Children were enrolled in the study after written informed consen

Children were enrolled in the study after written informed consent, that was obtained both from the respective parents and the institutional

ethics committee of the Faculty of Medicine and Surgery of the University of Bari Aldo Moro, Italy. Table 5 Demographic and clinical characteristic of the children included in the trial   Age Median (range) F/M Cesarean section Feeding habits IEC* Median (range) Marsh score* Celiac children 9.7 (6 – 12) years 11/8 68% Strict gluten free diet 34 (26-50) 3c Non-celiac children 10.4 (6 – 12) years 8/7 60% Unrestricted 5 (0-12) 0 *At diagnosis Collection of duodenal biopsies, faecal and urine samples Each child had fasted overnight, and biopsies, which were taken always from the second duodenum, faecal and urine were collected in the morning pre-prandial. Urine

samples were collected after the second mittus. Each child provided a duodenal biopsy and three faecal and urine samples over the see more time. Duodenal biopsy specimens were obtained from the second duodenum by upper intestinal endoscopy, frozen immediately at -80°C and kept until further processing. After collection, faeces (ca. 15 g), contained in sterile plastic box, were immediately mixed (1:1 wt/wt) with the Amies Transport medium (Oxoid LTD, Basingstoke, Hampshire, England) under anaerobic conditions (AnaeroGen, Oxoid LTD). Samples were immediately buy CB-5083 subjected to analysis (plate counts) or frozen at -80°C (DNA extraction). The urine samples were collected into pre-labeled sterile collections cups. Three aliquots per patient were immediately frozen and stored at -80°C until use. DNA extraction from duodenal biopsies and faecal samples Biopsies specimens, the average weight was ca. 3.5 mg

(biopsies are not usually weighted, however all were taken by the same endoscopist using the same biopsy forceps), were homogenized using a sterile plastic pestle in 200 μl of 20 mM Tris-HCl, pH 8.0, 2 mM EDTA buffer. The homogenate was subjected to this website mechanical disruption in a FastPrep® instrument (BIO 101) and total DNA was extracted with a FastDNA® Pro Soil-Direct Kit (MP Biomedicals, CA., USA) according to the manufacturer’s instructions. Three samples of faecal slurry of each child were mixed Terminal deoxynucleotidyl transferase and used for DGGE analysis [43]. An aliquot of about 300 μl of each faecal slurry sample containing 150 μg of faeces was diluted in 1 ml of PBS-EDTA (phosphate buffer 0.01 M, pH 7.2, 0.01 M EDTA). After centrifugation (14,000 × g at 4°C for 5 min), the pellet was washed two times to decrease the content of PCR inhibitors. The resulting pellet was resuspended in 300 μl of PBS-EDTA and used for DNA extraction [44] with a FastPrep instrument as above. The final product was 100 μl of application-ready DNA both for stool and tissue samples [45]. Quality and concentration of DNA extracts were determined in 0.7% agarose-0.5X TBE gels stained with Gel Red ™ 10,000X (Biotium, Inc.

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