Selection of GERD patients
This is a cross-sectional study of patients with typical symptoms of gastroesophageal reflux disease (heartburn, regurgitation). This research was performed at the Department of Gastroenterology and Endoscopy at Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ). All patients who agreed to participate were subjected to superior endoscopy (already referred by their treating physician for this study) and were classified into 3 groups based on the endoscopy findings: BE (short segment <3 cm, long segment >3 cm), EE (Los Angeles Classification) and NERD. The last group was subclassified into abnormal acid exposure (AAE) or normal acid exposure (NAE) according to 24-h pH monitoring study results.
Selection criteria for patients with GERD included typical symptoms (heartburn and / or regurgitation) at least once a week for at least 1 year. Subjects were older than 18 years of age and included both genders. All of the subjects agreed to participate in the study by signing a consent form. The control group included patients with dyspepsia who at the time of their endoscopy presented a macroscopically gastric mucosa without lesions. The pH monitoring study results for control patients were subsequently negative for abnormal esophageal exposure to acid reflux.
All groups underwent biopsies of esophageal mucosa during the endoscopic procedure. If lesions were found (BE, EE), two biopsies were obtained: one from the injured region (BE, EE) and another from the adjacent mucosa without injury that was 5 cm above the esophageal mucosal junction when it was free of injured mucosa. If no lesions were found, a biopsy of the esophageal mucosa was obtained 5 cm above the esophageal mucosal junction (one biopsy for the non-erosive endoscopic phenotype and control group).
Prior to endoscopy, patients who did not present erosions underwent a pH monitoring study for classification according to the level of acid exposure in the esophagus. A 24-h esophageal pH monitoring study with a 1-sensor catheter (GeroFlex, Alpine Biomed, Fountain Valley CA, EU) placed 5 cm above the lower esophageal sphincter, which was located by esophageal manometry, was performed. A portable recording device (Digitrapper, Medtronic, Parkway, Minneapolis, MN, USA) was used. The pH monitoring study was performed when the patient was off proton pump inhibitors. These patients were subsequently subclassified with esophageal abnormal acid exposure (AAE) (presence of abnormal acid reflux) and esophageal normal acid exposure (NAE). Symptom analysis was not considered for the purposes of this study. According to the results of the percentage of exposure time at pH <4, patients were classified as AAE (exposure time percentage >4.2%) or NAE (percentage of exposure time <4.2%).
Operational definitions
BE: Patients with long segment (> 3 cm) and short segments (<3 cm) of the epithelial column located between the upper border of the gastric folds and the proximal part of the Z line and the presence of intestinal metaplasia was histopathologically confirmed in biopsies of the Barrett epithelium segment.
EE: Patients with GERD symptoms with erosions or disruptions of the esophageal mucosa of different degrees by Los Angeles Classification as follows: Grade A, one (or more) mucosal break less than 5 mm that does not extend between the tops of two mucosal folds; Grade B, one (or more) mucosal break greater than 5 mm long that does not extend between the tops of two mucosal folds; Grade C, one (or more) mucosal break that is continuous between the tops of two or more mucosal folds but involves less than 75% of the circumference; and Grade D, one (or more) mucosal break that involves at least 75% of esophageal circumference. (8)
Non-erosive phenotype: GERD symptoms but no lesions identified by endoscopy.
Abnormal acid exposure (AAE): GERD symptoms, no lesions identified by endoscopy and a pH monitoring study with >4.2% exposure time at pH <4.
Normal acid exposure (NAE): GERD symptoms, no lesions at endoscopy and a pH monitoring study with <4.2% exposure time at pH <4.
Control group (C): Patients without pathology involving their immunity (neoplasms, celiac disease, rheumatic diseases) who present dyspepsia under study with normal endoscopy (without organic disease) and with normal pH monitoring study results (which excludes gastroesophageal reflux).
Sample Processing and Gene Expression Analysis.
Based on previous studies, the expression of the following cytokines and inflammation mediators were analyzed: IL1B, IL-6, IL-8, IL-10, TNF-α, IFN-γ, MMP3 and MMP9.
The esophageal mucosal biopsies obtained from endoscopy were immediately placed in RNA later (Ambion, Austin, TX, USA) and stored at -70 °C (short-term; <6 months) until use. Then, total RNA was isolated using High Pure RNA Tissue (Roche Diagnostics, Mannheim, Germany) following the manufacturer’s guidelines. Two hundred nanograms of total RNA was reverse transcribed into cDNA with random hexamer primers (Roche Diagnostics, Mannheim, Germany). The methodology employed was based on the previous studies of gene expression (9, 10, 11). PCR amplification was performed with 20 ng of cDNA, 200 nM forward and reverse primers, and Taqman Master Mix (Roche Diagnostics, Mannheim, Germany Roche Diagnostics, Mannheim, Germany) in a final volume of 10 µl (Table 1). PCR reactions were run in a Light Cycler 480 (Roche Diagnostics, Mannheim, Germany) for 45 cycles. Each cycle consisted of denaturation for 15 seconds at 95°, primer annealing for 15 seconds at 55°C, extension for 30 seconds at 72°C and cooling 30 seconds at 40°C.
To ensure quality control of q-PCR assays, linearity and reproducibility were determined (VC<10%). The relative quantification of mRNA of target genes was conducted using the LightCycler software 4.1 according to the 2-delta Ct method.
GAPDH mRNA levels were used to standardize esophageal tissues from patients with disease and samples without intestinal inflammation.
Changes in gene expression were assessed and represented by relative gene expression units of target/housekeeping gene in each disease phenotype. The following inflammatory molecules were assessed and compared with the control group: IL-1B, IL-6, IL-8, IL-10, TNF-α, IFN-γ, MMP3, MMP9.
Statistical Analysis
Acid exposure times in each subgroup of the non-erosive phenotype and controls were compared using the Kruskal-Wallis test.
Patients with lesions (EE and BE) had 2 biopsies: one biopsy of the lesion and another biopsy of healthy mucosa. Expression of each gene was analyzed using the Wilcoxon's test, and each patient served as his/her own control. For analysis purposes, biopsies of the lesion tissue (EE, BE) were used for comparisons with the control group.
Statistical analysis of differential gene expression was performed using the Mann-Whitney U non-parametric test. A p-value < 0.05 was considered significant. Analysis was performed using SPSS version 20 and Prism GraphPad version 6.