Non-colitis Controls Research Articles

P0102 Isatin: An indole alkaloid with intestinal anti-inflammatory activity in the trinitrobenzene sulfonic acid colitis model

Abstract Background Inflammatory bowel disease (IBD) is a complex and recurring inflammatory disorder that affects the gastrointestinal tract.1 Various drugs have been developed for treating IBD, but there are still limitations due to adverse effects experienced by patients.2 Plant-derived molecules with anti-inflammatory properties represent a valuable alternative as a complementary therapy for IBD.3 Isatin is a compound found in several medicinal plant species with various biological activities, including a modulatory effect on pro-inflammatory mediators. This study aimed to evaluate the effect of isatin in experimental colitis. Methods Rats were divided into three groups (10 animals/group): Saline (non-colitic control), TNBS (colitic untreated), and isatin (colitic treated with isatin 6 mg/kg). After anesthesia with ketamine (50 mg/kg) and xylazine (10 mg/kg), the TNBS and isatin groups received a single dose of TNBS (10 mg) dissolved in 0.25 mL of 50% ethanol (v/v) by rectal administration. To evaluate the intestinal anti-inflammatory activity of isatin, animals were treated 2, 24, and 48 hours after the TNBS challenge and euthanized after 72 hours by CO2 overdose. The colons were removed, the macroscopic inflammation and the colon weight/length ratio were measured and the cytokines IL-1β, IL-12, IFN-γ, and TNF-α were quantified by ELISA. Median (minimum-maximum) or mean ± SEM values were compared through the Kruskal-Wallis test (Dunn’s test) or two-way ANOVA (Tukey’s test). The experiments were approved by the Ethics Commission in Animal Use (Protocol number 2773-1). Results The TNBS group showed increased colonic wall thickness and inflammatory cell infiltration. Isatin reduced the macroscopic inflammation score [Isatin: 6 (6-7) versus TNBS: 9 (8-10); p < 0.01] promoted by TNBS instillation. Oral administration of isatin prevented the increase in the colon weight/length ratio (0.151 ± 0.017 versus 0.192 ± 0.015; p < 0.01). The colonic damage caused by TNBS instillation was also characterised by an increase in the levels of pro-inflammatory cytokines compared to the Saline group. In contrast, isatin reduced the levels of TNF-α (2.42 ± 0.72 versus 6.32 ± 1.91; p<0.001), IFN-γ (4.26 ± 1.15 versus 7.34 ± 0.48; p<0.01), IL-1β (16.54 ± 4.76 versus 35.91 ± 4.11; p<0.001), and IL-12 (13.38 ± 3.65 versus 28.87 ± 8.39; p<0.01) while increased the levels of IL-10 (6.41 ± 0.60 versus 4.82 ± 0.26; p<0.01) when compared with TNBS group. Conclusion Isatin decreased the macroscopic inflammation score and the colon weight/length ratio. The action of isatin on colitis involves the reduction of pro-inflammatory mediators such as TNF-α and IL-1β and increase of the anti-inflammatory cytokine IL-10, suggesting a potential curative role of isatin in colitis therapy.

Preventive effect on intestinal inflammation and modulation of the microbiota of ‘Nordestino’ donkey milk in experimental DNBS-induced colitis in mice

Preventive effect on intestinal inflammation and modulation of the microbiota of ‘Nordestino’ donkey milk in experimental DNBS-induced colitis in mice

Quantitative Evaluation of NOD2 Promoter Methylation Profiling in Colon Biopsy Samples from Ulcerative Colitis Patients and Non-Colitis Controls

Background: Ulcerative colitis (UC) is an idiopathic chronic inflammatory disease of the colon with evidence addressing the role of epigenetic changes. The intention of this study was to detect the association of DNA methylation levels of NOD2 gene with UC and to evaluate whether any of these changes might be a useful biomarker for detecting patients with UC.
 Methods: The methylation status of the promoter CpG islands (CGIs) of NOD2 gene was examined in the colonic mucosae of 15 cancer-free patients with UC and 15 age- and sex-matched healthy controls by the real-time quantitative multiplex-methylation specific PCR (QM-MSP) assay. Methylation-specific melting curve analysis (MS-MCA) were used to analyze the data.
 Results: The median unmethylated DNA index was significantly higher in cases compared to controls, and hypormethylation of NOD2 gene was significantly correlated with UC (Unmethylated DNA in UC vs. HC; 0.102±0.055 vs. 0.025±0.016, P = 0.000).
 Conclusion: The NOD2 gene that was differentially methylated in UC patients, providing new insights into the pathogenesis of UC, with a view to making steps toward the development of accurate biomarkers for diagnostic tools in UC.

Markers of Hypoxia Correlate with Histologic and Endoscopic Severity of Colitis in Inflammatory Bowel Disease.

BackgroundInflammation results in significant shifts in tissue metabolism. Recent studies indicate that inflammation and hypoxia occur concomitantly. We examined whether circulating and tissue markers of hypoxia could serve as surrogate indicators of disease severity in adult and pediatric patients with inflammatory bowel disease (IBD).MethodsSerum and colonic biopsies were obtained from pediatric subjects with active IBD colitis and adult subjects with active and inactive ulcerative colitis, along with healthy non-colitis controls of all ages. Disease activity was evaluated by endoscopy and histopathology. Levels of serum hypoxia markers (macrophage inflammatory protein-3α [MIP-3α], vascular endothelial growth factor [VEGF], and erythropoietin [EPO]) were measured.ResultsChildren with active IBD colitis had higher levels of serum MIP-3α and VEGF compared to non-colitis controls (p<0.01 and p<0.05, respectively). In adult subjects with endoscopically active ulcerative colitis, serum MIP-3α and EPO were significantly elevated compared to non-colitis controls (both p<0.01). In parallel, analysis of colon tissue MIP-3α mRNA and protein in pediatric subjects revealed increased expression in those with IBD colitis compared to controls (p<0.05 and p<0.01 for mRNA and protein, respectively). Serum MIP-3α and VEGF significantly increased with histology grade.ConclusionPeripheral blood hypoxia markers may be useful indicators of disease activity for pediatric and adult IBD patients.

Potential effects of xylopic acid on acetic acid-induced ulcerative colitis in rats.

Xylopic acid (XA) has been reported to exhibit analgesic activity, alleviate neuropathic pain in rodents, and demonstrate anti-inflammatory effects. Intrarectal challenge of rats with acetic acid induces colitis that bears resemblance in terms of its pathogenesis, histopathology, and inflammatory profile to that in humans. Reactive oxygen species are implicated as the main driving force in this inflammatory bowel disease. This study aimed to investigate the anti-colitic potential of XA. We investigated the effect of XA on body weight, disease activity, inflammatory cell infiltration, and generation of reactive oxygen species. Rats were treated with XA or sulphasalazine, challenged intrarectally with acetic acid with macroscopic and microscopic findings made after eight days. Administration of XA to rats with colitis resulted in an increase in body weight with significant (p<0.05) improvement of the disease profile macroscopically. We observed decreased gross mucosal injury, reduced inflammation, and cellular proliferation with XA administration. Treatment with XA also resulted in decreased colonic epithelial expression of argyrophylic nucleolar organizer regions (AgNORs) with significant decrease (p<0.0001) in the quantitative expression of AgNORs/nucleus ratio to levels comparable with non-colitic control. We also observed reduced proliferation of mucosal mast cell in the colonic segment of the rats treated with XA. Treatment with XA also significantly (p<0.0001) increased the activity of SOD, CAT, and APx while it decreased the activity of MPO and MDA levels. Xylopic acid possesses anti-colitic activity in rats induced with acetic acid colitis.

Adenylyl cyclase 6 is involved in the hyposecretory status of experimental colitis.

One of the cardinal symptoms of intestinal inflammation is diarrhea. Acute intestinal inflammation is associated with inhibition of ion absorption and increased secretion, along with fluid leakage due to epithelial injury and changes in permeability. However, in the chronic situation, a downregulation of both absorptive and secretory transport has been reported. We investigated how experimental colitis reduces cAMP levels in intestinal epithelial cells through modulation of adenylyl cyclases (AC). Primary colonic epithelial cells obtained from rats with trinitrobenzenesulfonic acid colitis and non-colitic controls were analyzed for AC expression by RT-qPCR and Western blot, following a preliminary microarray analysis. AC6 and AC5 were found to be expressed in colonocytes, and downregulated by inflammation, with the former exhibiting considerably higher mRNA levels in both cases. To test the hypothesis that inflammatory cytokines may account for this effect, Caco 2 cells were treated with IL-1β, TNF-α, or IFN-γ. All three cytokines inhibited forskolin evoked short-circuit currents in Ussing chambers and lowered intracellular cAMP, but failed to alter AC6 mRNA levels. AC5/AC6 expression was however inhibited in mouse jejunal organoids treated with IFN-γ and TNF-α, but not IL-1β. Gene knockdown of AC6 resulted in a significant decrease of ion secretion in T84 cells. We conclude that the disturbances in ion secretion observed in rat TNBS colitis are associated with low intracellular levels of cAMP in the epithelium, which may be explained in part by the downregulation of AC5/AC6 expression by proinflammatory cytokines.

Effect of xylopic acid isolated from Xylopia aethiopica on acetic acid‐induced ulcerative colitis in rats

Xylopic acid (XA) is the principal constituent isolated by bio-fractionation from the dried fruit of Xylopia aethiopica, a known anti-inflammatory plant [1, 2]. The anti-inflammatory activity of xylopic acid has been investigated. In a previous publication, we established that the observed anti-inflammatory effect of Xylopic acid in H2S-induced paw oedema was as a result of the arachidonic acid pathway Osafo et al. [3]. This study establishes the anti-colitic activity of XA through inhibition of reactive oxygen species generation in vivo. Intrarectal challenge of rats with acetic acid induces colitis, which bears resemblance, in terms of its pathogenesis, histopathology and inflammatory profile, to that in humans. Rats were treated with xylopic acid or sulphasalazine and challenged intrarectally with 4 ml 1% acetic acid and monitored over 8-day period. Macroscopic and microscopic findings were then made after the 8 day study period. The rats treated with XA showed a decreased gross mucosal injury caused by the acetic acid at 10, 30 and 100 mg kg−1. Treatment with XA also resulted in decreased colonic epithelial expression of argyrophylic nucleolar organiser regions (AgNORs) at 10, 30 and 100 mg kg−1 respectively with significant decrease (P < 0.0001) in the quantitative expression of AgNORs/nucleus ratio to levels comparable with non-colitic control. Treatment with XA also significantly (P < 0.0001) increased the activity of SOD, CAT and APx at 10 – 100 mg kg−1 while decreasing the activity of myeloperoxidase (MPO). There was also reduced expression of MDA at the same doses. This study establishes that xylopic acid possesses anti-colitic activity in rats. Support or Funding Information No funding was obtain for this work. Control (untreated) (A), acetic acid control (B), sulphasalazine 500 mg kg−1 (C), XA 10 mg kg−1(D), XA 30 mg kg−1 (E) and XA 100 mg kg−1 (F). (a) Representative pictomicrographs and (b) Average count per 10 nuclei determined at ×10 magnification. [insert: × 40]. Non-colitic control (A), colitic control (B) sulphasalazine 500 mg kg−1 (C), XA 10 mg kg−1 (D), XA 30 mg kg−1 (E) and XA 100 mg kg−1 (F). *P < 0.0001 when compared with colitic control. #P < 0.0001 when compared with non-colitic control; N = 3. Assay of SOD (A), CAT (B), APx (C) and MPO (D) in XA-treated rats. ###P < 0.0001, φP = 0.012, φφP = 0.0012, ttP < 0.001, tttP < 0.0001, ddP = 0.01, dddP = 0.001, aaP < 0.002, rP < 0.0001, vP = 0.00012, xP < 0.0001, zP < 0.05, nsP > 0.05 when compared with non-colitic control; N = 3. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ITraq‐based Proteomics analysis of colon mucosal proteins in a dextran sulfate sodium (DSS)‐induced colitis mouse model and the effects of dietary treatments with edible mushroom Pleurotus eryngii

Our previous studies have demonstrated the protective effects of an edible mushroom Pleurotus eryngii against the colitis in mice. Herein, an itraq‐based proteomics analysis has been utilized to establish the potential mechanism of actions of the mushroom. Three groups of mice (10 mice per group) were treated with DSS in drinking water (the colitic positive control group), DSS in drinking water plus Pleurotus eryngii (1.5% in diet, the treatment group), or regular drinking water (the negative control group), respectively, for 40 days as we described previously. The colon mucosa of mice were collected and subjected to an itraq‐based proteomic analysis. In total, 5794 proteins were identified by LC‐ESI‐MS/MS analysis in the colon mucosal of the mice. Among them, the expression levels of 626 proteins were different (392 up‐regulated proteins and 234 down‐regulated proteins) in the colitic positive control mice in comparison with the noncolitic negative control mice. There were 303 differentially expressed proteins (107 up‐regulated and 196 down‐regulated) in the treatment group compared to that of the positive control t group. The KEGG pathways that these differentially expressed proteins involved in were mainly: Steroid hormone, Arachidonic acid, Basal cell carcinoma, Linoleic acid metabolism, Phospholipase D signaling, viral myocarditis, chemical carcinogenesis, natural killer cell mediated, thyroid hormone signaling pathways. Twenty‐five proteins that were highly upregulated in the colitic positive control group (vs. the negative control group) were found significantly down‐regulated in the treatment group (vs. the colitic positive control group), whereas, twenty‐one proteins that were highly down‐regulated in the colitic positive control group (vs. the negative control group) were found significantly up‐regulated in the treatment group (vs. the colitic positive control group). Importantly, western blot analysis validated the changes in the protein expression described above. These results demonstrated that dietary mushroom treatment reversed, at least partially, the abnormal expression of proteins in the colon mucosa of colitic mice, which may be accounted for the protective effects of Pleurotus eryngii mushroom against pathogenesis of colitis in mice. Overall, our results provided preliminary but critical information on the potential mechanism of Pleurotus eryngii mushroom in inhibiting colitis.Support or Funding InformationThis research work was funded by the National Natural Science Foundation of China under Grant No. 31471927.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intestinal anti-inflammatory activity of the seeds of Raphanus sativus L. in experimental ulcerative colitis models

Intestinal anti-inflammatory activity of the seeds of Raphanus sativus L. in experimental ulcerative colitis models

Expression of alternatively spliced variants of Na–Ca-exchanger-1 in experimental colitis: role in reduced colonic contractility

Inflammation-induced colonic motility dysfunction is associated with a disturbance in Ca(2+) ion transporting mechanisms. The main objective of this study was to identify the types of Na-Ca-exchanger-1 (NCX-1) variants expressed in the rat colon, and how this was affected by colitis. In addition, the effect of colitis on the possible involvement of NCX-1 in the reduced carbachol-induced contraction of the rat colon was examined. Colitis was induced in male Sprague-Dawley rats by intra-rectal instillation of trinitrobenzenesulphonic acid (TNBS). Animals were killed on day 5. Colitis was characterized by estimating myeloperoxidase (MPO) activity, body weight, and histological scores. NCX-1 mRNA and protein variants were confirmed by RT-PCR coupled nucleotide sequencing and by Western blot analysis, respectively. Contractility of the colon segments was studied using standard procedure. There was a significant reduction in body weight of TNBS-treated rats. A significant increase in MPO activity and infiltration of inflammatory cells were observed in the inflamed rat colon. RT-PCR coupled nucleotide sequencing identified NCX-1.3 mRNA variant containing exons B and D. Western blot analysis confirmed 70 and 120 kDa molecular mass NCX-1 protein variants in rat colon. There was no significant difference (p > 0.05) in the level of NCX-1 protein variants in inflamed colon as compared to non-colitis controls. Functional experiments demonstrated that NCX in reverse mode played a role in carbachol-induced contraction of colon, and this was not affected by colitis. These findings demonstrated expression of a NCX-1.3 mRNA splice variant, and 70 and 118 kDa protein variants. Inhibition of the reverse mode of NCX-1 was not different in reduced carbachol-induced contraction between the groups. These findings are interpreted to suggest that NCX-1, though expressed did not play a role in reduced contractility in experimental colitis.

Partial normalization of pubertal timing in female mice with DSS colitis treated with anti-TNF-α antibody.

Inflammatory bowel disease (IBD) and resultant colitis occurring prior to puberty are frequently associated with delayed puberty and losses of growth and bone mineralization. Some of this delay may be due to colonic inflammation and associated systemic inflammation. To date no treatments for IBD have been shown to normalize the timing of puberty. Our objective in this study was to determine whether there is a normalization of the timing of puberty during treatment of colitis using monoclonal antibodies (abs) to tumor necrosis factor (TNF)-α. We induced colitis in 23-day-old C57Bl6 female mice using 3% dextran sodium sulfate (DSS) for 7 days, followed by removal of DSS for an additional 3 days, resulting in 10 days of worsening colitis. DSS-treated mice received either TNF-α ab or Control ab on days 4 and 8 of colitis, while non-colitic Control mice received injections of TNF-α ab (Control + TNF-α ab). All groups were followed for the timing of vaginal opening until day of life 33, when they were euthanized for serum and colon collection. The DSS + TNF-α ab group had lower levels of systemic interleukin (IL)-6 and a partial normalization of the timing of vaginal opening compared to the DSS + Control ab group. There were no differences in weight gain, growth, or colon histological inflammatory scores between the DSS + TNFα ab and DSS + Control ab groups over the course of the experiment. We conclude that anti-TNF-α ab treatment causes a partial normalization of pubertal timing coincident with decreased systemic inflammation in DSS colitis. These data may have implications regarding growth and bone mineralization outcomes in pediatric IBD.

Colonic mucosal lesions associated with low-dose aspirin: A case control study

Objective. Low-dose aspirin (LDA) is widely used because it reduces the risk of vascular events in patients with atherosclerosis. Recently, there has been a substantial increase in prescriptions for LDA. We analyzed the risk of colonic mucosal lesions associated with the long-term use of LDA. Material and methods. Among Japanese patients who underwent a colonoscopy between January 2004 and December 2006, 199 colitis cases and 5764 non-colitis controls were identified after excluding 749 patients based on study criteria. The history of LDA use was compared between the cases and controls and the multivariate (age-, sex- and underlying diseases-) adjusted odds ratio (OR) was estimated using a multiple logistic regression model. Results. The adjusted OR for colonic mucosal lesions associated with LDA use versus non-use was 1.45 [95% confidence interval (CI), 0.87–2.42; p = 0.152]. In terms of gender differences, the OR for LDA-induced colitis in females was significantly increased at 2.55 (95% CI, 1.31–4.94; p = 0.006) but was not significantly increased in males at 0.70 (95% CI, 0.34–1.45; p = 0.334). Conclusions. In females, LDA increased the risk of colonic mucosal lesions, suggesting that LDA may contribute to the pathogenesis of colonic ulceration or colitis. Therefore, it is essential that prescribing physicians be aware of the risk of LDA-induced colonic lesions.

Expression of Na–H exchanger-8 isoform is suppressed in experimental colitis in adult rat: lack of reversibility by dexamethasone

Objectives. Mechanism of the apical transporter Na–H exchanger-8 (NHE-8) regulation was investigated by examining the effects of anti-inflammatory dexamethasone in experimental colitis. In addition, its localization was investigated in the lipid rich membrane domain called membrane rafts. Material and methods. Colitis was induced by trinitrobenzene sulfonic acid (TNBS) and colon segments were removed from 5 day post-TNBS and used to estimate the levels of NHE-8 protein and mRNA using ECL western blot analysis and a competitive RT-PCR method. Myeloperoxidase activity, malondialdehyde levels and histologic changes were evaluated. Results. NHE-8 protein level was decreased in inflamed colon and was not reversed by dexamethasone. However, mRNA levels remained unchanged in inflamed colon. The levels of NHE-8 protein and mRNA were not significantly different in non-colitic control as compared to dexamethasone treated non-colitis. Elevation of myeloperoxidase activity, malondialdehyde and infiltration of inflammatory cells in inflamed colon were suppressed by dexamethasone treatment of colitis significantly. Furthermore, NHE-8 protein was not detected in the detergent resistant membrane (DRM) or lipid rafts, but was present in the detergent sensitive membrane (DRS) fractions. Actin showed its partition similar to NHE-8. On the contrary, NHE-3 was present in both DRM and DRS fractions. Flotillin-1 and caveolin were enriched in the fractions designated as lipid rafts. Conclusions. These findings demonstrate the suppression of NHE-8 protein in inflamed adult rat colon, which seems to be regulated post-transcriptionlly. Furthermore, the absence of NHE-8 in lipid rafts suggests its regulation independent of cAMP or recycling through endocytosis unlike NHE-3 isoform.

Colonic mucosal lesions associated with long-term or short-term administration of nonsteroidal anti-inflammatory drugs

The effects of short- or long-term administration of nonsteroidal anti-inflammatory drugs (NSAIDs) on the colon have not been well characterized. We assessed the risk of developing colonic mucosal lesions according to the duration of exposure to NSAIDs: short-term and/or long-term use. A case-controlled study was performed by reviewing medical records for endoscopic findings, underlying disease, pre-endoscopic symptoms, category of NSAIDs used and duration of use. The patients underwent colonoscopy between January and October 2004, and 75 colitis cases and 1801 non-colitis controls were identified. The prevalence of NSAID use was compared between the cases and controls. The age- and sex- adjusted odds ratios (OR) were estimated using multiple logistic regression models. NSAIDs had been used in colitis cases and non-colitis controls for over six months in 20.0% and 12.7%, and for one week in 4.0% and 2.1%. Overall 76.0% and 85.2% had not received NSAIDs. The adjusted OR (95% confidence interval) for colonic mucosal lesions with short- and long term NSAID administration combined vs. non-use was 2.04 (1.16-3.61). When determined separately for short- and long-term NSAID users, the adjusted ORs were 1.48 (0.42-5.25) and 2.21 (1.19-4.11), compared to non-users. These values signify a trend toward an increased frequency of colonic mucosal lesions with longer use of NSAIDs (P=0.011 for trend). Long-term use of NSAIDs increased the risk of colonic mucosal lesions, suggesting that NSAIDs may contribute to the pathogenesis of colonic ulcer or colitis.

A Short Treatment With an Antibody to Sclerostin Can Inhibit Bone Loss in an Ongoing Model of Colitis

Chronic inflammation leads to bone loss, and increased fracture rates have been reported in a number of human chronic inflammatory conditions. The study reported here investigates the skeletal effects of dosing a neutralizing antibody to the bone regulatory protein sclerostin in a mouse model of chronic colitis. When dosed prophylactically, an antibody to sclerostin (Scl-AbI) did not reduce the weight loss or histological changes associated with colitis but did prevent inflammation-induced bone loss. At the end of the experiment, Scl-AbI-treated animals had a significantly higher femoral BMD (+27%, p < 0.05) than control antibody (Cntrl-Ab)-treated animals. In a second experiment, treatment with Scl-AbI was delayed until colitis had developed, by which time the mechanical properties of femurs in colitic animals were significantly worse than those of healthy age-matched control mice (maximum load, -26%, p < 0.05; energy, -37%, p < 0.05; ultimate strength, -33%, p < 0.05; elastic modulus, -17%, p < 0.05). A short treatment with Scl-AbI halted bone loss and reversed the decline of both intrinsic and extrinsic mechanical properties of the femur such that, after 19 days of treatment, the bone mechanical properties in the Scl-AbI-treated animals were not significantly different from those of noncolitic age-matched controls. Serum markers of bone formation and resorption suggested that the antibody to sclerostin stimulated osteoblast activity and inhibited osteoclast-mediated bone resorption.

Probiotic administration alters the gut flora and attenuates colitis in mice administered dextran sodium sulfate

Probiotics are used in the therapy of inflammatory bowel disease. This study aimed to determine whether prior administration of probiotic lactobacilli and bifidobacteria would prevent disease and change gut flora in an animal model of colitis. Swiss albino mice received a probiotic mixture (four Lactobacillus and four Bifidobacterium species) or medium (control) for a week prior to induction of colitis by oral 4% dextran sodium sulfate (DSS) for seven days. Appropriate non-colitis controls were used. Histological damage was assessed (n = 5 per group), as was expression of mRNA for tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, transforming growth factor (TGF)-beta1 and SOCS-1 in the colonic mucosa (n = 6 per group). Secretion of TNF-alpha was measured in distal colon organ culture (n = 5-6 per group). Levels of Bacteroides, Bifidobacterium, and Lactobacillus acidophilus in feces were quantified by real time polymerase chain reaction (PCR) targeting 16S rDNA. Compared to untreated DSS colitis, probiotic treatment significantly reduced weight loss (P < 0.05), shifted histological damage to lesser grades of severity (P < 0.001), reduced mRNA expression of TNF-alpha and TGF-beta1 (P < 0.05), and down-regulated production of TNF-alpha from distal colon explants (P < 0.05). Colitis induced a significant reduction in the relative proportions of Bifidobacterium, Bacteroides and Lactobacillus acidophilus group bacteria in feces, and these levels were significantly increased in probiotic-treated mice compared to DSS mice (P < 0.001). Prior administration of probiotic bacteria reduced mucosal inflammation and damage in DSS-induced colitis. DSS colitis was associated with significant changes in the fecal anaerobic bacterial flora and these changes were modulated by administration of probiotic bacteria.

Quantitive Cytokine mRNA Expression Profiles in the Colonic Mucosa of Patients with Steroid Naïve Ulcerative Colitis During Active and Quiescent Disease

Cytokines have validated roles in the immunopathogenesis of inflammatory bowel disease (IBD). This study was to investigate the expressions of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-8, and IL-10 mRNAs in the colonic mucosa of patients with ulcerative colitis (UC) during active and quiescent UC. At colonoscopy, biopsies were taken from inflamed and non-inflamed mucosa of patients with steroid-naive UC (n = 15), non-IBD inflammatory colitis controls (ICC, n = 6), and non-colitis controls (NCC, n = 14). The presence of extensive mononuclear cells and neutrophils infiltrate in the lamina propria, cryptitis, and epithelial damage defined an inflammatory lesion in the mucosa. Quantitative cytokine mRNA expressions in biopsies were measured by real-time polymerase chain reaction (PCR). Of 15 UC patients, 3 remitted with 5-aminosalicylate and 11 received granulocytapheresis; of these, 10 remitted. At baseline, IL-6, IL-8, TNF-alpha, and IL-10 mRNAs were high in inflamed mucosa compared with NCC (P < 0.01). In active UC, IL-6, IL-8 and IL-10 mRNAs were high compared with non-inflamed mucosa (P = 0.03, P = 0.03, P < 0.05, respectively). Both TNF-alpha mRNA (P = 0.03) and IL-6 mRNA (P = 0.04) were higher in UC compared with ICC. Even in non-inflamed mucosa, IL-8 and TNF-alpha mRNA expressions were high compared with NCC. Both IL-6 and IL-8 mRNAs decreased to normal levels after granulocytapheresis. During active UC, all 4 cytokine mRNA levels were high; only IL-6 and IL-8 mRNAs decreased to normal levels during remission. IL-8 mRNA was high even at sites of endoscopically quiescent UC during active disease. Steroid naïve patients respond well to granulocytapheresis.

Extended lifespan and long telomeres in rectal fibroblasts from late-onset ulcerative colitis patients

Ulcerative colitis (UC) is characterized by damage to the intestinal epithelium and connective tissue. The causes of this damage could include changes in the ability of colonic fibroblasts to heal wounds and maintain epithelial cell proliferation. Telomeres shorten with each cell division and eventually signal senescence. The aim of this study is to investigate whether the impaired function of rectal fibroblasts in UC is due to accelerated telomere shortening, oxidative stress and premature senescence. We isolated rectal fibroblasts from eight UC patients and nine non-colitis controls, and recorded their in-vitro lifespans. Telomere lengths and superoxide dismutase mRNA expression were also measured by real-time polymerase chain reaction and peroxide levels were measured by flow cytometry. The fibroblast lifespan decreased as patient age increased (R2=0.68, P=0.003) in control patients, but this relationship was absent in UC fibroblasts. We identified a group of patients who were diagnosed later in life than a second group (59 versus 35 years, P=0.002). Fibroblasts from these late-onset UC patients underwent significantly more population doublings before senescence than age-matched controls (25 versus 15, P=0.02). Slower in-vitro telomere shortening rates (32 versus 344, P=0.006) and trends towards longer telomeres at explant were also observed in late-onset UC fibroblasts. Peroxide levels correlated positively with telomere shortening rate (r=0.581, P=0.078). Some UC-predisposed individuals may have more efficient antioxidant systems that protect the telomeres from oxidative damage. This may allow their rectal fibroblasts to live longer, function better and thus delay the onset of the disease until later life.

Dietary Antigen‐Specific T‐Cell Responses: Switch from an Interleukin‐10‐Dominated Response in Normal Mice to a T‐Helper 1 Cytokine Profile in Gαi2‐Deficient Mice prior to Colitis

We investigated dietary antigen-specific T-cell responses in mesenteric lymph nodes (MLN) and Peyer's patches (PP) in noncolitic control mice as well as in colitis-prone mice prior to onset of histological active colitis. T cells were restimulated in vitro with constituents isolated from the mouse diet. Interestingly, MLN T cells of littermate G(alpha)i2+/- control mice responded to soya with high production of interleukin (IL)-10, but did not produce proinflammatory T-helper 1 (Th1) cytokines. Recall dietary antigen stimulation of G(alpha)i2+/- PP T cells did not result in increased IL-10 production above the spontaneous production in the absence of antigenic stimulation. In strong contrast, MLN T cells from precolitic G(alpha)i2-/- mice produced high levels of interferon-gamma (IFN-gamma) upon restimulation with soya, which could be abolished using a major histocompatibility complex class II-blocking antibody. In conclusion, the present study demonstrates that MLN T lymphocytes in normal healthy mice respond with a significantly increased production of the regulatory cytokine IL-10 on re-encounter with dietary proteins in vitro. In marked contrast precolitic G(alpha)i2-/- mice respond to dietary antigens with a Th1-dominated cytokine response in the mucosa, prior to onset of colitis, with excessive IFN-gamma production. These results suggest that aberrant immune responses to dietary antigens could contribute as a potential pathogenic mechanism in the onset of colitis in G(alpha)i2-deficient mice.

Cytomegalovirus infection in steroid-refractory ulcerative colitis: a case-control study.

Cytomegalovirus (CMV) infection is reported to be a cause of steroid-refractory ulcerative colitis (UC), but the strength of this association has not been tested in a case control study. Controlled studies have also not been performed to determine the sensitivity of available immunohistochemical techniques to detect CMV in this setting. The pathology database at Stanford Hospital was searched for UC patients with a diagnosis of "severe colitis" between the years 1992 and 2002 and medical records were reviewed. Forty patients were identified with refractory UC, defined as poor response to highdose systemic steroids for >2 weeks. Another group of 40 patients with severe, but nonrefractory, UC was case-matched for age and year of biopsy. A series of 40 patients who underwent colectomy for reasons other than inflammatory bowel disease with representative sections of "normal" colon were selected as noncolitis controls. CMV inclusions were detected on hematoxylin and eosin (H&E) in 2 of 40 patients with refractory UC, but not in other patients. Immunohistochemistry (IHC) detected CMV in 10 of 40 (25%) patients with refractory UC and 1 of 40 (2.5%) patients with nonrefractory UC (P = 0.007). The CMV-positive cases initially identified on IHC but not on H&E were re-reviewed for viral inclusions on H&E: 3 had rare, but typical, inclusions; 3 had atypical inclusions; and 3 had no inclusions. CMV was not detected by H&E or IHC in 40 noncolitis controls. Of 10 steroid-refractory UC patients with CMV detected, 7 were refractory to cyclosporin or 6-mercaptopurine/azathioprine (70%) and 6 had undergone proctocolectomy (60%) prior to detection of the CMV. Two patients with recognized CMV infection were treated with gancyclovir, improved, and were able to taper off steroids and avoid proctocolectomy. This study provides evidence that unrecognized and therefore untreated CMV infection is significantly associated with steroid-refractory UC. Moreover, IHC is more sensitive than H&E for detection of CMV and should be considered as part of the routine evaluation of steroid-refractory UC patients, before proceeding with other medical or surgical therapy that may be unnecessary once the CMV is treated.