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sTu1702

Vitamin K Deficiency Deteriorates Murine DSS Colitis Through IL-6Production From B CellsEri Shiraishi, Hideki Iijima, Manabu Araki, Shoichiro Kawai, Satoshi Hiyama, TakahiroInoue, Shinichiro Shinzaki, Masahiko Tsujii, Tetsuo Takehara

Objective: Bone mineral density (BMD) is decreased in patients with inflammatory boweldisease (IBD). Reduced BMD in these patients may be multifactorial. Our recent researchdemonstrated that serum undercarboxylated osteocalcin (ucOC) levels, which are widelyused as a surrogate marker of vitamin K deficiency, were significantly higher in patientswith Crohn's disease (CD) than healthy subjects. Vitamin K has been shown to ameliorateliver injury through inhibition of inflammatory cytokine production. We aimed to evaluatethe effect of vitamin K on inflammatory responses in the intestine in mice. Methods: CD4-, CD11b- and CD19-positive subpopulations were isolated from splenocytes using theMagnetic Cell Sorting (MACS) separator and these cells were incubated in vitro in thepresence or absence of vitamin K2 for 24 h. The mRNA and the culture supernatant of thepurified cells were collected. The levels of IL-6 and TNF-α were measured by quantitativeRT-PCR and ELISA. Male 8-week-old C57/BL6J mice were fed with the vitamin K deficientdiets (K-def) or supplemented diets (K-sup) for 10 days, and then the mice were administeredorally with dextran sulfate sodium (DSS) in drinking water for 7 days. Histologic scores ofthe colon were analyzed 3 days after the cessation of DSS administration. The level of IL-6 production from the colonic lamina propria (LP) mononuclear cells was measured byELISA in the K-def and the K-sup groups. Proportion of the cells costained with CD4 andCD25 was analyzied by flow cytometry. Results: Vitamin K2 reduced the production of IL-6 by the splenocytes cultured in vitro, while the expression of TNF-α showed no significantdifference. Expression of IL-6 in CD19-positive B cells, but not in CD4- and CD11b-positivecells, was significantly decreased in the presence of vitamin K2. The histologic score of DSScolitis was significantly lower in K-sup group than in K-def group. In addition, the expressionof IL-6 from LP cells was significantly lower in K-sup group than in the K-def group. Theproportion of CD4 and CD25-positive regulatory T cells did not significantly differ betweenthe K-sup and K-def groups. Conclusion: Vitamin K has a suppressive effect on IL-6 produc-tion especially in B cells. Vitamin K deficiency deteriorates murine DSS colitis through IL-6 production from B cells. Vitamin K may have a potential for the treatment target of IBD.

Tu1703

Analysis of the Protease MT1-MMP and Its Substrates As Biomarkers andTherapeutic Targets in Inflammatory Bowel DiseaseAgnieszka Koziol, Angela Pollán, Pilar Gonzalo, Norman Nuñez Andrade, Pablo M.Linares, Maria Encarnacion Fernandez Contreras, Maria Chaparro, Ana Urzainqui,Francisco Sánchez-Madrid, Javier P. Gisbert, Alicia G. Arroyo

Background: We have recently reported that the protease MT1-MMP is up-regulated inTNFα-activated endothelial cells where it can process substrates normally bound to theextracellular matrix, such as thrombospondin1 (TSP1) and nidogen1 (NID1), inducing theirrelease and thus modulating the angiogenic response. Our aim was to analyse the expressionof MT1-MMP and its substrates TSP1 and NID1 as potential biomarkers of inflammatorybowel disease (IBD), a chronic disorder involving inflammation and angiogenesis, in mousemodels and human samples. Methods: A mouse model of dextran sodium sulphate (DSS)-induced colitis was used. Immunostaining (IS) in colon sections was performed to analyse:(i) the angiogenic response with the specific endothelial cell marker CD31/PECAM1, and(ii) the expression of MT1-MMP, TSP1 and NID1, in either control mice, mice treated with1%DSS (moderate colitis) or 4%DSS (severe colitis). TSP1, NID1 and VEGF levels werealso measured by enzyme-linked immunosorbent assay in serum from patients with activeulcerative colitis (UC) or Crohn's disease (CD). The in vivo function of endothelial MT1-MMP in mouse colitis was analysed using recently generated conditional mice with endothe-lium-specific deletion of MT1-MMP (MT1 flox/flox x VECadhERT2-Cre). Results: Serum levelsof the proinflammatory cytokine TNFα and of the proangiogenic factor VEGF-A wereupregulated in the DSS-induced colitis model in a dose response manner. This correlatedwith increased presence of new vessels (CD31/PECAM1+) in the lamina propria /(LP) andwith MT1-MMP upregulated expression in colonic vascular cells of mice with moderatecolitis when compared to either control or mice with severe colitis. Soluble serum levels ofTSP1 and NID1 also increased gradually from moderate to severe mouse colitis. In IBDpatients, serum levels of TSP-1 and NID1 were significantly higher in UC and CD patientswith low disease activity compared to high activity. Preliminary results show that endothe-lium-specific deletion of MT1-MMP prevents development of DSS-induced colitis in mice.

Tu1704

Effect of Chondroitin Sulphate on Pro-Inflammatory Mediators and DiseaseActivity in Patients With Inflammatory Bowel Disease (IBD)Pablo M. Linares, Maria Chaparro, Alicia Algaba, Manuel Román, M. Isabel Moreno Arza,Francisco Abad-Santos, Dolores Ochoa, Fernando Bermejo, Javier P. Gisbert

Background: Chondroitin sulphate, a glycosaminoglycan that modulates NF-κB, mightmodulate several pro-inflammatory proteins involved in the pathogenesis of IBD. The aimof our study was to evaluate the incidence rate of relapse in patients with IBD underchondroitin sulphate treatment and its effect on the concentrations of diverse pro-inflamma-tory mediators in serum and urine. Methods: Prospective observational 12-month (m)follow-up study in patients with IBD in remission for at least 6 m, starting chondroitinsulphate treatment (Condrosan®, CS Bio-ActiveTM, Bioibérica S.A., Barcelona, Spain) forosteoarthritis (OA) (dose: 800 mg o.d.). Visits were as follows: Baseline, 3rd, 6th, 9th and12th m. CDAI and modified Truelove-Witts clinical indexes were calculated for Crohn'sdisease (CD) and ulcerative colitis (UC) respectively. C-reactive protein (CRP), orosomucoid,and erythrocyte sedimentation rate (ESR) were also determined. Levels of VEGFA, VEGFC,FGF2, HGF, Ang1, Ang2, TGFβ, TNFα, IL1β, -6, -12, -17, -23, ICAM1, VCAM1, MMP3and PGE2 were quantified by ELISA. OA joint pain was evaluated by a visual analogue scale(VAS). Results: 37 patients with IBD (19 UC, 18 CD) were included. Mean age was 59.8

S-822AGA Abstracts

years, and 70% were women. The mean disease duration was 12.7 years. 62% of patientswere under mesalazine, 5% with sulfasalazine, 22% with thiopurines, and 3% with methotrex-ate. There was only one patient (with UC) that had a flare during follow-up (6th m visit).The incidence rate of relapse was 3.4% per patient-year of follow-up. That figure is lowerthan the relapse rate previously reported for IBD patients. 12 UC and 11 CD patientscompleted the 12 m follow-up. In all patients with IBD, mean serum VEGFA levels werehigher after 12 months of CS treatment (799 pg/mL) as compared to baseline (492 pg/mL)(P<0.05). Further differences regarding the other studied pro-inflammatory markers werenot found. At 12th m, the OA joint pain had improved in all but four patients (from 5.9to 3.0) (P<0.01). 43% of patients suffered adverse events, but only 5% were related to thedrug. Conclusions: The incidence of IBD relapse in patients under chondroitin sulphatetreatment was lower than the generally reported. This treatment might modulate VEGFAserum levels, but it is not associated with modifications in the concentrations of the otherstudied pro-inflammatory mediators. Chondroitin sulphate decreases pain related to OA inpatients with IBD.

Tu1705

Role of Allograft Inflammatory Factor 1 (AIF-1) in Inflammatory BowelDiseaseD. Cano-Martínez, Jorge Monserrat, Borja Hernandez-Breijo, Patricia Sanmartín-Salinas,Carolina García-Torrijos, Irene de los Dolores Román Curto, Dolores Fernández-Moreno,Pablo M. Linares, Maria Chaparro, Antonio Julià, Sara Marsal, Javier P. Gisbert, LuisGonzález Guijarro

Background: Inflammatory bowel disease (IBD) is characterized by chronic inflammationof the gastrointestinal tract and it includes two pathologies, Ulcerative Colitis (UC) andCrohn's disease (CD). The immune response of the last one, exhibits a Th-1 phenotype.Newly, Genome-wide association studies (GWAS) have associated Allograft InflammatoryFactor 1 (AIF-1) with risk of CD. The purpose of this study was to elucidate the role ofAIF-1 in IBD. Methods: To determine the presence of AIF-1 in human peripheral mononu-clear blood cells (PBMC), cell sorting was carried out to separate the different types of cellspopulation. Thus, the expression of AIF-1 messenger RNA levels was detected and evaluatedby RT- qPCR. The study was completed with clonal expansion and in vitro Th-1 polarization,assessing the effect of AIF-1 on proliferation (IL-2) and activation (INFγ). Furthermore, theprotein levels of AIF-1 were quantificated by Western blot in cell lysates and culture medium.Moreover, serum levels of AIF-1 were determined in 47 subjects (46.4 ±13.5 years; 19 male/28 female) by Western blot. There were three experimental groups available: 10 healthycontrols, 18 patients with active IBD (9 with CD/ 9 with UC) and 19 patients with inactiveIBD (10 with CD/ 9 with UC). The activity in CD was determined by Harvey- Bradshawindex and in UC by the partial Mayo score. Results: AIF-1 expression was predominant inT lymphocytes in peripheral blood mononuclear cells (PBMC), detecting the highest levelsof AIF-1 in Naive cells CD4+. Naive cells activated with a physiological stimulant (CD3-CD28) enhanced the expression of AIF-1, as well as IL-2 and INFγ. Also, the response toPBMC stimulation with CD3-CD28, in the absence and presence of exogenous AIF-1, showedan increased transcription of AIF-1, IL-2 and INFγ. But this increase was greater in polarizationto Th-1 (CD3-CD28, IL-12, anti-IL-4) in the presence of exogenous AIF-1. Furthermore,an increase of AIF-1 levels in the culture medium of activated cells was observed. Regardingto the serum levels of AIF-1 in patients with IBD, significant differences at 99 percentconfidence interval were found among patients with active IBD and inactive IBD (p value=0,002). Conclusion: AIF-1 is found predominantly in CD4+ Naive T cells and plays a rolein the differentiation of Th1. It is a secreted protein, which has higher serum levels in patientswith active IBD, and therefore it could be used as a biomarker for disease activity monitoring.

Tu1706

The TLR-9 Agonist DIMS0150 Leads to the Induction of IL-10 PositiveMucosal Cells in Ulcerative Colitis PatientsUlrike Billmeier, Charlotte Admyre, Thomas Knittel, Arezou Zargari, Markus F. Neurath,Raja Atreya

INTRODUCTION The oligonucleotide DIMS0150 acts as a Toll like receptor 9 (TLR-9)agonist and has shown beneficial effects on response and remission rates in treatmentrefractory patients with ulcerative colitis. It has been demonstrated that DIMS0150 inducesa variety of anti-inflammatory cytokines including IL-10 in cultured PBMCs. In order togain deeper insights into the mechanism of action in vivo we analyzed the expression ofIL-10 in colon biopsies taken at different time points after local application of DIMS0150in ulcerative colitis patients. METHODS A randomized, double-blind, multicenter phase IItrial was performed with DIMS0150 in steroid refractory patients with ulcerative colitis ofmoderate degree (EudraCT number: 2006-001846-15). Patients were randomized to a singlerectal administration of either 30 mg of DIMS0150 or placebo. Clinical response (DAI scoredecrease of at least 3 points from baseline) at week 1 and 4 was 36% and 53% in theDIMS0150 and 9% and 41% in the placebo treated group. 28 days after DIMS0150 (n=14)or placebo treatment (n=4) cross-sections from colon biopsies of ulcerative colitis patientswere taken and stained for IL-10 by immunofluorescence. The quantification of IL-10 positivecells was conducted by blindly analyzing the number of IL-10 positive mucosal immunecells among a total of 300 counted cells in each sample. Additionally, IL-10 expression wassimilarly determined at day 7 after DIMS0150 (n=10) and placebo (n=7) treatment. RESULTSAnalysis of IL-10 expression in colonic biopsies in ulcerative colitis patients at day 28 afterDIMS0150 treatment interestingly indicated a significantly increased number of IL-10 positiveimmune cells (mean 108/300 positive cells; SEM±28.9) compared to placebo treated patients(mean 67/300 positive cells; SEM±10.8) (p=0.02). Additionally performed analysis at day7 further implicated that the observed enhanced expression of IL-10 in colon infiltratingimmune cells is an early occurring and direct effect of locally applied DIMS0150 (mean of114/300 IL-10 positive cells; SEM±34.2 in DIMS0150 versus 70/300 IL-10 positive cellsSEM±10.2 in placebo treated patients). CONCLUSION The TLR9 agonist DIMS0150 hasshown promising effects in the treatment of patients with ulcerative colitis. Our data revealedfor the first time in vivo a significantly higher expression of the anti-inflammatory cytokineIL-10 in the gut mucosa of ulcerative colitis patients who received DIMS0150 in comparison