC. J. Nutr. 141: 1306‐1311, 2011.

Objective To investigate the suitability of multimarker detection of DTCs in PB and BM of GC patients. Design and method A qRT-PCR assay was developed to estimate the number of CEA, CK20, TFF1 and MUC2 transcripts in PB and BM samples of 35 GC patients prior to the initiation of therapy. PB samples from healthy volunteers and BM from patients with hematological malignancies were used as negative controls. Results In PB analysis; 22.9%, 37.1%, 31.4%, and 22.9% of GC patients and in BM analysis; 20%, 28.6%, 45.7%, and 22.9% of GC patients were positive for CEA, CK20, TFF1 and MUC2 mRNAs, respectively. Samples from the control group were negative for the expression of all the markers tested in this study. A higher positive ratio was obtained with the multimarker detection in comparison to the single marker detection. There was a significant correlation between the PB and BM samples for DTC detection. Conclusion Multimarker detection assay is a reliable and powerful tool for the early detection of DTCs in GC patients.

Cholangiocarcinoma (CC) is a highly malignant epithelial cancer of the biliary tract, the cellular and molecular pathogenesis of which remains unclear. Malignant transformation of glandular epithelial cells is associated with the altered expression of mucin. We investigated the type of mucins expressed in CC. Twenty-six patients with histologically confirmed CC were included in this study. The expression of mucin was studied by immunohistochemistry using antibodies to MUC1, MUC1 core, MUC2, MUC3, MUC4, MUC5AC, and MUC6. There was extensive (>50%) expression of mucin, mainly MUC1 in 11/25 and MUC5AC in 12/26 cases. In the case of MUC3, 6/26 cases expressed mucin extensively, whilst only 1/26 had MUC2, MUC4, and MUC6 expression. Well-differentiated tumors significantly expressed MUC3 extensively compared to poor or moderately differentiated tumors (p=0.003). Fifteen of 25 cases had metastatic disease. MUC1 was extensively expressed in 9/15 cases with metastatic disease. In contrast, MUC1 expression was present in 2/10 cases where metastases were absent. Hilar lesions were less likely to express MUC1, but this was not statistically significant. Fifteen of 25 cases had metastatic disease. Extensive MUC3 expression was significantly associated with well-differentiated tumors, whilst there was an approaching significance between the extensive expression of MUC1 and metastasis in cholangiocarcinoma.