Microalterations of esophagus in patients with non-erosive reflux disease: in-vivo diagnosis by confocal laser endomicroscopy and its relationship with gastroesophageal reflux.

OBJECTIVES: Objectively diagnosing non-erosive reflux disease (NERD) is still a challenge. We aimed to evaluate the use of in-vivo confocal laser endomicroscopy (CLE) to examine the microalterations of the esophagus in patients with NERD and its relationship with reflux episodes monitored by multiple intraluminal impedance-pH (MII-pH). METHODS: Patients with gastroesophageal reflux symptoms completed reflux disease questionnaires. NERD was determined by negative gastroscopy. Patients without reflux symptoms were recruited as controls. Pilot clinical study was followed by prospective controlled blinded study. All subjects were examined by white-light mode of the endoscopy followed by the standard CLE mode and then MII-pH monitoring. The microalterations seen on CLE images and the correlation between CLE features and reflux episodes were evaluated, the correlation between CLE and transmission electron microscope (TEM) data was also analyzed. RESULTS: On CLE images, NERD patients had more intrapapillary capillary loops (IPCLs) per image than did controls (8.29 ± 3.52 vs. 5.69 ± 2.31, P=0.010), as well as the diameter of IPCLs (19.48 ± 3.13 vs. 15.87 ± 2.21 µm, P=0.041) and intercellular spaces of squamous cells (3.40 ± 0.82 vs. 1.90 ± 0.53 µm, P=0.042). The receiver operating characteristic analysis indicated that IPCLs number (optimal cutoff >6 per image, area under the curve (AUC) 0.722, 95% confidence interval (CI) 0.592-0.853, sensitivity 67.7%, specificity 71.6%), IPCLs diameter (optimal cutoff >17.2 µm, AUC 0.847, 95% CI 0.747-0.947, sensitivity 81%, specificity 76%), and the intercellular spaces of squamous cells (optimal cutoff >2.40 µm, AUC 0.935, 95% CI 0.875-0.995, sensitivity 85.7%, specificity 90.5%) diagnosed NERD with reasonable accuracy. Combined features of dilatation of intercellular space plus increased IPCLs provided 100% specificity in the diagnosis of NERD patients. The intercellular spaces of squamous cells observed on CLE were highly related to that on TEM findings (r=0.75, P<0.001). Multivariate progressive regression analysis showed that acidic reflux, especially in the supine position, was related to the increased number and dilation of IPCLs in the squamous epithelium (ß=0.063, t=2.895, P=0.038 and ß=0.156, t=1.023, P=0.04). CONCLUSIONS: CLE represents a useful and potentially significant improvement over standard endoscopy to examine the microalterations of the esophagus in vivo. Acidic reflux is responsible for the microalterations in the esophagus of patients with NERD.

Impacts of different promoters on the mammalian one-hybrid assay for detecting nuclear receptor agonists.

Nuclear receptors are a superfamily of ligand-activated transcription factors that play key roles in many biological processes, and have become one class of the most important targets in drug discovery. Mammalian one-hybrid system has been used to develop a cell-based functional transactivation high-throughput screening (HTS) assay for detecting nuclear receptors ligands. In the present study, we proved that different promoters used in the reporter vector had significant different impacts on the performance of HTS assays. The assay using the SV40 promoter in the reporter vector showed the characteristics of much higher signal/noise ratios, acceptable Z' factors (>0.6), low coefficient variation (<12.5%) and higher hits rate, which could be more robust, reproducible, and sensitive. In contrast, utilizing a TATA box promoter in the assay resulted in higher variance and low sensitivity. In addition, it was found that the assay using SV40 had longer signal decay time and was easier to be miniaturized in 384-well format. It has been confirmed that the choice of a promoter is a critical factor in developing a reporter gene HTS assay. However, the SV40 promoter used in the present study has been shown to be more adaptable than the minimal promoter TATA box in the Mammalian one-hybrid HTS assays for detecting nuclear receptor agonists.

A cell-based high-throughput screening assay for Farnesoid X receptor agonists.

OBJECTIVE: To develop a high-throughput screening assay for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system (a chimera receptor gene system) for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library. METHODS: cDNA encoding the human FXR ligand binding domain (LBD) was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain (DBD) of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD (189-472) chimera expression plasmid. RESULTS: After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z' factor value of 0.65. CONCLUSION: A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.

Visceral hypersensitivity following cold water intake in subjects with irritable bowel syndrome.

BACKGROUND: Visceral hypersensitivity has been shown to be present in irritable bowel syndrome (IBS). This study sought to investigate rectal sensitivity and abdominal symptoms in IBS patients before and after 220 ml cold water intake. METHODS: A total of 60 IBS patients and 18 healthy controls participated in this study. Both the perception thresholds and defecation thresholds to rectal balloon distension were measured. Then, all subjects were asked to drink 220 ml 37 degrees C warm water or 4 degrees C cold water, and these steps were repeated 20 min later. Symptoms including abdominal pain/discomfort, bloating, and diarrhea were recorded during the study. RESULTS: Compared with the controls, the thresholds of initial sensation to rectal balloon distention in IBS patients were significantly lower while the defecation thresholds were higher in constipation-predominant IBS patients. After drinking cold water, the perception thresholds in IBS patients and the defecation thresholds in diarrhea-predominant IBS patients were further decreased. However, warm water intake did not change the perception thresholds significantly in either IBS patients or controls. A negative linear correlation was found between the symptoms and the visceral perception thresholds in diarrhea-predominant IBS patients who showed significant symptoms after cold water intake. CONCLUSION: The results indicated that cold water intake leads to lowered visceral perception thresholds in IBS patients that were inversely relevant to the abdominal symptoms in symptomatic diarrhea-predominant IBS patients. The alteration of rectal sensitivity and abdominal symptoms following cold water stimulation provided further objective evidence for visceral hypersensitivity in IBS patients.

Tegaserod for dyspepsia and reflux symptoms in patients with chronic constipation: an exploratory open-label study.

OBJECTIVE: To evaluate the potential role of tegaserod in the management of functional dyspepsia (FD) and gastroesophageal reflux disease (GERD) in patients with chronic constipation and to determine the possible efficacy of tegaserod on solid-phase gastric emptying and gastric hypersensitivity. METHOD: This was an exploratory open-label trial of tegaserod therapy for dyspepsia and reflux symptoms in patients with chronic constipation. The study cohort consisted of 90 patients randomized to three treatment groups for a study period of 4 weeks (tegaserod 6 mg, twice daily; esomeprazole 40 mg, once daily; tegaserod 6 mg, twice daily plus esomeprazole 40 mg, once daily). Twenty healthy volunteers provided control values. Clinical symptoms were evaluated by one of the investigators using a Gastrointestinal Symptom Rating Scale (GSRS). Solid-phase gastric emptying and colonic transit were measured by the radiopaque barium marker method, and the water load test (WLT) was used to evaluate gastric sensation and the function of proximal stomach. The proportions of patients with complete relief of epigastric pain /discomfort, epigastric fullness, early satiety and heartburn in the tegaserod group and the tegaserod plus esomeprazole group were compared with the esomeprazole group, respectively. RESULTS: The mean global gastrointestinal (GI) scores of all three treatment groups reported using the GSRS showed the same trend, with decreasing scores over the 4-week study period indicating a reported decreasing severity of symptoms that was significantly different from baseline values. Patients in the tegaserod plus esomeprazole group reported the lowest global GI scores after 4 weeks, as expected. Solid-phase gastric emptying (GER) and colonic transit (CTT) increased significantly in the tegaserod 6 mg twice daily group compared with baseline. These parameters did not change in the esomeprazole group at week 4 compared with baseline. In terms of gastric sensation, in the tegaserod group, the proportions of patients with hypersensitivity of the first perception threshold did not change at week 2 or week 4 compared with baseline; however, in this group and in the tegaserod plus esomeprazole group, the proportions of patients with hypersensitivity of discomfort threshold decreased significantly at week 4 compared with baseline. In the esomeprazole group, there were no changes in the proportions of patients with hypersensitivity of the first perception threshold and discomfort threshold at week 2 or 4 compared with baseline. No severe adverse events were recorded, and the medications were in general well-tolerated. CONCLUSION: Tegaserod is effective and safe at improving dyspepsia and reflux symptoms in patients with chronic constipation, and tegaserod plus esomeprazole is superior to esomeprazole alone in the resolution of epigastric pain/discomfort and heartburn.

Alterations in cerebral potentials evoked by rectal distention and drinking ice water in patients with irritable bowel syndrome.

AIM: Visceral hypersensitivity has been found to be present in irritable bowel syndrome (IBS). The current study sought to study visceral afferent hypersensitivity in IBS patients and obtain further objective evidence of alterations in intestinal afferent pathways in IBS patients by cerebral evoked potentials (CEP). METHOD: We studied 30 female IBS patients and 12 female healthy subjects. Rectal perception thresholds to balloon distention were measured and CEP was recorded in response to rhythmic rectal distention (two distention series, each of 100 repetitions at a frequency of 1 Hz) at the volume of perception thresholds. All subjects were then asked to drink 220 mL 4 degrees C ice water and the above steps were repeated 20 min later. RESULTS: Rectal distention led to recognizable and reproducible CEP. Compared to healthy subjects, IBS patients demonstrated significantly shorter N1, P1 and N2 latencies (P < 0.05). After drinking ice water, IBS patients exhibited further shortened N1, P1 and N2 latencies (P < 0.05), but drinking did not alter the latencies of healthy controls and the amplitudes of both IBS patients and healthy controls. CONCLUSION: The shorter latency of cerebral potentials evoked by rectal distention and ice water stimulation in IBS patients provided further objective evidence for defective visceral afferent transmission in IBS patients.