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IL-1R Signaling in Human Enteric Glial Cells - Induction of a Reactive Phenotype and Disruption of Mechanosensitivity, Purinergic Signaling and Ca2 + Waves
AGA Abstracts
difference: -61.6 ± 50.8, p<0.01, and -63.3 ± 43.1, p<0.01, respectively). No significant differences in the placebo group compared to baseline were observed, and neither between treatment and placebo. The GSRS-IBS total score significantly decreased for both the treatment and the placebo group 2 weeks after FMT (-0.46 ± 0.32, p<0.01, and -0.71 ± 0.73, p<0.05, respectively) and 4 weeks after FMT compared to baseline (-0.79 ± 0.57, p<0.01, and -0.57 ± 0.63, p<0.05, respectively). Again, no differences between the two intervention groups were observed. The IBS-QoL total score was significantly increased in the treatment but not in the placebo group at 8 weeks (10.0 ± 6.3, p<0.01). Similar results were observed in three out of eight SF-36 subscores, especially in the general health subscore. This score was significantly increased in the treatment compared to the placebo group 8 weeks after FMT (11.8 ± 8.3 compared to -8.1 ± 11.3, p<0.01). Conclusions These data showed that there is a beneficial effect of FMT on symptom scores and quality of life in IBS patients. This effect is also observed in the placebo group, although to a lesser extent, indicating that placebo-controlled studies are essential in IBS patients. The bowel cleansing and the processing of the autologous fecal material might have contributed to the placebo effect. Further analysis on individual basis, separation into non-responders and responders as well as correlation with additional outcomes such as microbiota composition will provide more insight.
ICC was detected in 8 (61.5%) GP and none of the CUNV patients. Mean M2 macrophages count/HPF in the antrum of GP and CUNV patients was 3.5 ± 2.3 and 3.8 ± 2.7, respectively. In the pylorus of GP and CUNV groups, mean M2 count was 3.0 ± 2.1 and 2.0 ± 1.4. M2 count was not associated with the ICC count or ICC depletion in GP or CUNV. Nine GP (69.2%) and 1 (16.7%) CUNV patients had pyloric fibrosis. Mean M2 count in the antrum of fibrotic and non-fibrotic groups was 3.6 ± 2.8 and 3.6 ± 2.3, while mean M2 count in the pylorus of patients with fibrosis (3.8 ± 1.7) was significantly higher than those without pyloric fibrosis (1.5 ± 1.4) (t-test; p=0.04). Conclusions: Pyloric ICC loss and fibrosis are significant findings in GP compared to CUNV and the presence of increased pyloric M2 macrophages were associated with pyloric fibrosis. While by definition, M2 macrophages are anti-inflammatory, they may pave the way for a tissue injury pathway leading to smooth muscle fibrosis and pyloric dysfunction in GP patients.
433 OPTOGENETIC INDUCTION OF PROPAGATING COLONIC MOTOR COMPLEXES AND SILENCING OF COLONIC MOTILITY USING CREINDUCIBLE ACTIVATION AND INACTIVATION OF CALRETININEXPRESSING NEURONS Jing Feng, Timothy J. Hibberd, Jialie Luo, Pu Yang, Nick J. Spencer, Hongzhen Hu
431 IL-1 β / IL-1R Signaling in Human Enteric Glial Cells - Induction of a Reactive Phenotype and Disruption of Mechanosensitivity, Purinergic Signaling and Ca2+ Waves Alix Zuleta-Alarcon, Sven Wehner, Mahmoud Abdel-Rasoul, Paolo Fadda, Iveta Grants, Fabio Turco, Alan Harzman, Sergio D. Bergese, Fievos L. Christofi
The ability to selectively control gastrointestinal motility without using therapeutic agents that can have off-target effects offers great hope for patients with dysfunctional gastrointestinal transit. Optogenetics is an exciting new technique to control specific neural pathways in the central nervous system (CNS), but has not been demonstrated to control motor patterns generated by the enteric nervous system (ENS). Calretinin (CAL), a calcium-binding protein, is a well-established marker for both interneurons and muscle motor neurons in the ENS, and absence of CAL staining is an important criterion for the diagnosis of Hirschsprung disease (HD) in humans. Our aim was to demonstrate that optogenetic activation of CALexpressing neurons or selective ablation of CAL-expressing neurons could modify propulsive motor patterns in the colon. We generated a novel transgenic mouse using Cre-driven expression of the light-gated cation channel, channelrhodopsin-H134R (ChR2-H134R) in neurons expressing CAL. Immunohistochemical analysis of colonic myenteric neurons revealed 97% of the cholinergic CAL-immunoreactive neurons expressed detectable ChR2(H134R)-eYFP. Mechanical recordings were made from intact whole colons in vitro (n= 5). Both CAL-ChR2(H134R) mice and wild-type littermates generated ongoing propagating colonic motor complexes (CMCs, mean interval 227±46s, n=5), which were always blocked by tetrodotoxin (TTX). Focal illumination (5-10Hz, 10-60s) of the proximal, mid or distal colon using focused blue light evoked premature CMCs in CAL-ChR2(H134R) mice (mean latency from previous contraction 102±14s, p<0.001, n=5, Bonferroni post-test, two-way ANOVA), but not in wild-type littermates. TTX prevented optogenetic activation of CMCs (7/7 times tested, n=4). To investigate if loss of CAL-positive neurons mimics the dysmotility phenotype in HD we used a method for inducible cell-type-specific ablation of CAL neurons involving Cre-dependent expression of the diphtheria toxin receptor (DTR) (CAL-DTR) followed by administration of diphtheria toxin (DTX). Remarkably, CMCs were either abolished or severely disrupted in CAL-DTR mice compared to littermate controls (n=3 for each group). In conclusion, we generated transgenic mice specifically expressing opsins or human DTR in a major neurochemical class of enteric neurons with high efficacy. We provide the first demonstration that optogenetic stimulation of a specific class of neuron in the ENS can evoke propagating CMCs. Also, that selective depletion of the CAL neurons can severely impair the CMC motor pattern. This study demonstrates that neurogenetics could prove a very exciting technique to selectively control the enteric neural circuits and modulate gastrointestinal motility.
Introduction: Glial Ca2+waves are essential for normal intestinal motility. Inflammation provoked by bacterial LPS induces a reactive human EGC (hEGC) phenotype and disrupts Ca2+ waves and mechanosensitivity. Interleukin-1β (IL-1β) and IL1R1 signaling in enteric glial cells (EGC) is a potential contributing mechanism in disrupting motility and postoperative ileus in response to surgical intestinal manipulation/trauma. The functional role of IL1β in hEGC is unknown. Aim: The aim was to probe the role of IL-1β in hEGC and test the hypothesis that IL-1β/IL-1R1 signaling induces a reactive hEGC phenotype and disrupts Ca2+waves, mechanosensory and purinergic Ca2+signaling. Experimental Design: Eight human colon surgical specimens were used to isolate, purify and culture hEGC from myenteric ganglia for molecular (nanostring, IHC) and functional studies (Ca2+waves, mechanosensitivity). Acute (3 min) or chronic (24 h) effects of IL-1β were tested in hEGC using realtime fluo-4/AM Ca2+imaging. Mechanical stimulation (MS) by increasing fluid-flow (2 to 10ml/min) or a purinergic agonist UTP was used to trigger Ca2+responses. Results: Acute exposure to 30ng/ml IL-1β induces Ca2+transients, oscillations or Ca2+waves in 36% and 100ng/ml in 65% of hEGC (n=162). Ca2+responses to MS were disrupted by chronic exposure to IL-1β. MS triggers gadolinium-sensitive Ca2+oscillations (3 to 25 oscillations/10 min, p<0.0001) and Ca2+waves. IL-1β inhibits mechanically evoked responses (p<0.0001), but increases basal Ca2+responses (p=0.04, n=288 cells). UTP triggers a monophasic Ca2+response (lasting~30 sec), a biphasic Ca2+response with a sustained plateau phase (lasting 3-5 min) or Ca2+oscillations. Chronic exposure to IL-1β disrupts UTP Ca2+responses by preventing the plateau phase [3.9% vs 67% of cells]. Most responsive cells treated with IL-1β only had monophasic Ca2+responses (87% vs 13% in control). Fewer treated cells had Ca2+oscillations compared to control (6% vs 18%, N=16 cultures; p<0.0001). IL-1β (24 h) induces a reactive hEGC phenotype with up regulation in 14 of 36 gene transcripts for CxCl2 (264 fold), IP10 (50 fold), IL-8 (1580 fold), SOD2 (17 fold, p<0.00001 each), nfkB (3 fold), connexins (3 of 18, hCx26, 5.6 fold; hCx30, 8.3 fold, p<0.0001), AMPD3 (4 fold) and Cav (L-type α1S; 33 fold, p<0.00001); and no effect on Piezo channels. The gene induction profile for IL-1β differed significantly from that of LPS (10µg/ml). LPS increased IL1R-ir and IL-1β mRNA expression. IL-1β had no effect on viability (ENZO). Conclusions: IL-β/IL1R signaling leads to a reactive glial phenotype, disrupts Ca2+waves, purinergic and mechanosensory signaling and alters expression of mechanogated and Cav channels. AMPD3 is a biomarker of high pro-inflammatory levels of purines. IL-1β induction and glial disruption has potential implications for motility disorders (DK 093499).
434 EVIDENCE FOR TRP CHANNEL SENSITIZATION IN IBS WITH HISTAMINE 1 RECEPTOR ANTAGONISM AS EFFECTIVE TREATMENT Dafne Balemans, Javier Aguilera-Lizarraga, Morgane Florens, Stavroula Theofanous, Eluisa Perna, Schalk Van Der Merwe, Mira M. Wouters, Guy E. Boeckxstaens Background & Aims: Increased abdominal pain perception or visceral hypersensitivity (VHS) is the hallmark of irritable bowel syndrome (IBS). We previously showed histamine-1receptor (Hrh1)-mediated sensitization of TRPV1, TRPA1 and TRPV4 in patients with IBS. In the present study we investigated the prevalence of TRP channel sensitization and its correlation with symptoms in IBS patients versus healthy volunteers (HV). In addition, we evaluated the effect of treatment with the Hrh1 antagonist ebastine on TRP channel sensitization and symptom scores in IBS patients. Methods: 25 HV (11M, 28 yrs IQR [23-53]) and 34 IBS patients (9M, 35 yrs IQR [25-53]) were invited to provide rectal biopsies for live Ca2+ imaging. Submucosal neurons were loaded with Fluo-4 and their response to the TRP channel agonists capsaicin (1nM, TRPV1), cinnamaldehyde (1µM, TRPA1) and/or GSK1016790A (1nM, TRPV4) was assessed. Thereafter, a subgroup of IBS patients (n=22) was treated (open label) with ebastine (20-40 mg) and invited to provide rectal biopsies for live Ca2+ imaging after 8-12 weeks of treatment. Finally, all subjects filled out validated symptom questionnaires (ROME III, GSRS and numeric scale for pain (0: no pain, 10: worst possible)). Correlations between symptoms and neuronal data were calculated using Spearman correlations and Fishers exact tests. Results: Sensitization of TRPV1, TRPA1 or TRPV4 was significantly more prevalent in rectal submucosal neurons of IBS patients compared to those of HV (Fig. 1A). TRP channel sensitization of one or more TRP channels was detected in 68% of IBS patients compared to 7% in HV (p<0.001,Fishers exact test). 60% of IBS patients with positive symptom scores for pain, bloating, urgency, flatulence and diarrhea had sensitized TRP channels compared to 30% of patients with normal TRP sensitivity. Within the IBS group, no significant correlation was detected between the response of submucosal neurons and abdominal symptoms, including pain, urgency, bloating, flatulence and diarrhea. Ebastine however significantly improved IBS symptoms resulting in 63% pain responders (>30% reduction in pain score). Of note, patients responding to ebastine had a normalization of their neuronal Ca2+ response to capsaicin, cinnamaldehyde or GSK1016790A (n=3). Conclusion: Our data demonstrate that sensitization of submucosal neuronal TRP channels is prevalent and can be detected in up to 68% of IBS patients.
432 M2 MACROPHAGES ARE LINKED WITH PYLORIC FIBROSIS IN PATIENTS WITH GASTROPARESIS SYMPTOMS Richard W. McCallum, Alireza Torabi, Mohammad Bashashati, Daniel Welder, Irene Sarosiek, Jesus R. Diaz, Brian R. Davis, Dolgor Baatar Background: Fibrogenesis has been preserved throughout development based on its benefits in the wound healing process that happens after injury. Meanwhile, fibrosis contributes to several chronic and progressive diseases. While the polarization of macrophages toward the M1 pro-inflammatory phenotype induces phagocytosis and neutrophil apoptosis, the contribution of anti-inflammatory M2 macrophages in fibrosis has been shown in previous studies. It is believed that M2 macrophages are protective in diabetic gastroparesis (GP) and in preserving interstitial cells of Cajal (ICC); however, we hypothesized that these macrophages may also have deleterious effects. Our study was designed to understand whether gastric M2 macrophages predict the severity of gastric emptying, and the histological findings of GP particularly the presence of gastric smooth muscle fibrosis. Methods: Full-thickness antral and pyloric biopsies were obtained from 13 GP (10 diabetic, 11 F; age: 25-77 years) and 6 chronic unexplained nausea and vomiting (CUNV) patients (no diabetic, 5 F; age: 22-36 years) during abdominal surgery to place a gastric electrical stimulator and/or pyloroplasty or J-Tube insertion. The diagnosis of GP was based on the presence of typical GP symptoms and >10% retention of a radiolabeled meal in the stomach after 4 hours. CUNV had similar clinical findings except for a normal retention (<10% at 4 h). The tissues were stained with anti-c-Kit and anti-CD206 antibodies as well as H&E and trichrome. Based on our previous control data, an ICC count of <10 per high power field (HPF) in the muscularis propria of antrum and/or pylorus was considered depletion. The results are presented as frequency (%) or mean ± standard deviation. Results: Antral ICC depletion was present in 3 (23.1%) and 2 (33.3%) of GP and CUNV patients, respectively. Depletion of the pyloric
AGA Abstracts
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difference: -61.6 ± 50.8, p<0.01, and -63.3 ± 43.1, p<0.01, respectively). No significant differences in the placebo group compared to baseline were observed, and neither between treatment and placebo. The GSRS-IBS total score significantly decreased for both the treatment and the placebo group 2 weeks after FMT (-0.46 ± 0.32, p<0.01, and -0.71 ± 0.73, p<0.05, respectively) and 4 weeks after FMT compared to baseline (-0.79 ± 0.57, p<0.01, and -0.57 ± 0.63, p<0.05, respectively). Again, no differences between the two intervention groups were observed. The IBS-QoL total score was significantly increased in the treatment but not in the placebo group at 8 weeks (10.0 ± 6.3, p<0.01). Similar results were observed in three out of eight SF-36 subscores, especially in the general health subscore. This score was significantly increased in the treatment compared to the placebo group 8 weeks after FMT (11.8 ± 8.3 compared to -8.1 ± 11.3, p<0.01). Conclusions These data showed that there is a beneficial effect of FMT on symptom scores and quality of life in IBS patients. This effect is also observed in the placebo group, although to a lesser extent, indicating that placebo-controlled studies are essential in IBS patients. The bowel cleansing and the processing of the autologous fecal material might have contributed to the placebo effect. Further analysis on individual basis, separation into non-responders and responders as well as correlation with additional outcomes such as microbiota composition will provide more insight.
ICC was detected in 8 (61.5%) GP and none of the CUNV patients. Mean M2 macrophages count/HPF in the antrum of GP and CUNV patients was 3.5 ± 2.3 and 3.8 ± 2.7, respectively. In the pylorus of GP and CUNV groups, mean M2 count was 3.0 ± 2.1 and 2.0 ± 1.4. M2 count was not associated with the ICC count or ICC depletion in GP or CUNV. Nine GP (69.2%) and 1 (16.7%) CUNV patients had pyloric fibrosis. Mean M2 count in the antrum of fibrotic and non-fibrotic groups was 3.6 ± 2.8 and 3.6 ± 2.3, while mean M2 count in the pylorus of patients with fibrosis (3.8 ± 1.7) was significantly higher than those without pyloric fibrosis (1.5 ± 1.4) (t-test; p=0.04). Conclusions: Pyloric ICC loss and fibrosis are significant findings in GP compared to CUNV and the presence of increased pyloric M2 macrophages were associated with pyloric fibrosis. While by definition, M2 macrophages are anti-inflammatory, they may pave the way for a tissue injury pathway leading to smooth muscle fibrosis and pyloric dysfunction in GP patients.
433 OPTOGENETIC INDUCTION OF PROPAGATING COLONIC MOTOR COMPLEXES AND SILENCING OF COLONIC MOTILITY USING CREINDUCIBLE ACTIVATION AND INACTIVATION OF CALRETININEXPRESSING NEURONS Jing Feng, Timothy J. Hibberd, Jialie Luo, Pu Yang, Nick J. Spencer, Hongzhen Hu
431 IL-1 β / IL-1R Signaling in Human Enteric Glial Cells - Induction of a Reactive Phenotype and Disruption of Mechanosensitivity, Purinergic Signaling and Ca2+ Waves Alix Zuleta-Alarcon, Sven Wehner, Mahmoud Abdel-Rasoul, Paolo Fadda, Iveta Grants, Fabio Turco, Alan Harzman, Sergio D. Bergese, Fievos L. Christofi
The ability to selectively control gastrointestinal motility without using therapeutic agents that can have off-target effects offers great hope for patients with dysfunctional gastrointestinal transit. Optogenetics is an exciting new technique to control specific neural pathways in the central nervous system (CNS), but has not been demonstrated to control motor patterns generated by the enteric nervous system (ENS). Calretinin (CAL), a calcium-binding protein, is a well-established marker for both interneurons and muscle motor neurons in the ENS, and absence of CAL staining is an important criterion for the diagnosis of Hirschsprung disease (HD) in humans. Our aim was to demonstrate that optogenetic activation of CALexpressing neurons or selective ablation of CAL-expressing neurons could modify propulsive motor patterns in the colon. We generated a novel transgenic mouse using Cre-driven expression of the light-gated cation channel, channelrhodopsin-H134R (ChR2-H134R) in neurons expressing CAL. Immunohistochemical analysis of colonic myenteric neurons revealed 97% of the cholinergic CAL-immunoreactive neurons expressed detectable ChR2(H134R)-eYFP. Mechanical recordings were made from intact whole colons in vitro (n= 5). Both CAL-ChR2(H134R) mice and wild-type littermates generated ongoing propagating colonic motor complexes (CMCs, mean interval 227±46s, n=5), which were always blocked by tetrodotoxin (TTX). Focal illumination (5-10Hz, 10-60s) of the proximal, mid or distal colon using focused blue light evoked premature CMCs in CAL-ChR2(H134R) mice (mean latency from previous contraction 102±14s, p<0.001, n=5, Bonferroni post-test, two-way ANOVA), but not in wild-type littermates. TTX prevented optogenetic activation of CMCs (7/7 times tested, n=4). To investigate if loss of CAL-positive neurons mimics the dysmotility phenotype in HD we used a method for inducible cell-type-specific ablation of CAL neurons involving Cre-dependent expression of the diphtheria toxin receptor (DTR) (CAL-DTR) followed by administration of diphtheria toxin (DTX). Remarkably, CMCs were either abolished or severely disrupted in CAL-DTR mice compared to littermate controls (n=3 for each group). In conclusion, we generated transgenic mice specifically expressing opsins or human DTR in a major neurochemical class of enteric neurons with high efficacy. We provide the first demonstration that optogenetic stimulation of a specific class of neuron in the ENS can evoke propagating CMCs. Also, that selective depletion of the CAL neurons can severely impair the CMC motor pattern. This study demonstrates that neurogenetics could prove a very exciting technique to selectively control the enteric neural circuits and modulate gastrointestinal motility.
Introduction: Glial Ca2+waves are essential for normal intestinal motility. Inflammation provoked by bacterial LPS induces a reactive human EGC (hEGC) phenotype and disrupts Ca2+ waves and mechanosensitivity. Interleukin-1β (IL-1β) and IL1R1 signaling in enteric glial cells (EGC) is a potential contributing mechanism in disrupting motility and postoperative ileus in response to surgical intestinal manipulation/trauma. The functional role of IL1β in hEGC is unknown. Aim: The aim was to probe the role of IL-1β in hEGC and test the hypothesis that IL-1β/IL-1R1 signaling induces a reactive hEGC phenotype and disrupts Ca2+waves, mechanosensory and purinergic Ca2+signaling. Experimental Design: Eight human colon surgical specimens were used to isolate, purify and culture hEGC from myenteric ganglia for molecular (nanostring, IHC) and functional studies (Ca2+waves, mechanosensitivity). Acute (3 min) or chronic (24 h) effects of IL-1β were tested in hEGC using realtime fluo-4/AM Ca2+imaging. Mechanical stimulation (MS) by increasing fluid-flow (2 to 10ml/min) or a purinergic agonist UTP was used to trigger Ca2+responses. Results: Acute exposure to 30ng/ml IL-1β induces Ca2+transients, oscillations or Ca2+waves in 36% and 100ng/ml in 65% of hEGC (n=162). Ca2+responses to MS were disrupted by chronic exposure to IL-1β. MS triggers gadolinium-sensitive Ca2+oscillations (3 to 25 oscillations/10 min, p<0.0001) and Ca2+waves. IL-1β inhibits mechanically evoked responses (p<0.0001), but increases basal Ca2+responses (p=0.04, n=288 cells). UTP triggers a monophasic Ca2+response (lasting~30 sec), a biphasic Ca2+response with a sustained plateau phase (lasting 3-5 min) or Ca2+oscillations. Chronic exposure to IL-1β disrupts UTP Ca2+responses by preventing the plateau phase [3.9% vs 67% of cells]. Most responsive cells treated with IL-1β only had monophasic Ca2+responses (87% vs 13% in control). Fewer treated cells had Ca2+oscillations compared to control (6% vs 18%, N=16 cultures; p<0.0001). IL-1β (24 h) induces a reactive hEGC phenotype with up regulation in 14 of 36 gene transcripts for CxCl2 (264 fold), IP10 (50 fold), IL-8 (1580 fold), SOD2 (17 fold, p<0.00001 each), nfkB (3 fold), connexins (3 of 18, hCx26, 5.6 fold; hCx30, 8.3 fold, p<0.0001), AMPD3 (4 fold) and Cav (L-type α1S; 33 fold, p<0.00001); and no effect on Piezo channels. The gene induction profile for IL-1β differed significantly from that of LPS (10µg/ml). LPS increased IL1R-ir and IL-1β mRNA expression. IL-1β had no effect on viability (ENZO). Conclusions: IL-β/IL1R signaling leads to a reactive glial phenotype, disrupts Ca2+waves, purinergic and mechanosensory signaling and alters expression of mechanogated and Cav channels. AMPD3 is a biomarker of high pro-inflammatory levels of purines. IL-1β induction and glial disruption has potential implications for motility disorders (DK 093499).
434 EVIDENCE FOR TRP CHANNEL SENSITIZATION IN IBS WITH HISTAMINE 1 RECEPTOR ANTAGONISM AS EFFECTIVE TREATMENT Dafne Balemans, Javier Aguilera-Lizarraga, Morgane Florens, Stavroula Theofanous, Eluisa Perna, Schalk Van Der Merwe, Mira M. Wouters, Guy E. Boeckxstaens Background & Aims: Increased abdominal pain perception or visceral hypersensitivity (VHS) is the hallmark of irritable bowel syndrome (IBS). We previously showed histamine-1receptor (Hrh1)-mediated sensitization of TRPV1, TRPA1 and TRPV4 in patients with IBS. In the present study we investigated the prevalence of TRP channel sensitization and its correlation with symptoms in IBS patients versus healthy volunteers (HV). In addition, we evaluated the effect of treatment with the Hrh1 antagonist ebastine on TRP channel sensitization and symptom scores in IBS patients. Methods: 25 HV (11M, 28 yrs IQR [23-53]) and 34 IBS patients (9M, 35 yrs IQR [25-53]) were invited to provide rectal biopsies for live Ca2+ imaging. Submucosal neurons were loaded with Fluo-4 and their response to the TRP channel agonists capsaicin (1nM, TRPV1), cinnamaldehyde (1µM, TRPA1) and/or GSK1016790A (1nM, TRPV4) was assessed. Thereafter, a subgroup of IBS patients (n=22) was treated (open label) with ebastine (20-40 mg) and invited to provide rectal biopsies for live Ca2+ imaging after 8-12 weeks of treatment. Finally, all subjects filled out validated symptom questionnaires (ROME III, GSRS and numeric scale for pain (0: no pain, 10: worst possible)). Correlations between symptoms and neuronal data were calculated using Spearman correlations and Fishers exact tests. Results: Sensitization of TRPV1, TRPA1 or TRPV4 was significantly more prevalent in rectal submucosal neurons of IBS patients compared to those of HV (Fig. 1A). TRP channel sensitization of one or more TRP channels was detected in 68% of IBS patients compared to 7% in HV (p<0.001,Fishers exact test). 60% of IBS patients with positive symptom scores for pain, bloating, urgency, flatulence and diarrhea had sensitized TRP channels compared to 30% of patients with normal TRP sensitivity. Within the IBS group, no significant correlation was detected between the response of submucosal neurons and abdominal symptoms, including pain, urgency, bloating, flatulence and diarrhea. Ebastine however significantly improved IBS symptoms resulting in 63% pain responders (>30% reduction in pain score). Of note, patients responding to ebastine had a normalization of their neuronal Ca2+ response to capsaicin, cinnamaldehyde or GSK1016790A (n=3). Conclusion: Our data demonstrate that sensitization of submucosal neuronal TRP channels is prevalent and can be detected in up to 68% of IBS patients.
432 M2 MACROPHAGES ARE LINKED WITH PYLORIC FIBROSIS IN PATIENTS WITH GASTROPARESIS SYMPTOMS Richard W. McCallum, Alireza Torabi, Mohammad Bashashati, Daniel Welder, Irene Sarosiek, Jesus R. Diaz, Brian R. Davis, Dolgor Baatar Background: Fibrogenesis has been preserved throughout development based on its benefits in the wound healing process that happens after injury. Meanwhile, fibrosis contributes to several chronic and progressive diseases. While the polarization of macrophages toward the M1 pro-inflammatory phenotype induces phagocytosis and neutrophil apoptosis, the contribution of anti-inflammatory M2 macrophages in fibrosis has been shown in previous studies. It is believed that M2 macrophages are protective in diabetic gastroparesis (GP) and in preserving interstitial cells of Cajal (ICC); however, we hypothesized that these macrophages may also have deleterious effects. Our study was designed to understand whether gastric M2 macrophages predict the severity of gastric emptying, and the histological findings of GP particularly the presence of gastric smooth muscle fibrosis. Methods: Full-thickness antral and pyloric biopsies were obtained from 13 GP (10 diabetic, 11 F; age: 25-77 years) and 6 chronic unexplained nausea and vomiting (CUNV) patients (no diabetic, 5 F; age: 22-36 years) during abdominal surgery to place a gastric electrical stimulator and/or pyloroplasty or J-Tube insertion. The diagnosis of GP was based on the presence of typical GP symptoms and >10% retention of a radiolabeled meal in the stomach after 4 hours. CUNV had similar clinical findings except for a normal retention (<10% at 4 h). The tissues were stained with anti-c-Kit and anti-CD206 antibodies as well as H&E and trichrome. Based on our previous control data, an ICC count of <10 per high power field (HPF) in the muscularis propria of antrum and/or pylorus was considered depletion. The results are presented as frequency (%) or mean ± standard deviation. Results: Antral ICC depletion was present in 3 (23.1%) and 2 (33.3%) of GP and CUNV patients, respectively. Depletion of the pyloric
AGA Abstracts
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