Application of Lactoferrin and α1-Antitrypsin in Gingival Retention Fluid to Diagnosis of Periodontal Disease

Objectives. Periodontal disease is prevalent and has an inflammation associated with not only oral but also systemic pathologies. The diagnosis by biomarkers is required for clinical practice on periodontal disease. The lactoferrin and α1-antitrypsin were both inflammation-related molecules. The present study investigated the relationship between the periodontal status and the two biomarkers in gingival retention fluid (GRF). Patients and Methods. In 63 subjects with periodontitis, the GRF was sampled from maxillary anterior gingiva using a microbrush for 30 seconds. The lactoferrin and α1-antitrypsin levels in GRF were measured by an enzyme-link solvent immunoassay. Periodontal status was evaluated by probing pocket depth (PD) and bleeding on probing (BOP). Results. There was a higher level of these biomarkers in saliva (median (ng/mL), lactoferrin: 3611.9, α1-antitrypsin: 4573.3) than in GRF (lactoferrin: 61.0, α1-antitrypsin: 54.7). There was a mild-to-moderate but significantly positive correlation in lactoferrin or α1-antitrypsin between GRF and saliva. There was a positively mild-to-moderate accuracy (area under the curve: 0.60–0.81) of lactoferrin or α1-antitrypsin in GRF or in saliva to distinguish the severity of periodontal status. The cutoff level (ng/mL) of lactoferrin in GRF for detecting ≥30% of PD ≥ 4 mm (moderate periodontitis) was 68.6 and for detecting ≥20% of BOP (clinically active periodontitis) was 61.2. The cutoff level (ng/mL) of α1-antitrypsin in GRF for detecting ≥30% of PD ≥ 4 mm was 54.5 and for detecting ≥20% of BOP was 35.3. Conclusions. The data can promote an application of the measurements of lactoferrin and α1-antitrypsin in GRF to clinical practice on periodontal disease

Nicotine-induced expression of low-density lipoprotein receptor in oral epithelial cells.

BACKGROUND: Nicotine use is one of the most important risk factors for the development of cardiovascular and periodontal diseases. Numerous reports have suggested the possible contribution of disturbed lipid metabolism for the development of both disease groups. Despite these observations, little is known about the relationship between tobacco smoking and the development of these diseases. Our previous microarray data revealed that nicotine induced low-density lipoprotein receptor (LDLR) expression in oral epithelial cells (OECs). The aim of the present study was to confirm nicotine-mediated LDLR induction and to elucidate the signaling mechanisms leading to the augmented expression of LDLR in OECs. METHODS AND RESULTS: LDLR and nicotinic acetylcholine receptor (nAChR) subunit expression was detected by real-time PCR. The production of LDLR was demonstrated by immunofluorescence staining. nAChR-mediated LDLR induction was examined by pre-incubation of the cells with its specific inhibitor, α-bungarotoxin (α-BTX). The functional importance of transcription factor specific protein 1 (Sp1) was examined by luciferase assay, mithramycin pre-incubation or by small interfering RNA (siRNA) transfection. The specific binding of Sp1 to R3 region of LDLR 5'-untranslated region was demonstrated with electrophoretic mobility shift assay (EMSA) and streptavidin-agarose precipitation assay followed by western blotting. The results confirmed that nicotine induced LDLR expression at the transcriptional level. Nicotine was sensed by nAChR and the signal was transduced by Sp1 which bound to the R3 region of LDLR gene. Augmented production of LDLR in the gingival epithelial cells was further demonstrated by immunofluorescence staining using the gingival tissues obtained from the smoking patients. CONCLUSIONS: Taken together, the results suggested that nicotine might contribute to the development of both cardiovascular and periodontal diseases by inducing the LDLR in OECs thereby disturbing lipid metabolism

Sp1 plays an important role in nicotine-mediated LDLR expression.

<p>(A) Ca9-22 cells were pre-treated with or without mithramycin for 1 h. After washes, the cells were stimulated with 100 μM of nicotine for 3 h and luciferase activity was measured. (B) Ca9-22 cells were transfected with various concentrations of siRNA against Sp1 or control siRNA (con) for 3 h. After transfection, the cells were stimulated with or without 100 μM of nicotine for 3 h. The expression level of LDLR mRNA was examined by real-time PCR. (C) The silencing effect of siRNA transfection on the expression of Sp1 was assessed by real-time PCR. Sp1 expression level of control siRNA-transfected Ca9-22 cell was set as 1. The data are presented as mean ± SD of at least 3 separate experiments. *p < 0.05.</p

Nicotine stimulation leads to Sp1 binding to R3.

<p>(A) Left panel: Ca9-22 cells were stimulated with (+) or without (-) nicotine for 3 h. After stimulation, the cells were harvested and the nuclear extracts were prepared. Twenty μg of nuclear extract was incubated with biotinylated R3 probe with (+) or without (-) cold R3 probe. The samples were separated by native PAGE and transferred to a nylon membrane. The retarded band was detected by incubating the membrane with streptavidine-HRP followed by ECL. The representative of 3 separate experiments was shown. Right panel: Nicotine-stimulated nuclear extract was incubated with or without R2 or R3 probe and the retarded bands were detected as described above. (B) Nicotine-stimulated nuclear extracts were prepared from Ca9-22 cells and subjected to Stre-Av precipitation assays. Upper panel: lane 1: Stre-Av precipitation assay performed without nuclear extract. Lane 2: Stre-Av precipitation assay with nuclear extract. Lane 3: Stre-Av precipitation assay with biotinylated and non-labeled R3 probes. Lower panel: lane 1: Stre-Av precipitation assay performed without probe. Lane 2: Stre-Av precipitation assay with R2 probe. Lane 3: Stre-Av precipitation assay with R3 probe.</p

Distinct contributions of R1, R2, and R3 of LDLR gene regulatory regions to nicotine-mediated LDLR expression.

<p>(A) Schematic illustration of the 271 bp of the 5ʹ-UTR of the LDLR gene. Nucleotide numbering is relative to the translation initiation site AUG where A is +1. The positions of R1 (-103), R2, and R3 (-68) were indicated as boxes. WT indicates the wild type structure. R1, R2, and R3 represent the mutant constructs lacking each sequence. Each fragment was subcloned in the pGL4-basic vector and used for luciferase assays. Ca9-22 cells were transfected with WT (B) or with R1, R2, or R3 (C) along with normalized pRL-CMV vector for 3 h. After transfection, the cells were stimulated with or without nicotine for 3 h and luciferase activity was measured. At least 3 independent experiments were performed. The data are presented as mean ± SD. *p < 0.05.</p

Increased expression of LDLR in the gingival epithelium from smoking patients.

<p>(A) The gingival tissues were excised from smoking (lower panel) or non-smoking patients (upper panel) and fixed with 5% acetic acid-ethanol. The tissues were embedded in paraffin and 4 μm sections were prepared. The specimens were incubated with anti-LDLR Ab followed by FITC-conjugated goat anti-rabbit IgG Ab. Images were viewed and photographed using a LSM510 confocal laser microscope (Carl Zeiss, Heidelberg, Germany). Green, LDLR; red, nuclei with monomeric cyanine nucleic acid stain. Scale bar (white line): 50 μm.</p