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Mo2041 Effect of Ibodutant, a Neurokinin NK2 Receptor Antagonist, on Gender-Related Influences Following Induction of Trinitrobenzenesulfonic Acid-Induced Colitis in the Guinea Pig
Methods: All procedures followed guidelines and were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. TNBS (30mg/ml in 30% ethanol) was rectally instilled into male and female Hartley GPs under isoflurane anesthesia. Animals receiving sham catheterization were compared to groups receiving TNBS alone versus those pretreated with Ibodutant (0.65mg/ml and 1.9mg/ml) thirty minutes prior to TNBS instillation. Two hours after TNBS instillation L6-S1 spinal cord was isolated and proteins extracted. The immediate early gene product c-fos, a surrogate marker for nociceptive neural activity, was examined using western blot using nuclear protein extracts from the L6/S1 spinal cord (SC). We also used RT-PCR to examine changes in inflammatory markers in the urinary bladder (UB) epithelium. Results: We find that c-fos was increased in male (187%) to a greater extent than female (46%) as compared to sham control. There was also a significant increase in inflammatory markers (TGF-b, IL-1b, COX and PGE2) in bladder urothelium that was more evident in the male. However, ibodutant reduced colitis-induced c-fos expression at a lower dose in female (normalized to baseline) versus male (65% reduction). Gross observation in both gender also revealed a visual reduction in colonic inflammation with ibodutant. Conclusions: Ibodutant was effective in reducing colonic-irritation induced visceral hyperalgesia (measured by c-fos induction) at a lower dose in female GPs as compared to male. It has also been shown that afferent sensitization of the colon can also adversely impact UB function resulting in a clinical overlap in many visceral disorders. The elevation in UB epithelial inflammatory markers suggests involvement of non-neuronal cells in events underlying activation of afferent nerves. Taken together with clinical evidence, we conclude that blockade of NK2 receptors may be a viable therapy for decreasing painful symptoms, with a selectivity in female IBS patients. Mo2042 TNFα in Irritable Bowel Syndrome (IBS): From Gene Polymorphisms to Circulating Levels Mohammad Bashashati, Nima Rezaei, Eamonn M. Quigley, Max J. Schmulson, Keith A. Sharkey Background: IBS is a functional gastrointestinal disorder of unknown etiology. Low-grade, residual or reactivated microscopic inflammation has been proposed in the pathophysiology of IBS. TNFα is a cytokine involved in the initiation and amplification of inflammatory reactions. In a recent meta-analysis, we have shown that circulating TNFα is significantly elevated in all IBS subtypes (1). Here we hypothesized that TNFα (-308 G/A) polymorphism is relevant to the higher serum/plasma TNFα levels in IBS. To test this hypothesis, we conducted a meta-analysis on TNFα (-308 G/A) in IBS patients compared to healthy controls. Methods: A search on TNFα (-308 G/A) gene polymorphism studies in IBS vs. control, was performed on PubMed and EMBASE through December 2014. Pooled odds ratio (OR) and 95%CI was calculated for the minor allele frequency (MAF; high producer allele A) and genotypes. Heterogeneity was assessed based on I2 values. Results: Seven studies were identified, 3 from Europe and USA and one each from Mexico, Iran, India and Korea. TNFα (-308 G/A) was not associated with IBS in the whole meta-analysis; however, after excluding studies from Europe and USA, low producer TNFα (-308 G/G) showed a significant association with an increased risk of IBS, OR: 1.78 (95%CI: 1.17-2.72), while high producer TNFα (-308G/A), OR: 0.54 (95%CI: 0.35-0.82) decreased the risk of IBS. Moreover, MAF was significantly associated with the decreased risk of IBS, OR: 0.65 (95%CI: 0.45-0.95) in nonEuropean studies. Detailed analysis of two European studies which had categorized patients based on the presence of post-infectious IBS (PI-IBS), showed that the minor high producer A allele was more prevalent in PI-IBS compared to controls, OR: 1.76 (95%CI: 1.14-2.72). Conclusion: There is geographical variation in the findings of studies on TNFα (-308G/A) polymorphism and IBS. PI-IBS patients are genetically prone to have higher TNFα levels. The findings in the TNFα (-308 G/A) polymorphism in idiopathic IBS are in contrast to those reported in inflammatory bowel disease (IBD) and PI-IBS, suggesting that while TNFα (-308 G/A) gene polymorphism may explain higher TNFα levels in IBD and PI-IBS, increased serum levels of TNFα in idiopathic IBS may be due to environmental insults or polymorphisms in other loci of the TNF gene, which requires further investigation. This work was supported by the Canadian Institutes of Health Research (to KAS) Reference: (1) Bashashati M, Rezaei N, Shafieyoun A, et al. Cytokine imbalance in irritable bowel syndrome: a systematic review and meta-analysis. Neurogastroenterol Motil. 2014;26(7):1036-48.
Mo2040 Experimentally Induced Visceral Pain, Variation in the Microbiome, and Host Gene Expression in Irritable Bowel Syndrome Nicolaas H. Fourie, Dan Wang, Sarah Abey, Eric Ferguson, LeeAnne Sherwin, Bridgett Rahim-Williams, Wendy A. Henderson Background: Abdominal pain is a principal symptom of irritable bowel syndrome (IBS). It is an inducible and measurable symptom which may be used to gain insights into the etiology of the syndrome. Subclinical inflammation is implicated in the etiology of IBS. Evidence suggests systemic inflammatory dysregulation which may be linked to dysregulation of the microbiome. We examined the relationship between severity of induced abdominal pain, variation in peripheral blood gene expression, and the oral microbiome in order to identify possible biological pathways underlying IBS and abdominal pain. Methods: A four probe sugar test solution was orally administered to 18 healthy volunteers (HV) and 16 IBS patients. Abdominal pain was assessed via self-report (100 point scale) using the Gastrointestinal Pain Pointer. RNA was isolated from whole blood. Amplified cRNA was hybridized to Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays for whole genome gene expression determination. DNA was isolated from buccal mucosa. The bacterial 16s RNA gene was amplified and hybridized to PhyloChip™ Version G3 arrays for microbiome characterization. Results: 81% of IBS patients and 11% of HV reported pain in response to the test solution. 258 bacteria were significantly correlated to pain severity. Negative correlations were dominated by Lachnospiraceae (25%). Positive correlations were dominated by Lachnospiraceae (18%), Prevotellaceae (12%) and Veillonellaceae (11%). 508 genes were correlated to pain. Pathways relating to stem cell pluripotency and inflammation were significantly represented. Seven bacterial families (Lachnospiraceae, Prevotellaceae, Rikenellaceae, Bacillaceae, Veillonellaceae, Moraxellaceae and Psuedomonodaceae) dominated correlations with pain correlated genes. Bacteria-gene expression correlation coefficients often exceeded 50%. Common molecular pathways included cytokine pathways, Rho signaling and the PI3K/AKT pathway. The expression of several known opportunistic pathogens associated with extra-oral inflammatory pathologies were significantly associated with inflammation associated genes and pain. Discussion: Over expression of Prevotellaceae and Veillonellaceae in IBS and inflammatory bowel disease have been previously described. Their positive relationship with abdominal pain and strong correlation (>50%) to inflammation associated genes suggest that they may be key drivers of systemic inflammation and gastrointestinal symptoms. Decreased expression of Lachnospiraceae with increasing pain severity may point to changing availability of butyrate, which is essential to epithelial health. Epithelial dysregulation facilitates translocation of pathogens and toxins. Bacteria-gene-symptom relationships suggest functional inflammatory associations, which remain to be further explored as diagnostic markers and targets for intervention.
Mo2043 Increased Pain Hypersensitivity in Women With Irritable Bowel Syndrome and Functional Dyspepsia Explained by Psychological Factors: A Prospective Birth Cohort Population Olli-Pekka Koivurova, Timo Blomster, Juha Auvinen, Sauli Herrala, Jaro Karppinen, Nicholas J. Talley, Jukka Ronkainen Background: The pathogenesis of irritable bowel syndrome (IBS) and functional dyspepsia (FD) is likely multifactorial. Altered serotonin metabolism and changes in pain signal processing in the central nervous system have been described. Psychological factors such as depression and anxiety are known to enhance pain sensation. However, whether peripheral pain sensitivity is altered in IBS and FD is controversial. We aimed to determine whether nociceptive pressure pain threshold (PPT) or tolerance (PPTo) is decreased in IBS and FD indicating pain hypersensitivity. Methods: The prospectively collected Northern Finland Birth Cohort 1966 (NFBC 1966) population (http://www.oulu.fi/nfbc/) is comprised of children from the two northernmost provinces of Finland, Oulu and Lapland with a date of birth Jan 1st Dec 31st, 1966 (12 231 children, 96.3% of all births during 1966 in that area). Between years 2013 and 2014, at the age of 47 years, a large health examination including both questionnaires (n=6868/10282, 67% response rate) and clinical examination (n=5851/10282, 57% response rate) was performed. For this study, 3969 subjects (44% male) were available. Uncomfortable pressure, i.e. PPT and intolerable pressure, i.e. PPTo were tested using an algometer (Somedic AB, Hörby, Sweden). The pressure was increased from 0 kPa at a constant rate of 50 kPa/s until the safety maximum of 1200 kPa. The measurements were done for four anatomical sites in the following order: 1) the upper trapezius muscle, 2) the tibialis anterior muscle, 3) the dorsal aspect of the wrist joint line, and 4) the L5/S1 interspinous space. IBS and FD were defined according to the Rome III definition. Anxiety
Mo2041 Effect of Ibodutant, a Neurokinin NK2 Receptor Antagonist, on GenderRelated Influences Following Induction of Trinitrobenzenesulfonic AcidInduced Colitis in the Guinea Pig Amanda S. Wolf-Johnston, Florenta A. Kullmann, Jonathan M. Beckel, Anthony J. Kanai, Sttefania Meini, Carlo A. Maggi, Lori A. Birder Introduction: Neurokinin NK2 receptors are expressed in the gastrointestinal tract of animals and humans and play a role in the regulation of motility and visceral sensitivity. Antagonists to NK2 receptors reduce hypersensitivity responses in animals following induction of colonic inflammation (TNBS). Taken together, available data suggest that a block of NK2 receptors could be a viable therapeutic option for treatment of patients with irritable bowel syndrome (IBS). In support is recent evidence in humans, which has shown that the NK2 antagonist, Ibodutant, a selective and potent NK2 antagonist, demonstrates a greater efficacy in female patients with IBS with diarrhea (IBS-D). The present study used the TNBS-induced colitis model (in guinea pigs-GP) to investigate possible gender influences on NK2-related responses.
S-777
AGA Abstracts
AGA Abstracts
hyperalgesia to examine the relationship the microbiome and host gene expression. Methods: Male Wistar rats were exposed to 1 hour of water avoidance stress (WA) each day over a 10 day period (12 stressed and 12 controls). Visceral hypersensitivity was determined using colorectal distension and electromyographic recording. Animals were sacrificed at the end of the experimental protocol and colonic tissue was dissected out. The colonic epithelium was mechanically separated and DNA and RNA was isolated. Hypervariable bacterial 16S RNA regions were amplified (V3 and V4) and sequenced on an IonTorrent PGM. Host colonic epithelial mRNA (13 stressed and 12 controls) was hybridized to Affymetrix GeneChip® Rat Gene 2.0 ST Arrays for whole genome gene expression determination. Results: WA animals showed increased bacterial richness compared to healthy controls (HC). Proteobacteria and Actinobacteria were significantly enriched in WA animals. 2991 bacterial operational taxonomic units (OTUs) were significantly differentially expressed. Of these 61% were over expressed in WA animals. All differentially expressed OTUs belonging to the families Rikenellaceae and Porphyromonadaceae (20% of OTUs) were down regulated in WA animals. Almost all differentially expressed OTUs belonging to the families Weeksellaceae, S24-7, Comamonadaceae, Lachnospiraceae, Moraxellaceae and Pseudomonadaceae (39% of OTUs) were over expressed in WA animals. Using a novel analytical methodology (maximum parsimony analysis) 32 changes in gene expression which are implicated or associated with inflammation were identified. Genes that showed the most uniform change in expression included Anxa5, Csnk2a1, Ddx19a, Hnf1a, Hoxc4, Igfbp7, Kcnh2, Map6d1, RT-M6-2, Stard10 and Vtcn1. Discussion: Bacterial richness showed the opposite of what is generally observed in disease conditions. WA animals were characterized by bacterial over expression. Specifically, families which contain butyrate producing taxa are over expressed. Although butyrate is essential for colonic mucosal health it is known to cause concentration dependent colonic hypersensitivity in rats. The expression of the proinflammatory gene Vtcn1 can be modulated by butyrate suggesting a functional link with changes in microbial expression. Microbial dysbiosis in chronically stressed animals is associated with gene expression changes in the colonic mucosa which implicate inflammatory pathways. Possible relationships between the identified genes and specific bacterial groups and species remain to be further elucidated and offer a rich source of future research.
Mo2040 Experimentally Induced Visceral Pain, Variation in the Microbiome, and Host Gene Expression in Irritable Bowel Syndrome Nicolaas H. Fourie, Dan Wang, Sarah Abey, Eric Ferguson, LeeAnne Sherwin, Bridgett Rahim-Williams, Wendy A. Henderson Background: Abdominal pain is a principal symptom of irritable bowel syndrome (IBS). It is an inducible and measurable symptom which may be used to gain insights into the etiology of the syndrome. Subclinical inflammation is implicated in the etiology of IBS. Evidence suggests systemic inflammatory dysregulation which may be linked to dysregulation of the microbiome. We examined the relationship between severity of induced abdominal pain, variation in peripheral blood gene expression, and the oral microbiome in order to identify possible biological pathways underlying IBS and abdominal pain. Methods: A four probe sugar test solution was orally administered to 18 healthy volunteers (HV) and 16 IBS patients. Abdominal pain was assessed via self-report (100 point scale) using the Gastrointestinal Pain Pointer. RNA was isolated from whole blood. Amplified cRNA was hybridized to Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays for whole genome gene expression determination. DNA was isolated from buccal mucosa. The bacterial 16s RNA gene was amplified and hybridized to PhyloChip™ Version G3 arrays for microbiome characterization. Results: 81% of IBS patients and 11% of HV reported pain in response to the test solution. 258 bacteria were significantly correlated to pain severity. Negative correlations were dominated by Lachnospiraceae (25%). Positive correlations were dominated by Lachnospiraceae (18%), Prevotellaceae (12%) and Veillonellaceae (11%). 508 genes were correlated to pain. Pathways relating to stem cell pluripotency and inflammation were significantly represented. Seven bacterial families (Lachnospiraceae, Prevotellaceae, Rikenellaceae, Bacillaceae, Veillonellaceae, Moraxellaceae and Psuedomonodaceae) dominated correlations with pain correlated genes. Bacteria-gene expression correlation coefficients often exceeded 50%. Common molecular pathways included cytokine pathways, Rho signaling and the PI3K/AKT pathway. The expression of several known opportunistic pathogens associated with extra-oral inflammatory pathologies were significantly associated with inflammation associated genes and pain. Discussion: Over expression of Prevotellaceae and Veillonellaceae in IBS and inflammatory bowel disease have been previously described. Their positive relationship with abdominal pain and strong correlation (>50%) to inflammation associated genes suggest that they may be key drivers of systemic inflammation and gastrointestinal symptoms. Decreased expression of Lachnospiraceae with increasing pain severity may point to changing availability of butyrate, which is essential to epithelial health. Epithelial dysregulation facilitates translocation of pathogens and toxins. Bacteria-gene-symptom relationships suggest functional inflammatory associations, which remain to be further explored as diagnostic markers and targets for intervention.
Mo2043 Increased Pain Hypersensitivity in Women With Irritable Bowel Syndrome and Functional Dyspepsia Explained by Psychological Factors: A Prospective Birth Cohort Population Olli-Pekka Koivurova, Timo Blomster, Juha Auvinen, Sauli Herrala, Jaro Karppinen, Nicholas J. Talley, Jukka Ronkainen Background: The pathogenesis of irritable bowel syndrome (IBS) and functional dyspepsia (FD) is likely multifactorial. Altered serotonin metabolism and changes in pain signal processing in the central nervous system have been described. Psychological factors such as depression and anxiety are known to enhance pain sensation. However, whether peripheral pain sensitivity is altered in IBS and FD is controversial. We aimed to determine whether nociceptive pressure pain threshold (PPT) or tolerance (PPTo) is decreased in IBS and FD indicating pain hypersensitivity. Methods: The prospectively collected Northern Finland Birth Cohort 1966 (NFBC 1966) population (http://www.oulu.fi/nfbc/) is comprised of children from the two northernmost provinces of Finland, Oulu and Lapland with a date of birth Jan 1st Dec 31st, 1966 (12 231 children, 96.3% of all births during 1966 in that area). Between years 2013 and 2014, at the age of 47 years, a large health examination including both questionnaires (n=6868/10282, 67% response rate) and clinical examination (n=5851/10282, 57% response rate) was performed. For this study, 3969 subjects (44% male) were available. Uncomfortable pressure, i.e. PPT and intolerable pressure, i.e. PPTo were tested using an algometer (Somedic AB, Hörby, Sweden). The pressure was increased from 0 kPa at a constant rate of 50 kPa/s until the safety maximum of 1200 kPa. The measurements were done for four anatomical sites in the following order: 1) the upper trapezius muscle, 2) the tibialis anterior muscle, 3) the dorsal aspect of the wrist joint line, and 4) the L5/S1 interspinous space. IBS and FD were defined according to the Rome III definition. Anxiety
Mo2041 Effect of Ibodutant, a Neurokinin NK2 Receptor Antagonist, on GenderRelated Influences Following Induction of Trinitrobenzenesulfonic AcidInduced Colitis in the Guinea Pig Amanda S. Wolf-Johnston, Florenta A. Kullmann, Jonathan M. Beckel, Anthony J. Kanai, Sttefania Meini, Carlo A. Maggi, Lori A. Birder Introduction: Neurokinin NK2 receptors are expressed in the gastrointestinal tract of animals and humans and play a role in the regulation of motility and visceral sensitivity. Antagonists to NK2 receptors reduce hypersensitivity responses in animals following induction of colonic inflammation (TNBS). Taken together, available data suggest that a block of NK2 receptors could be a viable therapeutic option for treatment of patients with irritable bowel syndrome (IBS). In support is recent evidence in humans, which has shown that the NK2 antagonist, Ibodutant, a selective and potent NK2 antagonist, demonstrates a greater efficacy in female patients with IBS with diarrhea (IBS-D). The present study used the TNBS-induced colitis model (in guinea pigs-GP) to investigate possible gender influences on NK2-related responses.
S-777
AGA Abstracts
AGA Abstracts
hyperalgesia to examine the relationship the microbiome and host gene expression. Methods: Male Wistar rats were exposed to 1 hour of water avoidance stress (WA) each day over a 10 day period (12 stressed and 12 controls). Visceral hypersensitivity was determined using colorectal distension and electromyographic recording. Animals were sacrificed at the end of the experimental protocol and colonic tissue was dissected out. The colonic epithelium was mechanically separated and DNA and RNA was isolated. Hypervariable bacterial 16S RNA regions were amplified (V3 and V4) and sequenced on an IonTorrent PGM. Host colonic epithelial mRNA (13 stressed and 12 controls) was hybridized to Affymetrix GeneChip® Rat Gene 2.0 ST Arrays for whole genome gene expression determination. Results: WA animals showed increased bacterial richness compared to healthy controls (HC). Proteobacteria and Actinobacteria were significantly enriched in WA animals. 2991 bacterial operational taxonomic units (OTUs) were significantly differentially expressed. Of these 61% were over expressed in WA animals. All differentially expressed OTUs belonging to the families Rikenellaceae and Porphyromonadaceae (20% of OTUs) were down regulated in WA animals. Almost all differentially expressed OTUs belonging to the families Weeksellaceae, S24-7, Comamonadaceae, Lachnospiraceae, Moraxellaceae and Pseudomonadaceae (39% of OTUs) were over expressed in WA animals. Using a novel analytical methodology (maximum parsimony analysis) 32 changes in gene expression which are implicated or associated with inflammation were identified. Genes that showed the most uniform change in expression included Anxa5, Csnk2a1, Ddx19a, Hnf1a, Hoxc4, Igfbp7, Kcnh2, Map6d1, RT-M6-2, Stard10 and Vtcn1. Discussion: Bacterial richness showed the opposite of what is generally observed in disease conditions. WA animals were characterized by bacterial over expression. Specifically, families which contain butyrate producing taxa are over expressed. Although butyrate is essential for colonic mucosal health it is known to cause concentration dependent colonic hypersensitivity in rats. The expression of the proinflammatory gene Vtcn1 can be modulated by butyrate suggesting a functional link with changes in microbial expression. Microbial dysbiosis in chronically stressed animals is associated with gene expression changes in the colonic mucosa which implicate inflammatory pathways. Possible relationships between the identified genes and specific bacterial groups and species remain to be further elucidated and offer a rich source of future research.