| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2002: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2001: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
The existence of calprotectim (CPT), a leukocyte protein, in gingival tissue with periodontal disease and the effect of lipopolysaccharide (LPS) from periodontopahic bacteria on CPT expression were investigated to elucidate the regulatory mechanism of CPT in periodontal diseases. Gingival tissue was obtained from a periodontitis parient and immunostained using anti-CPT antibody (Ab). Neutrophils were separated from human healthy donors and incubated with LPS from P. gingivalis. CPT in cell and medium fractions was identified by immunobolotting analysis, and its amount was determined by ELISA. CPT was detected in the connective tissue with inflammatory cells and epithelium. P-LPS increased CPT release from neutrophils after 30 min incubation in a dose-dependent manner (0.1-1,000 ng/ml), and the released amount was increased to about 16 times that of control level by 1,000 ng/ml P-LPS. LPSs from A. actinomycelemcomitans, P. intermedia, F. nucleatum also induced calprotectin release, and P-LPS produced the maximal effect. It is known that CD14, Toll-like receptor (TLR) 2&4 and NF-_KB play roles in LPS signal transduction. To study the mechanism of P-LPS-induced CPT release, the effects of anti-CD14 and TLR Abs and NF-_KB inhibitors on P-LPS-induced CPT release were investigated. The anti-CD14 and TLR2 Abs significantly inhibited P-LPS-induced CPT release, but anti-TLR4 Abs had no effect. Five NF-_KB inhibitors blocked P-LPS-induced NF-_KB binding activity (Gel shift assay) and CPT release from neutrophils. Futhermore, P-LPS did not increase the expression of MRP8/14, subunits of CRT, mRNAs when they were investigated by RT-PCR. From these results, it is suggested that LPSs from periodontopahic bacteria including P. gingivalis induce CTP release from neutrophils and that CPT release is induced by P-LPS via the CD14-TLR2-NF_KB signal pathway
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