CSA-13 was ABT-263 research buy prepared as previously

described [34]. Amoxicillin (AMX), clarithromycin (CLR), tetracycline (TET) and metronidazole were purchased from Sigma. Collection of gastric mucosal and bile samples During gastroscopy, performed with either a GIF V2 or Q145 (Olympus) gastroscope, several gastric mucosal slices were taken from the prepyloric and corpus regions of the stomach. H. pylori infection was established in the biopsy specimens using a urease test (CLO-test). Human bile was obtained from the gallbladder of patients undergoing cholecystectomy. Samples were filter-sterilized through a 0.45 μm membrane before being diluted in PBS (1:1) and mixed with antibacterial agents used in bacteria killing assays. The studies were

approved by Medical University of Bialystok Ethics Committee for Research on Humans and Animals, and all patients gave informed written consent for participation in the study. Immunohistochemical studies Immunohistochemical studies were performed on formalin-fixed, paraffin-embedded human gastric mucosal sections using a rabbit anti-LL-37 antibody (H-075-06, used at 1:100 dilution; Phoenix Pharamceuticals Inc.). Paraffin-embedded materials were sectioned to 5 μm 3-Methyladenine price thickness and floated on distilled water at 45°C. Sections were then mounted on slides and placed in 57°C oven overnight. The sections were deparaffinized according Linsitinib chemical structure to standard procedures and quenched with 0.9% hydrogen peroxide in methanol for 30 minutes. The sections were incubated with primary antibody at 37°C for 60 minutes, washed with 1% PBSA (1% BSA in PBS), and subjected to binding with secondary antibody (biotinylated goat anti-Rabbit IgG, 1:400 dilution). Amplification was performed with a Vectastain ABC kit, and

an HRP detection system was used to colocalize peroxidase activity with a DAB substrate. The sections were counterstained with hematoxylin. Samples were viewed with a Nikon Eclipse 80 microscope under 40× magnification. Evaluation of MIC and MBC The minimal inhibitory concentration (MIC) of conventional antibiotics against seven different clinical isolates of H. pylori (9 × 108 CFU/ml) was determined using Muller-Hinton agar (MH) containing 5% sheep blood. The incubation was continued for 4 days at 35°C in microaerophilic P-type ATPase conditions maintained with use of a Gas Pack-Campylobacter gas generating kit BR60. Clinical isolates of H. pylori were considered resistant to respective antibiotics when the MIC values were above 4 μg/ml for AMX, 1 μg/ml for CLR and 16 μg/ml for TET and Metronidazole. The minimal bactericidal concentration (MBC) of antibacterial agents was evaluated using an inoculum at 108 CFU/ml. After a 6-hour incubation at 37°C, 10 μl aliquots of the suspensions were spotted on Columbia agar supplemented with sheep blood (5%). Bacteria killing assay The bactericidal activities of LL-37, WLBU2 peptides and ceragenin CSA-13 against E.