During the procedure, subjects were instructed to rinse their mouth with water and chew a piece of sterilized rubber tourniquet to stimulate saliva, which was collected to yield

a total 1.0 mL. Samples were centrifuged CX-5461 price for 10 min at 15,000 × g at 4 °C, and the supernatants were immediately stored at −80 °C. The quantification of HBD-2 in saliva was done by an Enzyme Linked Immunosorbent Assay – ELISA (Peprotech, Rocky Hill, NJ, USA) according to manufacturer’s instructions. The process was carried as follows: 100 μL (0.25 μg/mL) of specific antibody (anti-HBD-2) was added to the 96-well polystyrene ELISA plates and incubated overnight (4 °C); after being washed four times with PBST (PBS with 0.05% Tween-20), 300 μL of a blocking solution (1% BSA in PBST) was added to the wells and incubated for 1 h at room temperature. Plates were then washed and 100 μL of the samples or standards were added into the respective PD0325901 purchase wells in duplicate and these plates were incubated for 2 h. After washing,

100 μL of detection antibody (0.5 μg/mL) was applied to the wells and plates were incubated for 2 h. After this period, plates were washed and 100 μL of streptavidin-conjugated horseradish peroxidase (1:2000 in PBST) was added to the respective wells and incubated for 30 min. Colorimetric reactions were developed using o-phenylenediamine in the presence of 0.02% H2O2. Reaction was stopped using H2SO4 (2N) and measured by an ELISA reader (OD 490 nm). One-way analysis of variance was used to compare means among groups. In case of significant differences among groups, post hoc two-group comparisons were assessed with a Tukey–Kramer test. The prevalence of P. gingivalis among groups was analysed using Dichloromethane dehalogenase the chi-square test. A p value < 0.05 was considered statistically significant. Data are expressed as mean ± SE. Mean pocket depth (PD) and mean clinical attachment loss (CAL) were significantly higher (p < 0.05) in subjects in the chronic periodontitis group than in those

in control. Clinical parameters were significantly (p < 0.05) improved by conventional periodontal treatment ( Table 1). Patients with chronic periodontitis showed a significant increase (p < 0.001) in the mean PAR2 mRNA expression relative to the GAPDH RT-PCR signal. Moreover, conventional periodontal treatment significantly (p < 0.05) decreased PAR2 mRNA expression ( Fig. 1A). Although being significantly (p < 0.05) more prevalent in patients with chronic periodontitis than in those in the control group, the levels of P. gingivalis decreased after periodontal therapy (p < 0.0001) ( Fig. 1B). Levels of TNF-α, that were also higher (p < 0.01) in chronic periodontitis patients also decreased after periodontal therapy (p < 0.001) ( Fig. 2A).