Irritable bowel syndrome (IBS) is a common gastrointestinal disorder in which the underlying pathophysiology is poorly understood; however, increased intestinal permeability in diarrhea-predominant IBS patients has been reported. Here we demonstrate diarrhea-predominant IBS patients (D-IBS) that display increased intestinal permeability. We have also found that increased intestinal membrane permeability is associated with visceral and thermal hypersensitivity in this subset of D-IBS patients. We evaluated 54 D-IBS patients and 22 controls for intestinal membrane permeability using the lactulose / mannitol method. All subjects ingested 5 g laclulose and 2 g mannitol in 100 ml of water after which their urine was collected. We also evaluated the mean mechanical visual analogue (MVAS) pain rating to nociceptive thermal and visceral stimulation in all subjects. All study participants also completed the FBDSI scale. Approximately 39% of diarrhea-predominant IBS patients have increased intestinal membrane permeability as measured by the lactulose / mannitol ratio. These IBS patients also demonstrated higher M-VAS pain intensity reading scale. Interestingly, the IBS patients with hypersensitivity and increased intestinal permeability had a higher FBDSI score (100.8±5.4) compared to IBS patients with normal membrane permeability and sensitivity (51.6±12.7) and controls (6.1 ± 5.6) (p<0.001). A subset of D-IBS patients have increased intestinal membrane permeability that is associated with an increased FBDSI score and increased hypersensitivity to visceral and thermal nociceptive pain stimuli. Thus, increased intestinal membrane permeability in D-IBS patients may lead to more severe IBS symptoms and hypersensitivity to somatic and visceral stimuli.

Background-The molecular mechanisms underlying the pathophysiology of irritable bowel syndrome (IBS) are poorly understood. One mechanism may involve increased intestinal permeability that is reversed with glutamine supplementation. Our goal was to evaluate the expression of glutamine synthetase and its complementary miRNA in blood microvesicles and gut tissues of IBS patients with increased intestinal membrane permeability.

BACKGROUND & AIMS Some patients with irritable bowel syndrome with diarrhea (IBS-D) have intestinal hyperpermeability, which contributes to their diarrhea and abdominal pain. MicroRNA 29 (MIR29) regulates intestinal permeability in patients with IBS-D. We investigated and searched for targets of MIR29 and investigated the effects of disrupting Mir29 in mice. METHODS We investigated expression MIR29A and B in intestinal biopsies collected during endoscopy from patients with IBS (n = 183) and without IBS (controls) (n = 36). Levels were correlated with disease phenotype. We also generated and studied Mir29−/− mice, in which expression of Mir29a and b, but not c, is lost. Colitis was induced by administration of 2,4,6-trinitrobenzenesulfonic acid; intestinal tissues were collected and permeability was assessed. Microarray analysis was performed using tissues from Mir29−/− mice. Changes in levels of target genes were measured in human colonic epithelial cells and small intestinal epithelial cells after knockdown of MIR29 with anti-MIRs. RESULTS Intestinal tissues from patients with IBS-D (but not IBS with constipation or controls) had increased levels of MIR29A and B, but reduced levels of Claudin-1 (CLDN1) and nuclear factor-κB–repressing factor (NKRF). Induction of colitis and water avoidance stress increased levels of Mir29a and Mir29b and intestinal permeability in wild-type mice; these increased intestinal permeability in colons of far fewer Mir29−/− mice. In microarray and knockdown experiments, MIR29A and B were found to reduce levels of NKRF and CLDN1 messenger RNA, and alter levels of other messenger RNAs that regulate intestinal permeability. CONCLUSIONS Based on experiments in knockout mice and analyses of intestinal tissue samples from patients with IBS-D, MIR29 targets and reduces expression of CLDN1 and NKRF to increase intestinal permeability. Strategies to block MIR29 might be developed to restore intestinal permeability in patients with IBS-D.

ObjectiveMany patients with irritable bowel syndrome IBS not only have abdominal pain but also may suffer from visceral hypersensitivity and heighted visceral nociception. Moreover, IBS has few effective therapeutic agents and mechanisms of disease are unclear. Our goals were to (i) identify microRNA (miRNA) expression, signalling and targets in human colon (controls; patients with IBS); (ii) verify in vitro, IBS-associated changes in miRNAs, especially miR-199, which is complementary to the transient receptor potential vanilloid type 1 (TRPV1) gene; and (iii) determine whether modulating the expression of miRNAs in vivo, especially miR-199, reverses associated changes and pathological hallmarks of visceral hypersensitivity via TRPV1 signalling.DesignWe evaluated 45 patients with diarrhoea-predominant IBS (IBS-D) and 40 controls with (1) visceral pain severity score and (2) colonoscopy with biopsies. miRNA expression was evaluated in human colon following miRNA array analysis. Luciferase assays were done to confirm relationships between miR-199 and TRPV1 expression. A rat model of visceral hypersensitivity was used to study miR-199 and its target gene (TRPV1) expression in dorsal root ganglion (DRG) and colon in vivo.ResultsGut miR-199a/b expression in IBS-D was significantly decreased, which correlated directly with both increased visceral pain scores and TRPV1 expression. In vivo upregulation of miR-199a by intraperitoneal injection of lenti-miR-199a precursors decreased visceral hypersensitivity via diminished TRPV1 signalling.ConclusionsDecreased colonic miR-199a/b correlates with visceral pain in patients with IBS-D. Similarly, reduced miR-199a expression in rat DRG and colon tissue is associated with heightened visceral hypersensitivity. In vivo upregulation of miR-199a decreases visceral pain via inhibition of TRPV1 signalling. Thus, miR-199 precursors may be promising therapeutic candidates for the treatment in patients with visceral pain.

Evidence for multiprotein complexes playing a role in DNA replication has been growing over the years. We have previously reported on a replication-competent multiprotein form of DNA polymerase isolated from human (HeLa) cell extracts. The proteins that were found at that time to co-purify with the human cell multiprotein form of DNA polymerase included: DNA polymerase alpha, DNA primase, topoisomerase I, RNase H, PCNA, and a DNA-dependent ATPase. The multiprotein form of the human cell DNA polymerase was further purified by Q-Sepharose chromatography followed by glycerol gradient sedimentation and was shown to be fully competent to support origin-specific and large T-antigen dependent simian virus 40 (SV40) DNA replication in vitro [Malkas et al. (1990b): Biochemistry 29:6362-6374]. In this report we describe the further characterization of the human cell replication-competent multiprotein form of DNA polymerase designated MRC. Several additional DNA replication proteins that co-purify with the MRC have been identified. These proteins include: DNA polymerase delta, RF-C, topoisomerase II, DNA ligase I, DNA helicase, and RP-A. The replication requirements, replication initiation kinetics, and the ability of the MRC to utilize minichromosome structures for DNA synthesis have been determined. We also report on the results of experiments to determine whether nucleotide metabolism enzymes co-purify with the human cell MRC. We recently proposed a model to represent the MRC that was isolated from murine cells [Wu et al. (1994): J Cell Biochem 54:32-46]. We can now extend this model to include the human cell MRC based on the fractionation, chromatographic and sedimentation behavior of the human cell DNA replication proteins. A full description of the model is discussed. Our experimental results provide further evidence to suggest that DNA synthesis is mediated by a multiprotein complex in mammalian cells.

This study was designed to systematically examine the effects of persistent orofacial tissue injury on prolonged neuronal activation in the trigeminal nociceptive pathways by directly comparing the effects of orofacial deep vs. cutaneous tissue inflammation on brainstem Fos protein expression, a marker of neuronal activation. Complete Freund's adjuvant (CFA) was injected unilaterally into the rat temporomandibular joint (TMJ) or perioral (PO) skin to produce inflammation in deep or cutaneous tissues, respectively. Rats were perfused 2 hours, 24 hours, 3 days, or 10 days following CFA injection. The TMJ and PO inflammation‐induced Fos expression paralleled the intensity and course of inflammation over the 10‐day observation period, suggesting that the increase in intensities and persistence of Fos protein expression may be associated with a maintained increase in peripheral input. Compared to PO CFA injection, the injection of CFA into the TMJ produced a significantly stronger inflammation associated with a greater Fos expression. In TMJ‐ but not in PO‐inflamed rats, Fos‐like immunoreactivity (LI) spread from superficial to deep upper cervical dorsal horn as the inflammation persisted and there was a dominant ipsilateral Fos‐labeling in the paratrigeminal nucleus. Common to TMJ and PO inflammation, Fos‐LI was induced in the trigeminal subnuclei interpolaris and caudalis, C1–2 dorsal horn, and other medullary nuclei. Substantial bilateral Fos‐LI was found in the interpolaris‐caudalis trigeminal transition zone. Further analysis revealed that Fos‐LI in the ventral transition zone was equivalent bilaterally, whereas Fos‐LI in the dorsal transition zone was predominantly ipsilateral to the inflammation. The differential induction of Fos expression suggests that an increase in TMJ C‐fiber input after inflammation and robust central neuronal hyperexcitability contribute to persistent pain associated with temporomandibular disorders. J. Comp. Neurol. 412:276–291, 1999. © 1999 Wiley‐Liss, Inc.

A subset of patients with IBS have visceral hypersensitivity and/or somatic hypersensitivity. Visceral hypersensitivity might have use as a clinical marker of IBS and could account for symptoms of urgency for bowel movements, bloating and abdominal pain. The mechanisms that lead to chronic visceral hypersensitivity in patients who have IBS are unclear. However, several working models may be considered, including: nociceptive input from the colon that leads to hypersensitivity; increased intestinal permeability that induces a visceral nociceptive drive; and alterations in the expression of microRNAs in gastrointestinal tissue that might be delivered via blood microvesicles to other target organs, such as the peripheral and/or central nervous system. As such, the chronic visceral hypersensitivity that is present in a subset of patients with IBS might be maintained by both peripheral and central phenomena. The theories underlying the development of chronic visceral hypersensitivity in patients with IBS are supported by findings from new animal models in which hypersensitivity follows transient inflammation of the colon. The presence of somatic hypersensitivity and an alteration in the neuroendocrine system in some patients who have IBS suggests that multisystemic factors are involved in the overall disorder. Thus, IBS is similar to other chronic pain disorders, such as fibromyalgia, chronic regional pain disorder and temporomandibular joint disorder, as chronic nociceptive mechanisms are activated in all of these disorders.

BackgroundMore effective treatments are needed for patients with postinfectious, diarrhoea-predominant, irritable bowel syndrome (IBS-D). Accordingly, we conducted a randomised, double-blind, placebo-controlled, 8-week-long trial to assess the efficacy and safety of oral glutamine therapy in patients who developed IBS-D with increased intestinal permeability following an enteric infection.MethodsEligible adults were randomised to glutamine (5 g/t.i.d.) or placebo for 8 weeks. The primary end point was a reduction of ≥50 points on the Irritable Bowel Syndrome Severity Scoring System (IBS-SS). Secondary endpoints included: raw IBS-SS scores, changes in daily bowel movement frequency, stool form (Bristol Stool Scale) and intestinal permeability.ResultsFifty-four glutamine and 52 placebo subjects completed the 8-week study. The primary endpoint occurred in 43 (79.6%) in the glutamine group and 3 (5.8%) in the placebo group (a 14-fold difference). Glutamine also reduced all secondary endpoint means: IBS-SS score at 8 weeks (301 vs 181, p<0.0001), daily bowel movement frequency (5.4 vs 2.9±1.0, p<0.0001), Bristol Stool Scale (6.5 vs 3.9, p<0.0001) and intestinal permeability (0.11 vs 0.05; p<0.0001). ‘Intestinal hyperpermeability’ (elevated urinary lactulose/mannitol ratios) was normalised in the glutamine but not the control group. Adverse events and rates of study-drug discontinuation were low and similar in the two groups. No serious adverse events were observed.ConclusionsIn patients with IBS-D with intestinal hyperpermeability following an enteric infection, oral dietary glutamine supplements dramatically and safely reduced all major IBS-related endpoints. Large randomised clinical trials (RCTs) should now be done to validate these findings, assess quality of life benefits and explore pharmacological mechanisms.Trial registration numberNCT1414244; Results.