Methods Patients Two groups of children referred to the Pediatric Gastroenterology and Liver Unit of the “”Sapienza”" University of Rome were included in this study: 20 CD (mean age 8.3 years, range 1.2-16.1 years) in active and in remission state

(at AZD5363 supplier diagnosis and after at least 9 months of gluten-free diet, respectively) and 10 controls undergoing upper gastrointestinal endoscopy for functional dyspepsia (mean age 11.7 years, range 7.8-20.8 years). The latter tested negative for antitransglutaminase and antiendomysial antibodies with normal IgA levels, while histology of duodenum did not reveal features of CD. Diagnosis of CD MI-503 had been performed according to ESPGHAN criteria [15]. Table 2 summarizes clinical features of the

studied population. selleck chemicals Size appropriate and well oriented endoscopic biopsy specimens were obtained from the second part of the duodenum. The histopathological diagnosis was based on typical mucosal lesions with crypt cell hyperplasia, villous atrophy, and increased number of intra-epithelial lymphocytes (IELs) [16]. All untreated CD patients were positive for antiendomysial and antitransglutaminase antibodies at the time of diagnosis. In all patients there was an endoscopic improvement of duodenal mucosa following gluten withdrawal, but only in 2 of them (patients number 12 and 19) there was also a full histological improvement. None of the children included in the study was treated with antibiotics for at least 3 months before the sampling time. The study protocol was approved by the Committee on Ethical Practice of the ‘Policlinico Umberto I’ hospital. Children were enrolled in the study after written informed consent from their parents. The biopsy

samples were placed in liquid nitrogen immediately after their emission and stored at -80°C until analysis. Bacterial strains The strains listed below were obtained from the American Type Culture Collection (ATCC) and used MTMR9 as marker on TTGE gel electrophoresis: Bacteroides fragilis ATCC 23745, Bacteroides thetaiotaomicron ATCC 29148, Bacteroides vulgatus ATCC 8482, Parabacteroides distasonis ATCC 8503, Escherichia coli MG1655. Bacterial DNA was extracted with UltraClean kit (MO BIO Laboratories, Solana Beach, California, USA) according to the manufacturer’s instructions. DNA extraction Duodenal biopsy specimens from CD and control patients were first quickly washed in 500 μL of physiologic saline with 0.016% dithiothreitol to remove luminal bacteria from the mucus, and then utilized for DNA extraction procedure by DNeasy tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. In order to obtain maximum yield of both Gram-positive and Gram-negative bacteria, a special step in DNA purification protocol was added, following DNeasy tissue kit manual.