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Background & Aims Visceral hypersensitivity is one feature of irritable bowel syndrome (IBS). Bacterial dysbiosis might be involved in the activation of nociceptive sensory pathways, but there have been few studies of the role of the mycobiome (the fungal microbiome) in the development of IBS. We analyzed intestinal mycobiomes of patients with IBS and a rat model of visceral hypersensitivity. Methods We used internal transcribed spacer 1-based metabarcoding to compare fecal mycobiomes of 18 healthy volunteers with those of 39 patients with IBS (with visceral hypersensitivity or normal levels of sensitivity). We also compared the mycobiomes of Long-Evans rats separated from their mothers (hypersensitive) with non-handled (normally sensitive) rats. We investigated whether fungi can cause visceral hypersensitivity using rats exposed to fungicide (fluconazole and nystatin). The functional relevance of the gut mycobiome was confirmed in fecal transplantation experiments: adult maternally separated rats were subjected to water avoidance stress (to induce visceral hypersensitivity), then given fungicide and donor cecum content via oral gavage. Other rats subjected to water avoidance stress were given soluble β-glucans, which antagonize C-type lectin domain family 7 member A (CLEC7A or DECTIN1) signaling via spleen-associated tyrosine kinase (SYK), a SYK inhibitor to reduce visceral hypersensitivity, or vehicle (control). The sensitivity of mast cells to fungi was tested with mesenteric windows (ex vivo) and the human mast cell line HMC-1. Results α diversity (Shannon index) and mycobiome signature (stability selection) of both groups of IBS patients differed from healthy volunteers, and the mycobiome signature of hypersensitive patients differed from that of normally sensitive patients. We observed mycobiome dysbiosis in rats that had been separated from their mothers compared with non-handled rats. Administration of fungicide to hypersensitive rats reduced their visceral hypersensitivity to normal levels of sensitivity. Administration of cecal mycobiomes from rats that had been separated from their mothers (but not non-handled mycobiome) restored hypersensitivity to distension. Administration of soluble β-glucans or a SYK inhibitor reduced visceral hypersensitivity, compared with controls. Particulate β-glucan (a DECTIN-1 agonist) induced mast cell degranulation in mesenteric windows and HMC-1 cells responded to fungal antigens by release of histamine. Conclusions In an analysis of patients with IBS and controls, we associated fungal dysbiosis with IBS. In studies of rats, we found fungi to promote visceral hypersensitivity, which could be reduced by administration of fungicides, soluble β-glucans, or a SYK inhibitor. The intestinal fungi might therefore be manipulated for treatment of IBS-related visceral hypersensitivity. © 2017 AGA Institute. Chemicals/CAS: protein kinase Syk, 138674-26-7; Antifungal Agents; beta-Glucans; Protein Kinase Inhibitors; Syk Kinase; Syk protein, rat

Aims. Cyclooxygenase isoforms (COX-1, COX-2) have been implicated in the development of fibrosis at gastrointestinal sites. Under bowel inflammation, transforming growth factor beta (TGF-beta) has been identified as the main regulator of fibrotic remodelling. The present study investigated the effects of cyclooxygenase inhibitors on pro-fibrotic signalling mediated by the TGF-beta/SMAD pathway in experimental colitis. Methods. Colitis was induced in rats by intrarectal 2,4-dinitrobenzenesulfonic acid (DNBS, 30 mg/rat in 0.25 ml ethanol 50%). After 6 days, systemic [body and spleen weight] and tissue inflammatory parameters [macroscopic and microscopic damage] were assessed. Three days before colitis assessment, the animals were treated daily with indomethacin (IND, non-selective COX-1/COX-2 inhibitor, 2 mg/kg), SC-560 (SC, selective COX-1 inhibitor, 2.5 mg/kg), or celecoxib (CEL, selective COX-2 inhibitor, 1 mg/kg) by intragastric gavage. At the time of sacrifice, functional and molecular tests were carried out. Peristaltic activity of control and inflamed colonic segments was studied in vitro by a modified Trendelenburg set-up. Collagen fibers (Van Gieson) and elastic fibers (orcein) were examined by histochemistry. The expression of molecular factors involved in TGF-beta signalling (TGF-beta, RhoA and SMAD6, as well as phosphorylated SMAD2, p38, ERKs and JNK), and cellular proliferation and apoptotis (PCNA, Akt and caspase-3) was analyzed by western blot. Results. Animals with colitis displayed an impairment of colonic peristalsis, as shown by a significant decrease in compliance and maximal ejection pressure, as well as an increase in the threshold pressure and luminal volume required to trigger peristalsis. Moreover, histochemistry showed an increased deposition of collagen fibers in parallel with a dramatic decrease in elastic fibers. IND, SC and CEL inhibited collagen deposition, and reverted the loss of elastic fibers. In the inflamed/fibrotic colon, western blot analysis revealed an increased expression of TGF-beta, PCNA, caspase-3, pAkt, p-p38, RhoA and pSMAD2. By contrast, the expression of SMAD6, pERKs and pJNK was reduced. In this setting, IND, SC and CEL counteracted the increased expression of TGF-beta, RhoA, pSMAD2 and p-p38, and partly reverted the reduced expression of SMAD6. Conclusions. In the DNBS model of colitis, bowel fibrosis is associated with impaired colonic peristalsis and alterations in the expression of factors driven by activation of TGF-beta signalling. The pharmacological blockade of COX-1 and COX-2 is able to counteract the fibrotic remodelling of inflamed colon, and this action appears to depend on the modulation of TGF-beta-dependent SMAD signalling, particularly through an induction of the inhibitory protein SMAD6 and a reduction of the phosphorylated status of SMAD2.

Parkinson’s disease (PD) is characterized by degeneration and loss of dopaminergic nigrostriatal neurons, responsible for neurologic symptoms. Besides motor signs, most PD patients experience gastrointestinal (GI) disturbances, including dyspepsia and constipation, which impair severely their quality of life. The pathophysiology of digestive disturbances in PD is largely unknown, but recently a link between PD and colonic inflammation has been observed in human biopsies [1]. Our study aims at evaluating some inflammatory patterns in colonic whole wall in a rat model of PD. PD was induced in rats by unilateral injection of 6-idroxydopamine (6-OHDA) into two sites of the right medial forebrain bundle. Studies were carried out 28 and 56 days after treatment on full-thickness samples from distal colon. The following parameters were examined: tissue malondialdehyde (MDA), an index of membrane lipid peroxidation; tumor necrosis factor (TNF), an index of inflammatory cytokine release; inflammatory cells (eosinophils, mast cells); glial fibrillar acid protein (GFAP) and substance P (SP) expression in myenteric ganglia by immunohistochemical staining. At both time points 6-OHDA-induced nigrostriatal denervation was associated with significant increase in: a) tissue levels of MDA (membrane oxidative stress) and TNF (tissue inflammation); b) density of eosinophil and mast cell in tunica mucosa and submucosa (inflammatory infiltration); b) GFAP expression in myenteric ganglia of tunica muscularis (enteric glia activation); c) SP expression in myenteric neurons, which is compatible with an enhanced tachykinenergic motor control as shown by preliminary functional studies on colonic muscle strips from PD rats. Our findings suggest that experimental PD, elicited by nigrostriatal dopaminergic degeneration, is associated with an inflammation of the colonic wall involving all layers. These results support the view that peripheral enteric neuroinflammation may account for GI motor dysfunctions in PD., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Parkinson’s disease (PD) is a degenerative neurological disorder, which is often associated with gastrointestinal symptoms. Among these disturbances, constipation has received considerable attention owing to its potential value as early marker of disease onset [1, 2]. Our study aims at evaluating motor dysfunctions and neurochemical coding of colonic neuromuscular compartment in a rat model of PD. Experimental PD was induced in rats by unilateral injection of 6-idroxydopamine (6-OHDA) into two sites of the right medial forebrain bundle. Functional and morphological studies were carried out 28 and 56 days after treatment. Colonic circular muscle contractions were revorded in organ baths after electrical stimulation or in the presence of exogenous substance P (SP). Colonic samples were examined by immunohistochemistry for the following parameters: density of myenteric HuC/D+ neurons and GFAP+ glial cells; distribution pattern and quantitative expression of SP, tyrosine hidroxylase (TH) and choline acethyltransferase (ChAT). Contractions elicited by electrically evoked tachykinin release as well as by exogenous SP were enhanced at both day 28 and 56, as compared to controls. Myenteric ganglia of PD animals displayed a significant increase in HuC/D+ neuron density, but no alteration in GFAP+ expression. SP+ myenteric neurons progressively increased over time, while the expression of TH and ChAT were significantly reduced at both time points. Experimental PD, elicited by nigrostriatal dopaminergic degeneration, is associated with functional and histopathological/neurochemical changes of the colonic neuromuscular compartment. The enhanced tachykinenergic motor control, associated with an upregulation of neuronal SP expression, together with reduced expression of TH and ChAT, may account for the colonic motor dysfunctions often associated to PD., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013