Transplantation of fecal microbiota from patients with irritable bowel syndrome alters gut function and behavior in recipient mice.

Irritable bowel syndrome (IBS) is a common disorder characterized by altered gut function and often is accompanied by comorbid anxiety. Although changes in the gut microbiota have been documented, their relevance to the clinical expression of IBS is unknown. To evaluate a functional role for commensal gut bacteria in IBS, we colonized germ-free mice with the fecal microbiota from healthy control individuals or IBS patients with diarrhea (IBS-D), with or without anxiety, and monitored gut function and behavior in the transplanted mice. Microbiota profiles in recipient mice clustered according to the microbiota profiles of the human donors. Mice receiving the IBS-D fecal microbiota showed a taxonomically similar microbial composition to that of mice receiving the healthy control fecal microbiota. However, IBS-D mice showed different serum metabolomic profiles. Mice receiving the IBS-D fecal microbiota, but not the healthy control fecal microbiota, exhibited faster gastrointestinal transit, intestinal barrier dysfunction, innate immune activation, and anxiety-like behavior. These results indicate the potential of the gut microbiota to contribute to both intestinal and behavioral manifestations of IBS-D and suggest the potential value of microbiota-directed therapies in IBS patients.

Technical Advance: Function and efficacy of an {alpha}4-integrin antagonist using bioluminescence imaging to detect leukocyte trafficking in murine experimental colitis.

Leukocyte trafficking is a therapeutic target in IBD. The integrins α4ß and α4ß1 regulate leukocyte migration into tissues and lymphoid organs. Current strategies rely on biologics, such as mAb, to inhibit leukocyte recruitment. Here we show the in vivo therapeutic effects of a small molecule α4-integrin antagonist (GSK223618A) in a leukocyte-trafficking model and a murine model of colitis. Leukocytes isolated from MLNs of transgenic ß-actin-luc+ mice were injected i.v. into recipients with DSS-induced colitis. Recipient mice were orally gavaged with vehicle or an α4-integrin antagonist 1 h pre-adoptive transfer, followed by bioluminescence whole body and ex vivo organ imaging 4 h post-transfer. To confirm its therapeutic effect, the α4-integrin antagonist was given orally twice daily for 6 days to mice with DSS-induced colitis, starting on Day 3. Clinical, macroscopic, and histological signs of inflammation were assessed and gene-expression profiles analyzed. Using bioluminescence imaging, we tracked and quantified leukocyte migration to the inflamed gut and demonstrated its inhibition by a small molecule α4-integrin antagonist. Additionally, the therapeutic effect of the antagonist was confirmed in DSS-induced colitis in terms of clinical, macroscopic, and histological signs of inflammation. Gene expression analysis suggested enhancement of tissue healing in compound-treated animals. Inhibition of leukocyte trafficking using small molecule integrin antagonists is a promising alternative to large molecule biologics. Furthermore, in vivo bioluminescence imaging is a valuable strategy for preclinical evaluation of potential therapeutics that target leukocyte trafficking in inflammatory diseases.

Targeting gut T cell Ca2+ release-activated Ca2+ channels inhibits T cell cytokine production and T-box transcription factor T-bet in inflammatory bowel disease.

Prolonged Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels is crucial in activating the Ca(2+)-sensitive transcription factor NFAT, which is responsible for directing T cell proliferation and cytokine gene expression. To establish whether targeting CRAC might counteract intestinal inflammation, we evaluated the in vitro effect of a selective CRAC inhibitor on T cell cytokine production and T-bet expression by lamina propria mononuclear cells (LPMC) and biopsy specimens from inflammatory bowel disease (IBD) patients. The inhibitory activity of the CRAC blocker was investigated through patch-clamp experiments on rat basophilic leukemia cells and fluorometric imaging plate reader intracellular Ca(2+) assays using thapsigargin-stimulated Jurkat T cells and its detailed selectivity profile defined using a range of in vitro radioligand binding and functional assays. Anti-CD3/CD28-stimulated LPMC and biopsy specimens from 51 patients with IBD were cultured with a range of CRAC inhibitor concentrations (0.01-10 microM). IFN-gamma, IL-2, IL-8, and IL-17 were analyzed by ELISA. T-bet was determined by immunoblotting. We found that the CRAC blocker concentration-dependently inhibited CRAC current in rat basophilic leukemia cells and thapsigargin-induced Ca(2+) influx in Jurkat T cells. A concentration-dependent reduction in T-bet expression and production of IFN-gamma, IL-2, IL-17, but not IL-8, was observed in IBD LPMC and biopsy specimens treated with the CRAC inhibitor. In conclusion, we provide evidence that the suppression of CRAC channel function may dampen the increased T cell response in the inflamed gut, thus suggesting a promising role for CRAC inhibitor drugs in the therapeutic management of patients with IBD.

Characterization of a calcitonin gene-related peptide release assay in rat isolated distal colon.

The release of calcitonin gene-related peptide (CGRP) plays a key role gastrointestinal tract homeostasis. We aimed to investigate mechanisms that mediate CGRP release from the rat colon in vitro. Colon segments were stimulated and the amount of CGRP released was measured using an enzyme immunoassay. Capsaicin and low pH induced significant increases in CGRP release which was shown to be mediated by TRPV1 activation as demonstrated with the TRPV1 antagonists CTPC and capsazepine. The mast cell degranulator, compound 48/80 significantly increased CGRP release an effect that was blocked in the presence of the mast cell stabilizer, ketotifen and the selective Gi inhibitor benzalkonium chloride. The addition of a mixture of inflammatory mediators containing pro-inflammatory cytokines, 5HT, bradykinin and PGE2 showed no effect at neutral pH but at low pH a significant additive effect was observed. We conclude that CGRP release in the rat distal colon occurs in response to mast cell degranulation, inflammatory mediators, low pH and capsaicin and describe a role for TRPV1 receptors in mediating the response.

Validation of a conscious rat model for the discovery of novel agents that inhibit gastric acid secretion.

Identification of novel drug molecules requires the extensive evaluation in vitro and in vivo. Following in vitro evaluation it is necessary to efficiently screen numerous novel molecules in vivo using relatively simple methodology that requires small numbers of animals, is rapid to perform, and provides results that can definitely discriminate potential candidates for further investigation. Herein, we describe the results of three standard compounds (omeprazole, a proton pump inhibitor; cimetidine, an histamine H(2) receptor antagonist; and AR-H047108, a potassium competitive acid blocker) in the rat aspiration model (under both basal and stimulated conditions), and compared the effects with those in the pyloric ligation model with a view to comparing the results in terms of sensitivity, robustness and simplicity of the methodology. In the aspiration model, drug or vehicle was administered orally 1 h prior to administration of pentagastrin or dimaprit. Ten minutes later 0.9% NaCl was administered orally and immediately recovered by aspiration. In the pyloric ligation model, drugs or vehicle were administered orally 2 h before ligation in a volume of 10 ml/kg. For each model, the volume of each sample was measured and the acidity was determined. In the aspiration model under basal acid secretion or following stimulation with pentagastrin omeprazole, cimetidine and AR-H047108 produced dose related inhibition of acidity. Omeprazole and cimetidine inhibited acid secretion following stimulation with dimaprit. In the pyloric ligation model omeprazole, cimetidine and AR-H047108 inhibited acid secretion. The profile of each of 3 inhibitors of acid secretion exhibited similar effects irrespective of the degree of stimulation (dimaprit, pentagastrin or pyloric ligation). Thus, based on these robust effects and ease of methodology we would recommend the use of the rat aspiration model with pentagastrin stimulation of gastric acid secretion as the primary in vivo methodology to screen novel inhibitors of acid secretion.

5-HT in the enteric nervous system: gut function and neuropharmacology.

In recent times, the perception of functional gastrointestinal disorders such as irritable bowel syndrome (IBS) has shifted fundamentally. Such disorders are now thought of as serious diseases characterized by perturbations in the neuronal regulation of gastrointestinal function. The concept of visceral hypersensitivity, the characterization of neuronal networks in the 'brain-gut axis' and the identification of several novel 5-HT-mediated mechanisms have contributed to this shift. Here, we review how some of the more promising of these new mechanisms (e.g. those involving 5-HT transporters and the 5-HT(2B), 5-HT(7) and putative 5-HT(1p) receptors) might lead to a range of second-generation therapies that could revolutionize the treatment of functional gastrointestinal disorders, particularly IBS.

Dual role of 5-HT3 receptors in a rat model of delayed stress-induced visceral hyperalgesia.

Despite its beneficial effect in IBS patients, the mechanism of action of the 5-HT3 receptor (5-HT3R) antagonist alosetron is still incompletely understood. We aimed to characterize the effect and site(s) of action in a model of stress-induced sensitization of visceral nociception in rats. Adult male Wistar rats were equipped for recording of visceromotor response (VMR) to phasic colorectal distension (CRD; 10-60 mmHg). VMR to CRD was recorded 24 h after an acute session of water avoidance (WA) stress (post-WA). Baseline and post-WA responses were measured in rats exposed to WA or sham-WA, treated with alosetron at 0.3 mg/kg subcutaneously (s.c.) 25 nmol intrathecally (i.t.) or vehicle before post-WA CRD. Some rats were treated with capsaicin/vehicle on the cervical vagus nerve and received alosetron (0.3 mg/kg, s.c.) 15 min before post-WA CRD. WA stress led to visceral hyperalgesia 24 h later. Alosetron (0.3 mg/kg, s.c.), failed to inhibit WA-induced exacerbation of VMR to CRD. Stress-induced visceral hyperalgesia was abolished when alosetron was injected intrathecally (P<0.05) in intact rats or subcutaneously (0.3 mg/kg) in capsaicin-pretreated animals (P<0.05). Capsaicin-pretreatment did not affect the exacerbating effect of stress on visceral sensitivity. Alosetron had no inhibitory effect on normal visceral pain responses when administered subcutaneously or intrathecally. We demonstrated that 5-HT3Rs on central terminals of spinal afferents are engaged in the facilitatory effect of stress on visceral sensory information processing. In addition, we showed that stress-induced sensitization of visceral nociception is independent of 5-HT3R activation on vagal afferents.

Effects of the 5-HT3 receptor antagonist, alosetron, in a rat model of somatic and visceral hyperalgesia.

Conflicting results exist regarding the role of 5-HT3 receptors in somatic and visceral nociceptive processing. We aimed to investigate the effects of the 5-HT3 receptor antagonist, alosetron, in a rat model of somatic and visceral hyperalgesia. Two injections (100 microl) of either pH 4.0 or 7.2 saline were given unilaterally in the gastrocnemius (GN) muscle. In all groups, the paw withdrawal thresholds (PWT) to von Frey filaments and the visceromotor responses (VMR) to colorectal distension (CRD) were recorded before the saline injections and 72 h, and 1 week after the second injection. Intrathecal (i.t.) (25 nmol) or intravenous (i.v.) (100 microg/kg/day) alosetron was given daily following the second injection and compared to either i.v. or i.t. saline (vehicle). There was a significant decrease in the mean PWT bilaterally in all groups following pH 4.0 injections (p<0.05). Intravenous alosetron resulted in a significant increase in the PWT bilaterally on days 2 and 3. Intrathecal alosetron resulted in significant increase in the PWT starting at day 3 and was significantly higher than baseline on days 4-7 (p<0.05). At CRD pressures 30 mmHg, the VMR of pH 4.0 injected rats was significantly increased at 72 h and 1 week (p<0.05). Both i.v. and i.t. alosetron treated rats failed to demonstrate any alteration in the VMR. Control rats (pH 7.2) failed to show any alteration in the VMR and were unaffected by alosetron. Both, systemically and centrally administered alosetron, reversed the mechanical somatic hypersensitivity and prevented the development of visceral hyperalgesia, suggesting a centrally mediated effect.

Antiinflammatory activity of soluble guanylate cyclase: cGMP-dependent down-regulation of P-selectin expression and leukocyte recruitment.

Nitric oxide (NO) production by the vascular endothelium maintains an essential antiinflammatory, cytoprotective influence on the blood vessel wall. A key component of this activity is attributed to prevention of leukocyte-endothelial cell interactions, yet the underlying mechanisms remain unclear. The NO receptor, soluble guanylate cyclase (sGC), is expressed in endothelial cells but fulfils an unknown function. Therefore, we used intravital microscopy in mesenteric postcapillary venules from WT and endothelial nitric oxide synthase (eNOS) knockout (eNOS(-/-)) mice, and an sGC activator (BAY 41-2272), to investigate a potential role for sGC in the regulation of adhesion molecule expression and leukocyte recruitment. Leukocyte rolling and adhesion was 6-fold greater in eNOS(-/-) than WT animals. BAY 41-2272 and the NO-donor, diethylamine-NONOate, reduced leukocyte rolling and adhesion in eNOS(-/-) mice to levels observed in WT animals. These effects were blocked by the sGC inhibitor ODQ [1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one], which itself caused a 6-fold increase in leukocyte rolling and adhesion in WT mice. Increased leukocyte rolling and adhesion in IL-1beta-treated mice was also inhibited by BAY 41-2272. Fluorescence-activated cell sorting analysis in vitro and a specific P-selectin neutralizing antibody in vivo revealed that selective down-regulation of P-selectin expression accounted for the antiadhesive effects of sGC activation. These data demonstrate that sGC plays a key antiinflammatory role by inhibiting P-selectin expression and leukocyte recruitment.

Protease-activated receptor-2 activation causes EDHF-like coronary vasodilation: selective preservation in ischemia/reperfusion injury: involvement of lipoxygenase products, VR1 receptors, and C-fibers.

Activation of protease-activated receptor (PAR)-2 has been proposed to be protective in myocardial ischemia/reperfusion (I/R) injury, an effect possibly related to an action on the coronary vasculature. Therefore, we investigated the effects of PAR2 activation on coronary tone in isolated perfused rat hearts and elucidated the mechanisms of any observed effects. Although having a negligible effect on ventricular contractility, the PAR2 activating peptide SLIGRL produced an endothelium-dependent coronary vasodilatation (ED(50)=3.5 nmol). Following I/R injury, the response to SLIGRL was selectively preserved, whereas the dilator response to acetylcholine was converted to constriction. Trypsin also produced a vasodilator dose-response curve that was biphasic in nature (ED(50-1)=0.36 U, ED(50-2)=38.71 U). Desensitization of PAR2 receptors indicated that the high potency phase was mediated by PAR2. Removal of the endothelium but not treatment with L-NAME (300 micromol/L), indomethacin (5 micromol/L), or oxyhemoglobin (10 micromol/L) inhibited the response to SLIGRL and trypsin. Treatment with the K(+)-channel blockers TEA (10 mmol/L), charybdotoxin (20 nmol/L)/apamin (100 nmol/L), or elevated potassium (20 mmol/L) significantly suppressed responses. Similarly, inhibition of lipoxygenase with nordihydroguaiaretic acid (1 micromol/L), eicosatetraynoic acid (1 micromol/L), or baicalein (10 micromol/L), desensitization of C-fibers using capsaicin (1 micromol/L, 20 minutes), or blockade of vanilloid (VR1) receptors using capsazepine (3 micromol/L) inhibited the responses. This study shows, for the first time, that PAR2 activation causes endothelium-dependent coronary vasodilation that is preserved after I/R injury and is not mediated by NO or prostanoids, but involves the release of an endothelium-derived hyperpolarizing factor (EDHF), possibly a lipoxygenase-derived eicosanoid, and activation of VR1 receptors on sensory C-fibers.