Downexpression of miR-200c-3p Contributes to Achalasia Disease by Targeting the PRKG1 Gene.

Achalasia is an esophageal smooth muscle motility disorder with unknown pathogenesis. Taking into account our previous results on the downexpression of miR-200c-3p in tissues of patients with achalasia correlated with an increased expression of PRKG1, SULF1, and SYDE1 genes, our aim was to explore the unknown biological interaction between these genes and human miR-200c-3p and if this relation could unravel their functional role in the etiology of achalasia. To search for putative miR-200c-3p binding sites in the 3'-UTR of PRKG1, SULF1 and SYDE1, a bioinformatics tool was used. To test whether PRKG1, SULF1, and SYDE1 are targeted by miR-200c-3p, a dual-luciferase reporter assay and quantitative PCR on HEK293 and fibroblast cell lines were performed. To explore the biological correlation between PRKG1 and miR-200c-3p, an immunoblot analysis was carried out. The overexpression of miR-200c-3p reduced the luciferase activity in cells transfected with a luciferase reporter containing a fragment of the 3'-UTR regions of PRKG1, SULF1, and SYDE1 which included the miR-200c-3p seed sequence. The deletion of the miR-200c-3p seed sequence from the 3'-UTR fragments abrogated this reduction. A negative correlation between miR-200c-3p and PRKG1, SULF1, and SYDE1 expression levels was observed. Finally, a reduction of the endogenous level of PRKG1 in cells overexpressing miR-200c-3p was detected. Our study provides, for the first time, functional evidence about the PRKG1 gene as a direct target and SULF1 and SYDE1 as potential indirect substrates of miR-200c-3p and suggests the involvement of NO/cGMP/PKG signaling in the pathogenesis of achalasia.

Accuracy and inter-observer agreement of the nice and kudo classifications of superficial colonic lesions: a comparative study.

PURPOSE: For superficial colonic lesions, the NICE and Kudo classifications are used in the in vivo prediction of histology and as guide to therapy. The NICE system derives information from unmagnified NBI endoscopic images. The Kudo one necessitates a magnification, but, as this tool is not commonly available, it is applied also to characterize unmagnified pictures to compare their diagnostic performances. METHODS: We conducted a prospective comparison of the NICE versus the Kudo classification for the differential diagnosis of colonic polyps taking histology as the gold standard. The inter-observer agreement for both classifications among 11 colonoscopists was also evaluated. Short unmagnified NBI videoclips of 64 colonic polyps were sent twice to the participants. In the first round, they classified the lesions according to the NICE classification; 4 months later, the same videos were assessed with the Kudo system. The diagnosis provided by the participants was grouped in non-neoplastic, non-invasive neoplasia, invasive neoplasia. RESULTS: Overall, the diagnostic accuracy was 82% (95%CI: 79-85) with the NICE system and 81% (95%CI: 78-84) with the Kudo one (ρ = 0.78). The accuracy of the NICE classification for non-neoplastic lesions was greater compared with the Kudo's (ρ = 0.03). Sensitivity sub-analyses revealed a higher ability of the NICE in distinguishing between neoplastic vs. non-neoplastic lesions (ρ = 0.01). The overall inter-rater agreement did not differ when the classifications were compared. CONCLUSION: The NICE and the Kudo classifications might be considered comparable. Our data could allow the use of the NBI Kudo classification even in those centers where magnification is not available.

Interobserver agreement of the Paris and simplified classifications of superficial colonic lesions: a Western study.

Background and study aims The Paris classification of superficial colonic lesions has been widely adopted, but a simplified description that subgroups the shape into pedunculated, sessile/flat and depressed lesions has been proposed recently. The aim of this study was to evaluate the accuracy and inter-rater agreement among 13 Western endoscopists for the two classification systems. Methods Seventy video clips of superficial colonic lesions were classified according to the two classifications, and their size estimated. The interobserver agreement for each classification was assessed using both Cohen k and AC1 statistics. Accuracy was taken as the concordance between the standard morphology definition and that made by participants. Sensitivity analyses investigated agreement between trainees (T) and staff members (SM), simple or mixed lesions, distinct lesion phenotypes, and for laterally spreading tumors (LSTs). Results Overall, the interobserver agreement for the Paris classification was substantial (κ = 0.61; AC1 = 0.66), with 79.3 % accuracy. Between SM and T, the values were superimposable. For size estimation, the agreement was 0.48 by the κ-value, and 0.50 by AC1. For single or mixed lesions, κ-values were 0.60 and 0.43, respectively; corresponding AC1 values were 0.68 and 0.57. Evaluating the several different polyp subtypes separately, agreement differed significantly when analyzed by the k-statistics (0.08-0.12) or the AC1 statistics (0.59-0.71). Analyses of LSTs provided a κ-value of 0.50 and an AC1 score of 0.62, with 77.6 % accuracy. The simplified classification outperformed the Paris classification: κ = 0.68, AC1 = 0.82, accuracy = 91.6 %. Conclusions Agreement is often measured with Cohen's κ, but we documented higher levels of agreement when analyzed with the AC1 statistic. The level of agreement was substantial for the Paris classification, and almost perfect for the simplified system.

Long-term results of the European achalasia trial: a multicentre randomised controlled trial comparing pneumatic dilation versus laparoscopic Heller myotomy.

OBJECTIVE: Achalasia is a chronic motility disorder of the oesophagus for which laparoscopic Heller myotomy (LHM) and endoscopic pneumodilation (PD) are the most commonly used treatments. However, prospective data comparing their long-term efficacy is lacking. DESIGN: 201 newly diagnosed patients with achalasia were randomly assigned to PD (n=96) or LHM (n=105). Before randomisation, symptoms were assessed using the Eckardt score, functional test were performed and quality of life was assessed. The primary outcome was therapeutic success (presence of Eckardt score ≤3) at the yearly follow-up assessment. The secondary outcomes included the need for re-treatment, lower oesophageal sphincter pressure, oesophageal emptying and the rate of complications. RESULTS: In the full analysis set, there was no significant difference in success rate between the two treatments with 84% and 82% success after 5 years for LHM and PD, respectively (p=0.92, log-rank test). Similar results were obtained in the per-protocol analysis (5-year success rates: 82% for LHM vs. 91% for PD, p=0.08, log-rank test). After 5 years, no differences in secondary outcome parameter were observed. Redilation was performed in 24 (25%) of PD patients. Five oesophageal perforations occurred during PD (5%) while 12 mucosal tears (11%) occurred during LHM. CONCLUSIONS: After at least 5 years of follow-up, PD and LHM have a comparable success rate with no differences in oesophageal function and emptying. However, 25% of PD patients require redilation during follow-up. Based on these data, we conclude that either treatment can be proposed as initial treatment for achalasia. TRIAL REGISTRATION NUMBERS: Netherlands trial register (NTR37) and Current Controlled Trials registry (ISRCTN56304564).

Role of interleukin polymorphisms in gastric cancer: "Pros and cons".

Helicobacter pylori (H. pylori) infection is the leading cause of gastric cancer worldwide. Infection with this bacterium causes a chronic active immune response that persists for the life of the host. The combination of bacterial factors, environmental insults, and the host immune response drives the initiation and progression of mucosal atrophy, metaplasia, and dysplasia toward GC. Among the host factors, IL-1 gene cluster polymorphisms (IL-1B encoding IL-1ß and IL-1RN encoding IL-1ra, its naturally occurring receptor antagonist) play a decisive role in modulating the risk of developing hypochlorhydria, gastric atrophy and GC in the presence of H. pylori infection. In particular, one single nucleotide polymorphism in the IL-1B promoter (IL-1B-511C/T), and the short allele of a 86-bp variable number of tandem repeats polymorphism in the IL-1RN second intron (IL-1RN*2) are associated with an increased risk for GC. However this hypothesis is still to be fully confirmed. This review focuses on the divergent results obtained by several epidemiological and functional in vitro and in vivo studies and show that IL-1 genotyping has still no role in the clinical management of patients with H. pylori infection.

How evidence-based are current guidelines for managing patients with peptic ulcer bleeding?

Current guidelines for managing ulcer bleeding state that patients with major stigmata should be managed by dual endoscopic therapy (injection with epinephrine plus a thermal or mechanical modality) followed by a high dose intravenous infusion of proton pump inhibitors (PPIs). This paper aims to review and critically evaluate evidence supporting the purported superiority of a continuous infusion over less intensive regimens of PPIs administration and the need for adding a second hemostatic endoscopic procedure to epinephrine injection. Systematic searches of PubMed, EMBASE and the Cochrane library were performed. There is strong evidence for an incremental benefit of PPIs over H2-receptor antagonists or placebo for the outcome of patients with peptic ulcer bleeding following endoscopic hemostasis. However, the benefit of PPIs is unrelated to either the dosage (intensive vs standard regimen) or the route of administration (intravenous vs oral). There is significant heterogeneity among the 15 studies that compared epinephrine with epinephrine plus a second modality, which might preclude the validity of reported summary estimates. Studies without second look endoscopy plus re-treatment of re-bleeding lesions showed a significant benefit of adding a second endoscopic modality for hemostasis, while studies with second-look and re-treatment showed equal efficacy between endoscopic mono and dual therapy. Inconclusive experimental evidence supports the current recommendation of the use of dual endoscopic hemostatic means and infusion of high-dose PPIs as standard therapy for patients with bleeding peptic ulcers. Presently, the combination of epinephrine monotherapy with standard doses of PPIs constitutes an appropriate treatment for the majority of patients.

Promoter methylation correlates with reduced NDRG2 expression in advanced colon tumour.

BACKGROUND: Aberrant DNA methylation of CpG islands of cancer-related genes is among the earliest and most frequent alterations in cancerogenesis and might be of value for either diagnosing cancer or evaluating recurrent disease. This mechanism usually leads to inactivation of tumour-suppressor genes. We have designed the current study to validate our previous microarray data and to identify novel hypermethylated gene promoters. METHODS: The validation assay was performed in a different set of 8 patients with colorectal cancer (CRC) by means quantitative reverse-transcriptase polymerase chain reaction analysis. The differential RNA expression profiles of three CRC cell lines before and after 5-aza-2'-deoxycytidine treatment were compared to identify the hypermethylated genes. The DNA methylation status of these genes was evaluated by means of bisulphite genomic sequencing and methylation-specific polymerase chain reaction (MSP) in the 3 cell lines and in tumour tissues from 30 patients with CRC. RESULTS: Data from our previous genome search have received confirmation in the new set of 8 patients with CRC. In this validation set six genes showed a high induction after drug treatment in at least two of three CRC cell lines. Among them, the N-myc downstream-regulated gene 2 (NDRG2) promoter was found methylated in all CRC cell lines. NDRG2 hypermethylation was also detected in 8 out of 30 (27%) primary CRC tissues and was significantly associated with advanced AJCC stage IV. Normal colon tissues were not methylated. CONCLUSION: The findings highlight the usefulness of combining gene expression patterns and epigenetic data to identify tumour biomarkers, and suggest that NDRG2 silencing might bear influence on tumour invasiveness, being associated with a more advanced stage.

Aberrant DNA methylation in non-neoplastic gastric mucosa of H. Pylori infected patients and effect of eradication.

BACKGROUND: Gene promoter methylation is an epigenetic event leading to gene silencing. This mechanism is particularly relevant in cancer since it can interfere with the activity of specific "suppressor" genes. AIM: To evaluate promoter methylation of CDH1, p16, APC, MLH1, and COX2 in patients with H. pylori (Hp) infection before and after eradication. METHODS: Fifty-seven dyspeptic outpatients who had never performed previous endoscopy or Hp testing and treatment underwent clinical interview, endoscopy with three paired gastric biopsy specimens from the antrum, angulus, and corpus, and (13)C-urea breath test (UBT). Biopsies were scored for the presence of Hp and intestinal metaplasia (IM). DNA methylation of five tumor-related genes (CDH1, p16, MLH1, APC, and COX2) was evaluated by methylation-specific PCR in each biopsy. Infected patients were given a standard eradicating treatment and, after 1 yr, underwent endoscopy with biopsies and UBT. RESULTS: Hp infection was found in 45 patients. IM was detected in 17 out of 45 (38%) infected patients. Mean number of methylated genes was 0, 1.1 +/- 0.9, and 1.6 +/- 0.9 among the 12 Hp-/IM-, the 28 Hp+/IM-, and the 17 Hp+/IM+ patients, respectively (P < 0.0001). Specifically, promoter hypermethylation of CDH1, p16, APC, MLH1, and COX2 was found in 68%, 25%, 7%, 0%, and 14% of Hp+/IM- patients and in 71%, 29%, 35%, 12%, and 12% of Hp+/IM+ patients. No significant difference was found among the three groups of patients as far as age, smoking, alcohol, meat and vegetable consumption, and family history of gastric cancer were considered. Twenty-three out of 45 (51%) infected patients underwent the 1-yr follow-up endoscopy: 17 out of 23 (74%) were successfully eradicated. After Hp eradication, CDH1, p16, and APC methylation significantly decreased while COX2 methylation completely disappeared. Conversely, MLH1 methylation did not change significantly in patients with IM. CONCLUSION: Hp infection is associated with promoter methylation of genes which are relevant in the initiation and progression of gastric carcinogenesis. While CDH1 methylation seems to be an early event in Hp gastritis, MLH1 methylation occurs late along with IM. Hp eradication is able to significantly reduce gene methylation thus delaying or reversing Hp-induced gastric carcinogenesis.

Genotyping of the lactase-phlorizin hydrolase c/t-13910 polymorphism by means of a new rapid denaturing high-performance liquid chromatography-based assay in healthy subjects and colorectal cancer patients.

Adult-type hypolactasia results from the progressive decline of lactase-phlorizin hydrolase activity in enterocytes after weaning. Lactase nonpersistence may determine a primary lactose intolerance with reduced diary product consumption, which is possibly related to an increased risk of colon cancer. Recently, a genetic variant C/T(-13910) upstream of the lactase-phlorizin hydrolase (LCT) gene has been strongly correlated with the lactase persistence/nonpersistence trait in both family and case-control studies. The authors validate a denaturing high-performance liquid chromatography (dHPLC)-based assay versus conventional genotype sequencing in detecting the C/T(-13910) polymorphism of LCT and evaluate its prevalence in 2 different Italian geographical areas and in colorectal cancer patients. DNA samples of 157 healthy subjects and 124 colon cancer patients from Apulia and of 97 healthy subjects from Sardinia were evaluated for the C/T(-13910) polymorphism by dHPLC, sequencing, and restriction fragment length polymorphism (RFLP). Under optimized conditions, dHPLC was as sensitive as DNA sequencing and detected a new genetic variant (T/C(-13913)) in 2 individuals that was not identified by RFLP assay. Frequency of lactase nonpersistence genotype (C/C(-13910)) was similar in healthy subjects from 2 different Italian geographical areas and not increased in patients with colorectal cancer. The results indicate that the dHPLC method may be used as a rapid, noninvasive, and labor-saving screening tool for genotyping C/T(-13910) polymorphism, with high success, low cost, and reproducibility.

Mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the cationic trypsinogen (PRSS1) gene, and the serine protease inhibitor, Kazal type 1 (SPINK1) gene in patients with alcoholic chronic pancreatitis.

Susceptibility to alcoholic chronic pancreatitis (ACP) could be genetically determined. Mutations in cationic trypsinogen (PRSS1), cystic fibrosis transmembrane conductance regulator (CFTR), and serine protease inhibitor, Kazal type 1 (SPINK1) genes have been variably associated with both the hereditary and the idiopathic form of chronic pancreatitis (CP). Our aim was to analyze the three genes in ACP patients. Mutational screening was performed in 45 unrelated ACP patients and 34 patients with alcoholic liver disease (ALD). No mutation of PRSS1 was found in ACP and ALD patients. Three mutations of CFTR were detected in four ACP patients with a prevalence (8.9%) not significantly different from that observed (3.0%) in ALD patients and from that expected (3.2%) in our geographical area. Neither compound heterozygotes for CFTR nor trans-heterozygotes for CFTR/SPINK1 were found. One ACP patient (2.2%) was found to carry the most common mutation (N34S) of SPINK1 compared to none of the ALD patients (P=NS). In five other patients (two with ACP and three with ALD) other rare variants, including P55S, were found. In contrast with the hereditary and the idiopathic forms of CP, in which mutations of PRSS1, CFTR, and SPINK1 genes may occur, ACP is still a "gene(s)-orphan" disease. The supposed genetic susceptibility to ACP relies on other yet unknown gene(s) which could affect the alcohol metabolism or modulate the pancreatic inflammatory response to alcohol abuse.