Synthesis of secretory leukocyte protease inhibitor using cell-free protein synthesis system.

Secretory leukocyte protease inhibitor (SLPI) functions as a protease inhibitor that modulates excessive proteolysis in the body, exhibits broad-spectrum antimicrobial activity, regulates inflammatory responses, and plays an important role in the innate immunity. The purpose of the study was to artificially synthesize a SLPI, an antimicrobial peptide, and investigate its effect on antimicrobial activity against Porphyromonas gingivalis and interleukin-6 (IL-6) production. SLPI protein with a molecular weight of approximately 13 kDa was artificially synthesized using a cell-free protein synthesis (CFPS) system and investigated by western blotting and enzyme-linked immunosorbent assay (ELISA). Disulfide bond isomerase in the protein synthesis mixture increased the amount of SLPI synthesized. The synthesized SLPI (sSLPI) protein was purified and its antimicrobial activity was investigated based on the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells. The effect of sSLPI on IL-6 production in human periodontal ligament fibroblasts (HPLFs) was examined by ELISA. Our results showed that sSLPI significantly inhibited the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells and further inhibited IL-6 production by HPLFs. These results suggested that SLPI artificially synthesized using the CFPS system may play a role in the prevention of periodontal diseases through its antimicrobial and anti-inflammatory effects.

ß-defensin 2 synthesized by a cell-free protein synthesis system and encapsulated in liposomes inhibits adhesion of Porphyromonas gingivalis to oral epithelial cells.

ß-defensin 2 (BD-2), an antimicrobial peptide (AMP), is expressed by oral epithelial cells and plays an important role in innate immunity of the oral cavity. Cell-free protein synthesis (CFPS) systems have been studied for the synthesis of various proteins, however, the synthesis of BD-2 by a CFPS system has not been extensively explored. Liposomes have been developed as tools for drug delivery. A delivery of liposome-encapsulated AMP to oral epithelium may be useful to prevent oral infectious diseases. In the present study, we investigated the antimicrobial activity of the BD-2 protein, artificially synthesized using a CFPS system and encapsulated in liposomes. BD-2 protein was artificially synthesized using template DNA and a reconstituted CFPS system and was identified by western blotting. Bilayer liposomes were prepared using 1,2-dioleoyl-sn-glycero-3-phospho-choline and 3-sn-phosphatidylcholine from egg yolk. The artificially synthesized BD-2 was encapsulated in liposomes, collected by ultrafiltration, and detected by western blotting. Human oral epithelial cells were cultured with the liposome-encapsulated BD-2 and the concentration of BD-2 in the cell lysate of the culture with the synthesized BD-2 was higher than that of the control cultures. The antimicrobial activity of the synthesized BD-2 was investigated by an adhesion assay of Porphyromonas gingivalis to oral epithelial cells. The artificially synthesized BD-2 and its liposome significantly inhibited adhesion of P. gingivalis to oral epithelial cells. These results suggest that artificially synthesized BD-2 and liposome-encapsulated BD-2 show antimicrobial activity and can potentially play a role in oral healthcare for periodontal diseases.

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.

Utility of a haemoglobin test of gingival crevicular fluid: A multicentre, observational study.

OBJECTIVE: The purpose of this study was to verify the accuracy and utility of clinical parameters (plaque index, gingival crevicular fluid volume, probing depth, clinical attachment level, bleeding on probing and gingival index) and biochemical parameters (aspartate aminotransferase, protein and haemoglobin) in a longitudinal analysis during the supportive periodontal therapy period. SUBJECTS AND METHODS: A total of 279 test sites of 128 patients were investigated clinically and biochemically. After the first examination of clinical and biochemical parameters, periodontal support treatments were administered immediately and performed once every three months up to the second examination. RESULTS: All of the clinical and biochemical parameters were significantly lower at the second examination than at the first, except for the plaque index and bleeding on probing. Of these parameters, in particular, aspartate aminotransferase and haemoglobin in the gingival crevicular fluid were significantly reduced compared to those of the first examination in both the ≤4 and ≥5 mm probing depth groups, and they clearly suggested that periodontitis tended to recover. CONCLUSION: Adding the haemoglobin test to the bleeding on probing test strongly improves the accuracy of measurement of clinical parameters after periodontal treatment.

Lipocalin 2, synthesized using a cell-free protein synthesis system and encapsulated into liposomes, inhibits the adhesion of Porphyromonas gingivalis to human oral epithelial cells.

BACKGROUND AND OBJECTIVE: Lipocalin 2 (LCN2), a glycoprotein expressed in epithelial cells and leukocytes, has an antibacterial effect and plays a role in innate immunity. The delivery of LCN2 encapsulated in liposomes to oral epithelium may be useful to prevent oral infectious diseases. This study aimed to investigate the inhibitory effect of LCN2, artificially synthesized using a cell-free protein synthesis (CFPS) system, on the adhesion of Porphyromonas gingivalis to oral epithelial cells in order to approach oral healthcare using LCN2. METHODS: LCN 2 was synthesized using a CFPS system and assayed by Western blotting, mass spectrometry and enzyme-linked immunosorbent assay (ELISA). The bilayer liposomes were prepared by the spontaneous transfer method using 1,2-dioleoyl-sn-glycero-3 phosphocholine (DOPC), 3-sn-phosphatidylcholine from Egg Yolk (Egg-PC), and 1,2-dioleoyl-sn-glycero-3 phosphoethanolamine (DOPE). The cellular and medium fractions derived from the culture of oral epithelial cells with liposome-encapsulated LCN2 were assayed by Western blotting and ELISA. The effect of the synthesized LCN2 on adhesion of the labeled P. gingivalis to oral epithelial cells was investigated as an evaluation of its antibacterial activity. RESULTS: The synthesized LCN2 protein was identified by Western blotting; its amino acid sequence was similar to that of recombinant LCN2 protein. The additions of DOPE and octa-arginine in the outer lipid-layer components of liposome significantly increased the delivery of liposomes to epithelial cells. When oral epithelial cells were cultured with the synthesized and liposome-encapsulated LCN2, LCN2 was identified in the cellular and medium fractions by Western blotting and its concentration in the cellular fraction from the culture with the synthesized LCN2 was significantly higher than that of a template DNA-free protein. The synthesized LCN2 and liposome-encapsulated LCN2 significantly inhibited the adhesion of P. gingivalis to oral epithelial cells compared with template DNA-free protein. CONCLUSION: LCN2 was artificially synthesized by a CFPS system, encapsulated in liposomes, and delivered to oral epithelial cells, and demonstrated an antibacterial action against P. gingivalis. This approach may become a useful model for oral healthcare.

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.

Advanced glycation end-products increase lipocalin 2 expression in human oral epithelial cells.

BACKGROUND AND OBJECTIVES: Diabetes mellitus (DM), a risk factor of periodontal diseases, exacerbates the pathological condition of periodontitis. A major factor for DM complications is advanced glycation end-products (AGEs) that accumulate in periodontal tissues and cause inflammatory events. Lipocalin 2 (LCN2) is an antimicrobial peptide and inflammation-related factor, and LCN2 levels increase in DM. In this study, the effects of AGEs and lipopolysaccharide of Porphyromonas gingivalis (P g-LPS) on LCN2 expression in human oral epithelial cells (TR146 cells) and the role of secreted LCN2 in periodontitis with DM were investigated. MATERIAL AND METHODS: TR146 cells were cultured with AGEs (AGE2) and control BSA and cell viability was estimated, or with P g-LPS. Conditioned medium and cell lysates were prepared from cultures of epithelial cells and used for Western blotting and ELISA to analyze LCN2, RAGE, IL-6, MAPK, and NF-κB. RNA was isolated from AGE-treated TR146 cells and differentiated HL-60 (D-HL-60) cells and used for quantitative real-time PCR to examine the expression of LCN2 and interleukin-6 (IL-6) mRNAs. RAGE- and LCN2-siRNAs (siRAGE, siLCN2) were transfected into epithelial cells, and AGE-induced LCN2 expression was investigated. D-HL-60 cells were co-cultured with TR146 cells that were transfected with siLCN2 and treated with AGEs, and IL-6 mRNA expression in D-HL-60 cells and cell migration was investigated. RESULTS: AGEs increased the expression levels of LCN2 and IL-6 in oral epithelial cells. siRAGE and a neutralizing antibody for RAGE inhibited AGE-induced LCN2 expression. AGEs stimulated the phosphorylation of ERK, p38, and NF-κB in epithelial cells, and their inhibitors suppressed AGE-induced LCN2 expression. In contrast, P g-LPS did not show a significant increase in LCN2 level in TR146 cells that expressed Toll-like receptor 2. In co-culture experiments, AGE-induced LCN2 inhibited IL-6 mRNA expression in D-HL-60 cells, and LCN2 knockdown in epithelial cells suppressed HL-60 cell migration. CONCLUSION: These results suggested that AGEs increase LCN2 expression via RAGE, MAPK, and NF-κB signaling pathways in oral epithelial cells, and secreted LCN2 may influence the pathological condition of periodontitis with DM.

S100A9 Increases IL-6 and RANKL Expressions through MAPKs and STAT3 Signaling Pathways in Osteocyte-Like Cells.

OBJECTIVE: Calprotectin is a heterocomplex of S100A8 and S100A9 and is mainly secreted from neutrophils, monocytes, and chondrocytes in inflammatory condition. Calprotectin binds to RAGE and TLR4 and induces the expression of proinflammatory chemokines and cytokines in various cells. Periodontitis is a chronic inflammatory disease that leads to gingival inflammation and alveolar bone resorption. Calprotectin levels in gingival crevicular fluid of periodontitis patients are higher than healthy patients. In the present study, the effects of S100A8 and S100A9 on the expressions of proinflammatory cytokines and bone metabolism-related factors in mouse osteocyte-like cells (MLO-Y4-A2) were investigated. DESIGN: MLO-Y4-A2 cells were treated with S100A8 and S100A9, and the expressions of RAGE, TLR4, RANKL, and several inflammatory cytokines were analyzed by PCR and Western blotting or ELISA methods. To investigate the intracellular signaling pathways, phosphorylation of MAPK and STAT3 was determined by Western blotting, and chemical specific inhibitors and siRNAs were used. RESULTS: Expressions of IL-6 and RANKL were increased by treatment with S100A9 but not S100A8. However, both S100A8 and S100A9 did not change expression of IL-1ß, IL-8, and TNF-α. Although RAGE and TLR4 expressions were not upregulated by S100A9 treatment, transfection of siRNA for RAGE and TLR4 significantly decreased IL-6 and RANKL expressions. In addition, S100A9 activated p38, ERK, and STAT3 signaling pathways, and inhibitors for these factors significantly decreased S100A9-induced IL-6 and RANKL expressions. CONCLUSIONS: These results indicated that S100A9 induces IL-6 and RANKL production via engagement with RAGE and TLR4 signalings in osteocytes and suggested that S100A9 may play important roles in the periodontal alveolar bone destruction.

6-Shogaol Inhibits Advanced Glycation End-Products-Induced IL-6 and ICAM-1 Expression by Regulating Oxidative Responses in Human Gingival Fibroblasts.

Advanced glycation end-products (AGEs) cause diabetes mellitus (DM) complications and accumulate more highly in periodontal tissues of patients with periodontitis and DM. AGEs aggravate periodontitis with DM by increasing the expression of inflammation-related factors in periodontal tissues. 6-Shogaol, a major compound in ginger, has anti-inflammatory and anti-oxidative activities. However, the influence of shogaol on DM-associated periodontitis is not well known. In this study, the effects of 6-shogaol on AGEs-induced oxidative and anti-oxidative responses, and IL-6 and ICAM-1 expression in human gingival fibroblasts (HGFs) were investigated. When HGFs were cultured with 6-shogaol and AGEs, the activities of reactive oxygen species (ROS) and antioxidant enzymes (heme oxygenase-1 [HO-1] and NAD(P)H quinone dehydrogenase 1 [NQO1]), and IL-6 and ICAM-1 expressions were investigated. RAGE expression and phosphorylation of MAPKs and NF-κB were examined by western blotting. 6-Shogaol significantly inhibited AGEs-induced ROS activity, and increased HO-1 and NQO1 levels compared with the AGEs-treated cells. The AGEs-stimulated expression levels of receptor of AGE (RAGE), IL-6 and ICAM-1 and the phosphorylation of p38, ERK and p65 were attenuated by 6-shogaol. These results suggested that 6-shogaol inhibits AGEs-induced inflammatory responses by regulating oxidative and anti-oxidative activities and may have protective effects on periodontitis with DM.

Nucleic acid purification from dried blood spot on FTA Elute Card provides template for polymerase chain reaction for highly sensitive Plasmodium detection.

Polymerase chain reaction (PCR) is an essential diagnostic method for highly sensitive detection of Plasmodium-infected erythrocytes in patients with malaria. This study compared the performance of filter papers used for the preparation of dried blood spots (DBS) in detecting Plasmodium by PCR. Whole blood spiked with P. falciparum-infected erythrocytes to obtain samples with various levels of parasitemia were applied to Whatman 3MM Chr papers, FTA Cards, or FTA Elute Cards to prepare the DBS. DNA was purified from the DBS using a DNA purification kit and used as the template for nested PCR. In probit analysis, the estimated limit of detection (LoD) was 5.5 parasites/µL blood for Whatman 3MM Chr papers and FTA Cards and 1.6 parasites/µL blood for the FTA Elute Card. This result suggested that the DBS prepared on an FTA Elute Card yield the best template DNA for subsequent high-sensitivity PCR-based detection of P. falciparum-infected erythrocytes. This finding can help improve the accuracy of malarial diagnostic tests.

Advanced glycation end-product 2 and Porphyromonas gingivalis lipopolysaccharide increase sclerostin expression in mouse osteocyte-like cells.

Sclerostin is a secreted glycoprotein that is mainly expressed in osteocytes, exerts negative effects on bone formation, and is present at elevated levels in diabetes mellitus (DM). Periodontitis is an infectious disease caused by periodontopathic bacteria, a complication of DM, and sometimes associated with severe inflammation and alveolar bone resorption. Advanced glycation end-products (AGEs) are a major pathogen in DM complications and adversely influence periodontitis in DM patients. In the present study, the effects of AGE2 and Porphyromonas gingivalis lipopolysaccharide (P-LPS) on the expression of sclerostin in mouse osteocyte-like cells (MLO-Y4-A2 cells) and its function in osteoblast differentiation were investigated. AGE2 and P-LPS up-regulated the expressions of receptor of AGE (RAGE) and Toll-like receptor 2 (TLR2), respectively, and significantly up-regulated that of sclerostin and interleukin 6 (IL-6) in osteocytes. Sclerostin, RAGE and TLR2 levels were synergistically increased by AGE2 and P-LPS. The siRNAs of RAGE and TLR2 significantly inhibited AGE2- and P-LPS-induced sclerostin expression. AGE2 up-regulated sclerostin expression in osteocyte-like cells via the RAGE, ERK and JNK, and NF-κB signal pathways. On the other hand, P-LPS elevated sclerostin levels via the TLR2, JNK and p38, and NF-κB signal pathways. When osteocytes pre-treated with AGE2 and P-LPS and osteoblastic cells (MC3T3-E1) were co-cultured in the medium with a sclerostin-neutralizing antibody, AGE2- and P-LPS-induced decreases in alkaline phosphatase activity and Runx2 expression in osteoblastic cells were significantly inhibited by the sclerostin-neutralizing antibody. These results suggest that AGE2 and P-LPS influence bone metabolism and inflammation through the regulation of sclerostin expression, and may aggravate periodontitis with DM.

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.

Calprotectin and cross-linked N-telopeptides of type I collagen levels in crevicular fluid from implant sites with peri-implant diseases: a pilot study.

BACKGROUND: Peri-implant crevicular fluid (PICF) contains calprotectin and NTx, which are markers for inflammation and bone resorption, respectively. The aims of this pilot study were to compare calprotectin and NTx levels in PICF from implant sites with or without peri-implant diseases and to evaluate the usefulness of calprotectin and NTx as diagnostic markers for peri-implant diseases. METHODS: Thirty-five patients with dental implants participated in this pilot study. PICF samples were collected from peri-implant disease sites (n = 40) and non-diseased (healthy) sites (n = 34) after clinical indicators including probing depth (PD), bleeding on probing (BOP), gingival index (GI), and bone loss (BL) rate were investigated. Calprotectin and NTx amounts in PICF were measured using their respective ELISA kits and then compared between diseased and healthy samples. The relationship between PICF calprotectin or NTx levels and clinical indicator levels was investigated. A receiver operating characteristic (ROC) curve analysis of calprotectin and NTx was performed to predict peri-implant diseases. RESULTS: Calprotectin and NTx levels in PICF were significantly higher from peri-implant disease sites than from healthy sites. PICF calprotectin amounts correlated with PD, and its levels were significantly higher in the GI-1 and GI-2 groups than in the GI-0 group. PICF NTx amounts correlated with PD and the BL rate. ROC curves indicated that PICF calprotectin and NTx are useful biomarkers for peri-implant diseases. CONCLUSIONS: Calprotectin and NTx in PICF have potential as biomarkers for the diagnosis of peri-implant diseases.

Clinical effects of low body mass index on geriatric status in elderly patients.

BACKGROUND: There is limited information on the relationship of low body mass index (BMI) to the geriatric conditions in elderly patients. OBJECTIVE: The objective of this study is to investigate whether low BMI associates with geriatric status in elderly patients by calculating suitable cut-off point of BMI for assessment of geriatric conditions. METHOD: A total of 1223 elderly patients was enrolled (male/female: N = 472/751), and cut-off point of the BMI values to assess the geriatric status such as aspiration pneumonia, cognitive impairment was determined by receiver operating characteristic (ROC) analyses. Logistic regression analyses were performed to examine the association between several geriatric status and low BMI. Of these patients, 262 patients (male/female: 101/161) had received standard rehabilitation treatment. Functional Independence Measure (FIM) scores were measured both at admission and discharge to calculate FIM gain and efficiency, and retrospective cohort study was performed. RESULTS: Cut-off point of BMI value to assess the geriatric status was determined (19.0 kg/m2). Significant associations of low BMI to several geriatric factors such as loss of posterior occlusion, cognitive impairment were observed in both male and female. FIM scores in above cut-off point group were significantly higher than in below cut-off point group in female (FIM gain, P = 0.0005; FIM efficiency, P = 0.0025, Mann-Whitney U test). On the other hand, there were no significant differences between low and above BMI cut-off point in FIM scores of male patients. CONCLUSION: Low BMI might be a useful parameter to evaluate the geriatric status, and the viewpoint would contribute to decide the care plan for the good end-of-life of elderly.

The KCNQ1 gene polymorphism as a shared genetic risk for rheumatoid arthritis and chronic periodontitis in Japanese adults: A pilot case-control study.

BACKGROUND: A number of studies have suggested a bidirectional relationship of periodontitis with rheumatoid arthritis (RA) and type 2 diabetes mellitus (T2DM). However, the genetic factors that underlie these relationships have not been elucidated. METHODS: We conducted a multicenter case-control study that included 185 patients with RA and chronic periodontitis (CP), 149 patients with T2DM and CP, 251 patients with CP, and 130 systemically and periodontally healthy controls from a cohort of Japanese adults to assess the shared genetic risk factors for RA and CP as well as for T2DM and CP. A total of 17 candidate single nucleotide polymorphisms (SNPs) associated with RA, T2DM, and CP were genotyped. RESULTS: Multiple logistic regression analyses revealed that the KCNQ1 rs2237892 was significantly associated with comorbidity of RA and CP (P = 0.005) after adjustment for age, sex, and smoking status. The carriers of the T allele among patients with RA and CP showed significantly higher disease activity scores including 28 joints using C-reactive protein values than the non-carriers (P = 0.02), although the age, female percentage, and smoking status were comparable. Other SNPs were not associated with comorbidity of RA and CP, T2DM and CP, or susceptibility to CP. CONCLUSION: The results of the present pilot study suggest for the first time that the KCNQ1 rs2237892 may constitute a shared genetic risk factor for RA and CP, but not for T2DM and CP in Japanese adults.

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.

Useful immunochromatographic assay of calprotectin in gingival crevicular fluid for diagnosis of diseased sites in patients with periodontal diseases.

BACKGROUND: Calprotectin, an inflammation-related protein, is present in gingival crevicular fluid (GCF), and the determination of calprotectin is useful for diagnosing periodontal diseases. The authors have recently developed a novel immunochromatographic (IC) chip system to determine calprotectin levels in GCF. In the present study, the usefulness of this diagnostic system is investigated in patients with periodontal diseases. METHODS: Thirty-six patients with periodontal diseases participated in this clinical test at multiple centers. Periodontitis sites (n = 118) and non-periodontitis (healthy) sites (n = 120) were selected after periodontal examination. GCF collection and periodontal examination were performed at baseline, after supragingival and subgingival scaling and root planing. Calprotectin levels in GCF were determined using a novel IC chip system and evaluated as a visual score and an IC reader value. Correlations between GCF calprotectin levels, clinical indicators, and changes in calprotectin levels by periodontal treatments were investigated. Receiver operating characteristic (ROC) analysis of IC reader value for GCF calprotectin was performed to predict periodontal diseases. RESULTS: The visual score of GCF calprotectin was highly correlated with the IC reader value. IC reader values of GCF calprotectin in the periodontitis group were higher than those of the healthy group at three dental examination stages, and they significantly decreased with periodontal treatments. Visual scores and IC reader values of GCF calprotectin were correlated to levels of clinical indicators. ROC analysis for GCF calprotectin showed an optimal cutoff value to predict periodontal diseases. CONCLUSION: Determination of GCF calprotectin using a novel IC chip system is useful for diagnosis of periodontal diseases.

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.

Advanced glycation end-products and Porphyromonas gingivalis lipopolysaccharide increase calprotectin expression in human gingival epithelial cells.

Accumulation of advanced glycation end-products (AGEs) in periodontal tissues of patients with diabetes mellitus aggravates periodontitis, but the mechanisms are unknown. Calprotectin, a heterocomplex of S100A8 and S100A9 proteins, is a constitutive cytoplasmic component of healthy gingival epithelial cells. This study aimed at investigating the effects of AGE and Porphyromonas gingivalis lipopolysaccharide (PgLPS) on calprotectin expression in the human gingival epithelial cell line OBA-9. AGE and PgLPS increased the expression of S100A8 and S100A9 mRNAs, and AGE+PgLPS co-stimulation amplified their expression in OBA-9 cells. A higher concentration of calprotectin in cell lysates was also induced by stimulation with AGE and/or PgLPS. S100A8 was mainly translocated from the nucleus to the cytoplasm by AGE stimulation, while cytoplasmic localization of S100A9 was not altered following stimulation with AGE and/or PgLPS. Calprotectin was found in the cytoplasm of BSA-treated cells, but cytoplasmic and nuclear localization was observed following stimulation with AGE and/or PgLPS. AGE-induced S100A8, and S100A9 mRNA expression was partially suppressed by RAGE-specific siRNA. In contrast, PgLPS-induced S100A8 and S100A9 mRNA expression was strongly suppressed by TLR2-specific siRNA. Furthermore, the inhibition of p38, JNK MAPK, and NF-κB attenuated AGE- and PgLPS-induced S100A8 and S100A9 mRNA expression. Taken together, these results demonstrate that AGE acts in synergy with PgLPS to stimulate RAGE and TLR2 expression and activate p38, JNK MAPK, and NF-κB signaling pathways, resulting in increased activation of calprotectin (S100A8/S100A9) in human gingival epithelial cells. Our results suggest that calprotectin may be involved in the pathogenesis of diabetic periodontitis.