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P070 Genetic polymorphisms of interleukin 15 (IL-15) in patients with ulcerative colitis
Basic Science
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which are major component proteins of tight junction are identified twenty four subtypes in human. However some previous reports show claudin-2 decrease epithelial barrier function in increasing paracellular permeability, and claudin-2 over-expression was implicated in exacerbation of active Crohn’s disease. On the other hand, it is becoming apparent that amino acids are not only a material of protein synthesis but also own some pharmacological activity by itself. Thus we sought to determine whether any amino acids modulate barrier function and claudin expressions in colonic epithelial cell monolayers. Methods: T84 cells which are human colonic epithelial cells were cultured as confluent monolayer models for a week in insert well of transwell assay. The epithelial barrier function of the cells was determined by monitoring transepithelial electrical resistance (TER) with administration of pro-inflammatory cytokines and amino acids into basal surface and apical surface of the well respectively. Furthermore protein assay was performed by Western blotting assay. Results: TER of the monolayers was significantly reduced when incubated with 10 ng/ml of IL-6 (533±42 W·cm2 ) compared with that of untreated monolayers (1,074±54 W·cm2 ). Further, the monolayers were incubated with 2 mM of 20 kinds of each amino acid (pH = 7.4) with or without IL-6. Notably, treatment with glycine and glutamine significantly increased TER of the IL-6-untreated monolayers (1,205±118, 1,151±113 W·cm2 , respectively), whereas other amino acids had minimum effects. Moreover, glycine and glutamine neutralized IL-6-induced decrease in TER (1,054±103, 924±91 W·cm2 , respectively). The protein level of claudin-2 that deteriorates barrier function was selectively and significantly increased when the cells were incubated with IL-6 (829±161%) compared with that of the untreated monolayers (100%). Intriguingly, treatment with glycine and glutamine selectively and remarkably downregulated IL-6-induced claudin-2 protein expressions (83±8.8%, 60±1.4%, respectively). Conclusions: This study demonstrated a novel physiological effect of glycine and glutamine, both of which enhanced barrier function of human colonic epithelial cell monolayers by a selective down-regulation of claudin-2 proteins.
polymorphisms and haplotype reconstruction were performed with Haploview version 3:32 (Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA). Results: UC patients and healthy controls showed similar distribution of the rs3806798, rs10833, rs4956403, rs2857261, rs10519613, and rs1057972 polymorphisms. A moderate increased frequency of the rs2254514 CC genotype was observed in UC patients when compared to healthy controls (p = 0.04, OR = 1.62) and was associated with young age at diagnosis <40 years (p = 0.03, OR = 3.67). Five polymorphisms (rs1051613, rs2254514, rs2857261, rs1057972, and rs10833) were in strong linkage disequilibrium and were included in six haplotypes: H1 (ACAAC), H2 (CCGTC), H3 (CTAAT), H4 (CCAAT), H5 (CTAAC) and H6 (CCAAC). UC patients showed an increased frequency of the H6 haplotype (P = 0.005, OR = 3.2) and a decreased frequency of the H5 haplotype (P = 0.031, OR = 0.40). In the subgroup analysis, the rs2254514 CC genotype was associated with young age at diagnosis <40 years (p = 0.03, pC=0.05, OR = 3.67 95% CI: 1.13 15.9). Conclusions: These results suggest that IL-15 rs2254514 polymorphism might have an important role in the development of UC. In our study was possible to distinguish one risk and one protective uncommon haplotypes for developing UC.
P070 Genetic polymorphisms of interleukin 15 (IL-15) in patients with ulcerative colitis J. Yamamoto-Furusho1 *, J. De-Leon-Rendon1 , G. VargasAlarcon2 . 1 IBD Clinic, Instituto Nacional de Ciencias Medicas y Nutricion, Gastroenterology, Mexico, Mexico, 2 Instituto Nacional de Cardiologia, Molecular Biology, Mexico
Background: Gelenterum, a gelatin powder containing Tannic Acids, is used for diarrhea in children. Few information exist on its mechanisms of action, involving gel formation and bacterial toxin sequestration. Aim of this study was to evaluate the effect and mechanisms of action of Gelenterum in the murine model of acute colitis by DSS. Methods: C57BL/6 mice receiving 2.5% DSS in drinking water ad libitum for 8 days, at day 4, 5, 6 and 7, were treated with gelenterum 1 mg or 10 mg in 200 ml of tap water, or saline solution, given orally by gavage starting day 3. Body weight, occult blood test and stool consistency were measured every other day to calculate Disease Activity Index (DAI), as assessment of severity of colitis. Mice were sacrificed at day 9, serum samples were collected, colon length measured for histology assessment by Rachmilewitz score. To characterize gut microbiota modulation, stools were collected at day 0, 5 and 9 and cultured in selective media. RT-PCR was also performed on fecal as well as on intestinal samples. Colonic mucus layer were analysed at confocal microscopy at 2 photon and atomic force microscope. Finally, LPS and peptidoglycan were evaluated by ELISA test in peripheral blood. Results: Gelenterum reduced DAI and body weight loss in treated mice, being 10 mg more efficacious than 1 mg dose. Gelenterum treated mice displayed a longer colon. In fecal culture, Acinetobacteria, Enterobacteria, Enterococci and Lactobacilli grew from stool as well as from intestinal culture. Treated mice showed a lower concentration of Enterobacteria and Enterococci, and higher concentration of lactobacilli. At confocal microscopy, intestinal samples from healthy and
Background: Interleukin 15 (IL-15) is a Th1-related cytokine that triggers inflammatory cell recruitment with implications for pathogenesis in ulcerative colitis. The interleukin 15 gene is located within a 35 kb region of q28 31 locus of chromosome 4. No previous studies have reported this novel association between UC and IL-15 polymorphisms. In the present work, the role of IL-15 gene polymorphisms as susceptibility markers for UC was evaluated. Methods: Seven polymorphisms of IL-15 (rs3806798, rs10833, rs4956403, rs2254514, rs2857261, rs10519613, and rs1057972) were genotyped by 5’ exonuclease TaqMan genotyping assays in a group of 199 Mexican patients with UC and 698 Mexican Mestizo healthy unrelated individuals. Polymorphisms (rs3806798, rs10833, rs4956403, rs2254514, rs2857261, rs10519613, and rs1057972) were genotyped using 5’ exonuclease TaqMan genotyping assays on an ABI Prism 7900 HT Fast Real-Time PCR System. Gene frequencies of IL-15 polymorphisms on patients and controls were obtained by direct counting. The Hardy-Weinberg equilibrium was evaluated by chi-square test. Statistical significance was considered as p < 0.05. Pair wise linkage disequilibrium (LD, D’) estimations between
P071 Gelenterum ameliorates murine colitis while modulating gut microbiota and intestinal mucus layer F. Scaldaferri1 , L.R. Lopetuso1 *, V. Petito1 , V. Gerardi1 , M. Bilotta2 , A. Poscia3 , M. Papi4 , G. Maolucci5 , V. Cufino6 , E. Stigliano6 , V. Arena6 , G. Delogu2 , M. Sanguinetti2 , M. De Spirito4 , A. Sgambato6 , A. Gasbarrini1 . 1 Catholic University of Sacred Hearth, Internal Medicine, Gastroenterology Division, Rome, Italy, 2 Catholic University of Sacred Hearth, Istitute of Microbiology, Rome, Italy, 3 Catholic University of Sacred Hearth, Insitute of Hygiene, Rome, Italy, 4 Catholic University of Sacred Hearth, Istitute of Phisics, Rome, Italy, 5 Catholic University of Sacred Hearth, Institute of Phisics, Rome, Italy, 6 Catholic University of Sacred Hearth, Institute of Pathology, Rome, Italy
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which are major component proteins of tight junction are identified twenty four subtypes in human. However some previous reports show claudin-2 decrease epithelial barrier function in increasing paracellular permeability, and claudin-2 over-expression was implicated in exacerbation of active Crohn’s disease. On the other hand, it is becoming apparent that amino acids are not only a material of protein synthesis but also own some pharmacological activity by itself. Thus we sought to determine whether any amino acids modulate barrier function and claudin expressions in colonic epithelial cell monolayers. Methods: T84 cells which are human colonic epithelial cells were cultured as confluent monolayer models for a week in insert well of transwell assay. The epithelial barrier function of the cells was determined by monitoring transepithelial electrical resistance (TER) with administration of pro-inflammatory cytokines and amino acids into basal surface and apical surface of the well respectively. Furthermore protein assay was performed by Western blotting assay. Results: TER of the monolayers was significantly reduced when incubated with 10 ng/ml of IL-6 (533±42 W·cm2 ) compared with that of untreated monolayers (1,074±54 W·cm2 ). Further, the monolayers were incubated with 2 mM of 20 kinds of each amino acid (pH = 7.4) with or without IL-6. Notably, treatment with glycine and glutamine significantly increased TER of the IL-6-untreated monolayers (1,205±118, 1,151±113 W·cm2 , respectively), whereas other amino acids had minimum effects. Moreover, glycine and glutamine neutralized IL-6-induced decrease in TER (1,054±103, 924±91 W·cm2 , respectively). The protein level of claudin-2 that deteriorates barrier function was selectively and significantly increased when the cells were incubated with IL-6 (829±161%) compared with that of the untreated monolayers (100%). Intriguingly, treatment with glycine and glutamine selectively and remarkably downregulated IL-6-induced claudin-2 protein expressions (83±8.8%, 60±1.4%, respectively). Conclusions: This study demonstrated a novel physiological effect of glycine and glutamine, both of which enhanced barrier function of human colonic epithelial cell monolayers by a selective down-regulation of claudin-2 proteins.
polymorphisms and haplotype reconstruction were performed with Haploview version 3:32 (Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA). Results: UC patients and healthy controls showed similar distribution of the rs3806798, rs10833, rs4956403, rs2857261, rs10519613, and rs1057972 polymorphisms. A moderate increased frequency of the rs2254514 CC genotype was observed in UC patients when compared to healthy controls (p = 0.04, OR = 1.62) and was associated with young age at diagnosis <40 years (p = 0.03, OR = 3.67). Five polymorphisms (rs1051613, rs2254514, rs2857261, rs1057972, and rs10833) were in strong linkage disequilibrium and were included in six haplotypes: H1 (ACAAC), H2 (CCGTC), H3 (CTAAT), H4 (CCAAT), H5 (CTAAC) and H6 (CCAAC). UC patients showed an increased frequency of the H6 haplotype (P = 0.005, OR = 3.2) and a decreased frequency of the H5 haplotype (P = 0.031, OR = 0.40). In the subgroup analysis, the rs2254514 CC genotype was associated with young age at diagnosis <40 years (p = 0.03, pC=0.05, OR = 3.67 95% CI: 1.13 15.9). Conclusions: These results suggest that IL-15 rs2254514 polymorphism might have an important role in the development of UC. In our study was possible to distinguish one risk and one protective uncommon haplotypes for developing UC.
P070 Genetic polymorphisms of interleukin 15 (IL-15) in patients with ulcerative colitis J. Yamamoto-Furusho1 *, J. De-Leon-Rendon1 , G. VargasAlarcon2 . 1 IBD Clinic, Instituto Nacional de Ciencias Medicas y Nutricion, Gastroenterology, Mexico, Mexico, 2 Instituto Nacional de Cardiologia, Molecular Biology, Mexico
Background: Gelenterum, a gelatin powder containing Tannic Acids, is used for diarrhea in children. Few information exist on its mechanisms of action, involving gel formation and bacterial toxin sequestration. Aim of this study was to evaluate the effect and mechanisms of action of Gelenterum in the murine model of acute colitis by DSS. Methods: C57BL/6 mice receiving 2.5% DSS in drinking water ad libitum for 8 days, at day 4, 5, 6 and 7, were treated with gelenterum 1 mg or 10 mg in 200 ml of tap water, or saline solution, given orally by gavage starting day 3. Body weight, occult blood test and stool consistency were measured every other day to calculate Disease Activity Index (DAI), as assessment of severity of colitis. Mice were sacrificed at day 9, serum samples were collected, colon length measured for histology assessment by Rachmilewitz score. To characterize gut microbiota modulation, stools were collected at day 0, 5 and 9 and cultured in selective media. RT-PCR was also performed on fecal as well as on intestinal samples. Colonic mucus layer were analysed at confocal microscopy at 2 photon and atomic force microscope. Finally, LPS and peptidoglycan were evaluated by ELISA test in peripheral blood. Results: Gelenterum reduced DAI and body weight loss in treated mice, being 10 mg more efficacious than 1 mg dose. Gelenterum treated mice displayed a longer colon. In fecal culture, Acinetobacteria, Enterobacteria, Enterococci and Lactobacilli grew from stool as well as from intestinal culture. Treated mice showed a lower concentration of Enterobacteria and Enterococci, and higher concentration of lactobacilli. At confocal microscopy, intestinal samples from healthy and
Background: Interleukin 15 (IL-15) is a Th1-related cytokine that triggers inflammatory cell recruitment with implications for pathogenesis in ulcerative colitis. The interleukin 15 gene is located within a 35 kb region of q28 31 locus of chromosome 4. No previous studies have reported this novel association between UC and IL-15 polymorphisms. In the present work, the role of IL-15 gene polymorphisms as susceptibility markers for UC was evaluated. Methods: Seven polymorphisms of IL-15 (rs3806798, rs10833, rs4956403, rs2254514, rs2857261, rs10519613, and rs1057972) were genotyped by 5’ exonuclease TaqMan genotyping assays in a group of 199 Mexican patients with UC and 698 Mexican Mestizo healthy unrelated individuals. Polymorphisms (rs3806798, rs10833, rs4956403, rs2254514, rs2857261, rs10519613, and rs1057972) were genotyped using 5’ exonuclease TaqMan genotyping assays on an ABI Prism 7900 HT Fast Real-Time PCR System. Gene frequencies of IL-15 polymorphisms on patients and controls were obtained by direct counting. The Hardy-Weinberg equilibrium was evaluated by chi-square test. Statistical significance was considered as p < 0.05. Pair wise linkage disequilibrium (LD, D’) estimations between
P071 Gelenterum ameliorates murine colitis while modulating gut microbiota and intestinal mucus layer F. Scaldaferri1 , L.R. Lopetuso1 *, V. Petito1 , V. Gerardi1 , M. Bilotta2 , A. Poscia3 , M. Papi4 , G. Maolucci5 , V. Cufino6 , E. Stigliano6 , V. Arena6 , G. Delogu2 , M. Sanguinetti2 , M. De Spirito4 , A. Sgambato6 , A. Gasbarrini1 . 1 Catholic University of Sacred Hearth, Internal Medicine, Gastroenterology Division, Rome, Italy, 2 Catholic University of Sacred Hearth, Istitute of Microbiology, Rome, Italy, 3 Catholic University of Sacred Hearth, Insitute of Hygiene, Rome, Italy, 4 Catholic University of Sacred Hearth, Istitute of Phisics, Rome, Italy, 5 Catholic University of Sacred Hearth, Institute of Phisics, Rome, Italy, 6 Catholic University of Sacred Hearth, Institute of Pathology, Rome, Italy