Colitis Scores Research Articles

Thymoquinone Prevents and Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice

Thymoquinone (TQ), an active ingredient of the seed oil extract of Nigella sativa Linn, has previously been shown to possess antitumor, antioxidant, and anti-inflammatory bioactivity. Whether TQ has any effect on colitis remains controversial. The aim of this study was to determine whether treatment with TQ prevents and ameliorates colonic inflammation in a mouse model of inflammatory bowel disease. C57BL/6 murine colitis was induced by the administration of dextran sodium sulfate (DSS) (3 % W/V) in the drinking water supplied to the mice for 7 consecutive days. The mice with colitis were treated with 5, 10, or 25 mg/kg TQ orally, and changes in body weight and macroscopic and microscopic colitis scores were examined. In addition, biochemical analyses were conducted. The treatment of mice with TQ prevented and significantly reduced the appearance of diarrhea and body weight loss. These results were associated with amelioration of colitis-related damage, as measured by macroscopic and microscopic colitis scores. In addition, there was a significant reduction in colonic myeloperoxidase activity and malondialdehyde levels and an increase in glutathione levels. These results indicate that TQ administration can prevent and improve murine DSS-induced colitis. These findings suggest that TQ could serve as a potential therapeutic agent for the treatment of patients with inflammatory bowel disease.

Omega‐3 supplementation prevents intestinal inflammation by inhibiting the expansion of an intestinal pathobiont in IL10−/− mice

The human microbiome of Western populations has changed over the past century, brought on by new environmental triggers that often have a negative impact on human health. Previously we showed consumption of a diet high in saturated (anhydrous milkfat‐derived) fat (MF) but not polyunsaturated (safflower oil‐derived)‐fat (PUFA) changes the conditions for microbial assemblage and promotes expansion of a rare, immunogenic, sulfite‐reducing pathobiont, Bilophila wadsworthia. This was associated with a proinflammatory TH1 immune response and development of colitis in genetically susceptible IL‐10−/− mice in both specific pathogen free (SPF) and germ‐free environments. The current study examines whether omega‐3 supplementation can attenuate the effect of the MF diet by reducing the bacterial load of B. wadsworthia and subsequent inflammation. SPF IL10−/− mice were adapted to either a low‐fat (LF), high saturated milk‐fat (MF), fish oil supplemented (FO), or safflower oil supplemented (SO) diets. All three high fat diets were isocaloric with fat comprising 37% total kcal, with the supplemented groups representing 32% MF + 5% FO or SO. After 10wk, 454 pyrosequencing of cecal contents and subsequent principal component analysis revealed colonic bacterial communities of mice on the FO diet were significantly different than those on MF and SO diets. Particularly noticeable was the decrease in abundance of B. wadsworthia in the FO treated mice. This observation was accompanied by reduced levels of IL‐6, IL‐17, IL12p70, and IFNγ, as well as reduced histological colitis scores compared to MF and SO fed mice. Together these data suggest omega‐3 supplementation may decrease risk for inflammatory bowel diseases by suppressing pro‐inflammatatory pathobionts such as B. wadsworthia. Supported by NIH F31.Grant Funding Source : NIH

P404 Acute severe ulcerative colitis: The last twelve years in Edinburgh

Background Acute severe ulcerative colitis (ASUC) remains a common reason for urgent colectomy, yet there are relatively few large cohort studies exploring prognosis and outcome. This study aims to examine presentation and management of ASUC in the Western General Hospital, a tertiary referral centre in Edinburgh, UK and to identify prognostic factors. Methods Patients were identified from a large database of participants in genetic studies in Edinburgh, as well as from two previous small cohorts of ASUC studied in Edinburgh. More recent cases were found from minutes of a weekly IBD meeting. Cases were included if they met the standard clinical, radiological and pathological criteria for ulcerative colitis and required an admission for 3 days or more requiring intravenous corticosteroids and/or colectomy. Two cohorts were analysed, one with full clinical detail (97 admissions in 86 patients) and one with more basic detail (444 admissions in 323 patients). Results Overall colectomy rate was 31.8%. Haemoglobin, C-reactive protein and albumin at days 0 and 3 were significant predictors of colectomy in both cohorts (p<0.05 in each case), while in the detailed cohort day 3 but not day 0 stool frequency was predictive (p<0.001 and 0.81 respectively). A simple score was derived to predict colectomy at admission based on disease extent, albumin and CRP. Scores of 0, 1, 2 and 3 corresponded to risks for colectomy of 10%, 31%, 61% and 75%. For day 3 parameters, both the Edinburgh acute colitis (Ho) score (figure 1) and Travis criteria performed well. Conclusions ASUC remains an important cause of colectomy. This study confirms the prognostic value of the Ho score and Travis criteria at day 3, but also indicates that day 0 CRP and albumin are strong predictors of outcome.

P073 ADAMDEC1: A novel molecule in inflammation and bowel disease

Background: Innate immunity is attenuated in patients with Crohn's disease (CD) with impaired neutrophil recruitment to skin and bowel, delayed clearance of E. coli from the skin, and impaired secretion of pro-inflammatory cytokines from macrophages (Marks et al, 2006; Smith et al, 2009). The primary defect of acute inflammation results in failure to eradicate bacterial flora entering the bowel wall leading to the chronic granulomatous inflammation characteristic of CD. Microarray analysis of monocyte derived macrophage mRNA expression, confirmed by qPCR, revealed that ADAMDEC1 (ADAM like Decysin1) was under-expressed in 10% (6/60) of CD patients. ADAMDEC1, a Metalloprotease and Decysin, is part of a family of proteins involved in wound healing and tissue repair, and is almost exclusively expressed in macrophages, dendritic cells and the gastrointestinal tract. To determine the role of this protein we examined E. coli induced inflammation and Dextran Sodium Sulphate (DSS) colitis in Adamdec1 knockout (KO) mice. Method: In an acute colitis model, Adamdec1 KO mice were exposed to 2% DSS for 7 days. Controls, wild type (WT) litter mates, were age, weight and sex matched (n=11 per group). Clinical colitis scores (weight loss, PR blood, loose stool) were recorded daily. Histology was obtained from small and large bowels. For bacterial inflammation, 5x108 heat killed E. coli (HkEc) were injected subcutaneously (SC) into two sites on the backs of KO andWTmice (n=8 per group). Mice were weighed, injection sites inspected for ulceration and subcutaneous inflammatory nodules measured daily. Injection sites were excised at different times for histology and identification of infiltrating cells by FACS. Results: Adamdec1 KOmice were more susceptible to DSS colitis. They demonstrated higher clinical colitis scores with an earlier and more dramatic weight loss (p<0.001). A more florid inflammatory response was seen on histology. In response to a subcutaneous injection of E. coli, Adamdec1 KOmice had smaller inflammatory nodules and less ulceration at the injection sites after 48-72 hours, than WT mice (p<0.001). Conclusion: Mice lacking Adamdec1 develop a phenotype that closely mirrors that observed in patients with CD, an attenuated and delayed E.coli induced acute inflammatory response and an increased susceptibility to bowel inflammation. These results suggest ADAMDEC1 plays an important role in the acute inflammatory response to bacteria and has a protective role within the intestine; reduced levels may have a pivotal role in the development and persistence of CD. Studies are currently underway to further investigate the impaired cellular recruitment and potentially defective bacterial clearance at these early stages of inflammation in our model which could predispose to a more exuberant secondary response and chronic inflammation.

Differential effects of energy balance on experimentally-induced colitis

To characterize the influence of diet-induced changes in body fat on colitis severity in SMAD3-/- mice. SMAD3-/- mice (6-8 wk of age) were randomly assigned to receive a calorie restricted (30% of control; CR), control (CON), or high fat (HF) diet for 20 wk and were gavaged with sterile broth or with Helicobacter hepaticus (H. hepaticus) to induce colitis. Four weeks after infection, mice were sacrificed and the cecum and colons were processed for histological evaluation. Dietary treatment significantly influenced body composition prior to infection (P < 0.05), with CR mice having less (14% ± 2%) and HF-fed mice more body fat (32% ± 7%) compared to controls (22% ± 4%). Differences in body composition were associated with alterations in plasma levels of leptin (HF > CON > CR) and adiponectin (CON > HF ≥ CR) (P < 0.05). There were no significant differences in colitis scores between CON and HF-fed mice 4 wk post-infection. Consistent with this, differences in proliferation and inflammation markers (COX-2, iNOS), and infiltrating cell types (CD3+ T lymphocytes, macrophages) were not observed. Unexpectedly, only 40% of CR mice survived infection with H. hepaticus, with mortality observed as early as 1 wk following induction of colitis. Increased adiposity does not influence colitis severity in SMAD3-/- mice. Importantly, caloric restriction negatively impacts survival following pathogen challenge, potentially due to an impaired immune response.

Characterization of the APC presenting a microbial polysaccharide to regulatory T cells

Commensal bacteria harbored in the mammalian gut are possibly reservoirs of immunomodulatory molecules having profound effects on the host immune system. Polysaccharide A (PSA) from the human symbiont Bacteroides fragilis is one such molecule and has been demonstrated to have potent anti-inflammatory properties in various murine models. Induced IL-10 liberated from CD4+T cells is a key feature of this immunoregulatory activity. However, little is known about how these immunoregulatory T cells are induced in order to control inflammation. We hypothesized that a subset of Dendritic cells (DCs) may provide the cellular platform for PSA action. This hypothesis was supported by our initial finding that following oral gavage with B fragilis in healthy mice, PSA dependent augmentation of CD4+CD25+FOXP3+ Tregs correlated with the frequency of B220+CD11cint nonconventional DCs(R2=0.6993, p=0.038) in the MLN. The B220+CD11cint cell population is primarily Plasmacytoid DCs (PDCs), which characteristically have surface expression of PDCA-1, Siglec H, and Ly6c. These cells have been implicated as tolerogenic DCs in other inflammatory murine models. Interestingly, the tolerogenic role of PDCs has been described in humans as well. However, while TLR2 ligation has not been felt to be important to PDC activation, TLR2 ligation by PSA has been described to be essential for this polysaccharide to activate the immune system. To investigate whether PDCs respond to PSA in a TLR2 dependent way, we investigated the in vitro interaction between PSA and DCs derived from bone marrow cells stimulated with Flt3L. After 36 hours of culture, PDCA-1+B220+CD11c+ PDCs in the presence of PSA showed substantially higher expression of TLR2 compared to media controls. PDCs isolated with PDCA-1 microbeads from a pool of bone marrow derived DCs, significantly augmented the liberation of IL-10 from CD4T cells in presence of PSA. In a co-culture assay, PSA stimulated liberation of IL-10 from CD4T cells was significantly reduced when compared to PDCs derived from TLR2-/- mice (p=0.0046). In the intra-rectal TNBS colonic inflammation model, the frequency of Siglec H+B220+CD11b-CD11c+ PDCs and the GMFI of Siglec H correlated inversely with cumulative colitis score (R2=0.4377, p=0.0002 and R2=0.4255, p=0.0003) suggesting a possible immunoregulatory role for PDCs in PSA mediated protection. Interestingly, in TNBS treated TLR2-/-mice who were not protected by PSA, no increase in GMFI of Siglec H was seen. In order to confirm a role for PDCs in PSA mediated protection in this colitis model, we depleted PDCs with a monoclonal antibody (anti-PDCA-1) and compared disease score to isotype controls. We observed that among control animals not treated with PSA, neither the cumulative disease score nor the accumulation of potentially pathogenic CD11c-CD11b+ myeloid cells in the colon was reduced in anti-PDCA-1 treated animals compared to isotype controls. Both these parameters (pathogenic CD11c-CD11b+ myeloid cells in the colon and clinical score) were significantly reduced in PSA treated animals compared to controls (p= < 0.01). However, PSA failed to protect mice when PDCs were depleted with PDCA-1 mAb treatment. This suggests that in PSA-induced immunoregulation of a model colitis, PDCs have a tolerogenic or immunoregulatory role which may be critical in generating regulatory features and function in T cells.

Pretreatment with Interferon-γ Enhances the Therapeutic Activity of Mesenchymal Stromal Cells in Animal Models of Colitis

Mesenchymal stromal cells (MSCs) are currently under investigation for the treatment of inflammatory disorders, including Crohn's disease. MSCs are pluripotent cells with immunosuppressive properties. Recent data suggest that resting MSCs do not have significant immunomodulatory activity, but that the immunosuppressive function of MSCs has to be elicited by interferon-γ (IFN-γ). In this article, we assessed the effects of IFN-γ prestimulation of MSCs (IMSCs) on their immunosuppressive properties in vitro and in vivo. To this end, we pretreated MSCs with IFN-γ and assessed their therapeutic effects in dextran sodium sulfate (DSS)- and trinitrobenzene sulfonate (TNBS)-induced colitis in mice. We found that mice treated with IMSCs (but not MSCs) showed a significantly attenuated development of DSS-induced colitis. Furthermore, IMSCs alleviated symptoms of TNBS-induced colitis. IMSC-treated mice displayed an increase in body weight, lower colitis scores, and better survival rates compared with untreated mice. In addition, serum amyloid A protein levels and local proinflammatory cytokine levels in colonic tissues were significantly suppressed after administration of IMSC. We also observed that IMSCs showed greater migration potential than unstimulated MSCs to sites within the inflamed intestine. In conclusion, we show that prestimulation of MSCs with IFN-γ enhances their capacity to inhibit Th1 inflammatory responses, resulting in diminished mucosal damage in experimental colitis. These data demonstrate that IFN-γ activation of MSCs increases their immunosuppresive capacities and importantly, their therapeutic efficacy in vivo.

P-Selectin Glycoprotein Ligand-1 Is Needed for Sequential Recruitment of T-Helper 1 (Th1) and Local Generation of Th17 T Cells in Dextran Sodium Sulfate (DSS) Colitis

Activated effector T cells contribute to tissue injury observed in inflammatory bowel disease. T cells are recruited to effector sites after activation in peripheral lymph nodes directs expression of tissue-specific homing receptors. One such mechanism for effector T cell recruitment employs activation-induced fucosylation of P-selectin glycoprotein ligand (PSGL)-1 that mediates binding to endothelial P-selectin. Here we examine the differential role of PSGL-1 in recruiting effector T-cell subsets in colitis. C57BL/6 wildtype and PSGL-1 mice received 2.5% dextran sodium sulfate (DSS) for 6 days and were euthanized 7 and 14 days after the initiation of DSS. Disease activity was monitored throughout. Histologic colitis scores, colonic CD4+ accumulation, and cytokine production were assessed at days 7 and 14. Recruitment of T-helper (Th) subsets was assessed by enumerating adoptively transferred Th1 or Th17 CD4+ cells 2 days after transfer to DSS-treated mice. DSS colitis increases CD4+ T cells in colonic tissue and induces Th1 (interferon gamma [IFN-γ], tumor necrosis factor [TNF]) and Th17 (interleukin [IL]-17, IL-22) cytokines. Loss of PSGL-1 attenuates DSS colitis, decreases colonic CD4+ T cell numbers, and reduces both Th1 and Th17 cytokine production. Colitis increases recruitment of Th1 (19-fold) and Th17 (2.5-fold) cells. PSGL-1 deficiency in transferred T cells abrogates colonic recruitment of Th1 cells in DSS colitis, whereas Th17 recruitment is unaffected. PSGL-1 selectively controls Th1 recruitment in colitis. Whereas Th17 recruitment is independent of PSGL-1, generation of colonic Th17 cytokine requires initial Th1 recruitment. Therefore, attenuating PSGL-1 binding may prevent colonic recruitment of disease-causing Th1 cells that promote local Th17 generation.

Changes of colonic endocrine cells in trinitrobenzene sulfonic acid (TNBS)-induced rat colitis

In this study, immunohistochemistry was used to examine the changes in the density of colonic endocrine cells - argyrophil and argentaffin cells, chromogranin A (CGA), serotonin, somatostatin and glucagon-containing cells in trinitrobenzene sulfonic acid (TNBS)-induced rat colitis. Ulcerative colitis was induced by the instillation of 10 mg of TNBS into the colonic lumen through the anus. To confirm the inducement of ulcerative colitis, the macroscopic and microscopic scores as well as the colonic myeloperoxidase (MPO) activities were monitored for 8 days after TNBS instillation in the colonic lumens. In addition, the number of argyrophil and argentaffin cells, CGA, serotonin, somatostatin and glucagon-immunoreactive cells were counted in the colonic mucosa, respectively. After TNBS instillation into the lumen of the colon from the anus in rats, increases in macroscopic and microscopic scores in the colon tissues were observed along with increases in the colonic MPO activities. Therefore, ulcerative colitis was relatively well induced by the TNBS instillations. Marked decreases in the number of colonic endocrine cells were detected in the TNBS-treated animal compared to the sham control. These results suggest that colonic endocrine cells were also disrupted by TNBS-induced ulcerative colitis.

The MUC13 cell-surface mucin protects against intestinal inflammation by inhibiting epithelial cell apoptosis

Background and AimsThe MUC13 transmembrane mucin is highly and constitutively expressed in the small and large intestine. Although MUC13 polymorphisms have been associated with human inflammatory bowel diseases and susceptibility...

Mechanisms underlying the development of inflammatory bowel disease: the role of rankl in dendritic cell activity

IntroductionUp to 40% of patients with inflammatory bowel disease (IBD) also suffer with osteoporosis.1 Receptor activator of NF-kB ligand (RANKL) promotes the formation of bone resorbing osteoclasts and is linked...

Fish oil modulates cytolytic lymphocytes during H. hepaticus‐induced colitis in mice

Increasing evidence suggests an important role for natural killer (NK) cells in providing innate defense against pathogenic bacteria. We reported that feeding fish oil enriched with decosahexaenoic acid (DFO) exacerbated colitis and promoted colonic dysplasia in SMAD3−/− mice following infection with H. hepaticus. Flow cytometric analysis of secondary lymphoid tissues suggested alterations in NK cell populations. The objective of this study was to determine the effect of DFO on cytolytic lymphocyte infiltration at the local site of inflammation/dysplasia by immunohistochemistry. Mice were fed AIN‐93G diets modified to include corn oil (CO) or DFO (0.75–6.0 %) as the fatty acid source for 8 wks, gavaged with H. hepaticus to induce colitis, then euthanized 4 wks post‐infection. Colons were removed and processed for analysis of cytolytic cells (DX5 and CD3) as well as the granzyme B by immunohistochemistry. We found a progressive increase in tumor associated CD3+ and DX5+ lymphocytes as well as granzyme B staining as dietary DFO increased, consistent with increased colitis scores. However, despite significant infiltration of cytolytic cells in the 6% DFO group, the intensity of granzyme B staining was reduced. These data imply that high levels of DFO may impair activation of cytolytic lymphocytes potentially contributing to accelerated tumor formation previously reported.Grant Funding Source: n/a

Fish oil increases tumor associated T regulatory cells in experimentally induced colitis

Our laboratory recently reported the unexpected observation that feeding fish oil enriched with docosahexaenoic acid (DFO) exacerbated colitis and colonic dysplasia in SMAD3−/− mice. These effects were associated with reduced T cell populations in secondary lymphoid tissue. The objective of this study was to determine the effect of DFO on immune cell populations and markers of inflammation at the local site of inflammation/dysplasia by immunohistochemistry. Mice were fed AIN‐93G diets modified to include corn oil (CO), safflower oil (SF), or DFO (0.75–6.00%) as the fatty acid source for 8 wks, gavaged with H. hepaticus to induce colitis, then sacrificed 4 wks post‐infection. We found significant differences between dietary treatments for multiple T cell markers in the proximal colon. Compared to controls, total T lymphocytes as well as FoxP3 positive cells were more prominent in the 3.75 and 6% DFO diets, which corresponded with the highest colitis scores. Staining for iNOS and COX‐2, markers associated with colon carcinogenesis, did not differ across treatments. These data suggest that DFO promotes an immunosuppressive environment at the tumor site with strong direction by T regulatory cells, shifting from a Th1 type to Th2 type immune response. The observed changes in immune cell populations and suppressed cell‐mediated immunity may begin to explain the exacerbated colitis.Grant Funding Source: n/a

Salmonella-mediated gene therapy in experimental colitis in mice

Bacterial gene therapy - bactofection is a simple and effective method to deliver plasmid DNA into target tissue. We hypothesize that oral in vivo bactofection can be an interesting approach to influence the course of inflammatory bowel diseases. The aim of this study was to prove the effects of antioxidative and anti-inflammatory bactofection in dextran sulfate sodium (DSS)-treated mice. Attenuated bacteria Salmonella Typhimurium SL7207 carrying plasmids with genes encoding Cu-Zn superoxide dismutase and an N-terminal deletion mutant of monocyte chemoattractant protein-1 were prepared. Male Balb/c mice had ad libitum access to 1% DSS solution in drinking water during 10 days (mild model of colitis). The animals were daily fed with 200 Mio bacteria via gastric gavage during the experiment. Fecal consistency, clinical status, food and water intake were monitored. After 10 days samples were taken and markers of oxidative stress and inflammatory cytokine levels were measured. Colonic tissue was scored histologically by a blinded investigator. DSS treatment significantly increased the levels of inflammatory cytokines and malondialdehyde as a marker of lipoperoxidation in the colon. Anti-inflammatory gene therapy improved the total antioxidative capacity. In comparison with the untreated group, bacterial gene therapy lowered the histological colitis score. Salmonella-mediated antioxidative and anti-inflammatory gene therapy alleviated colitis in mice. The effect seems to be mediated by increased antioxidative status. Further studies will show whether recombinant probiotics expressing therapeutic gene might be used for the therapy of inflammatory bowel diseases.

Intestinal alkaline phosphatase has beneficial effects in mouse models of chronic colitis

The brush border enzyme intestinal alkaline phosphatase (IAP) functions as a gut mucosal defense factor and is protective against dextran sulfate sodium (DSS)-induced acute injury in rats. The present study evaluated the potential therapeutic role for orally administered calf IAP (cIAP) in two independent mouse models of chronic colitis: 1) DSS-induced chronic colitis, and 2) chronic spontaneous colitis in Wiskott-Aldrich Syndrome protein (WASP)-deficient (knockout) mice that is accelerated by irradiation. The wildtype (WT) and IAP knockout (IAP-KO) mice received four cycles of 2% DSS ad libitum for 7 days. Each cycle was followed by a 7-day DSS-free interval during which mice received either cIAP or vehicle in the drinking water. The WASP-KO mice received either vehicle or cIAP for 6 weeks beginning on the day of irradiation. Microscopic colitis scores of DSS-treated IAP-KO mice were higher than DSS-treated WT mice (52±3.8 versus 28.8±6.6, respectively, P<0.0001). cIAP treatment attenuated the disease in both groups (KO=30.7±6.01, WT=18.7±5.0, P<0.05). In irradiated WASP-KO mice cIAP also attenuated colitis compared to control groups (3.3±0.52 versus 6.2±0.34, respectively, P<0.001). Tissue myeloperoxidase activity and proinflammatory cytokines were significantly decreased by cIAP treatment. Endogenous IAP appears to play a role in protecting the host against chronic colitis. Orally administered cIAP exerts a protective effect in two independent mouse models of chronic colitis and may represent a novel therapy for human IBD.

Microencapsulation of budesonide with dextran by spray drying technique for colon-targeted delivery: an in vitro/in vivo evaluation in induced colitis in rat

The aim of this study was developing colon targeted-delivery of budesonide for ulcerative colitis. Microcapsules were prepared using spray drying technique by different drug-to-dextran ratios and three molecular weights (MWs) of polymer. Differential scanning calorimetry, X-ray diffraction (XRD), drug release and loading efficiency of microcapsules were studied. In vivo efficacy of the selected formulation prepared by 1 : 10 drug-to-polymer ratio and dextran with MW 500 000 (D10M500) against acetic acid-induced colitis in rats was evaluated and compared to the control and reference groups (mesalasine and budesonide suspensions). The results showed that D10M500 microcapsules could target the drug to colon and its efficacy in reducing macroscopic damage score was higher than mesalasine suspension. Treatment with D10M500 decreased the scores of crypt damage and total colitis significantly compared to the control group which just received the vehicle and the groups treated with mesalasine and budesonide suspension which could not reduce the colitis parameters significantly.

The phytogenic feed additive Sangrovit modulates dextran sulfate sodium-induced colitis in rats

The alkaloids of Macleaya cordata (Papaveracae) are active components of the phytogenic feed additive Sangrovit. The present study was conducted to evaluate the effects of Sangrovit on dextran sulfate sodium (DSS)-induced colitis using rats as an experimental model. Thirty-five male rats were randomly assigned to a control group (Group 1, n = 5), a Sangrovit group (Group 2, n = 20) and a DSS group (Group 3, n = 10). Group 1 received standard diet and tap water for 14 days. Group 2 received 500 ppm Sangrovit in their feed for 14 days and in the second week 5% DSS was added to the tap water. The animals in Group 3 were fed for seven days with standard diet and tap water and for the next seven days with standard diet and 5% DSS added to their tap water. The rats were sacrificed on day 14 and the following parameters were measured: disease activity (body and organ weight, colon length, presence of blood in stool), colon myeloperoxidase activity, expression of colon cyclooxygenase-2 (cox-2), hematological parameters, histological colitis score and selected parameters of oxidative stress. The animals treated with DSS for seven days (Groups 2 and 3) showed increases in liver and cecum weight, leukocyte count and colon shortening, decreases in hemoglobin and hematocrit associated with hematochezia. In comparison with Group 3 where DSS caused mucosal edema, cellular infiltration and epithelial disruption, the Sangrovit group showed less severe damage to the colon mucosa and decreased histological colitis scores. The Sangrovit group also showed diminished expression of DSS-induced COX-2, significantly mitigated myeloperoxidase activity in colon tissue and level of reduced glutathione in erythrocytes. In conclusion, some parameters of DSS-induced colitis were modulated by 500 ppm Sangrovit added to feed.

Gut microbial diversity is reduced by the probiotic VSL#3 and correlates with decreased TNBS-induced colitis

Compositional changes within the normal intestinal microbiota have been associated with the development of various intestinal inflammatory disorders such as pouchitis and inflammatory bowel diseases (IBD). Therefore, it has been speculated that manipulation of a dysbiotic intestinal microbiota has the potential to restore microbial homeostasis and attenuate inflammation. We performed community composition analyses by terminal restriction fragment length polymorphism (T-RFLP) of the bacterial 16S ribosomal RNA gene to investigate the impact of the probiotic VSL#3 on colonic microbial community composition and development of trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. TNBS-induced chronic colitis was significantly reduced in VSL#3-fed rats compared to controls (P < 0.05). T-RFLP analysis revealed distinct microbial communities at luminal versus mucosal sites. Within the luminal microbiota, chronic colitis was associated with an overall decrease in bacterial richness and diversity (Margalef's richness, P < 0.01; Shannon diversity, P < 0.01). This decrease in luminal microbial diversity was enhanced in TNBS-treated rats fed VSL#3 (Margalef's richness, P < 0.001; Shannon diversity, P < 0.001) and significantly correlated with reduced clinical colitis scores (Pearson correlation P < 0.05). Our data demonstrate that the probiotic VSL#3 alters the composition of the intestinal microbiota and these changes correlate with VSL#3-induced disease protection.

The atypical cannabinoid O-1602 protects against experimental colitis and inhibits neutrophil recruitment

Cannabinoids are known to reduce intestinal inflammation. Atypical cannabinoids produce pharmacological effects via unidentified targets. We were interested in whether the atypical cannabinoid O-1602, reportedly an agonist of the putative cannabinoid receptor GPR55, reduces disease severity of dextran sulfate sodium (DSS) and trinitrobenzene sulfonic acid (TNBS)-induced colitis in C57BL/6N and CD1 mice. DSS (2.5% and 4%) was supplied in drinking water for 1 week while TNBS (4 mg) was applied as a single intrarectal bolus. Both treatments caused severe colitis. Injection of O-1602 (5 mg/kg intraperitoneally) significantly reduced macroscopic and histological colitis scores, and myeloperoxidase activity. The protective effect was still present in cannabinoid receptor 1 (CB₁) and 2 (CB₂) double knockout mice and mice lacking the GPR55 gene. To investigate a potential mechanism underlying the protection by O-1602 we performed neutrophil chemotactic assays. O-1602 concentration-dependently inhibited migration of murine neutrophils to keratinocyte-derived chemokine (KC), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the N-formyl-peptide receptor ligand WKYMVm. The inhibitory effect of O-1602 was preserved in neutrophils from CB₁/CB₂ double knockout and GPR55 knockout mice. No differences were seen in locomotor activity between O-1602-treated and control mice, indicating lack of central sedation by this compound. Our data demonstrate that O-1602 is protective against experimentally induced colitis and inhibits neutrophil recruitment independently of CB₁, CB₂, and GPR55 receptors. Thus, atypical cannabinoids represent a novel class of therapeutics that may be useful for the treatment of inflammatory bowel diseases.

Krüppel-Like Factor 5 Protects Against Dextran Sulfate Sodium−Induced Colonic Injury in Mice by Promoting Epithelial Repair

Krüppel-like factor 5 (KLF5) is a transcription factor that promotes proliferation, is highly expressed in dividing crypt cells of the gastrointestinal epithelium, and is induced by various stress stimuli. We sought to determine the role of KLF5 in colonic inflammation and recovery by studying mice with dextran sulfate sodium (DSS)-induced colitis. Wild-type (WT) and Klf5(+/-) mice were given DSS in the drinking water to induce colitis. For recovery experiments, mice were given normal drinking water for 5 days after DSS administration. The extent of colitis was determined using established clinical and histological scoring systems. Immunohistochemical and immunoblotting analyses were used to examine proliferation, migration, and expression of the epidermal growth factor receptor. Klf5 expression was increased in colonic tissues of WT mice given DSS; induction of Klf5 was downstream of mitogen-activated protein kinase signaling. In DSS-induced colitis, Klf5(+/-) mice exhibited greater sensitivity to DSS than WT mice, with significantly higher clinical and histological colitis scores. In recovery experiments, Klf5(+/-) mice showed poor recovery, with continued weight loss and higher mortality than WT mice. Klf5(+/-) mice from the recovery period had reduced epithelial proliferation and cell migration at sites of ulceration compared to WT mice; these reductions correlated with reduced expression of epidermal growth factor receptor. Epithelial repair is an important aspect of recovery from DSS-induced colitis. The transcription factor KLF5 regulates mucosal healing through its effects on epithelial proliferation and migration.