Background Irritable bowel syndrome (IBS) is a disorder with multifactorial pathophysiology. Intestinal barrier may be altered, especially in diarrhea-predominant IBS (IBS-D). Several mediators may contribute to increased intestinal permeability in IBS. Aim We aimed to assess effects of tryptase and LPS on in vitro permeability using a 3-dimensional cell model after basolateral cell exposure. Furthermore, we assessed the extent to which these mediators in IBS plasma play a role in intestinal barrier function. Materials and Methods Caco-2 cells were grown in extracellular matrix to develop into polarized spheroids and were exposed to tryptase (10 - 50 mU), LPS (1 - 50 ng/mL) and two-fold diluted plasma samples of 7 patients with IBS-D, 7 with constipation-predominant IBS (IBS-C) and 7 healthy controls (HC). Barrier function was assessed by the flux of FITC-dextran (FD4) using live cell imaging. Furthermore, plasma tryptase and LPS were determined. Results Tryptase (20 and 50 mU) and LPS (6.25 – 50 ng/mL) significantly increased Caco-2 permeability versus control (all P< 0.05). Plasma of IBS-D only showed significantly elevated median tryptase concentrations (7.1 [3.9 – 11.0] vs. 4.2 [2.2 – 7.0] vs. 4.2 [2.5 – 5.9] μg/mL; P<0.05) and LPS concentrations (3.65 [3.00 – 6.10] vs. 3.10 [2.60-3.80] vs. 2.65 [2.40 – 3.40] EU/ml; P< 0.05) vs. IBS-C and HC. Also, plasma of IBS-D increased Caco-2 permeability versus HC (0.14450 ± 0.00472 vs. 0.00021 ± 0.00003; P < 0.001), which was attenuated by selective inhibition of tryptase and LPS (P< 0.05). Conclusion Basolateral exposure of spheroids to plasma of IBS-D patients resulted in a significantly increased FD4 permeation, which was partially abolished by selective inhibition of tryptase and LPS. These findings point to a role of systemic tryptase and LPS in the epithelial barrier alterations observed in patients with IBS-D.

development of GI symptoms and alteration in intestinal permeability.Methods: In a prospective, longitudinal study, urinary metabotyping was conducted on 38 male soldiers (ages 19-23) during combat training and the subsequent rest period using gas chromatographymass spectrometry.Stress was measured using the perceived stress scale-10 item (PSS-10) questionnaire, while incidence and severity of GI symptoms were assessed using the irritable bowel syndrome symptom severity score (IBS-SSS).Whole gut intestinal permeability was evaluated by quantifying the 24h urinary excretion of sucralose as a percentage of the orally administered 1g dose.Results: PSS-10 stress and IBS-SSS scores were higher during the combat-training period than at rest [p<0.05].The urinary metabotype was clearly distinct from the rest period [partial least squares discriminant analysis (PLSDA) R2X=0.395,R2Y= 0.716, Q2 (cumulative)=0.581],confirming the presence of a unique stress-induced metabotype.Based on PLSDA, differential metabolites related to combat stress were uncovered (e.g.elevated pyroglutamate and fructose; reduced gut microbial metabolites such as hippurate and m-hydroxyphenylacetate) [p<0.05].The extent of pyroglutamate upregulation exhibited a positive correlation with the increase in IBS-SSS in soldiers during combat-training [r= 0.5, p<0.05].Additionally, the rise in fructose levels during combat-training was positively correlated with an increase in intestinal permeability [r=0.5, p<0.05].Conclusion: Protracted and mixed psychological and physical combat-training stress yielded unique metabolic changes that corresponded with the incidence and severity of GI symptoms and alteration in intestinal permeability.Taken together, our data provided new insights into the molecular changes underlying stress-induced GI perturbations which could be exploited for future biomarker research or therapeutic strategies.

Partially Hydrolyzed Guar Gum (PHGG) Inhibits Castor Oil-Induced Diarrhea in Rats Through the Inhibition of Substance P Production Kentaro Suzuki, Yuji Naito, Kazuhiro Kamada, Syunsuke Kishimoto, Yukiko Uehara, Hideki Horie, Wataru Fukuda, Yutaka Inada, Takaya Iida, Munehiro Kugai, Toshifumi Tsuji, Hiroyuki Yoriki, Akifumi Fukui, Yasuki Higashimura, Katsura Mizushima, Kazuhiro Katada, Kazuhiko Uchiyama, Osamu Handa, Tomohisa Takagi, Nobuaki Yagi, Zenta Yasukawa, Makoto Tokunaga, Tsutomu Okubo, Lekh R. Juneja, Toshikazu Yoshikawa

Background: Little is known about the spinal cord dorsal horn (DH) neurons activated by colonic mechanical stimuli.Even less is known about how chronic hypersensitivity of colonic nociceptors effects DH neuron activity.Methods: Experiments were performed in healthy mice (N=4) and in a TNBS model of post-inflammatory chronic visceral hypersensitivity (CVH; N=4).TNBS (130μL/ml) was administered by colorectal enema and after 28 days mice underwent 80 mmHg of colorectal distension (CRD).By this time inflammation is resolved and the peripheral endings of colonic nociceptors are mechanically hypersensitive1.Following CRD, mice were perfused fixed, spinal cord T10-L1 removed and processed for phosphorylated MAP kinase ERK 1/2 (pERK) immunohistochemistry to identify DH neurons activated by CRD.Spinal sections were co-labeled for calcitonin gene related peptide (CGRP), isolectin B4 (IB4), calbindin, GABA and NMDA receptor subunit 2B (NMDAr2B).The average number of pERK-immunoreactive (IR) neurons from 6-10 spinal sections was compared between healthy and post-inflamed mice using Two-way ANOVA with Bonferroni's posttests whilst unpaired student t-tests determined differences in co-labelling.Results: Significantly more DH neurons were pERK-IR following CRD in CVH mice compared to healthy mice (P<0.0001),specifically in T12-T13 (P<0.05) and T13-L1 (P<0.001).In healthy mice, pERK-IR neurons were located in CGRP-IR laminae I (LI), but not in the IB4-IR substantia gelatinosa.37±3% of pERK-IR neurons were calbindin-IR, which accounted for 3±0.8% of all calbindin-IR DH neurons, classified as projection neurons in LI and a subtype of excitatory interneuron in LI-LII.All pERK/calbindin-IR neurons were located in LI and NMDAr2B-IR.3.8±2% of pERK-IR neurons were GABA-IR, which accounted for 0.9±0.6% of all GABA-IR DH inhibitory interneurons.In CVH mice, pERK-IR neurons were located in CGRP-IR LI and ventral to the IB4-IR substantia gelatinosa.A smaller proportion of pERK-IR neurons were calbindin-IR (14±5%, P=0.07), which was a consequence of more pERK-IR neurons not containing calbindin-immunoreactivity and the number of pERK/calbindin-IR neurons unchanged.Significantly more pERK-IR neurons were GABA-IR in CVH mice (13.45±3%,P<0.05), with pERK-IR neurons accounting for a greater number of GABA-IR neurons (8±1%, P<0.05).Conclusion: We identified DH neurons responsive to colonic mechanical stimulation.We also show that the chronic hypersensitivity of colonic afferent endings following inflammation leads to a significant increase in the number of DH neurons responsive to noxious colonic stimulation.These data also indicate a change in the types of DH neurons activated by colonic mechanical stimuli post-inflammation.Such DH changes may correlate with increased pain perception associated with CVH. 1) Hughes & Brierley, et al.Gut.2009.

Background: Select perinatal and early life experiences-such as low birth weight, nasogastric tube placement, and maternal separation-have been linked to irritable bowel syndrome (IBS) or IBS-like symptoms in humans and animals.However, confirmatory data regarding these events are lacking.Specific Aims: To determine whether there are specific perinatal and early life experiences associated with adult IBS.Methods: Participants in a previously constructed family case-control study were mailed new questionnaires asking about early life events.Subjects contacted included 506 cases and 462 controls.Continuous variables were compared between IBS-affecteds and controls using the t-test; categorical variables were compared with the Pearson chi-square test.Results: 642 (66%) responded.Median age for cases and controls was 49 years (range: 18.0-70.0)and consisted of 82% females, 98% were Caucasian.The distribution of IBS subtypes among cases was: 23% IBS-D, 9% IBS-C, 26% IBS-M, and 42% other.Cases had similar birth weights to controls; low birthweight (≤2500g) was not associated with IBS status.Cases and controls reported similar gestational age at birth (median 40.0 wks), were equally likely to have reported health problems at delivery (8%) and to have received breast milk during infancy and for a similar duration.However, cases were more likely to have experienced infant colic (22% v. 11%, p<0.001).By IBS subtype, 13% of IBS-D (p=0.64,compared to controls), 28% of IBS-C (p=0.01),25% of IBS-M (p<0.01), and 25% of other IBS (p<0.01)cases reported colic.Conclusions: Infancy health factors-such as birth weight, gestational age, health problems at delivery, and breast milk-were not predictive of adult IBS.However, infantile colic was more common among IBS patients, raising the question as to whether colic may be an early symptom of IBS or an early life event that results in the development of visceral hypersensitivity.

Background: Provision of adequate nutrition is a major determinant of clinical outcome for critically ill patients.However, effective delivery of enteral feeds is frequently hampered by glucose malabsorption [1], which may arise secondary to cellular defects in the small intestine.While no human data are available, levels of the sodium-dependent glucose transporter (SGLT-1) are reduced in animal models of critical illness [2].Moreover, it is now established that the lingual G-protein coupled sweet taste receptors, T1R2 and T1R3, are expressed in animal and human small intestine [3], where they may act to increase SGLT-1 levels.We sought to determine whether expression of intestinal glucose transporters and sweet taste receptors was (i) dynamically regulated by luminal glucose in healthy humans, and (ii) whether this was impaired in critical illness.Methods: Healthy subjects (n = 8) were studied during euglycemia (~5mmol/l), while critically ill patients (n = 5 mechanically ventilated) were studied under non-clamp conditions.Endoscopic biopsies were collected from the duodenum at baseline and after glucose perfusion (30g/150ml water, 30 min).Expression levels of SGLT-1, the facilitative glucose transporter (GLUT-2) and T1R2 were quantified by RT PCR in biopsy tissue.Results: SGLT-1 and T1R2 expression was significantly lower in the duodenum of critically ill patients (22%, 76%, p < 0.05 respectively vs. healthy subjects), while GLUT2 levels were higher (91%, p < 0.001).Duodenal glucose perfusion did not change expression of SGLT-1 in critically ill patients, while GLUT2 levels were increased (33%, p < 0.05).In comparison, glucose perfusion reduced levels of both transporters in healthy subjects (20%, 17% respectively, p < 0.05).T1R2 expression in critically ill patients further decreased (42%, p < 0.05) in the presence of glucose, whereas levels were increased (59%, p < 0.01) in healthy subjects.Conclusions: Duodenal expression of the primary glucose transporter SGLT-1 is significantly reduced in critical illness, which may lead to glucose malabsorption.In contrast, GLUT2-dependent glucose transport may increase in these patients.Duodenal expression of T1R2 is markedly reduced in critically ill patients at baseline and reduced further by luminal glucose.Reduced T1R2 signaling in critical illness may explain why control of SGLT-1 expression is uncoupled from luminal glucose signals in these patients, in contrast to health.Increasing intestinal SGLT-1 levels may improve clinical outcomes in critically ill patients.

patients with CC.METHODS: The AEs from 14 Phase II/III, double-blind, placebo (PLA)controlled trials (duration 4-12 weeks; PRU dose 0.5-4mg q.d.) in patients with CC were pooled, and different AE categories were compared between elderly and adults.RESULTS: 2717 patients were treated with PRU (exposure: 406 patient-yrs), of which 564 were ≥65 yrs (exposure: 63 patient-yrs).Exposure time to 2mg (recommended dose for adults) was 165 pt-yrs (n=938); exposure to 1mg (recommended dose for elderly) was 8 pt-yrs (n= 113).In the elderly, the incidence of the different AE categories was comparable between PRU and PLA, and comparable to those seen in adults except for serious AEs (SAEs).SAEs were reported at a higher rate in elderly (most commonly infections, infestations or respiratory disorders), with no difference observed between PRU and PLA (Table ).Three elderly patients died, 2 on PRU, due to pneumonia or bronchitis (not considered related to study medication) and 1 on PLA due to arrhythmia/myocardial infarction.Most common AEs with PRU in both age groups were gastrointestinal symptoms (nausea, diarrhea, abdominal pain) and headache.Results in subgroups of patients 65-75 yrs and patients ≥75 yrs were similar to those observed in the whole group of elderly patients.The proportion of patients on PRU discontinuing therapy among the elderly and adults was similar.The incidence of cardiovascular AEs of interest (palpitations, corrected QT interval-related AEs, and ventricular arrhythmias) was comparable in both age groups, as well as for PRU and PLA.Cardiac ischemia and atrial arrhythmia-related AEs were more common in elderly, but their incidence was not different for PRU and PLA.The majority of ischemic and atrial rhythm-related AEs were reported by the investigator as unrelated to the study medication or doubtful.CONCLUSIONS: In 14 double-blind, PLA-controlled, Phase II/III trials of PRU, the safety and AE profiles were similar relative to PLA in adult and elderly patients with CC.

1).This profile was similar to the spinal thoracic dorsal root ganglia (DRG) TRPV1-positive nociceptors innervating the esophagus (also derived from neural crest).In stark contrast, the putative placodes-derived JNG nociceptors (TRPV1+/P2X2+) lacked PPTA, GFRα3 and TrkA , but expressed TrkB.We conclude that both the neural crest-and placodes-derived vagal nociceptive neurons innervate the mouse esophagus.The phenotype of neural crestderived nociceptors in the vagal and spinal (DRG) pathways is similar, but distinct from the phenotype of the vagal placodes-derived nociceptors.Supported by DK074480 and MZdSR 2007/54UK15.Table 1.Phenotypes of the TRPV1-positive neurons retrogradely labeled from the mouse esophagus TRPV1 was detected in 59/172 of the JNG and 19/80 of the DRG neurons retrogradely labeled from the mouse esophagus.

treated with URB597 [3'-(aminocarbonyl) biphenyl-3-yl cyclohexylcarbamate] (3 mg/kg, SC route) or vehicle (PO route). Two hours later, rats were treated with cortagine (10 μg/kg, IP route). After 15 min, a second CRD was performed. Thirty min after the second CRD, rats were anesthetized with isoflurane, decapitated, and brains collected along with those from naive rats (no CRD, no treatment). AEA and 2-AG levels were quantified in brain extracts by LC-MS/MS. Results: In vehicle-treated rats, cortagine increased the VMR to CRD at 40 and 60 mmHg to 130 ± 20% and 144 ± 14% of baseline, respectively. URB597 blocked the cortagine-induced increase in VMR at 60 mmHg (95 ± 20% vs. 144 ± 14%, p<0.05) and showed a trend for reduction at 40 mmHg. URB597 also decreased the percentage of cortagine treated rats with an increased VMR (45 and 36% of URB597-treated rats at 40 and 60 mmHg vs. 84 and 92% with vehicle, p<0.05). URB597 increased brain AEA levels 5.7-fold (p<0.001) in rats compared to vehicle treatment and significantly elevated levels of other FAAH substrates. AEA levels in vehicle-treated rats were similar to those of naive rats indicating that vehicle, CRD, and cortagine did not affect the basal levels of AEA in the brain. 2-AG levels showed little or no significant change among all 3 groups of rats. Conclusions: These data support a role of eCBs in the control of visceral hypersensitivity induced by CRF1 signaling activation and suggest that FAAH inhibitors may represent a promising therapy for IBS-D.