Lps from p gingivalis

KSHV-infected PEL cells (BCBL-1) were originally purchased from ATCC (kindly provided by Dr. Dean Kedes, University of Virginia) and maintained in RPMI 1640 media (Gibco) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES (pH 7.5), 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 0.05 mM β-mercaptoethanol, and 0.02% (wt/vol) sodium bicarbonate. Primary human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PDLF) were originally purchased from ScienCell by Dr. Amy Bradshaw (Medical University of South Carolina) and kindly provided to our laboratory. These cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Mediatech) supplemented with 10% FBS, 10 mM HEPES (pH 7.5), 100 U/mL of penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B. HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% FBS, 100 U/ml of penicillin, and 100 µg/ml streptomycin. The LTA derived from S. aureus and LPS from P. gingivalis were purchased from InvivoGen, and the purities were more than 99.5% as described by the manufacturer.

The following materials and antibodies were purchased: LPS from Escherichia coli 0128:B12 (Sigma-Aldrich); LPS from P. gingivalis (InvivoGen); recombinant human HSP70 (Stressgen; endotoxin activity, <0.05 endotoxin units (EU)/μg); recombinant bovine HSC70 (Stressgen; <0.05 EU/μg); recombinant bovine HSC70 ATPase fragment (Enzo Life Sciences; 0.1 EU/μg); V8 protease (Roche); 15-deoxyspergualin (DSG) (Spanidin® Inj.; Nippon Kayaku); mouse monoclonal anti-TLR4 (HTA125) and anti-TLR2 (TL2.1) antibodies (Hycult Biotechnology); mouse monoclonal anti-CD14 (MY4) antibody (Beckman Coulter); rabbit polyclonal anti-p44/42, anti-p38, anti-phosphorylated p38, anti-phosphorylated IκB-α, mouse monoclonal anti-phosphorylated p44/42 antibodies (Cell Signaling); mouse monoclonal anti-JUN-N-terminal protein kinase (JNK) (D-2), anti-phosphorylated JNK (G-7), anti-IκB-α (H-4) antibodies (Santa Cruz Biotechnology); and mouse monoclonal anti-β-actin antibody (Abcam). Endotoxin levels of all materials used were determined using a ToxinSensor chromogenic LAL endotoxin assay kit (GenScript), and the resultant level was low (~0.1 EU/μg). The LPS, bovine serum albumin (BSA), HSP70, HSC70, and the HSC70 ATPase fragment were heated at 95°C for 20 min.

Determination of anti-P. gingivalis and anti-Escherichia coli LPS-specific antibodies by enzyme-linked immunosorbent assay (ELISA). To optimise our evaluation anti-P. gingivalis antibody assessed by ELISA., we performed two standardised ways for coating: Whole extract or lipopolysaccharide (LPS) components. LPS from P. gingivalis (InvivoGen, Toulouse, France) was coated on 96-well plates (Nunc, Dominique Dutscher, Brumath, France) at 10 µg/mL (diluted in carbonate buffer, pH 9.6) and incubated overnight at 4 °C. We used LPS from Escherichia coli (E. coli, InvivoGen, Toulouse, France) as control with the same dilution. Wells were washed three times with phosphate buffered saline (PBS)-Tween (0.005%). Plasma were diluted to 1:600 in PBS containing 1% of bovine serum albumin (BSA) and incubated (in duplicate) for 2 h at room temperature. Plates were washed as described above and incubated with peroxidase-conjugated goat anti-human IgG H + L (Jackson ImmunoResearch, West Grove, PA, USA) (diluted 1:50 000 in PBS) for 2 h at room temperature. After a final wash, detection was made by tetramethylbenzidine substrate (R&D Systems, Minneapolis, MN., USA). The reaction was stopped by the addition of H₂SO₄ (1M) solution and absorbances were measured at 450 nm.

Three µl of 1 µg/ml of CpG ODN (InvivoGen, San Diego, CA), 1 µg/ml of LPS from P. gingivalis (InvivoGen) or 1 µg/ml of control ODN (InvivoGen) were topically applied to the palate as described above, and 100 µl of 10 µM OFS solution was injected through the retro-orbital venous plexus one day prior to euthanasia. At 4 days after the topical application of CpG ODN, P. gingivalis LPS or control ODN, the OFS fluorescent signal was measured. Statistical analysis was performed using two-way analysis of variance with Tukey’s multiple-comparison test to assess the difference among multiple experimental groups. P < 0.05 was considered as statistically significant. In addition, the harvested maxillas were subjected to immunohistochemial staining for Ctsk and stained with HE as described above.