Neutrophils were preincubated with monoclonal antibodies (5 μg mL−1) or PP1 (10 μM) for 15 min. Culture supernatants were collected after 0.5, 2, and 4 h of incubation in these experiments. Concentrations of resistin and elastase in the sample supernatants were measured by ELISA (R&D Systems and Hycult Biotech, respectively). Cell-free supernatants were diluted in dilution buffer at a ratio of 1 : 10 for resistin and 1 : 20 for elastase measurements. Both resistin and elastase were quantified with reference to a standard curve generated by serial dilution of recombinant proteins provided

by the manufacturer. TSA HDAC The lower limit of detection was in pg mL−1 for resistin and in ng mL−1 for elastase. Relative release of elastase is expressed as a percentage of the total elastase obtained by lysing the cells with 0.1% Triton X-100 (Promega) for 1 h. All samples were measured in duplicate. The level of cytolysis was determined by the amount of the cytosolic enzyme lactate

dehydrogenase (LDH) that was released, as measured using an LDH detection kit (Takara) according to the manufacturer’s instructions. Relative cytolysis is expressed as a percentage of the GSK J4 molecular weight total LDH activity obtained by lysing the cells with 0.1% Triton X-100 for 1 h. Data are presented as the means ± SD. Student’s t-test was used for comparisons between two groups. One-way anova was used to test whether the means of four groups were equal. When there was a statistical difference in anova, post hoc comparisons were assessed using Scheffe’s test or Dunnett’s test for multiple comparisons, as appropriate. Differences were considered statistically significant when P<0.05. The amount of resistin in the supernatant of the neutrophils incubated with HK 921 increased significantly with an increase in the ratio of bacteria to neutrophils in a dose-dependent manner (Fig. 1). The ratio of 1000 bacteria per neutrophil was used in subsequent experiments. The amounts of resistin and elastase in the supernatant

of the neutrophils incubated with HK921 Teicoplanin for 2 h or longer were significantly higher than those with the two minimally leukotoxic strains, HK912 and HK1604 (Fig. 2a and b). Leukotoxin was detected on Western blots of protein samples from the wild-type HK921 strain using rabbit antiserum against A. actinomycetemcomitans leukotoxin. No leukotoxin was detected in the HK921 strain with an insertional mutation of the ltxA gene (Fig. 3), confirming that leukotoxin was not expressed by the mutated strain. We measured the resistin, elastase, and LDH released from neutrophils after stimulation with the wild-type and mutant HK921 strains for 0.5, 2, and 4 h. The resistin level in the supernatant of the neutrophils incubated with wild-type HK921 was significantly higher than the level after incubation with the mutant strain (Fig. 4a).