Role of TGFβ1/Smads pathway in the pathogenesis of intestinal fibrosis in Crohn’s disease

Inflammatory bowel diseases (IBD) are characterized by an intestinal fibrosis that may lead to stenosis and obstruction (Burke et al., 2007) and by disfuntions of gastrointestinal (GI) motility associated with altered functions of enteric nerves, interstitial cells of Cajal or smooth muscle (Vetuschi et al., 2006). In experimental model TGFβ1/ Smad3 signalling plays a major role in tissue fibrogenesis (Latella et al, 2009). Aim of this study was to evaluate the potential role of the TGFβ1/Smads pathway in intestinal fibrosis and to explore the possible mechanisms by which fibrogenesis induces alterations of GI motility in patients affected by CD. Evaluation of TGFβ1, CTGF, collagen I-III, Smad3/7, PDGF, C-Kit, α-SMA, and a neuronal cocktail expression and a morphometrical analysis were performed in human CD terminal ileum samples; human smooth muscle cells (HSMC) were cultured for morphofuncional and mRNA expression (RT-PCR). Histo-morphometrical evaluation of stenotic fragments showed a significantly increase of a) both intestinal fibrosis and inflammation; b) mucosa, submucosa and muscle layer thickness and c) expression of TGFβ1, CTGF, collagen I-III, Smad3, PDGF, C-Kit and α-SMA staining. HSMC obtained from stenotic tracts showed an increase of PDGF-β and collagen I-III types mRNA and an inhibition in contractile response to acetylcholine compared to pre-stenotic tracts. These data support the hypothesis that TGFβ1/Smads pathway play a central role in development and differentiation of intestinal mesenchymal cells in sustaining intestinal fibrosis in CD and could be responsible for alteration of GI motility

The gut brain axis. interactions between enteric microbiota, central and enteric nervous systems

The gut-brain axis (GBA) consists of bidirectional communication between the central and the enteric nervous system, linking emotional and cognitive centers of the brain with peripheral intestinal functions. Recent advances in research have described the importance of gut microbiota in influencing these interactions. This interaction between microbiota and GBA appears to be bidirectional, namely through signaling from gut-microbiota to brain and from brain to gut-microbiota by means of neural, endocrine, immune, and humoral links. In this review we summarize the available evidence supporting the existence of these interactions, as well as the possible pathophysiological mechanisms involved. Most of the data have been acquired using technical strategies consisting in germ-free animal models, probiotics, antibiotics, and infection studies. In clinical practice, evidence of microbiota-GBA interactions comes from the association of dysbiosis with central nervous disorders (i.e. autism, anxiety-depressive behaviors) and functional gastrointestinal disorders. In particular, irritable bowel syndrome can be considered an example of the disruption of these complex relationships, and a better understanding of these alterations might provide new targeted therapies

Effect of Short-Time Exposures to Nickel and Lead on Brain Monoamine Oxidase from Danio rerio and Poecilia reticulata

The aim of this work was to verify, in two small size freshwater teleosts Danio rerio and Poecilia reticulata, the effects of short-time exposures (24 and 72 h) to a sublethal dose (500 mu g/L) of nickel and lead, on brain monoamine oxidase (MAO), an important neural enzyme. The 24-h treatment using both metals caused a strong reduction of MAO activity in D. rerio brain, whereas causing a slight MAO activity stimulation in P reticulata brain. The same treatment in both species did not affect the brain MAO mRNA production as showed by RT-PCR. Extending the duration of treatment as far as 72 h, partly (D. rerio) or completely (P. reticulata) reversed the metal effects on brain MAO activity suggesting that mechanisms to neutralize the metals had been activated. (C) 2008 Wiley Periodicals, Inc. Environ Toxicol 24: 309-313, 2009

Microbiota, Innate Immune System, and Gastrointestinal Muscle Ongoing Studies

Aim: To test the activities of culture-extracted or commercially available toll-like receptors (TLRs) ligands to establish their direct impact on target gastrointestinal motor cells. Methods: Short-term and long-term effects of Shigella flexneri M90T and Escherichia coil K-2 strains-extracted lipopolysaccharides (LPS), commercially highly purified LPS (E. coli 0111:B4 and EH100), and Pam2CSK4 and Pam3CSK4, which bind TLR2/6 and TLR1/2 heterodimers, respectively, have been assessed on pure primary cultures of colonic human smooth muscle cells (HSMC). Results: Pathogenic Shigella-LPS and nonpathogenic E. coli K-2-LPS induced a time-dependent decrease of resting cell length and acetylcholine-induced contraction, with both alterations occurring rapidly and being more pronounced in response to the former. However, their effects differed, prolonging HSMC exposure with Shigella-LPS effects maintained throughout the 4 hours of observation compared with E. coli K-2-LPS, which disappeared after 60 minutes of incubation. Similar differences in magnitude and time dependency of myogenic effects were observed between pure TLR4 and TLR2/1 or TLR2/6 ligands. The specific activation of TLR4 with LPS from pathogen or nonpathogen E. coli, 0111:B4 and EH100, respectively, induced smooth muscle alterations that progressively increased, prolonging incubation, whereas TLR2 ligands induced short-term alterations, of a lesser magnitude, which decreased over time. The real-time polymerase chain reaction analysis showed that HSMC express mRNA for TLR1, 2, 4, and 6, substantiating a direct effect of TLR ligands on human colonic smooth muscle. Conclusions: This study highlights that bacterial products can directly affect gastrointestinal motility and that TLRs subtypes may differ in their cellular activity

Gender-differences of in vitro colonic motility after chemo- and radiotherapy in humans

Abstract Background The aim of the present in vitro study was to investigate, in different genders, motor responses in surgical colonic specimens from patients with rectal cancer undergoing and not undergoing chemotherapy with capecitabine and radiotherapy. Methods This in vitro study was conducted from October 2015 to August 2017 at the Experimental Pharmacology Laboratory at the National Institute “S. de Bellis” after collecting samples at the Department of Surgery. Segments of sigmoid colon were obtained from 15 patients (Male (M)/Female (F) = 8/7; control group, CG) operated on for elective colorectal resection for rectal cancer without obstruction and 14 patients (M/F = 7/7; study group, SG) operated on for elective colorectal resection for rectal cancer who also received chemotherapy, based on capecitabine twice daily, and radiotherapy. Isometric tension was measured on colonic circular muscle strips exposed to increasing carbachol or histamine concentrations to obtain concentration-response curves. The motor responses to electrically evoked stimulation were also investigated. Results In males, carbachol and histamine caused concentration-dependent contractions in the CG and SG. An increased sensitivity and a higher response to carbachol and histamine were observed in SG than CG (P < 0.01). On the contrary, in females, the response to carbachol was not significantly different in CG from the SG and the maximal responses to carbachol were greater in CG than in SG (P < 0.001). The same applied to histamine for half-maximal effective concentrations and maximal response in that they were not significantly different in CG from the SG. Electrically evoked contractions were significantly more pronounced in males, especially in the SG (P < 0.05). Conclusions This preliminary in vitro study has shown gender differences in motor responses of colonic circular muscle strips in patients who had received chemotherapy with capecitabine and radiotherapy

Myogenic regional responsiveness to cholinergic and vipergic stimulation in human colon

Background Differences in the actions of enteric neurotransmitters on colonic circular and longitudinal muscle layers have not been clearly determined, nor the possible existence of intrinsic myogenic phenotypes that might contribute to regional differences in human colon motor activity. The aim of this study was to analyze the direct pharmaco-mechanical coupling of carbachol (CCh) and vasoactive intestinal polypeptide (VIP) on human colonic smooth muscle strips and cells. Methods Circular and longitudinal muscle strips and cells were obtained from 15 human specimens of ascending and sigmoid colon. Both isometric tension on muscle strips and contraction and relaxation on cells were measured in response to increasing CCh and VIP concentrations. Key Results Circular muscle strips of ascending colon were more sensitive to the effect of CCh than that of sigmoid colon, EC50 values being, respectively, 4.15 mu mol L-1 and 8.47 mu mol L-1 (P < 0.05), although there were no differences in maximal responses. No regional differences were observed in longitudinal muscle strips or in smooth muscle cells. Maximal responses to CCh were higher on circular than longitudinal muscle strips and cells throughout the colon. A greater sensitivity to VIP was observed in ascending colon compared with sigmoid colon, both in circular (EC50: 0.041 and 0.15 mu mol L-1, respectively, P < 0.01) and longitudinal (EC50: 0.043 and 0.09 mu mol L-1, respectively, P < 0.05) strips, and similar differences were observed in longitudinal smooth muscle cells (EC50: 44.85 and 75.24 nmol L-1, respectively, P < 0.05). Conclusions & Inferences Regional myogenic differences in pharmaco-mechanical coupling between the enteric neurotransmitters and smooth muscle contribute to the complex regional motor patterns of human colon