RhoA downregulation in the murine intestinal epithelium results in chronic Wnt activation and increased tumorigenesis.

Rho GTPases are molecular switches regulating multiple cellular processes. To investigate the role of RhoA in normal intestinal physiology, we used a conditional mouse model overexpressing a dominant negative RhoA mutant (RhoAT19N) in the intestinal epithelium. Although RhoA inhibition did not cause an overt phenotype, increased levels of nuclear ß-catenin were observed in the small intestinal epithelium of RhoAT19N mice, and the overexpression of multiple Wnt target genes revealed a chronic activation of Wnt signaling. Elevated Wnt signaling in RhoAT19N mice and intestinal organoids did not affect the proliferation of intestinal epithelial cells but significantly interfered with their differentiation. Importantly, 17-month-old RhoAT19N mice showed a significant increase in the number of spontaneous intestinal tumors. Altogether, our results indicate that RhoA regulates the differentiation of intestinal epithelial cells and inhibits tumor initiation, likely through the control of Wnt signaling, a key regulator of proliferation and differentiation in the intestine.

Acute psychological stress increases paracellular permeability and modulates immune activity in rectal mucosa of healthy volunteers.

BACKGROUND: Psychological stress and increased permeability are implicated as contributing factors in the initiation and worsening of gastrointestinal diseases. A link between stress and intestinal permeability has been shown in animal models as well as in human small intestine, but stress effects on the human colorectal mucosal barrier has not been reported. OBJECTIVE: To investigate the potential effects of acute psychological stress on colorectal mucosal barrier function and to explore stress-induced molecular events in the rectal mucosa under healthy conditions. METHODS: Endoscopic biopsies were taken from the rectosigmoid region of healthy volunteers, who had been subjected to dichotomous listening stress and after a control session, respectively. Paracellular and transcellular permeability were assessed in modified Ussing chambers. RNA expression (microarray technology confirmed by quantitative real-time polymerase chain reaction) and biological pathway analysis were used to investigate the local mucosal response to acute stress. RESULTS: Dichotomous listening stress induced a subjective and objective stress response, and significantly increased paracellular but not transcellular permeability. We also identified a stress-induced reduction in RNA expression of genes related to immune cell activation and maturation (CR2, CD20, TCLA1, BANK1, CD22, FDCSP), signaling molecules of homing of immune cells to the gut (chemokines: CCL21, CXCL13, and CCL19, and receptors: CCR7, CXCR5), and innate immunity (DUOX2). Eight of the 10 top down-regulated genes are directly involved in B cell activation, signaling and migration. The systemic stress response correlated positively with paracellular permeability and negatively with DUOX2 expression. CONCLUSION: Dichotomous listening stress increases paracellular permeability and modulates immune cell activity in the rectal mucosa. Further studies are warranted to identify the primary mechanisms of stress-mediated reduction of mucosal defensive activity and barrier dysfunction, and their potential implications for gastrointestinal disorders.

Mucosal Plasma Cell Activation and Proximity to Nerve Fibres Are Associated with Glycocalyx Reduction in Diarrhoea-Predominant Irritable Bowel Syndrome: Jejunal Barrier Alterations Underlying Clinical Manifestations.

Irritable bowel syndrome (IBS) is a disorder of brain-gut interaction characterised by abdominal pain and changes in bowel habits. In the diarrhoea subtype (IBS-D), altered epithelial barrier and mucosal immune activation are associated with clinical manifestations. We aimed to further evaluate plasma cells and epithelial integrity to gain understanding of IBS-D pathophysiology. One mucosal jejunal biopsy and one stool sample were obtained from healthy controls and IBS-D patients. Gastrointestinal symptoms, stress, and depression scores were recorded. In the jejunal mucosa, RNAseq and gene set enrichment analyses were performed. A morphometric analysis by electron microscopy quantified plasma cell activation and proximity to enteric nerves and glycocalyx thickness. Immunoglobulins concentration was assessed in the stool. IBS-D patients showed differential expression of humoral pathways compared to controls. Activation and proximity of plasma cells to nerves and IgG concentration were also higher in IBS-D. Glycocalyx thickness was lower in IBS-D compared to controls, and this reduction correlated with plasma cell activation, proximity to nerves, and clinical symptoms. These results support humoral activity and loss of epithelial integrity as important contributors to gut dysfunction and clinical manifestations in IBS-D. Additional studies are needed to identify the triggers of these alterations to better define IBS-D pathophysiology.

Overexpression of corticotropin-releasing factor in intestinal mucosal eosinophils is associated with clinical severity in Diarrhea-Predominant Irritable Bowel Syndrome.

Corticotropin-releasing factor (CRF) has been identified in intestinal mucosal eosinophils and associated with psychological stress and gut dysfunction. Irritable bowel syndrome (IBS) is commonly characterized by altered intestinal motility, immune activation, and increased gut barrier permeability along with heightened susceptibility to psychosocial stress. Despite intensive research, the role of mucosal eosinophils in stress-associated gut dysfunction remains uncertain. In this study, we evaluated eosinophil activation profile and CRF content in the jejunal mucosa of diarrhea-predominant IBS (IBS-D) and healthy controls (HC) by gene/protein expression and transmission electron microscopy. We also explored the association between intestinal eosinophil CRF and chronic stress, and the potential mechanisms underlying the stress response by assessing eosinophil response to neuropeptides. We found that mucosal eosinophils displayed higher degranulation profile in IBS-D as compared to HC, with increased content of CRF in the cytoplasmic granules, which significantly correlated with IBS clinical severity, life stress background and depression. Eosinophils responded to substance P and carbachol by increasing secretory activity and CRF synthesis and release, without promoting pro-inflammatory activity, a profile similar to that found in mucosal eosinophils from IBS-D. Collectively, our results suggest that intestinal mucosal eosinophils are potential contributors to stress-mediated gut dysfunction through CRF production and release.

Duodenal acidification induces gastric relaxation and alters epithelial barrier function by a mast cell independent mechanism.

Duodenal hyperpermeability and low-grade inflammation in functional dyspepsia is potentially related to duodenal acid exposure. We aimed to evaluate in healthy volunteers the involvement of mast cell activation on the duodenogastric reflex and epithelial integrity during duodenal acidification. This study consisted of 2 parts: (1) Duodenal infusion of acid or saline during thirty minutes in a randomized, double-blind cross-over manner with measurement of intragastric pressure (IGP) using high resolution manometry and collection of duodenal biopsies to measure epithelial barrier function and the expression of cell-to-cell adhesion proteins. Mast cells and eosinophils were counted and activation and degranulation status were assessed. (2) Oral treatment with placebo or mast cell stabilizer disodiumcromoglycate (DSCG) prior to duodenal perfusion with acid, followed by the procedures described above. Compared with saline, acidification resulted in lower IGP (P < 0.01), increased duodenal permeability (P < 0.01) and lower protein expression of claudin-3 (P < 0.001). Protein expression of tryptase (P < 0.001) was increased after acid perfusion. Nevertheless, an ultrastructural examination did not reveal degranulation of mast cells. DSCG did not modify the drop in IGP and barrier dysfunction induced by acid. Duodenal acidification activates an inhibitory duodenogastric motor reflex and, impairs epithelial integrity in healthy volunteers. However, these acid mediated effects occur independently from mast cell activation.

Peripheral Corticotropin-Releasing Factor Triggers Jejunal Mast Cell Activation and Abdominal Pain in Patients With Diarrhea-Predominant Irritable Bowel Syndrome.

INTRODUCTION: To determine the effect of peripheral CRF on intestinal barrier function in diarrhea-predominant IBS (IBS-D). Irritable bowel syndrome (IBS) pathophysiology has been linked to life stress, epithelial barrier dysfunction, and mast cell activation. Corticotropin-releasing factor (CRF) is a major mediator of stress responses in the gastrointestinal tract, yet its role on IBS mucosal function remains largely unknown. METHODS: Intestinal response to sequential i.v. 5-mL saline solution (placebo) and CRF (100 µg) was evaluated in 21 IBS-D and 17 healthy subjects (HSs). A 20-cm jejunal segment was perfused with an isosmotic solution and effluents collected at baseline, 30 minutes after placebo, and 60 minutes after CRF. We measured water flux, albumin output, tryptase release, stress hormones, cardiovascular and psychological responses, and abdominal pain. A jejunal biopsy was obtained for CRF receptor expression assessment. RESULTS: Water flux did not change after placebo in IBS-D and HS but significantly increased after CRF in IBS-D (P = 0.007). Basal luminal output of albumin was higher in IBS-D and increased further after CRF in IBS-D (P = 0.042). Basal jejunal tryptase release was higher in IBS-D, and CRF significantly increased it in both groups (P = 0.004), the response being higher in IBS-D than in HS (P = 0.0023). Abdominal pain worsened only in IBS-D after CRF and correlated with jejunal tryptase release, water flux, and albumin output. IBS-D displayed jejunal up-regulation of CRF2 and down-regulation of CRF1 compared with HS. DISCUSSION: Stress via CRF-driven mast cell activation seems to be relevant in the pathophysiology of IBS-D.

Transcriptomic identification of TMIGD1 and its relationship with the ileal epithelial cell differentiation in Crohn's disease.

Crohn's disease (CD) is a complex and multifactorial illness. There are still considerable gaps in our knowledge regarding its pathophysiology. A transcriptomic approach could shed some light on little-known biological alterations of the disease. We therefore aimed to explore the ileal transcriptome to gain knowledge about CD. We performed whole transcriptome gene expression analysis on ileocecal resections from CD patients and inflammatory bowel disease-free controls, as well as on a CD-independent cohort to replicate selected results. Normalized data were hierarchically clustered, and gene ontology and the molecular network were studied. Cell cultures and molecular methods were used for further evaluations. Genome-wide expression data analysis identified a robust transmembrane immunoglobulin domain-containing 1 (TMIGD1) gene underexpression in CD tissue, which was even more marked in inflamed ileum, and which was replicated in the validation cohort. Immunofluorescence showed TMIGD1 to be located in the apical microvilli of well-differentiated enterocytes but not in intestinal crypt. This apical TMIGD1 was lower in the noninflamed tissue and almost disappeared in the inflamed mucosa of surgical resections. In vitro studies showed hypoxic-dependent TMIGD1 decreased its expression in enterocyte-like cells. The gene enrichment analysis linked TMIGD1 with cell recovery and tissue remodeling in CD settings, involving guanylate cyclase activities. Transcriptomics may be useful for finding new targets that facilitate studies of the CD pathology. This is how TMIGD1 was identified in CD patients, which was related to multiciliate ileal epithelial cell differentiation.NEW & NOTEWORTHY This is a single-center translational research study that aimed to look for key targets involved in Crohn's disease and define molecular pathways through different functional analysis strategies. With this approach, we have identified and described a novel target, the almost unknown TMIGD1 gene, which may be key in the recovery of injured mucosa involving intestinal epithelial cell differentiation.

Increased Colonic Epithelial Permeability and Mucosal Eosinophilia in Ulcerative Colitis in Remission Compared With Irritable Bowel Syndrome and Health.

BACKGROUND: Barrier dysfunction is recognized as a pathogenic factor in ulcerative colitis (UC) and irritable bowel syndrome (IBS), but it is unclear to what extent the factors related to barrier dysfunction are disease-specific. The aim of this study was to compare these aspects in UC patients in remission, IBS patients, and healthy controls (HCs). METHODS: Colonic biopsies were collected from 13 patients with UC in remission, 15 patients with IBS-mixed, and 15 HCs. Ulcerative colitis patients had recently been treated for relapse, and biopsies were taken from earlier inflamed areas. Biopsies were mounted in Ussing chambers for measurements of intestinal paracellular permeability to 51chromium (Cr)-ethylenediaminetetraacetic acid (EDTA). In addition, biopsies were analyzed for mast cells and eosinophils by histological procedures, and plasma tumor necrosis factor (TNF)-α was assessed by ELISA. RESULTS: Ussing chamber experiments revealed an increased 51Cr-EDTA permeability in UC and IBS (P < 0.05). The 51Cr-EDTA permeability was higher in UC compared with IBS (P < 0.005). There were increased numbers of mucosal mast cells and eosinophils in UC and IBS and more eosinophils in UC compared with IBS (P < 0.05). Also, increased extracellular granule content was found in UC compared with HCs (P < 0.05). The 51Cr-EDTA permeability correlated significantly with eosinophils in all groups. Plasma TNF-α concentration was higher in UC compared with IBS and HCs (P < 0.0005). CONCLUSIONS: Results indicate a more permeable intestinal epithelium in inactive UC and IBS compared with HCs. Ulcerative colitis patients, even during remission, demonstrate a leakier barrier compared with IBS. Both eosinophil numbers and activation state might be involved in the increased barrier function seen in UC patients in remission.

Toll-like receptors-mediated pathways activate inflammatory responses in the esophageal mucosa of adult eosinophilic esophagitis.

OBJECTIVES: Esophageal microbiota and regulation of adaptive immunity are increasingly being investigated in eosinophilic esophagitis (EoE). Toll-like receptors (TLRs) play a central role in the initiation and maintenance of innate immune activity. Our objective was to characterize the esophageal and duodenal innate immune response in EoE and its modulation by dietary therapy. METHODS: Esophageal and duodenal biopsy samples were collected from 10 adults with untreated EoE, before and after effective treatment with a six-food elimination diet (SFED), and 10 controls with normal esophagus. In all cases, bacterial load (by mRNA expression of 16S), TLRs, mucins, transcription factors, interleukins, components of the NKG2D system, and innate immunity effectors were assessed by qPCR. Protein expression of TLRs were also determined by immunofluorescence. RESULTS: Bacterial load and TLR1, TLR2, TLR4, and TLR9 were overexpressed on biopsies with active EoE compared with controls. Muc1 and Muc5B genes were downregulated while Muc4 was overexpressed. Upregulation of MyD88 and NFκB was found together with IL-1ß, IL-6, IL-8, and IL-10 mediators and PER-1, iNOS, and GRZA effectors. NG-K2D components (KLRK1, IL-15, MICB) were also upregulated. In all cases, changes in active EoE were normalized following SFED and mucosal healing. Duodenal samples also showed increased expressions of TLR-1, TLR-2, and TLR-4, but not 16S or any other mediators nor effectors of inflammation. CONCLUSIONS: Esophageal TLR-dependent signaling pathways in EoE support the potential implication of microbiota and the innate immune system in the pathogenesis of this disease.

Decreased TESK1-mediated cofilin 1 phosphorylation in the jejunum of IBS-D patients may explain increased female predisposition to epithelial dysfunction.

Disturbed intestinal epithelial barrier and mucosal micro-inflammation characterize irritable bowel syndrome (IBS). Despite intensive research demonstrating ovarian hormones modulation of IBS severity, there is still limited knowledge on the mechanisms underlying female predominance in this disorder. Our aim was to identify molecular pathways involved in epithelial barrier dysfunction and female predominance in diarrhea-predominant IBS (IBS-D) patients. Total RNA and protein were obtained from jejunal mucosal biopsies from healthy controls and IBS-D patients meeting the Rome III criteria. IBS severity was recorded based on validated questionnaires. Gene and protein expression profiles were obtained and data integrated to explore biological and molecular functions. Results were validated by western blot. Tight junction signaling, mitochondrial dysfunction, regulation of actin-based motility by Rho, and cytoskeleton signaling were differentially expressed in IBS-D. Decreased TESK1-dependent cofilin 1 phosphorylation (pCFL1) was confirmed in IBS-D, which negatively correlated with bowel movements only in female participants. In conclusion, deregulation of cytoskeleton dynamics through TESK1/CFL1 pathway underlies epithelial intestinal dysfunction in the small bowel mucosa of IBS-D, particularly in female patients. Further understanding of the mechanisms involving sex-mediated regulation of mucosal epithelial integrity may have significant preventive, diagnostic, and therapeutic implications for IBS.

Epithelial immunity: priming defensive responses in the intestinal mucosa.

As the largest interface between the outside and internal milieu, the intestinal epithelium constitutes the first structural component facing potential luminal threats to homeostasis. This single-cell layer is the epicenter of a tightly regulated communication network between external and internal factors that converge to prime defensive responses aimed at limiting antigen penetration and the maintenance of intestinal barrier function. The defensive role developed by intestinal epithelial cells (IEC) relies largely on the variety of receptors they express at both extracellular (apical and basolateral) and intracellular compartments, and the capacity of IEC to communicate with immune and nervous systems. IEC recognize pathogen-associated molecules by innate receptors that promote the production of mucus, antimicrobial substances, and immune mediators. Epithelial cells are key to oral tolerance maintenance and also participate in adaptive immunity through the expression of immunoglobulin (Ig) receptors and by promoting local Ig class switch recombination. In IEC, different types of antigens can be sensed by multiple immune receptors that share signaling pathways to assure effective responses. Regulated defensive activity maintains intestinal homeostasis, whereas a breakdown in the control of epithelial immunity can increase the intestinal passage of luminal content and microbial invasion, leading to inflammation and tissue damage. In this review, we provide an updated overview of the type of immune receptors present in the human intestinal epithelium and the responses generated to promote effective barrier function and maintain mucosal homeostasis.

Vasoactive Intestinal Polypeptide and Mast Cells Regulate Increased Passage of Colonic Bacteria in Patients With Irritable Bowel Syndrome.

BACKGROUND & AIMS: Irritable bowel syndrome (IBS) is associated with intestinal dysbiosis and symptoms of IBS develop following gastroenteritis. We aimed to study the passage of live bacteria through the colonic epithelium, and determine the role of mast cells (MCs) and vasoactive intestinal polypeptide (VIP) in barrier regulation in IBS and healthy individuals. METHODS: Colon biopsies from 32 women with IBS and 15 age-matched healthy women (controls) were mounted in Ussing chambers; we measured numbers of fluorescently labeled Escherichia coli HS and Salmonella typhimurium that passed through from the mucosal side to the serosal side of the tissue. Some biopsies were exposed to agents that block the VIP receptors (VPAC1 and VPAC2) or MCs. Levels of VIP and tryptase were measured in plasma and biopsy lysates. Number of MCs and MCs that express VIP or VIP receptors were quantified by immunofluorescence. Biopsies from an additional 5 patients with IBS and 4 controls were mounted in chambers and Salmonella were added; we studied passage routes through the epithelium by transmission electron microscopy and expression of tight junctions by confocal microscopy. RESULTS: In colon biopsies from patients with IBS, larger numbers of E coli HS and S typhimurium passed through the epithelium than in biopsies from controls (P < .0005). In transmission electron microscopy analyses, bacteria were found to cross the epithelium via only the transcellular route. Bacterial passage was reduced in biopsies from patients with IBS and controls after addition of antibodies against VPACs or ketotifen, which inhibits MCs. Plasma samples from patients with IBS had higher levels of VIP than plasma samples from controls. Biopsies from patients with IBS had higher levels of tryptase, larger numbers of MCs, and a higher percentage of MCs that express VPAC1 than biopsies from controls. In biopsies from patients with IBS, addition of Salmonella significantly reduced levels of occludin; subsequent addition of ketotifen significantly reversed this effect. CONCLUSIONS: We found that colonic epithelium tissues from patients with IBS have increased translocation of commensal and pathogenic live bacteria compared with controls. The mechanisms of increased translocation include MCs and VIP.

Towards a new paradigm of microscopic colitis: Incomplete and variant forms.

Microscopic colitis (MC) is a chronic inflammatory bowel disease that has emerged in the last three decades as a leading cause of chronic watery diarrhoea. MC classically includes two main subtypes: lymphocytic colitis (LC) and collagenous colitis (CC). Other types of histopathological changes in the colonic mucosa have been described in patients with chronic diarrhoea, without fulfilling the conventional histopathological criteria for MC diagnosis. Whereas those unclassified alterations remained orphan for a long time, the use of the term incomplete MC (MCi) is nowadays universally accepted. However, it is still unresolved whether CC, LC and MCi should be considered as one clinical entity or if they represent three related conditions. In contrast to classical MC, the real epidemiological impact of MCi remains unknown, because only few epidemiological studies and case reports have been described. MCi presents clinical characteristics indistinguishable from complete MC with a good response to budesonide and cholestiramine. Although a number of medical treatments have been assayed in MC patients, currently, there is no causal treatment approach for MC and MCi, and only empirical strategies have been performed. Further studies are needed in order to identify their etiopathogenic mechanisms, and to better classify and treat MC.

Increased humoral immunity in the jejunum of diarrhoea-predominant irritable bowel syndrome associated with clinical manifestations.

BACKGROUND AND AIMS: Altered intestinal barrier is associated with immune activation and clinical symptoms in diarrhoea-predominant IBS (IBS-D). Increased mucosal antigen load may induce specific responses; however, local antibody production and its contribution to IBS aetiopathogenesis remain undefined. This study evaluated the role of humoral activity in IBS-D. METHODS: A single mucosal jejunal biopsy, luminal content and blood were obtained from healthy volunteers (H; n=30) and IBS-D (n=49; Rome III criteria) participants. Intraepithelial lymphocytes, mast cells, B lymphocytes and plasma cells were studied by imaging techniques. Differential gene expression and pathway analysis were assessed by microarray and PCR techniques. Blood and luminal immunoglobulins (Igs) were quantified. Gastrointestinal symptoms, respiratory atopy and stress and depression were also recorded. RESULTS: Patients with IBS-D showed a higher number and activation of mucosal B lymphocytes and plasma cells (p<0.05). Mast cell density was increased in patients with IBS-D (non-atopic) and in close proximity to plasma cells (p<0.05). Microarray profiling identified differential humoral activity in IBS-D, involving proliferation and activation of B lymphocytes and Igs production (p<0.001). Mucosal humoral activity was higher in IBS-D, with upregulation of germline transcripts and Ig genes (1.3-fold-1.7-fold increase; p<0.05), and increased IgG(+) cells and luminal IgG compared with H (p<0.05), with no differences in blood. Biological markers of humoral activity correlated positively with bowel movements, stool form and depression. CONCLUSIONS: Enhanced small bowel humoral immunity is a distinctive feature of IBS-D. Mucosal Ig production contributes to local inflammation and clinical manifestations in IBS-D.

Impaired duodenal mucosal integrity and low-grade inflammation in functional dyspepsia.

OBJECTIVE: Functional dyspepsia (FD) is an extremely common functional gastrointestinal disorder, the pathophysiology of which is poorly understood. We hypothesised that impaired intestinal barrier function is involved in the onset and persistence of this disorder by inducing low-grade inflammation. Therefore, our aim was to evaluate duodenal mucosal integrity and low-grade inflammation in patients with FD. DESIGN: Duodenal biopsy specimens were obtained from 15 patients with FD fulfilling the Rome III criteria and 15 age- and gender-matched healthy volunteers. Transepithelial electrical resistance (TEER) and paracellular permeability were measured in Ussing chambers. Expression of cell-to-cell adhesion proteins was evaluated by real-time PCR, western blot and/or immunofluorescence. Numbers of mast cells, eosinophils and intraepithelial lymphocytes were assessed by immunohistochemistry. RESULTS: Patients with FD displayed lower TEER and increased paracellular passage compared with healthy controls, which is indicative of impaired mucosal integrity. In addition, abnormal expression of cell-to-cell adhesion proteins at the level of tight junctions, adherens junctions and desmosomes was shown. Furthermore, patients were characterised by the presence of low-grade inflammation, as demonstrated by increased infiltration of mucosal mast cells and eosinophils. A significant association between the expression level of several cell-to-cell adhesion proteins, the extent of increased permeability and the severity of low-grade inflammation was found. CONCLUSIONS: These findings challenge the classical paradigm that patients with FD show no structural changes in the gastrointestinal tract. We suggest that impaired intestinal barrier function is a pathophysiological mechanism in FD. Thus, restoration of intestinal barrier integrity may be a potential therapeutic target for treating patients with FD.

Diarrhoea-predominant irritable bowel syndrome: an organic disorder with structural abnormalities in the jejunal epithelial barrier.

OBJECTIVE: Recently, the authors demonstrated altered gene expression in the jejunal mucosa of diarrhoea-predominant irritable bowel syndrome patients (IBS-D); specifically, the authors showed that genes related to mast cells and the intercellular apical junction complex (AJC) were expressed differently than in healthy subjects. The aim of the authors here was to determine whether these alterations are associated with structural abnormalities in AJC and their relationship with mast cell activation and IBS-D clinical manifestations. DESIGN: A clinical assessment and a jejunal biopsy were obtained in IBS-D patients (n=45) and healthy subjects (n=30). Mucosal mast cell number and activation were determined by quantifying CD117(+) cells/hpf and tryptase expression, respectively. Expression and distribution of AJC specific proteins were evaluated by western blot and confocal microscopy. AJC ultrastructure was assessed by transmission electron microscopy. RESULTS: Compared with healthy subjects, IBS-D patients exhibited: (a) increased mast cell counts and activation; (b) increased protein expression of claudin-2, reduced occludin phosphorylation and enhanced redistribution from the membrane to the cytoplasm; and (c) increased myosin kinase expression, reduced myosin phosphatase and, consequently, enhanced phosphorylation of myosin. These molecular alterations were associated with ultrastructural abnormalities at the AJC, specifically, perijunctional cytoskeleton condensation and enlarged apical intercellular distance. Moreover, AJC structural alterations positively correlated both with mast cell activation and clinical symptoms. CONCLUSION: The jejunal mucosa of IBS-D patients displays disrupted apical junctional complex integrity associated with mast cell activation and clinical manifestations. These results provide evidence for the organic nature of IBS-D, a heretofore model disease of functional gastrointestinal disorders.

The jejunum of diarrhea-predominant irritable bowel syndrome shows molecular alterations in the tight junction signaling pathway that are associated with mucosal pathobiology and clinical manifestations.

OBJECTIVES: Diarrhea-predominant irritable bowel syndrome (IBS-D) patients show altered epithelial permeability and mucosal micro-inflammation in both proximal and distal regions of the intestine. The objective of this study was to determine the molecular events and mechanisms and the clinical role of upper small intestinal alterations. METHODS: Clinical assessment and a jejunal biopsy was obtained in IBS-D patients and healthy subjects. Routine histology and immunohistochemistry was performed in all participants to assess the number of mast cells (MCs) and intraepithelial lymphocytes. RNA in tissue samples was isolated to identify genes showing consistent differential expression by microarray analysis followed by pathway and network analysis in order to identify the biological functions of the differentially expressed genes in IBS-D. Gene and protein expression of tight junction (TJ) components was also assessed by quantitative real-time polymerase chain reaction and confocal microscopy to evaluate the pathways identified by gene expression analysis. RESULTS: The analysis reveals a strong association between the transcript signature of the jejunal mucosa of IBS-D and intestinal permeability, MC biology, and TJ signaling. The expression of zonula occludens 1 (ZO-1) was reduced in IBS-D at both gene and protein level, with protein redistribution from the TJ to the cytoplasm. Remarkably, our analysis disclosed significant correlation between ZO proteins, MC activation, and clinical symptoms. CONCLUSIONS: IBS-D manifestations are linked to molecular alterations involving MC-related dysregulation of TJ functioning in the jejunal mucosa.

Eosinophils express muscarinic receptors and corticotropin-releasing factor to disrupt the mucosal barrier in ulcerative colitis.

BACKGROUND & AIMS: Altered intestinal barrier function has been implicated in the pathophysiology of ulcerative colitis (UC) in genetic, functional, and epidemiological studies. Mast cells and corticotropin-releasing factor (CRF) regulate the mucosal barrier in human colon. Because eosinophils are often increased in colon tissues of patients with UC, we assessed interactions among mast cells, CRF, and eosinophils in the mucosal barrier of these patients. METHODS: Transmucosal fluxes of protein antigens (horseradish peroxidase) and paracellular markers ((51)Cr-EDTA, fluorescein isothiocyanate-dextran 4000) were studied in noninflamed, colonic mucosal biopsy samples collected from 26 patients with UC and 53 healthy volunteers (controls); samples were mounted in Ussing chambers. We also performed fluorescence and electron microscopy of human tissue samples, assessed isolated eosinophils, and performed mechanistic studies using in vitro cocultured eosinophils (15HL-60), mast cells (HMC-1), and a colonic epithelial cell line (T84). RESULTS: Colon tissues from patients with UC had significant increases in permeability to protein antigens compared with controls. Permeability was blocked by atropine (a muscarinic receptor antagonist), α-helical CRF(9-41) (a CRF receptor antagonist), and lodoxamide (a mast-cell stabilizer). Eosinophils were increased in number in UC tissues (compared with controls), expressed the most M2 and M3 muscarinic receptors of any mucosal cell type, and had immunoreactivity to CRF. In coculture studies, carbachol activation of eosinophils caused production of CRF and activation of mast cells, which increased permeability of T84 epithelial cells to macromolecules. CONCLUSIONS: We identified a neuroimmune intercellular circuit (from cholinergic nerves, via eosinophils to mast cells) that mediates colonic mucosal barrier dysfunction in patients with UC. This circuit might exacerbate mucosal inflammation.

[Trends in drug consumption among immigrants between 2004 and 2008]. / Evolución del consumo de drogas por inmigrantes entre los años 2004 y 2008.

OBJECTIVE: To determine the sociodemographic and drug use profile of immigrants attended in Castile-Leon (Spain). METHODS: We performed a retrospective descriptive study comparing sociodemographic profiles and drug use variables through Pearson's chi-square test. RESULTS: A total of 80.8% of drug users were men, with a mean age 33.8 years (SD: 9.0); 72.3% were from Latin America, Portugal and eastern Europe and 51.6% had lived for 5 years or less in Spain. The main drug used was heroine (43.8%), via smoking (43.5%); most drug users started using in the country of origin (64.3%). Comparisons between 2008 and 2004 showed the following significant differences: for men: mean age (33.8 vs 30.9); length of main drug use: > or =21 years (19.2% vs 8.3%); for women: main drug use: heroin plus cocaine (25.6% vs 3.6%); length of main drug use: 16-20 years (27.9% vs 4.0%). CONCLUSIONS: The pattern of drug use differed by country of origin. The most commonly used drug was heroin, and injection was a frequent route of administration. We identified a need to strengthen harm-reduction interventions in this collective, enhance surveillance and adapt health services.