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Gene Expression Profiling Identifies CDKN2B-AS1 as a Long Non-Coding RNA Associated with IBD and Regulated by TGF-Beta
AGA Abstracts
colonic NCM460-NTR1 cells (Bakirtzi et al., Gastroenterology, 2011). In addition, these lncRNAs also regulate MAPK and AKT pathways, representing established pathways of NT/ NTR1 signaling associated with the pathophysiology of NT-regulated colitis. Summary and Conclusions: Expression of the human lncRNAs UCA1 and CCAT1 are upregulated by NT/ NTR1 activation in human colonocytes and overexpressed in the colonic mucosa from UC patients. This is the first study of lncRNA expression in human colonic epithelial cells regulated by NT/NTR1 signaling, suggesting potential lncRNA-miR axes involved in IBD pathophysiology. Supported by NIH grant DK60729 (CP), and The Crohn's & Colitis Foundation of America (IKML).
dysregulated in colitis such as the formation of a barrier, proliferation and apoptosis. Supported by NIH grant DK60729 (CP) and T32 DK07180-41 (DP)
660 REVERSAL OF A VIRTUAL LESION IN HEALTHY HUMAN PHARYNGEAL MOTOR CORTEX BY HIGH-FREQUENCY RTMS OVER THE CEREBELLUM Masahiro Watanabe, Emilia Michou, Alicja Raginis-Zborowska, Jin Magara, Makoto Inoue, Shaheen Hamdy Introduction Previous reports have revealed that excitation of human pharyngeal motor cortex can be induced by repetitive high frequency (10Hz) transcranial magnetic stimulation (rTMS) applied to the cerebellum. In order to further assess its therapeutic potential in neurologic dysphagia, we explored whether cerebellar rTMS can reverse cortical suppression of the healthy human pharyngeal motor system induced by a virtual lesion as a prelude to treatment. Materials & Methods Fifteen healthy volunteers (7 males, age range 19-49 yrs) were intubated with an intraluminal catheter to record pharyngeal electromyography (EMG). Each participant underwent baseline cortical and cerebellar excitability measurements assessed through motor evoked potentials (MEP) at three cortical sites (dominant and nondominant pharyngeal cortices and hand motor cortex) and in each cerebellar hemisphere. Thereafter, each subject received 1 Hz rTMS (for 10 minutes at an intensity of 120% of resting motor threshold) to dominant pharyngeal motor cortex to transiently suppress cortical swallowing activity (the virtual lesion). After cortical suppression, subjects were randomized to receive one of three cerebellar 10Hz rTMS interventions (ipsilesional cerebellar hemisphere, contralesional cerebellar hemisphere and ipsilesional sham). Post-intervention, cortical (pharynx and hand) and cerebellar MEPs were re-measured for up to 60 minutes and compared to sham using repeated measures and 1-way ANOVA. Results As the pattern of excitability for both cortical hemispheres was similar, data from the 2 pharyngeal hot spots were combined and compared to sham. Post hoc 1-way ANOVAs comparing each treatment to sham confirmed that rTMS to either the ipsilesional or contralesional cerebellar hemisphere significantly increased excitability, so reversing the virtual lesion effect, which lasted up to 60 minutes ( P<0.05) , Figure 1. Cerebellar excitability itself was unchanged. Conclusions Ten Hz rTMS to either cerebellar hemisphere was able to bilaterally reverse the inhibition provoked in pharyngeal motor cortex after a virtual lesion. Our data suggest that 10 Hz cerebellar rTMS may have a therapeutic role in recovery from dysphagia with potential brain targeting advantages over other neurostimulation techniques.
658 A DOWN REGULATION OF SEMAPHORIN3E (SEMA3E) IN ACTIVE ULCERATIVE COLITIS AND EXPERIMENTAL COLITIS IS ASSOCIATED TO AN ALTERATION OF SPLENIC CD11C+ T-CELL PRIMING Laëtitia Kermarrec, Nour Eissa, Charles N. Bernstein, Jean-Eric Ghia Introduction: Inflammatory bowel diseases (IBD) involve an increase of dendritic cells (DC) infiltration and cytokines production. Recently, semaphorins (Sema) has emerged as an essential axis in DC immune responses. Previously, using an acute model of injury repair, we demonstrated that Sema3E regulates the intestinal inflammation via the modulation of DC-IL-12p40 release. This study aims to determine the role of Sema3E on colitis and on DC regulation and T-cell priming. Methodology: mRNA expression level of Sema3E was determined in human rectal biopsies collected from healthy (n=7) and active UC patients (n=7) using RT-qPCR, and an absolute correlation analysis was conducted against pro- and anti-inflammatory markers. Colitis was induced by dextran sulfate sodium (DSS 5%) for 5 days in Sema3E-/- and WT mice treated or not with recombinant Sema3E-Fc. Disease activity index (DAI), macro- and microscopic scores were determined. Colonic, Sema3E, INF-γ, TNF-α, IL-4, IL-17, IL-23, IL-12p70 and IL-12p40 were quantified using ELISA. IL-12p70, p40 and IL-23 levels in isolated splenic DCs were determined in the presence of absence of Nk-B inhibitor (BAY 11-7082) or activator (betulinic acid). IFN-γ, IL-17 levels from DC/ CD4+CD25- T cell co-culture were determined in the presence or absence of anti p19-mAb, p35-mAb or IL-12p70 or IL-23 recombinant proteins. Results: In active UC biopsies, Sema3E mRNA was decreased and levels of Sema3E were negatively and positively correlated with pro- and anti-inflammatory cytokines respectively. In colitic WT mice, Sema3E level was significantly decreased. In colitic Sema3E-/- mice, clinical score, IL-17, IL-12p40, p70, IL23, IFN-γ were significantly increased compared to WT with no effect on IL-4. Colitic SemaE-/- splenic CD11C+ cells showed an increased production of IL-12p70, p40 and IL23, with no effect on CD11C+ proliferation and IL-4; BAY 11-7082 abrogated the increase. Moreover, addition of rSema3E-FC to the culture medium decrease the three markers, and betulinic acid partially revered the effect. Colitic SemaE-/- splenic CD11C+ cells showed a higher effective T-cell priming reflected by an increase of IFN-γ and IL-17 release from CD4+CD25- T cells. Addition of the anti p19-mAb in the medium abolished the deleterious effect on IL-17 in CD4+CD25- T cells conditioned with colitic SemaE-/- splenic CD11C+ cells, whereas, in the presence of anti p35-mAb only IFN-g levels were affected. Finally, in vivo treatment with rSema3E-Fc reversed all the effects studied with no effect on IL-4. Conclusions: Suppression of splenic immune cell activation and altered interaction between DCs and T cells are important aspects of the deleterious effect of the lack of Sem3E in the context of acute colitis. These findings may lead to improved therapeutic strategies in the treatment of IBD by restoring Sema3E level in IBD patients.
659 GENE EXPRESSION PROFILING IDENTIFIES CDKN2B-AS1 AS A LONG NON-CODING RNA ASSOCIATED WITH IBD AND REGULATED BY TGFBETA Carl R. Rankin, Dimitrios Iliopoulos, Charalabos Pothoulakis, David M. Padua
661 EFFECT OF AGING ON UES PRESSURE RESPONSE TO SLOW AND ULTRA-SLOW SIMULATED REFLUX EVENTS: NOT ALL IS LOST WITH AGING Ling Mei, Arshish Dua, Mark Kern, Siyuan Gao, Francis O. Edeani, Kulwinder S. Dua, Amy Wilson, Patrick Sanvanson, Sudarshan Jadcherla, Reza Shaker
Background: Inflammatory bowel disease (IBD) consists of Crohn's disease (CD) and ulcerative colitis (UC). The etiology of IBD continues to be an area of active research. New therapies and new modes of understanding IBD pathogenesis are needed to alleviate this disease burden. Recent research is assessing aspects of gene regulation from previously uninvestigated portions of the genome, namely long non-coding RNAs (lncRNAs). These non-coding transcripts have been implicated in a variety of cellular processes including cell growth, differentiation and embryonic development however their role in inflammatory bowel disease is only now being uncovered. Methods: Gene expression profiling of 8 UC and 7 control samples were profiled for lncRNA expression using Arraystar lncRNA arrays (Padua et al., Am J Physiol Gastrointest Liver Physiol, 2016). A validation cohort of 16 control and 15 UC patient colonic samples were used for qRT-PCR gene expression analysis. The human colon cell lines NCM460, Hct116, and DLD-1 were assayed for lncRNA expression using qRT-PCR. Cells were treated with TGF-beta, TNF-alpha, and IL-1b and assayed for gene expression. In silico analysis was performed on the promoter to determine relevant transcription factors that may regulate CDKN2B-AS1 expression. Results: The lncRNA, CDKN2B-AS1, was significantly downregulated by 12-fold (p-value: 0.0009) in UC and by 4.9 fold (p-value: 0.041) in CD patient samples when compared to controls. A separate clinical validation cohort confirmed these expression changes in UC (fold change 8.24, p-value: 0.034). We observed that CDKN2B-AS1 was expressed in multiple colonic epithelial cell lines including NCM460, HCT116, and DLD-1 cells. In silico analysis of the promoter identified N-myc as a possible regulatory transcription factor and found it to positively correlate with CDKN2B-AS1 expression in IBD patient samples (R2: 0.691, p-value: 0.004304). We tested the expression of NMyc in colonic epithelial cell lines and observed a positive correlation of N-Myc expression with CDKN2B-AS1. In addition, NCM460 cells were treated with TNF-alpha and IL1-beta and found not to influence the levels of CDKN2B-AS1. When TGF-beta was added, CDKN2BAS1 was significantly downregulated by 2-fold (p-value: 0.0289). Analyzing the clinical data found that CDKN2B-AS1 was inversely correlated with TGF-beta1 expression in human samples (R2: 0.573, p-value: 0.0255). Conclusions: LncRNA biology has been implicated in multiple cellular processes including inflammatory diseases. Through this study, a novel lncRNA was found to be associated with IBD pathogenesis. Further studies are needed to identify the influence of CDKN2B-AS1 on colonic epithelial cell functions that could be
AGA Abstracts
Background: Upper esophageal sphincter (UES) and its reflex responses to reflux events constitute one of the major mechanisms preventing pharyngeal reflux and aspiration. Clinically, gastroesophageal reflux events may occur with various rates, volumes and different chemical compositions. Since prior studies have documented deterioration of a number of airway protective reflexes in the elderly, we hypothesized that aging is associated with deterioration of UES response to low threshold stimuli by slow occurring fluid reflux events. Aim: To determine the effect of aging on the UES and esophageal body pressure responses to well-defined slow and ultra-slow occurring simulated acid and non-acid reflux events. Methods: We studied 11 elderly (74±9yr) and 10 young (28±7yr) healthy volunteers in supine position using a concurrent high-resolution manometry, impedance and infusion techniques. We tested two types of perfusates: 0.1N HCl (pH 1.2-1.4) and half saline (pH 5.8), two perfusion rates X3 (slow: 1ml/s and ultra-slow: 0.05ml/s) as well as two time intervals (active perfusion time of 60 sec followed by passive dwell time of 60 sec). Subjects refrained from swallowing for these periods. Ten-second time epochs of UES High-Pressure Zone Contractile Integrals (UESHPZ CI) were measured 30 seconds prior to as well as during perfusion and during 60 sec after perfusion. Repeated Measures ANOVA, Fisher Exact or c2 test were used for comparisons. Results: Both young and elderly showed significant increase in UES pressure represented by UESHPZ CI during the entire period of active infusion at the rate of 1ml/s as well as during the entire passive dwell interval compared to baseline (p<0.01, fig 1). For the 0.05ml/s infusions, significant increase of UESHPZ Cl was seen in the young group beginning at the late infusion period and into the dwell interval (p<0.01, fig 1). In contrast, the elderly showed no appreciable UES effect during or after the 0.05ml/s infusion either for acid or saline (fig 1). In the elderly, the average UESHPZ CI across the active 0.05ml/s infusion interval and during the dwell interval was similar to baseline. Importantly, there was no difference in UES contractile response to acid infusion and half saline infusion in both groups (fig 2). There was no significant difference between young and elderly in the number of infusions resulting in secondary peristalsis. There were more secondary peristalsis during 1ml/s active infusions than dwell intervals (p<0.05).
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colonic NCM460-NTR1 cells (Bakirtzi et al., Gastroenterology, 2011). In addition, these lncRNAs also regulate MAPK and AKT pathways, representing established pathways of NT/ NTR1 signaling associated with the pathophysiology of NT-regulated colitis. Summary and Conclusions: Expression of the human lncRNAs UCA1 and CCAT1 are upregulated by NT/ NTR1 activation in human colonocytes and overexpressed in the colonic mucosa from UC patients. This is the first study of lncRNA expression in human colonic epithelial cells regulated by NT/NTR1 signaling, suggesting potential lncRNA-miR axes involved in IBD pathophysiology. Supported by NIH grant DK60729 (CP), and The Crohn's & Colitis Foundation of America (IKML).
dysregulated in colitis such as the formation of a barrier, proliferation and apoptosis. Supported by NIH grant DK60729 (CP) and T32 DK07180-41 (DP)
660 REVERSAL OF A VIRTUAL LESION IN HEALTHY HUMAN PHARYNGEAL MOTOR CORTEX BY HIGH-FREQUENCY RTMS OVER THE CEREBELLUM Masahiro Watanabe, Emilia Michou, Alicja Raginis-Zborowska, Jin Magara, Makoto Inoue, Shaheen Hamdy Introduction Previous reports have revealed that excitation of human pharyngeal motor cortex can be induced by repetitive high frequency (10Hz) transcranial magnetic stimulation (rTMS) applied to the cerebellum. In order to further assess its therapeutic potential in neurologic dysphagia, we explored whether cerebellar rTMS can reverse cortical suppression of the healthy human pharyngeal motor system induced by a virtual lesion as a prelude to treatment. Materials & Methods Fifteen healthy volunteers (7 males, age range 19-49 yrs) were intubated with an intraluminal catheter to record pharyngeal electromyography (EMG). Each participant underwent baseline cortical and cerebellar excitability measurements assessed through motor evoked potentials (MEP) at three cortical sites (dominant and nondominant pharyngeal cortices and hand motor cortex) and in each cerebellar hemisphere. Thereafter, each subject received 1 Hz rTMS (for 10 minutes at an intensity of 120% of resting motor threshold) to dominant pharyngeal motor cortex to transiently suppress cortical swallowing activity (the virtual lesion). After cortical suppression, subjects were randomized to receive one of three cerebellar 10Hz rTMS interventions (ipsilesional cerebellar hemisphere, contralesional cerebellar hemisphere and ipsilesional sham). Post-intervention, cortical (pharynx and hand) and cerebellar MEPs were re-measured for up to 60 minutes and compared to sham using repeated measures and 1-way ANOVA. Results As the pattern of excitability for both cortical hemispheres was similar, data from the 2 pharyngeal hot spots were combined and compared to sham. Post hoc 1-way ANOVAs comparing each treatment to sham confirmed that rTMS to either the ipsilesional or contralesional cerebellar hemisphere significantly increased excitability, so reversing the virtual lesion effect, which lasted up to 60 minutes ( P<0.05) , Figure 1. Cerebellar excitability itself was unchanged. Conclusions Ten Hz rTMS to either cerebellar hemisphere was able to bilaterally reverse the inhibition provoked in pharyngeal motor cortex after a virtual lesion. Our data suggest that 10 Hz cerebellar rTMS may have a therapeutic role in recovery from dysphagia with potential brain targeting advantages over other neurostimulation techniques.
658 A DOWN REGULATION OF SEMAPHORIN3E (SEMA3E) IN ACTIVE ULCERATIVE COLITIS AND EXPERIMENTAL COLITIS IS ASSOCIATED TO AN ALTERATION OF SPLENIC CD11C+ T-CELL PRIMING Laëtitia Kermarrec, Nour Eissa, Charles N. Bernstein, Jean-Eric Ghia Introduction: Inflammatory bowel diseases (IBD) involve an increase of dendritic cells (DC) infiltration and cytokines production. Recently, semaphorins (Sema) has emerged as an essential axis in DC immune responses. Previously, using an acute model of injury repair, we demonstrated that Sema3E regulates the intestinal inflammation via the modulation of DC-IL-12p40 release. This study aims to determine the role of Sema3E on colitis and on DC regulation and T-cell priming. Methodology: mRNA expression level of Sema3E was determined in human rectal biopsies collected from healthy (n=7) and active UC patients (n=7) using RT-qPCR, and an absolute correlation analysis was conducted against pro- and anti-inflammatory markers. Colitis was induced by dextran sulfate sodium (DSS 5%) for 5 days in Sema3E-/- and WT mice treated or not with recombinant Sema3E-Fc. Disease activity index (DAI), macro- and microscopic scores were determined. Colonic, Sema3E, INF-γ, TNF-α, IL-4, IL-17, IL-23, IL-12p70 and IL-12p40 were quantified using ELISA. IL-12p70, p40 and IL-23 levels in isolated splenic DCs were determined in the presence of absence of Nk-B inhibitor (BAY 11-7082) or activator (betulinic acid). IFN-γ, IL-17 levels from DC/ CD4+CD25- T cell co-culture were determined in the presence or absence of anti p19-mAb, p35-mAb or IL-12p70 or IL-23 recombinant proteins. Results: In active UC biopsies, Sema3E mRNA was decreased and levels of Sema3E were negatively and positively correlated with pro- and anti-inflammatory cytokines respectively. In colitic WT mice, Sema3E level was significantly decreased. In colitic Sema3E-/- mice, clinical score, IL-17, IL-12p40, p70, IL23, IFN-γ were significantly increased compared to WT with no effect on IL-4. Colitic SemaE-/- splenic CD11C+ cells showed an increased production of IL-12p70, p40 and IL23, with no effect on CD11C+ proliferation and IL-4; BAY 11-7082 abrogated the increase. Moreover, addition of rSema3E-FC to the culture medium decrease the three markers, and betulinic acid partially revered the effect. Colitic SemaE-/- splenic CD11C+ cells showed a higher effective T-cell priming reflected by an increase of IFN-γ and IL-17 release from CD4+CD25- T cells. Addition of the anti p19-mAb in the medium abolished the deleterious effect on IL-17 in CD4+CD25- T cells conditioned with colitic SemaE-/- splenic CD11C+ cells, whereas, in the presence of anti p35-mAb only IFN-g levels were affected. Finally, in vivo treatment with rSema3E-Fc reversed all the effects studied with no effect on IL-4. Conclusions: Suppression of splenic immune cell activation and altered interaction between DCs and T cells are important aspects of the deleterious effect of the lack of Sem3E in the context of acute colitis. These findings may lead to improved therapeutic strategies in the treatment of IBD by restoring Sema3E level in IBD patients.
659 GENE EXPRESSION PROFILING IDENTIFIES CDKN2B-AS1 AS A LONG NON-CODING RNA ASSOCIATED WITH IBD AND REGULATED BY TGFBETA Carl R. Rankin, Dimitrios Iliopoulos, Charalabos Pothoulakis, David M. Padua
661 EFFECT OF AGING ON UES PRESSURE RESPONSE TO SLOW AND ULTRA-SLOW SIMULATED REFLUX EVENTS: NOT ALL IS LOST WITH AGING Ling Mei, Arshish Dua, Mark Kern, Siyuan Gao, Francis O. Edeani, Kulwinder S. Dua, Amy Wilson, Patrick Sanvanson, Sudarshan Jadcherla, Reza Shaker
Background: Inflammatory bowel disease (IBD) consists of Crohn's disease (CD) and ulcerative colitis (UC). The etiology of IBD continues to be an area of active research. New therapies and new modes of understanding IBD pathogenesis are needed to alleviate this disease burden. Recent research is assessing aspects of gene regulation from previously uninvestigated portions of the genome, namely long non-coding RNAs (lncRNAs). These non-coding transcripts have been implicated in a variety of cellular processes including cell growth, differentiation and embryonic development however their role in inflammatory bowel disease is only now being uncovered. Methods: Gene expression profiling of 8 UC and 7 control samples were profiled for lncRNA expression using Arraystar lncRNA arrays (Padua et al., Am J Physiol Gastrointest Liver Physiol, 2016). A validation cohort of 16 control and 15 UC patient colonic samples were used for qRT-PCR gene expression analysis. The human colon cell lines NCM460, Hct116, and DLD-1 were assayed for lncRNA expression using qRT-PCR. Cells were treated with TGF-beta, TNF-alpha, and IL-1b and assayed for gene expression. In silico analysis was performed on the promoter to determine relevant transcription factors that may regulate CDKN2B-AS1 expression. Results: The lncRNA, CDKN2B-AS1, was significantly downregulated by 12-fold (p-value: 0.0009) in UC and by 4.9 fold (p-value: 0.041) in CD patient samples when compared to controls. A separate clinical validation cohort confirmed these expression changes in UC (fold change 8.24, p-value: 0.034). We observed that CDKN2B-AS1 was expressed in multiple colonic epithelial cell lines including NCM460, HCT116, and DLD-1 cells. In silico analysis of the promoter identified N-myc as a possible regulatory transcription factor and found it to positively correlate with CDKN2B-AS1 expression in IBD patient samples (R2: 0.691, p-value: 0.004304). We tested the expression of NMyc in colonic epithelial cell lines and observed a positive correlation of N-Myc expression with CDKN2B-AS1. In addition, NCM460 cells were treated with TNF-alpha and IL1-beta and found not to influence the levels of CDKN2B-AS1. When TGF-beta was added, CDKN2BAS1 was significantly downregulated by 2-fold (p-value: 0.0289). Analyzing the clinical data found that CDKN2B-AS1 was inversely correlated with TGF-beta1 expression in human samples (R2: 0.573, p-value: 0.0255). Conclusions: LncRNA biology has been implicated in multiple cellular processes including inflammatory diseases. Through this study, a novel lncRNA was found to be associated with IBD pathogenesis. Further studies are needed to identify the influence of CDKN2B-AS1 on colonic epithelial cell functions that could be
AGA Abstracts
Background: Upper esophageal sphincter (UES) and its reflex responses to reflux events constitute one of the major mechanisms preventing pharyngeal reflux and aspiration. Clinically, gastroesophageal reflux events may occur with various rates, volumes and different chemical compositions. Since prior studies have documented deterioration of a number of airway protective reflexes in the elderly, we hypothesized that aging is associated with deterioration of UES response to low threshold stimuli by slow occurring fluid reflux events. Aim: To determine the effect of aging on the UES and esophageal body pressure responses to well-defined slow and ultra-slow occurring simulated acid and non-acid reflux events. Methods: We studied 11 elderly (74±9yr) and 10 young (28±7yr) healthy volunteers in supine position using a concurrent high-resolution manometry, impedance and infusion techniques. We tested two types of perfusates: 0.1N HCl (pH 1.2-1.4) and half saline (pH 5.8), two perfusion rates X3 (slow: 1ml/s and ultra-slow: 0.05ml/s) as well as two time intervals (active perfusion time of 60 sec followed by passive dwell time of 60 sec). Subjects refrained from swallowing for these periods. Ten-second time epochs of UES High-Pressure Zone Contractile Integrals (UESHPZ CI) were measured 30 seconds prior to as well as during perfusion and during 60 sec after perfusion. Repeated Measures ANOVA, Fisher Exact or c2 test were used for comparisons. Results: Both young and elderly showed significant increase in UES pressure represented by UESHPZ CI during the entire period of active infusion at the rate of 1ml/s as well as during the entire passive dwell interval compared to baseline (p<0.01, fig 1). For the 0.05ml/s infusions, significant increase of UESHPZ Cl was seen in the young group beginning at the late infusion period and into the dwell interval (p<0.01, fig 1). In contrast, the elderly showed no appreciable UES effect during or after the 0.05ml/s infusion either for acid or saline (fig 1). In the elderly, the average UESHPZ CI across the active 0.05ml/s infusion interval and during the dwell interval was similar to baseline. Importantly, there was no difference in UES contractile response to acid infusion and half saline infusion in both groups (fig 2). There was no significant difference between young and elderly in the number of infusions resulting in secondary peristalsis. There were more secondary peristalsis during 1ml/s active infusions than dwell intervals (p<0.05).
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