Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

Bacterial LPS stimulates human monocytes to secrete inflammatory cytokines, which are involved in several disease processes. However, the mechanism of LPS activation of cytokine expression and secretion is not completely understood. In this study, we investigated the signal transduction pathways involved in LPS-stimulated TNF-alpha and IL-1 beta secretion. TNF-alpha and IL-1 beta secretion were completely blocked by protein kinase C (PKC) and cyclic nucleotide-dependent protein kinase inhibitor, H-7, but were not affected by H-89, a specific cyclic nucleotide-dependent protein kinase inhibitor. In addition, LPS was found to induce activation of PKC, reaching maximal activity at 30 min and returning to unstimulated levels after 60 min. LPS stimulation only slightly increased intracellular levels of diacylglycerol, the natural activator of PKC, and pretreatment of monocytes with the diacylglycerol-kinase inhibitor, R59022, did not affect LPS-stimulated TNF-alpha secretion. LPS-induced PKC activation was found not to be affected by blocking of the LPS receptor, CD14, with mAb or by inhibition of protein tyrosine kinase with herbimycin A. However, these agents suppressed LPS-induced TNF-alpha secretion and TNF-alpha mRNA accumulation. The results suggest that TNF-alpha and IL-1 beta secretion after LPS stimulation of human monocytes requires the activation of protein tyrosine kinase and PKC, upstream to the activation of gene transcription. The activation of PKC by LPS is probably mediated by a diacylglycerol-independent pathway.

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Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Plasminogen serves as the precursor to plasmin, an essential element in the fibrinolytic process, and is synthesized primarily in the liver. Plasminogen activation occurs through the action of plasminogen activator, converting it into plasmin. This conversion greatly enhances the fibrinolytic system within tissues and blood vessels, facilitating the dissolution of fibrin clots. Consequently, congenital deficiency of plasminogen results in impaired fibrin degradation. Patients with plasminogen deficiency typically exhibit fibrin deposits in various mucosal sites throughout the body, including the oral cavity, eyes, vagina, and digestive organs. Behcet's disease is a chronic recurrent systemic inflammatory disease with four main symptoms: aphthous ulcers of the oral mucosa, vulvar ulcers, skin symptoms, and eye symptoms, and has been reported worldwide. This disease is highly prevalent around the Silk Road from the Mediterranean to East Asia. We report a case of periodontitis in a patient with these two rare diseases that worsened quickly, leading to alveolar bone destruction. Genetic testing revealed a novel variant characterized by a stop-gain mutation, which may be a previously unidentified etiologic gene associated with decreased plasminogen activity. CASE PRESENTATION: This case report depicts a patient diagnosed with ligneous gingivitis during childhood, originating from plasminogen deficiency and progressing to periodontitis. Genetic testing revealed a suspected association with the PLG c.1468C > T (p.Arg490*) stop-gain mutation. The patient's periodontal condition remained stable with brief intervals of supportive periodontal therapy. However, the emergence of Behçet's disease induced acute systemic inflammation, necessitating hospitalization and treatment with steroids. During hospitalization, the dental approach focused on maintaining oral hygiene and alleviating contact-related pain. The patient's overall health improved with inpatient care and the periodontal tissues deteriorated. CONCLUSIONS: Collaborative efforts between medical and dental professionals are paramount in comprehensively evaluating and treating patients with intricate complications from rare diseases. Furthermore, the PLG c.1468C > T (p.Arg490*) stop-gain mutation could contribute to the association between plasminogen deficiency and related conditions.

DOI： 10.1186/s12903-023-03586-8

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Language：English   Publishing type：Research paper (scientific journal)  

Dental caries are an oral infectious disease that can affect human health both orally and systemically. It remains an urgent issue to establish a novel antibacterial method to prevent oral infection for a healthy life expectancy. The aim of this study was to evaluate the inhibitory effects of novel iron chelators, super-polyphenols (SPs), on the cariogenic bacterium Streptococcus mutans, in vitro. SPs were developed to reduce the side effects of iron chelation therapy and were either water-soluble or insoluble depending on their isoforms. We found that SP6 and SP10 inhibited bacterial growth equivalent to povidone-iodine, and viability tests indicated that their effects were bacteriostatic. These results suggest that SP6 and SP10 have the potential to control oral bacterial infections such as Streptococcus mutans.

DOI： 10.3390/antibiotics12111562

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(一社)日本成人先天性心疾患学会  

歯科治療は,観血的処置にはみえなくても菌血症を起こすリスクが高い.今回,感染性心内膜炎(IE)高リスクに分類されるフォンタン手術後の患者が歯科治療に起因すると考えられる感染症を起こし,緊急入院に至る症例を経験した.患者は20歳の男性.多脾症候群,右室型単心室に対して,両側両方向性グレン手術とフォンタン手術の手術歴がある.2021年6月,近医で下顎左側第二大臼歯(#37)の慢性根尖性歯周炎の診断のもと,予防的抗菌薬の投与なく歯内治療を開始した.2021年7月,治療中の#37部の自発痛,悪寒,戦慄,発熱を自覚し,当院循環器内科を緊急受診した.履歴から歯性感染が疑われたため,当院歯周科へ緊急紹介され,#37急性根尖性歯周炎と診断した.IE高リスク患者のため緊急入院となり,経験的抗菌療法を開始した.入院5日目,抗菌薬持続投与下で#37の歯内治療を再開,入院12日目に歯内治療を終了,入院13日目に退院した.今回の症例を教訓に,患者自身が歯科治療に先立ち予防的抗菌薬投与の必要性を簡便に提示できる患者カードを作成した.本カードが適切に運用され,歯科治療由来のIE発症リスクが軽減されることを期待する.(著者抄録)

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Apical periodontitis is an inflammatory response caused by pulp infection. It induces bone resorption in the apical and periapical regions of the tooth. The most conservative approach to treat this condition is nonsurgical endodontic treatment. However, clinical failure has been reported with this approach; thus, alternative procedures are required. This review highlights recent literature regarding advanced approaches for the treatment of apical periodontitis. Various therapies, including biological medications, antioxidants, specialized pro-resolving lipid mediators, and stem cell therapy, have been tested to increase the success rate of treatment for apical periodontitis. Some of these approaches remain in the in vivo phase of research, while others have just entered the translational research phase to validate clinical application. However, a detailed understanding of the molecular mechanisms that occur during development of the immunoinflammatory reaction in apical periodontitis remains unclear. The aim of this review was to summarize advanced approaches for the treatment of apical periodontitis. Further research can confirm the potential of these alternative nonsurgical endodontic treatment approaches.

DOI： 10.3390/bioengineering10040488

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Current periodontal treatment focuses on the mechanical removal of the source of infection, such as bacteria and their products, and there is no approach to control the host inflammatory response that leads to tissue destruction. In order to control periodontal inflammation, we have previously reported the optimization of (+)-terrein synthesis methods and the inhibitory effect of (+)-terrein on osteoclast differentiation in vitro. However, the pharmacological effect of (+)-terrein in vivo in the periodontitis model is still unknown. In this study, we investigated the effect of synthetic (+)-terrein on inflammatory bone resorption using a ligature-induced periodontitis mouse model. Synthetic (+)-terrein (30 mg/kg) was administered intraperitoneally twice a week to the mouse periodontitis model. The control group was treated with phosphate buffer. One to two weeks after the induction of periodontitis, the periodontal tissues were harvested for radiological evaluation (micro-CT), histological evaluation (HE staining and TRAP staining), and the evaluation of inflammatory cytokine production in the periodontal tissues and serum (quantitative reverse-transcription PCR, ELISA). The synthetic (+)-terrein-treated group suppressed alveolar bone resorption and the number of osteoclasts in the periodontal tissues compared to the control group (p < 0.05). In addition, synthetic (+)-terrein significantly suppressed both mRNA expression of TNF-α in the periodontal tissues and the serum concentration of TNF-α (both p < 0.05). In conclusion, we have demonstrated that synthetic (+)-terrein abrogates alveolar bone resorption via the suppression of TNF-α production and osteoclast differentiation in vivo. Therefore, we could expect potential clinical effects when using (+)-terrein on inflammatory bone resorption, including periodontitis.

DOI： 10.3390/jof9030314

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Language：English   Publishing type：Research paper (scientific journal)  

The major active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D3), is known for its wide bioactivity in periodontal tissues. Although the exact mechanisms underlying its protective action against periodontitis remain unclear, recent studies have shown that 1,25D3 regulates autophagy. Autophagy is vital for intracellular pathogen invasion control, inflammation regulation, and bone metabolic balance in periodontal tissue homeostasis, and its regulation could be an interesting pathway for future periodontal studies. Since vitamin D deficiency is a worldwide health problem, its role as a potential regulator of autophagy provides new insights into periodontal diseases. Based on this premise, this narrative literature review aimed to investigate the possible connection between 1,25D3 and autophagy in periodontitis. A comprehensive literature search was conducted on PubMed using the following keywords (e.g., vitamin D, autophagy, periodontitis, pathogens, epithelial cells, immunity, inflammation, and bone loss). In this review, the latest studies on the protective action of 1,25D3 against periodontitis and the regulation of autophagy by 1,25D3 are summarized, and the potential role of 1,25D3-activated autophagy in the pathogenesis of periodontitis is analyzed. 1,25D3 can exert a protective effect against periodontitis through different signaling pathways in the pathogenesis of periodontitis, and at least part of this regulatory effect is achieved through the activation of the autophagic response. This review will help clarify the relationship between 1,25D3 and autophagy in the homeostasis of periodontal tissues and provide perspectives for researchers to optimize prevention and treatment strategies in the future.

DOI： 10.1186/s12903-023-02802-9

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DOI： 10.34376/jsachd.C-2022-0002

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Language：English   Publishing type：Research paper (scientific journal)  

UNLABELLED: Periodontal disease often develops in patients with diabetes, and further exacerbated with diabetic complications. It would be clinically important to clarify the relationship between diabetic microvascular diseases and periodontal disease. This study aimed to evaluate the association between periodontal disease and diabetic complications in patients with type 2 diabetes with poor glycemic control. A total of 447 patients with type 2 diabetes hospitalized at Rakuwakai Otowa Hospital, Japan, were initially recruited in this study. After excluding 134 patients who lacked clinical data or were edentulous, 312 were included in our study. The severity of periodontal disease was evaluated based on the average bone resorption rate. Patients with diabetic nephropathy developed severe periodontal disease (multivariate-adjusted odds ratio, 3.00 [95% CI 1.41-5.19]). Diabetic neuropathy was positively associated with the severity of periodontal disease; the multivariate-adjusted odds ratio (95% CI) was 1.62 (0.87‒2.99) for moderate and 4.26 (2.21‒8.20) for severe periodontal disease. In contrast, diabetic retinopathy was linked with moderate periodontal disease (multivariate-adjusted odds ratio 2.23 [95% CI 1.10-4.10]), but not with severe conditions (multivariate-adjusted odds ratio 0.92 [95% CI 0.67-3.07]). In conclusion, periodontal disease, evaluated by average bone resorption rate, was associated with diabetic nephropathy and neuropathy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13340-022-00591-0.

DOI： 10.1007/s13340-022-00591-0

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Publishing type：Research paper (scientific journal)   Publisher：Tokyo Dental College  

DOI： 10.2209/tdcpublication.2022-0019

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Language：English   Publishing type：Research paper (scientific journal)  

The failure of endodontic treatment is directly associated with microbial infection in the root canal or periapical areas. An endodontic sealer that is both bactericidal and biocompatible is essential for the success of root canal treatments. This is one of the vital issues yet to be solved in clinical dental practice. This in vitro study assessed the effectiveness of graphene oxide (GO) composites GO-CaF2 and GO-Ag-CaF2 as endodontic sealer materials. Dentin slices were coated with either the GO-based composites or commonly used root canal sealers (non-eugenol zinc oxide sealer). The coated slices were treated in 0.9% NaCl, phosphate-buffered saline (PBS), and simulated body fluid (SBF) at 37˚C for 24 hours to compare their sealing effect on the dentin surface. In addition, the radiopacity of these composites was examined to assess whether they complied with the requirements of a sealer for good radiographic visualization. Scanning electron microscopy showed the significant sealing capability of the composites as coating materials. Radiographic images confirmed their radiopacity. Mineral deposition indicated their bioactivity, especially of GO-Ag-CaF2, and thus it is potential for regenerative application. They were both previously shown to be bactericidal to oral microbes and cytocompatible with host cells. With such a unique assemblage of critical properties, these GO-based composites show promise as endodontic sealers for protection against reinfection in root canal treatment and enhanced success in endodontic treatment overall.

DOI： 10.18926/AMO/64122

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Language：Japanese   Publisher：岡山歯学会  

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Language：Japanese   Publisher：岡山歯学会  

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Language：Japanese   Publisher：岡山歯学会  

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Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Periodontal disease is associated with non-alcoholic fatty liver disease (NAFLD). We evaluated periodontal treatment efficacy in patients with NAFLD and periodontal disease. METHODS: This multicenter, 2-arm, randomized study recruited adult patients with NAFLD and periodontitis, alanine aminotransferase levels ≥40 U/L, and equivalent steatosis grade ≥1. Forty eligible patients (18 men and 22 women) were randomly assigned to 2 groups (scaling and root planning [SRP; n = 20] and tooth-brushing [n = 20] groups) stratified by age and sex. The primary and secondary endpoints were changes in alanine aminotransferase levels and serum Porphyromonas gingivalis IgG-antibody titers from baseline to 12 weeks, respectively. Efficacy analysis was performed using an intention-to-treat approach (t-test). This trial was registered with the University Hospital Medical Information Network Clinical Trials Registry (UMINXXXXXXX). RESULTS: We observed a significantly higher decrease in absolute alanine aminotransferase levels and P. gingivalis IgG-antibody titers in the SRP group than in the tooth-brushing group (-12 vs 1 U/L; mean difference [δ], -12; 95% confidence interval [CI], -20 to -5; P = 0.002). The decrease in P. gingivalis IgG-antibody titer was significantly higher in the SRP group than in the tooth-brushing group (FDC381, -1.6 [2.5]; δ, -1.6; 95% CI, -2.7 to -0.4; P = 0.0092; SU63, -1.7 [2.0]; δ, -1.7; 95% CI, -2.7 to -0.7). No life-threatening events or treatment-related deaths occurred. CONCLUSION: Periodontal treatment induced significant short- and mid-term reductions in liver enzyme levels and antibody titers. Further research is warranted to clearly define SRP efficacy and tolerability in patients with NAFLD and periodontitis.

DOI： 10.14309/ctg.0000000000000520

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Language：Japanese   Publisher：(一社)日本口腔検査学会  

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

The risk factors for non-alcoholic fatty liver disease (NAFLD) progression are not completely known. Porphyromonasgingivalis infection is a risk factor for systemic diseases. We investigated the association of P.gingivalis infection with the risk of non-alcoholic steatohepatitis progression. Here, hematological tests, periodontal examination, and saliva collection were performed for 164 patients with NAFLD. P.gingivalis was identified in saliva using polymerase chain reaction. Hepatic steatosis and stiffness were evaluated using vibration-controlled transient elastography (VCTE) and magnetic resonance imaging. In patients with NAFLD, P.gingivalis positivity (P.gingivalis ratio ≥ 0.01%) in saliva correlated with liver stiffness determined using magnetic resonance elastography (MRE; p < 0.0001). A P.gingivalis ratio of 0.01% corresponds to 100,000 cells/mL and indicates the proportion of P.gingivalis in the total number of bacteria in the oral cavity. Patients with NAFLD and advanced fibrosis on MRE showed significantly elevated endotoxin activity; those who had > 10 periodontal pockets with depths ≥ 4 mm had significantly increased hepatic stiffness on both VCTE and MRE.

DOI： 10.1038/s41598-022-17917-2

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Other Link： https://www.nature.com/articles/s41598-022-17917-2

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Recent studies have shown that periodontitis is associated with rheumatoid arthritis (RA) and periodontal bacteria, such as Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) are involved in the pathogenesis of RA via citrullinated proteins. Smoking has also been shown to be involved in the pathogenesis of RA; however, the extent of this involvement is still poorly understood. In addition, RA and polymyalgia rheumatica (PMR) are sometimes difficult to differentiate; however, the relationship between PMR and the factors from smoking and periodontal bacteria is unclear. The aim of this study was to clarify the relationship between periodontal pathogenic bacterial infections and smoking in patients with RA or PMR. This case-control study included 142 patients with untreated RA or PMR. This study evaluated the serum antibody titers against periodontal pathogenic bacterial antigens and an anti-citrullinated peptide antibody (ACPA). In patients with RA, the relationship between antibody titers and disease activity of RA and response after 3 months of treatment was also investigated. Additionally, the effects of smoking were evaluated. Although there was no significant difference in serum antibody titer against periodontal pathogenic bacteria between the ACPA-positive RA group and the ACPA-negative PMR group, we found an association between the elevated antibody titer against Pg and the degree of ACPA value, especially between negative group and high-value positive group (≥ 100 U/mL). The antibody titers against Aa and Pg did not differ depending on disease activity score 28 (DAS28) at baseline; however, patients with high antibody titers had poor RA therapeutic response as judged by DAS28 after 3 months. We could not find any association between smoking and any of these parameters. Periodontal pathogenic bacteria, especially Pg, are associated with elevated ACPA levels. Our findings suggest that Pg and Aa infections interfere with the therapeutic response of RA.

DOI： 10.1038/s41598-022-16279-z

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Authorship：Last author   Language：English  

The study aims to reveal the composition of subgingival bacteria in monozygotic twins with discordant in severity and progression risk of periodontitis. Microbiome analysis indicated that most bacteria were heritable but differed in their abundance and immune response. The dysbiotic bacteria can be considered as risk markers for periodontitis progression.

DOI： 10.1002/ccr3.5725

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Periodontal disease is the most common dental disease in dogs. Although the systemic effects of periodontal disease have not been clarified in veterinary science, it is necessary to evaluate the effects of periodontal disease in clinical trials in the future. There have been a few clinical attempts made, however, to assess the severity of periodontal inflammation and its impact on the systemic health of dogs. Meanwhile, in the field of dentistry for humans, the periodontal inflamed surface area (PISA) and periodontal epithelial surface area (PESA) have been used to quantitatively assess the degree of periodontal disease affecting a single tooth as well as the overall extent of periodontitis. Recent studies have also suggested the use of these assessments to examine the relationship between periodontal inflammation and systemic health. RESULTS: The estimation formula for a dog's periodontal pocket surface area (PPSA), an alternative to PISA and PESA in humans, was established using body weight and periodontal pocket depth. Actual values were measured using extracted teeth from various dog breeds and sizes (2.3-25.0 kg of body weight) to obtain universal regression equations for PPSA. Altogether, 625 teeth from 73 dogs of 16 breeds were extracted and subsequently analyzed for morphological information. PPSA was measured in 61 dogs of 10 breeds with periodontal disease using the established estimation formulas, and the correlation between PPSA and preoperative blood chemistry data was analyzed accordingly. A strong correlation was found between PPSA and serum globulin (r = 0.71) while moderate correlations were found for C-reactive protein (r = 0.54) and serum albumin (r = -0.51). CONCLUSIONS: Estimation formulas using body weight and the 6-point probing depth were established for determining PPSA. Direct correlations between PPSA and several blood test results were observed in the study sample. Taken together, these results suggest that PPSA could be useful for evaluating the effects of periodontitis on systemic conditions in dogs.

DOI： 10.1186/s12917-021-03116-0

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Periodontal disease is a chronic inflammatory condition caused by periodontal pathogens in the gingival sulcus. Short-chain fatty acids (SCFAs) produced by causal bacteria are closely related to the onset and progression of periodontal disease and have been reported to proliferate in the periodontal sulcus of patients experiencing this pathology. In such patients, propionic acid (C3), butyric acid (C4), isobutyric acid (IC4), valeric acid (C5), isovaleric acid (IC5), and caproic acid (C6), henceforth referred to as [C3-C6], has been reported to have a detrimental effect, while acetic acid (C2) exhibits no detrimental effect. In this study, we established an inexpensive and simple enzymatic assay that can fractionate and measure these acids. The possibility of applying this technique to determine the severity of periodontal disease by adapting it to specimens collected from humans has been explored. We established an enzyme system using acetate kinase and butyrate kinase capable of measuring SCFAs in two fractions, C2 and [C3-C6]. The gingival crevicular fluid (GCF) and saliva of 10 healthy participants and 10 participants with mild and severe periodontal disease were measured using the established enzymatic method and conventional gas chromatography-mass spectrometry (GC-MS). The quantification of C2 and [C3-C6] in human GCF and saliva was well correlated when using the GC-MS method. Furthermore, both C2 and [C3-C6] in the GCF increased with disease severity. However, while no significant difference was observed between healthy participants and periodontal patients when using saliva, [C3-C6] significantly differed between mild and severe periodontal disease. The enzymatic method was able to measure C2 and [C3-C6] separately as well as using the GC-MS method. Furthermore, the C2 and [C3-C6] fractions of GCF correlated with disease severity, suggesting that this method can be applied clinically. In contrast, the quantification of C2 and [C3-C6] in saliva did not differ significantly between healthy participants and patients with periodontal disease. Future studies should focus on inflammation rather than on tissue destruction.

DOI： 10.1371/journal.pone.0268671

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Language：Japanese   Publisher：(一社)日本未病学会  

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

This case report highlights the importance of using a dental operating microscope (DOM) and ultrasonic endodontic tips (UETs) to locate all root canals in the lower first premolar. A 53-year-old woman presented to our clinic with pain in the lower right first premolar. After a detailed search using a DOM and UETs, three root canals were found, prepared with rotary HyFlex endodontic files, and obturated using the lateral condensation technique. At the five-year follow-up after treatment, the tooth was completely restored and fulfilling its function, with no signs or symptoms of any post-treatment flare-up.

DOI： 10.18926/AMO/62778

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Malnutrition causes prolonged inflammation, resulting in delayed wound healing. High mobility group box-1 (HMGB1) is a damage-associated molecular pattern that is present in the nuclei of macrophages and is secreted into the extracellular milieu in response to stimuli. It stimulates the production of interleukin-1β (IL-1β) through the receptors for advanced glycation end products (RAGE), inducing an inflammatory response, which is an essential response to initiate wound healing. We hypothesized that malnutrition may interfere with this cascade, causing abnormal inflammation and ultimately delaying wound healing. We used tooth-extracted mice with malnutrition fed with low-casein diet for two weeks. On days 3 and 7 after tooth extraction, the wound tissue was histologically observed and analyzed for several factors in the inflammation-regeneration lineage, including IL-1β, mesenchymal stem cells, myeloperoxidase activity, HMGB1, macrophage polarization, and adenosine 5-triphosphate (ATP). On day 7, delayed wound healing was observed with the following findings under malnutrition conditions: decreased mRNA expression of genes for regeneration and mesenchymal stem cell (MSC) accumulation, an obvious increase in myeloperoxidase and IL-1β mRNA expression, an increase in HMGB1 levels, and an increase in ATP concentration in tissues with elevated proportion of M2 macrophages. These results suggest that the significantly increased secretion of HMGB1 associated with the upregulated production of ATP and IL-1β secretion via the RAGE pathway may interfere with the resolution of inflammation and wound healing under the state of malnutrition.

DOI： 10.1016/j.intimp.2021.107772

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Osteoporosis is a common disease characterized by a systemic impairment of bone mass and microarchitecture that results in fragility fractures. Severe bone loss due to osteoporosis triggers pathological fractures and consequently decreases the daily life activity and quality of life. Therefore, prevention of osteoporosis has become an important issue to be addressed. We have reported that the fungal secondary metabolite (+)-terrein (TER), a natural compound derived from Aspergillus terreus, has shown receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation by suppressing nuclear factor of activated T-cell 1 (NFATc1) expression, a master regulator of osteoclastogenesis. TER has been shown to possess extensive biological and pharmacological benefits; however, its effects on bone metabolism remain unclear. In this study, we investigated the effects of TER on the femoral bone metabolism using a mouse-ovariectomized osteoporosis model (OVX mice) and then on RANKL signal transduction using mouse bone marrow macrophages (mBMMs). In vivo administration of TER significantly improved bone density, bone mass, and trabecular number in OVX mice (p < 0.01). In addition, TER suppressed TRAP and cathepsin-K expression in the tissue sections of OVX mice (p < 0.01). In an in vitro study, TER suppressed RANKL-induced phosphorylation of PKCα/βII, which is involved in the expression of NFATc1 (p < 0.05). The PKC inhibitor, GF109203X, also inhibited RANKL-induced osteoclastogenesis in mBMMs as well as TER. In addition, TER suppressed the expression of osteoclastogenesis-related genes, such as Ocstamp, Dcstamp, Calcr, Atp6v0d2, Oscar, and Itgb3 (p < 0.01). These results provide promising evidence for the potential therapeutic application of TER as a novel treatment compound against osteoporosis.

DOI： 10.3389/fphar.2021.674366

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Language：English   Publishing type：Research paper (scientific journal)  

Oral health is closely related to subjective general health and systemic diseases. This cross-sectional study aimed to identify the factors related to oral symptoms and their worsening in relation to psychosocial factors after the Great East Japan Earthquake. In this study, 64,186 residents aged 15-101 years old, who experienced the earthquake on 11 March 2011, were surveyed regarding their oral symptoms; psychological factors, such as post-traumatic reactions and psychological distress; and social factors such as evacuation, work change, and loss of a close person; history of systemic diseases; and lifestyle. Binomial logistic regression analysis was used to calculate odds ratios, and 95% confidence intervals were established for each factor associated with prevalent and exacerbated oral symptoms. The proportions of participants with prevalent and exacerbated oral symptoms were 10.3% and 1.6%, respectively. The multivariate odds ratios and 95% CI of psychosocial factors associated with exacerbated oral symptoms were as follows: post-traumatic stress disorder symptoms, 2.24 (1.64-3.06); work changes, 1.88 (1.34-2.65); history of dyslipidemia, 1.74 (1.27-2.39); and subjective current poor health condition, 2.73 (2.00-3.75). Psychological factors, social factors, and physical factors were associated with both prevalent and exacerbated oral symptoms.

DOI： 10.3390/ijerph18116054

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Periodontal disease is a chronic inflammatory disease of the periodontal tissue. The periodontal inflamed surface area (PISA) is a proposed index for quantifying the inflammatory burden resulting from periodontitis lesions. This study aimed to investigate longitudinal changes in the periodontal status as evaluated by the PISA following the active periodontal treatment. To elucidate the prognostic factors of PISA, mixed-effect modeling was performed for clinical parameters, tooth-type, and levels of periodontal pathogens as independent variables. One-hundred-twenty-five patients with chronic periodontitis who completed the active periodontal treatment were followed-up for 24 months, with evaluations conducted at 6-month intervals. Five-times repeated measures of mean PISA values were 130+/-173, 161+/-276, 184+/-320, 175+/-417, and 209+/-469 mm2. Changes in clinical parameters and salivary and subgingival periodontal pathogens were analyzed by mixed-effect modeling. Plaque index, clinical attachment level, and salivary levels of Porphyromonas gingivalis were associated with changes in PISA at the patient- and tooth-level. Subgingival levels of P. gingivalis and Prevotella intermedia were associated with changes in PISA at the sample site. For most patients, changes in PISA were within 10% of baseline during the 24-month follow-up. However, an increase in the number of bleeding sites in a tooth with a deep periodontal pocket increased the PISA value exponentially.

DOI： 10.3390/jcm10061165

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The periodontal inflamed surface area (PISA) is a useful index for clinical and epidemiological assessments, since it can represent the inflammation status of patients in one contentious variable. However, calculation of the PISA is difficult, requiring six point probing depth measurements with or without bleeding on probing on 28 teeth, followed by data input in a calculation program. More simple methods are essential for screening periodontal disease or in epidemiological studies. In this study, we tried to establish a convenient partial examination method to estimate PISA. Cross-sectional data of 254 subjects who completed active periodontal therapy were analyzed. Teeth that represent the PISA value were selected by an item response theory approach. The maxillary second molar, first premolar, and lateral incisor and the mandibular second molar and lateral incisor were selected. The sum of the PISAs of these teeth was significantly correlated with the patient's PISA (R2 = 0.938). More simply, the sum of the maximum values of probing pocket depth with bleeding for these teeth were also significantly correlated with the patient's PISA (R2 = 0.6457). The simple model presented in this study may be useful to estimate PISA.

DOI： 10.3390/jcm10040723

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There is no conclusive evidence regarding a causal relationship between periodontitis and atherosclerosis. In this study, we examined the microbiome in the oral cavity and atheromatous plaques from atherosclerosis patients with or without periodontitis to investigate the role of oral bacteria in the formation of atheromatous plaques. We chose four patients with and without periodontitis, who had undergone carotid endarterectomy. Bacterial samples were extracted from the tongue surface, from periodontal pocket (during the oral examination), and from the atheromatous plaques (APs). We investigated the general and oral conditions from each patient and performed next-generation sequencing (NGS) analysis for all bacterial samples. There were no significant differences between both groups concerning general conditions. However, the microbiome patterns of the gingival pocket showed differences depending on the absence or presence of periodontitis, while those of the tongue surface were relatively similar. The microbiome pattern of the atheromatous plaques was entirely different from that on the tongue surface and gingival pocket, and oral bacteria were seldom detected. However, the microbiome pattern in atheromatous plaques was different in the presence or absence of periodontitis. These results suggested that oral bacteria did not affect the formation of atheromatous plaques directly.

DOI： 10.1007/s10266-020-00524-w

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DOI： 10.1007/s42399-020-00671-9

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Other Link： http://link.springer.com/article/10.1007/s42399-020-00671-9/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)  

Periodontal examination data have a complex structure. For epidemiological studies, mass screenings, and public health use, a simple index that represents the periodontal condition is necessary. Periodontal indices for partial examination of selected teeth have been developed. However, the selected teeth vary between indices, and a justification for the selection of examination teeth has not been presented. We applied a graded response model based on the item response theory to select optimal examination teeth and sites that represent periodontal conditions. Data were obtained from 254 patients who participated in a multicenter follow-up study. Baseline data were obtained from initial follow-up. Optimal examination sites were selected using item information calculated by graded response modeling. Twelve sites-maxillary 2nd premolar (palatal-medial), 1st premolar (palatal-distal), canine (palatal-medial), lateral incisor (palatal-central), central incisor (palatal-distal) and mandibular 1st premolar (lingual, medial)-were selected. Mean values for clinical attachment level, probing pocket depth, and bleeding on probing by full mouth examinations were used for objective variables. Measuring the clinical parameters of these sites can predict the results of full mouth examination. For calculating the periodontal index by partial oral examination, a justification for the selection of examination sites is essential. This study presents an evidence-based partial examination methodology and its modeling.

DOI： 10.3390/jcm9113754

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Salivary glands (SGs) are very important for maintaining the physiological functions of the mouth. When SGs regenerate and repair from various damages, including mechanical, radiological, and immune diseases, acinar and granular duct cells originate from intercalated duct cells. However, the recovery is often insufficient because of SGs' limited self-repair function. Furthermore, the precise repair mechanism has been unclear. Here, we focused on CD49f, one of the putative stem cell markers, and characterized CD49f positive cells (CD49f+ cells) isolated from male murine SGs. CD49f+ cells possess self-renewal ability and express epithelial and pluripotent markers. Compared to CD49f negative cells, freshly isolated CD49f+ cells highly expressed inhibin beta A and beta B, which are components of activin that has anti-proliferative effects. Notably, an inhibitor of activin, follistatin was expressed in mechanically-damaged SGs, meanwhile no follistatin was expressed in normal SGs in vivo. Moreover, sub-cultured CD49f+ cells highly expressed both Follistatin and a series of proliferative genes, expressions of which were decreased by Follistatin siRNA. These findings indicated that the molecular interaction between activin and follistatin may induce CD49f+ cells proliferation in the regeneration and repair of mouse SGs.

DOI： 10.1038/s41598-020-77004-2

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In this study, we focus on the antimicrobial properties of tempeh, a soybean fermented food, against oral bacteria. Tempeh showed antimicrobial activity against dental caries pathogenic bacterium Streptococcus mutans at a final concentration of 1 mg/mL. An antimicrobial substance contained in tempeh was present in the 100 kDa or greater fraction generated by ultrafiltration, but it was found not to be proteinaceous by native-PAGE, SDS-PAGE and protein degradation tests. Next, when the fraction was purified with an ODS column, the 80% and 100% methanol eluates showed antimicrobial activity against S. mutans. The 100% methanol eluate was further subjected to a 2nd column purification, and isolation of the target was confirmed by HPLC. When the isolated material was analyzed by ESI-MS, the m/z was 279.234. Further analysis by Raman spectroscopy revealed a peak similar to linoleic acid. This substance also possessed antimicrobial properties equivalent to linoleic acid.

DOI： 10.1016/j.ijfoodmicro.2020.108645

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Publishing type：Research paper (scientific journal)   Publisher：The Society for Chromatographic Sciences  

DOI： 10.15583/jpchrom.2019.025

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Chronic periodontitis is an inflammatory disease caused by the formation of oral microbial biofilms. Periodontitis is associated with general health and not only oral diseases. Porphyromonas gingivalis is a well-known keystone pathogen for periodontitis and is associated with several systemic diseases, such as diabetes mellitus and Alzheimer's disease. We previously developed a system for screening periodontitis using P. gingivalis-specific serum immunoglobulin G (IgG) in an enzyme-linked immunosorbent assay with a sensitivity of 0.774 and a specificity of 0.586 and an area under the receiver operating characteristic curve of 0.708. However, the antigens elicited non-specific responses, since they were obtained from whole extracts of sonicated cultured bacteria. The purpose of this study was to identify antigens ideal for a sensitive and specific serum test. We identified the specific antigens using immunoaffinity columns immobilized with IgG antibodies from periodontitis patients. Liquid chromatography-tandem mass spectrometry identified 29 antigens from the elutes. Recombinant proteins for these candidates were synthesized using the wheat germ cell-free translation system and screened by dot blot analysis with serum from the columns. Three of the 16 candidates that reacted showed strongest affinities upon dot blot analysis; they included outer membrane protein 28, cysteine proteases, lysine gingipain Kgp, and arginine gingipain RgpA. Outer membrane protein 28 was not suitable for screening P. gingivalis infection because of its high false-negative rates. Kgp and RgpA were unstable antigens since they underwent self-digestion. They were made stable by substituting the active cysteine residues in Kgp and RgpA with alanine using site-directed mutagenesis. Using the modified antigens, we demonstrated that the patient serum IgG level against RgpA was the highest among all the antigens expressed in P. gingivalis. Moreover, the N-terminus of recombinant RgpA was excellent in differentiating between diseased and non-diseased states (with sensitivity of 0.85, specificity of 0.9, and area under the curve of 0.915). Although dot blot analysis was the only experiment used, the N-terminus of RgpA is an excellent antigen to immunologically test for P. gingivalis infection, especially for estimating the risks for periodontitis-associated systemic diseases. In conclusion, we have developed a P. gingivalis antigen for screening periodontitis.

DOI： 10.3389/fimmu.2020.01017

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Pathophysiological bone resorption is commonly associated with periodontal disease and involves the excessive resorption of bone matrix by activated osteoclasts. Receptor activator of nuclear factor (NF)-κB ligand (RANKL) signaling pathways have been proposed as targets for inhibiting osteoclast differentiation and bone resorption. The fungal secondary metabolite (+)-terrein is a natural compound derived from Aspergillus terreus that has previously shown anti-interleukin-6 properties related to inflammatory bone resorption. However, its effects and molecular mechanism of action on osteoclastogenesis and bone resorption remain unclear. In the present study, we showed that 10 µM synthetic (+)-terrein inhibited RANKL-induced osteoclast formation and bone resorption in a dose-dependent manner and without cytotoxicity. RANKL-induced messenger RNA expression of osteoclast-specific markers including nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), the master regulator of osteoclastogenesis, cathepsin K, tartrate-resistant acid phosphatase (Trap) was completely inhibited by synthetic (+)-terrein treatment. Furthermore, synthetic (+)-terrein decreased RANKL-induced NFATc1 protein expression. This study revealed that synthetic (+)-terrein attenuated osteoclast formation and bone resorption by mediating RANKL signaling pathways, especially NFATc1, and indicated the potential effect of (+)-terrein on inflammatory bone resorption including periodontal disease.

DOI： 10.1016/j.intimp.2020.106429

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The prevention of nosocomial infections is an imperative task. The dental chair unit (DCU) is an indispensable device used in dental treatment. However, it is known that the dental unit water line (DUWL) can become contaminated with biofilm, consisting mainly of heterotrophic bacteria (HB). Recently, the International Organization for Standardization specified the methods for testing DUWL contamination management. On these grounds, a simulator reproducing DUWL was prepared to standardize the examination method of the DUWL contamination. Objectives: To evaluate the reproducibility of the DUWL simulator, monitor the DUWL contamination states, and test the efficacy of a commercial decontaminant for DUWL. Methods: The DUWL simulator was assembled by a DCU manufacturing company. The simulator's DUWL was filled with tap water (TW), and left for approximately one year. Neutral electrolyzed water (NEW) was used as a decontaminant for DUWL. Both TW and NEW were passed through DUWL in a timely manner simulating daily dental treatment. Water was sampled from the air turbine hand piece weekly for 4 weeks and used for HB culture. Contamination status was evaluated by measuring bacterial adenosine triphosphate release and by culturing on Reasoner's 2A medium. Results: The DUWL released contaminated water had a bacterial count of over 6 × 104 cfu/mL. After passing NEW through DUWL for 1 week, the count drastically decreased to its basal level and remained steady for 4 weeks. However, TW showed no effect on DUWL decontamination throughout the examination periods. Conclusions: The DUWL simulator could be useful to examine the efficacy of the decontaminant for DUWL and development of new methods in DUWL contamination management.

DOI： 10.1016/j.heliyon.2020.e04132

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This inclusive review summarizes the recent advances in the application of graphene oxide (GO) and functionalized GO in oral and dental research. GO possesses several extraordinary physical, chemical, optical, electrical, and mechanical properties. Because of its high surface area and oxygenated functional groups, GO exhibits excellent interaction ability with metals and ions as well as organic species. The current review reveals that GO has been used to produce a variety of functionalized nanocomposites, scaffolds, and advanced nanoparticle carriers. Accordingly, GO shows potential in a variety of research fields, such as tissue engineering, materials engineering, biomaterials, and drug delivery, indicating that the application of GO to biomedicine is particularly promising. More specifically, the recent application of GO in dentistry has provided outstanding results in antimicrobial action, regenerative dentistry, bone tissue engineering, drug delivery, physicomechanical property enhancement of dental biomaterials, and oral cancer treatment. The biocompatibilities of GO and its nanocomposites make them potential units in bone regeneration, osseointegration, and cell proliferation. Furthermore, its antibiofilm and antiadhesion properties have inspired researchers to develop GO for biofilm and caries prevention, as well as implant surface modification and as a quorum sensing inhibitor. This updated review is wide-ranging and provides a useful source for additional information on GO and its composites in dental research and applications.

DOI： 10.1016/j.apmt.2020.100576

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Nanocarbons, especially two-dimensional carbons, have received considerable attention due to their unique structure and physical and chemical properties, which make them promising candidate materials for biomedical applications. In this study, we focus on graphene oxide (GO), which has many oxygenated functional groups and high affinity with water and biomaterials, and the synthesis of GO complexes with antibacterial agents, like cetylpyridinium chloride (CPC) and its derivatives. We found that the sustained release of CPCs from GO can be controlled by changing the terminal functional group of CPC. The prepared GO-CPC complexes were subjected to antibacterial tests against S. mutans. CPC with the carboxy group was degraded by the oxidizing property of GO, resulting in the loss of antibacterial properties. On the other hand, the other CPC derivatives were released from GO and showed antibacterial activities. Finally, we propose a new mechanism describing how GO and CPC form a functional complex, and how CPC is released from this complex. These findings will lead to pioneering the carbon-based functional antibacterial agents designed at the molecular level.

DOI： 10.1016/j.carbon.2019.11.094

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BACKGROUND: We report the first protocol for a multicenter, randomized comparison study to compare the efficacies of periodontal scaling and root-planing treatment against that of tooth-brushing treatment for nonalcoholic fatty liver disease (NAFLD) (PERION: PERIOdontal treatment for NAFLD). Nonalcoholic steatohepatitis (NASH) is an advanced form of NAFLD, which can progress to cirrhosis and hepatocellular carcinoma. Increased endotoxemia is associated with the progression of NAFLD. Periodontal bacteria possess endotoxins; Porphyromonas gingivalis is well-known as a major pathogenic bacterium in periodontitis, and serum antibody levels for P. gingivalis are high in patients with periodontitis. Several reports have indicated that P. gingivalis is related to NAFLD. This study aims to investigate the effect of periodontal treatment for liver damage, P. gingivalis infection, and endotoxemia on patients with NAFLD. METHODS: We will include adult patients (20-85 years old) with NAFLD, alanine aminotransferase (ALT) ≥ 40 IU/L, and equivalent steatosis grade ≥ 1 (target sample size, n = 40 patients; planned number of patients with outcome data, n = 32). Participants will be randomly assigned to one of two groups: a scaling and root-planing group or tooth-brushing as the usual group. The primary outcome will be the change in ALT levels from baseline to 12 weeks; the key secondary outcome will be the change in the serum immunoglobulin G (IgG) antibody titer for P. gingivalis at 12 weeks. DISCUSSION: This study should determine whether periodontal treatment decreases liver damage, P. gingivalis infection, and endotoxemia in patients with NAFLD. TRIAL REGISTRATION: University Hospital Medical Information Network (UMIN) Clinical Trials Registry, ID: UMIN000022079.

DOI： 10.1186/s13063-020-4201-y

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Dental caries is a type of oral microbiome dysbiosis and biofilm infection that affects oral and systemic conditions. For healthy life expectancy, natural bacteriostatic products are ideal for daily and lifetime use as anti-oral infection agents. This study aimed to evaluate the inhibitory effects of abietic acid, a diterpene derived from pine rosin, on the in vitro growth of cariogenic bacterial species, Streptococcus mutans. The effective minimum inhibitory concentration of abietic acid was determined through observation of S. mutans growth, acidification, and biofilm formation. The inhibitory effects of abietic acid on the bacterial membrane were investigated through the use of in situ viability analysis and scanning electron microscopic analysis. Cytotoxicity of abietic acid was also examined in the context of several human cell lines using tetrazolium reduction assay. Abietic acid was found to inhibit key bacterial growth hallmarks such as colony forming ability, adenosine triphosphate activity (both planktonic and biofilm), acid production, and biofilm formation. Abietic acid was identified as bacteriostatic, and this compound caused minimal damage to the bacterial membrane. This action was different from that of povidone-iodine or cetylpyridinium chloride. Additionally, abietic acid was significantly less cytotoxic compared to povidone-iodine, and it exerted lower toxicity towards epithelial cells and fibroblasts compared to that against monocytic cells. These data suggest that abietic acid may prove useful as an antibacterial and antibiofilm agent for controlling S. mutans infection.

DOI： 10.1007/s10266-019-00456-0

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High mobility group box 1 (HMGB1) is a non-histone DNA-binding protein of about 30 kDa. It is released from a variety of cells into the extracellular milieu in response to inflammatory stimuli and acts on specific cell-surface receptors, such as receptors for advanced glycation end-products (RAGE), Toll-like receptor (TLR)2, TLR4, with or without forming a complex with other molecules. HMGB1 mediates various mechanisms such as inflammation, cell migration, proliferation, and differentiation. On the other hand, HMGB1 enhances chemotaxis acting through the C-X-C motif chemokine ligand (CXCL)12/C-X-C chemokine receptor (CXCR)4 axis and is involved in regeneration. In the oral cavity, high levels of HMGB1 have been detected in the gingival tissue from periodontitis and peri-implantitis patients, and it has been shown that secreted HMGB1 induces pro-inflammatory cytokine expression, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, which prolong inflammation. In contrast, wound healing after tooth extraction or titanium dental implant osseointegration requires an initial acute inflammation, which is regulated by secreted HMGB1. This indicates that secreted HMGB1 regulates angiogenesis and bone remodeling by osteoclast and osteoblast activation and promotes bone healing in oral tissue repair. Therefore, HMGB1 can prolong inflammation in the periodontal tissue and, conversely, can regenerate or repair damaged tissues in the oral cavity. In this review, we highlight the role of HMGB1 in the oral cavity by comparing its function and regulation with its function in other diseases. We also discuss the necessity for further studies in this field to provide more specific scientific evidence for dentistry.

DOI： 10.3389/fimmu.2020.01461

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We report a case of acute prevertebral abscess caused by traumatic tooth fractures in a 77-year-old Japanese man. After being transferred to our hospital the patient was initially diagnosed with a neck hematoma; however, blood culture showed Streptococcus parasanguinis, an oral bacterium, and an MRI examination suggested prevertebral abscesses. Tooth fractures, severe periodontitis, and peri-implantitis with Streptococcus parasanguinis were observed. Antibiotics were administered and fractured teeth were extracted. The patient's condition then gradually improved. We concluded that bacteremia caused by traumatic tooth fractures induced the acute prevertebral abscesses.

DOI： 10.18926/AMO/57376

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The periodontal pathogen Porphyromonas gingivalis shows colonial pigmentation on blood agar and produces gingipains (Kgp, RgpA, and RgpB), cysteine proteases involved in an organism's virulence and pigmentation. We showed previously that deletion of the PGN_0300 gene abolished the pigmentation activity and reduced the proteolytic activity of gingipains. The role of the PGN_0297 gene, which consists of an operon with the PGN_0300 gene, is unclear. Herein we examined the effect of PGN_0297 gene deletion on the pigmentation and proteolytic activities and transcriptional levels of gingipains. A PGN_0297 gene deletion mutant (ΔPGN_0297) did not exhibit the pigmentation. The proteolytic activity of the gingipains was decreased in the culture supernatant and on the cell surface of ΔPGN_0297. The mutant ΔPGN_0297 failed to attenuate Akt phosphorylation at Thr308 and Ser473, but both phosphorylations were attenuated in the wild-type and its complementation strain. The deletion of PGN_0297 gene did not substantially affect the transcriptional levels of the gingipain genes kgp, rgpA, and rgpB. Taken together, these results indicate that PGN_0297 is closely involved in the secretion and maturation of gingipains.

DOI： 10.18926/AMO/56933

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Contamination of dental unit waterlines (DUWL) with heterotrophic bacteria can cause problems in immune compromised patients (aged, tumor and organ transplantation-patients). We focused on the use of low-concentrated ozonized water (OZW) as the biofilm formation restraint system for DUWL. Here, we examined the effects of low-concentrated OZW on the growth of bacteria and related biofilm formation and harmfulness to dental unit components (DUCs) in vitro. Objectives: To evaluate the bactericidal effects of OZW on biofilms in DUWL and DUC in vitro. Methods: Low-concentrated OZW (0.4 mg/L) was generated using an OZS-PTDX generator. Heterotrophic bacterial biofilms in old DUWL tubes and Candia albicans solution (control microbe) were treated with OZW for 1 h with gentle agitation before static culturing for 96 h in Reasoner's 2A liquid media. The control solutions were 0.1% cetylpyridinium chloride (CPC), chlorinated tap water (TW), and phosphate-buffered saline (PBS). Adenosine triphosphate (ATP) amounts of the microbes were measured and the biofilms of these microbes were observed using scanning electron microscopy (SEM). Moreover, surfaces of DUC soaked in OZW and TW were observed by SEM. Results: The OZW reduced ATP levels in microbes to 50% compared to TW and PBS treatment, although CPC reduced it below detection limits. SEM observation revealed deformation of microbes cultured with OZW, whereas no changes were seen on DUC surfaces. Conclusions: Low-concentrated OZW is bactericidal against heterotrophic bacteria biofilms and it is not harmful to DUC, suggesting that it might be useful in preventing DUWL contamination.

DOI： 10.1016/j.heliyon.2019.e02306

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Taste alteration is one of the complications of severe diabetes. It is important in diabetes treatment to assess taste alteration and perform dietary counseling, therapeutic exercise, and oral care. In this case, multidisciplinary clinical approach by medical staff was successful for a severely diabetic patient with dysgeusia.

DOI： 10.1002/ccr3.2111

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Objective: We aimed to evaluate molecular imaging as a novel diagnostic tool for mice periodontitis model induced by ligature and Porphyromonas gingivalis (Pg) inoculation. Materials and methods: Twelve female mice were assigned to the following groups: no treatment as control group (n = 4)
periodontitis group induced by ligature and Pg as Pg group (n = 4)
and Pg group treated with glycyrrhizinic acid (GA) as Pg + GA group (n = 4). All mice were administered a myeloperoxidase (MPO) activity-specific luminescent probe and observed using a charge-coupled device camera on day 14. Image analysis on all mice was conducted using software to determine the signal intensity of inflammation. Additionally, histological and radiographic evaluation for periodontal inflammation and bone resorption at the site of periodontitis, and quantitative enzyme-linked immunosorbent assay (ELISA) were conducted on three mice for each group. Each experiment was performed three times. Results: Levels of serum IgG antibody against P. gingivalis were significantly higher in the Pg than in the Pg + GA group. Histological analyses indicated that the number of osteoclasts and neutrophils were significantly lower in the Pg + GA than in the Pg group. Micro-CT image analysis indicated no difference in bone resorption between the Pg and Pg + GA groups. The signal intensity of MPO activity was detected on the complete craniofacial image
moreover, strong signal intensity was localized specifically at the periodontitis site in the ex vivo palate, with group-wise differences. Conclusions: Molecular imaging analysis based on MPO activity showed high sensitivity of detection of periodontal inflammation in mice. Clinical relevance: Molecular imaging analysis based on MPO activity has potential as a diagnostic tool for periodontitis.

DOI： 10.1007/s00784-018-2510-2

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The recruitment of tissue-resident stem cells is important for wound regeneration. Periodontal ligament cells (PDL cells) are heterogeneous cell populations with stemness features that migrate into wound sites to regenerate periodontal fibres and neighbouring hard tissues. Cell migration is regulated by the local microenvironment, coordinated by growth factors and the extracellular matrix (ECM). Integrin-mediated cell adhesion to the ECM provides essential signals for migration. We hypothesized that PDL cell migration could be enhanced by selective expression of integrins. The migration of primary cultured PDL cells was induced by platelet-derived growth factor-BB (PDGF-BB). The effects of blocking specific integrins on migration and ECM adhesion were investigated based on the integrin expression profiles observed during migration. Up-regulation of integrins α3, α5, and fibronectin was identified at distinct localizations in migrating PDL cells. Treatment with anti-integrin α5 antibodies inhibited PDL cell migration. Treatment with anti-integrin α3, α3-blocking peptide, and α3 siRNA significantly enhanced cell migration, comparable to treatment with PDGF-BB. Furthermore, integrin α3 inhibition preferentially enhanced adhesion to fibronectin via integrin α5. These findings indicate that PDL cell migration is reciprocally regulated by integrin α3-mediated inhibition and α5-mediated promotion. Thus, targeting integrin expression is a possible therapeutic strategy for periodontal regeneration.

DOI： 10.1111/jcmm.14023

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Background: The aim of this study was to investigate the protein and trace element components of PM2.5 and their contribution to the allergic airway inflammation in BALB/c mice. Methods: PM2.5, treated at high temperature and with a strong acid to hydrolyze any protein content and remove trace elements, was administered to BALB/c mice. Allergic airway inflammation was compared between the three groups (saline, pure PM2.5 and treated PM2.5) by evaluating airway hyperresponsiveness (AHR), bronchoalveolar lavage fluid (BALF) cells, serum IgE, the mRNA of various cytokine (IL-4, IL-5, IL-13, eotaxin-1 and CXCL3), mucus protein mRNA (MUC5ac and MUC5b) and the filtration of inflammatory cells in the lung. Results: The treatment of PM2.5 with a strong acid at a high temperature attenuated AHR, eosinophil percentage in BALF, mRNA levels of IL-13 and CXCL3 and peribronchial inflammation. On the contrary, the percentage of neutrophils in BALF, mRNA expression of MIP2α, EGFR, Nrf2, and TLR4 and 4-OH-2-nonenal levels in the lung was increased. Moreover, the treatment of the PM2.5 reduced PM2.5-bound proteins as well as the percentages of the trace elements in PM2.5 in the order Zn > Cu > Pb > P > S > Mn > Fe > Ca > Ni, whereas the percentage of C, Si and Cl increased. Conclusions: PM2.5 collected by of the cyclone system induced allergic airway inflammation in mice. PM2.5-bound proteins and acid-soluble metals may be involved in the pathogenesis of PM2.5-induced allergic airway inflammation.

DOI： 10.1080/08958378.2018.1561769

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Control of bacterial infection-induced inflammatory responses is one of the effective therapeutic approaches of periodontal diseases. Natural products such as lipid mediators and metabolites from microorganisms have been used for decreasing inflammation. We previously reported that (+)-terrein inhibited activation of STAT3 and ERK1/2 in interleukin-6 (IL-6) signaling cascade, leading to prevent vascular endothelial growth factor (VEGF) secretion in human gingival fibroblasts (HGFs). However, little is still known about the role of (+)-terrein on inflammatory responses. In this study, we provided the possibility of novel action that (+)-terrein inhibits activation of Janus-activated kinase 1 (JAK1), which has a central function in IL-6 signaling cascade, and alters expression of mRNAs and proteins induced by IL-6/soluble IL-6 receptor (sIL-6R) stimulation in HGFs. First, we performed PCR array to examine IL-6/sIL-6R-induced mRNA expression, and then expression of mRNA and protein of colony stimulating factor-1 (CSF1) and VEGF were clearly determined by quantitative RT-PCR and ELISA, respectively. Treatment with (+)-terrein suppressed expression of mRNA and protein of CSF1 and VEGF by IL-6/sIL-6R stimulation. Next, to test the effect of (+)-terrein on IL-6/sIL-6R signaling cascade, we demonstrated whether (+)-terrein affects phosphorylation of JAK1 and its downstream proteins, Akt and SHP-2. Western blotting revealed that (+)-terrein inhibited IL-6/sIL-6R-induced phosphorylation of JAK1, Akt, and SHP-2. Therefore, (+)-terrein suppresses IL-6/sIL-6R-induced expression of CSF1 and VEGF via inhibition of JAK1, Akt, and SHP-2. Based on our results, we suggest that (+)-terrein is a candidate compound for anti-inflammatory effect associated with IL-6 signaling.

DOI： 10.1016/j.heliyon.2018.e00979

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Control of bacterial infection-induced inflammatory responses is one of the effective therapeutic approaches of periodontal diseases. Natural products such as lipid mediators and metabolites from microorganisms have been used for decreasing inflammation. We previously reported that (+)-terrein inhibited activation of STAT3 and ERK1/2 in interleukin-6 (IL-6) signaling cascade, leading to prevent vascular endothelial growth factor (VEGF) secretion in human gingival fibroblasts (HGFs). However, little is still known about the role of (+)-terrein on inflammatory responses. In this study, we provided the possibility of novel action that (+)-terrein inhibits activation of Janus-activated kinase 1 (JAK1), which has a central function in IL-6 signaling cascade, and alters expression of mRNAs and proteins induced by IL-6/soluble IL-6 receptor (sIL6R) stimulation in HGFs. First, we performed PCR array to examine IL-6/sIL6R-induced mRNA expression, and then expression of mRNA and protein of colony stimulating factor-1 (CSF1) and VEGF were clearly determined by quantitative RT-PCR and ELISA, respectively. Treatment with (+)-terrein suppressed expression of mRNA and protein of CSF1 and VEGF by IL-6/sIL-6R stimulation. Next, to test the effect of (+)-terrein on IL-6/sIL-6R signaling cascade, we demonstrated whether (+)-terrein affects phosphorylation of JAK1 and its downstream proteins, Akt and SHP-2. Western blotting revealed that (+)-terrein inhibited IL-6/sIL-6R-induced phosphorylation of JAK1, Akt, and SHP-2. Therefore, (+)-terrein suppresses IL-6/sIL-6R-induced expression of CSF1 and VEGF via inhibition of JAK1, Akt, and SHP-2. Based on our results, we suggest that (+)-terrein is a candidate compound for anti-inflammatory effect associated with IL-6 signaling.

DOI： 10.1016/j.heliyon.2018.e00979

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley-Liss Inc.  

High mobility group box 1 (HMGB1) is a non-histone DNA-binding protein that is secreted into the extracellular milieu in response to inflammatory stimuli. The secreted HMGB1 has been suggested to mediate various inflammatory diseases. However, it is still unknown whether HMGB1 is involved in a healing process in the tooth extraction socket, the tissue containing gingival epithelium, and alveolar bone that is exposed to oral bacteria. In this study, we constructed a murine tooth extraction model with anti-HMGB1 neutralization antibody administration and observed the inflammatory response and bone healing process in tooth extraction sockets by molecular imaging of myeloperoxidase (MPO) activity, histological analysis, and quantitative RT-PCR. The translocation of HMGB1 from the nucleus to the cytoplasm in gingival epithelial cells and inflammatory cells was inhibited by anti-HMGB1 antibody administration. The MPO activity around the tooth extraction socket was significantly reduced, and the numbers of CD31- and CD68-positive cells were significantly lower in the anti-HMGB1 antibody treatment samples than in the control samples. The TRAP-positive cells, osteocalcin positive cells, and the neoplastic bone area were significantly lower in anti-HMGB1 antibody treatment samples than in control samples. The expression levels of IL-1β and VEGF-A were also decreased in anti-HMGB1 antibody treatment samples compared to that in control samples. Secreted HMGB1 induced initial acute inflammation and inflammatory cells recruitment after tooth extraction. HMGB1 was associated with angiogenesis and bone remodeling by osteoclast and osteoblast activation and promoted bone healing in the tooth extraction socket.

DOI： 10.1002/jcb.26710

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Unfortunately, in Table-5 of the original article, the parameter in the 5th row was published incorrectly as “LDL-C (mg/dL)”. The correct parameter should read as “HDL-C (mg/dL)”.

DOI： 10.1007/s10266-018-0346-5

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Unfortunately, in Table-5 of the original article, the parameter in the 5th row was published incorrectly as “LDL-C (mg/dL)”. The correct parameter should read as “HDL-C (mg/dL)”.

DOI： 10.1007/s10266-018-0346-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

High mobility group box 1 (HMGB1) is a non-histone DNA-binding protein that is secreted into the extracellular milieu in response to inflammatory stimuli. The secreted HMGB1 mediates various inflammatory diseases, including periodontitis
however, the underlying mechanisms of HMGB1-induced periodontal inflammation are not completely understood. Here, we examined whether anti-HMGB1 neutralizing antibody inhibits periodontal progression and investigated the molecular pathology of HMGB1 in vitro and in vivo. In vitro analysis indicated that HMGB1, granulocytemacrophage colony-stimulating factor (GM-CSF), and interleukin-1β (IL-1β) were secreted in response to tumor necrosis factor-α (TNF-α) stimuli in human gingival epithelial cells (HGECs) and human monocytic leukemia cells (THP-1) treated with phorbol myristate acetate. Increased levels of GM-CSF and IL-1β were observed in the conditioned media from TNF-α-stimulated HGECs and THP-1 in vitro. Simultaneous stimulation with TNF-α and anti-HMGB1 antibody significantly decreased TNF-α- induced inflammatory cytokine secretion. Experimental periodontitis was induced in mice using Porphyromonas gingivalis-soaked ligatures. The extracellular translocation was confirmed in gingival epithelia in the periodontitis model mice by immunofluorescence analysis. Systemic administration of anti-HMGB1 neutralizing antibody significantly inhibited translocation of HMGB1. The anti-HMGB1 antibody inhibited periodontal inflammation, expression of IL-1β and C-X-C motif chemokine ligand 1 (CXCL1), migration of neutrophils, and bone resorption, shown by bioluminescence imaging of myeloperoxidase activity, quantitative reverse transcription-PCR (RT-PCR), and micro-computed tomography analysis. These findings indicate that HMGB1 is secreted in response to inflammatory stimuli caused by periodontal infection, which is crucial for the initiation of periodontitis, and the anti-HMGB1 antibody attenuates the secretion of a series of inflammatory cytokines, consequently suppressing the progression of periodontitis.

DOI： 10.1128/IAI.00111-18

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In the original publication of the article, one of the author name was published incorrectly as “Keisuke Yamashairo” and correct name should be “Keisuke Yamashiro”.

DOI： 10.1007/s10266-017-0334-1

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We previously isolated rat 14.7K-interacting protein-2 (rFIP-2) from the rat-wounded pulp. The protein, homologous to human FIP-2, is known as optineurin and was initially identified as a novel tumor necrosis factor-α (TNF-α)-inducible protein, and more recently, as an autophagy receptor. However, the biological role of optineurin in dental pulp remains elusive. We hypothesized that optineurin has a crucial role in regulating molecular processes during pulp inflammatory responses induced by TNF-α. We examined the kinetics of optineurin expression in pulp inflammation. Optineurin localization and expression were examined using rat pulp fibroblasts. The cells were treated with pharmacological inhibitors for TNF-α-induced inflammatory signals or with hydrogen peroxide as apoptotic stimuli. Stable optineurin-knockdown cells (OPTN-KD cells) were established by transfecting normal rat kidney cells with a vector expressing optineurin-specific small interfering RNA. Cell proliferation and the profiles of cytokines and intracellular signaling molecules were examined using OPTN-KD cells stimulated by TNF-α. Optineurin was localized in the cytoplasm and then translocated into the nucleus upon apoptotic stimuli. Optineurin expression was increased by TNF-α and decreased by a specific inhibitor of c-Jun N-terminal kinase. The OPTN-KD cells secreted smaller amounts of monocyte chemotactic protein-1 (MCP-1) and intracellular MCP-1 mRNA, and cell proliferation was significantly increased. Apoptosis-related signaling molecules were downregulated in OPTN-KD cells. These results demonstrated that optineurin is a crucial molecule mediated by TNF-α, which induces the production of inflammatory factors and apoptosis signaling, suggesting the presence of signaling interactions between optineurin and a transcription factor for MCP-1.

DOI： 10.1007/s10266-017-0314-5

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Cells behave in a variety of ways when they perceive changes in their microenvironment
the behavior of cells is guided by their coordinated interactions with growth factors, niche cells, and extracellular matrix (ECM). Modulation of the microenvironment affects the cell morphology and multiple gene expressions. Rho/Rho-associated coiled-coil-containing protein kinase (ROCK) signaling is one of the key regulators of cytoskeletal dynamics and actively and/or passively determines the cell fate, such as proliferation, migration, differentiation, and apoptosis, by reciprocal communication with the microenvironment. During periodontal wound healing, it is important to recruit the residential stem cells into the defect site for regeneration and homeostasis of the periodontal tissue. Periodontal ligament (PDL) cells contain a heterogeneous fibroblast population, including mesenchymal stem cells, and contribute to the reconstruction of tooth-supporting tissues. Therefore, bio-regeneration of PDL cells has been the ultimate goal of periodontal therapy for decades. Recent stem cell researches have shed light on intrinsic ECM properties, providing paradigm shifts in cell fate determination. This review focuses on the role of ROCK activity and the effects of Y-27632, a specific inhibitor of ROCK, in the modulation of ECM-microenvironment. Further, it presents the current understanding of how Rho/ROCK signaling affects the fate determination of stem cells, especially PDL cells. In addition, we have also discussed in detail the underlying mechanisms behind the reciprocal response to the microenvironment.

DOI： 10.1007/s12079-017-0425-3

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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.

DOI： 10.1002/JPER.17-0412

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Oral bacteria initiate biofilm formation by attaching to tooth surfaces via an interaction of a lectin-like bacterial protein with carbohydrate chains on the pellicle. This study aimed to find naturally derived lectins that inhibit the initial attachment of a cariogenic bacterial species, Streptococcus mutans (S. mutans), to carbohydrate chains in saliva in vitro. Seventy kinds of lectins were screened for candidate motifs that inhibit the attachment of S. mutans ATCC 25175 to a saliva-coated culture plate. The inhibitory effect of the lectins on attachment of the S. mutans to the plates was quantified by crystal violet staining, and the biofilm was observed under a scanning electron microscope (SEM). Surface plasmon resonance (SPR) analysis was performed to examine the binding of S. mutans to carbohydrate chains and the binding of candidate lectins to carbohydrate chains, respectively. Moreover, binding assay between the biotinylated-lectins and the saliva components was conducted to measure the lectin binding. Lectins recognizing a salivary carbohydrate chain, Galβ1-3GalNAc, inhibited the binding of S. mutans to the plate. In particular, Agaricus bisporus agglutinin (ABA) markedly inhibited the binding. This inhibition was confirmed by SEM observation. SPR analysis indicated that S. mutans strongly binds to Galβ1-3GalNAc, and ABA binds to Galβ1-3GalNAc. Finally, the biotinylated Galβ1-3GalNAc-binding lectins including ABA demonstrated marked binding to the saliva components. These results suggest that ABA lectin inhibited the attachment of S. mutans to Galβ1-3GalNAc in saliva and ABA can be useful as a potent inhibitor for initial attachment of oral bacteria and biofilm formation.

DOI： 10.1007/s10719-017-9795-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Luigi Ponzio e figlio Editori  

An insertion sequence, IS1598 (IsPg4) has been found in virulent strains of Porphyromonas gingivalis in a murine abscess model. The present study was performed to investigate the effects of genetic rearrangements by IS1598 on the phenotypic characteristics of the virulent strains. For this purpose, we searched for a common insertion site of IS1598 among the virulent strains. Through cloning and database search, a common insertion site was identified beside an nrdD-like gene in the virulent FDC 381, W83 and W50 strains. In this region, predicted promoters of the nrdD-like gene and IS1598 are located in tandem, and accumulation of nrdD-like gene mRNA was 5-fold higher in virulent strains (W83, W50, FDC 381) than avirulent strains (ATCC33277, SU63, SUNY1021, ESO59 without IS1598). The role of the nrdD-like gene in virulence of P. gingivalis was investigated by constructing a nrdD-deficient mutant. In the murine abscess model, the parental W83 strain produced necrotic abscesses, while the nrdD-deficient mutant had almost lost this ability. Insertion of IS1598 into the nrdD-like gene promoter region may be related to the phenotypic differences in virulence among P. gingivalis strains through upregulation of the expression of this gene.

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DOI： 10.1371/journal.pone.0188014

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DOI： 10.1002/ccr3.751

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BACKGROUND: The diagnosis of the progression of periodontitis presently depends on the use of clinical symptoms (such as attachment loss) and radiographic imaging. The aim of the multicenter study described here was to evaluate the diagnostic use of the bacterial content of subgingival plaque recovered from the deepest pockets in assessing disease progression in chronic periodontitis patients. METHODS: This study consisted of a 24-month investigation of a total of 163 patients with chronic periodontitis who received trimonthly follow-up care. Subgingival plaque from the deepest pockets was recovered and assessed for bacterial content of Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans using the modified Invader PLUS assay. The corresponding serum IgG titers were measured using ELISA. Changes in clinical parameters were evaluated over the course of 24 months. The sensitivity, specificity, and prediction values were calculated and used to determine cutoff points for prediction of the progression of chronic periodontitis. RESULTS: Of the 124 individuals who completed the 24-month monitoring phase, 62 exhibited progression of periodontitis, whereas 62 demonstrated stable disease. The P. gingivalis counts of subgingival plaque from the deepest pockets was significantly associated with the progression of periodontitis (p < 0.001, positive predictive value = 0.708). CONCLUSIONS: The P. gingivalis counts of subgingival plaque from the deepest pockets may be associated with the progression of periodontitis.

DOI： 10.1186/s12903-017-0337-x

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DOI： 10.1111/jre.12353

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.archoralbio.2016.06.025

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.oooo.2016.04.006

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This OTC medication is biochemically effective for steady chronic periodontitis in the supportive periodontal therapy phase.

DOI： 10.1111/jre.12311

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DOI： 10.1002/jbmr.2738

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

<title>ABSTRACT</title>

The oral Gram-negative anaerobic bacterium
<named-content content-type="genus-species">Porphyromonas gingivalis</named-content>
is an important pathogen involved in chronic periodontitis. Among its virulence factors, the major extracellular proteinases, Arg-gingipain and Lys-gingipain, are of interest given their abilities to degrade host proteins and process other virulence factors. Gingipains possess C-terminal domains (CTDs) and are translocated to the cell surface or into the extracellular milieu by the type IX secretion system (T9SS). Gingipains contribute to the colonial pigmentation of the bacterium on blood agar. In this study, Omp17, the PGN_0300 gene product, was found in the outer membrane fraction. A mutant lacking Omp17 did not show pigmentation on blood agar and showed reduced proteolytic activity of the gingipains. CTD-containing proteins were released from bacterial cells without cleavage of the CTDs in the
<italic>omp17</italic>
mutant. Although synthesis of the anionic polysaccharide (A-LPS) was not affected in the
<italic>omp17</italic>
mutant, the processing of and A-LPS modification of CTD-containing proteins was defective. PorU, a C-terminal signal peptidase that cleaves the CTDs of other CTD-containing proteins, was not detected in any membrane fraction of the
<italic>omp17</italic>
mutant, suggesting that the defective maturation of CTD-containing proteins by impairment of Omp17 is partly due to loss of function of PorU. In the mouse subcutaneous infection experiment, the
<italic>omp17</italic>
mutant was less virulent than the wild type. These results suggested that Omp17 is involved in
<named-content content-type="genus-species">P. gingivalis</named-content>
virulence.

DOI： 10.1128/iai.01308-15

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It has been revealed that atherosclerosis and periodontal disease may have a common mechanism of "chronic inflammation". Several reports have indicated that periodontal infection is related to atherosclerosis, but none have yet reported such an investigation through the cooperation of local clinics. This study was performed in local Japanese clinics to examine the relationship between periodontal disease and atherosclerosis under collaborative medical and dental care. A pilot multicenter cross-sectional study was conducted on 37 medical patients with lifestyle-related diseases under consultation in participating medical clinics, and 79 periodontal patients not undergoing medical treatment but who were seen by participating dental clinics. Systemic examination and periodontal examination were performed at baseline, and the relationships between periodontal and atherosclerosis-related clinical markers were analyzed. There was a positive correlation between LDL-C level and plasma IgG antibody titer to Porphyromonas gingivalis. According to the analysis under adjusted age, at a cut-off value of 5.04 for plasma IgG titer to Porphyromonas gingivalis, the IgG titer was significantly correlated with the level of low-density lipoprotein cholesterol (LDL-C). This study suggested that infection with periodontal bacteria (Porphyromonas gingivalis) is associated with the progression of atherosclerosis. Plasma IgG titer to Porphyromonas gingivalis may be useful as the clinical risk marker for atherosclerosis related to periodontal disease. Moreover, the application of the blood examination as a medical check may lead to the development of collaborative medical and dental care within the local medical clinical system for the purpose of preventing the lifestyle-related disease.

DOI： 10.1007/s10266-014-0172-3

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BACKGROUND AND OBJECTIVE: Tumor necrosis factor alpha (TNF-α) is a major cytokine implicated in various inflammatory diseases. The nature of the nuclear factors associated with human TNF-α gene regulation is not well elucidated. We previously identified a novel region located from -550 to -487 in human TNF-α promoter that did not contain the reported binding sites for nuclear factor kappa B (NF-κB) but showed lipopolysaccharide (LPS)-induced transcriptional activity. The purpose of this study is to identify novel factors that bind to the promoter region and regulate TNF-α expression. MATERIAL AND METHODS: To identify DNA-binding proteins that bound to the target region of TNF-α promoter, a cDNA library from LPS-stimulated human monocytic cell line THP-1 was screened using a yeast one-hybrid system. Cellular localizations of the DNA-binding protein in the cells were examined by subcellular immunocytochemistry. Nuclear amounts of the protein in LPS-stimulated THP-1 cells were identified by western blot analysis. Expression of mRNA of the protein in the cells was quantified by real-time polymerase chain reaction. Electrophoretic mobility shift assays were performed to confirm the DNA-binding profile. Overexpression of the protein and knockdown of the gene were also performed to investigate the role for TNF-α expression. RESULTS: Several candidates were identified from the cDNA library and transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) was focused on. Western blot analysis revealed that nuclear TDP-43 protein was increased in the LPS-stimulated THP-1 cells. Expression of TDP-43 mRNA was already enhanced before TNF-α induction by LPS. Electrophoretic mobility shift assay analysis showed that nuclear extracts obtained by overexpressing FLAG-tagged TDP-43 bound to the -550 to -487 TNF-α promoter fragments. Overexpression of TDP-43 in THP-1 cells resulted in an increase of TNF-α expression. Knockdown of TDP-43 in THP-1 cells downregulated TNF-α expression. CONCLUSION: We identified TDP-43 as one of the novel TNF-α factors and found that it bound to the LPS-responsive element in the TNF-α promoter to increase TNF-α expression.

DOI： 10.1111/jre.12227

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DOI： 10.1111/ggi.12430

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DOI： 10.1007/s00520-014-2432-8

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Publishing type：Research paper (scientific journal)   Publisher：Informa UK Limited  

DOI： 10.2147/cia.s73388

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DOI： 10.1002/ccr3.112

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DOI： 10.1002/ccr3.114

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DOI： 10.1016/j.bmc.2014.07.047

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DOI： 10.1007/s00520-014-2151-1

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BACKGROUND AND OBJECTIVE: Spatiotemporal inhibition of apical migration and proliferation of gingival epithelium are significant factors involved in periodontal regeneration. Transforming growth factor β (TGF-β) is important in multiple aspects of wound healing, and Smad2, a downstream transcription factor of TGF-β, has an inhibitory effect on re-epithelialization during gingival wound healing. Therefore, we investigated the effects on migration and proliferation status, and intra/extracellular signaling regulated by Smad2 overexpression in gingival epithelial cells. MATERIAL AND METHODS: Gingival epithelial cells were isolated from the palatal gingival tissue of transgenic mice overexpressing Smad2 driven by the Keratin14 promoter. Smad2 expression was identified by western blotting and immunofluorescence analysis. Scratch assay and 5-bromo-2'-deoxyuridine staining were performed to assess cell migration and proliferation. To inactivate TGF-β type I receptor, the cultures were supplemented with SB431542. Secreted TGF-β was quantified by ELISA. Smad2 target gene expression was examined by real-time RT-PCR and in vivo immunofluorescence analysis of gingival junctional epithelium. RESULTS: Smad2-overexpressing cells were confirmed to have significant phosphorylated Smad2 in the nucleus. Scratch assay and 5-bromo-2'-deoxyuridine staining indicated that Smad2-overexpressing cells showed no significant differences in migration, but had reduced proliferation rates compared to wild-type controls. SB431542 significantly inhibited Smad2 phosphorylation, which coincided with restoration of the proliferation rate in Smad2-overexpressing cells. ELISA of TGF-β release did not show any differences between genotypes. The cell cycle inhibitors, p15 and p21, showed significant upregulation in Smad2-overexpressing cells compared to wild-type controls. Moreover, junctional epithelium of the transgenic mice showed increased expression of P-Smad2, p15 and p21. CONCLUSION: The signaling activation triggered by overexpression of Smad2 was dependent on TGF-β type I receptor, and the activated Smad2 increased p15 and p21 expression, responsible for inhibiting cell cycle entry, resulting in antiproliferative effects on gingival epithelial cells. Understanding of Smad2-induced signaling would be useful for possible clinical application to regulate gingival epithelial downgrowth.

DOI： 10.1111/jre.12106

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DOI： 10.1016/j.micinf.2013.10.005

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DOI： 10.1111/2049-632x.12041

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Authorship：Last author   Publishing type：Research paper (scientific journal)   Publisher：Allergy, Asthma, and Immunology Association of Thailand  

DOI： 10.12932/ap0287.31.4.2013

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DOI： 10.1007/s00520-012-1602-9

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publisher：JAPANESE SOCIETY OF PERIODONTOLOGY  

DOI： 10.2329/perio.54.237

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Chronic periodontitis is a silent infectious disease prevalent worldwide and affects lifestyle-related diseases. Therefore, efficient screening of patients is essential for general health. This study was performed to evaluate prospectively the diagnostic utility of a blood IgG antibody titer test against periodontal pathogens. Oral examination was performed, and IgG titers against periodontal pathogens were measured by ELISA in 1,387 individuals. The cut-off value of the IgG titer was determined in receiver operating characteristic curve analysis, and changes in periodontal clinical parameters and IgG titers by periodontal treatment were evaluated. The relationships between IgG titers and severity of periodontitis were analyzed. The best cut-off value of IgG titer against Porphyromonas gingivalis for screening periodontitis was 1.682. Both clinical parameters and IgG titers decreased significantly under periodontal treatment. IgG titers of periodontitis patients were significantly higher than those of healthy controls, especially in those with sites of probing pocket depth over 4 mm. Multiplied cut-off values were useful to select patients with severe periodontitis. A blood IgG antibody titer test for Porphyromonas gingivalis is useful to screen hitherto chronic periodontitis patients (ClinicalTrials.gov number NCT01658475).

DOI： 10.1177/0022034512461796

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During periodontal regeneration, inhibition of gingival downgrowth is necessary to promote migration of mesenchymal cells into the defects. Transforming growth factor (TGF)-β is a pleiotropic cytokine that has numerous cell functions, including regulation of epithelial growth. Recent studies have shown that Smad2, a downstream transcription factor of TGF-β, plays crucial roles in wound healing in the epithelia. Therefore, we investigated the effects of Smad2 overexpression on re-epithelialization of gingival wounds. Transgenic mice overexpressing smad2 driven by the keratin 14 promoter ( k14-smad2) were confirmed to have significant Smad2 phosphorylation in gingival basal epithelia. Punch wounds were made in the palatal gingiva, and wound healing was assessed histologically for 7 days. Re-epithelialization was significantly retarded on day 2, while collagen deposition was enhanced on day 7 in k14-smad2 compared with wild-type mice. Moreover, expression of keratin 16 (K16), an indicator of keratinocyte migration, was significantly inhibited in wound-edge keratinocytes in k14-smad2. The inhibition of K16 coincided with the induction of Smad2 in the corresponding epithelia, while BrdU incorporation was unaffected. These results indicated that Smad2 has inhibitory effects in regulating keratinocyte migration during gingival wound healing. TGF-β/Smad2 signaling mediating alteration of K16 expression must be tightly regulated during periodontal regeneration.

DOI： 10.1177/0022034512451449

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DOI： 10.1371/journal.pone.0040570

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DOI： 10.1007/s10266-011-0051-0

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publisher：JAPANESE SOCIETY OF PERIODONTOLOGY  

DOI： 10.2329/perio.54.3

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DOI： 10.4012/dmj.2011-164

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DOI： 10.1371/journal.pone.0026163

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DOI： 10.1007/s00520-010-0923-9

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OBJECTIVE: Multiple risk factor syndrome is a clustering of cardiovascular risk factors, such as diabetes, dyslipidemia, hypertension, and obesity associated epidemiologically with insulin resistance. This report describes the clinical course of a patient suffering from severe periodontitis with multiple risk factor syndrome, and discusses the association between periodontal infection and systemic health. METHODS: The patient had a history of type 2 diabetes, dyslipidemia, and hypertension for over 10 years. At baseline, her hemoglobin A1 c was 8.1%. However, she had no diabetic complications except periodontitis. The IgG antibody titers against Porphyromonas gingivalis FDC 381 and SU63 were elevated above the mean of healthy subjects +2 standard deviations. Intensive periodontal treatment, including periodontal surgery, was performed to reduce periodontal infection and bacteremia. Her systemic and periodontal conditions were evaluated longitudinally for 10 years. RESULTS: Following periodontal treatment, antibody titers against Porphyromonas gingivalis and hemoglobin A1c values were significantly improved. The other clinical data and medication for her systemic condition also remained stable during supportive periodontal therapy. However, she developed myocardial infarction, and showed continuous deterioration of hemoglobin A1 c level and periodontitis. CONCLUSION: The long-term clustering of risk factors, such as diabetes, dyslipidemia, hypertension, and periodontitis, are associated with the development of myocardial infarction. Treatment of systemic conditions in combination with comprehensive periodontal treatment is important in management of patients with multiple risk factor syndrome.

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The enzyme-labeled antigen method was applied to visualize plasma cells producing antibodies to Porphyromonas gingivalis, flora of the human oral cavity. Antibodies to P. gingivalis have reportedly been detected in sera of patients with periodontitis. Biotinylated bacterial antigens, Ag53, and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro, and Lys-hgp) were prepared by the cell-free protein synthesis system using the wheat germ extract. In paraformaldehyde-fixed frozen sections of rat lymph nodes experimentally immunized with Ag53-positive and Ag53-negative P. gingivalis, plasma cells were labeled with biotinylated Arg-hgp and Lys-hgp. Antibodies to Ag53 were detected only in the nodes immunized with Ag53-positive bacteria. In two of eight lesions of gingival radicular cyst with inflammatory infiltration, CD138-positive plasma cells in frozen sections were signalized for Arg-hgp and Lys-hgp. An absorption study using unlabeled antigens confirmed the specificity of staining. The AlphaScreen method identified the same-type antibodies in tissue extracts but not in sera. Antibodies to Ag53, Arg-pro, and Lys-pro were undetectable. In two cases, serum antibodies to Arg-hgp and Lys-hgp were AlphaScreen positive, whereas plasma cells were scarcely observed within the lesions. These findings indicate the validity of the enzyme-labeled antigen method. This is the very first application of this novel histochemical technique to human clinical samples.

DOI： 10.1369/0022155411408906

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DOI： 10.1111/j.1601-5037.2010.00448.x

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Periodontitis is localized infectious disease caused by periodontopathic bacteria such as Porphyromonas gingivalis (T: gingivalis), and the severity correlates to significance of immune responses. Recently, it has been reported that periodontitis is associated with the development of systemic disease such as diabetes and atherosclerosis because of increasing invasion of oral pathogens to the circulation. However, the association between local and systemic infectious responses is still unclear. In the present study, we examined the differences of biological responses in animals with or without bacterial infection. After Balb/c mice were infected subcutaneously with live P gingivalis W83, serum, skin and liver were collected according to experimental protocol. The skin and liver tissues were observed pathologically by haematoxylin-eosin staining, and serum IL-6 levels were measured using ELISA method. Throughout the experimental period, conditions of the mice were observed continuously. As expected, severe infiltration of leukocytes were observed at inflamed skin corresponding to the number of bacterial challenges. Although no inflammatory appearance of skin was observed, serum IL-6 levels were increased dramatically (P &lt; 0.01, Student&apos;s t-test) and liver tissues were injured in the mice without bacterial challenge. Interestingly, although severe inflammatory appearance of the skin was observed, serum IL-6 levels were not increased and no inflammatory responses were observed in the liver of the 3-times bacterially challenged group. Importantly, immunoglobulin G against P gingivalis W83 was detected in the blood of mice with 3-times bacterial challenge corresponding to improvement of weight loss and survival. In conclusion, although multiple infections develop severe localized inflammation, the immune system should be sufficient to protect the systemic inflammatory responses.

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DOI： 10.1007/s00520-010-1002-y

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The efficacy of the local application of recombinant human fibroblast growth factor-2 (FGF-2) in periodontal regeneration has been investigated. In this study, a randomized, double-blind, placebo-controlled clinical trial was conducted in 253 adult patients with periodontitis. Modified Widman periodontal surgery was performed, during which 200 µL of the investigational formulation containing 0% (vehicle alone), 0.2%, 0.3%, or 0.4% FGF-2 was administered to 2- or 3-walled vertical bone defects. Each dose of FGF-2 showed significant superiority over vehicle alone (p &lt; 0.01) for the percentage of bone fill at 36 wks after administration, and the percentage peaked in the 0.3% FGF-2 group. No significant differences among groups were observed in clinical attachment regained, scoring approximately 2 mm. No clinical safety problems, including an abnormal increase in alveolar bone or ankylosis, were identified. These results strongly suggest that topical application of FGF-2 can be efficacious in the regeneration of human periodontal tissue that has been destroyed by periodontitis.

DOI： 10.1177/0022034510384616

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DOI： 10.1002/jcla.20381

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL INST INFECTIOUS DISEASES  

Periodontitis is a chronic inflammatory disease caused by the infection of periodontopathic bacteria in dental plaque However, an individual's susceptibility to this disease appears to be associated with multiple genetic factors, as seen in the case of leprosy In order to gain a better understanding of the pathophysiology of periodontal disease in subjects with leprosy, we investigated the clinical features of periodontitis and the immunological responses against periodontopathic bacteria in 382 subjects with a history of leprosy and 451 age-matched control subjects The prevalence of periodontitis and the degree of periodontal pocket depth were found to be significantly higher in leprosy patients than in age-matched controls Furthermore, a comparison of the clinical parameters of lepromatous leprosy (L-lep) and tuberculoid leprosy (T-lep) patients showed that the probing pocket depth of L-lep patients with periodontal disease was significantly higher than that for T-lep patients In contrast, serum IgG titers against Porphyromonas gingivalis in L-lep patients were significantly lower than in T-lep patients These results imply that L-lep patients show more severe periodontal disease than T-lep patients or age-matched control subjects, and that low humoral immunity against P gingivalis might be one of the genetic factors determining periodontal disease susceptibility in leprosy patients

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DOI： 10.1111/j.1600-0765.1998.tb02289.x

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DOI： 10.1111/j.2041-1014.2009.00548.x

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Aim:
To develop a detection assay for staphylococcal mecA and spa by using loop-mediated isothermal amplification (LAMP) method.
Methods and Results:
Staphylococcus aureus and other related species were subjected to the detection of mecA and spa by both PCR and LAMP methods. The LAMP successfully amplified the genes under isothermal conditions at 64 degrees C within 60 min, and demonstrated identical results with the conventional PCR methods. The detection limits of the LAMP for mecA and spa, by gel electrophoresis, were 102 and 10 cells per tube, respectively. The naked-eye inspections were possible with 103 and 10 cells for detection of mecA and spa, respectively. The LAMP method was then applied to sputum and dental plaque samples. The LAMP and PCR demonstrated identical results for the plaque samples, although frequency in detection of mecA and spa by the LAMP was relatively lower for the sputum samples when compared to the PCR methods.
Conclusion:
Application of the LAMP enabled a rapid detection assay for mecA and spa. The assay may be applicable to clinical plaque samples.
Significance and Impact of the Study:
The LAMP offers an alternative detection assay for mecA and spa with a great advantage of the rapidity.

DOI： 10.1111/j.1472-765x.2010.02806.x

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DOI： 10.1111/j.1574-695x.2009.00624.x

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DOI： 10.1007/s00520-009-0789-x

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DOI： 10.1007/s00520-009-0637-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE Publications  

Genetic variants at multiple loci have been shown to be associated with susceptibility to periodontitis. To better assess the genetic risk factors for periodontitis, we performed a case-control study in 319 Japanese individuals with periodontitis (172 aggressive and 147 chronic disease) and 303 race-matched healthy control individuals. Thirty-five functional gene polymorphisms that had been previously associated with immune responses were genotyped. For all gene polymorphisms tested, no significant differences were observed in the allele frequencies of persons with aggressive, chronic, and combined (aggressive and chronic) periodontitis, compared with control individuals. Multiple logistic regression analysis revealed a significant association of the vitamin D receptor +1056 T/C polymorphism with susceptibility to chronic periodontitis, after adjustment for age, gender, and smoking status (P = 0.002). These results suggest that none of the polymorphisms tested was strongly associated with periodontitis in a Japanese population. However, the vitamin D receptor +1056 polymorphism may be related to chronic periodontitis.

DOI： 10.1177/0022034509350037

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DOI： 10.1111/j.1600-0765.2008.01150.x

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DOI： 10.1007/s00520-008-0532-z

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DOI： 10.1016/j.dental.2008.08.012

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DOI： 10.1902/jop.2009.080516

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DOI： 10.1111/j.1600-0765.2008.01097.x

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DOI： 10.1002/jcp.21517

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DOI： 10.1007/s00520-008-0470-9

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DOI： 10.1111/j.1574-6968.2008.01304.x

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DOI： 10.1111/j.1574-695x.2008.00417.x

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DOI： 10.1371/journal.pone.0002611

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DOI： 10.1111/j.1601-0825.1999.tb00100.x

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DOI： 10.1007/s00520-007-0391-z

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DOI： 10.1111/j.1600-051x.2008.01208.x

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DOI： 10.1902/jop.2008.070205

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Language：English   Publisher：Japan Society of Ningen Dock  

Background Periodontal disease is a multiple infection caused by bacteria represented by Porphyromonas gingivalis (Pg). The prevalence of periodontal disease is high, as it predominantly affects the people in the same age range as diabetes mellitus, but it is often overlooked because of the lack of subjective symptoms. There has been a need for the introduction of self-sampled device-treated plasma using fingertip capillary blood in routine tests.<br>Methods Based on the report by Kudo et al. (Beppu Conference 2006, p.46, 2006) on enzyme-linked immunosorbent assay (ELISA) as a blood test for periodontal disease bacteria, the precision and disease specificity were examined towards the establishment of a methodology for routine tests.<br>Results The intra-assay reproducibility of the measurements using Pg and 3 other types of antigens, Prevotella intermedia (Pi), Eikenella corroders (Ec), and Actinobacillus actinomycetemcomitans (Aa), was coefficient of variation (CV) 4-7%, and the results from capillary blood and those from venous blood were closely analogous to each other with a correlation (r) of 0.900 or more. The difference between the non-periodontal control subjects group and the periodontal disease group in the distribution of data on histograms using the Pg antigen was approximately 7-fold. In addition, the correlation with mean periodontal pocket depth was significant (r=0.597, p<0.001) in the group showing the test results (SD index; SDI) of 20 or more. These results substantiated the specificity of this method.<br>Conclusion The ability to detect periodontal disease in the setting of "Human Dry Dock" screening examinations using the sampling and test methods proposed by us would be an important means to improve services. This study established the practical feasibility of this approach.

DOI： 10.11320/ningendock2005.22.6_35

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DOI： 10.1111/j.1600-0765.2007.00985.x

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DOI： 10.1038/oby.2007.305

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DOI： 10.1007/s00441-007-0457-8

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Diabetic subjects are susceptible to atherosclerosis. It has been postulated that inflammation plays a crucial role in atherogenesis. Since previous studies suggested persistent low-grade infection by Gram-negative bacteria such as Chlamydia spp. and/or periodontal infection is associated with increased atherogenesis among diabetic subjects, we hypothesized that macrophages under hyperglycemia respond to lipopolysaccharide (LPS) challenge in a more exaggerated manner than under normal glucose conditions. Therefore, we examined cytokine productivity and associated signal transduction molecules in LPS-stimulated the monocytic cell line THP-1, under conditions of hyperglycemia. Differentiated THP-1 cells were cultured under normal and high glucose conditions without fetal bovine serum, and were stimulated with Escherichia coli LPS in the presence of LPS binding protein. Following stimulation, activated signal transduction molecules were detected by protein microarray and confirmed thereafter. Results indicated that c-jun N-terminal kinase (JNK) was highly-phosphorylated at high glucose concentrations, and this was confirmed by Western-immunoblotting. Tumor necrosis factor-α and monocyte chemo-attractant protein-1 production were significantly enhanced under these conditions. SP600125, a selective inhibitor of JNK, dose-dependently suppressed the production of these cytokine. Therefore, we suggest that this may be one of the mechanisms by which sub-clinical infection by Gram-negative bacteria promotes atherosclerosis in diabetic subjects.

DOI： 10.1177/0968051907082608

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DOI： 10.1016/j.joen.2007.04.016

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DOI： 10.1111/j.1365-2249.2007.03450.x

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DOI： 10.1016/j.biocel.2007.01.015

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DOI： 10.1111/j.1365-4632.2006.02900.x

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DOI： 10.1111/j.1574-695x.2006.00094.x

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DOI： 10.1111/j.1600-0757.2005.00142.x

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DOI： 10.1111/j.1574-6968.1997.tb10187.x

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DOI： 10.1007/s10103-005-0350-1

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α2 integrin on fibroblasts is reported to play an important role in the induction of drug-induced gingival overgrowth, which is characterized by excessive accumulation of type I collagen in gingival connective tissue. Silent polymorphism 807 T/C within the α2 integrin gene is associated with high/low α2 integrin expression. The aim of this study was to test the hypothesis that expression of α2 integrin 807 T/C polymorphism correlates with drug-induced gingival overgrowth. A case-control study comparing 136 subjects taking calcium channel blockers (72 with vs. 64 without drug-induced gingival overgrowth) demonstrated that the frequency of the +807 C allele was significantly higher in the case group than in the controls (odds ratio, 3.61; 95% confidence interval, 2.14 – 6.10; P &lt; 0.05). The present findings suggest that the α2 +807 C allele is one of the genetic risk factors for drug-induced gingival overgrowth.

DOI： 10.1177/154405910508401217

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Pulpal wound healing followed by cavity preparation may involve reactionary or reparative dentinogenesis in relation to the cavity position; however, little is known about the molecular responses. We aimed to isolate and analyze genes induced or suppressed in the wounded pulp to identify molecular processes involved in the pulp responses to injury. Twenty-three cDNAs were isolated by cDNA subtraction between healthy and wounded pulp of rats. By library screening, we identified rat 14.7K-interacting protein (rFIP)-2A and B genes homologous to human FIP-2, being involved in regulating membrane trafficking and cellular morphogenesis. RT-PCR analysis showed induction for only rFIP-2B in the wounded pulp. In situ hybridization analysis revealed that both rFIP-2s were expressed strongly in condensing mesenchymal cells of the palatal process and surrounding Meckel’s cartilage, but not in intramembranous chondrogenic cells. Thus, up-regulated rFIP-2B expression may play a role in regulating membrane trafficking or cellular morphogenesis of these embryonic and wounded pulpal cells.

DOI： 10.1177/154405910508400912

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DOI： 10.1016/j.femsim.2005.01.008

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Background: Aplastic anemia (AA) is a rare hematologic disease characterized by hypo-cellular bone marrow. The clinical features include fatigue, increased bruising, and gingival bleeding caused by anemia, leukopenia, and thrombocytopenia. A patient with AA is at high risk for infection because of leukopenia. The risk of systemic infection is especially high in AA patients with severe local infections, including periodontitis. Accordingly, periodontal treatment should include antibiotic prophylaxis to reduce the risk of systemic infection. However, treatment of periodontitis in the AA patient is significantly complicated by the bleeding disorder. We present a case report of the successful periodontal treatment of an AA patient with spontaneous gingival bleeding.
Methods: The patient was closely monitored for platelet and neutrophil counts before every treatment. The patient's platelet count was always under 10,000/mu l. Therefore, it was necessary to increase platelet counts to over 25,000/mu l by transfusion, after which subgingival scaling with anesthesia was performed. When the neutrophil count was less than 2,000/mu l, local minocycline chemotherapy was applied to the pockets. Periodontal infection was monitored by detection of bacterial DNA and measurement of serum immunoglobulin (Ig) G titer against periodontal bacteria.
Results: Following the physical and chemical treatment, the gingival appearance improved dramatically and the spontaneous gingival bleeding disappeared. Moreover, the IgG titer against periodontal bacteria decreased to normal range and specific periodontal pathogens were no longer detectable in the tested pockets.
Conclusion: We believe that the treatment strategy in the present report provides new sight into treatment planning for severely medically compromised patients.

DOI： 10.1902/jop.2005.76.7.1211

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DOI： 10.1111/j.1399-302x.2004.00203.x

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An elevated level of C-reactive protein (CRP) predicts the future development of coronary heart disease. Periodontitis appears to up-regulate CRP. CRP is produced by hepatocytes in response to interleukin-6 (IL-6). A major source of IL-6 in obese subjects is adipocytes. We hypothesized that lipopolysaccharide (LPS) from periodontal pathogens stimulated adipocytes to produce IL-6, and that the production was suppressed by the drugs targeted against insulin resistance, thiazolidinedione (pioglitazone), since this agent potentially showed an anti-inflammatory effect. Mouse 3T3-L1 adipocytes were stimulated with E. coli, P. gingivalis, and F. nucleatum LPS. The IL-6 concentration in culture supernatants was measured. All LPS stimulated adipocytes to produce IL-6. Although pioglitazone changed adipocyte appearance from large to small, and completely suppressed P. gingivalis and F. nucleatum LPS-induced IL-6 production, E. coli LPS-induced IL-6 production was not efficiently blocked. Thus, pioglitazone completely blocked periodontal-bacteria-derived LPS-induced IL-6 production in adipocytes, a major inducer of CRP.

DOI： 10.1177/154405910508400306

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DOI： 10.1016/j.femsim.2004.08.005

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BACKGROUND: Gingival overgrowth is a common side-effect following administration of cyclosporin A. We reported previously that lysosomal protease cathepsin-L activity, but not cathepsin-B, was significantly suppressed by short-term cyclosporin A exposure in human gingival fibroblasts. Although this suppression may lead to decreased degradation of gingival connective tissue, a net increase in matrix proteins, and gingival overgrowth, the effects of cyclosporin A need to be more elucidated, considering the long-term use for patients following organ transplantation. OBJECTIVE: The aim of the present study was to evaluate the effects of clinically relevant doses of cyclosporin A on cultured human gingival fibroblasts. We evaluated the effects of long-term cyclosporin A exposure on cell proliferation, mRNA expression of various proteases and both cathepsin-B and -L activity in human gingival fibroblasts. MATERIALS AND METHODS: Human gingival fibroblasts were isolated from three donors' healthy gingiva and cultured from five to eight passages with or without 200 ng/ml of cyclosporin A. Proliferative activity of cyclosporin A-treated cells was examined using MTT assay. Total RNA and cellular proteins were collected for semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis and for measurement of the cathepsin-B and -L activity. RESULTS: Long-term cyclosporin A exposure had no effects on cell proliferation. Accumulation of cathepsin-B, -H and -L mRNA was markedly suppressed by long-term cyclosporin A exposure, whereas accumulation of another lysosomal enzyme N-acetyl-beta-D-glucosaminidase mRNA, which is involved in remodeling of gingival epithelium, was not apparently impaired in cyclosporin A-treated cells. Accumulation of matrix metalloprotease-1 (MMP-1) and tissue inhibitor of matrix metalloprotease-1 (TIMP-1) mRNA, which are involved in remodeling of extracellular matrix, also was not impaired. In addition, we demonstrated that long-term cyclosporin A exposure significantly suppressed not only the activity of the active form of cathepsin-(B + L) compared to the activity in non-treated cells (p = 0.0458), but also the activity of the active form of cathepsin-B (p < 0.0001) in human gingival fibroblasts. CONCLUSION: The decreased ability of protein degradation by not only cathepsin-L but also cathepsin-B is, at least, one of the several factors developing the cyclosporin A-induced gingival overgrowth.

DOI： 10.1111/j.1600-0765.2004.00746.x

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Periodontal healing requires the participation of regulatory molecules, cells, and scaffold or matrix. Here, we hypothesized that a certain set of genes is expressed in alveolar bone wound healing. Reciprocal subtraction gave 400 clones from the injured alveolar bone of Wistar rats. Identification of 34 genes and analysis of their expression in injured tissue revealed several clusters of unique gene regulation patterns, including the up-regulation at 1 wk of cytochrome c oxidase regulating electron transfer and energy metabolism, presumably occurring at the site of inflammation; up-regulation at 2.5 wks of pro-α-2 type I collagen involving the formation of a connective tissue structure; and up-regulation at 1 and 2 wks and down-regulation at 2.5 and 4 wks of ubiquitin carboxyl-terminal hydrolase l3 involving cell cycle, DNA repair, and stress response. The differential expression of genes may be associated with the processes of inflammation, wound contraction, and formation of a connective tissue structure.

DOI： 10.1177/154405910408300707

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DOI： 10.1074/jbc.m311688200

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DOI： 10.1016/j.yexmp.2003.09.002

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DOI： 10.1016/j.lfs.2003.07.018

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Ovid Technologies (Wolters Kluwer Health)  

DOI： 10.1097/01.tp.0000085661.52980.95

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DOI： 10.1034/j.1600-0765.2003.02607.x

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Genes expressed by human periodontal ligament fibroblasts (HPFs) are likely to be associated with specific functions of the ligament. The aim of this study is to profile genes expressed highly by HPFs. A library (6 × 10³pfu) was constructed, followed by subtraction of HPF cDNAs with human gingival fibroblast (HGF) cDNAs. Reverse-dot hybridization revealed that 33 clones expressed higher levels of specific mRNAs in HPFs than in HGFs. These were mRNAs for known genes, including several associated with maturation and differentiation of cells. None had been reported in PFs. One clone, PDL-29, identified as a COX assembly factor, showed much stronger mRNA expression in HPFs than in HGFs in culture. In rat periodontium, however, PDL-29 mRNA expression was similar in PFs and GFs. These results suggest that HPFs express many previously unreported genes associated with maturation and differentiation, but expression can differ in vitro and in vivo.

DOI： 10.1177/154405910308200814

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DOI： 10.1902/jop.2003.74.8.1231

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Objectives: Tumour necrosis factor-alpha (TNF-alpha ) and interleukin-1beta (IL-1beta ) participate in the establishment of inflammatory lesions in periodontitis. High production of these cytokines may relate to the severity of periodontitis. There have already been several studies examining the association between periodontitis and single nucleotide polymorphisms (SNPs) that affect cytokine productivity. Recently, new SNPs of TNF-alpha , -1031, -863 and -857, variants of which are observed in a relatively large proportion in Japanese, have been identified. The variant alleles of these SNPs have been suggested to be related to high TNF-alpha production. For a better understanding of the genetic factors associated with the severity of periodontitis, further analysis including these newly identified SNPs is essential. In addition, previous reports on TNF-alpha or IL-1beta SNPs associated with periodontitis were mainly for Caucasian populations. Therefore, the aim of this study is to examine the association between severe periodontitis in Japanese and the following SNPs: five in the TNF-alpha gene promoter (-1031, -863, -857, -308, -238) and three in the IL-1beta gene (-511, -31, +3953).
Material and Methods: A total of 128 Japanese individuals were enrolled in this study. They were 64 patients with severe adult periodontitis and 64 healthy subjects. TNF-alpha and IL-1beta SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism for all subjects. TNF-alpha and IL-1beta production from LPS-stimulated monocytes/macrophages was also measured for 15 healthy male subjects.
Results: TNF-alpha production in TNF-alpha -1031/-863 (linkage disequilibrated) or -857 SNP variant allele carriers tended to be elevated, and the frequency of subjects who carried at least one variant allele in TNF-alpha -1031, -863 or -857 SNPs among severe periodontitis patients was significantly higher than in healthy subjects.
Conclusion: Since the frequency of subjects who carried at least one variant allele in TNF-alpha -1031, -863 or -857 SNPs was higher in periodontitis patients than in healthy subjects, TNF-alpha -1031, -863 and -857 SNPs appear to be associated with severe adult periodontitis in Japanese populations.

DOI： 10.1034/j.1600-051x.2003.00287.x

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Objectives: The aim of this study is to isolate mechanical stress-induced genes (MSGens) from human periodontal ligament (PDL) cells and to analyze profiles of the mRNA expression of these genes.
Background: Differential expression of genes in PDL cells under physiological stress such as occlusal force is thought to be orchestrated not only for the remodeling of PDL itself but also for the repair and regeneration of periodontal tissues. However, little is known about the genes expressed in PDL cells under mechanical stress.
Methods: The cDNA from mechanical stress-applied human PDL cells was subtracted against the cDNA from static control cells. The subtracted cDNA was amplified by polymerase chain reaction (PCR) and cloned for further analysis.
Results: Among 68 independent clones isolated, 15 contained DNA fragments greater than 250 bp. Reverse Northern analysis revealed a marked induction of MSGen-15 and MSGen-28 mRNA expression in the mechanical stress-applied cells. However, little difference in the magnitude of expression for the other MSGens was detected between the stress-applied cells and the control cells. After nucleotide sequencing and the analysis of homology with known genes, five clones were identified; ribosomal protein S27 (MSGen-9), MRG 15 (MSGen-15), androgen-binding protein (MSGen-18), cathepsin H (MSGen-28), and cytochrome c (MSGen-47). Interestingly, it has been reported that MRG 15 is a novel transcription factor involved in the regulation of cell growth and senescence. The remaining 10 clones, classified into six sequence types, had no significant homology with any known genes.
Conclusions: These results suggest that many known and unknown genes are expressed in response to mechanical stress in PDL cells, and that a transcription factor, MRG 15, may be responsible for molecular events in PDL cells under mechanical stress.

DOI： 10.1034/j.1600-0765.2003.00602.x

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DOI： 10.1016/s1043-4666(03)00123-6

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The oral epithelium is continuously exposed to a variety of microbial challenges that can cause infectious diseases such as periodontal disease. Human B Defensin-2 (hBD-2) is a cationic antimicrobial peptide with low molecular weight, which is inducible from oral epithelial cells upon either bacterial infection or stimulation with inflammatory cytokines. This peptide has a broad antimicrobial spectrum that includes gram-positive bacteria, gram-negative bacteria, and fungi. Therefore, it is thought that hBD-2 plays an important role as one of natural immunities to bacterial infection. However, its activity is inhibited by body fluids such as serum. The aim of this study was to assess the antibacterial activity of synthetic hBD-2 against oral bacteria in the presence of saliva or serum. The antibacterial activity of synthetic hBD-2 was tested against Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Streptococcus mutans, and Escherichia coli. Antibacterial broth assay and diffusion assay were performed in vitro. The antibacterial activity of hBD-2 was approximately equal to that of minocycline at equimolar concentrations. Furthermore, the activity of hBD-2 remained at 60% in the presence of 80% saliva, whereas no activity remained in the presence of 20% serum. Our results suggest the possibility that synthetic hBD-2 could be useful to prevent infection by periodontal bacteria.

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DOI： 10.1902/jop.2003.74.1.103

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DOI： 10.1006/cyto.2001.0976

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DOI： 10.1016/s0264-410x(01)00354-1

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Periodontal disease is a complication of patients with type 1 diabetes mellitus (T1DM), although the mechanisms responsible for this relationship remain unclear. The aim of this study was to examine oral manifestations and the prevalence of periodontal pathogens from subgingival plaque samples and serum IgG antibody levels against them in young Japanese type 1 diabetic subjects. One hundred and seventeen Japanese T1DM subjects (53 male, 64 female, mean age +/- SD, 16 +/- 6.5 years) participated in this study. Thirty-nine periodontally healthy, age-matched nondiabetics served as controls. T1DM subjects were clinically assigned into three groups: 12 periodontitis, 32 gingivitis and 73 periodontally healthy. Microbiological tests for four periodontal pathogens, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia and Capnocytophaga ochracea were performed using 16S ribosomal RNA-based polymerase chain reaction methods. Serum IgG antibody levels against 12 periodontal bacteria including the four species assessed by polymerase chain reaction were measured by enzyme-linked immunosorbent assay. In the T1DM subjects, the Periodontitis group had a significantly longer mean duration of diabetes and a higher percentages of subjects harbouring P. gingivalis and P. intermedia than the Periodontally Healthy group. Serum IgG antibody levels against P. gingivalis were significantly elevated in the Periodontitis group compared with Gingivitis and Periodontally Healthy groups. These results indicate that Japanese T1DM subjects are a high-risk group for periodontal disease and both P. gingivalis infection and duration of T1DM are risk factors for the progression of periodontitis in patients with T1DM.

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DOI： 10.1111/j.1348-0421.2001.tb01307.x

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Interleukin-6 (IL-6) binds to human gingival fibroblasts (HGF) in the presence of a soluble form of IL-6 receptor (sIL-6R). We investigated the effects of IL-6 on the functions of HGF in the presence of sIL-6R. HGF changed their morphology from spindle-shaped to round, and detached from the culture dish by stimulation with IL-6/sIL-6R. In this condition, a signal transducer gpl30 and a transcription factor Stat3 were phosphorylated, resulting in activation of transcription factors Stat3 and C/EBPβ. Cytoskeletal β-actin and adhesion molecule integrin-a5, a subunit of α₅β₁ integrin (VLA-5), were found to possess potential binding domains for these transcription factors in their promoters. Accumulation of (3-actin and integrin-a5 mRNA decreased, contrary to the expectation of the induction of gene transcription. Furthermore, the decrease in their mRNAs was associated with reduced expression of both actin and VLA-5 proteins. These results suggest that the expression of VLA-5 and actin was down-regulated in HGF through an IL-6 signaling pathway, resulting in impairment of HGF adherence.

DOI： 10.1177/00220345010800050701

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DOI： 10.1034/j.1399-302x.2001.016002073.x

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Epileptic patients taking phenytoin with gingival-overgrowth and those without gingival-overgrowth were compared for daily drug dose, plasma total phenytoin concentration, plasma free-phenytoin concentration and serum IgG antibody titre against 13 periodontal bacteria. Significantly higher daily drug dose was noted in patients with gingival overgrowth (P < 0.05) when compared with those without overgrowth. In addition, both total and free forms of plasma phenytoin concentration were significantly higher in sera of patients with gingival growth than of those without overgrowth (P < 0.01). Strong positive correlation was found between daily drug dose and serum phenytoin concentration in patients with gingival overgrowth, while weak correlation was found in patients without gingival overgrowth, suggesting a difference in drug metabolism in these two groups. However, no differences were found in serum IgG antibody titres to 13 periodontal bacteria examined between two groups. These results suggest that metabolic ability of phenytoin is one of the factors for developing gingival overgrowth, and that periodontal infection may not be a primary causative factor for gingival overgrowth but act as an additive factor which increase tissue mass for this unwanted side effect.

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DOI： 10.1902/jop.2001.72.4.425

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DOI： 10.1902/jop.2001.72.2.243

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Mary Ann Liebert Inc  

Tumor necrosis factor-alpha (TNF-alpha) exerts its functions by binding two different receptors (TNFR55 and TNFR75), Both TNFR55 and TNFR75 exist in cell-associated and soluble forms. Soluble TNF receptors (sTNFR), sTNFR55 and sTNFR75, are proteolytically shed upon inflammatory stimuli and then modulate various TNF-alpha bioactivities. As human gingival fibroblasts (HGF) can be potential targets for TNF-alpha in inflamed gingiva, we hypothesized that HGF partially modulate the cellular responses to TNF-alpha by regulating their own TNFR. In this study, the kinetics of expression of cell-associated and soluble forms of both receptors from cultured HGF in response to proinflammatory cytokines TNF-alpha and interleukin-1 beta (IL-1 beta) were investigated in vitro. Both TNF-alpha and IL-1 beta upregulated the gene expression of TNFR75 and did not affect that of TNFR55. TNF-alpha and IL-1 beta decreased binding of [I-125]TNF-alpha to HGF. Moreover, TNF-alpha and IL-1 beta upregulated the release of sTNFR75 from HGF but not that of sTNFR55, These results suggest that HGF under inflammatory conditions may contribute to the inactivation of circulating TNF-a through the preferential induction and shedding of TNFR75.

DOI： 10.1089/107999000750053744

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We isolated and characterized a possible regulatory gene, designated actX gene, from Actinobacillus actinomyctemcomitans Y4, which defined the Actinobacillus pleuropneumoniae hlyX-like regulatory gene. DNA sequence analysis for plasmid clone pKM317 containing a 1.6-kb DNA insert indicated an open reading frame encoding a polypeptide of 257 amino acid residues. Analysis of the deduced amino acid sequence showed the presence of five characteristic cysteine residues in the N-terminal region and a putative DNA binding residue in the C-terminal region, indicating that actX might belong to a regulatory gene family. Escherichia coli DH5 alpha and a mutant strain JRG1728 transformed by plasmid carrying actX manifested apparent hemolytic activity on sheep blood agar and grew anaerobically, although the original strains did not. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

DOI： 10.1016/s0923-2508(00)01137-2

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Background, aims: The aim of this study was to evaluate antibody responses against Porphyromonas gingivalis (P. gingivalis) infection in early-onset periodontitis (EOP) patients to elucidate further the host-parasite interactions in the pathogenesis of EOP.
Method: 16 P. gingivalis-infected EOP and 20 adult periodontitis (AP) patients, and 18 periodontally healthy subjects (HS) participated in this study. Serum immunoglobulin G (IgG) antibody levels and avidities against extracted P. gingivalis whole cells were measured. The components of P. gingivalis outer membrane antigens (OMA) reacting to patients' sera were analysed from the molecular weights by Western blotting. Serum antibody levels against P. gingivalis lipopolysaccharide (LPS) were also measured. The ability of the patients' sera to block interleukin-1 beta (IL-1 beta) production by human mononuclear cells in response to P. gingivalis LPS was examined.
Results: Antibody levels were positively correlated with antibody avidities in both EOP and AP patients (r=0.91, r=0.72, p&lt;0.0005, respectively), while not significantly so in HS (r=0.09). There was variability in the antigen recognition of P. gingivalis OMA in EOP and AP patients. Smear and 53-kDa protein were more frequently recognized by sera of EOP and AP patients rather than that of HS (p&lt;0.05). The smear was partly diminished by absorption with P. gingivalis LPS, indicating the smear antigen was partly composed of LPS. There was high correlation between antibody levels against P. gingivalis whole-cell extracts and LPS in EOP and AP patients (r=0.81, p=0.0002, r=0.87, p&lt;0.0001, respectively), while not significant in HS (r=0.22). The sera of EOP and AP patients with high IgG titre to P. gingivalis LPS blocked IL-1 beta production more effectively than that of the patients with low IgG titre to P. gingivalis LPS.
Conclusions: These results indicate that EOP patients' antibody response against P. gingivalis infection does not differ significantly from that of AP patients. The person-to-person heterogeneous antibody production against P. gingivalis LPS could contribute to our understanding of the relationship between the defensive ability of EOP patients and their chronic infection with this pathogen.

DOI： 10.1034/j.1600-051x.2000.027010769.x

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The type II interleukin-1 receptor (IL-1RII) has been thought to be incapable of transducing signals to cells because of its short intracellular domain, while type I IL-1 receptor (IL-1RI) does transduce signals. Since over-expression of IL-1RII has been demonstrated to inhibit cytokine production in the fibroblastic cell line, it has been proposed to use IL-1RII to prevent IL-1-induced inflammation in connective tissue. In this study, trace amounts of IL-1RII mRNA expression were detected in human gingival fibroblasts (HGFs), which are affected by cytokines in inflammatory periodontal disease. Cloning of the cDNA encoding IL-1RII expressed in HGFs revealed 3 amino acid substitutions in the extracellular domain, when compared with the 408 residues predicted from human B-cells. Over-expression of IL-1RII on HGFs by gene transfer down-regulated the expression of IL-1β mRNA and IL-6 mRNA in response to IL-1β stimulation, while the expression of IL-8 mRNA was not affected. In the IL-1RII-transfected HGFs, phosphorylation of 25-and 74-kDa proteins was up-regulated upon IL-1β stimulation in the transfected HGFs. The phosphorylation of these proteins was suppressed by the addition of a neutralizing antibody against IL-1RII. These results suggest that the IL-1RII may regulate HGFs expression of cytokine mRNA upon IL-1β stimulation, possibly by altering the IL-1RI-dependent signals.

DOI： 10.1177/00220345000790090801

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The accumulation of extracellular matrices and integrins by high glucose has been reported in relation to diabetic complications. We previously reported that PDL cells expressed a higher amount of VLA-5 when cultured in high-glucose (4500 mg/L) medium than those cultured in low-glucose (1100 mg/L) medium. In this study, we aimed to address (1) whether this effect was mediated by the transcriptional level of the gene or the degradative level of the protein, and (2) whether this effect was mediated by TGF-β. The results indicated that the level of mRNA encoding a5 integrin did not change in PDL cells regardless of the concentration of glucose. Alternatively, high glucose suppressed cathepsin B+L activity. Additionally, the level of mRNA encoding TGF-β was not affected by high glucose, nor did an anti-TGF-β neutralizing antibody have an effect on the expression of β5 gene or cathepsin activity. Therefore, the effects of high glucose appeared to be mediated by impaired protein degradation, but not by autocrine TGF-β.

DOI： 10.1177/00220345000790081501

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DOI： 10.1111/j.1574-695x.2000.tb01480.x

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DOI： 10.1046/j.1472-765x.2000.00735.x

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Background: Fibroblasts are known not only to synthesize and secrete extracellular matrix proteins, but also to degrade them for connective tissue remodeling. Drug-induced gingival overgrowth is characterized by a massive accumulation of extracellular matrix components in gingival connective tissues. Although some previous reports suggested that causative drugs stimulated the fibroblast proliferation, the results are not conclusive yet. In this study, we hypothesized that drug-induced gingival overgrowth could be a consequence of impaired ability of matrix degradation rather than an enhanced proliferation of gingival fibroblasts induced by these drugs.
Methods: Normal human gingival fibroblasts were cultured with or without either 20 mug/ml of phenytoin or 200 ng/ml of cyclosporin A. Total RNA and cellular proteins were collected every day for RT-PCR analyses and for measuring lysosomal enzyme activity. In addition, an immunohistochemical study was performed to detect lysosomal enzymes in cells from enlarged gingiva of the patients with phenytoin-induced gingival overgrowth.
Results: RT-PCR analyses revealed that these drugs suppressed the expression of MMP-1, TIMP-1, and cathepsin L, but not that of cathepsin B in a time-dependent manner. Then, we measured the activity of lysosomal enzymes and cathepsin B and L. The results indicated that although cathepsin B activity was not observed to be impaired, regardless of the drugs used in these cells, both total and active forms of combined activity of cathepsins B and L were suppressed in a time-dependent manner.
Conclusions: The results indicate that, besides suggested effects of these drugs on gingival fibroblasts and/or on accumulated cells in the gingival tissues, extracellular matrix-degrading ability, particularly that by cathepsin L, is also suppressed by cyclosporin A and phenytoin in gingival fibroblasts, and that lysosomal enzyme plays an important role in the pathogenesis of drug-induced gingival hyperplasia.

DOI： 10.1902/jop.2000.71.6.955

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DOI： 10.1111/j.1348-0421.2000.tb02505.x

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

<title>ABSTRACT</title>
During infection, circulating blood monocytes migrate from the vasculature to the extravascular compartments where they mature into tissue macrophages. The maturation process prepares the cell to actively participate in the inflammatory and the immune responses, and many transcription factors have been found to be involved. Here we report on a novel role for nuclear factor κB (NF-κB) in this process. Its accumulation in the cytoplasm of differentiated macrophages is responsible for the enhanced ability of the cell to respond to lipopolysaccharide (LPS) stimulation, as determined by tumor necrosis factor alpha (TNF-α) secretion. Differentiation of the human monocytic cell line THP-1 into macrophage-like cells was induced by exposure of the cells to phorbol myristate acetate. DNA-bindable NF-κB was not detected in the cytoplasm of undifferentiated THP-1 cells but accumulated in the cytoplasm of the cells following differentiation. No TNF-α was detected in the media of resting differentiated and nondifferentiated THP-1 cells. Stimulation with LPS of differentiated cells induced the production of higher levels of TNF-α than stimulation of nondifferentiated cells. This hyperresponsiveness to LPS was found in the mRNA and secreted TNF-α levels. Furthermore, stimulation with LPS induced the translocation of NF-κB from the cytoplasm into the nucleus. This translocation process was more rapid in the differentiated cells than in the nondifferentiated cells, and the resultant accumulated levels of NF-κB in the nucleus were higher. The DNA-bindable NF-κB was identified as a heterodimer of p65 and p50. The results suggest that NF-κB accumulation in the cytoplasm during maturation of monocytes to macrophages primes the cells for enhanced responsiveness to LPS and results in the rapid secretion of inflammatory mediators, such as TNF-α, by mature macrophages following LPS challenge.

DOI： 10.1128/iai.67.11.5573-5578.1999

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<title>ABSTRACT</title>

Subtractive hybridization was employed to isolate specific genes from virulent
<italic>Porphyromonas gingivalis</italic>
strains that are possibly related to abscess formation. The genomic DNA from the virulent strain
<italic>P. gingivalis</italic>
W83 was subtracted with DNA from the avirulent strain ATCC 33277. Three clones unique to strain W83 were isolated and sequenced. The cloned DNA fragments were 885, 369, and 132 bp and had slight homology with only
<italic>Bacillus stearothermophilus</italic>
IS
<italic>5377</italic>
, which is a putative transposase. The regions flanking the cloned DNA fragments were isolated and sequenced, and the gene structure around the clones was revealed. These three clones were located side-by-side in a gene reported as an outer membrane protein. The three clones interrupt the open reading frame of the outer membrane protein gene. This inserted DNA, consisting of three isolated clones, was designated IS
<italic>1598</italic>
, which was 1,396 bp (i.e., a 1,158-bp open reading frame) in length and was flanked by 16-bp terminal inverted repeats and a 9-bp duplicated target sequence. IS
<italic>1598</italic>
was detected in
<italic>P. gingivalis</italic>
W83, W50, and FDC 381 by Southern hybridization. All three
<italic>P. gingivalis</italic>
strains have been shown to possess abscess-forming ability in animal models. However, IS
<italic>1598</italic>
was not detected in avirulent strains of
<italic>P. gingivalis</italic>
, including ATCC 33277. The IS
<italic>1598</italic>
may interrupt the synthesis of the outer membrane protein, resulting in changes in the structure of the bacterial outer membrane. The IS
<italic>1598</italic>
isolated in this study is a novel insertion element which might be a specific marker for virulent
<italic>P. gingivalis</italic>
strains.

DOI： 10.1128/iai.67.11.5621-5625.1999

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Human leukocyte antigens (HLA) are essential in the recognition of foreign antigens in humoral immune response, which is genetically predetermined. Susceptibility to certain diseases that involve the immune response has been studied in relation to distinct HLA types. Although some diseases have been found Co correlate to specific HLA loci positively, it has been difficult to isolate HLA types that predispose patients to periodontal destruction. Here. we review the current knowledge and recent advances in HLA genetics and its biology, which determine susceptibility to early-onset periodontitis (EOP). The HLA-DRB1*1501-DQB1*0602 genotype has been found with increasing frequency in EOP patients. This HLA genotype expresses aspartic acid at position 57 and glycine at position 70 on the DQ beta chain, suggesting a capability to bind certain bacterial antigens. The T cell response against the outer membrane protein (Ag53) of Porphyromonas against gingivalis was examined via this HLA genotype. Strong T cell response against Ag53 p141-161 was inhibited partially by anti-DR antibody, but not by anti-DQ antibody, possible host and bacterial peptides capable of binding DRB1*1501 were elucidated when the peptide sequence was compared to gene and protein databases. These results suggest that patients who have the HLA-DRB1*1501-DQB1*0602 genotype may have an accelerated T cell response to certain periodontapathic bacteria such as P. gingivalis in hyperimmune reactions and thus increased susceptibility to EOP.

DOI： 10.1111/j.1600-0765.1999.tb02269.x

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Interleukin-6 (IL-6), frequently detected in periodontitis, is known to mediate important signals in the inflammatory cytokine network. Gingival fibroblasts (GF) secrete cytokines upon stimulation with inflammatory mediators. However, it is not clear if GF respond to IL-6. We examined the lL-6 receptor gene expression in GF. Furthermore, we tested whether GF are target cells for IL-6 by examination of binding of IL-6. GF were found to contain trace amounts of mRNA for IL-6 receptor (IL-GR), but had high levels of mRNA for 130-kDa glycoprotein (gp130), which is a signal transducer for IL-6/IL-6R complex. Based on this observation, we hypothesized that IL-6 could bind GF if exogenous soluble forms of IL-6R (sIL-6R) existed in the gingiva or culture condition. Thus, we investigated the existence of sIL-6R in gingiva using enzyme-linked immunosorbent assay and whether sIL-6R influenced the binding of IL-6 to GF in vitro. In inflamed gingiva, sIL-6R was detected and its concentration ranged from 150 to 700 pg/mu g protein. The sIL-6R enhanced the binding of IL-6 to GF in a dose-dependent manner. This enhancement was inhibited by an antibody against gp130, suggesting that the IL-6/sIL-6R complex bound to the fibroblasts via gp130. These data demonstrated that gingival fibroblasts can be target cells for IL-6 in the presence of appropriate amounts of sIL-6R. This situation may exist during inflammation in periodontal tissue.

DOI： 10.1111/j.1600-0765.1999.tb02257.x

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Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

DOI： 10.1073/pnas.96.8.4518

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<title>ABSTRACT</title>
Periodontitis is a chronic inflammatory disease which gradually destroys the supporting tissues of the teeth, leading to tooth loss in adults. The lesions are characterized by a persistence of inflammatory cells in gingival and periodontal connective tissues. To understand what mechanisms are involved in the establishment of chronic lesions, we hypothesized that infiltrating lymphocytes might be resistant to apoptosis. However, both Bcl-2 and Bcl-xL were weakly detected in lymphocytes from the lesions, compared with those from peripheral blood, suggesting that these cells are susceptible to apoptosis. Nevertheless, very few apoptotic cells were observed in tissue sections from the lesions. Lymphocytes from the lesions expressed mRNA encoding Fas, whereas Fas-ligand mRNA was very weakly expressed in lymphocytes from the lesions and in periodontal tissues. Since the results indicated that lymphocytes in the lesions might be susceptible to Fas-mediated apoptosis but lack the death signal, we next investigated if these lymphocytes actually undergo apoptosis by the addition of anti-Fas antibodies in vitro. Fas-positive lymphocytes from the lesions underwent apoptosis by these antibodies, but Fas-negative lymphocytes and Fas-positive peripheral lymphocytes did not undergo apoptosis by these antibodies. These results indicate that lymphocytes in the lesions are susceptible to activation-induced cell death and are induced to die by apoptosis after the addition of exogenous Fas ligand.

DOI： 10.1128/iai.67.3.1450-1454.1999

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The nearly complete 16S rRNA gene sequences for oral Gram-negative anaerobic motile bacteria, Centipeda periodontii, Selenomonas sputigena and Selenomonas species (formerly S. sputigena type strain), were determined in order to unveil their relationship to other oral motile bacteria. To determine the phylogenetic characterization of these bacteria, their 16S rRNA gene sequences were obtained and compared with those from the ribosomal sequence databases previously reported. The 16S rRNA gene sequences of these bacteria were similar to those of Selenomonas ruminantium and Schwartzia succinivorans isolated from rumens, and to Pectinatus cerevisiiphilus isolated from spoiled beer. Among oral bacteria, the nucleotide sequence analysis of these bacteria revealed high nucleotide similarity to Veillonella species, whereas low similarity to oral motile bacteria such as Campylobacter species. Phylogenetic analysis clearly confirmed that C. periodontii and two Selenomonas species were classified as relatives of a group besides Selenomonas, Schwartzia, and Pectinatus species, and not as close relatives to oral motile bacteria, such as Campylobacter species. These results suggest that such oral Gram-negative anaerobic motile bacteria are close relatives of oral bacteria.

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Patients with early-onset periodontitis (EOP) are susceptible to infection with periodontopathic bacteria, such as Porphyromonas gingivalis. Ag53, 53-kDa outer membrane protein of P. gingivalis, evokes strong humoral immune responses in EOP patients. In a first step to clarify how host immune cells recognize Ag53, we established Ag53-specific short-term T cell lines from 22 subjects including 6 EOP patients and 16 healthy donors, using overlapping peptides based on Ag53 amino acid sequences. All T cell lines from active EOP patients recognized a common region (p141-181, especially p141-161) on Ag53, while those from healthy donors showed heterogeneous specificity. p141-181 was not recognized by T cell lines established from EOP patients following therapy. A monoclonal antibody to HSA-DRB1 inhibited Ag53-induced proliferation of most of the T cell lines. Our observations suggest that, although antigen-presenting molecules are common in EOP patients and in healthy individuals, p141-161 includes a major T cell epitope(s) on Ag53 for active EOP patients but not for healthy individuals or inactive EOP patients. (C) American Society for Histocompatibility and Immunogenetics, 1998 Published by Elsevier Science Inc.

DOI： 10.1016/s0198-8859(98)00064-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

The gene encoding the crystalline surface layer (S-layer) protein from Campylobacter rectus, designated sip, was sequenced and the recombinant gene product was expressed in Escherichia coli. The gene consisted of 4086 nucleotides encoding a protein with 1361 amino acids. The N-terminal amino acid sequence revealed that Sip did not contain a signal sequence, but that the initial methionine residue was processed. The deduced amino acid sequence displayed some common characteristic features of S-layer proteins previously reported. A homology search showed a high similarity to the Campylobacter fetus S-layer proteins, especially in their N-terminus. The C-terminal third of Sip exhibited homology with the RTX toxins from Gram-negative bacteria via the region including the glycine-rich repeats. The Sip protein had the same N-terminal sequence as a 104-kDa cytotoxin isolated from the culture supernatants of C. rectus. However, neither native nor recombinant Sip showed cytotoxicity against HL-60 cells or human peripheral white blood cells. These data support the idea that the N-terminus acts as an anchor to the cell surface components and that the C-terminus is involved in the assembly and/or transport of the protein. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

DOI： 10.1111/j.1574-6968.1998.tb13901.x

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Based on our clinical observations that patients with insulin-dependent diabetes mellitus (IDDM) are subject to periodontal disease, we developed the hypothesis that hyper- or hypoglycemia might contribute to the pathogenesis of diabetic periodontitis. In this article, experimental facts that substantiate this hypothesis are presented on the basis of our studies and then discussed. Hyperglycemia progressively glycates body proteins, forming advanced glycation end products (AGE), which stimulate phagocytes to release inflammatory cytokines such as TNF-alpha and IL-6. In this context, to understand the effects of hyperglycemic episodes on periodontal health, 24 adolescent IDDM patients were examined for their periodontal status, and 3 of them were found to have periodontitis. Laboratory analyses on these 3 patients revealed that 2 had elevated serum TNF-alpha levels. These results may partly support the current hypothesis of a mechanism of diabetic complications in which abnormal cytokine levels induced by AGE could exacerbate inflammatory responses. In IDDM patients, the diabetes is often accompanied not only by hyperglycemic episodes but also by iatrogenic hypoglycemia. Periodontal ligament cells (PDL) cultured under hyperglycemic conditions were impaired in such biological functions as adhesion and motility, while cells cultured under hypoglycemic conditions (10 mg/dL) gradually dissociated from their anchor and underwent cell death. These phenomena correlated well with the expression profile of fibronectin receptor. Interestingly, these changes due to the different glucose levels were observed more intensively in PDL than in other fibroblastic cells, suggesting that the biological functions of PDL are easily led to impairment by variation or rapid fluctuation of glucose levels. These observations suggest that hyperglycemia could indirectly exacerbate inflammatory tissue destruction through the body's scavenger system against AGE, and that both hyper- and hypoglycemia might directly impair the biological functions of periodontal connective tissues through cell-matrix interactions.

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Language：English   Publisher：AMER ASSOC DENTAL RESEARCH  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FACULTY PRESS  

A periodontal pathogen, Porphyromonas gingivalis possesses either a 53 kD (Ag53) or a 67 kD (Ag67) outer membrane protein (OMP). Almost all sera from patients with periodontal diseases reacted strongly with either Ag53 or Ag67. In previous work the cloning and sequencing of the 53 kD outer membrane protein gene designated pga53 from Fl gingivalis FDC381, was reported and the presence of a gene homologous to pga53 in Fl gingivalis ATCC 33277 demonstrated. In the present work this pga53-homologous gene from Fl gingivalis ATCC 33277 was isolated and characterized. Nucleotide sequence analysis revealed that this gene encoded Ag67, and the gene was designated pga67. The deduced amino acid sequence and composition of pga67 was similar to the amino acid composition and N-terminal partial sequence of Ag67. An open reading frame of pga67 consisted of 1,692 nucleotides encoded as 564 amino acids, including a 49 amino acid signal sequence. The comparative analysis between pga67 and pga53 revealed that (1) the deduced amino acid sequence showed a 30.1% homology; (2) signal sequence and proline-rich regions at the C-terminus were the most conserved regions; (3) considerable differences were found mainly in the middle part of the OMPs; and (4) obvious differences in the two-dimensional models were evoked. These differences between pga67 and pga53 may explain the antigenic diversity between Ag67 and Ag53 OMPs.

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Language：English   Publisher：AMER ASSOC DENTAL RESEARCH  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

VIRUS-ASSOCIATED HEMOPHAGOCYTIC SYNDROME (VAHS) is a disorder characterized by benign generalized histiocytic proliferation and marked hemophagocytosis associated with systemic viral infection. An immunodeficiency which includes an extremely decreased leukocyte and platelet count together with abnormalities in the CD4/CD8 ratio are the most common features of VAHS. Here we report an early-onset periodontitis (EOP) patient with VAHS from the standpoint of host-parasite interaction to understand the effect of this systemic disorder which might possibly influence susceptibility to periodontal disease. The patient is a 16-year-old Japanese male clinically diagnosed as having generalized EOP with slight gingival inflammation and moderate bone loss. This patient manifested VAHS at 3 years of age, and then had an unusual 4 recurrences (at 5, 7, 11, and 14 years old). Laboratory tests conducted include: 1) complete blood analyses; 2) peripheral neutrophil functions (chemotaxis, phagocytosis, superoxide production, and adherence); 3) peripheral lymphocyte subpopulations and functions, T-cell proliferative activity and productivity of cytokines (interleukin-2 [IL-2], interferon gamma [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]); 4) serum cytokine levels cn. Ip, IL-2, soluble IL-2 receptor [sIL-2R], IL-4, IL-6, IFN-gamma, and TNF-alpha; 5) serum immunoglobulin G (IgG) antibody titers against periodontopathic bacteria; 6) serological human leukocyte antigen (HLA) typing; and 7) determination of bacterial flora of the periodontal pockets. The results indicated that the patient's neutrophil chemotaxis and random migration were below the normal range. In lymphocyte examinations, T-cell proliferative activity, IL-2, and IFN-gamma productivity were elevated. Serum IFN-gamma level was also significantly higher than normal range. No specific periodontopathic bacteria were predominant in the periodontal pockets, however, the serum IgG titer against Porphyromonas gingivalis was elevated throughout the examination period. It is suggested that VAHS might be a possible risk factor for periodontal disease, and hence may serve as a model in understanding the role of host defense mechanisms in the establishment of inflammatory periodontal disease.

DOI： 10.1902/jop.1997.68.12.1223

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：3  

OBJECTIVE: The aim of this study is to compare the in vitro proliferative capacity of periodontal ligament (PDL) cells from aged and juvenile donors. MATERIALS AND METHODS: Flow-cytometric analysis of the cell cycle was used to compare the length of each cell cycle, and the ratio of the cells progressing through the cycles between four PDL cells from juvenile donors and four cells from aged donors. Then, replicative capacity of the PDL cells from three juvenile and three aged donors was compared by serial cultures. Finally, expression of c-fos was compared between cells proliferating and cells which had reached senescent. RESULTS: Flow- cytometric analysis of the cell cycle had revealed that although there were no differences in the length of each phase of the cell cycle, significant differences were found in the ratio of the cells entering from Gap I to DNA synthesis phase of the cell cycle (P &lt
0.025). Replicative capacity was much longer in two cells from juvenile donors (about 20 population doublings), while all cells from aged donors showed short dividing abilities (less than eight population doublings), hence entered senescent phases shortly. Additionally, no c-fos was detected in cells which had reached senescence upon stimulation with serum. CONCLUSIONS: It is generally believed that aged humans have an impaired wound healing ability. We believe that more fibrotic PDL tissues seen in aged humans might be the reason for this, and suggest that this phenomena might be due to the progressive accumulation of senescent cell populations.

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Language：Japanese   Publisher：The Japanese Society of Periodontology  

Tumor necrosis factor-α (TNF-α) is one of proinflammatory cytokines produced as early inflammatory responses, and is suggested to participate in the establishment of inflammatory lesions. To understand the regulatory role of TNF-α on periodontal connective tissue, we evaluated the effect of TNF-α on DNA synthesis, collagenous and non-collagenous protein synthesis, prostaglandin E_2 (PGE_2) productivity, and platelet-derived growth factor (PDGF)-A chain mRNA expression in human gingival fibroblasts (HGF). Results indicated that 1) TNF-α promoted DNA synthesis, both collagenous and non-collagenous protein synthesis in HGF, 2) TNF-α markedly enhanced PGE_2 production in HGF, 3) inhibition of PGE_2 synthesis by the addition of indomethacin, further augmented the ability of DNA synthesis and collagenous and non-collagenous protein synthesis in HGF, and 4) TNF-α enhanced the expression of PDGF-A chain mRNA in HGF. These results suggest that TNF-α enhances the DNA synthesis and extracellular matrix protein synthesis in HGF, but endogenous PGE_2 which is induced by TNF-α, partially blocks these effects. Furthermore, the enhancement of DNA synthesis in response to TNF-α could be possibly mediated by autocrine PDGF.

DOI： 10.2329/perio.39.264

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Other Link： http://search.jamas.or.jp/link/ui/1997240849

Language：English   Publishing type：Research paper (scientific journal)   Publisher：FACULTY PRESS  

The pga53 gene which encoded the antigenic 53 kD outer membrane protein (Ag53) was isolated from a genomic DNA library of Porphyromonas gingivalis FDC381 by using an Ag53-immunized rabbit serum. Determination of its complete nucleotide sequence revealed that the precursor of Ag53 had a 50 amino-acid putative signal sequence and the mature protein of 448 amino acids. The deduced amino acid sequence after a 50 amino-acid putative signal sequence was in complete agreement with the first 20 N-terminal amino acids of purified Ag53. Analysis of the deduced amino acid sequence revealed the presence of a highly hydrophilic proline-rich region at the C-terminal of Ag53. The deduced amino acid sequence showed 29.9% homology with that of a 72 kD cell-surface protein in P. gingivalis. Southern hybridization revealed that pga53 was specific to several P. gingivalis strains and that P. gingivalis strains which did not possess Ag53 had genes homologous to pga53.

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Language：English   Publisher：AMER ASSOC DENTAL RESEARCH  

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/j.1399-302x.1996.tb00205.x

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DNA TYPING WAS PERFORMED on 24 Japanese patients with early-onset periodontitis (EOP) using the PCR-RFLP method to investigate an association of the susceptibility to EOP with the particular HLA class II alleles (HLA-DRB1, -DQA1, and -DQB1), DRB1*1401, DRB1*1501, DQB1*0503, and DQB1*0602 were found more frequently (''susceptible'') in the EOP patients than in healthy controls. In contrast, DRB1*0405 and DQB1*0401 were found less frequently (''resistant'') in EOP patients, All patients carrying DQB1*0602 had an atypical BamHI site in the intron upstream of the third exon of the DQB1 gene, which in our previous studies appeared to be a susceptible marker for EOP. A comparative analysis of the amino acid sequences of these susceptible and resistant HLA-DRB1 and DQB1 alleles elucidated some differences in antigen-derived peptide binding sites related to the susceptible or resistant alleles. Especially, DQB1*0503 and DQB1*0602 alleles carrying aspartic acid at position 57 and glycine at position 70 are increased significantly in EOP. Since amino acid residues at positions 57 and 70 on the DQB1 molecule are supposed to be involved in antigen binding, amino acid substitutions at these positions may affect the immune responsiveness to the periodontopathic antigen. Our results suggest that the DQB1 molecule plays a crucial role in the pathogenesis of EOP and that the susceptibility to EOP may be determined by the binding ability between the peptide and HLA-DQ antigens.

DOI： 10.1902/jop.1996.67.9.888

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FAMILY CASE STUDIES HELP US IDENTIFY host risk factors in periodontal disease. In this study we examine a family consisting of a mother (40 years old, with rapidly progressive periodontitis), her elder daughter (14 years old, with localized juvenile periodontitis), and younger daughter (13 years old, with simple gingivitis). We examined 1) the peripheral neutrophil functions (chemotactic migration, phagocytosis, superoxide production); 2) lymphocyte functions (proliferative activity and cytokine productivity of T cells, immunoglobulin [Ig] M productivity of B cells when stimulated with pokeweed mitogen); 3) phenotypic analyses of peripheral lymphocyte subpopulations; 4) serum IgG antibody titers against periodontopathic bacteria; and 5) serological type of HLA class IS. All the subjects exhibited high T4/T8 ratios due to high percentage of CD4-positive cells, showed high IgG titers to Actinobacillus actinomycetemcomitans, and had a HLA DQw1 in common. The mother showed a slight deficiency of neutrophil chemotactic migration to N-formyl methyonyl leucyl phenylalanin (fMLP), raised interleukin-2 productivity of T cell, and high levels of IgG titers to Porphyromonus gingivalis and Fusobacterium nucleatum. Both daughters showed weak T cell proliferative response to anti-CD3 monoclonal antibody and low IgM productivity. Low lymphocyte responsiveness may be involved in the pathogenesis of periodontal disease of these daughters; therefore, the lymphocyte dysfunctions shown should be considered in relation to the progression of periodontal disease.

DOI： 10.1902/jop.1996.67.4.433

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DOI： 10.1902/jop.1995.66.5.403

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

Lipopolysaccharide (LPS) is one of the most potent trigger substances for monocytes and macrophages causing secretion of tumor necrosis factor alpha (TNF-alpha) and other inflammatory mediators. The nature of the nuclear factors involved in human TNF-alpha gene regulation is still unknown. Nuclear factor kappa B (NF-kappa B) proteins have been suggested to play an important role in gene transcription of inflammatory mediators when monocytes are stimulated with LPS. However, it remains unclear whether these nuclear factors are the only ones involved in human TNF-alpha gene regulation. In this report, to further the identification of nuclear factor(s) involved in TNF-alpha gene regulation, human monocytic THP-1 cells were transfected with a series of truncated versions of human TNF-alpha promoter. A 98-bp region located from nucleotides -584 to -487 demonstrated strong promoter activity. Electrophoretic mobility shift assays demonstrated that a 64-bp fragment located within the 98-bp region and lacking any potential NF-kappa B-binding sites avidly bound LPS-challenged THP-1 nuclear protein. Although this binding was inhibited in salicylate-treated cells, as was binding of NF-kappa B, the pattern of binding was found to differ from that noted for NF-kappa B. Analysis of this 64-bp fragment disclosed the absence of an NF-kappa B consensus sequence, suggesting a novel nuclear DNA-binding protein necessary for the initiation of human TNF-alpha transcription other than, or in addition to, NF-kappa B.

DOI： 10.1128/iai.63.4.1529-1534.1995

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INFLAMMATORY MEDIATORS PRODUCED BY HUMAN GINGIVAL FIBROBLASTS (HGF) have been implicated in the initiation and progression of periodontal disease. The purpose of this study was to examine whether prostaglandin E(2) (PGE(2)), which is produced in abundance from HGF after stimulation with interleukin (IL)-1 beta or tumor necrosis factor-alpha (TNF-alpha), could regulate IL-6 production by HGF. HGF stimulated with either IL-1 beta or TNF-alpha showed a rapid and dose-dependent increase in IL-6 mRNA accumulation and IL-6 secretion, as demonstrated by reverse transcription-polymerase chain reaction analysis and bioassay. IL-6 secretion from either IL-1 beta- or TNF-alpha-stimulated HGF was enhanced by the inhibition of PGE(2) synthesis with indomethacin. Furthermore, the addition of PGE(2) inhibited IL-6 secretion from these cells. In contrast, indomethacin or PGE(2) did not affect the accumulation of IL-6 mRNA in IL-1 beta-stimulated HGF. These data indicate that IL-6 production by HGF is up-regulated by specific cytokines, IL-1 beta and TNF-alpha, and suggest that this production may be partially down-regulated by endogenous and exogenous PGE(2) at the post-transcriptional level.

DOI： 10.1902/jop.1994.65.12.1122

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HUMAN GINGIVAL FIBROBLASTS (HGF) may have an important role in the orchestration of immuno participant cells infiltrating the gingiva in response to continuously recurring bacterial infection. To examine the cytokine network regulating HGF-derived interleukin (IL)-8, a potent neutrophil chemotactic cytokine, we analyzed the effects of inflammatory cytokines alone and iii combination on IL-8 production by HGF. IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), Interferon-gamma (IFN-gamma), IL-6, and IL-8 were used as stimulants. HGF secreted IL-8 in a dose-dependent manner after stimulation with either IL-1 beta or TNF-alpha, but not with IFN-gamma or IL-6. Furthermore, IL-8 itself did not affect IL-8 mRNA accumulation in HGF in an autocrine manner, The combination of IL-1 beta and TNF-alpha synergistically enhanced the secretion of IL-8, whereas IFN-gamma suppressed IL-8 secretion by IL-1 beta- or TNF-alpha-stimulated HGF. These effects were also observed at each level of IL-8 mRNA expression in HGF. IL-8 secretion by cytokine-stimulated HGF was not influenced my the inhibition of PGE(2) synthesis with indomethacin, indicating that endogenous PGE(2) was not involved in IL-8 production by HGF. These results indicate that IL-8 production by HGF is synergistically stimulated by specific cytokines, IL-1 beta and TNF-alpha, and suggest that these stimulatory effects are down-regulated by IFN-gamma at the transcriptional let ei through PGE(2)-independent pathways. Thus, neutrophil-mediated processes in periodontal disease may be regulated in part by HGF in the cytokine network of immuno-participant cells.

DOI： 10.1902/jop.1994.65.11.1002

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WE ANALYZED THE CELL-CELL ADHERENCE RELATED to CD11/CD18 and CD18 mRNA in individuals with decreased CD11/CD18 expression on their neutrophil surface. Epstein Barr virus-transformed B cell lines were developed from one localized juvenile periodontitis (LJP) patient with decreased CD11/CD18 in the peripheral blood neutrophils and without systemic diseases; two siblings with generalized prepubertal periodontitis (GPP) caused by leukocyte adhesion deficiency (LAD); another LJP patient; one localized prepubertal periodontitis (LPP) patient; and two healthy subjects. Adhesion of leukocytes to each other was measured as cluster formation by aggregation assay. The length and the amount of CD18 mRNA expressed in the cell lines were analyzed by Northern blotting using the P-32-labeled CD18 cDNA. The coding region of the mRNA was analyzed by the reverse transcription-polymerase chain reaction method. Base-mismatches between CD18 mRNA and the P-32-labeled RNA probe synthesized from CD18 cDNA were analyzed by RNase protection assay. In the adherence assay, cells from the LJP patients with decreased CD11/CD18 formed more clusters of smaller size and fewer cells than those of the other subjects. The cells from GPP and LAD patients did not aggregate and did not form clusters either in the absence or presence of PMA. There were no differences in the length and the amount of mRNA between the LJP patients and the other subjects, while GPP-LAD patients expressed a small amount of long mRNA. The whole coding region (2,313 base pairs) of all subjects was amplified except for the GPP-LAD patients, and the 5'-region (1,119 base pairs) was amplified from all subjects. Base-mismatches were detected on the coding region from 965 to 1,450 nucleotides of CD18 mRNA in GPP-LAD patients. No mismatch was detected on other regions of CD18 mRNA in any subject. These results suggest that the LJP patient with an anomaly of qualitative aggregation related to CD11/CD18 is not related to sequence abnormalities of CD18 mRNA suggesting other mechanisms. In contrast, the adherence defect in two GPP-LAD patients was related to heterogeneous molecular mutations on CD18.

DOI： 10.1902/jop.1994.65.10.949

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Language：English   Publisher：AMER LEPROSY MISSION  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

HUMAN LEUKOCYTE ANTIGEN (HLA) class II beta chain plays an important role in the recognition of foreign antigens in immune reactions. Different forms of immune reaction may be concerned with initiation and progression of infectious diseases such as periodontitis. In this study we examined the frequency of HLA class II serotype and the variation of HLA class II beta gene in periodontitis patients. HLA serotypic frequencies in 70 Japanese patients with periodontitis and 26 individuals with periodontal health were examined. No HLA serotype specific to any type of periodontitis was observed. In order to detect differences among some HLA serotypes, restriction fragment length polymorphism (RFLP) analysis was undertaken with cDNA probes for HLA-DR beta and HLA-DQ beta genes in 20 subjects (15 patients and 5 healthy individuals). Atypical BamHI and EcoRI restriction sites were found in the HLA-DQ beta gene from 3 patients with early-onset periodontitis. In addition to these 20 subjects, an additional 80 subjects (40 patients and 40 healthy individuals) were screened for the atypical BamHI restriction site using the polymerase chain reaction method. It was detected in 7 patients with early-onset periodontitis, 1 patient with adult periodontitis, and 3 healthy subjects. No clinical differences except age were found between patients with this gene variation and other patients. Interestingly, all 3 healthy subjects with this gene variation were from subjects whose family members developed early-onset periodontitis with the gene variation. Atypical BamHI and EcoRI restriction sites and 41-nt repeated sequence were found in the intron before the third exon of HLA-DQB gene. These results suggest that these intronic gene variations may be useful as gene markers for a subpopulation of early-onset periodontitis and might affect immune reactions such as antigen recognition.

DOI： 10.1902/jop.1994.65.5.379

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The purpose of this study was to investigate factors influencing the ability of lipopolysaccharide (LPS) derived from Porphyromonas gingivalis to elicit secretion of tumor necrosis factor-alpha (TNFalpha) from human monocytes (adherent mononuclear cells). The results indicate that P gingivalis LPS stimulation of TNFalpha from monocytes is comparable to LPS from Escherichia coli. Both LPS, although structurally different, increased TNFalpha secretion in a dose-dependent manner. In serum-free conditions, TNFalpha secretion was relatively low, but it dramatically increased at human serum concentrations as low as 1%. Maximal secretion was observed in the presence of 10% serum, with a slight decrease at higher serum concentrations. The CD14 molecule is a putative monocyte LPS receptor. When cells were pre-incubated with a blocking monoclonal antibody (My4) to CD14, TNFalpha-mRNA accumulation and TNFalpha secretion were reduced to control levels at LPS concentrations of up to 10 ng/ml. At higher LPS concentrations, the blocking effect was only partial, in spite of 50-fold excess antibody concentration. The blocking effect was observed only in the presence of serum. The effect of the CD14 antibody was dose-dependent with saturation at 2.5 mug/ml. The results suggest that CD14 is one of the major receptors for P gingivalis LPS but highlight the necessity to investigate other cell-surface receptors mediating P gingivalis-LPS interactions. These interactions are believed to be important in the pathogenesis of periodontal destruction.

DOI： 10.1111/j.1399-302x.1994.tb00044.x

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THIS STUDY WAS UNDERTAKEN IN AN EFFORT to understand the role of cytokines on human gingival fibroblasts and T lymphocyte trafficing into inflamed gingival tissue. Using flow cytometry we examined gingival fibroblasts to determine the level of cell surface expression and the percentage of cells positive for intercellular adhesion molecule 1 (ICAM-1), the HLA-DR antigen, lymphocyte function-associated antigen 3 (LFA-3), and the CD44 molecule, which are involved in antigen presentation. The following cytokines were used: interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-6, and IL-8. The levels of ICAM-1 expression were enhanced in a dose- and time-dependent manner by IL-1 beta, TNF-alpha, or IFN-gamma, but not by IL-6 or IL-8. HLA-DR surface expression was induced only by IFN-gamma in a dose- and time-dependent manner, but not by the other cytokines tested. In contrast, the expression of LFA3 and the CD44 molecule could be detected without the stimulation of any cytokine, but the levels of their expression were not significantly changed by any cytokines. The enhanced ICAM-1 expression by cytokines was reduced in a time-dependent manner following the removal of cytokines from the reaction mixture, while IFN-gamma-induced HLA-DR expression was maintained even 7 days after the removal of IFN-gamma. These data support an interactive role for inflammatory cytokines and the expression of adhesion molecules on gingival fibroblasts in the pathogenesis of gingival inflammation in periodontal disease.

DOI： 10.1902/jop.1994.65.3.230

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