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Elafin is an antimicrobial and anti-inflammatory protein. We hypothesize that elafin expression correlates with diabetes. Among non-diabetic and prediabetic groups, men have significantly higher serum elafin levels than women. Men with type 2 diabetes mellitus (T2DM) have significantly lower serum elafin levels than men without T2DM. Serum elafin levels are inversely correlated with fasting blood glucose and hemoglobin A1c levels in men with T2DM, but not women with T2DM. Lentiviral elafin overexpression inhibited obesity, hyperglycemia, and liver steatosis in high-fat diet (HFD)-treated male mice. Elafin-overexpressing HFD-treated male mice had increased serum leptin levels, and serum exosomal miR181b-5p and miR219-5p expression. Transplantation of splenocytes and serum exosomes from elafin-overexpressing HFD-treated donor mice reduced food consumption and fat mass, and increased adipose tissue leptin mRNA expression in HFD-treated recipient mice. Elafin improved leptin sensitivity via reduced interferon-gamma expression and induced adipose leptin expression via increased miR181b-5p and miR219-5p expression. Subcutaneous and oral administration of modified elafin inhibited obesity, hyperglycemia, and liver steatosis in the HFD-treated mice. Circulating elafin levels are associated with hyperglycemia in men with T2DM. Elafin, via immune-derived miRNAs and cytokine, activates leptin sensitivity and expression that subsequently inhibit food consumption, obesity, hyperglycemia, and liver steatosis in HFD-treated male mice.

Background & Aims Clostridium difficile induces intestinal inflammation by releasing toxins A and B. The antimicrobial compound cationic steroid antimicrobial 13 (CSA13) has been developed for treating gastrointestinal infections. The CSA13-Eudragit formulation can be given orally and releases CSA13 in the terminal ileum and colon. We investigated whether this form of CSA13 reduces C difficile infection (CDI) in mice. Methods C57BL/6J mice were infected with C difficile on day 0, followed by subcutaneous administration of pure CSA13 or oral administration of CSA13-Eudragit (10 mg/kg/d for 10 days). Some mice were given intraperitoneal vancomycin (50 mg/kg daily) on days 0–4 and relapse was measured after antibiotic withdrawal. The mice were monitored until day 20; colon and fecal samples were collected on day 3 for analysis. Blood samples were collected for flow cytometry analyses. Fecal pellets were collected each day from mice injected with CSA13 and analyzed by high-performance liquid chromatography or 16S sequencing; feces were also homogenized in phosphate-buffered saline and fed to mice with CDI via gavage. Results CDI of mice caused 60% mortality, significant bodyweight loss, and colonic damage 3 days after infection; these events were prevented by subcutaneous injection of CSA13 or oral administration CSA13-Eudragit. There was reduced relapse of CDI after administration of CSA13 was stopped. Levels of CSA13 in feces from mice given CSA13-Eudragit were significantly higher than those of mice given subcutaneous CSA13. Subcutaneous and oral CSA13 each significantly increased the abundance of Peptostreptococcaceae bacteria and reduced the abundance of C difficile in fecal samples of mice. When feces from mice with CDI and given CSA13 were fed to mice with CDI that had not received CSA13, the recipient mice had significantly increased rates of survival. CSA13 reduced fecal levels of inflammatory metabolites (endocannabinoids) and increased fecal levels of 4 protective metabolites (ie, citrulline, 3-aminoisobutyric acid, retinol, and ursodeoxycholic acid) in mice with CDI. Oral administration of these CSA13-dependent protective metabolites reduced the severity of CDI. Conclusions In studies of mice, we found the CSA13-Eudragit formulation to be effective in eradicating CDI by modulating the intestinal microbiota and metabolites.

Background & Aims: Cathelicidin (LL-37 in human and mCRAMP in mice) represents a family of endogenous antimicrobial peptides with anti-inflammatory effects. LL-37 also suppresses collagen synthesis, an important fibrotic response, in dermal fibroblasts. Here, we determined whether exogenous cathelicidin administration modulates intestinal fibrosis in two animal models of intestinal inflammation and in human colonic fibroblasts. Methods: C57BL/6J mice (n = 6 per group) were administered intracolonically with a trinitrobenzene sulphonic acid (TNBS) enema to induce chronic (6â7 weeks) colitis with fibrosis. We administered mCRAMP peptide (5 mg/kg every 3 day, week 5â7) or cathelicidin gene (Camp)-expressing lentivirus (107 infectious units week 4) intracolonically or intravenously, respectively. We then infected 129Sv/J mice with Salmonella typhimurium orally to induce cecal inflammation with fibrosis. Camp-expressing lentivirus (107 infectious units day 11) was administered intravenously. Results: TNBS-induced chronic colitis was associated with increased colonic collagen (col1a2) mRNA expression. Intracolonic cathelicidin (mCRAMP peptide) administration or intravenous delivery of lentivirus-overexpressing cathelicidin gene significantly reduced colonic col1a2 mRNA expression in TNBS-exposed mice compared with vehicle administration. Salmonella infection also caused increased cecal inflammation associated with collagen (col1a2) mRNA expression that was prevented by intravenous delivery of Camp-expressing lentivirus. Exposure of human primary intestinal fibroblasts and human colonic CCD-18Co fibroblasts to transforming growth factor-β1 (TGF-β1) and/or insulin-like growth factor 1 induced collagen protein and mRNA expression, which was reduced by LL-37 (3â5 μM) through a MAP kinase-dependent mechanism. Conclusions: Cathelicidin can reverse intestinal fibrosis by directly inhibiting collagen synthesis in colonic fibroblasts. Keywords: Antimicrobial Peptide, Collagen, Inflammatory Bowel Disease

TL1A is a member of the TNF superfamily, and its expression is increased in the mucosa of inflammatory bowel disease patients. Moreover, patients with certain TNFSF15 variants over-express TL1A and have a higher risk of developing strictures in the small intestine. Consistently, mice with sustained Tl1a expression in either lymphoid or myeloid cells develop spontaneous ileitis and increased intestinal collagen deposition. Transgenic (Tg) mice with constitutive Tl1a expression in both lymphoid and myeloid cells were generated to assess their in vivo consequence. Constitutive expression of Tl1a in both lymphoid and myeloid cells showed increased spontaneous ileitis and collagen deposition than WT mice. T cells with constitutive expression of Tl1a in both lymphoid and myeloid cells were found to have a more activated phenotype, increased gut homing marker CCR9 expression, and enhanced Th1 and Th17 cytokine activity than WT mice. Although no differences in T cell activation marker, Th1 or Th17 cytokine activity, ileitis, or collagen deposition were found between constitutive Tl1a expression in lymphoid only, myeloid only, or combined lymphoid and myeloid cells. Double hemizygous Tl1a-Tg mice appeared to have worsened ileitis and intestinal fibrosis. Our findings confirm that TL1A–DR3 interaction is involved in T cell-dependent ileitis and fibrosis.

Michelle Cheng,1,* Samantha Ho,1,* Jun Hwan Yoo,1,2,* Deanna Hoang-Yen Tran,1,* Kyriaki Bakirtzi,1 Bowei Su,1 Diana Hoang-Ngoc Tran,1 Yuzu Kubota,1 Ryan Ichikawa,1 Hon Wai Koon1 1Center for Inflammatory Bowel Diseases, Division of Digestive Diseases, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA; 2Digestive Disease Center, CHA University Bundang Medical Center, Seongnam, Republic of Korea *These authors share co-first authorship Background: Cathelicidin (LL-37 in humans and mCRAMP in mice) represents a family of endogenous antimicrobial and anti-inflammatory peptides. Cancer-associated fibroblasts can promote the proliferation of colon cancer cells and growth of colon cancer tumors. Methods: We examined the role of cathelicidin in the development of colon cancer, using subcutaneous human HT-29 colon-cancer-cell-derived tumor model in nude mice and azoxymethane- and dextran sulfate-mediated colon cancer model in C57BL/6 mice. We also determined the indirect antitumoral mechanism of cathelicidin via the inhibition of epithelial–mesenchymal transition (EMT) of colon cancer cells and fibroblast-supported colon cancer cell proliferation. Results: Intravenous administration of cathelicidin expressing adeno-associated virus significantly reduced the size of tumors, tumor-derived collagen expression, and tumor-derived fibroblast expression in HT-29-derived subcutaneous tumors in nude mice. Enema administration of the mouse cathelicidin peptide significantly reduced the size and number of colonic tumors in azoxymethane- and dextran sulfate-treated mice without inducing apoptosis in tumors and the adjacent normal colonic tissues. Cathelicidin inhibited the collagen expression and vimentin-positive fibroblast expression in colonic tumors. Cathelicidin did not directly affect HT-29 cell viability, but did significantly reduce tumor growth factor-ß1-induced EMT of colon cancer cells. Media conditioned by the human colonic CCD-18Co fibroblasts promoted human colon cancer HT-29 cell proliferation. Cathelicidin pretreatment inhibited colon cancer cell proliferation mediated by media conditioned by human colonic CCD-18Co fibroblasts. Cathelicidin disrupted tubulin distribution in colonic fibroblasts. Disruption of tubulin in fibroblasts reduced fibroblast-supported colon cancer cell proliferation. Conclusion: Cathelicidin effectively inhibits colon cancer development by interfering with EMT and fibroblast-supported colon cancer cell proliferation. Keywords: colon cancer, epithelial–mesenchymal transition, fibroblasts

© 2014 Society for Mucosal Immunology. Intestinal fibrostenosis is among the hallmarks of severe Crohn's disease. Patients with certain TNFSF15 (gene name for TL1A) variants over-express TL1A and have a higher risk of developing strictures in the small intestine. In addition, sustained Tl1a expression in mice leads to small and large intestinal fibrostenosis under colitogenic conditions. The aim of this study was to determine whether established murine colonic fibrosis could be reversed with Tl1a antibody (Ab). Treatment with neutralizing Tl1a Ab reversed colonic fibrosis back to the original pre-inflamed levels, potentially as a result of lowered expression of connective tissue growth factor, Il31Ra, transforming growth factor β1 and insulin-like growth factor-1. In addition, blocking Tl1a function by either neutralizing Tl1a Ab or deletion of death domain receptor 3 (Dr3) reduced the number of fibroblasts and myofibroblasts, the primary cell types that mediate tissue fibrosis. Primary intestinal myofibroblasts expressed Dr3 and functionally responded to direct Tl1a signaling by increasing collagen and Il31Ra expression. These data demonstrated a direct role for TL1A-DR3 signaling in tissue fibrosis and that modulation of TL1A-DR3 signaling could inhibit gut fibrosis.

Substance P (SP) and the neurokinin-1 receptor (NK-1R) are involved in the development of colitis and mucosal healing after colonic inflammation. We studied whether SP modulates colonic fibrosis by using a chronic model of trinitrobenzenesulfonic acid (TNBS)-induced colitis in wild-type (WT) and NK-1R-deficient (NK-1R KD) mice. We found increased mRNA expression levels of collagen, vimentin, and the fibrogenic factors transforming growth factor β1 and insulin-like growth factor 1 in the chronically inflamed colons of WT mice treated with repeated intracolonic TNBS administrations. Fibrosis in TNBS-treated mice was also evident immunohistochemically by collagen deposition in the colon. Treatment of TNBS-exposed WT mice with the NK-1R antagonist CJ-12255 reduced colonic inflammation, colonic fibrosis, fibroblast accumulation, and expression levels of the fibrogenic factors. NK-1R knockout mice chronically exposed to TNBS had similar colonic inflammation compared with WT, but reduced colonic fibrosis, fibroblast accumulation, and expression levels of fibrogenic factors. Immunohistochemical staining also showed co-localization of NK-1R with fibroblasts in inflamed colons of mice and in colonic mucosa of patients with Crohn's disease. Exposure of human colonic CCD-18Co fibroblasts to SP (10 nmol/L) increased cell migration. SP stimulated collagen synthesis in CCD-18Co fibroblasts in the presence of transforming growth factor β1 and insulin-like growth factor 1, and this effect was reduced by Akt inhibition. Thus, SP, via NK-1R, promotes intestinal fibrogenesis after chronic colitis by stimulating fibrotic responses in fibroblasts.

Background: IL-17 has been thought to be pathogenic in IBD. However, a recent clinical trial using IL-17 blockade unexpectedly worsened IBD (Hueber, et al. Gut. 2012). A proportion of trial patients who responded to IL-17 blockade were found to carry a risk TL1A IBD polymorphism that predicted elevated expression of TL1A. The mechanism of IL-17 and its interaction with TL1A on mucosal inflammation has not been found. Aim: To determine the mechanism of TL1A mediated differential effects of IL-17 on mucosal inflammation Methods: Naive (CD4+CD45RBhi) T-cells were isolated from WT, Tl1a-Tg, Il-17a-/-, and Tl1a-Tg with deficiency in Il-17a (Tl1a-Tg/Il-17a-/-) mice and adoptively transferred into Rag-/mice. Naive (CD4+CD25loCD44loCD62hi) T-cells were differentiated in vitro into T helper (Th) effector cells. Intestinal inflammation was evaluated by disease activity index (DAI) and histological analyses. Flow cytometry was used to determine CD4+ T-cell infiltration, activation, and cytokine expression in the LPMC from the colon. Results: Consistent with the human IL-17 trial, we found that mice that received Il-17a-/Naive cells had worsened colitis (increased DAI scores and cecal inflammation) as compared to WT. Comparable results were seen with Tl1a-Tg Naive cells. Similar to the human IL-17 trial, Il-17a deficiency under Tl1a driven conditions (Tl1a-Tg/Il-17a-/-) ameliorated colitis (reduced DAI and cecal inflammation). Mucosal CD4+ T-cell infiltration and activation (CD69 and CD44) were increased in both Il-17a-/and Tl1a-Tg, but reduced with Tl1a-Tg/Il-17a-/-. Analysis of Th effector pathways demonstrated a shift towards Th-1 (increased Ifn-γ) and Th-9 (increased Il-9), and reduced regulatory cytokine Il-10 production with both Il-17a-/and Tl1a-Tg. In contrast, Il-17 deficiency under Tl1a driven conditions (Tl1a-Tg/Il-17a-/-) had a reduction in Th1 (reduced Ifn-γ) and Th9 (reduced Il-9) responses, and increased regulatory responses (increased Il-10). To assess whether intrinsic cellular differences exist in Naive T-cells between WT, Il-17a-/-, Tl1a-Tg, and Tl1a-Tg/Il-17a-/-, in vitro differentiation assays were performed. Consistently, in vitro differentiated Naive Il-17a-/and Tl1a-Tg T-cells expressed increased Ifn-γ and Il-9, whereas Tl1a-Tg/Il17a-/T-cells expressed lower Il-9 but higher Il-10 levels. Conclusion: IL-17 blockade induces mucosal inflammation by enhancing Th1 and Th9 effector pathways and reducing regulatory response. However under TL1A driven conditions, inhibiting IL-17 may be beneficial by reducing Th1 and Th9 effector responses and enhancing regulatory responses. This may be one mechanism of why the subset of IBD patients with TL1A polymorphisms improved with IL-17 blockade, while most trial patients did not. This highlights the importance of understanding pathway-pathway interactions in designing clinical trials. Table 1: Inflammatory Markers

No final conclusion has yet been achieved on whether the area postrema (AP) is involved in the regulation of cardiovascular activity in the rats. The aim of the present study was to investigate the role of the AP in the regulation of basal blood pressure under normal as well as abnormal (cold stress) conditions in Sprague-Dawley rats. The lesion of AP was performed by the electrolytic-lesion method. Stressed animals were subjected to chronic intermittent cold stress (2 degrees C, 3 h/day for 14 days). The systolic blood pressure was measured by the indirect tail-cuff transducer method. The results showed that no significant difference was found between systolic blood pressure measured before and after AP-lesion surgery. The AP-lesion group had similar systolic blood pressure to both sham-operation and the control groups under normal environmental conditions. However, it was found that cold stress resulted in a significant increase in systolic blood pressure in the AP-lesion rats, but not in sham-lesion animals, within two weeks. Furthermore, there was no significant difference between blood pressures of sham-lesion rats with or without cold stress and the control animals. These results support the view that the AP plays no role in keeping basal blood pressure under normal condition and indicate as well that the AP is important in maintaining normal blood pressure under the conditions of stress (cold).

After the publication of this article (EurJ Microbiol Immunol (Bp) 2013;3: , 13 Mar), we discovered that the version of Figure 3b included in the article is incorrect due to it being done for a separate project. Please see the correct Figure 3b file here. This change does not affect the summary data for Figure 3c, statistical analysis, or figure legend. Fig. 3b. Sustained Tl1a expression led to an increased expression of activation and gut homing marker on Tl1a-Tg T cells.

Introduction: Faecal Calprotectin (FC) is a cytosolic protein belonging to the S-100 family of calcium binding proteins found in neutrophils. It is excreted in the intestinal lumen in inflammatory conditions of the gut and can be used to distinguish irritable bowel syndrome (IBS) from other inflammatory bowel conditions such as colitis, diverticulosis, etc. Pointof-contact qualitative FC tests are now available and can be used in primary care to aid decision making for referrals to gastroenterologists for young patients presenting with chronic diarrhoea. Aims: To assess the feasibility and cost effectiveness of a primary care Pathway using a point-of-contact FC Test (Caldetect®) to aid decision making for referrals to gastroenterology in young patients presenting to their primary physicians with chronic diarrhoea. Methods: Primary Care data indicated that approximately 253 referrals are made annually to gastroenterologists from Primary Care to assess patients ,60years presenting with diarrhoea, costing approx. £119,000 for investigations and consultations. Using a Caldetect® (Preventis, GmbH) point of contact FC test, it was estimated that a saving of £89,000 could be achieved. A pathway for investigating chronic diarrhoea using Caldetect® was designed and implemented in the community (population 150,000) between September 2011-March 2012. (this will be presented). FC results were categorised using manufacturer cut-offs of ,15ug/ g, 15-60ug/g and .60ug/g. Patients with FC results of 15-60 and .60 were deemed to have an inflammatory process and referred to Gastroenterology Clinics. Cost analysis was carried out using the 2010-11 tariffs for the NHS. Results: 142 Caldetect® tests were carried out in Primary Care during this pilot phase. Of these, a negative result ( ,15ug/g) was present in 89, with 36 tests being .60ug/g. 3 tests were at the intermediate level and 14 tests could not be accurately reported. Negative results were managed in primary care as IBS. A monthly cost savings of £6100 was calculated taking consultation and endoscopy tariffs into account. Conclusion: This pilot study demonstrates the feasibility and cost effectiveness of a Pathway for decision making and a point-of-care faecal calprotectin test in rationalising referrals to Gastroenterologists for chronic diarrhoea.

Endothelin-1 (ET-1) is a potent vasoconstrictor involved in the regulation of vascular tone and implicated in hypertension. However, the role of small blood vessels endothelial ET-1 in hypertension remains unclear. The present study investigated the effect of chronic over-expression of endothelial ET-1 on arterial blood pressure and vascular reactivity using transgenic mice approach. Transgenic mice (TET-1) with endothelial ET-1 over-expression showed increased in ET-1 level in the endothelial cells of small pulmonary blood vessels. Although TET-1 mice appeared normal, they developed mild hypertension which was normalized by the ET A receptor (BQ123) but not by ET B receptor (BQ788) antagonist. Tail-cuff measurements showed a significant elevation of systolic and mean blood pressure in conscious TET-1 mice. The mice also exhibited left ventricular hypertrophy and left axis deviation in electrocardiogram, suggesting an increased peripheral resistance. The ionic concentrations in the urine and serum were normal in 8-week old TET-1 mice, indicating that the systemic hypertension was independent of renal function, although, higher serum urea levels suggested the occurrence of kidney dysfunction. The vascular reactivity of the aorta and the mesenteric artery was altered in the TET-1 mice indicating that chronic endothelial ET-1 up-regulation leads to vascular tone imbalance in both conduit and resistance arteries. These findings provide evidence for the role of spatial expression of ET-1 in the endothelium contributing to mild hypertension was mediated by ET A receptors. The results also suggest that chronic endothelial ET-1 over-expression affects both cardiac and vascular functions, which, at least in part, causes blood pressure elevation. © 2011 Leung et al., published_or_final_version

Background and Aims: Clostridium difficile infection is the major cause of nosocomial antibiotic-associated colitis. C. difficile produces toxin A, a proinflammatory enterotoxin involved in the pathophysiology of C. difficile infection. Cathelicidin is an endogenous antimicrobial peptide that protects the host from infections. Recent findings also support the ability of cathelicidin to modulate innate immunity and alter the development of intestinal inflammation. Here we explored whether toxin Amodifies the expression of cathelicidin inmouse ileum and cultured human colonocytes. We also determined the anti-inflammatory mechanism of cathelicidin in toxin A-mediated intestinal inflammation using a mouse model of enteritis and human primary peripheral blood monocytes. Methods: Ileal loops were prepared in anesthetized mice and injected with either toxin A (10 microg/20g mouse) or saline (control) (n= 6 loops per group). A separate group was treated with cathelicidin (200 microg/mouse) administered into the ileal loops. After 4 hrs, levels of cathelicidin and TNFalpha and mucosal histological changes of ileal loops were evaluated. Results: Toxin A significantly increased cathelicidin mRNA and protein expression (up to 5 fold, P=0.005) in mouse ileum. In human colonic epithelial NCM460 cells, toxin A induced cathelicidin protein (by ~2 fold, P 50%, P 50%, P 80%, P 50%, P