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Biomarkers are needed in inflammatory bowel disease (IBD) to help define disease activity and identify underlying pathogenic mechanisms. We hypothesized that serum metabolomics, which produces unique metabolite profiles, can aid in this search.The aim of this study was to characterize serum metabolomic profiles in patients with IBD, and to assess for differences between patients with ulcerative colitis (UC), Crohn's disease (CD), and non- IBD subjects.Serum samples from 20 UC, 20 CD, and 20 non-IBD control subjects were obtained along with patient characteristics, including medication use and clinical disease activity. Non-targeted metabolomic profiling was performed using ultra-high performance liquid chromatography/mass spectrometry (UPLC-MS/MS) optimized for basic or acidic species and hydrophilic interaction liquid chromatography (HILIC/UPLC-MS/MS).In total, 671 metabolites were identified. Comparing IBD and control subjects revealed 173 significantly altered metabolites (27 increased and 146 decreased). The majority of the alterations occurred in lipid-, amino acid-, and energy-related metabolites. Comparing only CD and control subjects revealed 286 significantly altered metabolites (54 increased and 232 decreased), whereas comparing UC and control subjects revealed only 5 significantly altered metabolites (all decreased). Hierarchal clustering using significant metabolites separated CD from UC and control subjects.We demonstrate that a number of lipid-, amino acid-, and tricarboxylic acid (TCA) cycle- related metabolites were significantly altered in IBD patients, more specifically in CD. Therefore, alterations in lipid and amino acid metabolism and energy homeostasis may play a key role in the pathogenesis of CD.

Background L-arginine (L-Arg) is the substrate for both inducible nitric oxide (NO) synthase (NOS2) and arginase (ARG) enzymes. L-Arg is actively transported into cells by means of cationic amino acid transporter (SLC7) proteins. We have linked L-Arg and arginase 1 activity to epithelial restitution. Our aim was to determine if L-Arg, related amino acids, and metabolic enzymes are altered in ulcerative colitis (UC). Methods Serum and colonic tissues were prospectively collected from 38 control subjects and 137 UC patients. Dietary intake, histologic injury, and clinical disease activity were assessed. Amino acid levels were measured by high-performance liquid chromatography. Messenger RNA (mRNA) levels were measured by real-time PCR. Colon tissue samples from 12 Crohn's disease patients were obtained for comparison. Results Dietary intake of arginine and serum L-Arg levels were not different in UC patients versus control subjects. In active UC, tissue L-Arg was decreased, whereas L-citrulline (L-Cit) and the L-Cit/L-Arg ratio were increased. This pattern was also seen when paired involved (left) versus uninvolved (right) colon tissues in UC were assessed. In active UC, SLC7A2 and ARG1 mRNA levels were decreased, whereas ARG2 and NOS2 were increased. Similar alterations in mRNA expression occurred in tissues from Crohn's disease patients. In involved UC, SLC7A2 and ARG1 mRNA levels were decreased, and NOS2 and ARG2 increased, when compared with uninvolved tissues. Conclusions Patients with UC exhibit diminished tissue L-Arg, likely attributable to decreased cellular uptake and increased consumption by NOS2. These findings combined with decreased ARG1 expression indicate a pattern of dysregulated L-Arg availability and metabolism in UC.

Objective The myeloid translocation genes (MTGs) are transcriptional corepressors with both Mtg8 −/− and Mtgr1 −/− mice showing developmental and/or differentiation defects in the intestine. We sought to determine the role of MTG16 in intestinal integrity. Methods Baseline and stress induced colonic phenotypes were examined in Mtg16 −/− mice. To unmask phenotypes, we treated Mtg16 −/− mice with dextran sodium sulphate (DSS) or infected them with Citrobacter rodentium and the colons were examined for ulceration and for changes in proliferation, apoptosis and inflammation. Results Mtg16 −/− mice have altered immune subsets, suggesting priming towards Th1 responses. Mtg16 −/− mice developed increased weight loss, diarrhoea, mortality and histological colitis and there were increased innate (Gr1 + , F4/80 + , CD11c + and MHCII + ; CD11c + ) and Th1 adaptive (CD4) immune cells in Mtg16 −/− colons after DSS treatment. Additionally, there was increased apoptosis and a compensatory increased proliferation in Mtg16 −/− colons. Compared with wild-type mice, Mtg16 −/− mice exhibited increased colonic CD4;IFN-γ cells in vehicle-treated and DSS-treated mice. Adoptive transfer of wild-type marrow into Mtg16 −/− recipients did not rescue the Mtg16 −/− injury phenotype. Isolated colonic epithelial cells from DSS-treated Mtg16 −/− mice exhibited increased KC (Cxcl1) mRNA expression when compared with wild-type mice. Mtg16 −/− mice infected with C rodentium had more severe colitis and greater bacterial colonisation. Last, MTG16 mRNA levels were reduced in human ulcerative colitis versus normal colon tissues. Conclusions These observations indicate that MTG16 is critical for colonocyte survival and regeneration in response to intestinal injury and provide evidence that this transcriptional corepressor regulates inflammatory recruitment in response to injury.

until used for cytokine determination. The concentration of intestine mucosa cytokines involved in the Th1 (IL-2, IL-12p70, IFN-γ), Th2 (IL-4), Th17 (IL-6, IL-17A, IL-23) and Treg (IL-10, TGF-β) cell commitment and IL-1 β and TNF-α (cytokines of the innate immune response) was assayed in duplicate by ELISA (Biosource, Camarillo, CA) and the results were expressed as picogram of cytokine per milligram of protein. Data were analyzed with SPSS. Comparisons between the groups were done by the two-tailed Mann-Whitney U-test. The ileal and colonic concentration of all proinflammatory cytokines was significantly higher in the patients with CD and also UC than in control group (p

Background: In a previous study utilizing multiplex Luminex technology in a cohort of control and ulcerative colitis (UC) patients, we identifiedmultiple proinflammatory cytokines/ chemokines that were increased in the serum and tissue of patients with active UC, determined by histology, compared to control patients. In order to better assess the potential for Luminex profiling for clinical trials, our aim was to investigate the relationship between a validated clinical disease activity tool, the Mayo Disease Activity Index (DAI) and the endoscopy score (ES) subset of the DAI, and the cytokine/chemokine profile in UC patients. Methods: Human serum and colon tissue samples were collected prospectively from 40 control subjects undergoing screening colonoscopy and 137 UC patients. The DAI (a combination of stool frequency, blood in the stool, physician global assessment, and ES) was assessed for UC patients at the time of colonoscopy. Serum and tissue cytokines/chemokines were measured by a 39-plex MILLIPLEX bead assay. We classified UC patients by DAI or ES alone into inactive (DAI 0-2; ES 0) and active (DAI 3-12; ES 1-3) disease. Cytokine/chemokine levels were analyzed based upon the DAI and ES classifications and were compared to control patients. We also assessed the correlation of DAI with the serum and tissue cytokine/ chemokine levels. Results: When active versus inactive UC was defined by a DAI cutoff of 3 or greater, patients with active UC had increased serum levels of eotaxin-1, G-CSF, GMCSF, IL-8, and TGF-α compared to control patients (all with at least p,0.05). None of the tissue or serum cytokine/chemokine levels resulted in a correlation greater than an r2 value of 0.18 with DAI as a continuous variable. When active disease was defined by an ES of 1 or greater, serum eotaxin-1, G-CSF, GM-CSF, IL-8, and IL-9 levels were increased in active UC (all p,0.05). Eotaxin-1, GM-CSF, and IL-8 were also increased in tissues when compared to controls when active UC was defined by either increased DAI or ES. In total, there were 21 of 39 cytokines/chemokines increased in tissues from patients with a DAI of 3 or greater vs. normal control subjects, and 20 were increased when ES was used. In active vs. inactive UC by DAI or ES, there were increases in serum levels of G-CSF, IFNγ, and IL-8; IL-15 and TGF-α were also increased when active UC was defined by DAI. In active vs. inactive UC defined by DAI, there were 17 analytes increased in tissues. When defined by ES, there were 22 analytes increased, including eotaxin-1, G-CSF, and IL-8. Conclusions: Luminex profiling was robust in identifying active disease defined by using either the DAI or the ES. Testing for serum cytokines/chemokines, especially G-CSF, IL-8, and eotaxin-1, may represent a non-invasive strategy for detecting active UC that compares favorably with current clinical tools.