Fabio Turco

Palmitoylethanolamide improves colon inflammation through an enteric glia/toll like receptor 4-dependent PPAR-α activation

Objective Enteric glia activation has been reported to amplify intestinal inflammation via the enteroglial-specific S100B protein. This neurotrophin promotes macrophage recruitment in the mucosa, amplify colonic inflammation and interacts with toll-like receptors (TLR). Molecules inhibiting S100B-driven enteric activation might mitigate the course of ulcerative colitis (UC). This study aims to investigate the effects of palmitoylethanolammide (PEA), a drug able to counteract astroglial activation in the central nervous system, on intestinal inflammation, in humans and mice. Design Mouse models of dextran sodium sulphate (DSS)-induced colitis, colonic biopsies deriving from UC patients and primary cultures of mouse and human enteric glial cells (EGC), have been used to assess the effects of PEA, alone or in the presence of specific PPARα or PPARγ antagonists, on: macroscopic signs of UC (DAI score, colon length, spleen weight, macrophages/neutrophils infiltration); the expression and release of proinflammatory markers typical of UC; TLR pathway in EGCs. Results PEA treatment improves all macroscopic signs of UC and decreases the expression and release of all the proinflammatory markers tested. PEA anti-inflammatory effects are mediated by the selective targeting of the S100B/TLR4 axis on ECG, causing a downstream inhibition of nuclear factor kappa B (NF-kB)-dependent inflammation. Antagonists at PPARα, but not PPARγ, abolished PEA effects, in mice and in humans. Conclusions Because of its lack of toxicity, its ability in reducing inflammation and its selective PPARα action, PEA might be an innovative molecule to broaden pharmacological strategies against UC.

Enteroglial-derived S100B protein integrates bacteria-induced Toll-like receptor signalling in human enteric glial cells

Objective Enteric glial cells (EGC) have been suggested to participate in host–bacteria cross-talk, playing a protective role within the gut. The way EGC interact with microorganisms is still poorly understood. We aimed to evaluate whether: EGC participate in host–bacteria interaction; S100B and Toll-like receptor (TLR) signalling converge in a common pathway leading to nitric oxide (NO) production. Design Primary cultures of human EGC were exposed to pathogenic (enteroinvasive Escherichia coli; EIEC) and probiotic (Lactobacillus paracasei F19) bacteria. Cell activation was assessed by evaluating the expression of cFos and major histocompatibility complex (MHC) class II molecules. TLR expression in EGC was evaluated at both baseline and after exposure to bacteria by real-time PCR, fluorescence microscopy and western blot analysis. S100B expression and NO release from EGC, following exposure to bacteria, were measured in the presence or absence of specific TLR and S100B pathway inhibitors. Results EIEC activated EGC by inducing the expression of cFos and MHC II. EGC expressed TLR at baseline. Pathogens and probiotics differentially modulated TLR expression in EGC. Pathogens, but not probiotics, significantly induced S100B protein overexpression and NO release from EGC. Pretreatment with specific inhibitors of TLR and S100B pathways abolished bacterial-induced NO release from EGC. Conclusions Human EGC interact with bacteria and discriminate between pathogens and probiotics via a different TLR expression and NO production. In EGC, NO release is impaired in the presence of specific inhibitors of the TLR and S100B pathways, suggesting the presence of a novel common pathway involving both TLR stimulation and S100B protein upregulation.

Proinflammatory stimuli activates human-derived enteroglial cells and induces autocrine nitric oxide production.

Background  Enteric glial cells (EGCs) have been recently indicated as key regulators of intestinal inflammation in animals. Whether or not this is true and how these cells participate to inflammatory responses in humans is unknown.

S100B protein in the gut: The evidence for enteroglial- sustained intestinal inflammation

Glial cells in the gut represent the morphological and functional equivalent of astrocytes and microglia in the central nervous system (CNS). In recent years, the role of enteric glial cells (EGCs) has extended from that of simple nutritive support for enteric neurons to that of being pivotal participants in the regulation of inflammatory events in the gut. Similar to the CNS astrocytes, the EGCs physiologically express the S100B protein that exerts either trophic or toxic effects depending on its concentration in the extracellular milieu. In the CNS, S100B overexpression is responsible for the initiation of a gliotic reaction by the release of pro-inflammatory mediators, which may have a deleterious effect on neighboring cells. S100B-mediated pro-inflammatory effects are not limited to the brain: S100B overexpression is associated with the onset and maintenance of inflammation in the human gut too. In this review we describe the major features of EGCs and S100B protein occurring in intestinal inflammation deriving from such.

The Bitter Taste Receptor Agonist Quinine Reduces Calorie Intake and Increases the Postprandial Release of Cholecystokinin in Healthy Subjects.

Background/Aims Bitter taste receptors are expressed throughout the digestive tract. Data on animals have suggested these receptors are involved in the gut hormone release, but no data are available in humans. Our aim is to assess whether bitter agonists influence food intake and gut hormone release in healthy subjects. Methods Twenty healthy volunteers were enrolled in a double-blind cross-over study. On 2 different days, each subject randomly received an acid-resistant capsule containing either placebo or 18 mg of hydrochloride (HCl) quinine. After 60 minutes, all subjects were allowed to eat an ad libitum meal until satiated. Plasma samples were obtained during the experiment in order to evaluate cholecystokinin (CCK) and ghrelin levels. Each subject was screened to determine phenylthiocarbamide (PTC) tasting status. Results Calorie intake was significantly lower when subjects received HCl quinine than placebo (514 ± 248 vs 596 ± 286 kcal; P = 0.007). Significantly higher CCK ΔT90 vs T0 and ΔT90 vs T60 were found when subjects received HCl quinine than placebo (0.70 ± 0.69 vs 0.10 ± 0.86 ng/mL, P = 0.026; 0.92 ± 0.75 vs 0.50 ± 0.55 ng/mL, P = 0.033, respectively). PTC tasters ingested a significantly lower amount of calories when they received HCl quinine compared to placebo (526 ± 275 vs 659 ± 320 kcal; P = 0.005), whereas no significant differences were found for PTC non-tasters (499 ± 227 vs 519 ± 231 kcal; P = 0.525). Conclusions This study showed that intra-duodenal release of a bitter compound is able to significantly affect calorie intake and CCK release after a standardized meal. Our results suggest that bitter taste receptor signaling may have a crucial role in the control of food intake.

Molecular Signaling and Dysfunction of the Human Reactive Enteric Glial Cell Phenotype: Implications for GI Infection, IBD, POI, Neurological, Motility, and GI Disorders.

Background:Clinical observations or animal studies implicate enteric glial cells in motility disorders, irritable bowel syndrome, inflammatory bowel disease, gastrointestinal (GI) infections, postoperative ileus, and slow transit constipation. Mechanisms underlying glial responses to inflammation in human GI tract are not understood. Our goal was to identify the “reactive human enteric glial cell (rhEGC) phenotype” induced by inflammation, and probe its functional relevance. Methods:Human enteric glial cells in culture from 15 GI-surgical specimens were used to study gene expression, Ca2+, and purinergic signaling by Ca2+/fluo-4 imaging and mechanosensitivity. A nanostring panel of 107 genes was designed as a read out of inflammation, transcription, purinergic signaling, vesicular transport protein, channel, antioxidant, and other pathways. A 24-hour treatment with lipopolysaccharide (200 &mgr;g/mL) and interferon-&ggr; (10 &mgr;g/mL) was used to induce inflammation and study molecular signaling, flow-dependent Ca2+ responses from 3 mL/min to 10 mL/min, adenosine triphosphate (ATP) release, and ATP responses. Results:Treatment induced a “rhEGC phenotype” and caused up-regulation in messenger RNA transcripts of 58% of 107 genes analyzed. Regulated genes included inflammatory genes (54%/IP10; IFN-&ggr;; CxCl2; CCL3; CCL2; C3; s100B; IL-1&bgr;; IL-2R; TNF-&agr;; IL-4; IL-6; IL-8; IL-10; IL-12A; IL-17A; IL-22; and IL-33), purine-genes (52%/AdoR2A; AdoR2B; P2RY1; P2RY2; P2RY6; P2RX3; P2RX7; AMPD3; ENTPD2; ENTPD3; and NADSYN1), channels (40%/Panx1; CHRNA7; TRPV1; and TRPA1), vesicular transporters (SYT1, SYT2, SNAP25, and SYP), transcription factors (relA/relB, SOCS3, STAT3, GATA_3, and FOXP3), growth factors (IGFBP5 and GMCSF), antioxidant genes (SOD2 and HMOX1), and enzymes (NOS2; TPH2; and CASP3) (P < 0.0001). Treatment disrupted Ca2+ signaling, ATP, and mechanical/flow-dependent Ca2+ responses in human enteric glial cells. ATP release increased 5-fold and s100B decreased 33%. Conclusions:The “rhEGC phenotype” is identified by a complex cascade of pro-inflammatory pathways leading to alterations of important molecular and functional signaling pathways (Ca2+, purinergic, and mechanosensory) that could disrupt GI motility. Inflammation induced a “purinergic switch” from ATP to adenosine diphosphate/adenosine/uridine triphosphate signaling. Findings have implications for GI infection, inflammatory bowel disease, postoperative ileus, motility, and GI disorders.

Increased severity of dyspeptic symptoms related to mental stress is associated with sympathetic hyperactivity and enhanced endocrine response in patients with postprandial distress syndrome

Background  Mental stress (MS) may alter gastric sensory‐motor function. The aim of the study was to assess postprandial autonomic nervous system activity and stress hormones in response to acute mental stress in dyspeptic patients.

Bacterial stimuli activate nitric oxide colonic mucosal production in diverticular disease. Protective effects of L. casei DG® (Lactobacillus paracasei CNCM I-1572)

Background Micro-inflammation and changes in gut microbiota may play a role in the pathogenesis of diverticular disease (DD). Objective The objective of this article is to evaluate the expression of nitric oxide (NO)-related mediators and S100B in colonic mucosa of patients with DD in an ex vivo model of bacterial infection. Methods Intestinal biopsies obtained from patients with diverticulosis, symptomatic uncomplicated diverticular disease (SUDD) and SUDD with previous acute diverticulitis (SUDD+AD) were stimulated with the probiotic L. casei DG® (LCDG) and/or the pathogen enteroinvasive Escherichia coli (EIEC). S100B, NO release and iNOS expression were then evaluated. Results Basal iNOS expression was significantly increased in SUDD and SUDD+AD patients. Basal NO expression was significantly increased in SUDD+AD. No differences in S100B release were found. In all groups, iNOS expression was significantly increased by EIEC and reduced by LCDG. In all groups, except for SUDD+AD, EIEC significantly increased NO release, whereas no increase was observed when LCDG was added to biopsies. EIEC did not induce significant changes in S100B release. Conclusions Colonic mucosa of patients with DD is characterized by a different reactivity toward pathogenic stimuli. LCDG plays a role in counteracting the pro-inflammatory effects exerted by EIEC, suggesting a beneficial role of this probiotic in DD.

Acetonic Extract from the Feijoa sellowiana Berg. Fruit Exerts Antioxidant Properties and Modulates Disaccharidases Activities in Human Intestinal Epithelial Cells

Feijoa sellowiana fruit has been shown to possess various biological activities, such as anti‐bacterial and anti‐cancer properties, in a variety of cellular models, but its activity on human intestinal epithelial cells has never been tested. The purpose of this study was to investigate the effects of the acetonic extract of F. sellowiana fruits on the viability, membrane peroxidation, disaccharidases activities and proliferation of in vitro models of human intestinal epithelial cells. To obtain this goal, Caco‐2 and HT‐29 cells were exposed to the acetonic extract for 24 h. Cell proliferation, viability, lactase and sucrase‐isomaltase activity and H2O2‐induced membrane lipid peroxidation were tested. We found that, compared to control conditions, the acetonic extract significantly increased lactase and sucrase‐isomaltase activity in Caco‐2, but not HT‐29, cells, decreased proliferation, had no effects on viability and restored lipid peroxidation in both cell models. This study suggests that the acetonic extract improves lactase and sucrase‐isomaltase activity, inhibits cell proliferation, have no cytotoxic effects and prevent lipid peroxidation of intestinal epithelial cells. These effects may be exploited in case of disaccharidases deficit and also as an adjuvant treatment of diseases related to oxidative stress. Copyright

Tu1452 Enteroglial-Derived S100B Protein Modulates Differentiation and Proliferation of Human Intestinal Epithelial Cells in a RAGE-Dependent Manner

Retrograde axonal transport was impaired in the gastric branch of the vagus nerve in animals with type 1 diabetes mellitus (DM). These studies evaluated active, retrograde axonal transport of the neural tracer fluorogold (FG) in gastric enteric neurons in KKAy mice 4 weeks prior to and at the onset of type 2 DM, and after 8, 16, and 24 weeks of uncontrolled DM. We hypothesized that the number of FG-labeled enteric neurons would progressively decrease with increasing durations of uncontrolled DM as compared to control KK mice. Under anesthesia, FG was injected into the ventral fundus or antrum. After 5-6 days, the stomach was removed and the numbers of FG-labeled enteric neurons with cell bodies in 2.8 mm2 of fundus, 1.7 mm2 of corpus, and 1.7 mm2 of antrum were counted. The total number of FG-labeled neurons in ascending pathways (cell bodies in corpus or antrum with nerve terminals in fundus), descending pathways (cell bodies in the fundus or corpus with nerve terminals in antrum), the local fundic pathway (cell body and nerve terminals in fundus), and the local antral pathway (cell body and nerve terminals in antrum) were compared in control and diabetic mice of different ages by 2-way ANOVA. Only main effects were significant; no interactions were significant. Data are mean±SEM with n representing the number of mice. In the ascending pathway, fewer FG-labeled neurons were observed in diabetic as compared to control mice [F(1,116)=10.668; control (n=68), 179±11; diabetic (n= 58), 145±11*; *p<0.05]. In contrast, similar numbers of FG-labeled neurons were observed in descending pathways [F(1,124)=0.489; p=0.486], local fundic pathways [F(1,53)=2.464; p=0.122], and local antral pathways [F(1,57)=0.728; p=0.397] of control and diabetic mice. Overall, the number of FG-labeled neurons decreased significantly with age in all pathways [ascending: F(4,116)=42.862; descending: F(4,124)=2.456; local fundic: F(4,53)=5.195; local antral: F(4,57)=8.920]. In the ascending pathway, the number of FG-labeled neurons was significantly less at 16 weeks of age [n=28, 146±12] as compared to 4 weeks [n=24, 246±8] or 8 weeks [n=28, 229±12] of age, reaching its lowest value at 24 weeks of age [n= 24, 80±10]. In conclusion, DM did not uniformly impair active tracer transport in all gastric enteric neurons suggesting specific axonal targets in ascending enteric neurons rendering them more susceptible to hyperglycemia and low effective insulin. Supported by ORSP at MWU.