High Resolution Oesophageal Manometry: Addressing Thermal Drift

AGA Abstracts

epithelial cells causes production and release of platelet activating factor (PAF) and IL-8. PAF and IL-8 act as chemokines, attracting peripheral blood leukocyte (PBL), and PAF increases hydrogen peroxide release from PBL. Because epithelial cells release both inflammatory mediators and cytokines in response to HCl, we examined whether they may also release additional pro-inflammatory cytokines, further contributing to the activation of the inflammatory infiltrates in GERD mucosa. METHODS: HET-1A cells were exposed to acidified cell culture medium (pH 5) for 12 min, 6 times in 48h, to simulate In Vivo events, such a recurrent exposure to acid in GERD. After 48h cells were collected, total RNA extracted, and cytokine mRNA measured by real-time PCR. Cell supernatant was collected and examined for the presence of cytokine protein by ELISA, using a human multiplex cytokine/chemokine assay. Expression of Lyso-PAF acetyltransferase, the enzyme responsible for the production of PAF, was measured by Western blot, and its mRNA was examined by real-time PCR in the cultured cells. RESULTS: We have previously demonstrated that mucosal exposure to HCl results in formation of PAF in cat, rabbit and human esophageal mucosa, and involves HCl-induced activation of TRPV1 receptors in esophageal epithelial cells. Similarly, in HET1A cells HCl caused upregulation of Lyso-PAF acetyltransferase mRNA and protein expression. In addition HCl exposure increased mRNA of several chemokines. These include interleukin 8 (IL-8) a known chemoattractant of neutrophils; eotaxin-1, -2, and -3, known chemoattractants for eosinophils, basophils and mast cells; macrophage inflammatory protein-1α (MIP-1α) and monocyte chemoattractant protein-1 (MCP-1), both being chemoattractants for monocytes/macrophages and T cells. The multiplex assay detected increased protein levels of IL-8; eotaxin-1, and -2; MIP-1α; and MCP-1. In contrast, RANTES mRNA levels and protein expression were not affected. CONCLUSION: These findings provide a direct link between acid exposure and the recruitment and activation of immune cells by products derived from esophageal epithelial cells. The data demonstrate that epithelial cells, not only constitute the first barrier to diffusion of refluxate, but also behave as active participants in inflammation as they respond to HCl by releasing selected cytokines and inflammatory mediators. These, in turn, may attract and activate immune cells that initiate an inflammatory process leading to damage of the esophageal squamous epithelial layer. Supported by NIDDK RO1 57030

capacity in 4sec followed by forced expiratory vital capacity in 6 sec at a frequency of 0.1Hz (6 breaths / minute, over 5 min). ANS was monitored throughout in both studies. Results: The majority of volunteers were ‘agreeable extroverts’, which are protective personality factors. There was only 10% depression and 20% anxiety, showing our exclusion criteria effective. 70% of the subjects rated significant for alexithymia, and showed a weak nonsignificant correlation with the level of sensitisation to acid (r=0.49 p=0.14). In study 1 in comparison to baseline, we observed a reduction in CVT during acid exposure while in study 2 the CVT increased during acid exposure i.e. (study 1 difference of -0.09 vs. baseline; study 2 difference of 4.93 vs. baseline (p=0.035). In study 2, all 10 volunteers reduced their level of esophageal sensitisation to acid in comparison to study 1 with 50% failing to sensitise completely. Overall there was a reduction in sensitisation of 16mA between study 1 and 2 (mean PT±SEM; 16.24±3.28, p=0.001). Conclusion: This preliminary study suggests that the parasympathetic nervous system has anti-hyperalgesic properties in human esophageal VPH. This data points to a possible change in the way we treat visceral pain, highlighting likely new targets for the development of novel treatments. Further research is now required with a powered study focusing on exploring the mechanism of desensitization during deep breathing. 1012 TRPV1-Induced ATP Production by Human Esophageal Epithelial Cells Mediates Release of CGRP and Substance P From Esophageal Mucosa Jie Ma, Florian Rieder, Claudio Fiocchi, Jose Behar, Piero Biancani, Karen M. Harnett INTRODUCTION: Exposure of esophageal mucosa to hydrochloric acid (HCl) is a crucial event in the pathogenesis of GERD. HCl-induced activation of transient receptor potential channel vanilloid subfamily member-1 (TRPV1) in esophageal mucosa causes release of substance P (subP) and calcitonin gene-related peptide (CGRP) from submucosal neurons and release of platelet activating factor (PAF) from epithelial cells. PAF release from epithelial cells, however, plays no role in neural release of subP and CGRP. We therefore investigated whether ATP acts as a possible second messenger responsible for the release of these two sensory neurotransmitters from submucosal neurons. METHODS: A tubular segment of rabbit esophageal mucosa was tied at both ends to form a sac which was filled with HClacidified Krebs buffer at pH 5 (or plain Krebs buffer as control) and kept oxygenated at 37° C. The medium around the sac was collected after 3 hours and examined for presence of subP and CGRP. The human esophageal epithelial cell line HET-1A was used to examine mechanisms mediating release of ATP from epithelial cells. RESULTS: In the rabbit mucosal sac HCl induced release of subP and CGRP that was blocked by the purinergic antagonist suramin, suggesting that release of subP and CGRP from submucosal neurons may be mediated by ATP. We therefore used the human esophageal epithelial cell line HET-1A to identify esophageal epithelial cells as a possible source of ATP. When exposed to pH 5 for 5 min HET-1A cells released ATP, that was significantly reduced by the TRPV1 antagonist IRTX, indicating HCl-induced activation of TRPV1. Real time PCR demonstrated the presence of mRNA for the purinergic receptors P2X4, P2X5, P2Y14 , in order of relative expression, and of other purinergic receptors at significantly lower levels. Repeated exposure of HET1A cells to HCl over two days caused a significant upregulation of mRNA for Lyso-PAF acetyltransferase, the enzyme responsible for production of PAF. The upregulation was blocked by suramin, suggesting ATP as a possible second messenger responsible for HClinduced upregulation. Similarly, repeated exposure to ATP over two days caused a significant increase in Lyso-PAF acetyltransferase mRNA that was significantly reduced by suramin, further supporting ATP as a possible second messenger for upregulation of the enzyme. CONCLUSION: HCl-induced activation of TRPV1 triggers release of ATP from esophageal epithelial cells. ATP in turn causes release of the sensory neurotransmitters CGRP and subP from esophageal mucosa. If repeatedly applied, ATP also causes upregulation of Lyso-PAF acetyltransferase in epithelial cells. Thus ATP acts as a second messenger causing release of sensory neurotransmitters from esophageal mucosa and upregulating the enzyme responsible for production of PAF in epithelial cells. Supported by NIDDK RO1 57030

1010 High Resolution Oesophageal Manometry: Addressing Thermal Drift Elaine V. Robertson, Yeong Yeh Lee, Mohammad H. Derakhshan, James Whiting, Angela A. Wirz, John P. Seenan, Kenneth E. McColl Background: High resolution manometry (HRM) is a sophisticated and widely used technology allowing detailed examination of oesophageal function A described limitation of the HRM system is its propensity to ‘thermal drift’. Thermal drift is a false change in the measured pressure attributed to the change from ambient to body temperature. In prolonged studies this effect can be marked. We sought to investigate the nature and magnitude of this phenomenon and to validate the currently employed corrections. Methods: Six experiments were performed with the HRM catheter placed in a water bath at a constant depth and temperature of 37oC. Recordings were carried out for two hours. Pressure readings for the thirty-six sensors were plotted against time. Results: The mean pressure change for six experiments and thirty six sensors was 13.1mmHg (Range 1.9-44.7mmHg). The rate of drift varied between sensors and for an individual sensor between experiments. For an individual sensor within an experiment the pressure increase was linear (R2 > 0.9 in 211 of 216 graphs). In the standard correction for thermal drift, the pressure increase in each sensor at the end of the study is subtracted from the data set to reset the baseline. This was replicated in the recorded pressures and the residual error calculated. The mean error increased with study duration and for a given study was maximal for the early data. For data captured at the start of a fifteen minute study the mean error with 95% confidence intervals was 1.4 +/- 0.12mmHg. Corresponding values for a thirty minute study were 2.8 +/- 0.24 mmHg and for a sixty minute study 6.1 +/- 0.52mmHg. The distal border of the lower oesophageal sphincter, used to calculate sphincter length, is defined by an increase in pressure of 2mmHg from intragastric pressure. Errors of this magnitude therefore have the potential to affect measured physiological parameters. A linear correction was then applied to the data, using the best fit line for each sensor within each experiment. For this tailored correction the mean error with 95% confidence intervals was 0.4 +/- 0.017mmHg and was independent of study duration. Conclusions: Thermal drift is better considered as ‘baseline drift’ a continuous upward drift of the baseline pressure with time rather than a ‘once and for all’ baseline change. A correction process which takes into account the ongoing and linear nature of the drift reduces the error associated with this phenomenon. Incorporating this correction into existing software would improve the accuracy of the system without impact on ease of use

1013 HDAC-Mediated Gene Silencing of AKT1 by the Tumor Suppressor KLF11: Implications in Growth Regulation and Bile Salt Mediated Neoplastic Transformation in Barrett's Esophagus Sonia Chowdhury, Alireza Faridar, Cathrine J. DeMars, Abhayjit Singh, Gwen A. Lomberk, Ganapathy A. Prasad, Raul A. Urrutia, Navtej Buttar BACKGROUND/AIM: Serine/threonine kinase AKT, a signal transducer is being increasingly recognized to play key role in growth and metabolism. Our previously published studies demonstrate that carcinogenic bile salts, by activating AKT, regulate cyclooxygenase 2, the enzyme involved in arachidonic acid metabolism to prostaglandin E2 thus outlining an important pro-oncogenic pathway in Barrett's esophagus (Gut 2007, Cancer Res 2008). While most AKT related research, including our work has remained focused on phosphorylation and signaling, it is plausible that baseline expression of AKT also impacts the growth regulatory function of AKT. Our recent work (JBC 2010) demonstrating the transcriptional regulation of arachidonic acid metabolism by KLF11 and the down regulation of AKT by KLF11 in expression arrays prompted us to investigate the relevance of AKT expression to neoplastic transformation, the mechanisms of and the growth regulatory consequences of AKT expression. METHODS & RESULTS: Using qPCR, Western blot and IHC, we found overexpression of AKT, particularly isoform AKT1 during neoplastic progression in Barrett's esophagus. ChIP assays, promoter reporter constructs, qPCR and Western blot combined with KLF11 mutant/deletion constructs, showed that KLF11 decreases AKT-1 expression, binds to endogenous AKT1 promoter, represses AKT1 promoter activity in esophageal neoplastic cells via SID (SIN3A-HDAC interacting domain) within KLF11, indicating that histone deacetylation underlies this mechanism. Co-immunoprecipitation assays revealed a decreased recruitment of SIN3A-HDAC to KLF11 by bile salts (Mixture of CDCA, DCA, LCA resembling human bile) suggesting that, pathophysiologically, bile salts may interfere with KLF11 dependent regulation of AKT-1, a novel mechanism for the promotion of cell growth by endogenous carcinogens. The increase in cell growth and colony formation by SKGT esophageal cell lines transduced with either AKT-1 wt or constitutively active AKT-1 adenovirus, to a similar

1011 Effect of Autonomic Modulation on Human Esophageal Pain Hypersensitivity Claude A. Botha, Hira Naqvi, Yang C. Chua, Charles H. Knowles, Qasim Aziz Introduction: Patients with functional gastrointestinal disorders (FGID) often display persistent and heightened pain sensitivity to experimental gut stimulation, a phenomenon known as visceral pain hypersensitivity (VPH). Current management of pain in FGID involves the use of either antispasmodics or antidepressants; both are often unsatisfactory. The role of the autonomic nervous system (ANS) in modulating pain is well known however the effect of ANS modulation on VPH has not been studied. Aim: to determine whether physiological modulation of the autonomic nervous system (ANS) influences the degree of esophageal VPH in healthy volunteers using a validated model. Methods: 10 healthy volunteers (mean age 30.5yrs; 8 male, 6 W-British) underwent psychological profiling for stress, anxiety and personality type. Study 1: Pain thresholds (PT) to proximal and distal oesophageal electrical stimulation were measured. Thereafter the subjects underwent hydrochloric acid infusion (0.15M) in the distal esophagus for 30 minutes. This was followed by measurements of esophageal pain threshold (using visual analogue scales) to electrical stimulation in the proximal, unexposed esophagus at 30, 60 and 90 minutes. Study 2 was performed at an interval of 2 weeks and the protocol was identical to that of study 1 except that during the acid infusion cardiac vagal tone (CVT) was increased by deep breathing at full inspiratory

AGA Abstracts

S-164