M D Barrachina

The stiffening of the extracellular matrix, and changes in its cellular and molecular composition, have been reported in the pathogenesis of fibrosis. We analyze the mechanisms that perpetuate ileal fibrosis in surgical resections of complicated Crohn's disease patients.Ileal resections were obtained from affected and non-affected tissue of stenotic or penetrating Crohn's disease behavior. Ilea from non-IBD patients were used as control tissue. All samples underwent RNA sequencing. Human small intestinal fibroblasts were treated for 48 h with IL-1β, TFGβ1, PDGFB or TNF-α. Resistance to apoptosis was analysed by RT-PCR, western blot and immunohistochemistry in ileal tissue and by RT-PCR and FACS in cultured cells.Growth factor-driven signaling pathways and increased RAS GTPase activity were up-regulated in affected ilea in which we found expression of both the antiapoptotic molecule MCL1 and the transcription factor ETS1 in submucosal fibroblasts, and a senescence-associated secretory phenotype. In cultured intestinal fibroblasts, PDGFB induced an ETS1-mediated resistance to apoptosis that was associated with the induction of both of TGFB1 and IL1B, a cytokine that replicated the expression of SASP detected in ileal tissue. ETS1 drove fibroblast polarization between inflammatory and fibrogenic phenotypes in IL1β-treated cells.Our data show resistance to apoptosis in complicated ileal CD, and demonstrate that PDGFB induce an ETS1-mediated resistance to apoptosis associated with an inflammatory and fibrogenic pattern of expression in intestinal fibroblasts. Results point to PDGFRB, IL1R1 or MCL1 as potential targets against ileal fibrosis.

A single intraperitoneal injection of endotoxin (40 microg/kg) significantly delayed gastric emptying of a solid nutrient meal. Blockade of nitric oxide synthase (NOS) with 30 mg/kg ip N(G)-nitro-L-arginine methyl ester or 20 mg/kg ip 7-nitroindazole [neuronal NOS (nNOS) inhibitor] significantly delayed gastric emptying in control animals but failed to modify gastric emptying in endotoxin-treated rats. Administration of 2.5, 5, and 10 mg/kg ip N(6)-iminoethyl-L-lysine [inducible NOS (iNOS) inhibitor] had no effect in either experimental group. Indomethacin (5 mg/kg sc), NS-398 (cyclooxygenase-2 inhibitor; 10 mg/kg ip), and dexamethasone (10 mg/kg sc) but not quinacrine (20 mg/kg ip) significantly prevented delay in gastric emptying induced by endotoxin but failed to modify gastric emptying in vehicle-treated animals. Ca(2+)-dependent NOS activity in the antrum pylorus of the stomach was diminished by endotoxin, whereas Ca(2+)-independent NOS activity was not changed. In addition, decreased nNOS mRNA and protein were observed in the antrum pylorus of endotoxin-treated rats. Our results suggest that downregulation of nNOS in the antrum pylorus of the stomach and synthesis of prostaglandins mediate the delay in gastric emptying of a solid nutrient meal induced by endotoxin.

Moderate somatic stress inhibits gastric acid secretion. We have investigated the role of endogenously released NO in this phenomenon. Elevation of body temperature by 3°C or a reduction of 35 mmHg (1 mmHg = 133 Pa) in blood pressure for 10 min produced a rapid and long-lasting reduction of distension-stimulated acid secretion in the rat perfused stomach in vivo . A similar inhibitory effect on acid secretion was produced by the intracisternal (i.c.) administration of oxytocin, a peptide known to be released during stress. Intracisternal administration of the NO-synthase inhibitor, N G -nitro- l -arginine methyl ester ( l -NAME) reversed the antisecretory effect induced by all these stimuli, an action prevented by intracisternal coadministration of the NO precursor, l -arginine. Furthermore, microinjection of l -NAME into the dorsal motor nucleus of the vagus nerve reversed the acid inhibitory effects of mild hyperthermia, i.v. endotoxin, or i.c. oxytocin, an action prevented by prior microinjection of l -arginine. By contrast, microinjection of l -NAME into the nucleus tractus solitarius failed to affect the inhibitory effects of hyperthermia, i.v. endotoxin, or i.c. oxytocin. Immunohistochemical techniques demonstrated that following hyperthermia there was a significant increase in immunoreactivity to neuronal NO synthase in different areas of the brain, including the dorsal motor nucleus of the vagus. Thus, our results suggest that the inhibition of gastric acid secretion, a defense mechanism during stress, is mediated by a nervous reflex involving a neuronal pathway that includes NO synthesis in the brain, specifically in the dorsal motor nucleus of the vagus.

Intestinal epithelial cells (IECs) constitute a defensive physical barrier in mucosal tissues and their disruption is involved in the etiopathogenesis of several inflammatory pathologies, such as Ulcerative Colitis (UC). Recently, the succinate receptor SUCNR1 was associated with the activation of inflammatory pathways in several cell types, but little is known about its role in IECs. We aimed to analyze the role of SUCNR1 in the inflammasome priming and its relevance in UC. Inflammatory and inflammasome markers and SUCNR1 were analyzed in HT29 cells treated with succinate and/or an inflammatory cocktail and transfected with SUCNR1 siRNA in a murine DSS model, and in intestinal resections from 15 UC and non-IBD patients. Results showed that this receptor mediated the inflammasome, priming both in vitro in HT29 cells and in vivo in a murine chronic DSS-colitis model. Moreover, SUNCR1 was also found to be involved in the activation of the inflammatory pathways NFкB and ERK pathways, even in basal conditions, since the transient knock-down of this receptor significantly reduced the constitutive levels of pERK-1/2 and pNFкB and impaired LPS-induced inflammation. Finally, UC patients showed a significant increase in the expression of SUCNR1 and several inflammasome components which correlated positively and significantly. Therefore, our results demonstrated a role for SUCNR1 in basal and stimulated inflammatory pathways in intestinal epithelial cells and suggested a pivotal role for this receptor in inflammasome activation in UC.

Chronic treatment with leptin regulates body weight and energy balance and reduces food intake in obese and lean mice. In 18- to 20-h fasted lean mice (C57BL/6, +/+), we examined the acute effect of a single intraperitoneal injection of recombinant mouse leptin (0.12 mg/kg) on food intake and gastric emptying. Leptin reduced food intake, with a peak inhibition at the 5th h postinjection (69 +/- 12%/h), although there was no change in food consumption at the 1st h. Leptin did not alter the 4-h rate of gastric emptying of a solid nutrient meal (free access to Purina chow for either 1-, 2-, or 4-h period). In normal Sprague-Dawley rats fasted for 18-20 h, a single intraperitoneal injection of recombinant mouse leptin (0.2 or 1.2 mg/kg) did not modify the 7-h cumulative or hourly food intake. These results show that a single intraperitoneal injection of recombinant mouse leptin reduces food intake within 5 h while not influencing gastric emptying of ingested food in lean mice. Sprague-Dawley rats are unresponsive to the food intake-reducing effect of a single intraperitoneal injection of mouse leptin at a dose 10-fold higher than that shown to be effective in mice within the first 4-7 h postinjection.

Fibrosis is a common complication of Crohn's disease (CD) in which macrophages play a central role. Epithelial-mesenchymal transition (EMT) and the WNT pathway have been associated with fibrosis. We aim to analyse the relevance of the tissue microenvironment in macrophage phenotype and the EMT process.Intestinal surgical resections are obtained from control and CD patients with stenotic or penetrating behaviour. Cytokine's expression, macrophage phenotype, EMT markers and WNT signalling pathway are determined by WB, RT-PCR, ELISA or Cytometry. U937 cells are treated with IFNγ, TNFα, IL1β, IL4 or IL10 and co-cultured with HT29 cells and, in some cases, are treated with XAV939 or miFZD4. The expression of macrophage, EMT and WNT pathway markers in U937 or HT29 cells is analysed by WB or RT-PCR.IFNγ, WNT6, CD16 and CD86 are increased in the intestinal tissue of CD patients. IFNγ-treated U937 activated the EMT process and WNT pathway in HT29 cells, and the EMT process is mediated by FZD4.An IFNγ-rich microenvironment polarises macrophages, which induces EMT through the WNT pathway.

In inflammatory bowel disease (IBD), chronic inflammation in the gastrointestinal tract can lead to tissue damage and remodelling, which can ultimately result in fibrosis. Prolonged injury and inflammation can trigger the activation of fibroblasts and extracellular matrix (ECM) components. As fibrosis progresses, the tissue becomes increasingly stiff and less functional, which can lead to complications such as intestinal strictures, obstructive symptoms, and eventually, organ dysfunction. Epithelial cells play a key role in fibrosis, as they secrete cytokines and growth factors that promote fibroblast activation and ECM deposition. Additionally, epithelial cells can undergo a process called epithelial-mesenchymal transition, in which they acquire a more mesenchymal-like phenotype and contribute directly to fibroblast activation and ECM deposition. Overall, the interactions between epithelial cells, immune cells, and fibroblasts play a critical role in the development and progression of fibrosis in IBD. Understanding these complex interactions may provide new targets for therapeutic interventions to prevent or treat fibrosis in IBD. In this review, we have collected and discussed the recent literature highlighting the contribution of epithelial cells to the pathogenesis of the fibrotic complications of IBD, including evidence of EMT, the epigenetic control of the EMT, the potential influence of the intestinal microbiome in EMT, and the possible therapeutic strategies to target EMT. Finally we discuss the pro-fibrotic interactions epithelial-immune cells and epithelial-fibroblasts cells.

We have investigated the mechanisms underlying acute changes in gastric motor function triggered by endotoxemia. In fundal strips from rats pre-treated with endotoxin (40 microg/kg, i.p. 30 min), mechanical activity was analyzed and the source of nitric oxide (NO) was visualized by confocal microscopy of tissue loaded with the fluorescent dye DAF-FM. NOS expression was determined by quantitative RT-PCR and Western blot, and enzyme activity by the citrulline assay. Strips from endotoxin-treated rats were hypo-contractile. This was prevented by pre-incubation with the neurotoxin tetrodotoxin, the gangliar blocker hexamethonium, or non-selective and neuronal-specific NOS inhibitors (L-NOARG and TRIM, respectively). The soluble guanylyl cyclase (sGC) inhibitor ODQ and the inhibitor of small conductance Ca2+-activated K+ channels apamin prevented relaxation induced by endotoxin, nicotine, exogenous NO (DETA-NONOate), and the NO-independent sGC activator BAY 41-2272. NO synthesis was observed in neuronal soma, axons, and nerve endings of the myenteric plexus in the fundus of endotoxin-treated rats and was prevented by L-NAME, tetrodotoxin, and hexamethonium. nNOS and iNOS mRNA and protein contents were unchanged. Our findings demonstrate synthesis of NO in post-ganglionic myenteric neurons during early endotoxemia that mediates gastric hypo-contractility. The effect of NO is mediated via sGC and small conductance Ca2+-activated K+channels.

Mechanisms involved in the cephalic phase of gastric acid secretion were studied in awake fasted rats with chronic gastric fistula and exposed to the sight and smell of chow for 30 min. Acid secretion was monitored using constant intragastric perfusion and automatic titration. Sham feeding induced a peak acid response reaching 82 ± 7 μmol/10 min within 20 min compared with the average 22 ± 2 μmol/10 min in controls. The sham-feeding response was abolished by intracisternal pretreatment with the TRH 1 -receptor antisense oligodeoxynucleotides or subcutaneous injection of atropine, whereas TRH 1 mismatch oligodeoxynucleotides had no effect. Serum gastrin was not altered by the sham feeding and increased by refeeding. Gastrin antibody did not block the rise in acid during sham feeding, although the net acid response was reduced by 47% compared with the control group. Glycine-gastrin antibody, indomethacin and nitro-l-arginine methyl ester had no effect. Atropine and gastrin antibody decreased basal acid secretion by 98 and 75%, respectively, whereas all other pretreatments did not. These results indicate that the cholinergic-dependent acid response to sham feeding is mediated by brain medullary TRH 1 receptors in rats.

Abstract Background Fibrosis represent the main complications related to Crohn's disease (CD). Notch signalling mediate fibrogenic process, including epithelial-mesenchymal transition (EMT) and fibroblast senescence but its role in CD fibrosis is currently unknown. Previous studies have shown a high expression of NOTCH4, NOTCH3, DLL3 and DLL4 in CD fibrotic tissue. DLL4 mainly activates transcription factors involved in EMT, and macrophages could act as a possible source of DLL4 (Edo, et al., 2022. JCC (i200)).The general aim of the present study is to determine the possible potential of Notch pathway as a therapeutic target in intestinal fibrosis associated with CD. Specifically, we pretend: to analyze the localization of NOTCH3/4 receptors in the intestinal tissue of patients with CD complicated; to study the relevance of NOTCH3/4 receptors in the EMT; to study the relevance of Notch pathway in the senescence of intestinal fibroblast. Methods We have analyzed in intestinal samples from CD patients with complicated lesions: the localization of NOTCH3/4 receptors by IH and the protein expression of senescence markers (BCL2 and P53) by WB. We carry out in vitro studies and analyze: the protein expression of HES1 (effector Notch pathway) and EMT markers in DLL4-HT29 treated cells transfected with miNOTCH3 or miNOTCH4; and the protein expression of senescence proteins in HSIF fibroblasts treated with DLL4 or DLL3. Results NOTCH3 was located preferentially in muscular areas -muscularis mucosa, endothelium and muscularis externa- with a striking staining of infiltrated cells in the mucosa of the unaffected area. NOTCH4 is found more specifically in the crypts of the mucosa, as well as in cells of the lamina propria of the unaffected mucosa. The expression of senescence proteins in the tissue showed elevated levels of BCL2 and P53, compared to the unaffected tissue of the same patient. DLL4 increased the protein expression of EMT markers in HT29 cells, and NOTCH4 silencing significantly reverted the expression of these EMT markers. NOTCH3 silencing produced no significant changes after DLL4-HT29 treatment. DLL3, and not DLL4, produced in intestinal fibroblasts a significant increase in the protein expression levels of BCL2, and P53, compared to the vehicle (Figure). Conclusion NOTCH pathway is involved in the regulation of key cellular functions and processes essential for the pathogenesis of intestinal fibrosis in CD patients. DLL4-NOTCH4 interaction triggers transcription factors involved in mesenchymal epithelial transition in colonic epithelial cells, while DLL3 seems to have a more relevant role in activation of senescence in fibroblasts.

This study examines the role of a central pathway involving glutamate receptors, nitric oxide (NO) and cyclic GMP in the acute inhibitory effects of low doses of peripheral endotoxin on pentagastrin‐stimulated acid production. Vagotomy or intracisternal (i.c.) microinjections of the NO‐inhibitor, N G ‐nitro‐ L ‐arginine methyl esther ( L ‐NAME; 200 μg rat −1 ) restored acid secretory responses in endotoxin (10 μg kg −1 , i.v.)‐treated rats. The acid‐inhibitory effect of i.v. endotoxin (10 μg kg −1 , i.v.) was prevented by prior i.c. administration of the NMDA receptor antagonists, dizocilpine maleate (MK‐801; 10 nmol rat −1 ) and D‐2‐amino‐5‐phosphono‐valeric acid (AP‐5; 20 nmol rat −1 ), or the AMPA/kainate antagonist 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX; 10 nmol rat −1 ). However, the competitive metabotropic glutamate receptor antagonist (+)‐α‐methyl‐4‐carboxyphenylglycine (MCPG; 20–1000 nmol rat −1 ) did not antagonize the effects of endotoxin. I.c. administration of L ‐glutamate (0.1 nmol rat −1 ) inhibited pentagastrin‐stimulated gastric acid secretion. Coadministration with L ‐NAME (200 μg rat −1 ) prevented the inhibition of gastric acid secretion by the aminoacid. I.c. administration of 1H‐[1,2,4]Oxazodiolo[4,3‐a]quinoxalin‐1‐one (ODQ; 100 nmol rat −1 ), a soluble guanylyl cyclase (sGC) blocker, reversed the hyposecretory effect of endotoxin. I.c. administration of the cyclic GMP analogue 8‐Bromoguanosine‐3,5‐cyclic monophosphate (8‐Br‐cGMP; 100–300 nmol rat −1 ) reduced gastric acid production in a dose‐dependent manner. We conclude that central NMDA and AMPA/kainate receptors are involved in the acid inhibitory effect of peripherally administered endotoxin. This central pathway involves synthesis of NO, which acts on the enzyme sGC. British Journal of Pharmacology (2000) 130 , 1283–1288; doi: 10.1038/sj.bjp.0703436

It has been suggested that the peripheral sensory neurons are involved in the splanchnic hemodynamic changes of portal hypertension. Therefore the influence of permanent ablation of sensory neurons by neonatal capsaicin pretreatment (50 mg/kg, subcutaneously) on the development of the hyperdynamic splanchnic circulation in portal-hypertensive rats was studied. In adulthood, portal hypertension was induced with partial portal vein ligation. In study 1, systemic and splanchnic hemodynamics were measured by means of a radiolabeled-microsphere technique in portal-hypertensive rats, under ketamine anesthesia, pretreated with capsaicin or vehicle. Mean arterial pressure, heart rate, cardiac index, systemic and splanchnic vascular resistance, portal pressure, portal venous inflow, portal-collateral resistance and portalsystemic shunting were not significantly different between capsaicin-pretreated and vehicle-pretreated rats. In study 2, gastric mucosal blood flow, measured by means of hydrogen gas clearance, and the hemoglobin and oxygen content of the gastric mucosa, as assessed with reflectance spectrophotometry, were not significantly different in the two groups of anesthetized portal-hypertensive rats pretreated with capsaicin or vehicle. In study 3, we confirmed the effectiveness of neonatal capsaicin pretreatment by measuring calcitonin gene—related peptide content of the gastric corpus wall. Capsaicin pretreatment caused a depletion of calcitonin gene—related peptide by at least 98% compared with that in vehicle-pretreated rats. These results do not support a role of capsaicin-sensitive sensory neurons that innervate the gastrointestinal tract in the development of the splanchnic vasodilatation characteristically observed in chronic portal hypertension. (Hepatology 1994;20:1609–1614).

Abstract Background Fibrosis constitute an important complication of CD. MicroRNAs (miRNAs), which are small RNA molecules that regulate gene expression, have been shown to participate in the molecular interactions of both inflammation and fibrosis. Lower levels of miR-378a-3p have been reported associated to murine liver fibrosis (Hyun et al. DOI: 10.1038/ncomms10993) and we analyze here the relevance of miR-378a-3p on intestinal fibrosis. Methods B57BL/6 mice were intravenously injected with 2,5mg/kg of negative control (NC) or mm-miR-378a-3p mimic, twice a week and received vehicle or Dextran Sulfate Sodium (DSS) for 2 cycles (7 days drinking DSS 2% in water solution followed by 10 days drinking water). Body weight and DAI score was obtained every day and the colon was collected after sacrifice. Sirius and hematoxylin-eosin dyes were employed to determine the fibrosis and structural state in 5µm slides of intestinal tissue. Gene expression and miRNA profiles were analyzed by RT-qPCR. Human small intestinal fibroblasts (HSIF; P10760, Innoprot, Spain) were transfected with 20nM of NC or hsa-miR-378a-3p mimic during 24h. Results No significant changes in body weight, DAI score, and colon length were detected between mice receiving NC and those receiving mimic, all along the two DSS cycles. Colon of mice treated with DSS, exhibited a significant diminution in the mRNA expression of mm-miR-378a-3p compared with naïve samples. In DSS-treated mice, the iv administration of the mimic compared with the NC: a) significantly increased levels of miR-378a-3p in the colon; b) increased the number of neutrophils, and heightened changes in the glandular epithelia and architectural distortion (figure 1a) C) decreased the number of lymphoid follicles (figure 1a); d) slightly increased collagen deposition, as analyzed by sirius red (figure 1b) and e) significantly increased the mRNA expression of Tgfb1, Il1b, and Mmp2. Treatment of human intestinal fibroblasts with hsa-miR-378a-3p mimic did not significant modify the mRNA expression of markers of fibrosis but it significantly increased the mRNA expression of two antiapoptotic molecules BCL2 and MCL1. Conclusion Levels of mm-miR-378a-3p are diminished in a murine model of intestinal fibrosis; The exogenous administration of miR-378a-3p, increased the acute inflammatory response and the expression of fibrosis markers in murine fibrotic colon and the expression of antiapoptotic molecules in human intestinal fibroblasts.

Abstract Background Fibrosis is a complication commonly present in Crohn’s disease (CD) patients with a structuring (B2) or penetrating (B3) behaviour, with no available treatment. This process is characterized by an excessive extracellular matrix deposition, mainly associated with a dysregulated function of myofibroblasts. We analyse here, the expression of markers of senescence in human intestinal fibroblasts. Methods Human small intestinal fibroblasts (HSIF; P10760, Innoprot, Spain) were treated during 48h with 2µg/mL of DLL4, 20ng/ml of TNFα, 2ng/ml of IL-1β, 5ng/ml of TGFβ1, and 100ng/ml of. In some cases, fibroblasts were treated with 20nM of siRNA to ETS1 gene (siETS21) or negative control (NC). Gene expression profiles were analysed by RT-qPCR. Results Treatment of fibroblasts with IL-1β significantly increased the mRNA expression of a) different senescence-associated secretory phenotype (SASP) factors (IL-1α, IL-1β, IL-8, and Serpine1); b) a metalloprotease ADAM12 and the chitinase CHI3L1 and c) the transcription factor related to senescence ETS proto-oncogene 1 (ETS1), all compared with vehicle treatment. Treatment with PDGF significantly increased the mRNA expression of ADAM12, ETS1, PTEN and the transcription factor, E2F5, while it induced a non-significant increase in the expression of senescence markers such as P16, P21, P53 and MCL1. Treatment with TGFβ1 did not significantly alter the expression of markers reported above and it only significantly increased the expression of IGFBP3 gene, another SASP factor. Both DLL4 and TNFα failed to significantly modify the expression of any of the above markers. Finally, a cellular model of depletion of ETS1 showed that siETS1 fibroblasts in basal conditions exhibited decreased levels of IL-8 and BCL2. Conclusion In human primary intestinal fibroblasts, treatment with IL1b increased the expression of senescence associated secretory phenotype while treatment with PDGF increased the expression of markers involved in cell cycle arrest. The induction of ETS1 by both treatments and the involvement of this transcription factor regulating gene expression in basal conditions, suggest a relevant role for ETS1 in the activation of intestinal fibroblasts

Abstract Background Crohn’s Disease (CD) is a subtype of IBD characterized by a chronic transmural inflammation of the gastrointestinal tract associated with several complications being intestinal fibrosis the most frequent. CD patients present microbiota dysbiosis and altered metabolomic profiles. GPCRs constitute a family of receptors which could be involved in inflammatory and fibrotic processes associated to CD. We aim to characterize microbiota composition, tissue metabolomic profile and metabolite-sensing GPCRs expression in ileal resections from fibrotic CD patients. Methods Ileal resections from B2-CD (n=21) and non-IBD (n=13) patients were obtained. Microbiota characterization was performed by 16S rRNA gene Illumina Miseq sequencing. Bioinformatic analysis of sequencing data was performed using constrained correspondence analysis and non-parametric Wilcoxon test to compare species proportions. Bacterial load was estimated by qPCR. Metabolomic analysis was performed by NMR. Results are expressed as μg metabolite/g tissue. Murine intestinal fibrosis was induced in C67BL/6 mice by: a) the heterotopic intestinal transplant model and b) chronic administration of 4 cycles of increasing DSS percentages. Gene expression of GPCRs was analyzed by qPCR. Data were expressed as fold induction vs control (mean±SEM) and compared by a t-test. Correlations were analyzed with the Spearman coefficient. Results First, microbiota analysis revealed a reduction in bacterial diversity and load in fibrotic CD patients. Then, in B2-CD samples we found at genus level Enterococcus genera significantly decreased and at species level Ruminococcus bromii and Faecalibacterium prausnitzii also reduced compared to controls. From the metabolomic analysis, altered levels of metabolites were found in ileal resections from fibrotic CD patients as summarized in Table 1. Next, B2-CD patients exhibited differential expression of metabolite-sensing GPCRs vs non-IBD as shown in Table 2. Moreover, gene expression of fibrotic markers was analyzed in B2-CD patients and significantly increased levels of COL1A1 (13.22±4.38), COL3A1 (1.84±0.52), and COL4A1(7.75±2.19), were found vs controls. Of interest, GPR81, GPR84, GPR4 and GPR68 positively correlated with profibrotic markers, specifically with COL1A1 and COL4A1. Finally, in line with human results, we also analyzed the expression of metabolite-sensing GPCRs in two different murine colitis models and results obtained are represented in Table 3. Conclusion Fibrotic CD patients exhibit microbial dysbiosis, joined with altered levels of metabolites and gene expression of metabolite-sensing GPCRs, which are also affected in murine colitis models. Their correlation with profibrotic markers points them as protagonists of intestinal fibrosis.

Introduction: We have recently shown that membrane fatty acids are key modulators of megakaryocyte differentiation and platelet formation. In obesity, patients have an altered plasma lipid composition, which coincides with enhanced platelet responsiveness to activation. However, the mechanism underlying this platelet phenotype remains unknown. Hypothesis: We hypothesized that the obese plasma lipidome alters the composition of the megakaryocyte membrane, which ultimately leads to the generation of hyper-reactive platelets. Aim: To investigate the impact of high fat diets with different fatty acid compositions on megakaryocyte development and platelet reactivity. Results: Mice were fed chow or high fat diets enriched in either saturated fatty acids (50% SFAs) or polyunsaturated fatty acids (50% PUFAs, omega-6-enriched). Neither high fat diet resulted in changes in megakaryocyte numbers, but both caused a significant increase in the size of bone marrow megakaryocytes, suggesting diet-derived lipid uptake into megakaryocytes. When examining platelet reactivity, we found that mice fed the SFA-enriched high fat diet exhibited increased expression of the active form of integrin αIIbβ3 (JON/A) in resting platelets and that both JON/A and P-selectin expression were significantly decreased after platelets were stimulated with ADP and the thromboxane A 2 analogue U46619. In contrast, platelets from mice fed the PUFA omega-6-enriched high fat diet had no differences in platelet reactivity compared to mice fed normal chow. Conclusions: Our data reveal that dietary fatty acid saturation status affects platelet reactivity, consistent with previous human studies showing variability in platelet reactivity in obese individuals. Further explorations will elucidate if plasma and/or platelet lipid content may be an indicator of risk for thrombosis or cardiovascular disease.

BackgroundMacrophages contribute to fibrosis through the release of different mediators and the pattern of secretion may vary according to their phenotype. The expression of WNT ligands has been related with the macrophage phenotype and strong evidence identifies the WNT signalling pathway as an emerging modulator of fibrosis.

Abstract Background Ulcerative colitis (UC) is characterized by a diffuse, continuous, and chronic inflammation of mucosa and submucosa layers in the colon whose etiology is still unknown. Intestinal microbiota dysbiosis and alterations in the metabolomic profile have been reported in mucosal biopsies from Inflammatory Bowel Disease (IBD) patients. We aim to characterize the microbiota composition and metabolomic profile in colonic resections of UC patients. Methods Colonic resections from UC (n=18) and non-IBD (n=20) patients were obtained. Microbiota identification, composition and classification was performed by 16S rRNA gene Illumina Miseq sequencing. The bioinformatic analysis of sequencing data was performed using constrained correspondence analysis (CCA) and non-parametric Wilcoxon test to compare species and genera proportions. Bacterial load was estimated by qPCR. Metabolomic analysis was performed using Nuclear Magnetic Resonance (NMR) to study polar metabolites and Gas-Chromatography or Liquid-Chromatography Mass-Spectrometry to study non-polar metabolites. Results Microbiota analysis revealed differences at genus and species level between UC and non-IBD patients. Cellulosimicrobium was found at 10-fold higher levels in UC samples while Escherichia genus was 3.88 times more abundant in controls. A reduction in bacterial load was seen in UC samples. Levels of short-chain fatty acids (FA), medium-chain FA, long-chain FA, carboxylic acids and amino acids were obtained from the metabolomics analysis and a positive correlation between propionic, aspartic and hydroxybutyric acids and Cellulosimicrobium and Pseudomonas was found (Fig1B). Gut commensals such as unclassified Agathobacter, Blautia faecis, Oscillospiraceae or Faecalibacterium prausnitzii positively correlated with the abundance of butyric acid, acetic acid and succinic acid; meanwhile Subdoligranulum, Lachnospireace or unclassified Blautia negatively correlated with some medium or long chain fatty acids (Fig1B). Fig1. Correlations between bacterial species and metabolites in (A) controls and (B) UC patients. Performed with mixOmics library in R. Conclusion Significant differences in microbiota composition and abundance are found in UC patients, which undergo a clear gut dysbiosis in colonic tissue. Correlation analysis suggests Cellulosimicrobium and Pseudomonas as sources of propionic, aspartic and hydroxybutyric acid. These results point at Cellulosimicrobium as a potential candidate in UC pathology.

Abstract Background Ulcerative colitis (UC) is characterized by a diffuse, continuous, and chronic inflammation of mucosa and submucosa layers in the colon1. Inflammasome complex is involved in the intestinal homeostasis regulation, but its role in UC has not been established yet. We have recently reported that SUCNR1 mediates intestinal inflammation and fibrosis2. We aim to analyze the role of SUCNR1 in inflammasome activation and UC. Methods Intestinal resections from UC and non-IBD patients were obtained. HT29 cells were treated with succinate 1mM and an inflammasome activator cocktail (TNF-α 25ng/ml, IFN-γ 20 ng/ml and LPS 1µg/ml) for 24 hours and transfected with SUCNR1 siRNA. Chronic DSS-colitis was induced in wild-type (WT) and SUNCR1-/- (KO) mice with 4 cycles of DSS. Gene expression and protein levels of SUCNR1 and inflammasome markers were analyzed by qPCR, WesternBlot and ELISA. Histology of murine tissue was analyzed by Hematoxylin-Eosin staining. Results were expressed as fold induction (mean±SEM, n≥5). Statistical analysis was performed with one-way ANOVA followed by Newman-Keuls test. Correlations were analyzed with the Spearman coefficient. Results In UC patients, gene expression of SUCNR1 (4.47±1.70), Nlrp3 (1.97±0.38), Caspase-1 (2.08±0.33) and IL-1β (6.90±2.02) were significantly increased vs non-IBD. SUCNR1 positively correlates with the expression of Caspase-1 (r=0.46) and ASC (r=0.45). Protein levels of SUCNR1 (151.00±8.29), Nlrp3 (215.20±53.35) and Caspase-1 (518.30±231.50) were increased in UC vs non-IBD. HT29 cells treated with succinate and inflammasome cocktail showed an increased gene expression of SUCNR1 (4.45±1.17), Nlrp3 (3.13±0.29), Caspase-1 (70.40±16.14), ASC (2.38±0.37), IL-18 (2.52±0.31) and IL-1β (2.32±0.18). Of interest, siSUCNR1 cells treated with the cocktail and succinate showed a significant reduction in the expression of Nlrp3 (1.00±0.15), IL-1β (0.98±0.21), and ASC (1.19±0.14). In parallel, protein levels of Caspase-1 in siSUCNR1 cells treated with cocktail and succinate were significantly reduced (609.00±116.20) vs non-transfected cells (1095.00±148.30). IL-1β levels were also significantly reduced in siSUCNR1 cells (148.60±16.13) vs non-transfected cells (744.70±94.06). Finally, WT-DSS mice exhibited a worse colon histology and an increased protein expression of Caspase-1 (170.60±32.20), compared with KO-DSS (61.39±4.59). Conclusion SUCNR1 is increased and positively correlates with the expression of inflammasome components in UC patients. Moreover, SUCNR1 mediates inflammasome activation and its absence ameliorates chronic DSS-colitis. Hence, SUCNR1 might be a potential pharmacological target for UC treatment. Reference

To investigate the eradication rate of Helicobacter pylori with omeprazole, amoxicillin and clarithromycin during 6 days in patients with duodenal ulcer. To compare the reliability of the analysis of the eradication with urea-13C breath test performed one month and 3 months after therapy. To evaluate the one-year reinfection rate.Prospective study including 99 patients with duodenal ulcer (65 with acute disease and 34 in maintenance treatment) infected by Helicobacter pylori (urease rapid test and urea-13C breath test positive). Patients were treated with omeprazole 20 mg, clarithromycin 500 mg and amoxicillin 1 g, b.i.d., during 6 days. The infection status was investigated 1 and 3 months after treatment by urea-13C breath test. The one-year reinfection rate was investigated using the same test.Per protocol eradication rates were 76% (95%-CI: 66-84) one month and 73% (95%-CI: 63-81) 3 months after treatment. In the intention to treat analysis, eradication rates were 74% (95%-CI: 64-82) and 70% (95%-CI: 60-79), respectively. Side effects were mild and uncommon. The rate of false negative urea-13C breath test results one month after therapy with respect to 3 months was 4.2% (95%-CI: 0.8-11.7). One-year reinfection rate determined in 56 patient was absent.The eradication of Helicobacter pylori with triple therapy for 6 days in patients with duodenal ulcer is not satisfactory. To investigate Helicobacter pylori infection with urea-13C breath test one month after treatment overestimates the results of the eradication. One-year reinfection rate is clinically irrelevant.

The present study analyses the analgesic activity and action on in vitro motility of methanol soluble and methanol insoluble fractions obtained from the hexane extract of fruits from Araujia sericifera. The methanol fraction did not show any pharmacological activity on the different tests evaluated. However, the insoluble methanol fraction exhibited an interesting analgesic effect in models of chemical and thermal stimulus and it reduced the Emax induced by histamine in vitro on guinea-pig ileum. This extract lacked any central depressor activity since it did not modify the number of mouse movements in the activity cage.

Background: Postoperative ileus follows every abdominal surgery.Recently, in vitro data suggested that impaired contractility of the rat small intestine following abdominal surgery resulted from late influx of inflammatory cells caused by intestinal manipulation (Kalff et al., 1998).Whether this inflammation is confined to the small intestine and is associated with changes in gastrointestinal motility in vivo in other parts of the gut remains unknown.Therefore, we evaluated the effect of intestinal manipulation and subsequent inflammation on gastric emptying in mice, Methods: Female Balb/C mice were anaesthetized with ketamine/xylazine.Animals underwent only anaesthesia (C, n=8), a skin incision (SI, u=8) or a laparotomy followed by manipulation of the small intestine (L+M, n=8).After 24 h and 48 h, gasl~ emptying was measured using pin-hole scintigraphic imaging after oral gavege of a 99Tc labeled semi-liquid meal (1.5 % methylcellulose) at 16 min intervals, After the gastric emptying study, the animals were killed and tissue (stomach, ileum, colon) was isolated and processed for immuno-histochemical staining of CD4, CD8, LFA-1 expressing cells.Results: After 24 h, gastric emptying was significantly delayed after L+M compared to L or C (C: 24 _+ 4 %, SI: 25 ± 4%, L+M: 53 _+ 4% of the residual activity present 48 min after gavaga, p<O.01).48 hours after surgery, gastric emptying recovered back to normal (C: 24 + 4%, SI: 19_+ 3%, L+ M: 31 _+ 7% of the residual activity present 48 min after gavaga).Inflammatory cells (CD4+ and LFA-I+, but no CD8+), were observed 24h and 48 h post-surgery in the muscularis of the small intestine, but not in that of the stomach or colon.Conclusions: Manipulation of the small intestine 1. induces influx of inflammatory cells in the muscularls of the small intestine, but not in the stomach or colon, and 2. results in prolonged gastric hypomotility in the mouse.These findings indicate that delayed gastric emptying following abdominal surgery does not result from local gastric inflammation.Whether it results horn activation of inhibitory neural pathways to the stomach triggered by the small intestinal inflammation remains to be studied.

Per acollir les families nouvingudes que s'incorporen al sistema educatiu catala, a la nostra escola, amb la col.laboracio de l'AMPA i dels professionals del centre, es promouen trobades d'acollida que afavoreixen la voluntat de crear una comunitat educativa a partir de la realitat plurilingue i multicultural on vivim. Aquesta es una aportacio mes per assolir la cohesio social, que es objectiu de tots.

Background: A defective autophagy is involved in the pathogenesis of inflammatory disorders such as IBD. Cross talk interactions between autophagy and inflammation have been reported and we analyse the effects of autophagy stimulators on murine colitis. Methods: Mice were treated with intrarectal administration of TNBS (3.5 mg/20 mg mice) and body weight was measured every day (and expressed as a percentage of starting weight), and histological damage score analysed two or four days after treatment. Some mice received trehalose (3% in drinking water three weeks before TNBS administration) or a daily administration of rapamycin (1.25 mg/kg, i.p.), betanin (1g/kg, i.p.) or betanin + 3MA (10mg/kg, i.p.). Mucosal protein levels of p-mTOR, p62, LC3, BCL10, NF-κB, IκBα and p-IκBα were determined by WB and mRNA expression of TNFα, IL1β, IL6, IL10, COX2, CCR7, CD11c, iNOS and CD86 by qRT-PCR. Results: An impaired autophagy associated with body weight loss and intestinal damage was detected in the mucosa of TNBS-treated mice. Administration of trehalose, rapamycin or betanin prevented the impaired autophagic flux induced by TNBS and decreased the expression of pro-inflammatory cytokines and M1 macrophage markers (Fig. 1A) and mucosal protein levels of BCL10, p-IκBα and NF-κBp65. Blockade of the autophagosome formation by treatment of mice with 3MA prevented the reduction in both body weight loss (Fig. 1B) and protein levels of p62, BCL10, p-IκBα and NF-κBp65 (Fig 1C) induced by betanin in TNBS-treated mice and weakened the protective effects of betanin on murine colitis. Figure 1 Conclusions: Our results demonstrate that pharmacological stimulation of mucosal autophagy reduces intestinal inflammation and ameliorates murine colitis.

Background: Vitamin D signaling modulates inflammation through the vitamin D receptor (VDR) which is a member of the nuclear receptor family of transcription factors. The presence of C instead of T in the single-nucleotide polymorphism (SNP) rs731236 in the VDR gene has been associated with a higher risk for Crohn's disease (CD). We analysed the relevance of the presence of risk allele C in the evolution of the disease. Methods: DNA was extracted from blood samples from 99 patients diagnosed with CD and 72 healthy donors from the Hospital of Manises (Valencia) and the SNP was genotyped using PCR-RFLP. We collected clinical data for each patient, including the Montreal classification in several phenotypes. Also, peripheral blood mononuclear cells (PBMCs) from 16 CD patients with the TT or CC genotype were obtained and gene expression of some cytokines was quantified in these cells by real-time RT-PCR. Results: The allelic frequency of the risk allele was higher in CD patients related to healthy controls (p=0.2881, Fisher's test) and it was significantly different when compared with patients showing a B3 phenotype (p=0.026, Fisher's test). In addition, CD patients homozygous for the risk allele C initiated with the disease at a lower age (Fig. 1; p=0.05, t-test CC vs TT), and exhibited a significant higher risk to have a B3-penetrating phenotype (Fig. 2; p=0.0018, Chi-square; p=0.0078, Fisher's test CC vs TT, OR=5.3) and to need surgery (Fig. 3; p=0.013, Chi-square; p=0.021, Fisher's test CC vs TT, OR=4.3). Finally, PBMCs from patients with the CC genotype showed a higher level of IL1 β (p=0.13, t-test), IL18 (p=0.05, t-test) and IFN γ (p=0.36, t-test) mRNA than patients with the TT genotype. Figure 1 Figure 2 Figure 3 Conclusions: Our study indicates that homozygosity for the allele C in the SNP rs731236 in the VDR gene confers a higher risk to develop a B3-penetrating phenotype in CD patients, associated with an elevated expression of pro-inflammatory cytokines in PBMCs.

Intestinal fibrosis is a common complication associated with Crohn’s Disease (CD) which cannot be reverted with any drug and forces repeated surgery. It has been reported that succinate, a metabolite accumulated in inflammatory pathologies, plays an important role in the activation of synovial fibroblasts and hepatic stellate cells through its receptor called SUCNR1 or GPR91. We aim to analyse the relevance of SUCNR1 receptor in intestinal fibrosis. Intestinal resections from CD patients and colon carcinoma patients were obtained and the expression of SUCNR1 and α-sma were analysed by immunostaining. Primary intestinal fibroblasts from human resections or from colon of wild-type (WT) or SUCNR1−/− (KO) mice were isolated, maintained in culture and treated with different concentrations (0, 0.1, 0.5, 1, and 5 mM) of succinate for 24 h. Intestinal fibrosis was induced in vivo introducing one intestinal graft from WT or KO mice into the neck of a receptor mice for 7 days. The expression of pro-fibrotic markers was analysed by qPCR. Sirius Red staining was performed and the collagen layer was quantified using ImageJ. Results are expressed by mean ± SEM (n ≥ 5). Statistical analysis was performed with one-way ANOVA followed by Newman–Keuls test. Correlations were analysed with the Spearman coefficient. SUCNR1 is expressed in epithelial cells and α-sma+ cells of intestinal resections from CD patients. The SUCNR1 expression positively and significantly correlates with the expression of α-sma (r = 0.759, p < 0.0001, n = 24) and colA1 (r = 0.82, p < 0.001, n = 24). In primary fibroblasts isolated from CD patients (13.54 ± 4.6) the expression of SUCNR1 was significantly higher than in those obtained from control patients (2.07 ± 0.86). In these cells, succinate induced the expression of profibrotic markers such as ColA1, α-sma, Tgfb, and TIMP1 in a dose-response manner. This profibrotic effect of succinate was also observed in fibroblasts from WT mice and it was completely reverted in fibroblasts obtained from KO mice. The murine model of intestinal fibrosis in vivo revealed that: (a) the thickness of the collagen layer was significantly reduced in colons from KO mice compared with those from WT mice; (b) the expression of pro-fibrotic markers such as colA1, α-sma and vimentin was also significantly reduced in colons from KO mice vs. colons from WT mice (19.7 ± 10.1 vs. 69.8 ± 26.6, 0.9 ± 0.1 vs. 2.5 ± 0.5, 1.7 ± 0 vs. 12.4 ± 0.3, respectively). An increased expression of SUCNR1 receptor is detected in fibroblasts from CD patients the activation of which induces a pro-fibrotic effect. This receptor mediates murine intestinal fibrosis and we propose its blockade as a new pharmacological target in CD treatment.

Vitamin D signals through the vitamin D receptor (VDR) which is a member of the nuclear receptor family of transcription factors that play an immunoregulatory role in the gut. Defective signalling due to vitamin D deficiency or decreased mucosal VDR levels has been related to Crohn’s disease (CD). We aim to analyse the acute effects of Vitamin D in the activation of human intestinal fibroblasts. Fibroblasts were isolated from non-damaged and damaged intestinal resection of CD patients and control patients (non-damaged intestine from colon cancer). Fibroblasts were treated with 1,25 Vitamin D3 (10 nM and 100 nM) for 24 h. Gene expression of pro-inflammatory cytokines and COL1A1 was quantified by qPCR and protein levels were determined by western blot. Statistical significance was measured by ANOVA. Vitamin D increased the mRNA expression of VDR in fibroblasts obtained from the inflamed and non-inflamed mucosa of CD patients (Figure 1A) and it increased the mRNA of CYP24A1, a VDR target (Figure 1B). Treatment with vitamin D rised in a dose-dependent manner COL1A1 mRNA expression in fibroblasts from CD patients (Figure 1C) and in parallel it induced the expression of pro-inflammatory cytokines (IL1β , IL6) (Figure 1D, 1E). Protein levels of phospho-NFκB and phospho-STAT3 were also higher in fibroblasts treated with Vitamin D from CD patients. Our study indicates that an acute treatment of Vitamin D activates an inflammatory pathway and a collagen I expression in human intestinal fibroblasts which may be involved in the initial response in the wound healing.

Abstract Background Fibrosis constitute the main complications associated to Crohn′s disease (CD). NOTCH signalling has been implicated in lung, kidney, liver and cardiac fibrosis. Macrophages contribute to fibrosis through the release of different mediators and the pattern of secretion may vary according to their microenvironment. The aim of the present study is to analyze the role of NOTCH ligands derived from macrophages in the complications of CD. Methods The aim of the present study is to analyze the role of NOTCH ligands derived from macrophages in the complications of CD. We have analyzed: the protein expression of NOTCH ligands and receptors in CD patients with fistulizing (B3) and stenting pattern (B2), the protein expression of NOTCH ligands in macrophages treated with the main cytokines present in CD patients (IFNγ-, IL10-, IL4, TNFα-U937 treated cells), the protein expression of HES1 and fibrosis markers in DLL4-HT29 and DLL3-HT29 treated cells. Results are expressed as fold induction (mean±SEM). Statistical analysis was performed with one-way ANOVA followed by Newman-Keuls test or t-tet. Results The expression of DLL4 and NOTCH4 were significantly higher in intestinal samples from B3 CD patients (3,2 ± 0,6 N=4* and 3,8 ± 0,6 N=8*, respectively) than in B2 patients (1,6 ± 0,2 N=4 and 1,7 ± 0,3 N=8, respectively) and controls (1,0 ± 0,1N=3 and 1,0 ± 0,1 N=8, respectively). IFNγ-U937 treated cells increased significantly the protein expression of DLL3 and DLL4 (1,6 ± 0,09 N=6* and 1,3 ± 0,1 N=7*, respectively) respect vehicle; IL4 increased significantly the expression of DLL4 (1,4 ± 0,1 N=7*) and TNFα increased significantly the expression of DLL3 (1,4 ± 0,1 N=5*), respect vehicle. DLL4-HT29 treated cells increased significantly fibrosis markers (VIMENTIN: 1,7 ± 0,1 N=3*; SNAIL: 2,2 ± 0,2 N=3*) and HES1 (1,4 ± 0,06 N=3*), respect vehicle (1,0 ± 0,07 N=6; 1,0 ± 0,05 N=6; and 1,0 ± 0,05 N=6, respectively). DLL3-HT29 treated cells only produced a reduction in the protein expression of ECADHERIN (0,4 ± 0,1 N=3*), respect vehicle (1,0 ± 0,09 N=6). Conclusion Macrophages may act as a source of NOTCH ligands who could act as fibrosis mediators in CD patients with a fistulizing (B3) behavior. The microenvironment rich in IFNγ could activate the fibrosis process in epithelial cells by favoring the expression of DLL4 and DLL3 in macrophages. DLL4 mainly activates transcription factors involved in mesenchymal epithelial transition in colonic epithelial cells (SNAIL), while DLL3 seems to have a more relevant role in cell-cell junction modification (ECADHERIN).

Abstract Background Metabolomics is a recent technique that has bounced into Inflammatory Bowel Diseases (IBD) due to its capacity to elucidate specific metabolites involved in the pathology and changes in the metabolomic profile have been detected in urine, blood or feces from UC patients. G-protein coupled receptors (GPCRs) have been recently identified as promising pharmacological targets. We aim to characterize the metabolomic profile and metabolite-sensing GPCRs expression in colonic resections from UC patients. Methods Colonic resections from UC (n=18) and non-IBD (n=20) patients were obtained. Metabolomic analysis was performed using Nuclear Magnetic Resonance (NMR) to study polar metabolites and Gas-Chromatography or Liquid-Chromatography Mass-Spectrometry to study non-polar metabolites. Results are expressed as μg of metabolite per gram of tissue. Gene expression of GPCRs was analyzed by qPCR. Data were expressed as fold induction vs control (mean±SEM) and compared by a t-test. A p-value&amp;lt;0.05 was considered statistically significant. Pearson correlation matrix were performed with R or mixOmics library in R. Results Metabolomic analysis revealed in colon of UC patients significant increased levels of propionic acid (7.7±1.1), undecanoic acid (0.3±0.02) decanoic acid (4.5±0.9), myristic acid (64.5±8.7), α-linoleic acid (23.1±4.0), aspartic acid (33.9±3.4), phenylalanine (8.8±1.6), glutamic acid, (136.3±10.8), b-hydroxybutyric (8.3±0.9) acid and lactic acid (297.5±34.7) vs non-IBD patients. Gene expression of GPR43 (22.7±9.5), GPR41 (4.7±1.7), GPR109a (19.1±5.3), GPR109b (13.6±4.6), GPR91 (13.2±5.7), GPR84 (6.7±2.0), GPR40 (6.6±1.8), GPR65 (3.0±0.5) and GPR68 (4.6±1.0) were significantly increased in UC patients compared with controls. In contrast, GPR120 (0.9±0.3), GPR119 (0.6±0.2) and GPR35 (0.5±0.1) were significantly reduced in UC patients. N-oleylethanolamide positively correlates with most of GPCRs, especially GPR68, GPR84 and GPR91 while acetic and succinic acids negatively correlate with GPR4 (Fig.1A). In UC patients, medium-chain fatty acids (FA) and most long-chain FA positively correlate with GPR84, GPR142, GPR43, GPR109a, GPR109b, GPR91 and GPR132 (Fig. 1C). Fig1. Graphs show the correlations between data relative to mRNA expression of GPCRs (expressed as ΔCt) vs metabolites. (A) Data from controls and UC. (B) Only controls. (C) Only UC. Conclusion Increased levels of several metabolites and GPCRs are detected in the colon of UC patients and their correlations suggest potential candidates to be involved in UC pathophysiology.

Abstract Background Macrophages contribute to fibrosis by releasing different mediators and the pattern of secretion may vary depending on the surrounding environment. We previously described that the mRNA expression of IFNγ was significantly higher in intestinal samples from CD patients. Methods The aim of the present study is to analyze the role of IFNγ-treated macrophages in epithelial mesenchymal transition (EMT) through the WNT pathway. The mRNA and protein expression of IFNγ in surgical resections from Crohn′s disease. U937 were differentiated to macrophages and then treated with IFNγ (10 ng/ml) for 4 days, the mRNA expression of WNT2b, WNT6 and TGFβ were determined by RT-PCR and protein. IFNγ-U937 were coculture with HT29 cells for 3 days and the expression of EMT markers, βCATENIN and WNT2b in HT29 cells were analyzed by WB. In some cases, HT29 cells were treated with the inhibitor of the WNT-pathway, XAV939 (1 μM), or were transfected with vectors-targeting human FZD4 (miFzd4). Results are expressed as mean±SEM. Statistical analysis was performed by ANOVA + Newman-Keuls or unpaired t-test. Results The mRNA and protein expression of IFNγ were significantly higher in intestinal samples from B2 CD patients (11.4±1.6 fold induction and 70,0 ± 2,3 pg/mg, respectively) and B3 CD patients (14.2±1.8 fold induction and 80,4 ± 6,8 pg/mg, respectively) than in controls (1,0±0,1 fold induction and 18,9 ± 0,3 pg/mg, respectively). U937 cells treated with IFNγ increased significantly the protein expression of WNT2b (1,3 ± 0,07 N=13* vs vehicle) but not the expression of WNT6 or TGFβ. IFNγ-U937 co-cultured with HT29 increased significantly the protein expression of EMT markers, βCATENIN and WNT2b in HT29 cells (VIMENTIN: 2,7 ± 0,3 N=6* vs vehicle-HT29; SNAIL1: 1,5 ± 0,1 N=5* vs vehicle-HT29; βCATENIN: 1,4 ± 0,09 N=5* vs vehicle-HT29; WNT2b: 2,0 ± 0,3 N=8* vs vehicle-HT29). Treatment HT29 cells with XAV939 (VIMENTIN: 1,4 ± 0,4 N=6 vs vehicle-HT29 XAV; SNAIL1: 1,0 ± 0,1 N=5 vs vehicle-HT29 XAV; βCATENIN: 0,8 ± 0,1 N=5 vs vehicle-HT29 XAV) or with miFzd4 (VIMENTIN: 0,3 ± 0,07 N=12* vs IFNγ-HT29 mock; SNAIL1: 0,5 ± 0,06 N=12* vs IFNγ-HT29 mock; βCATENIN: 0,6 ± 0,04 N=12* vs IFNγ-HT29 mock; FZD4: 0,5 ± 0,04 N=12* vs IFNγ-HT29 mock) partially reversed the increases produced by IFNγ-U937 in HT29 cells. Conclusion IFNγ-macrophages may stimulate the expression of WNT ligands in an autocrine and paracrine manner. The expression of WNT ligands at the epithelial level could favor mesenchymal epithelial transition through WNT2b ligand and FZD4 receptor.

Abstract Background Crohn′s disease is a chronic inflammatory disorder of gastrointestinal tract that is classified into three different behaviours: the inflammatory (B1), the stenotic (B2) or the penetrating (B3). We pretend to identify differences in transcriptomic and non-long coding RNA expression profiles associated to damaged and no-damaged surgical ileal resections from complicated CD patients. Methods We conducted both RNA and small RNA sequencing profiling on ileal surgical resections from CD patients with structuring (n=10) or penetrating (n=10) behaviour; from each patient we obtained a sample from affected tissue (B2A and B3A, respectively) and the paired non-affected ileum (B2C and B3C, respectively). Ten ileal resections (control samples) were obtained from non-affected ileum of patients with right colorectal cancer (IL). Enrichment scores of the GO terms and KEGG pathways were used to functional analysis investigated in this study. Gene expression and miRNA profiles were validated by RT-qPCR. Results Our comparative bioinformatic analysis of RNA sequencing showed that the comparation of IL and the non-affected ileum from CD patients (B2C+B3C) revealed that only 11 genes were significantly altered and any miRNA with significative expression. The comparative analysis between B2A+B3A vs B2C+B3C showed: a) 2562 genes differentially regulated and the GO enrichment analysis determined that inflammation, cell activation, and regulation of the extracellular matrix (EM) were the main biological processes altered (Figure 1a). KEGG analysis showed that cytokine-receptor interaction and MAPK and Ras signalling were the most affected pathways (Figure 1b). Among the top ten up-regulated genes we found several immunoglobulins (such as IGHG4, IGHG3, and IGHG1), proteins of the extracellular matrix EM (such as EPYC, COMP, and CHI3L1), and transcription factors (such as PRRX1). When the analysis was performed by CD behaviour, results revealed that most of these genes were significantly increased in both, B2 and B3 CD; b) 103 miRNAs with differential expression, 56 were up-regulated and 47 were down-regulated. Negative and significant correlations were detected between the RNA expression of ECM components and some miRNA expression. Figure 1. Bar plot of enrichment analysis with differential expressed genes between B2A+B3A vs B2C+B3C. A) Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) of Gene Ontology (GO) enrichment analysis. B) Enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Conclusion The correlation detected between extracellular matrix components and some miRNA suggest an epigenetic regulation in the affected ileum of complicated CD patients.

Intestinal fibrosis is a common complication of Crohn’s disease (CD) patients and it requires surgery. GWAS studies have identified several polymorphisms in genes involved in autophagy, which predispose to CD. It has been reported that this process is impaired in IBD patients, but the relevance of autophagy in intestinal fibrosis remains unclear. We aim to analyse the effect of pharmacological inhibition of autophagy in the development of murine intestinal fibrosis. Intestinal fibrosis was induced in vivo using the heterotopic transplant model. Segments of 1 cm colon from mice were subcutaneously transplanted into the neck of a recipient mice and collected after 7 days. Recipient mice were treated with a daily injection of 3-MA (10 mg/kg). Expression of intestinal inflammation, fibrosis, and EMT markers were analysed by qPCR and protein levels of autophagy markers by western blot. Collagen layer was evaluated by Sirius Red Staining. Intestinal resections from CD patients were obtained and expression of p62, Col1a1, α-SMA, Snail1, and Snail2 was analysed by qPCR. Results are expressed as fold induction (mean ± SEM, n ≥ 5). Statistical analysis was performed with one-way ANOVA followed by Newman–Keuls test. Correlations were analysed with the Spearman coefficient. Grafts obtained 7 days after surgery from 3-MA treated mice vs. vehicle-treated mice exhibited: (a) a significant increase in the expression of proinflammatory genes such as TNF-α (102.90 ± 22.94 vs. 50.46 ± 7.47), IL-1β (425.4 ± 84.92 vs. 243.70 ± 35.85), IL-6 (735.7 ± 235.0 vs. 339.90 ± 137.5) and INOS (325.7 ± 75.85 vs. 166.2 ± 23.64); (b) an increase in the expression of profibrotic genes such as Col1a1 (74.21 ± 9.18 vs. 41.27 ± 9.34), Vimentin (9.98 ± 4.54 vs. 6.73 ± 0.64) and TGF-β (6.69 ± 1.91 vs. 6.62 ± 0.60); (c) a significant increase in the expression of EMT genes such as Snail1 (21.10 ± 4.60 vs. 11.61 ± 1.49), Snail2 (7.32 ± 1.87 vs. 3.70 ± 0.73) and Itgb6 (7.70 ± 1.89 vs. 2.65 ± 0.43); (d) a significant thicker collagen layer after Sirius Red Staining. Autophagy inhibition by 3-MA was confirmed by western blot showing an increase of p62 and phospho-mTOR and a reduction in LC3. In intestinal resections from CD patients, the expression of p62 positively correlates with the expression of Col1a1 (rSpearman = 0.6098, p = 0.004), α-sma (rSpearman = 0.5168, p = 0.041), Snail1 (rSpearman = 0.4112, p = 0.0003) and Snail2 (rSpearman = 0.4410, p = 0.0009). Pharmacological inhibition of autophagy exacerbates murine intestinal inflammation, fibrosis, and EMT. In intestinal resections from CD patients the expression of autophagy markers correlates with the expression of pro-fibrotic and pro-EMT genes, which led us to suggest that pharmacological modulation of autophagy might be a new therapeutic option for intestinal fibrosis.

Fibrosis and fistula development constitute the main complications associated to Crohn’s disease. Notch signalling has been implicated in lung, kidney, liver, and cardiac fibrosis and in various disease conditions such as scleroderma. We aim to analyse here the pattern of NOTCH ligands, receptors, and effectors expression in surgical resections from stenotic and fistulizing CD patients and to determine the potential role of these ligands in favouring fistula and fibrosis. CD patients (n = 41) were categorised according to Montreal classification (age at diagnosis, location, and behaviour). mRNA was isolated from resections of patients presenting a stricturing (B2, n = 26) or a penetrating (B3, n = 15) behaviour or from unaffected mucosa of patients with colorectal cancer (control, n = 15). The expression of Notch ligands, receptors, and effectors (HES1 and MATH1) was determined by RT-PCR or WB. Correlations between data were analysed using Pearson’s correlation coefficient (*p < 0.05). A higher mRNA expression of NOTCH3 and NOTCH4 receptors was detected in CD patients compared with controls; in addition, the expression of these markers was higher in the fistulizing than in the stenotic behaviour (Table 1). The fistulizing group presented a generalised overexpression of NOTCH ligands (JAG2, DLL3, and DLL4) compared with controls and among them, only DLL3 expression was up-regulated in the stenotic group (Table 1). Similar levels of HES1 and MATH1 mRNA expression were detected between different groups while protein levels of HES1 were higher in the fistulising group than in control or stenotic groups (3.4 ± 0.1 A.U*#, 2.8 ± 0.2 A.U and 2.0 ± 0.1 A.U, respectively). The expression of DLL3 significantly correlated with FSP1 (r = 0.77, p = 0.04*), DESMIN (r = 0.80, p = 0.03*), and SNAIL1 (r = 0.59, p < 0.04*), only in intestinal tissue from the fistulizing CD group. Relative mRNA expression of NOTCH ligands and receptors vs. the housekeeping gene β-ACTIN in intestinal mucosa. Significant differences vs. the respective Non-IBD patients are shown by *p < 0.05 or **p < 0.05 and vs. B2 CD patients by #p < 0.05. Relative mRNA expression of NOTCH ligands and receptors vs. the housekeeping gene β-ACTIN in intestinal mucosa. Significant differences vs. the respective Non-IBD patients are shown by *p < 0.05 or **p < 0.05 and vs. B2 CD patients by #p < 0.05. Activation of the Notch signalling pathway is detected in Crohn’s disease patients presenting a penetrating (B3) behaviour compared with those with a structuring (B2) phenotype and it may be involved in fistula development over fibrosis.

Vitamin D deficiency and a defective signalling has been reported in Crohn’s disease (CD) patients. Vitamin D signals through the vitamin D receptor (VDR) which is a member of the nuclear receptor family of transcription factors that play an immunoregulatory role in the gut. We have previously demonstrated that a single-nucleotide polymorphism (SNP) in the VDR gene can modify the expression of this protein in peripheral blood mononuclear cells of CD patients. We aim to analyse the modulation of the VDR protein in human intestinal fibroblasts. We used intestinal fibroblasts isolated from intestinal tissue of the non-damaged mucosa and the damaged mucosa of CD patients. Control cells were obtained from the non-damaged intestine of patients with colorectal cancer. Fibroblasts were treated with 1,25 Vitamin D3 (100 nM) for 24 h. VDR protein levels were determined by western blot and VDR, CYP24A1, COL1A1 and αSMA gene expression by qPCR. Statistical significance was measured by t-test. VDR protein levels were significantly lower in fibroblasts obtained from the damaged intestine of CD patients than that obtained from controls (Figure 1A). In fibroblasts from CD patients, we detected lower VDR protein levels in those obtained from damaged mucosa than in those from the non-damaged. Treatment of these cells with vitamin D3 significantly increased VDR protein expression in all cases, but VDR protein levels were much lower in fibroblasts from damaged intestine (Figure 1B). The mRNA expression of VDR and its target, CPY24A1, was significantly lower in fibroblasts from the damaged tissue than in fibroblasts from the non-damaged. In contrast, the mRNA expression of collagen 1a1 and αSMA was higher in fibroblast from damaged intestine. When compared fibroblasts obtained from the non-damaged intestine of CD with control fibroblasts, the mRNA expression of CYP24A1 was significantly lower in cells from CD patients, suggesting that factors other than local inflammation may be involved (Figure 1C). Local inflammation, and probably genetic factors, are involved in the decrease in VDR protein levels detected in fibroblasts from CD patients.

Intestinal fistula is a common complication in CD patients whose aetiology is unknown. It is associated with an exacerbated inflammation and epithelial-to-mesenquimal transition (EMT), a process which allows a switch from epithelial towards a fibrotic phenotype. Under inflammatory conditions, succinate is accumulated and activates its receptor, SUCNR1, which has recently been related to intestinal fibrosis. We aim to analyse the role of succinate and SUCNR1 in EMT. HT-29 cells were treated with succinate (0, 0.1, 0.5, 1.5 mM) or TGF-β (5 ng/ml) during 48 h and transfected with SUCNR1 siRNA. Expression of EMT markers was analysed by qPCR and western blot. Intestinal fibrosis was induced in vivo using the heterotopic transplant model in WT and Sucnr1−/− mice and expression of EMT markers was analysed by qPCR and by confocal microscopy. Intestinal resections were obtained from CD and non-IBD patients. The expression of SUCNR1, Snail1, Snail2 and E-Cadherin was analysed by qPCR and succinate levels were quantified with a Succinate Assay Kit. Results are expressed as fold induction (mean ± SEM, n ≥5). Statistical analysis was performed with one-way ANOVA followed by Newman–Keuls test. Correlations were analysed with the Spearman coefficient. Succinate induces, in HT-29 cells a significant increase in Vimentin, Snail1, and Snail2 expression and a significant reduction in E-Cadherin expression compared with vehicle-treated cells and these changes were significantly prevented in cells transfected with SUCNR1 siRNA, (1.87 ± 0.09 vs. 1.12 ± 0.12, 1.85 ± 0.18 vs. 0.90 ± 0.09 and 2.57 ± 0.43 vs. 1.07 ± 0.26 vs.,, respectively). WT-grafts at Day 7 showed a significant increase in Vimentin expression (3.50 ± 0.48), Snail1 (4.87 ± 0.79) and Snail2 (2.45 ± 0.25) and a significant reduction in E-Cadherin expression (0.52 ± 0.07) vs. WT-grafts at day 0. KO-grafts at Day 7 showed a significant reduction in Vimentin expression (1.84 ± 0.14), Snail1 (1.91 ± 0.28) and Snail2 (1.07 ± 0.26) and an increase in E-Cadherin (0.94 ± 0.05) compared with WT-grafts at Day 7. Finally, in intestinal resections from B3-CD patients: (a) levels of succinate were higher than in that from B2-CD patients or non-IBD patients (244.90 ± 26.03 μM, 142.00 ± 21.66 μM and 99.73 ± 11.12 µM,, respectively); (b) SUCNR1 mRNA expression was significantly increased when compared with B2-CD or non-IBD controls. SUCNR1 mRNA expression correlates positively with Snail1 (r = 0.560) and Snail2 (r = 0.588) and negatively with E-cadherin (r = −0.714). Succinate activates EMT in intestinal epithelial cells through SUCNR1. Both succinate levels and SUCNR1 expression are increased in intestine from B3-CD patients and correlates with EMT markers, which points to a new possible target for fistula treatment.

Abstract Background Fibrosis is a common complication in Crohn’s disease (CD) patients and fibroblasts play an important role in the fibrogenic process. Low vitamin D (VD) levels and a defective VD-signalling pathway have been reported in CD. VD signals through both vitamin D receptor (VDR) and protein disulfide-isomerase A3 (PDIA3) and we have previously demonstrated that VDR protein levels are reduced in fibroblasts isolated from CD patients and that VD increased VDR expression in these cells (A-2080; ECCO 2019). We aim to analyse here the effect of VD on both PDIA3 protein levels and migration in CD fibroblasts. Methods We used intestinal fibroblasts isolated from surgical resections of the damaged mucosa of CD patients with stricturing behaviour (B2). Control fibroblasts were obtained from the non-damaged intestine of patients with colorectal cancer. Fibroblasts were treated with VD (100 nM) or its vehicle for 24 h and PDIA3 protein levels were measured by Western Blot. In the wound healing analysis, a single scraping was done in the centre of the fibroblasts monolayer and FBS-free medium was added to the cells, which allows us to determine the ability of fibroblasts to migrate and close the wound. Photos were taken at 0, 24 and 48 h. Results of wound healing were expressed as the percentage of the wound at each time point for the maximal wounded area (time 0, 100%). Statistical significance was measured by ANOVA or t-test. Results No significant differences in PDIA3 protein levels were detected between control and CD fibroblasts but VD significantly decreased PDIA3 expression in CD fibroblasts (Figure 1A). In the wound healing assay, we detected that CD-B2 fibroblasts migrate faster than control cells, resulting in a reduced wounding area, 48 h later (Figure 1B). Treatment of these CD-B2 cells with VD decreased their migration rate, and 48 h later cells exhibited a higher and significant wounding area than vehicle-treated cells (Figure 1C). Conclusion Vitamin D decreased PDIA3 expression and prevented the accelerated migration detected in intestinal fibroblasts from B2-CD patients which suggest an anti-fibrotic effect of VD mediated by a direct effect of this hormone on intestinal fibroblasts.

Abstract Background Macrophages contribute to fibrosis through the release of different mediators and the pattern of secretion may vary according to their phenotype. Methods The aim of the present study is to analyse the pattern of expression of macrophages, of EMT-related genes and cytokines in surgical resections from Crohn’s disease (CD, n = 43) patients which were categorised according to Montreal classification (B2 or B3); unaffected mucosa of patients with ileocecal cancer was used as control (n = 20). mRNA was isolated from intestinal samples and the expression of macrophage, EMT markers and cytokines were analysed by RT-PCR. PBMCS were isolated from healthy donors and treated during 5 days with secretomes, from control, B2 or B3 surgical resections; the mRNA expression of macrophage markers was determined by RT-PCR. U937 cells were differentiated to macrophages and then treated with IFNγ (20 ng/ml) for 4 days, the mRNA expression of macrophages markers were determined by RT-PCR. Results are expressed as mean ± SEM (n ≥ 5). Statistical analysis was performed by ANOVA + Newman–Keuls or t-test. Correlations between data were analysed using Pearson’s correlation coefficient (*p &amp;lt; 0.05). Results The expression of CD16 and CD86 was significantly higher in intestinal samples from B3 CD patients (7.2 ± 1.1 and 7.7 ± 1.3, respectively) than in controls (1.4 ± 0.2 and 2.5 ± 0.4, respectively) or B2 CD patients (4.8 ± 0.9 and 4.5 ± 0.6, respectively). The mRNA expression of CD16 and CD86 were significantly higher in PBMCS treated with B3-secretomes than in those treated with B2- or control secretomes. The expression of CD16 and CD86 significantly correlated with FSP1 (r = 0.74, p = 0.002* and r = 0.66, p = 0.003*, respectively), VIMENTIN (r = 0.60, p = 0.02* and r = 0.82, p = 0.001*, respectively), SNAIL1 (r = 0.61, p &amp;lt; 0.01* r = 0.52, p = 0.04*, respectively), IL4 (r = 0.63, p = 0.01* and r = 0.60, p = 0.02*, respectively) and IFNγ (r = 0.56, p = 0.001* and r = 0.58, p = 0.01*, respectively) in intestinal tissue from the fistulising CD group. U937 cells treated with IFNγ increased significantly the mRNA expression of CD16 (1.94 ± 0.24* vs. vehicle) and CD86 (1.60 ± 0.17* vs. vehicle). Conclusion A macrophage phenotype expressing CD86/CD16 may act as a source of EMT mediators in intestinal tissue from CD patients with a penetrating (B3) behaviour. IFNγ could be responsible for the increase in the number of CD86/CD16 macrophages in the B3 behaviour.

Abstract Background Intestinal fistula is a common complication in CD patients whose aetiology is still not well-characterised. It is associated with an exacerbated inflammation and epithelial-to-mesenchymal transition (EMT), a process which allows a switch from epithelial towards a fibrotic behaviour. We have recently reported that SUCNR1 mediates intestinal inflammation and fibrosis1 but its role in fistula has not yet been analysed. Therefore, we aim to analyse the role of SUCNR1 in EMT and in fistula formation. Methods Intestinal resections were obtained from CD and non-IBD patients. Fistula specimens were identified by the surgeons and collected from B3-CD patients. The expression of SUCNR1 and EMT markers was analysed by qPCR and the protein expression of SUCNR1 by immunohistochemistry. HT-29 cells were treated with succinate (0,0.1,0.5,1,5 mM) or TGF-β (5 ng/ml) during 48 h and transfected with SUCNR1 siRNA. Expression of EMT markers was analysed by qPCR and western blot. Intestinal fibrosis was induced in vivo using the heterotopic transplant model in WT and Sucnr1−/− mice and expression of EMT markers was analysed by qPCR and by confocal microscopy. Statistical analysis was performed with one-way ANOVA followed by Newman–Keuls test. Correlations were analysed with the Spearman’s coefficient. Results In intestinal resections from B3-CD patients, SUCNR1 mRNA expression was significantly increased when compared with B2-CD or non-IBD controls and it correlates positively with the mRNA expression of Snail1 and Snail2 and negatively with that of E-Cadherin. In the fistula tract, SUCNR1 is expressed in intestinal epithelial cells and lamina propria cells of the submucosa with a higher intensity in cells close to the fistula tract than in more distant areas. Succinate induced, in a dose–response manner, a significant increase in Vimentin, Snail1 and Snail2 expression and a reduction in E-cadherin expression. This effect was completely abolished when SUCNR1 was transiently knocked-down. WT-grafts 7 days after surgery exhibited: (a) an increase in gene and protein expression of SUCNR1, (b) an increase in the expression of Vimentin, Snail1 and Snail2 and a significant reduction in E-Cadherin compared with WT-grafts at day 0. Of interest, KO-grafts at day 7 failed to exhibited changes in the expression of Vimentin, Snail1, Snail2 or E-Cadherin. Conclusion SUCNR1 is increased specifically in the fistula tract of B3-CD patients. It mediates EMT in intestinal epithelial cells and in a murine model of fibrosis. Hence, SUCNR1 might be a potential pharmacological target for fistula treatment. Reference

appetite before and 1 rain after the infusion (VAS).Repeated measurements ANOVA was used for final analysis of spatial and infusion-induced differences.Results: Lipid showed a specific duodenal motor response in all volunteers (data:Ameans-+SEM vs. 5ml saline: number of contractions/5min 80.7-+ 19.3 p=O.O03 vs saline 0.8 -+2.6, motility index 3.7-+0.7 p=O.001 vs saline -0.56-+0.4),acid in 6 of 8 volunteers (contracrions/5min 15.4-+6.4,p=0.021 vs saline, MI 1.7-+0.8p= 0.02 vs saline).The motor response to lipid but not to acid was abolished by gramsetron (lipid/granisetron: contraction~5min 0.46-+ 5.2 p = 0.002, MI -0.08 + 0.8 p = 0.001 vs lipid/saline; acid: contractions/5min -0.63 -+ 6.1, p = 0.129, M1-0.17 -+ 0.9, p = O. 108).Perception was unaffected by all infusions.Basic motility was unchanged by granisetron.Conclusions: Lipid and acid elicit specific duodenal motility patterns in terms of the magnitude and timing of this response.The lipid induced duodenal motility is abolished by the 5-HT3 receptor antagonist granisetron indicating the involvement of the enterochromaffine cells in the sensing of lipids and the 5-HT3 receptor in mediating the motor response.The rapid local motor response to acid infusion is 5-HT3 receptor independent.

were inserted in pGk3-basic vector (Promega).pSV-beta-gaiactosidase control vector (Promega) was also trausfected for standardization.RESULTS: (1) In MKN45 cells, relative expression level of TFF1, TFF2, and TFF3 mRNA was 617:12:1 in the control condition.(2) Although indomethacin (1-250 uM) had no significant effect on the expression levels of TFF1 and TFF3 mRNA, it up-regulated the expression level of TFF2 mRNA in a dosedependent manner.24-h incubation with 125 uM indomethacin caused about 6-fold increase in the TFF2 mRNA level.(4) Luciferase reporter gene assay confirmed the stimulative effect of indomethacin on TFF2 expression.(5) Indomethacin-induced up-regulation of TFF2 expression was also observed in other gastric cell lines, AGS and JR. ( 6) Aspirin (2-10 raM), another non-selective COX-inhibitor, also up-regulated TFF2 expression.(7) Externally applied PGE2 did not antagonize the effect of indomethacin on TFF2 expression.CONCLU-SIONS: These results suggest that indomethacin up-regulates gastric epithelial cell TFF2 expression through COX-independent mechanism.Since TFF peptides play a critical role in gastric mucosal protection, indomethacin-induced TFF2 may reduce the degree of gastric raucosal damage induced by indoraethacin.

Abstract Background Vitamin D receptor (VDR) is a member of the nuclear receptor family of transcription factors that plays an immunomodulatory role in the gastrointestinal tract through binding Vitamin D. Single-nucleotide polymorphisms (SNPs) in the VDR gene have been related to inflammatory bowel disease. Indeed, Crohn′s disease (CD) patients carrying the Taq I polymorphism in VDR gene run a higher risk of developing a penetrating behaviour. We analyse here the association between the VDR SNPs Taq I, Bsm I, Apa I and Fok I and the clinical characteristics of CD. Methods DNA was extracted from blood samples from 80 patients diagnosed with CD from the Hospital of Manises (Valencia). Four polymorphisms identified in the VDR gene (Bsm I, Fok I, Apa I and Taq I) were genotyped using PCR-RFLP. Clinical data for each patient, including the Montreal classification was collected. Statistical significance was done using contingency tables and measured by Chi-squared or Fisher test. Results Results reveal a strong linkage disequilibrium between Apa I, Bsm I and Taq I polymorphisms. Apa I appears next to Bsm I and it is negatively associated with Taq I. The presence of at least a risk allele for Apa I is significantly associated with a stricturing behaviour in CD patients (P=0.014, Table 1), while the ancestral genotype (WT) of Apa I was significantly associated with a penetrating behaviour (P=0.062, Table 2). Finally, the presence of the risk genotype of Apa I is associated with a non-colonic location of the disease (P=0.059, Table 3). Fok I was not significantly associated with any of the parameters analysed. Conclusion Our results in CD patients reveal that the presence of the risk allele of Apa I in the VDR genotype is associated with a stricturing behaviour and tends towards a non-colonic location of the disease. This suggests that the analysis of this polymorphism may be useful for clinicians as a prognostic factor.

Abstract Background Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract whose etiology is unknown. CD is associated with complications such as fibrosis or fistula, which cannot be pharmacologically reversed, requiring repeated surgery. Although a profibrotic effect of the P2X7 receptor has been described in some scenarios such as lung, heart and liver, its role in intestinal fibrosis has not been analysed yet. Given the crosstalk between fibrosis and inflammasome, we aim to analyze the relevance of P2X7 in intestinal fibrosis and inflammasome activation. Methods Surgical intestinal resections of CD patients and healthy ileum of carcinoma patients were obtained. Murine chronic colitis was induced by 4 cycles of DSS in wild-type (WT) or P2X7-/- (KO) mice. HT29 cells were treated 24 hours with an inflammasome activator cocktail (LPS, TNF-α and IFN-γ) and the P2X7 antagonist A-80. Gene and protein expression of P2X7, inflammasome markers (NLRP3, ASC, CASPASE1, IL1β and IL18) and alternative inflammasome pathways (APIs) (NLRP1, NLRC4 and AIM2) were analysed by qPCR and Western Blot. The collagen layer was analysed by Sirius Red Staining. Results are expressed by mean±SEM. Statistical analysis was performed with one-way ANOVA and correlations were analysed with Spearman coefficient. Results In CD patients, the expression of P2X7 (2.97±0.50), Nlrp3 (2.53±0.41), Asc (5.61±0.76), Caspase1 (6.90±1.41), IL18 (4.17±0.89) and APIs Nlrp1 (3.07±0.40), Nlrc4 (6.99±1.19) is significantly increased vs non-IBD patients. Moreover, P2X7 expression positively and significantly correlates with the expression of the inflammasome markers NLRP3 (r=0.51), ASC (r=0.38), CASPASE1 (r=0.46), IL18 (r=0.36) and API such as NLRP1 (r=0.73), NRLC4 (r=0.67) and AIM2 (r=0.51) in CD patients (n≥45). The chronic murine model of DSS revealed that: a) KO-DSS showed more aggravated colitis with lower survival and greater weight loss compared with WT-DSS; b) the expression of NLRP3, IL18, IL1β and NLRP1 were significantly increased in KO-DSS (101.00±16.33, 3.28±1.49, 327.50±113.90, 4.92±1.00 respectively) vs WT-DSS; c) the thickness of the collagen layer in KO-DSS was increased vs WT-DSS. As expected, HT29 cells treated with the inflammasome cocktail increased protein expression of caspase-1 and the treatment with the P2X7 antagonist A-80 impaired the inflammasome activation since it significantly reduced the protein expression of caspase-1. Conclusion An increased expression of P2X7 receptor, the inflammasome and its APIs is detected in CD patients. Lack of P2X7 worsens chronic colitis associated with an increased activation of the inflammasome. Additional studies are needed in order to elucidate this dual role of P2X7 in intestinal fibrosis.

Abstract Background Fibrosis is a complication commonly present in Crohn’s disease (CD) patients with a structuring (B2) or penetrating (B3) phenotype, with no effective treatment. This process is characterized by a disequilibrium between the production and degradation of the extracellular matrix (ECM), mainly regulated by myofibroblasts. We aim to analyse here, the expression of markers of autophagy, apoptosis and proliferation in intestinal fibroblasts from CD patients. Methods Fibroblasts were isolated from the damaged intestinal mucosa of CD patients with a penetrating and stenotic behaviour. Control cells were obtained from the non-damaged intestine of patients with colorectal cancer. Protein levels of markers of autophagy and apoptosis were determined by Western Blot in isolated fibroblasts. The proliferation marker Ki67 was analysed by immunohistochemistry (IHC) in 5 µm slides of intestinal tissue from control or CD patients. Statistical significance was measured by t-test. Results In fibroblasts from CD patients, we detected a significant decrease in the ratio phospho-mTOR / mTOR (Fig. A) in parallel with a non-significant increment in the LC3 II / LC3 I protein ratio (174% ± 46.5), and a decrease in p62 protein levels (84.8% ± 5.5). When compared between CD behaviours, a significant decreased in the phospho-mTOR / mTOR protein ratio was detected in fibroblasts from B2- compared to that obtained in cells from B3-CD patients (Fig. B). The analysis of the expression of an apoptosis marker, Caspase 3, revealed a decreased of cleaved caspase 3 protein levels in CD fibroblasts compared to levels detected in control cells (Fig C). Finally, we observed in the lamina propria of the intestine from CD patients an increased number of Ki67 positive cells, compared to that detected in control tissue. Conclusion Our data show an increased autophagy and decreased apoptosis in isolated intestinal fibroblasts from CD patients; the high number of cells proliferating in the lamina propria of the intestinal tissue of these patients, strongly suggests a higher viability of these cells in the fibrotic context.

Abstract Background Fibrosis constitute the main complications associated to Crohn’s disease (CD). Notch signalling has been implicated in lung, kidney, liver and cardiac fibrosis. Macrophages contribute to fibrosis through the release of different mediators and the pattern of secretion may vary according to their microenvironment. The aim of the present study is to analyze the role of Notch ligands derived from macrophages in the complications of CD. Methods We have analyzed: the mRNA expression of cytokines and Notch ligands in CD patients with fistulizing and stenting pattern, the mRNA and protein expression of macrophage markers and Notch ligands in macrophages treated with the main cytokines present in CD patients (IFNγ-, IL10-, IL4, TNFα-U937 treated cells), the mRNA expression of fibrosis markers of DLL4-HT29 treated cells. Results are expressed as fold induction (mean±SEM, n≥4). Statistical analysis was performed with one-way ANOVA followed by Newman-Keuls test. Correlations were analyzed with the Spearman coefficient. Results The mRNA expression of IFNγ, TNFα, IL4, IL6 and IL10 was significantly higher in intestinal samples from B2- and B3-CD patients (11.4±1.6, 6.9±1.3, 16.4±3.9, 4.1±0.4, and 5.0±1.0 respectively for B2, and 14.2±1.8, 9.6±2.6, 21.5±4.1, 6.3±1.0 and 9.2±1.7 respectively for B3) than in controls (1.0±0.09, 1.1±0.2, 1.2±0.1, 1.1±0.1 and 1.0±0.1 respectively). The mRNA expression of DLL3 and DLL4 was significantly higher in intestinal samples from B2 (6.1±1.3 and 9.3±2.4, respectively) than in B3 CD patients (4.4±1.0 and 1.8±0.7, respectively) and in controls (0.9±0.2 and 0.7±1.2 respectively). IFNγ-U937 treated cells increased significantly the mRNA expression of DLL3 and DLL4 (2,9±0,6 N=5 N=4 and 3,7±1,1 N=4, respectively) respect vehicle; IL1β increased significantly the expression of DLL4 (1,8±0,01 N=4) and TNFα increased significantly the expression of DLL3 (7,1±2,2 N=4), respect vehicle. DLL4-HT29 treated cells increased significantly fibrosis markers (VIMENTIN (4,6±0,6 N=6), α-SMA (20,9±8,2 N=6), SNAIL1 (1,8±0,4 N=6), SNAIL2 (8,3±1,3 N=6), ZEB1 (8,2±4,3 N=6) and ZEB2 (4,3±0,2 N=6), respect vehicle. Conclusion Macrophages may act as a source of Notch ligands who could act as fibrosis mediators in CD patients with a stenting (B2) behavior. The microenvironment rich in IFNγ could activate the fibrosis process in epithelial cells by favoring the expression of DLL4 in macrophages.

Abstract Background Macrophages contribute to fibrosis by releasing different mediators and the pattern of secretion may vary depending on the surrounding environment. We previously described that the mRNA expression of IFNγ was significantly higher in intestinal samples from CD patients. The aim of the present study is to analyze the role of IFNγ-treated macrophages in epithelial mesenchymal transition (EMT). Methods The mRNA and protein expression of IFN in surgical resections from Crohn′s disease. U937 were differentiated to macrophages and then treated with IFNγ (2 ng/ml) for 4 days, the mRNA expression of macrophages markers were determined by RT-PCR. IFNγ-U937 were coculture with HT29 cells for 2 days and the expression of EMT markers in HT29 cells were analyzed by RT-PCR and WB. Results are expressed as mean±SEM (n≥5). Statistical analysis was performed by ANOVA + Newman-Keuls. Results The mRNA and protein expression of IFNγ were significantly higher in intestinal samples from B2 CD patients (11.4±1.6 fold induction and 3.5±0.3 pg/mg, respectively) and B3 CD patients (14.2±1.8 fold induction and 3.1±0.1 pg/mg, respectively) than in controls (1,0±0,1 fold induction and 0.8±0,1 pg/mg, respectively). U937 cells treated with IFNγ increased significantly the mRNA expression of CD16 (1.9±0.2* vs vehicle) and CD86 (1.6±0.1* vs vehicle). IFNγ-U937 cocultured with HT29 increased significantly the mRNA and protein expression of EMT markers in HT29 cells (Vimentin: 3.0±0.5* vs vehicle-HT29; αSMA: 24.4±8.3* vs vehicle-HT29; SNAIL1: 2.7±0.5* vs vehicle-HT29) respect IFNγ-HT29 cells (Vimentin: 0.6±0.01 vs vehicle-HT29; αSMA: 1.1±0.4 vs vehicle-HT29; SNAIL1: 0.7±0.06 vs vehicle-HT29). Conclusion IFNγ could be responsible for the increase in the number of CD86/CD16 macrophages in CD patients. A macrophage phenotype expressing CD86/CD16 may act as a source of EMT mediators in intestinal tissue from CD patients.

colon of severely affected cats.Diminished isometric stress responses in feline megacolonic smooth muscle are associated with reductions in the calcium-and calmodulin-dependent phosphorylation of the 2O-kD myosin light chains (MLCP).In this study, we identified cats in which clinical abnormalities were confined to the descending colon, and we have speculated that the pathogenesis of the generalized disorder may originate in the descending colon.Methods: Longitudinal and circular colonic smooth muscle strips from ascending, transverse, and descending colon of affected cats were attached to isometric force transducers at optimal muscle length.Peak active isometric stress (P,=; N = x 104/m z) and MLCP were determined following stimulation with ACh (10 .7 to 10 4 M), SP (10 -lO to 10 ~ M), or KCI (10 to 70 mM) in a 1.5 or 5.0 mM CaCI2 HEPES buffer solution, with or without 1 p.M bovine brain calmodulin.Results: Active isometric stress responses (ACh, SP, KCI) of descending colonic smooth muscle were significantly diminished (P,~ = 1.7-2.6N) compared to responses observed in ascending and transverse colonic smooth muscle (P,= = 7.5-14.5N).Diminished responses were observed in longitudinal and circular muscle layers of the descending colon.MLCP was diminished (20-25%) at all time points of contraction compared to ascending or transverse colon (MLCP = 57-62%).Raising the extracellular calcium concentration (5.0 mM), or incubating with 1 /~M calmodulin, did not induce further increases in P,r= or MLCP in the descending colon.Conclusions: 1) isometric stress output and MLCP are diminished in the descending colon of cats affected with severe constipation and idiopathic megacoion.2) Ascending and transverse colonic smooth muscle function (P,,= and MLCP) is normal in some cats with constipation and severe dysfunction of the descending colon.3) We speculate that the pathogenesis of this disorder may originate in the descending colon. 1701

Background:The neuropeptide bombesin inhibits gastric acid secretion (GAS) when administered centrally (1).Pentagastrin stimulates GAS acting on parietal cells of the stomach mucosa.Previous studies have shown that nitric oxide (NO) synthetised in the dorsal motor nucleus of the vagus (DMN) has an inhibitoy role on GAS stimulated by centrally acting agents such as distension or insulin (2).Aim: To analyse the implication of NO in the DMN in the inhibitory effects of bombesin on peripherallly stimulated GAS.Method: GAS was evaluated in rats perfused intragastrically with saline (0.9 ml min-l).Animals were placed in a stereotaxic apparatus and thereafter they received a bolus of pentagastrin (100 pg kg-l).Prior to this injection, L-NAME (80 pg kg -1) was administered locally in the DMN or in the nucleus of the tractus solitarius (NTS), and animals received an intracisternal (i.e.) administration of bombesin (40 ng kg-l).A bilateral vagotomy was performed in some experiments.Control animals were treated with vehicle (PBS) Results: (A~tEqH + 30 min -1 100 g-l; mean -+ s.e.m.; n > 6 per group)

Background: Cerebral administration of the endogenous neuropeptide bombesin inhibits gastric acid secretion (GAS).We have previously shown that synthesis of neuronal nitric oxide (NO) in the dorsal motor nucleus of the vagus (DMN) is implicated in the inhibition of GAS (2).Aim: To analyse the role of NO in the acid inhibitory effect of bombesin on two different stimulants of centrally mediated GAS.Methods: Gastric acid secretion was evaluated in urethane (1.5 mg kg -1) anaesthetized rats perfused intragastrically with saline (0.9 ml min-1).Once secretion had remained constant for 60 minutes, rats were placed in a stereotaxic apparatus and L-NAME (80 lag kg -1) or vehicle (PBS) were administered in the DMN.Animals received either an intracistemal (i.c.) microinjection of TRH (300 ng kg "1) or an i.p. bolus of insulin (0.75 u.i.kg-l).Previously bombesin was administered i.e.(40 ng kg-l).In the TRH stimulated group, some animals received a co-administration of L-NAME (80 lag kg q) and L-arginine (400 lag kgd).Results: Expressed as AlaEqH + 100 g-l during the appropriate experimental time in each experimental model (mean ± s.e.m., n>5):

The analgesic and central depressor effects of the dichloromethanol extract of Schinus molle L. were analysed in in vivo models. This extract showed low acute toxicity, CNS depressor activity and analgesic effect. Following further fractionation, the hexane/dichloromethane (75/25) fraction showed the most interesting results. Thus, this fraction caused a total inhibition of motor activity and significantly reduced the threshold of pain to chemical stimulus. © 1997 John Wiley & Sons, Ltd.

The effects on arterial blood pressure of the methanol and dichloromethanol extracts from Schinus molle L. were analysed in urethane anaesthetized rats. In normotensive rats, the mean arterial blood pressure was significantly reduced by the i.v. administration of both extracts. The dichloromethanol extract inhibited the effects of noradrenaline on arterial blood pressure in the anaesthetized rat and it reduced the maximal contractile effect (Emax) induced by noradrenaline on rat vas deferens in the organ bath. However, the methanol extract did not modify the effects of noradrenaline in the evaluated tests.