Project description:Oesophageal exposure to duodenogastroesophageal refluxate is implicated in the development of BarrettM-bM-^@M-^Ys Metaplasia, with increased risk of progression to oesophageal adenocarcinoma. The literature proposes that reflux exposure activates NF-kB, driving the aberrant expression of intestine-specific caudal-related homeobox genes. However, early events in the pathogenesis of BarrettM-bM-^@M-^Ys Metaplasia from a normal epithelium are poorly understood. To investigate this, our study subjected a 3D model of the normal human oesophageal mucosa to repeated, pulsatile exposure to specific bile components and examined changes in gene expression. Initial 2D experiments with a range of bile salts observed that taurochenodeoxycholate (TCDC) impacted upon NF-kB activation without causing cell death. Informed by this, the 3D human oesophageal model was repeatedly exposed to TCDC in the presence and absence of acid, and the epithelial cells underwent gene expression profiling. We identified ~300 differentially expressed genes following each treatment, with a large and significant overlap between treatments. Enrichment analysis (Broad GSEA, DAVID and MetacoreM-bM-^DM-", GeneGo Inc) identified multiple gene sets related to cell signalling, inflammation, proliferation, differentiation and cell adhesion. Specifically NF-kB activation, Wnt signalling, cell adhesion and targets for the transcription factors PTF1A and HNF4M-NM-1 were highlighted. CDX1/2 transcription factors are believed to play a role in BM development; however, in this study their targets were not enriched, suggesting that CDX1/2 activation may not be the one of the initial events for BM formation. Our findings highlight new areas for investigation in the earliest stages of BM pathogenesis of oesophageal diseases and new potential therapeutic targets. RNA was extracted from oesophageal epithelium grown in a 3D oesophageal mucosa model. The model received pulsatile exposure for 10 mins twice a day for 11 days to pH 7.4 (control, n=6), TCDC (n=6), pH 4 (n=6) or TCDC, pH 4 (n=6). In one experiment, four oesophageal constructs were grown using the epithelial cells from one patient and each construct was exposed to a different insult. The experiment was performed six times using cells from three different patients, repeating each experiment once to produce technical replicates. RNA was then pooled from one technical replicate per patient to produce 8 samples for array analysis. The three treatments were compared to the control.

Project description:The major aetiological risk factor for Barrett's oesophagus and oesophageal adenocarcinoma is gastroesophageal reflux. This study's aim was to identify genes involved in the celular response to reflux in vitro. The Barrettâ??s oesophagus cell line, CP-A hTERT, was exposed to media with acid, deoxycholic acid or a primary bile salt mixture. RNA expression was compared with controls on Affymetrix U133 Plus 2.0 arrays. In CP-A hTERT, the greatest number of changes in gene expression was observed after treatment with deoxycholic acid, pH 4.5; 152 genes were up-regulated at 2 hours (91 at 6 hours) and 10 down-regulated at 2 hours (34 at 6 hours). 12 genes were identified and were subsequently assessed in patients with non-erosive reflux disease, oesophagitis, Barrett's oesophagus and oesophageal adenocarcinoma; Background and Aims: The major etiological risk factor for Barrettâ??s esophagus and esophageal adenocarcinoma is gastro-esophageal reflux. This studyâ??s aim was to identify genes involved in the cellular response to components of reflux both in vitro and in patients with reflux-related disease. Methods: The Barrettâ??s cell line, CP-A hTERT, was exposed to media with acid, deoxycholic acid or a primary bile salt mixture. RNA expression was compared with controls on Affymetrix U133 Plus 2.0 arrays. 12 genes of interest were analysed by Real Time PCR both in cell line and biopsies from 110 patients with non-erosive reflux disease, esophagitis, Barrettâ??s esophagus and esophageal adenocarcinoma. Results: In CP-A hTERT, the greatest number of changes in gene expression was observed after treatment with deoxycholic acid, pH 4.5. Of 12 genes analysed in biopsies, 10 were significantly different between the 4 groups with the largest change for anterior gradient homolog 2, which may modulate p53 function. This had highest expression in biopsies from Barrettâ??s esophagus (median gene fold change for Barrettâ??s esophagus versus non-erosive reflux disease, 411.2 (95% CI 290.5-682.7; p<0.01); esophageal adenocarcinoma versus non-erosive reflux disease 68.1 (20.5-161.4; p<0.01)). In addition 4 genes associated with development/differentiation were upregulated in Barrettâ??s biopsies compared to those from non-erosive reflux disease (SEL1L, MFNG, CRIP1 and EFNA1). Conclusions: Novel genes have been identified, whose expression is altered after acid and bile exposure in vitro and in biopsies from patients with reflux related diseases. These genes may have utility as biomarkers of response to reflux and should be assessed in prospective studies. Experiment Overall Design: The Barrett's oesophagus cell line CP-A hTERT was treated with a 15 minute exposure of acid (pH 4.5), a mixture of primary bile acids (pH 4.5) or deoxycholic acid (pH 4.5). RNA extraction occurred in treatment and non-treated cells at 2 hours and 6 hours. The treatments were performed in duplicate on 2 different days. RNA was compared in each treatment to each control at the relevant time points, in a 2 x 2 manner by using Affymetrex U133 Plus 2.0 arrays. Results of 12 genes were confirmed by Real Time PCR and were subsequently assessed in patients with non-erosive reflux disease, oesophagitis, Barrett's oesophagus and oesophageal adenocarcinoma.

Project description:The major aetiological risk factor for Barrett's oesophagus and oesophageal adenocarcinoma is gastroesophageal reflux. This study's aim was to identify genes involved in the celular response to reflux in vitro. The Barrett’s oesophagus cell line, CP-A hTERT, was exposed to media with acid, deoxycholic acid or a primary bile salt mixture. RNA expression was compared with controls on Affymetrix U133 Plus 2.0 arrays. In CP-A hTERT, the greatest number of changes in gene expression was observed after treatment with deoxycholic acid, pH 4.5; 152 genes were up-regulated at 2 hours (91 at 6 hours) and 10 down-regulated at 2 hours (34 at 6 hours). 12 genes were identified and were subsequently assessed in patients with non-erosive reflux disease, oesophagitis, Barrett's oesophagus and oesophageal adenocarcinoma Background and Aims: The major etiological risk factor for Barrett’s esophagus and esophageal adenocarcinoma is gastro-esophageal reflux. This study’s aim was to identify genes involved in the cellular response to components of reflux both in vitro and in patients with reflux-related disease. Methods: The Barrett’s cell line, CP-A hTERT, was exposed to media with acid, deoxycholic acid or a primary bile salt mixture. RNA expression was compared with controls on Affymetrix U133 Plus 2.0 arrays. 12 genes of interest were analysed by Real Time PCR both in cell line and biopsies from 110 patients with non-erosive reflux disease, esophagitis, Barrett’s esophagus and esophageal adenocarcinoma. Results: In CP-A hTERT, the greatest number of changes in gene expression was observed after treatment with deoxycholic acid, pH 4.5. Of 12 genes analysed in biopsies, 10 were significantly different between the 4 groups with the largest change for anterior gradient homolog 2, which may modulate p53 function. This had highest expression in biopsies from Barrett’s esophagus (median gene fold change for Barrett’s esophagus versus non-erosive reflux disease, 411.2 (95% CI 290.5-682.7; p<0.01); esophageal adenocarcinoma versus non-erosive reflux disease 68.1 (20.5-161.4; p<0.01)). In addition 4 genes associated with development/differentiation were upregulated in Barrett’s biopsies compared to those from non-erosive reflux disease (SEL1L, MFNG, CRIP1 and EFNA1). Conclusions: Novel genes have been identified, whose expression is altered after acid and bile exposure in vitro and in biopsies from patients with reflux related diseases. These genes may have utility as biomarkers of response to reflux and should be assessed in prospective studies. Keywords: Acid (pH 4.5) and bile (mixture of primary bile salts or the secondary bile salt deoxycholic acid, both at pH 4.5) challenge to a Barrett's oesophagus cell line. RNA extraction at 2 and 6 hours. Comparison of treatment RNA to control (non-treatment) RNA,

Project description:Barrett's esophagus is characterized by the replacement of squamous epithelium with specialized intestinal metaplastic mucosa. The exact mechanisms of initiation and development of Barrett's metaplasia remain unknown, but a hypothesis of successful adaptation against noxious reflux components has been proposed. To search for the repertoire of adaptation mechanisms of Barrett's metaplasia, we employed high-throughput functional genomic and proteomic methods that defined the molecular background of metaplastic mucosa resistance to reflux. Transcriptional profiling was established for 23 pairs of esophageal squamous epithelium and Barrett's metaplasia tissue samples using Affymetrix U133A 2.0 GeneChips and validated by quantitative real-time polymerase chain reaction. Differences in protein composition were assessed by electrophoretic and mass-spectrometry-based methods. Among 2,822 genes differentially expressed between Barrett's metaplasia and squamous epithelium, we observed significantly overexpressed metaplastic mucosa genes that encode cytokines and growth factors, constituents of extracellular matrix, basement membrane and tight junctions, and proteins involved in prostaglandin and phosphoinositol metabolism, nitric oxide production, and bioenergetics. Their expression likely reflects defense and repair responses of metaplastic mucosa, whereas overexpression of genes encoding heat shock proteins and several protein kinases in squamous epithelium may reflect lower resistance of normal esophageal epithelium than Barrett's metaplasia to reflux components. Despite the methodological and interpretative difficulties in data analyses discussed in this paper, our studies confirm that Barrett's metaplasia may be regarded as a specific microevolution allowing for accumulation of mucosal morphological and physiological changes that better protect against reflux injury. Department of Gastroenterology, Medical Center for Postgraduate Education and Maria SkM-EM-^Bodowska-Curie Memorial Cancer Center and Institute of Oncology, 02-781 Warsaw, Poland 2 types of tissue sample derived from the same patient: > Normal squamous epithelium (NE) from patients with longsegment of BarretM-bM-^@M-^Ys epithelium > Metaplastic epithelium from BarrettM-bM-^@M-^Ys esophagus (BE)

Project description:Prolonged skeletal unloading through bedrest results in bone loss similar to that observed in elderly osteoporotic patients, but with an accelerated timeframe. This rapid effect on weight-bearing bones is also observed in astronauts who lose up to 2% of their bone mass per month spent in Space. Despite important implications for Spaceflight travellers and bedridden patients on Earth, the exact mechanisms involved in disuse osteoporosis have not been elucidated. Parathyroid hormone-related protein (PTHrP) regulates many physiological processes including skeletal development, and has been proposed as a gravisensor. To investigate the role of PTHrP in microgravity-induced bone loss, trabecular osteoblasts (TOs) from Pthrp+/+ and -/- mice were exposed to simulated microgravity for 6 days. Viability of TOs decreased in inverse proportion to PTHrP expression levels. Microarray analysis of Pthrp+/+ TOs after 6 days at 0g revealed expression changes in genes encoding prolactins,apoptosis and survival molecules, bone metabolism and extra-cellular matrix composition proteins, chemokines, IGF family and Wnt-related signalling molecules. Importantly, 88% of 0g-induced expression changes in Pthrp+/+ cells overlap those observed in Pthrp-/- cells in normal gravity. Pulsatile treatment with PTHrP1-36 peptide during microgravity exposure reversed a large proportion of 0g-induced changes in Pthrp+/+ TOs. Our results confirm PTHrP efficacy as an anabolic agent to prevent microgravity-induced cell death in TOs. Total RNA samples extracted from Pthrp+/+and -/- trabecular osteoblasts (TOs) exposed for 6 days to simulated 0g in Synthecon rotating cell, or left 6 days in culture at 1g. Cells had either been treated with a pulsatile treatment (2 h/day) of PTHrP1-36 peptide (10-8M) or received a change in growth medium. In total: 8 different conditions with 2 replicates each, i.e. Pthrp+/+ TOs at 0g or 1g with or without PTHrP1-36 treatment, and Pthrp-/- TOs at 0g or 1 g,with or without PTHrP1-36 treatment.

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