Experimental Model Of Ulcerative Colitis Research Articles

Thymol has beneficial effects on the experimental model of ulcerative colitis.

Objective:Thymol, a natural aromatic monoterpene phenol derived from thymus, possesses anti-inflammatory benefits. Here, we evaluated the potential of thymol therapy in improving an animal model of ulcerative colitis. Materials and Methods:Luminal instillation of acetic acid was used to induce colitis in male Wistar rats. Treatment groups daily received prednisolone (2 mg/kg, orally) or thymol (100 mg/kg, orally) for 10 consecutive days. Then, the rats were euthanized and tissue specimens were collected for evaluation of cyclooxygenase-2 (COX-2) expression by immunohistochemistry. Furthermore, the levels of total protein, nitric oxide, myeloperoxidase, malondialdehyde, IL-1, IL-6, and TNF-α were monitored in colonic homogenates. Eventually, the relative mRNA expression of IκBα and NF-κBp65 was investigated using reverse-transcriptase PCR (RT-PCR) in colonic homogenates. Results:Both medications could reduce the mortality rate and the clinical scores of ulcerative colitis. The COX-2 expression was significantly reduced in the colons of thymol-treated animals compared to the prednisolone group. Also, the myeloperoxidase activity, nitric oxide level and malondialdehyde intensity were decreased in the colons of thymol-treated animals to a greater extent compared to the prednisolone group. Moreover, the total protein content of guts showed significant increases in the guts of thymol-treated animals in comparison to the prednisolone group. Nonetheless, thymol significantly reduced the levels of IL-6, and IL-1 compared to prednisolone. Both medications caused a significant decrease in the mRNA level of NF-κBp65, though the mRNA level of IκBα did not show significant changes between the groups.Conclusion:Thymol may be a promising agent to ameliorate ulcerative colitis.

Olmesartan ameliorates chemically-induced ulcerative colitis in rats via modulating NFκB and Nrf-2/HO-1 signaling crosstalk

Alteration in the expression pattern of Nrf-2 and NFκB has been reported in ulcerative colitis (UC) in which functional crosstalk between these two critical pathways has been suggested. The ameliorative potential of the AT1R blocker olmesartan (OLM) on oxidative stress and inflammatory cytokines has received considerable attention in recent years. Acetic acid (AA)-induced UC demonstrates close resemblance to human UC regarding histopathological features and cytokine profile and is associated with local intense immune response, oxidative stress and release of inflammatory cytokines. Therefore, The effect of OLM (1, 5 and 10 mg/kg) administered orally to rats subjected to intra-rectal instillation of 2 ml of 3% AA in saline solution is investigated. The study revealed that OLM ameliorated colon injury and inflammatory signs as visualized by histopathological examination. Levels of colon IL-6, TNF-α, IL-1β, TGF-β, and serum CRP were down-regulated, while the level of colon IL-10 was up-regulated. In a dose-dependent manner, OLM suppressed AA-induced neutrophils accumulation and improved colon anti-oxidant defense machinery. Also, OLM repressed the Bax:BCL-2 ratio and caspase3 expression. The mechanism of these protective effects was found to lay behind its ability to down-regulate gene expression and inhibit phosphorylation and nuclear translocation of p65 subunits. On the other hand, OLM up-regulated gene expression of Nrf-2 and HO-1. In conclusion, our data show that OLM is an Nrf2 activator, NFkB inhibitor and apoptosis inhibitor in an experimental model of ulcerative colitis. Overall, the study indicates that OLM shows promise as a potential therapy for the treatment of human inflammatory bowel diseases.

Dimethyl Oxalyl Glycine a Prolyl Hydroxylase Inhibitor Decreases Early Gastrointestinal Gvhd Via Two Independent Pathways of Apoptosis after Allogeneic Hematopoietic Cell Transplantation

Introduction: Allogeneic hematopoietic cell transplantation (HCT) is increasingly utilized in the treatment of hematologic malignant and non-malignant diseases. Therapy-related toxicity and graft versus host disease (GVHD) remain major challenges as they significantly contribute to treatment related morbidity and mortality. Prolyl hydroxylase inhibitors (PHI) interfere with signaling cascades of inflammation and cell death. Their beneficial use in experimental models of ulcerative colitis and lung allograft rejection resulted in the hypothesis, that use of the PHI dimethyl oxalyl glycine (DMOG) reduces GVHD after murine allogeneic HCT. We already showed that DMOG treatment of allogeneic recipients resulted in significantly improved survival by day +50 compared to allogeneic controls, and was associated with significant decrease in organ pathology and proinflammatory cytokines (data not shown). We now expanded our study to better understand the underlying mechanisms for this observation.Methods: Acute GVHD was induced in lethally irradiated BALB/c mice followed by the infusion of 5 million bone marrow cells mixed with 4 million splenocytes from allogeneic C57BL/6 donors. DMOG dissolved in PBS was given at 8mg per mice intraperitoneally while controls received PBS only. Animals were monitored for clinical GVHD and survival and analyzed at day +7 and at day +50 post HCT. Formalin-fixed paraffin sections were processed for immunohistochemistry with CD3, Hif-1 alpha, or FADD pSer191 antibodies. Western blot analysis was performed after CD3/CD28 antibody activation of T cells to see the effect of DMOG treatment on Hif-1 α expression. Effect of DMOG on apoptosis and CD137 expression was analyzed by flow cytometry.Results: Our results demonstrate that the protective effect of DMOG is mainly caused by two independent pathways of apoptosis. DMOG treatment of allogeneic recipients resulted in decreased T cells infiltration (p=0.0534) and (Fas associated death domain) FADDpSer191 level (p<0.05) in small intestine sections at day +50. Decreased FADD pSer191 indicates decreased TNF-a induced epithelial cell apoptosis. This was associated with increased Hif1- α expression at day+7 (p<0.05) as well day+50 (p<0.05) as shown by histochemical analysis of small intestine sections. DMOG treatment of activated T cells showed increased expression of CD137 (p<0.005) and Annexin V FITC (p<0.005) staining suggesting that DMOG subjects T cells to activation induced cell death (AICD) via induced CD137 expression. Microscopic scanning of DMOG treated T cells showed condensed chromatin along with numerous fragmented nuclei, indicative of apoptotic cell. In vitro proliferative T cell responses were suppressed (p<0.05) in the presence of DMOG in a dose-dependent manner along with increase in Hif1- α level (p<0.05) as analyzed by western blot.Conclusion: Our data demonstrates protective effects of DMOG on the GI tract early after allogeneic HCT which is associated with improved survival and reduced lung injury at later time point. DMOG treatment results in the induction of CD137 on T cells subjecting them to AICD and represses FADD level in epithelial cells regulating their apoptosis. Taken together, DMOG treatment reduces GVHD via a two pronged mechanism, possibly allowing for novel strategies to prevent and treat this deleterious disease. DisclosuresPalaniyandi:Pharmacyclics: Research Funding; Takeda Pharmaceutical International, Inc.: Research Funding; JAZZ: Research Funding.

Arginine supplementation reduces colonic injury, inflammation and oxidative stress of DSS-induced colitis in mice

This study evaluated the mechanisms action of arginine in experimental model of ulcerative colitis. C57BL/6 mice were randomized into 4 groups: Control group (C): standard diet; Colitis group (Col): standard diet and DSS solution; Pretreated group (PT): diet supplementation with arginine before and during colitis induction; Treated group (T): diet supplementation with arginine during colitis induction. Colitis was induced by administration of 1.5% DSS for 5 days. Parameters such as intestinal permeability (IP), bacterial translocation (BT), histological and morphometric analysis, cytokines, immunoglobulin A, inflammatory infiltrate, oxidative stress, tight junction, iNOS1Nitric oxide indutase.1 were performed. The Col group showed increased IP, BT, oxidative stress and inflammatory infiltrate in the colon. Arginine decreased IP, BT, oxidative stress and inflammatory infiltrate in the colon. Only, the group T reduced iNOS, cytokine IL-17,2Interleukine 17.2 and increased collagen area and cytokine TGF-β.3Transforming growth factor beta.3 These results suggest that arginine can constitute in potential therapy for colitis.

Catestatin Regulates Epithelial Cell Dynamics to Improve Intestinal Inflammation.

Ulcerative colitis (UC) is characterized by aberrant regulation of tight junctions (TJ), signal transducer and activator of transcription 3 (STAT3), and interleukin (IL)-8/18, which lead to intestinal barrier defects. Catestatin (CST), an enterochromaffin-derived peptide, regulates immune communication and STAT-3 in the inflamed intestine. Here, we investigated the effects of CST during the development of inflammation using human biopsies from patients with active UC, human colonic epithelial cells (Caco2), and an experimental model of UC (dextran sulfate sodium [DSS]-colitis). In UC patients, the protein and mRNA level of CST was significantly decreased. Colonic expression of CST showed a strong positive linear relationship with TJ proteins and STAT3, and a strong negative correlation with IL-8 and IL-18. Intra-rectal administration of CST reduced the severity of experimental colitis, IL-18 colonic levels, maintained TJ proteins and enhanced the phosphorylation of STAT3. CST administration increased proliferation, viability, migration, TJ proteins, and p-STAT3 levels, and reduced IL-8 & IL-18 in LPS- & DSS-induced Caco2 cell epithelial injury, and the presence of STAT-3 inhibitor abolished the beneficial effect of CST. In inflammatory conditions, we conclude that CST could regulate intestinal mucosal dynamic via a potential STAT3-dependent pathway that needs to be further defined. Targeting CST in intestinal epithelial cells (IECs) should be a promising therapeutic approach such as when intestinal epithelial cell homeostasis is compromised in UC patients.

Magnolol, a Natural Polyphenol, Attenuates Dextran Sulfate Sodium-Induced Colitis in Mice.

Magnolol is a lignan with anti-inflammatory activity identified in Magnolia officinalis. Ulcerative colitis (UC), one of the types of inflammatory bowel disease (IBD), is a disease that causes inflammation and ulcers in the colon. To investigate the effect of magnolol in dextran sulfate sodium (DSS)-induced experimental UC model, male C57 mice were treated with 2% DSS drinking water for 5 consecutive days followed by intragastric administration with magnolol (5, 10 and 15 mg/kg) daily for 7 days. The results showed that magnolol significantly attenuated disease activity index, inhibited colonic shortening, reduced colonic lesions and suppressed myeloperoxidase (MPO) activity. Moreover, colonic pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) induced by colitis were dramatically decreased by magnolol. To further unveil the metabolic signatures upon magnolol treatment, mass spectrometry-based metabolomic analysis of the small molecular metabolites in mice serum were performed. Compared with controls, abnormality of serum metabolic phenotypes in DSS-treated mice were effectively reversed by different doses of magnolol. In particular, magnolol treatment effectively elevated the serum levels of tryptophan metabolites including kynurenic acid (KA), 5-hydroxyindoleacetic acid, indoleacetic acid (IAA), indolelactic acid and indoxylsulfuric acid, which are potential aryl hydrocarbon receptor (AHR) ligands to impact colitis. These findings suggest that magnolol exerts anti-inflammatory effect on DSS-induced colitis and its underlying mechanisms are associated with the restoring of tryptophan metabolites that inhibit the colonic inflammation.

Medicinal Plants in the Treatment of Colitis: Evidence from Preclinical Studies.

Ulcerative colitis is a chronic inflammatory condition whose treatment includes aminosalicylates, corticosteroids, and immunomodulators. Medicinal plants seem to be an important alternative treatment for this condition. They have been the subject of a great number of studies in recent years. This study was conducted to systematically review the medicinal plants tested in experimental models of ulcerative colitis. We conducted a systematic literature search through specialized databases (PUBMED, SCOPUS, EMBASE, MEDLINE, LILACS, SCIELO, and SCISEARCH) and selected articles published between January 2000 and June 21, 2016 by using "medicinal plants" and "ulcerative colitis" as key words. Sixty-eight studies were included, and the families Asteraceae and Lamiaceae presented the largest number of studies, but plants from several other families were cited; many of them have shown good results in experimental animals. However, only a few species (such as Andrographis paniculata and Punica granatum) have undergone clinical tests against ulcerative colitis, and the observation that many preclinical studies reviewed are purely descriptive has certainly contributed to this fact. Chemical constituents (mainly flavonoids and terpenes) seem to play a role in the effects of the plants. Thus, the data herein reviewed reinforce the potential of medicinal plants as a source of alternative approaches to the treatment of ulcerative colitis.

Synthesis and evaluation of anti-inflammatory properties of silver nanoparticle suspensions in experimental colitis in mice.

The aim of our study was to investigate the effect of newly developed silver nanoparticle aqueous suspensions NanoAg1 and NanoAg2 in the mouse models mimicking ulcerative colitis and Crohn's disease. NanoAg1 and NanoAg2 were synthesized in aqueous medium with the involvement of tannic acid. To elucidate their anti-inflammatory activity, semi-chronic mouse models of inflammation induced by dextrane sulfate sodium addition to drinking water and intracolonic (i.c.) administration of 2,4,6-trinitrobenzenesulfonic acid were used. NanoAg1 and NanoAg2 (500mg/dm3, 100μl/animal, i.c., once daily) significantly ameliorated colitis in dextrane sulfate sodium- and 2,4,6-trinitrobenzenesulfonic acid-induced mouse models of colonic inflammation, as indicated by reduced macroscopic, ulcer and microscopic scores. The anti-inflammatory effect was dependent on the shape and diameter of silver nanoparticles, as indicated by weaker effect of NanoAg1 than NanoAg2. In addition, administration of NanoAg2, but not NanoAg1, modulated colonic microbiota, as indicated by reduced number of Escherichia coli and Clostridium perfringens, and increased number of Lactobacillus sp. Summarizing, NanoAg1 and NanoAg2 after administered i.c. effectively alleviate colitis in experimental models of ulcerative colitis and Crohn's disease in mice. Therefore, NanoAg1 and NanoAg2 administered i.c. have the potential to become valuable agents for the treatment of inflammatory bowel diseases.

Involvement of PPARγ in the protective action of tropisetron in an experimental model of ulcerative colitis

Inflammatory bowel disease (IBD) is a chronic inflammation of the gastrointestinal (GI) tract. Tropisetron, a selective 5-HT3 receptor antagonist, is highly used to counteract chemotherapy-induced emesis. Previous studies revealed the anti-inflammatory properties of this drug. The aim of this study was to evaluate the role of peroxisome proliferator-activated receptor gamma (PPARγ) receptor in the protective effect of tropisetron in an animal model of ulcerative colitis. Experimental colitis was induced by a single intra-colonic instillation of 4% (V/V) acetic acid in male rats. Tropisetron (3 mg/kg) and GW9662 (PPARγ antagonist) (5 mg/kg) were given twice daily for 2 days after colitis induction. Forty-eight hours after induction of colitis, colon was removed and macroscopic and microscopic features were given. Moreover, colonic concentrations of malondialdehyde (MDA), nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) levels, myeloperoxidase (MPO), and PPARγ activity were assessed. Both macroscopic and histopathological features of colonic injury were markedly ameliorated by tropisetron. Likewise, levels of NO, MDA, TNF-α, and IL-1β diminished significantly (p < .05). GW9662 reversed the effect of tropisetron on these markers partially or completely. In addition, tropisetron increased the PPARγ and decreased the MPO activity (p < .05). Tropisetron exerts notable anti-inflammatory effects in acetic acid-induced colitis in rats, which is probably mediated through PPARγ receptors.

Enhance in pentylenetetrazole or maximal electro shock induced seizures in experimental models of ulcerative colitis: An interplay of altered cytokines level, adhesion molecules, blood brain barrier disruption

Enhance in pentylenetetrazole or maximal electro shock induced seizures in experimental models of ulcerative colitis: An interplay of altered cytokines level, adhesion molecules, blood brain barrier disruption

An inhibitor of the Keap1-Nrf2 protein-protein interaction protects NCM460 colonic cells and alleviates experimental colitis.

Ulcerative colitis (UC) is a chronic relapsing-remitting form of inflammatory bowel disease (IBD) that increases the risk of colorectal cancer, the third most common malignancy in humans. Oxidative stress is a risk factor for the development of UC. The Keap1-Nrf2-ARE pathway is one of the most important defensive mechanisms against oxidative and/or electrophilic stresses. In this study, we identified CPUY192018 as a potent small-molecule inhibitor of the Keap1-Nrf2 PPI, investigated the cyto-protective effects of CPUY192018 on the NCM460 colonic cells and evaluated whether treatment with the inhibitor of the Keap1-Nrf2 PPI exerts protection on an established experimental model of UC induced by dextran sodium sulfate (DSS). Our study clearly demonstrated that CPUY192018 had a cytoprotective effect against DSS in both NCM460 cells and mouse colon via the activation of Nrf2 signaling. These results suggested that activation of Nrf2 by directly inhibiting the Keap1-Nrf2 PPI may be beneficial as a treatment for UC.

Acute dextran sulfate sodium (DSS)-induced colitis promotes gut microbial dysbiosis in mice.

The most widely used and characterized experimental model of ulcerative colitis (UC) is the epithelial erosion, dextran sulfate sodium (DSS)-induced colitis, which is developed by administration of DSS in drinking water. We investigated fecal and colonic mucosa microbial composition and functional changes in mice treated with DSS. C57Bl/6 mice received 5% DSS in drinking water for 5 days. Inflammation was evaluated clinically and by analysis of colonic tissue cytokine levels and C-reactive protein (CRP) in the serum. Colonic mucosa and fecal samples were used for DNA extraction and the V4 region of bacterial 16S rRNA gene was subjected to MiSeq Illumina sequencing. Alpha- and beta-diversities, and compositional differences at phylum and genus levels were determined, and bacterial functional pathways were predicted. DSS increased disease severity, serum CRP and cytokines IL-1β and IL-6, but decreased bacterial species richness, and shifted bacterial community composition. Bacteroides, Turicibacter, Escherichia, Clostridium, Enterobacteriaceae, Clostridiaceae, Bacteroidaceae, Bacteroidales, among other taxa were associated with DSS treatment in fecal and colonic samples. Also, DSS altered microbial functional pathways in both colonic mucosa and fecal samples. The development of colitis in DSS model was accompanied with reduced microbial diversity and dysbiosis of gut microbiota at lower taxonomical levels.

Impact of probiotics on toll-like receptor 4 expression in an experimental model of ulcerative colitis

Toll-like receptors (TLRs) are key components of the innate immune system which trigger antimicrobial host defense responses. This study aimed to investigate the impact of probiotics (Lactobacillus, Bifidobacterium) on the expression of TLR4 and tumor necrosis factor-alpha (TNF-α) in the colon mucosa of rat experimental ulcerative colitis model induced by trinitrobenzene sulfonic acid (TNBS)/ethanol and immune complexes. The gross and histological changes of the colonic mucosa were observed and assessed by the means-standard deviation and independent samples t-test. The protein expression levels of TLR4 and TNF-α were detected by using immunohistochemistry and Western blotting, respectively. It was revealed that there was visible infiltration of inflammatory cells, formation of crypt abscess, and the reduction of goblet cells in the colon tissue of experimental models. As compared with the control group, the levels of TLR4 and TNF-α protein were significantly increased in the model group (P<0.01 for both). No significant difference was found in the expression of TLR4 and TNF-α between the two-week probiotics treatment group and the model group (P>0.05), whereas significant reductions were shown in rats which were treated with probiotics for four weeks as compared with the model group (P<0.01). There was no significant difference between two probiotics-treated groups. Our results implied that probiotics were likely to play a key role in protecting ulcerative colitis by reducing the inflammatory factor TNF-α expression through inhibiting the TLR4 expression in the colon tissue of experimental models.

Induction with NCB-02 (curcumin) enema for mild-to-moderate distal ulcerative colitis — A randomized, placebo-controlled, pilot study

Background and aimsCurcumin, an active ingredient of turmeric with anti-inflammatory properties, has been demonstrated to be useful in experimental models of ulcerative colitis (UC). It's efficacy in humans needs to be investigated. MethodsA randomized, double-blind, single-centre pilot trial was conducted in patients with distal UC (<25cm involvement) and mild-to-moderate disease activity. Forty-five patients were randomized to either NCB-02 (standardized curcumin preparation) enema plus oral 5-ASA or placebo enema plus oral 5-ASA. Primary end point was disease response, defined as reduction in Ulcerative Colitis Diseases Activity Index by 3 points at 8weeks, and secondary end points were improvement in endoscopic activity and disease remission at 8weeks. ResultsResponse to treatment was observed in 56.5% in NCB-02 group compared to 36.4% (p=0.175) in placebo group. At week 8, clinical remission was observed in 43.4% of patients in NCB-02 group compared to 22.7% in placebo group (p=0.14) and improvement on endoscopy in 52.2% of patients in NCB-02 group compared to 36.4% of patients in placebo group (p=0.29). Per protocol analysis revealed significantly better outcomes in NCB-02 group, in terms of clinical response (92.9% vs. 50%, p=0.01), clinical remission (71.4% vs. 31.3%, p=0.03), and improvement on endoscopy (85.7% vs. 50%, p=0.04). ConclusionIn this pilot study we found some evidence that use of NCB-02 enema may tend to result in greater improvements in disease activity compared to placebo in patients with mild-to-moderate distal UC. The role of NCB-02 as a novel therapy for UC should be investigated further.

The effect of red grape seed extract on the experimental model of ulcerative colitis in male albino rats

BackgroundUlcerative colitis (UC) is a chronic inflammatory disorder of the large bowel. Red grape seed extract (GSE) has antioxidative, anti-inflammatory and antimicrobial activities.Aim of the workThis study was designed to evaluate the protective properties of GSE on an animal model of acetic aci

Commensal Bacteria Play an Important Role in Ulcerative Colitis via Bacterial Sphingolipids-Mediated Regulation of Host Invariant Natural Killer T Cells

Our group has shown previously that microbiota can impact ulcerative colitis disease phenotypes by modulating invariant natural killer T (iNKT) cell functions. We further found that chemokine CXCL16 is responsible for this modulation, with germ-free (GF) mice having high CXCL16 level, large iNKT cell numbers and severe experimental ulcerative colitis outcome. In this study, we hypothesized that in addition to CXCL16 regulated functions, intestinal microbiota can directly impact iNKT cells by providing bacterial antigen, such as glycosphingolipids. We generated a Bacteroides fragilis mutant (del SPT) that no longer produces any sphingolipids. We then mono-colonized GF mice using wild-type or del SPT mutant. Theses mice were further challenged with an experimental ulcerative colitis model—oxazolone colitis model and the disease phenotypes were evaluated. Results: We found that the wild-type B. fragilis mono-colonized mice have significantly lower colonic iNKT cell numbers than the mutant mono-colonized mice, which are similar to the GF mice. CXCL16 levels in both of the mono-colonized mice are as low as that of the conventional mice, indicating the existence of a new pathway for colonic iNKT cell regulation. When challenged in the oxazolone colitis model, the mutant-colonized mice were much more susceptible than the wild-type-colonized mice. These phenotypes were diminished when the mice were treated with CD1d antibody, indicating iNKT cell-specific functions were involved. Our study demonstrated that microbiota regulate colonic iNKT cells via a new and unknown pathway. This pathway uses components in bacterial sphingolipid biosynthesis and it points to a new direction for therapeutic intervention of ulcerative colitis.

The Prolyl Hydroxylase Inhibitor Dimethyl Oxalyl Glycine Decreases the Severity of Acute Lung Injury After Allogeneic Hematopoietic Cell Transplantation.

AbstractAbstract 3006 Background:Allogeneic hematopoietic cell transplantation (HCT) is increasingly utilized in the treatment of hematologic malignant and non-malignant diseases. Therapy-related toxicity and graft versus host disease (GVHD) remain major challenges, with acute lung injury being associated with significant morbidity and mortality. Prolyl hydroxylase inhibitors (PHI) interfere with signaling cascades of inflammation and cell death. Their beneficial use in experimental models of ulcerative colitis and lung allograft rejection resulted in the hypothesis, that the PHI DMOG (Dimethyl oxalyl glycine) may reduce lung injury evolving after murine allogeneic HCT. Methods:14 week old BALB/c were conditioned with 750 cGy single dose TBI, followed by transplantation of 5×10E6 bone marrow cells and 4×10E6 splenocytes from either syngeneic BALB/c or allogeneic C57BL/6 donors. DMOG in PBS was given to both syngeneic and allogeneic recipients at 8mg per mice ip on alternate days for the first two weeks post-transplant and then twice a week till day 50, while syngeneic and allogeneic controls received PBS only. Animals were monitored for clinical GVHD and survival. At day 50 post HCT, gut, liver and lung pathology as well as BAL fluid T cell numbers were assessed by using a scoring system quantifying severity and extent of parenchymal and peribronchial/perivascular infiltrates and flow cytometry, respectively. In addition, T cell expansion was analyzed both in vivo in the spleen on day +7 and in vitro by mixed lymphocyte reaction (MLR). Results:Allogeneic HCT resulted in severe lung pathology in PBS treated animals (mean 118.8±47.19), but was significantly reduced when DMOG was given (36.0±17.99; p < 0.05), and absent in both PBS and DMOG treated syngeneic controls. Consistently, BAL CD4+ and CD8+ T cells in DMOG recipients were decreased by 53.7% and 68.4%, respectively, when compared to PBS controls. Decreased pulmonary injury was associated with a trend towards better overall survival by day 50 (allogeneic DMOG: 59.2% vs allogeneic PBS 40.7%) and all syngeneic recipients survived. GVHD related injury to liver and GI tract at time of analysis was not different between allogeneic groups. Alloreactive proliferative T cell responses in vitro were suppressed in the presence of DMOG at a concentration of 1mM (cpm after 96h: mean 29316±875.9 vs 41735±3085; p < 0.01). In vivo, splenic T cell expansion of CD4+ but not CD8+ T cells was reduced at day +7 as well (p < 0.05). Interestingly, when compared to PBS treated controls reduced mortality of DMOG treated allogeneic HCT recipients could already be seen soon after HCT (day +10: 26.0% vs 11.1%) in association with decreased CD4+ T cell proliferation, while lung injury in this model this early is rather marginal. Conclusion:DMOG treatment decreases the severity of acute noninfectious lung injury and improves overall survival after allogeneic HCT. While impaired early T cell responses likely contribute to improvement of both endpoints, altered recruitment of CD8+ T cells to the lung seems to uniquely affect the development of pulmonary but not gastrointestinal and hepatic injury at later time points. Current studies are ongoing to delineate the effects of DMOG on T cell proliferation and recruitment to different target organs, and to define the role of this novel approach in the prevention and treatment of HCT related complications. Disclosures:No relevant conflicts of interest to declare.

P002 The potential role of IL-33 in inflammatory-associated gut fibrosis: Induction of pro-fibrogenic gene expression and myofibroblast hypertrophy

Background: The pathogenesis of human ulcerative colitis (UC) is characterized by the presence of IL-13-producing Natural Killer T cells (NKT cells). One major complication of UC is the development of colitis-associated colorectal cancer. In these studies we determined the role of alternatively activated “M2 macrophages” in a new mouse model of colitis-associated carcinogenesis. Further, we verified these results through immunological analysis of surgical specimens obtained from patients with UC-associated colon cancer. Methods: Studies were performed using a mouse model for colitis-associated carcinogenesis based on chronic Oxazolonecolitis in combination with an initial i.p. azoxymethane (AOM) injection in female BALB/c mice. We observed chronic intestinal inflammation lasting for 12 weeks in mice repeatedly administered Oxazolone. Surgical specimens with UC-associated colon cancer were analyzed by flow cytometry and immunohistochemistry. Results: We could show that Oxazolone-colitis was mediated by colonic alpha-galactosyl-ceramide+CD1+ NKT-cells producing IL-13 and therefore resembles the immunological characteristics of human UC. Colon cancer development in this model was dependent on F4/80+CD11bhighGr1low macrophages producing IL-6 and EGF. Those cells inherited phenotypic characteristics of IL-13-stimulated alternativelyactivated “M2 macrophages”. To elucidate the importance of these “M2 macrophages”, we conducted bone marrow chimera studies and demonstrated that innate immune signaling through MyD88 in “M2 macrophages” is the key event for the production of tumor supporting factors like IL-6 and EGF. In surgical specimens obtained from patients with UC-associated colon cancer we verified those experimental findings. We consistently found a dense accumulation of IL-13-producing NKT cells inside the tumors. In addition, in this IL-13-dominated tumor micromilieu CD14+ and CD68+ antigen-presenting cells accumulated intensively and those antigen-presenting cells demonstrated an alternatively activated “M2 phenotype” by the expression of CD163 and CD205. Conclusions: We could demonstrate that in an experimental UC model, as well as in human UC, pathogenic IL-13producing NKT cells accumulate in the tumor micromilieu of UC-associated colon cancers. This immunologic situation inside the tumor microenvironment of mice and men induces tumor promoting M2 macrophages. Therefore M2 macrophages represent a potential target for future therapeutic strategies.

Zinc deficiency induces dysregulation of cytokine productions in an experimental colitis of rats

Dextran sulfate sodium (DSS)-induced colitis is an experimental model of ulcerative colitis, although the precise mechanism has not yet been elucidated. We investigate whether Zn deficiency affects the pathogenesis of colitis induced by DSS with a focus on immune responses. Male WKAH/Hkm Slc rats were fed either a Zn-adequate (ZA, 30 mg Zn/kg diet) as a control or Zndeficient (ZD, 5 mg Zn/kg diet) diet for 21 days and then treated with 2% DSS via deionized drinking water for 7 days. The disease activity index (DAI) was recorded daily throughout DSS treatment. Serum Zn concentrations were significantly lowered in rats fed the ZD diet than those fed the ZA diet at day 7 and 14. Surprisingly, DSS treatment considerably reduced the serum Zn in both groups. The rats fed the ZD diet showed exacerbated colitis based on clinical outcomes, including weight loss, increased DAI, and shortened colon length. An in vitro study corroborated these results, showing that a large amount of TNFα was induced by rat mesenteric leukocytes in response to lipopolysaccharide in ZD medium, but not in ZA medium. These results indicate that a modulation of TNFα production due to Zn deficiency influences disease activity in DSS-induced colitis. In addition, more attention should be given to Zn for prevention of colitis.

APicrorhiza kurroaDerivative, Picroliv, Attenuates the Development of Dextran-Sulfate-Sodium-Induced Colitis in Mice

Background. Free radicals and proinflammatory cytokines have been shown to play a critical role in the pathogenesis of ulcerative colitis (UC). Picroliv, a Picrorhiza kurroa derivative, has been demonstrated to have antioxidant and anti-inflammatory effect. The purpose of the study was to investigate the effects of picroliv on experimental model of UC in mice. Materials and Methods. Picroliv was administrated orally by gavage to mice with colitis induced by dextran sulfate sodium (DSS). Disease activity index (DAI), colon length, and histology score were observed. Myeloperoxidase (MPO) activity, and SOD, MDA concentrations were measured by enzyme-linked immunosorbent assay (ELISA) while the expression of cytokine mRNAs was studied by real-time-quantitative polymerase chain reaction and also ELISA. The expression of NF-κB p65 was observed by immunohistochemistry staining and western blotting. Results. A significant improvement was observed in DAI and histological score in mice treated with picroliv, and incerased MPO activity, MDA concentrations, and the expression of IL-1β, TNF-α, and NF-κB p65 in mice with DSS-induced colitis were significantly reduced while decreased SOD level increased following administration of picroliv. Conclusion. The administration of picroliv leads to an amelioration of DSS-induced colitis, suggesting administration of picroliv may provide a therapeutic approach for UC.