Proximal Colonic Mucosa Research Articles

Tumor suppressors miR-143 and miR-145 and predicted target proteins API5, ERK5, K-RAS, and IRS-1 are differentially expressed in proximal and distal colon.

The colon differs regionally in local luminal environment, excretory function, and gene expression. Polycistronic microRNA (miR)-143 and miR-145 are downregulated early in colon cancer. We asked if these microRNAs (miRNAs) might be differentially expressed in the proximal vs. the distal colon, contributing to regional differences in protein expression. Primary transcripts and mature miR-143 and miR-145 were quantified by real-time PCR, putative targets were measured by Western blotting, and DNA methylation was assessed by sequencing bisulfite-treated DNA in proximal and distal normal colonic mucosa as well as colon cancers. Putative targets of these miRNAs were assessed following transfection with miR-143 or miR-145. Mean expression of mature miR-143 and miR-145 was 2.0-fold (P < 0.001) and 1.8-fold (P = 0.03) higher, respectively, in proximal than distal colon. DNA methylation or primary transcript expression of these miRNAs did not differ by location. In agreement with increased expression of miR-143 and miR-145 in proximal colon, predicted targets of these miRNAs, apoptosis inhibitor 5 (API5), ERK5, K-RAS, and insulin receptor substrate 1 (IRS-1), which are cell cycle and survival regulators, were expressed at a lower level in proximal than distal colon. Transfection of HCA-7 colon cancer cells with miR-145 downregulated IRS-1, and transfection of HT-29 colon cancer cells with miR-143 decreased K-RAS and ERK5 expression. In conclusion, miR-143 and miR-145 and the predicted target proteins API5, ERK5, K-RAS, and IRS-1 display regional differences in expression in the colon. We speculate that differences in these tumor suppressors might contribute to regional differences in normal colonic gene expression and modulate site-specific differences in malignant predisposition.

DNA Methylation Status of Epithelial-Mesenchymal Transition (EMT) - Related Genes Is Associated with Severe Clinical Phenotypes in Ulcerative Colitis (UC)

BackgroundEpithelial-to-mesenchymal transition (EMT) is a phenomenon that allows the conversion of adherent epithelial cells to a mesenchymal cell phenotype, which enhances migratory capacity and invasiveness. Recent studies have suggested that EMT contributes to the pathogenesis of ulcerative colitis (UC). We investigated the promoter DNA methylation status of EMT-related genes in the colonic mucosa in UC.MethodsColonic biopsies were obtained from the rectal inflammatory mucosa of 86 UC patients and the non-inflammatory proximal colonic mucosa of 10 paired patients. Bisulfite pyrosequencing was used to quantify the methylation of 5 candidate CpG island promoters (NEUROG1, CDX1, miR-1247, CDH1, and CDH13) and LINE1.ResultsUsing an unsupervised hierarchical clustering analysis, inflamed rectal mucosa was well separated from mucosa that appeared normal. The CDH1 and CDH13 promoters were significantly associated with patient age (p = 0.04, 0.03, respectively). A similar trend was found between those genes and the duration of disease (CDH1: p = 0.07, CDH13: p = 0.0002, mean of both: p<0.00001). Several positive associations were found between hypermethylation and severe clinical phenotypes (CDX1 and miR-1247 and a refractory phenotype: p = 0.04 and 0.006, respectively. miR-1247 and CDH1 hyper methylation and a more severe Mayo endoscopic subscore: miR-1247: p = 0.0008, CDH1: p = 0.03, mean of both: p = 0.003). When the severe clinical phenotype was defined as having any of five phenotypes (hospitalized more than twice, highest Mayo endoscopic subscore, steroid dependence, refractory, or a history of surgery) miR-1247 hypermethylation was associated with the same phenotype (p = 0.008).ConclusionsOur data suggest that variability in the methylation status of EMT-related genes is associated with more severe clinical phenotypes in UC.

Mechanisms of activation of mouse and human enteroendocrine cells by nutrients

ObjectiveInhibition of food intake and glucose homeostasis are both promoted when nutrients stimulate enteroendocrine cells (EEC) to release gut hormones. Several specific nutrient receptors may be located on EEC that...

Telomere length in non-neoplastic colonic mucosa in ulcerative colitis (UC) and its relationship to the severe clinical phenotypes.

Telomere shortening occurs with human aging in many organs and tissues and is accelerated by rapid cell turnover and oxidative injury. To clarify the clinical importance of telomere shortening in colonic mucosa in ulcerative colitis (UC), we measured average telomere length using quantitative real-time PCR in non-neoplastic colonic mucosa in UC patients and assessed its relationship to various clinical subtypes. Relative telomere length in genomic DNA was measured in colonic biopsies obtained from rectal inflammatory mucosa from 86 UC patients as well as paired non-inflammatory proximal colonic mucosae from 10 patients. Data were correlated with various clinical phenotypes. In paired samples, average relative telomere length of rectal inflammatory mucosa was shortened compared to normal appearing proximal colon in eight out of ten cases (p=0.01). Telomere length shortening was significantly associated with more severe Mayo endoscopic subscore (p<0.0001) and cases needing surgery due to toxic megacolon or cancer occurrence (p=0.043). When the severe clinical phenotype was defined as having at least one of following phenotypes, more than two times of hospitalization, highest Mayo endoscopic subscore, steroid dependent, refractory, or needing operation, average relative telomere length was significantly shortened in the same phenotypes than the others (p=0.003). Telomere shortening is associated with more severe clinical phenotypes of UC, reflecting severe inflammatory state in the colonic mucosa.

Diets high in fermentable protein and fibre alter tight junction protein composition with minor effects on barrier function in piglet colon

Protein fermentation end products may damage the colonic mucosa, which could be counteracted by dietary inclusion of fermentable carbohydrates (fCHO). Although fermentable crude protein (fCP) and fCHO are known to affect microbial ecology, their interactive effects on epithelial barrier function are unknown. In the present study, in a 2×2 factorial experiment, thirty-two weaned piglets were fed low-fCP/low-fCHO (14·5% crude protein (CP)/14·5% total dietary fibre (TDF)), low-fCP/high-fCHO (14·8% CP/16·6% TDF), high-fCP/low-fCHO (19·8% CP/14·5% TDF) and high-fCP/high-fCHO (20·1% CP/18·0% TDF) diets. After 21-23d, samples of proximal and distal colonic mucosae were investigated in Ussing chambers with respect to the paracellular and transcytotic passages of macromolecules and epithelial ion transport. The high-fCHO diets were found to reduce the permeability of the distal colon to the transcytotic marker horseradish peroxidase (HRP, 44kDa; P<0·05) and also reduce the paracellular permeation of N-hydroxysuccinimide-biotin into the submucosa (443Da; P<0·05), whereas that of HRP was decreased by the high-fCP diets (P<0·01). Short-circuit current (active ion transport), transepithelial resistance (barrier function) and charge selectivity were largely unaffected in both the segments. However, the high-fCP diets were found to suppress the aldosterone-induced epithelial Na channel activity (P<0·01) irrespective of fCHO inclusion. The high-fCP diets generally reduced the expression of colonic claudin-1, claudin-2 and claudin-3 (P<0·01), while that of claudin-4 was increased by the high-fCHO diets (P<0·01). The high-fCHO diets also altered the ratio between occludin forms (P<0·05) and increased the expression of tricellulin in the proximal colon, which was not observed with high-fCP diets. In conclusion, dietary fCHO and fCP exerted few and largely independent effects on functional measurements, but altered tight junction protein composition in a compensatory way, so that colonic transport and barrier properties were only marginally affected.

Cancer-Predicting Gene Expression Changes in Colonic Mucosa of Western Diet Fed Mlh1+/- Mice

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in the Western world and interactions between genetic and environmental factors, including diet, are suggested to play a critical role in its etiology. We conducted a long-term feeding experiment in the mouse to address gene expression and methylation changes arising in histologically normal colonic mucosa as putative cancer-predisposing events available for early detection. The expression of 94 growth-regulatory genes previously linked to human CRC was studied at two time points (5 weeks and 12 months of age) in the heterozygote Mlh1 +/- mice, an animal model for human Lynch syndrome (LS), and wild type Mlh1 +/+ littermates, fed by either Western-style (WD) or AIN-93G control diet. In mice fed with WD, proximal colon mucosa, the predominant site of cancer formation in LS, exhibited a significant expression decrease in tumor suppressor genes, Dkk1, Hoxd1, Slc5a8, and Socs1, the latter two only in the Mlh1 +/- mice. Reduced mRNA expression was accompanied by increased promoter methylation of the respective genes. The strongest expression decrease (7.3 fold) together with a significant increase in its promoter methylation was seen in Dkk1, an antagonist of the canonical Wnt signaling pathway. Furthermore, the inactivation of Dkk1 seems to predispose to neoplasias in the proximal colon. This and the fact that Mlh1 which showed only modest methylation was still expressed in both Mlh1 +/- and Mlh1 +/+ mice indicate that the expression decreases and the inactivation of Dkk1 in particular is a prominent early marker for colon oncogenesis.

CMV Colitis in an HIV Positive Patient with CD4 Greater Than 200

Case Report A 28-year-old male known to have HIV was admitted with a one-week history of worsening fatigue, nausea, vomiting, 2 kg weight loss, and constipation. Blood pressure was 110/70 mmHg. Heart rate was 105 bpm and body temperature was 37.2 C. On physical examination, the patient looked dehydrated. His abdomen was tender to palpation without guarding. Bowel sounds were normal. Initial lab work showed sodium at 130 mEq/l, albumin at 2.2 g/dl, and white blood count at 15.9 K/cmm with normal differentials. Blood cultures were negative. On admission, his CD4 count was 223 cells/ml and viral load was 5350 copies per ml. Three months prior, his CD4 count was 666 cells/ml with 1960 HIV RNA copies per ml. A kidney, ureter, and bladder (KUB) xray study showed small bowel obstruction. A nasogastric tube was inserted. Computed tomography (CT) of the abdomen showed multiple air and fluid filled loops of small bowel with marked edema of distal small bowel. Colonoscopy revealed congested and erythematous mucosa with skip areas within the proximal, middle, and distal transverse colon and congestive mucosa within the terminal ileum with prominent Peyer’s patches. Serum cytomegalovirus (CMV) PCR was 3250 IU/mL. Histological findings in colonic biopsies revealed active inflammation with ulceration (Figure 1) and characteristic large cells (Figure 2). CMV infection could be confirmed immunohistochemically (Figure 3). The patient was started on IV ganciclovir. Highly active anti-retroviral therapy (HAART) was initiated with efavirenz, emtricitabine, and tenofovir. The patient improved. He was discharged on ganciclovir 450 PO BID and HAART. Three weeks later, the patient was seen at primary care physician’s office with clinical improvement and repeat serum CMV PCR was negative.

Abstract B18: Western diet-fed mice with inherited cancer predisposition reveal early epigenetic changes and protein markers for colon oncogenesis

Abstract Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in the Western world. Interactions between genetic and environmental factors, such as diet, are suggested to play a critical role in its etiology. However, the mechanisms that mediate the effects of diet on oncogenesis are largely unknown. We conducted a long-term feeding experiment in the mouse to address epigenetic changes arising in normal colonic mucosa and their functional consequences as putative cancer-predisposing events available for early detection. The quantitative analysis of proteomes and the expression of 94 growth-regulatory genes previously linked to human CRC and aberrant hypermethylation were studied at two time points (5 weeks and 12 months of age) in the heterozygote Mlh1+/- mice analogous to human Lynch syndrome, and wild type Mlh1+/+ littermates, fed with Western-style (WD) or AIN-93G control diet. In the proteome study the most significant expression changes were found in the Mlh1+/- WD mice compared to mice fed with AIN-93G control diet and the 19 identified proteins mainly point to abnormities in energy metabolism and protein unfolding. We further found that in mice carrying Mlh1 mutation and/or fed with WD, histologically normal proximal colonic mucosa exhibited a significant expression decrease in tumor suppressor genes, Dkk1, Slc5a8, Hoxd1, and Socs1. Especially in Dkk1, a secreted antagonist of the Wnt/β-catenin pathway the reduced expression was associated with its promoter hypermethylation. The fact that changes in Dkk1 seem to predispose to neoplasia in the proximal colon and that 5 out of 6 mice with neoplastic colonic lesions were fed with WD suggests that the found expression changes are early markers for oncogenesis. Citation Format: Denis Dermadi Bebek, Satu Valo, Marjaana Pussila, Nima Reyhani, Laura Sarantaus, Minna Nystrom. Western diet-fed mice with inherited cancer predisposition reveal early epigenetic changes and protein markers for colon oncogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B18.

An exploration of the microrheological environment around the distal ileal villi and proximal colonic mucosa of the possum (Trichosurus vulpecula).

Multiple particle-tracking techniques were used to quantify the thermally driven motion of ensembles of naked polystyrene (0.5 µm diameter) microbeads in order to determine the microrheological characteristics around the gut mucosa. The microbeads were introduced into living ex vivo preparations of the wall of the terminal ileum and proximal colon of the brushtail possum (Trichosurus vulpecula). The fluid environment surrounding both the ileal villi and colonic mucosa was heterogeneous; probably comprising discrete viscoelastic regions suspended in a continuous Newtonian fluid of viscosity close to water. Neither the viscosity of the continuous phase, the elastic modulus (G') nor the sizes of viscoelastic regions varied significantly between areas within 20 µm and areas more than 20 µm from the villous mucosa nor from the tip to the sides of the villous mucosa. The viscosity of the continuous phase at distances further than 20 µm from the colonic mucosa was greater than that at the same distance from the ileal villous mucosa. Furthermore, the estimated sizes of viscoelastic regions were significantly greater in the colon than in the ileum. These findings validate the sensitivity of the method and call into question previous hypotheses that a contiguous layer of mucus envelops all intestinal mucosa and restricts diffusive mass transfer. Our findings suggest that, in the terminal ileum and colon at least, mixing and mass transfer are governed by more complex dynamics than were previously assumed, perhaps with gel filtration by viscoelastic regions that are suspended in a Newtonian fluid.

Ulcerative colitis patients in remission have an altered secretory capacity in the proximal colon despite macroscopically normal mucosa

One of the hallmarks of acute colitis is loss of epithelial transport. For unknown reasons, many patients still suffer from GI symptoms during remission, indicating a sustained imbalance between absorption and secretion. We hypothesize that the colonic epithelium becomes more reactive to secretagogues to compensate for a failing barrier. Biopsies from ascending colon and sigmoid colon of UC patients in remission and controls were mounted in Ussing chambers. Membrane current (Im) and epithelial capacitance (Cp) were used as markers for anion secretion and mucus exocytosis. Carbachol (1 mmol L(-1) ) and forskolin (10 μmol L(-1) ) were used to study Ca(2+) and cAMP-mediated secretion. Baseline values showed segmental patterns with higher Im in ascending colon and higher Cp in sigmoid colon of both UC patients and controls, but the patterns did not differ between the groups. The Im response to forskolin was increased (+35%) in the ascending colon of UC patients and the Im response to carbachol was decreased (-40%) in the same segment. No group differences were seen in the distal colon for either the forskolin or carbachol-induced Im responses. The Cp response to carbachol was instead up-regulated in the distal colon of UC patients, but remained unaffected in the proximal colon. The proximal colonic mucosa of UC patients in remission seems to shift its reactivity to secretagogues, becoming more sensitive to cAMP-dependent secretion and less sensitive to Ca(2+) -dependent secretion. This phenomenon may contribute to residual diarrhea in this patient group, despite resolution of inflammation.

PMO-250 Quantitative proteomics in ulcerative colitis reveals mucosal inflammation reduces levels of keratins in the insoluble fraction of the intermediate filament proteome

IntroductionKeratins (K) are a key component of intermediate filaments (IF), primarily composed of K8, 18 and 19 in the intestinal epithelia. Apart from a structural role, they may play a...

Tu1311 Quantitative Proteomics in Ulcerative Colitis Reveals Mucosal Inflammation Reduces Levels of Keratins in the Insoluble Fraction of the Intermediate Filament Proteome

Introduction Keratins (K) are a key component of intermediate filaments (IF), primarily composed of K8, 18 and 19 in the intestinal epithelia. Apart from a structural role, they may play a role in moderating TNF effects, including cytotoxicity. K8-null mice develop colitis, a subset of patients with IBD have missense mutation in K8 gene. Colonic expression of K8/K18 has been shown to be regulated by IL-6. In order to examine the relationship between acute inflammation and alteration in levels of insoluble IFs in mucosa of patients with ulcerative colitis (UC), we undertook a quantitative proteomic approach using an iTRAQ (isobaric tagging for relative and absolute quantification)-based proteomic workflow. Methods Endoscopic biopsies were obtained from patients with UC, from actively inflamed rectum and from non-inflamed proximal colonic mucosa in the same patient (n=10), and from normal controls (n=10). Endoscopic (Baron) and histological assessment was made. An iTRAQ-compatible extraction protocol for insoluble IF proteins was developed. Labelled peptides from pooled patients were analysed by SCX-LC-MS/MS (strong cation exchange-reverse phase HPLC tandem mass spectrometry) and data reconstituted in GeneBio Phenyx. Inter-group comparisons were made using in-house algorithms based on t-testing with multiple test correction. Results Median age was 36 years (range 23–71). Endoscopic Baron score was ≥2 in inflamed mucosa for all patients. Tandem mass spectrometry (MS/MS) identified 52 proteins, 32 (61.5%) matched by two or more peptides, showing significant log fold change, with reduced levels of keratins and vimentin in inflamed mucosa compared to controls (Abstract PMO-250 table 1). Abstract PMO-250 figure 1 shows significantly reduced IF protein levels in inflamed mucosa compared to inactive mucosa. Cytokine proteins neutrophil defensin 1 and bone morphogenetic protein 4 were increased only in actively inflamed mucosa. Conclusion Using a quantitative proteomic approach, we have shown significantly decreased levels of keratins in the actively inflamed colonic mucosa in UC providing further evidence of interaction between keratins and inflammatory pathways—which requires further elucidation. Competing interests None declared.

Grape antioxidant dietary fibre prevents mitochondrial apoptotic pathways by enhancing Bcl-2 and Bcl-xLexpression and minimising oxidative stress in rat distal colonic mucosa

Grape antioxidant dietary fibre (GADF) is a grape product rich in dietary fibre and natural antioxidants. We reported previously that GADF intake reduced apoptosis and induced a pro-reducing shift in the glutathione (GSH) redox status of the rat proximal colonic mucosa. The aim of the study was to elucidate the molecular mechanisms responsible for the anti-apoptotic effect of GADF and their association with the oxidative environment of the distal colonic mucosa. The ability of GADF to modify colonic crypt cell proliferation was also investigated. Male Wistar rats (n 20) were fed with diets containing either cellulose (control group) or GADF (GADF group) as fibre for 4 weeks. GADF did not modify cell proliferation but induced a significant reduction of colonic apoptosis. The anti-apoptotic proteins Bcl-2 (B-cell lymphoma-2) and Bcl-xL (B-cell lymphoma extra large) were up-regulated in the mitochondria and down-regulated in the cytosol of the GADF mucosa, whereas the opposite was found for the pro-apoptotic protein Bax (Bcl-2-associated X protein), leading to an anti-apoptotic shift in the pattern of expression of the Bcl-2 family. Cytosolic cytochrome c and cleaved caspase-3 levels and caspase-3 activity were reduced by GADF. The modulation of the antioxidant enzyme system and the increase of the cytosolic GSH:glutathione disulfide (GSSG) ratio elicited by GADF helped to reduce oxidative damage. The cytosolic GSH:GSSG ratio was negatively related to apoptosis. These results indicate that GADF acts on the expression of the pro- and anti- apoptotic Bcl-2 proteins, attenuating the mitochondrial apoptotic pathway in the distal colonic mucosa. This effect appears to be associated with the antioxidant properties of GADF.

Phentermine Induced Ischemic Colitis

Purpose: Introduction: Weight loss medications have historically been cautiously supported due to adverse side effects such as pulmonary hypertension and valvular heart disease. In 1997 the FDA withdrew the combination of fenfluramine and dexfenfluramine from the market due to these adverse events. Phentermine, a commonly prescribed medication for short-term weight loss, works as a sympathomimetic and is an amphetamine derivative that remains available for prescription. We report a unique case of severe ischemic colitis following the use of phentermine in a patient with no other risk factors for ischemia. Case Presentation: A 57 year old female GI nurse presented to the emergency room complaining of bilateral lower quadrant abdominal pain followed by hematochezia two hours later. Digital rectal exam in the ER revealed bright red blood. Past medical history was unremarkable for any risk factors for gastrointestinal ischemia aside from well-controlled hypertension on a single antihypertensive. Phentermine had been prescribed five months prior at a dose of 30mg daily for weight loss. A colonoscopy was performed and showed diffuse contiguous erythema, superficial ulceration, and edema from the mid sigmoid colon to the mid transverse colon. A strictured area was also appreciated in the descending colon. The rectal and proximal colonic mucosa were normal. Biopsies obtained showed patchy mucosal hemorrhage and necrosis and confirmed the diagnosis of ischemic colitis. Phentermine had been held on admission and all symptoms resolved within 48 hours with supportive care. Two months later the patient remains asymptomatic and has not resumed her weight loss supplement. Discussion: There is one case report of ischemic colitis following fenfluramine in combination with dexfenfluramine. There is, however, only one published case report of phentermine in association with ischemic colitis that only involved a small segment at the splenic flexure. Gastrointestinal bleeding is not listed as a possible side effect of this commonly prescribed medication. Phentermine has been associated with ischemic neurologic events such as transient ischemic attacks and cerebral infarcts and could, through similar physiology, cause gastrointestinal ischemia. This unique case describes an instance of severe ischemic colitis following the use of phentermine involving almost the entire left colon and highlights the need for heightened awareness to identify patients and inform providers of the risk of gastrointestinal ischemia from this medication.

Regional Mucosa-Associated Microbiota Determine Physiological Expression of TLR2 and TLR4 in Murine Colon

Many colonic mucosal genes that are highly regulated by microbial signals are differentially expressed along the rostral-caudal axis. This would suggest that differences in regional microbiota exist, particularly mucosa-associated microbes that are less likely to be transient. We therefore explored this possibility by examining the bacterial populations associated with the normal proximal and distal colonic mucosa in context of host Toll-like receptors (TLR) expression in C57BL/6J mice housed in specific pathogen-free (SPF) and germ-free (GF) environments. 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis revealed significant differences in the community structure and diversity of the mucosa-associated microbiota located in the distal colon compared to proximal colon and stool, the latter two clustering closely. Differential expression of colonic TLR2 and TLR4 along the proximal-distal axis was also found in SPF mice, but not in GF mice, suggesting that enteric microbes are essential in maintaining the regional expression of these TLRs. TLR2 is more highly expressed in proximal colon and decreases in a gradient to distal while TLR4 expression is highest in distal colon and a gradient of decreased expression to proximal colon is observed. After transfaunation in GF mice, both regional colonization of mucosa-associated microbes and expression of TLRs in the mouse colon were reestablished. In addition, exposure of the distal colon to cecal (proximal) microbiota induced TLR2 expression. These results demonstrate that regional colonic mucosa-associated microbiota determine the region-specific expression of TLR2 and TLR4. Conversely, region-specific host assembly rules are essential in determining the structure and function of mucosa-associated microbial populations. We believe this type of host-microbial mutualism is pivotal to the maintenance of intestinal and immune homeostasis.

Toll-Like Receptor mRNA Expression Is Selectively Increased in the Colonic Mucosa of Two Animal Models Relevant to Irritable Bowel Syndrome

BackgroundIrritable bowel syndrome (IBS) is largely viewed as a stress-related disorder caused by aberrant brain-gut–immune communication and altered gastrointestinal (GI) homeostasis. Accumulating evidence demonstrates that stress modulates innate immune responses; however, very little is known on the immunological effects of stress on the GI tract. Toll-like receptors (TLRs) are critical pattern recognition molecules of the innate immune system. Activation of TLRs by bacterial and viral molecules leads to activation of NF-kB and an increase in inflammatory cytokine expression. It was our hypothesis that innate immune receptor expression may be changed in the gastrointestinal tract of animals with stress-induced IBS-like symptoms.Methodology/Principal FindingsIn this study, our objective was to evaluate the TLR expression profile in the colonic mucosa of two rat strains that display colonic visceral hypersensivity; the stress-sensitive Wistar-Kyoto (WKY) rat and the maternally separated (MS) rat. Quantitative PCR of TLR2-10 mRNA in both the proximal and distal colonic mucosae was carried out in adulthood. Significant increases are seen in the mRNA levels of TLR3, 4 & 5 in both the distal and proximal colonic mucosa of MS rats compared with controls. No significant differences were noted for TLR 2, 7, 9 & 10 while TLR 6 could not be detected in any samples in both rat strains. The WKY strain have increased levels of mRNA expression of TLR3, 4, 5, 7, 8, 9 & 10 in both the distal and proximal colonic mucosa compared to the control Sprague-Dawley strain. No significant differences in expression were found for TLR2 while as before TLR6 could not be detected in all samples in both strains.ConclusionsThese data suggest that both early life stress (MS) and a genetic predisposition (WKY) to stress affect the expression of key sentinels of the innate immune system which may have direct relevance for the molecular pathophysiology of IBS.

Corticotropin releasing factor in the rat colon: Expression, localization and upregulation by endotoxin

Little is known about CRF expression and regulation in the rat colon compared to the brain. We investigated CRF gene expression, cellular location, and regulation by endotoxin and corticosterone in the male rat colon at 6 h after intraperitoneal (ip) injection. CRF mRNA level, detected by reverse transcription-polymerase chain reaction (RT-PCR) was 1.3-fold higher in the distal than proximal colon and 3.4-fold higher in the proximal colonic submucosa plus muscle layers than in mucosa. CRF immunoreactivity was located in the epithelia, lamina propria and crypts, and co-localized with tryptophan hydroxylase, a marker for enterochromaffin (EC) cells, and in enteric neurons. Lipopolysaccharide (LPS, 100 μg/kg, ip) increased defecation by 2.9-fold and upregulated CRF mRNA by 2.5-fold in the proximal and 1.1-fold in the distal colon while there was no change induced by corticosterone as monitored by quantitative PCR. LPS-induced increased CRF mRNA expression occurred in the submucosa plus muscle layers (1.5-fold) and the mucosa of proximal colon (0.9-fold). LPS increased significantly CRF immunoreactivity in the submucosal and myenteric plexuses of proximal and distal colon compared to saline groups. These results indicate that in rats, CRF is expressed in both proximal and distal colon and more prominently in enteric neurons of the submucosa plus muscle layers and subject to upregulation at the gene and protein levels by LPS through corticosteroid independent pathways. These data suggests that colonic CRF may be part of the local effector limb of the CRF 1 receptor mediated colonic alterations induced by acute stress.

Grape antioxidant dietary fibre reduced apoptosis and induced a pro-reducing shift in the glutathione redox state of the rat proximal colonic mucosa

Grape antioxidant dietary fibre (GADF) is a grape product rich in dietary fibre and natural antioxidants. We showed previously that the GADF intake induced an epithelial hypoplasia in the rat colonic mucosa. In the present study, we propose that the antioxidant effect of GADF could modulate mucosal apoptosis via modulation of the cellular redox environment. Male Wistar rats (n 20) were fed with diets containing either cellulose (control diet group) or GADF (GADF diet group) as fibre for 4 weeks. The GSH:GSSG ratio, the redox state of the GSSG/2GSH couple (Ehc), the mitochondrial and/or cytosolic antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)), lipid peroxidation (LPO) and apoptosis were evaluated. GADF enhanced the cytosolic GSH:GSSG ratio, shifting the redox potential (Ehc) to a more pro-reducing status. Decreased Cu,ZnSOD:CAT, Cu,ZnSOD:GPx and MnSOD:GPx ratios could indicate an enhanced capacity for reducing H2O2, contributing to decreased cytosolic LPO. Reduced apoptosis in GADF-treated mucosa was inversely related to MnSOD activity. Furthermore, apoptosis increased directly as GSSG content increased. These results suggest that the reduction in apoptosis associated with GADF intake may be due to a modulation of the glutathione redox system and endogenous antioxidant enzymes.

The Intestinal Nuclear Receptor Signature With Epithelial Localization Patterns and Expression Modulation in Tumors

The WNT-adenomatous polyposis coli system controls cell fate in the intestinal epithelium, where compartment-specific genes tightly regulate proliferation, migration, and differentiation. Nuclear receptors are transcription factors functioning as sensors of hormones and nutrients that are known to contribute to colon cancer progression. Here we mapped the messenger RNA (mRNA) abundance and the epithelial localization of the entire nuclear receptor family in mouse and human intestine. We used complementary high-resolution in situ hybridization and systematic real-time quantitative polymerase chain reaction in samples of normal distal ileum and proximal colon mucosa and tumors obtained from mouse and human adenomatous polyposis coli-initiated tumor models (ie, Apc(Min/+) mice and familial adenomatous polyposis patients) and in cellular models of human colon cancer. We first defined for each receptor an expression pattern based on its transcript localization in the distal ileum and the proximal colon. Then, we compared the mRNA levels between normal intestinal epithelium and neoplastic intestinal tissue. After analyzing the correspondence between mouse and human tumor samples plus genetically modified human colon cancer cells, we used complementary graphic and statistical approaches to present a comprehensive overview with several classification trees for the nuclear hormone receptor intestinal transcriptome. We defined the intestinal nuclear hormone receptor map, which indicates that the localization pattern of a receptor in normal intestine predicts the modulation of its expression in tumors. Our results are useful to select those nuclear receptors that could be used eventually as early diagnostic markers or targeted for clinical intervention in intestinal polyposis and cancer.

Collagenous Colitis and Colonic Perforation During Colonoscopy

Collagenous colitis can be associated with “fractures” of the proximal colon mucosa seen during colonoscopy. These linear cracks in the mucosa might be