Effects of Candidalysin Derived from Candida albicans on the Expression of Pro-Inflammatory Mediators in Human Gingival Fibroblasts.

Candida albicans (Ca) is frequently detected in the peri-implant sulcus with peri-implantitis, a major postoperative complication after oral implant therapy. However, the involvement of Ca in the pathogenesis of peri-implantitis remains unclear. In this study, we aimed to clarify Ca prevalence in the peri-implant sulcus and investigated the effects of candidalysin (Clys), a toxin produced by Ca, on human gingival fibroblasts (HGFs). Peri-implant crevicular fluid (PICF) was cultured using CHROMagar and Ca colonization rate and colony numbers were calculated. The levels of interleukin (IL)-1ß and soluble IL-6 receptor (sIL-6R) in PICF were quantified by enzyme-linked immunosorbent assay (ELISA). Pro-inflammatory mediator production and intracellular signaling pathway (MAPK) activation in HGFs were measured by ELISA and Western blotting, respectively. The Ca colonization rate and the average number of colonies in the peri-implantitis group tended to be higher than those in the healthy group. IL-1ß and sIL-6R levels in the PICF were significantly higher in the peri-implantitis group than in the healthy group. Clys significantly induced IL-6 and pro-matrix metalloproteinase (MMP)-1 productions in HGFs, and co-stimulation with Clys and sIL-6R increased IL-6, pro-MMP-1, and IL-8 production levels in HGFs compared with Clys stimulation alone. These findings suggest that Clys from Ca plays a role in the pathogenesis of peri-implantitis by inducing pro-inflammatory mediators.

Diagnosis of inflammatory peri-implant diseases using an immunochromatographic assay for calprotectin in peri-implant crevicular fluid.

BACKGROUND: The incidence rate of peri-implant diseases is increasing with implant placement. Early detection of peri-implant diseases is important to prevent and treat these diseases, and a simple and objective diagnostic method is expected. Immunochromatographic (IC) assays are used for rapid diagnostic methods for some diseases. The aim of this clinical study was to determine the amount of calprotectin, an inflammatory marker, in peri-implant crevicular fluid (PICF) using an IC chip, and estimate the possibility of this diagnostic system. METHODS: Forty-six individuals with dental implants participated in a pilot study. PICF samples were collected from the peri-implant sites with or without inflammation after clinical examinations including probing depth (PD), bleeding on probing (BOP) and gingival index (GI). Calprotectin in PICF was determined by an IC chip and enzyme-linked immunosorbent assay (ELISA) for calprotectin. The density of calprotectin line on the IC chip was measured using an IC reader (IC reader value). The relationship between IC reader value and ELISA value or clinical parameters was investigated. A receiver operating characteristic (ROC) curve analysis of IC reader value of calprotectin was performed to predict inflammation in peri-implant diseases. RESULTS: IC reader value of calprotectin was significantly correlated with its ELISA value and PD. IC reader values of calprotectin in PICF samples from periodontal sites with GI-1 and GI-2, and with BOP-positive sites were significantly higher than those of PICF samples from GI-0 sites, and BOP-negative sites, respectively. The IC reader value for calprotectin in PICF samples from inflammatory diseased sites was significantly higher than that of non-diseased sites. ROC analysis suggested that the IC reader value of PICF calprotectin was useful for predicting inflammatory peri-implant diseases. CONCLUSION: IC assay for PICF calprotectin may be a possible system for diagnosing the inflammatory peri-implant diseases.

Gan-Lu-Yin (Kanroin), Traditional Chinese Herbal Extracts, Reduces Osteoclast Differentiation In Vitro and Prevents Alveolar Bone Resorption in Rat Experimental Periodontitis

Gan-Lu-Yin (GLY), a traditional Chinese herbal medicine, shows therapeutic effects on periodontitis, but that mechanism is not well known. This study aims to clarify the precise mechanism by investigating the inhibitory effects of GLY extracts on osteoclastogenesis in vitro and on bone resorption in periodontitis in vivo. RAW264.7 cells are cultured with soluble receptor activator of nuclear factor-kappa B (sRANKL) and GLY extracts (0.01-1.0 mg/mL), and stained for tartrate-resistant acid phosphatase (TRAP) to evaluate osteoclast differentiation. Experimental periodontitis is induced by placing a nylon ligature around the second maxillary molar in rats, and rats are administered GLY extracts (60 mg/kg) daily for 20 days. Their maxillae are collected on day 4 and 20, and the levels of alveolar bone resorption and osteoclast differentiation are estimated using micro-computed tomography (CT) and histological analysis, respectively. In RAW264.7 cells, GLY extracts significantly inhibit sRANKL-induced osteoclast differentiation at a concentration of more than 0.05 mg/mL. In experimental periodontitis, administering GLY extracts significantly decreases the number of TRAP-positive osteoclasts in the alveolar bone on day 4, and significantly inhibits the ligature-induced bone resorption on day 20. These results show that GLY extracts suppress bone resorption by inhibiting osteoclast differentiation in experimental periodontitis, suggesting that GLY extracts are potentially useful for oral care in periodontitis.

High Glucose-Mediated Cytokine Regulation in Gingival Fibroblasts and THP-1 Macrophage: a Possible Mechanism of Severe Periodontitis with Diabetes.

BACKGROUND/AIMS: Diabetic patients are susceptible to severe periodontitis, but the precise mechanism is not fully understood. Aim of this study was to explore the biological pathogenesis of severe periodontitis in diabetic patients focusing on the crosstalk of human gingival fibroblasts (HGFs) and macrophages. METHODS: A total of 70 periodontitis patients with or without diabetes mellitus (DM) were enrolled, and the statistical relationships of diabetic conditions to the periodontal inflammatory parameters were examined by cross-sectional study. In in vitro study, HGFs cell line CRL-2014® (ATCC) and differentiated THP-1 macrophages were cultured with normal glucose (NG: 5.5 mM) or high glucose (HG: 25 mM) condition, and treated with indicated inflammatory factors such as calprotectin (CPT), interleukin (IL)-1ß and IL-6. To examine the effects of HG on soluble IL-6 receptor (sIL-6R) production in THP-1 macrophages, the supernatants were collected and the sIL-6R levels were measured by ELISA. To examine the effects of HG on IL-1ß or IL-6-induced matrix metalloproteinase (MMPs) production in HGFs, the supernatants were collected. Levels of MMP-1 and tissue inhibitor of MMP-1 (TIMP-1) were measured by ELISA. Finally, after conditioned medium (CM) from THP-1 macrophages cultured with NG or HG conditions was collected, HGFs were treated with the CM. The supernatants were collected 24 hours later and the levels of MMP-1 and TIMP-1 were measured. To examine the specific effects of IL-1ß contained in CM on MMP-1 and TIMP-1 production in HGFs, IL-1 receptor antagonist (IL-1ra) was used. RESULTS: There were statistical correlation between IL-1ß and sIL-6R levels in gingival crevicular fluid (GCF) and HbA1c in periodontitis patients with DM (IL-1ß: P=0.035, sIL-6R: P=0.040). HG and CPT significantly induced sIL-6R production in THP-1 macrophages. HG significantly enhanced IL-1ß or IL-6/sIL-6R-induced MMP-1 production in HGFs. The increase of MMP-1 by both IL-1ß and IL-6/sIL-6R was significantly inhibited by specific ERK or IκB inhibitors. Corresponding to the regulation of MMP-1 production, HG condition increased the phosphorylation of p44/42 MAPK and IκBα in HGFs treated with IL-1ß or IL-6/sIL-6R. Finally, MMP-1 production in HGFs cultured with HG increased significantly by CM from THP-1 macrophages cultured with HG. The induction of MMP-1 by the CM from THP-1 macrophages cultured with HG was significantly inhibited by dose dependent of IL-1ra in HGFs cultured with HG. CONCLUSION: Diabetic conditions such as HG induce IL-1ß and sIL-6R production from macrophages in inflammatory periodontal tissues and may exacerbate the periodontitis synergistically via MMP-1 production from HGFs.

Relationship of aspiration pneumonia to cognitive impairment and oral condition: a cross-sectional study.

OBJECTIVES: The objective of this study was to investigate the relationship of the incidence of aspiration pneumonia to cognitive impairment and the oral condition. MATERIALS AND METHODS: A total of 1174 elderly patients were analyzed in a cross-sectional study. Cognitive function was evaluated by the Clinical Dementia Rating scale and the oral condition was evaluated by inspection and palpation. Swallowing was examined in 196 patients by video-endoscopic evaluation. The Mann-Whitney U test or chi-square test was used for statistical analysis. Conditional logistic regression analysis was performed to compute the odds ratio (OR) and 95% confidence interval (CI). RESULTS: Loss of posterior occlusion, impaired tongue movements, and impaired cognition were factors significantly related to aspiration pneumonia. The incidence of aspiration pneumonia was higher in patients with both cognitive impairment and loss of posterior occlusion compared with those having either factor alone (OR: 5.16). There was no statistical association between impaired swallowing and the incidence of aspiration pneumonia in elderly patients with normal cognitive function (cognitive impairment, OR: 3.45; normal function, OR: 0.94). CONCLUSION: Co-existence of cognitive impairment and oral frailty significantly enhances the risk of aspiration pneumonia. CLINICAL RELEVANCE: Early and simple evaluation of the oral condition and cognitive function can predict the risk of aspiration pneumonia.

ß-carotene suppresses Porphyromonas gingivalis lipopolysaccharide-mediated cytokine production in THP-1 monocytes cultured with high glucose condition.

Periodontitis is associated with development of diabetes mellitus. Although lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg), a major pathogen of periodontitis, may lead the progression of diabetes complications, the precise mechanisms are unclear. We, therefore, investigated the effects of ß-carotene on production of Pg LPS-induced inflammatory cytokines in human monocytes cultured high glucose (HG) condition. THP-1 cells were cultured under 5.5 mM or 25 mM glucose conditions, and cells were stimulated with Pg LPS. To investigate the productivity of TNF-α, IL-6, and MCP-1, cell supernatants were collected for ELISA. To examine the effects of NF-kB signals on cytokine production, Bay11-7082 was used. HG enhanced Pg LPS-induced production of TNF-α, IL-6, and MCP-1 via NF-kB signals in THP-1. ß-carotene suppressed the enhancement of the Pg LPS-induced cytokine production in THP-1 via NF-κB inactivation. Our results suggest that ß-carotene might be a potential anti-inflammatory nutrient for circulating Pg LPS-mediated cytokine production in diabetic patients with periodontitis.

Swallowing impairment is a significant factor for predicting life prognosis of elderly at the end of life.

BACKGROUND: In end-of-life care of elderly, the decision of care plan including gastrostomy is difficult frequently because of insufficient knowledge relating the life prognosis of elderly. It is important the families to decide correctly the life prognosis of elderly with geriatric diseases. Our purpose is to examine the significant factors for predicting life prognosis of elderly in end-of-life care. METHODS: A total of 320 elderly patients was enrolled (male/female 151/169; averaged age: male 84.7 ± 5.9 year, female 86.8 ± 6.3 year) and retrospective analyses were performed. The elderly patients were classified as either: (1) with or without past illness of aspiration pneumonia; (2) with or without incidence of cerebrovascular disorder; (3) impaired or normal cognitive function; (4) impaired or normal swallowing function, and performed Kaplan-Meier survival analysis. Swallowing function was examined using video endoscopic (VE) evaluation method. The Kaplan-Meier analysis of the number of days from implementation of VE test (day 0) to death was evaluated with the log-rank Mantel-Cox test. The maximum follow-up time recorded was 180 days. RESULTS: There were no significant differences in number of days when divided with or without past illness of aspiration pneumonia, cerebrovascular disorder and impaired cognitive function. The survival probabilities of elderly with impaired swallowing function were significant lower than in elderly with the normal function. CONCLUSIONS: For judgement of life prognosis, the condition of being frail such as impaired swallowing function might be a useful factor, and the viewpoint would contribute to decide the treatment plan for the good end-of-life care of elderly.

Calprotectin Induces IL-6 and MCP-1 Production via Toll-Like Receptor 4 Signaling in Human Gingival Fibroblasts.

Calprotectin, a heterodimer of S100A8 and S100A9 molecules, is associated with inflammatory diseases such as inflammatory bowel disease. We have reported that calprotectin levels in gingival crevicular fluids of periodontitis patients are significantly higher than in healthy subjects. However, the functions of calprotectin in pathophysiology of periodontitis are still unknown. The aim of this study is to investigate the effects of calprotectin on the productivity of inflammatory cytokines in human gingival fibroblasts (HGFs). The HGFs cell line CRL-2014® (ATCC) were cultured, and total RNAs were collected to examine the expression of TLR2/4 and RAGE mRNA using RT-PCR. After the cells were treated with S100A8, S100A9, and calprotectin, supernatants were collected and the levels of IL-6 and MCP-1 were measured using ELISA methods. To examine the intracellular signals involved in calprotectin-induced cytokine production, several chemical inhibitors were used. Furthermore, after the siRNA-mediated TLR4 down-regulated cells were treated with S100A8, S100A9, and calprotectin, the levels of IL-6 and MCP-1 were also measured. HGFs showed greater expression of TLR4 mRNA, but not TLR2 and RAGE mRNA compared with human oral epithelial cells. Calprotectin increased significantly the production of MCP-1 and IL-6 in HGFs, and the cytokine productions were significantly suppressed in the cells treated with MAPKs, NF-κB, and TLR4 inhibitors. Furthermore, calprotectin-mediated MCP-1 and IL-6 production were significantly suppressed in TLR4 down-regulated cells. Taken together, calprotectin induces IL-6 and MCP-1 production in HGFs via TLR4 signaling that involves MAPK and NF-κB, resulting in the progression of periodontitis. J. Cell. Physiol. 232: 1862-1871, 2017. © 2016 Wiley Periodicals, Inc.

Clinical significance of GCF sIL-6R and calprotectin to evaluate the periodontal inflammation.

Background Periodontitis is an inflammatory disease. The aim of this study was to investigate whether the soluble form of interleukin-6 receptor (sIL-6R) and calprotectin concentrations in gingival crevicular fluid are useful biomarkers in the evaluation of periodontitis. Methods First, a cross-sectional study was performed. A total of 34 periodontitis patients were enrolled and the gingival crevicular fluid samples were collected from the healthy and inflamed sites of periodontal pockets in each patient. The relationship between periodontal condition and gingival crevicular fluid sIL-6R and calprotectin concentrations was analysed statistically. The cut-off values of gingival crevicular fluid sIL-6R and calprotectin concentrations for the evaluation of periodontitis were determined using a receiver operating characteristic curve. Next, by using enzyme-linked immunosorbent assay, it was examined whether calprotectin induces sIL-6R production in THP-1 macrophages. Results Both gingival crevicular fluid sIL-6R and calprotectin concentrations were significantly higher in the inflamed sites than in the healthy sites ( P < 0.0001). The cut-off values of gingival crevicular fluid sIL-6R and calprotectin concentrations for the evaluation of periodontal inflammation were as follows: sIL-6R: 43.5 pg/site; calprotectin: 134.3 ng/site. In the in vitro study, calprotectin significantly induced sIL-6R production in THP-1 macrophages ( P < 0.01). Conclusions Both gingival crevicular fluid sIL-6R and calprotectin concentrations are significant biomarkers in the evaluation of periodontal inflammation.