Tryptase + mast cells in the colonic mucosa of a healthy control (a-c), an D-IBS patientwith LM (d-f ) and an D-IBS patient with LI ( g-i )

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Increased Kynurenine Production Following Toll-Like Receptor (TLR)Activation: A Novel Role for Tlr8 in Irritable Bowel Syndrome (IBS)Gerard Clarke, Declan P. McKernan, Gabor Gaszner, Eamonn M. Quigley, John F. Cryan,Timothy G. Dinan

Background: Central and peripheral serotonergic systems have been implicated in symptompathogenesis in Irritable Bowel Syndrome (IBS) on the basis of serotonin's involvement inregulating gastrointestinal tract (GIT) homeostasis, in addition to its central role in theregulation of mood; accumulating evidence also points to the presence of low level immuneactivation in the condition. We previously showed that the activity of indoleamine-2,3-dioxygenase (IDO), the immunoresponsive enzyme which is responsible for the degradationof tryptophan along the kynurenine pathway, is enhanced in IBS. The mechanism underpin-ning this phenomenon is not well understood. Recently, altered function in specific membersof the toll-like receptor (TLR) family (TLR1-10) has also been demonstrated in IBS. However,the relationship between TLR activation and IDO activity in IBS is unknown. Aim: Toinvestigate whether specific TLR activation elicits exaggerated kynurenine production in IBSpatients compared to controls. Methods: 25 IBS patients and 37 healthy controls (HC) wererecruited and venous blood was donated. Whole blood was cultured with a panel of ninedifferent TLR agonists for 24 hours. Cell culture supernatants were then analysed for bothtrytophan and kynurenine by HPLC in a single isocratic run using sequential fluorescentand UV detection. Results: Kynurenine concentrations were elevated to similar levels in bothIBS (240.9 ± 11.58 vs 322.4 ± 19.73ng/ml, p<0.001) and HC samples (224.2 ± 6.330 vs316.5 ± 13.60ng/ml, p<0.001) following treatment with the TLR4 agonist lipopolysaccheride(LPS). A corresponding elevation in the kynurenine:tryptophan ratio, an indicator of IDOactivity, was observed in both IBS patients (0.01774 ± 0.00089 vs 0.02367 ± 0.00137,p<0.001) and HCs (0.01681 ± 0.0006 vs 0.02354 ± 0.001117, p<0.001). In contrast, thisratio was elevated in IBS patients alone (0.01774 ± 0.00089 vs 0.02166 ± 0.00162, p<0.05)following treatment with the TLR8 ligand ssRNA40. Conclusion: These results demonstratethat TLR activation can drive elevations in IDO activity in a manner that corresponds topreviously described functional TLR-agonist mediated responses in IBS. Moreover, it implic-ates TLR8 in the differential IDO activity observed in IBS patients compared to controls.This provides novel evidence for a putative TLR-dependant mechanism underpinning IDOactivation in IBS and may point to new therapeutic avenues in this debilitating condition.

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Mutations in SCN5A From Patients With IBS Result in Abnormal NaV1.5FunctionArthur Beyder, Peter R. Strege, Amelia Mazzone, Cheryl E. Bernard, David J. Tester, YuriA. Saito, Michael J. Ackerman, Gianrico Farrugia

Background: Patients with long QT or Brugada syndrome-associated mutations in SCN5A(encoding a voltage gated Na+ channel Nav1.5) reported increased gastrointestinal symptoms.This generated the hypothesis that a Nav1.5 channelopathy may be present in a subset ofpatients with irritable bowel syndrome (IBS). We performed a comprehensive mutationalanalysis of SCN5A on 500 persons with Rome II IBS and compared results to 1500 controls.Any known non-disease causing mutation was also excluded. Missense mutations whosefunctional significance is unknown were found in 2.6% of the screened patients. Aim: Todetermine whether SCN5A missense mutations in IBS patients result in altered Nav1.5function. Methods: Three mutations (I94V, P648L and A1870D) were introduced into themost common SCN5A background(hH1c3). Wildtype Nav1.5 hH1c3(WT) and mutants wereseparately co-transfected with GFP into HEK-293 cells. One day after the transfection,fluorescent cells were voltage clamped in the whole cell mode. For each mutant the followingfunctional variables were compared to WT: current density (pA/pF), voltage dependence ofactivation & inactivation (half-point V1/2 and slope dV), time to peak (t), rates of fastinactivation (τf, τs), recovery from inactivation (τR), intermediate inactivation (τi), and late

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current (pA/pF). Five to seven cells were screened per mutation. Results: Peak currentdensity for WT was -122±16 pA/pF at -35 mV and no different for all 3 mutants. All mutantshad the same voltage dependence of activation and inactivation as WT (WT activation: V1/

2= -51±5 mV, dV=4.2±0.2 mV; WT inactivation: V1/2= -90±2.5 mV, dV=7.8±2.4 mV). Timeto peak was the same for all mutants compared to WT between voltages -55 and +20 mV.However, inactivation rates were substantially faster for I94V (τf faster by 20-30% comparedto WT between -35 and -10 mV, p<0.05) and P648L (τf and τs faster by 19 to 31% comparedto WT between -30 and 0 mV, p<0.05). The late current density and the intermediateinactivation were not different for the mutants compared to WT. For the two mutants withaccelerated inactivation (I94V and P648L), recovery from inactivation at -140 mV wassignificantly accelerated compared to WT (WT: τR=8.58±0.90 ms; I94V: τR=5.85±0.52 msand P648L: τR=4.87±0.37 ms P<0.05). Conclusions: Of the 3 mutations tested, 2 Nav1.5mutations (I94V and P648L) found in patients with IBS had significant functional differencescompared to WT Nav1.5 (hH1c3). While current density and activation kinetics wereconserved in these mutants, significant differences in the inactivation properties were found.Accelerated recovery from inactivation allows for higher density of Na+ current duringcyclical depolarization while accelerated fast inactivation decreases the total Na+ flux duringdepolarization, likely slowing the slow wave upstroke. Supported by NIH R0152766

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A Functional Promoter Variant in the Tryptophan Hydroxylase 1 Gene isAssociated With a Bowel Habit Phenotype in Patients With Irritable BowelSyndrome (IBS)Helmut Grasberger, Lin Chang, Wendy Shih, Angela P. Presson, Emeran A. Mayer, FrankConyers, Juanita L. Merchant

Background: Tryptophan hydroxylase 1 (TPH1) is the rate-limiting enzyme of 5-hydroxy-tryptamine (5-HT; serotonin) synthesis in enterochromaffin (EC) cells. Basolateral releaseof 5-HT from EC cells initiates peristaltic and secretory reflexes by acting on receptors onintrinsic primary afferent neurons that project to the lamina propria. Alterations in the 5-HT signaling system are believed to play a role in altered bowel habits in IBS. Postprandialplasma 5-HT levels are increased in IBS-D vs. IBS-C and controls. In a database search ofTPH1 promoter SNPs, we identified a proximal promoter SNP that exhibits differential InVitro activity in luciferase reporter assays. Aim: To assess whether this SNP -347C/A shownto be functional in tissue culture assays was associated with IBS status and correlated withbowel habit predominance in patients with IBS. Methods: 225 Rome III positive IBS patientsand 304 healthy controls underwent genetic salivary testing. Bowel habit predominance wassubclassified by Rome III criteria. The proximal promoter SNP -347C/A variant was typedusing an MslI restriction fragment length polymorphism and confirmed in a subset ofindividuals by direct sequencing. A logistic model predicting IBS status and a multinomiallogistic model predicting bowel habit were performed controlling for race and other clinicalvariables that showed a significant association (p <0.05) with SNP -347C/A in bivariateanalyses. Results: The genotype distribution of TPH1 C/A alleles within bowel habit sub-groups is shown in the table. The proximal promoter SNP was related to bowel habit, i.e.,IBS-D vs. IBS-C (p=0.0065) and remained significant when controlling for race and anxietysymptoms (HAD scale, p=0.0138). The additive effect of the C allele resulted in the oddsof having IBS-D being 2.41 times the odds of having IBS-C. Compared to IBS-D patients,IBS-C patients were significantly less likely to be homozygous for the C allele (54.5% vs20.5%) but more likely to be homozygous for the A allele (15.9% vs 9.1%) having reducedpromoter activity. The CC genotype frequencywas intermediate for IBS-M,which is consistentwith the IBS-M phenotype being intermediate between IBS-D and IBS-C. In both bivariateand multivariate analyses, there was no association between the -347C/A SNP and IBS status.Conclusion: The -347C/A SNP alleles are associated with IBS bowel habit subtypes. Sinceour recent results show increased promoter activity with the C allele, we propose thatincreased TPH1 expression and resulting increased intestinal 5-HT production correlateswith the IBS-D phenotype. Supported by NIH grants R01 DK55732, P50 DK64539, andR01 DK48351.

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Mitochondrial Inheritance in One-Sixth of Adults With Irritable BowelSyndromeMiranda A. Van Tilburg, Essam A. Zaki, Richard G. Boles

Background: Mitochondrial dysfunction has been implicated in various functional disorderssuch as migraine, depression and chronic fatigue syndrome. IBS commonly overlaps withthese disorders and bowel dysmotility is a common symptom of mitochondrial dysfunction.Therefore, it has been suggested that mitochondrial dysfunction may play a role in IBS. Arecent study found partial evidence for the role of two mtDNA SNPs, 3010A and 16519T,in functional gastrointestinal disorders but did not explore evidence of maternal inheritancepatterns in these patients (PMID19147801). The aim of the current study was to determineif functional symptoms in IBS show a maternally inheritance bias, and if the degree of thismaternal inheritance is related to these two mtDNA SNPs. Methods: Pedigrees were obtainedfrom N=308 adult IBS patients (mean age 40.1; 88.6% female) and N=102 controls (freeof mitochondrial and gastrointestinal disease; mean age 38.7; 91.2% female). Probablematernal inheritance (PMI) was determined by Quantitative Pedigree Analysis(PMID18192313). mtDNA SNPs 16519T and 3010A were assayed in mtDNA haplogroup HIBS subjects and compared to genetic data from N=437 published controls (PMID19220304).Results: PMI was found in 17.5% of the IBS sample versus in 2% of controls (p<0.0001).

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