The peptides

were eluted with 0–65% acetonitrile over 80 min. All MS and MS/MS spectra in the LCQ-Deca electron spray ion trap mass spectrometer were acquired in data-dependent mode. The MS/MS spectra were searched using mascot software (Matrix Science, Inc., San Jose, CA) using the genome data of K. pneumoniae ATCC 13883 from NCBI (http://www.ncbi.nlm.nih.gov/) and the decoy sequence database. Search parameters allowed for the oxidation of methionine, carbamidomethylation of cysteines, one missed selleck products trypsin cleavage and were within 1.5 Da for peptide tolerance and within 1.5 Da for fragment mass tolerance. The molecular percentage of proteins identified was calculated based on the exponentially NU7441 price modified protein abundance index, which was generated using mascot software (Ishihama et al., 2005). Growth of cells treated with different amounts of OMVs was measured with a Premix WST1 Cell Proliferation Assay System (TaKaRa, Ohtsu, Japan) (Choi et al., 2005). Cells were seeded at 2.0 × 105 mL−1 in a 96-well microplate. After treating with the K. pneumoniae OMVs

for 24 h, cellular growth was measured at 450 nm 2 h after treatment with WST1. Hep-2 cells were treated with different amounts of OMVs for 24 h. Total RNA was isolated from cells using the RNAzol B (Biotecx Laboratories, Houston, TX) according to the manufacturer’s instructions and quantified by spectrophotometry. Total RNA (1 μg) was reverse transcribed using M-MLV Reverse Transcriptase (Promega, Madison, WI). The PCR reaction was carried out following the manufacturer’s instructions (Takara). The primer sequences and product sizes were as follows: (1) glyceraldehyde 3-phosphate dehydrogenase (forward, 5′-CGTCTTCACCACCATGGAGA-3′, reverse, 5′-CGGCCATCACGCCACAGTTT-3′), 300 bp; (2) IL-1β (forward, 5′-AAAAGCTTGGTGATGTCT GG-3′, reverse, 5′-TTTCAACACGCAGGACAG G-3′), 179 bp; (3) IL-6 (forward, 5′-GTGTGAAAGCAGCAAAGAGGC-3, reverse, 5′-CTGGAGGTACTCTAGGTATAC-3′), 159 bp; (4) IL-8 (forward, 5′-ATGACTTCCAAGCTGGGCCGTG-3′, reverse, 5′-TATGAATTCTCAGCCCTCTTCAAAA-3′), 301 bp; (5) macrophage inflammatory protein (MIP)-1α (forward, 5′-ATGGAAACTCCAAACACCAC-3′,

reverse, 5′-CCCAGTCATCCTTCAACTTG-3′), STK38 298 bp (Cho et al., 2009). Seven-week-old female Balb/c mice were maintained under specific pathogen-free conditions. Neutropenic mice were induced with intraperitoneal injections of cyclophosphamide (150 mg kg−1) on days 4 and 3 before bacterial inoculation (van Faassen et al., 2007). Immunocompromised mice were anaesthetized with ketamine and then 100 μL of 1 × 108 CFU mL−1 of K. pneumoniae ATCC 13883 or 20 μg (protein concentration) of OMVs suspended in 100 μL of PBS were administered intratracheally. Control mice were inoculated with 100 μL PBS (pH 7.4). Mice were sacrificed 1 day after the challenge and their lungs were removed. Lung sections were stained with haematoxylin and eosin.