Anti-Inflammatory Activity of No-Ozone Cold Plasma in Porphyromonas gingivalis Lipopolysaccharide-Induced Periodontitis Rats.

Periodontitis is an inflammatory disease caused by Porphyromonas gingivalis (P. gingivalis) in the oral cavity. This periodontal disease causes damage to the periodontal ligament and alveolar bone and can cause tooth loss, but there is no definite treatment yet. In this study, we investigated the possibility of using no-ozone cold plasma to safely treat periodontitis in the oral cavity. First, human gingival fibroblasts (HGFs) were treated with P. gingivalis-derived lipopolysaccharide (PG-LPS) to induce an inflammatory response, and then the anti-inflammatory effect of NCP was examined, and a study was conducted to identify the mechanism of action. Additionally, the anti-inflammatory effect of NCP was verified in rats that developed an inflammatory response similar to periodontitis. When NCP was applied to PG-LPS-treated HGFs, the activities of inflammatory proteins and cytokines were effectively inhibited. It was confirmed that the process of denaturing the medium by charged particles of NCP is essential for the anti-inflammatory effect of NCP. Also, it was confirmed that repeated treatment of periodontitis rats with NCP effectively reduced the inflammatory cells and osteoclast activity. As a result, this study suggests that NCP can be directly helpful in the treatment of periodontitis in the future.

Comparison of genetic and epigenetic profiles of periodontitis according to the presence of type 2 diabetes.

Type 2 diabetes mellitus (T2DM) and periodontitis (PD) have intricated connections as chronic inflammatory diseases. While the immune response is a key factor that accounts for their association, the underlying mechanisms remain unclear. To gain a deeper understanding of the connection, we conducted research using a multiomics approach. We generated whole genome and methylation profiling array data from the periodontium of PD patients with DM (PDDM) and without DM to confirm genetic and epigenetic changes. Independent bulk and single-cell RNA sequencing data were employed to verify the expression levels of hypo-methylated genes. We observed a gradual rise in C>T base substitutions and hypomethylation in PD and PDDM patients compared with healthy participants. Furthermore, specific genetic and epigenetic alterations were prominently associated with the Fc-gamma receptor-mediated phagocytosis pathway. The upregulation of these genes was confirmed in both the periodontal tissues of PD patients and the pancreatic tissues of T2DM patients. Through single-cell RNA analysis of peripheral blood mononuclear cells, substantial upregulation of Fc-gamma receptors and related genes was particularly identified in monocytes. Our findings suggest that targeting the Fc-gamma signaling pathway in monocytes holds promise as a potential treatment strategy for managing systemic complications associated with diabetes.

Immunological link between periodontitis and type 2 diabetes deciphered by single-cell RNA analysis.

BACKGROUND: Type 2 diabetes mellitus (DM) is a complex metabolic disorder that causes various complications, including periodontitis (PD). Although a bidirectional relationship has been reported between DM and PD, their immunological relationship remains poorly understood. Therefore, this study aimed to compare the immune response in patients with PD alone and in those with both PD and DM (PDDM) to expand our knowledge of the complicated connection between PD and DM. METHODS: Peripheral blood mononuclear cells were collected from 11 healthy controls, 10 patients with PD without DM, and six patients with PDDM, followed by analysis using single-cell RNA sequencing. The differences among groups were then compared based on intracellular and intercellular perspectives. RESULTS: Compared to the healthy state, classical monocytes exhibited the highest degree of transcriptional change, with elevated levels of pro-inflammatory cytokines in both PD and PDDM. DM diminished the effector function of CD8+ T and natural killer (NK) cells as well as completely modified the differentiation direction of these cells. Interestingly, a prominent pathway, RESISTIN, which is known to increase insulin resistance and susceptibility to diabetes, was found to be activated under both PD and PDDM conditions. In particular, CAP1+ classical monocytes from patients with PD and PDDM showed elevated nuclear factor kappa B-inducing kinase activity. CONCLUSIONS: Overall, this study elucidates how the presence of DM contributes to the deterioration of T/NK cell immunity and the immunological basis connecting PD to DM.

Single-cell analysis of platelets from patients with periodontitis and diabetes.

Background: Blood platelets are known to play a role in the development of atherosclerotic disease, thrombi, and hemostasis. Investigation of blood platelet transcriptome could provide evidence of disorders that increase vulnerability to cardiovascular disease. However, research on the molecular insights of platelet activation in patients with periodontitis and patients with periodontitis and type 2 diabetes mellitus (DM) is still lacking. Objectives: In this study, we analyzed expression in blood platelets from patients with periodontitis and patients with concurrent periodontitis and DM to examine the transcriptomic profile of platelets induced by periodontitis and the modifying effects of DM. Methods: We obtained the transcriptional profiles of blood platelets from 11 healthy donors, 10 patients with periodontitis, and 6 patients with periodontitis and DM using single-cell RNA sequencing. The biological processes and coexpressed modules of transcriptionally altered genes were further explored. Results: Both the patients with periodontitis and DM and those with periodontitis without DM showed higher levels of platelet activation and coagulation signals than the healthy individuals. Platelets from the patients with periodontitis had higher expression levels of genes for RHO GTPase effectors, whereas platelets from the patients with periodontitis and DM demonstrated higher expression of genes involved in oxidative phosphorylation and cellular responses to stress than those from the controls. However, compared with the patients with only periodontitis, those with periodontitis and DM presented a lower expression level of genes for hemostasis and platelet receptors. Conclusion: These results suggest that periodontitis contributes to establishment of blood coagulation via platelet dysregulation, whereas the comorbidities of patients with periodontitis and DM impair the components of platelets, thus preventing normal functions.

Increased prevalence of periodontitis with hypouricemic status: findings from the Korean National Health and Nutrition Examination Survey, 2016-2018.

PURPOSE: The aim of this study was to investigate the relationship between serum uric acid (SUA) levels and the risk of periodontitis in Korean adults using data from the Korean National Health and Nutrition Examination Survey (KNHANES). METHODS: This cross-sectional study used data from the KNHANES 2016-2018 and analysed 12,735 Korean adults aged ≥19 years who underwent oral examinations. Hypouricemia was defined as SUA <3 mg/dL in men and <2 mg/dL in women, and hyperuricemia was defined as SUA ≥7 mg/dL in men and ≥6 mg/dL in women. RESULTS: The weighted prevalence of hypouricemia and hyperuricemia was 0.6% and 12.9%, respectively. The overall weighted periodontitis rate was 30.5%. The frequency of periodontitis in subjects with hypouricemia, normouricemia, and hyperuricemia were 51.1%, 30.3%, and 30.6%, respectively. Study participants with hypouricemia were significantly older, had significantly fasting blood glucose levels, and had better kidney function than non-hypouricemic participants. In univariate logistic regression analyses, hypouricemia was associated with periodontitis, but hyperuricemia was not. The fully adjusted model revealed that the adjusted odds ratio of hypouricemia for periodontitis was 1.62 (95% confidence interval, 1.13-2.33), while the relationship between hyperuricemia and periodontitis in the multivariable logistic regression model was not significant. CONCLUSIONS: The results of this study suggest that hypouricemia is associated with an increased risk of periodontitis.

Profiling of plasma-derived exosomal RNA expression in patients with periodontitis: A pilot study.

OBJECTIVE: This study aimed to profile differentially expressed (DE) exosomal RNAs in healthy subjects and periodontitis patients and compare their levels before and after treatment. MATERIALS AND METHODS: Plasma samples from healthy subjects and patients with periodontitis (pre-/post-periodontal treatment) were collected for this case-control study. After isolation of exosomes from the plasma, the RNA was extracted and small RNA sequencing was performed (3 healthy samples, 4 pre-treatment samples, and 5 post-treatment samples). Two-way analyses were conducted according to the treatment status in the periodontitis group, unpaired analysis (grouping as pre-/post-treatment) and paired analysis (matching pre- and post-treatment in the same subject). The DE exosomal RNAs were screened by sequencing and visualized using the R software. Gene Ontology analysis was performed, and target genes were identified. RESULTS: In both paired and unpaired analyses, two DE microRNAs (DEmiRs; miR-1304-3p and miR-200c-3p) and two DE small nucleolar RNAs (DEsnoRs; SNORD57 and SNODB1771) were common, and they were found to be downregulated during periodontitis and recovered to healthy levels after treatment. The top three target genes (NR3C1, GPR158, and CNN3) commonly regulated by DEmiRs were identified. CONCLUSIONS: Plasma-derived exosomal miRs (miR-1304-3p and miR-200c-3p) and snoRs (SNORD57 and SNODB1771) could be valuable biomarkers for periodontitis.

Vasohibin-1 promotes osteoclast differentiation in periodontal disease by stimulating the expression of RANKL in gingival fibroblasts.

Vasohibin-1 (VASH1) is a key inhibitor of vascular endothelial growth factor-induced angiogenesis. Although the involvement of VASH1 in various pathological processes has been extensively studied, its role in periodontal disease (PD) remains unclear. We aimed to investigate the role of VASH1 in PD by focusing on osteoclastogenesis regulation. We investigated VASH1 expression in PD by analyzing data from the online Gene Expression Omnibus (GEO) database and using a mouse ligature-induced periodontitis model. The effects of VASH1 on osteoclast differentiation and osteoclastogenesis-supporting cells were assessed in mouse bone marrow-derived macrophages (BMMs) and human gingival fibroblasts (GFs). To identify the stimulant of VASH1, we used culture broth from Porphyromonas gingivalis (Pg), a periopathogen. The GEO database and mouse periodontitis model revealed that VASH1 expression was upregulated in periodontitis-affected gingival tissues, which was further supported by immunohistochemistry and qRT-PCR analyses. VASH1 expression was significantly stimulated in GFs after treatment with the Pg broth. Direct treatment with recombinant VASH1 protein did not stimulate osteoclast differentiation in BMMs but did contribute to osteoclast differentiation by inducing RANKL expression in GFs through a paracrine mechanism. Small interfering RNA-mediated silencing of VASH1 in GFs abrogated RANKL-mediated osteoclast differentiation in BMMs. Additionally, VASH1-activated RANKL expression in GFs was significantly suppressed by MK-2206, a selective inhibitor of AKT. These results suggest that Pg-induced VASH1 may be associated with RANKL expression in GFs in a paracrine manner, contributing to osteoclastogenesis via an AKT-dependent mechanism during PD progression.

Effects of a simplified drilling protocol at 50 rpm on heat generation under water-free conditions: an in vitro study.

PURPOSE: In recent years, guided implant surgery has been widely used for the convenience of patients and surgeons. Further streamlining the surgical procedure would make implant surgery more convenient. Low-speed water-free conditions are often used in guided implant surgery. Therefore, in this study, we attempted to confirm once again whether drilling was safe at a low speed without water. The main purpose of this study was to evaluate whether a simplified drilling protocol that omits some intermediate steps in the drilling process was safe from the viewpoint of heat generation. METHODS: D1 density artificial bone blocks were drilled under 50 rpm, 10 N·cm water-free conditions, and the surface temperature was measured using a digital infrared camera. First, drilling was performed with the sequential drilling method, which is the most widely used technique. Second, for each drill diameter, the temperature change was measured while performing simplified drilling with omission of the previous 1, 2, or 3 steps. RESULTS: In sequential drilling, the heat generated during drilling at all diameters was less than the critical temperature of osteonecrosis (47°C) except for the ⌀2 drill. Statistical significance was observed in all groups when comparing sequential and simplified drilling in the ⌀3.2, ⌀3.8, and ⌀4.3 drills (P<0.001). However, in the simplified drilling procedures, the temperature was below the osteonecrosis threshold temperature (47°C) except for the ⌀4.3 drill with the omission of the previous 3 steps (⌀3.0, ⌀3.2, and ⌀3.8). CONCLUSIONS: In general, drilling under low-speed, water-free conditions has shown stable results in terms of heat generation. Simplified drilling showed statistically significantly greater heat generation than sequential drilling. However, most of the diameters and omitted steps seem to be clinically acceptable, so it will be useful if an appropriate selection is made according to the patient's clinical condition.

Microbial profiling of peri-implantitis compared to the periodontal microbiota in health and disease using 16S rRNA sequencing.

PURPOSE: The objective of this study was to analyze the microbial profile of individuals with peri-implantitis (PI) compared to those of periodontally healthy (PH) subjects and periodontitis (PT) subjects using Illumina sequencing. METHODS: Buccal, supragingival, and subgingival plaque samples were collected from 109 subjects (PH: 30, PT: 49, and PI: 30). The V3-V4 region of 16S rRNA was sequenced and analyzed to profile the plaque microbiota. RESULTS: Microbial community diversity in the PI group was higher than in the other groups, and the 3 groups showed significantly separated clusters in the buccal samples. The PI group showed different patterns of relative abundance from those in the PH and PT groups depending on the sampling site at both genus and phylum levels. In all samples, some bacterial species presented considerably higher relative abundances in the PI group than in the PH and PT groups, including Anaerotignum lactatifermentans, Bacteroides vulgatus, Faecalibacterium prausnitzii, Olsenella uli, Parasutterella excrementihominis, Prevotella buccae, Pseudoramibacter alactolyticus, Treponema parvum, and Slackia exigua. Network analysis identified that several well-known periodontal pathogens and newly recognized bacteria were closely correlated with each other. CONCLUSIONS: The composition of the microbiota was considerably different in PI subjects compared to PH and PT subjects, and these results could shed light on the mechanisms involved in the development of PI.

Single-cell RNA sequencing reveals rebalancing of immunological response in patients with periodontitis after non-surgical periodontal therapy.

BACKGROUND: Periodontitis is a major inflammatory disease of the oral mucosa that is not limited to the oral cavity but also has systemic consequences. Although the importance of chronic periodontitis has been emphasized, the systemic immune response induced by periodontitis and its therapeutic effects remain elusive. Here, we report the transcriptomes of peripheral blood mononuclear cells (PBMCs) from patients with periodontitis. METHODS: Using single-cell RNA sequencing, we profiled PBMCs from healthy controls and paired pre- and post-treatment patients with periodontitis. We extracted differentially expressed genes and biological pathways for each cell type and calculated activity scores reflecting cellular characteristics. Intercellular crosstalk was classified into therapy-responsive and -nonresponsive pathways. RESULTS: We analyzed pan-cellular differentially expressed genes caused by periodontitis and found that most cell types showed a significant increase in CRIP1, which was further supported by the increased levels of plasma CRIP1 observed in patients with periodontitis. In addition, activated cell type-specific ligand-receptor interactions, including the BTLA, IFN-γ, and RESISTIN pathways, were prominent in patients with periodontitis. Both the BTLA and IFN-γ pathways returned to similar levels in healthy controls after periodontal therapy, whereas the RESISTIN pathway was still activated even after therapy. CONCLUSION: These data collectively provide insights into the transcriptome changes and molecular interactions that are responsive to periodontal treatment. We identified periodontitis-specific systemic inflammatory indicators and suggest unresolved signals of non-surgical therapy as future therapeutic targets.

Retrospective analysis of the effects of non-communicable diseases on periodontitis treatment outcomes.

PURPOSE: We retrospectively analysed patients' dental and periodontal status according to the presence of non-communicable diseases (NCDs) and the effects of NCDs on periodontal treatment outcomes. Factors influencing disease recurrence were investigated using decision tree analysis. METHODS: We analysed the records of patients who visited the Department of Periodontology, Pusan National University Dental Hospital from June 2014 to October 2019. As baseline subjects, 1,362 patients with periodontitis and who underwent full-mouth periodontal examinations before periodontal treatment were selected. Among them, 321 patients who underwent periodontal examinations after the completion of periodontal treatment and 143 who continued to participate in regular maintenance were followed-up. RESULTS: Forty-three percent of patients had a NCD. Patients without NCDs had more residual teeth and lower sum of the number of total decayed, missing, filled teeths (DMFT) scores. There was no difference in periodontal status according to NCD status. Patients with a NCD showed significant changes in the plaque index after periodontal treatment. The decision tree model analysis demonstrated that osteoporosis affected the recurrence of periodontitis. CONCLUSIONS: The number of residual teeth and DMFT index differed according to the presence of NCDs. Patients with osteoporosis require particular attention to prevent periodontitis recurrence.

Prevalence and abundance of 9 periodontal pathogens in the saliva of periodontally healthy adults and patients undergoing supportive periodontal therapy.

PURPOSE: This study aimed to examine the prevalence and abundance of 9 representative periodontal pathogens in the saliva samples of periodontally healthy subjects (PH) and patients with periodontitis who underwent supportive periodontal therapy (SPT). The age-specific distribution of these pathogens in periodontally healthy individuals was also analyzed. METHODS: One hundred subjects (aged >35 years) were recruited (50 each in the PH and SPT groups) between August 2016 and April 2019. The prevalence and abundance of periodontal pathogens in the PH group were compared with those in periodontally healthy young subjects (94 subjects; aged <35 years), who were included in our previous study. DNA copy numbers of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), Prevotella intermedia (Pi), Fusobacterium nucleatum (Fn), Campylobacter rectus (Cr), Peptostreptococcus anaerobius (Pa), and Eikenella corrodens (Ec) were analyzed using real-time polymerase chain reaction. RESULTS: The detection frequencies of all pathogens, except Aa, were high in the PH and SPT groups. The ranking order of pathogen DNA copy numbers was similar in both groups. In both groups, Fn had the highest abundance, Aa had the lowest abundance. Additionally, Td was significantly more abundant in men than in women in both groups (P<0.05). Compared with the PH group, the SPT group exhibited significantly lower total bacteria and Fn abundance and higher Pg abundance (P<0.05). The age-specific pathogen distribution analysis revealed a significantly low Aa abundance and high Tf and Cr abundance in the PH group. CONCLUSIONS: The clinical parameters and microbial profiles were similar between the SPT and PH groups. However, patients with periodontitis require supportive care to prevent recurrence. As the abundance of some bacteria varied with age, future studies must elucidate the correlation between age-related physiological changes and periodontal bacterial composition.

A decision tree to identify the combinations of non-communicable diseases that constitute the highest risk for dental caries experience: A hospital records-based study.

To investigate whether dental status, represented by the DMFT score, was affected by the presence of NCDs and determined the NCDs that had a greater impact on the DMFT score. This retrospective cross-sectional study included a total of 10,017 individuals. The presence of NCDs was investigated based on self-reported medical history recorded on each patient's dental hospital record. Individual DMFT score was evaluated on the basis of the dental records and panoramic radiographs. The data were further analyzed using multiple regression analysis and chi-squared automatic interaction detection (CHAID) analysis. A total of 5,388 individuals had more than one NCD among hypertension (HT), diabetes mellitus (DM), hyperlipidemia, cardiovascular disease (CVD), and osteoporosis. The average DMFT score was 8.62 ± 7.10 in the NCD group, significantly higher than that in those without NCD (5.53 ± 5.48) (P < 0.001). In the regression analysis, age, NCDs, and psychiatric problems were selected as risk factors of DMFT score. In the CHAID decision tree analysis, age was the risk factor that most influenced the DMFT score. HT was the most influential factor in a newly generated decision tree excluding age, and osteoporosis, DM, and CVD were important risk factors acting in the subgroups. Patients with NCD had worse dental conditions than those who did not, and some combinations of NCDs related highest risk for a dental caries-related index. In clinical practice, dentists should provide meticulous care for dental caries in elderly patients with NCDs, especially when certain diseases, such as HT, osteoporosis, DM, and CVD, are present together.

Elevated Expression of Cathepsin K in Periodontal Ligament Fibroblast by Inflammatory Cytokines Accelerates Osteoclastogenesis via Paracrine Mechanism in Periodontal Disease.

Cathepsin K (CTSK) is a cysteine protease that is mainly produced from mature osteoclasts and contributes to the destruction of connective tissues and mineralized matrix as a consequence of periodontal disease (PD). However, few studies have reported its regulatory role in osteoclastogenesis-supporting cells in inflammatory conditions. Here, we investigated the role of CTSK in osteoclastogenesis-supporting cells, focusing on the modulation of paracrine function. Microarray data showed that CTSK was upregulated in PD patients compared with healthy individuals, which was further supported by immunohistochemistry and qPCR analyses performed with human gingival tissues. The expression of CTSK in the osteoclastogenesis-supporting cells, including dental pulp stem cells, gingival fibroblasts, and periodontal ligament fibroblasts (PDLFs) was significantly elevated by treatment with inflammatory cytokines such as TNFα and IL-1ß. Moreover, TNFα stimulation potentiated the PDLF-mediated osteoclastogenesis of bone marrow-derived macrophages. Interestingly, small interfering RNA-mediated silencing of CTSK in PDLF noticeably attenuated the TNFα-triggered upregulation of receptor activator of nuclear factor kappa-B ligand (RANKL), macrophage colony-stimulating factor, and RANKL/osteoprotegerin ratio, thereby abrogating the enhanced osteoclastogenesis-supporting activity of PDLF. Collectively, these results suggest a novel role of CTSK in the paracrine function of osteoclastogenesis-supporting cells in periodontal disease.

Identification of Shared Genes and Pathways in Periodontitis and Type 2 Diabetes by Bioinformatics Analysis.

Introduction: It is well known that the presence of diabetes significantly affects the progression of periodontitis and that periodontitis has negative effects on diabetes and diabetes-related complications. Although this two-way relationship between type 2 diabetes and periodontitis could be understood through experimental and clinical studies, information on common genetic factors would be more useful for the understanding of both diseases and the development of treatment strategies. Materials and Methods: Gene expression data for periodontitis and type 2 diabetes were obtained from the Gene Expression Omnibus database. After preprocessing of data to reduce heterogeneity, differentially expressed genes (DEGs) between disease and normal tissue were identified using a linear regression model package. Gene ontology and Kyoto encyclopedia of genes and genome pathway enrichment analyses were conducted using R package 'vsn'. A protein-protein interaction network was constructed using the search tool for the retrieval of the interacting genes database. We used molecular complex detection for optimal module selection. CytoHubba was used to identify the highest linkage hub gene in the network. Results: We identified 152 commonly DEGs, including 125 upregulated and 27 downregulated genes. Through common DEGs, we constructed a protein-protein interaction and identified highly connected hub genes. The hub genes were up-regulated in both diseases and were most significantly enriched in the Fc gamma R-mediated phagocytosis pathway. Discussion: We have identified three up-regulated genes involved in Fc gamma receptor-mediated phagocytosis, and these genes could be potential therapeutic targets in patients with periodontitis and type 2 diabetes.

Prediction of Chronic Periodontitis Severity Using Machine Learning Models Based On Salivary Bacterial Copy Number.

Periodontitis is a widespread chronic inflammatory disease caused by interactions between periodontal bacteria and homeostasis in the host. We aimed to investigate the performance and reliability of machine learning models in predicting the severity of chronic periodontitis. Mouthwash samples from 692 subjects (144 healthy controls and 548 generalized chronic periodontitis patients) were collected, the genomic DNA was isolated, and the copy numbers of nine pathogens were measured using multiplex qPCR. The nine pathogens are as follows: Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), Prevotella intermedia (Pi), Fusobacterium nucleatum (Fn), Campylobacter rectus (Cr), Aggregatibacter actinomycetemcomitans (Aa), Peptostreptococcus anaerobius (Pa), and Eikenella corrodens (Ec). By adding the species one by one in order of high accuracy to find the optimal combination of input features, we developed an algorithm that predicts the severity of periodontitis using four machine learning techniques. The accuracy was the highest when the models classified "healthy" and "moderate or severe" periodontitis (H vs. M-S, average accuracy of four models: 0.93, AUC = 0.96, sensitivity of 0.96, specificity of 0.81, and diagnostic odds ratio = 112.75). One or two red complex pathogens were used in three models to distinguish slight chronic periodontitis patients from healthy controls (average accuracy of 0.78, AUC = 0.82, sensitivity of 0.71, and specificity of 0.84, diagnostic odds ratio = 12.85). Although the overall accuracy was slightly reduced, the models showed reliability in predicting the severity of chronic periodontitis from 45 newly obtained samples. Our results suggest that a well-designed combination of salivary bacteria can be used as a biomarker for classifying between a periodontally healthy group and a chronic periodontitis group.

Atheroprotective nasal immunization with a heat shock protein 60 peptide from Porphyromonas gingivalis.

PURPOSE: Immunization with Porphyromonas gingivalis heat shock protein 60 (PgHSP60) may have an immunoregulatory effect on atherogenesis. The aim of this study was to determine whether nasal immunization with a PgHSP60 peptide could reduce atherosclerotic plaque formation in apolipoprotein E knockout (ApoE KO) mice. METHODS: Seven-week-old male ApoE KO mice were assigned to receive a normal diet, a Western diet, a Western diet and challenge with PgHSP60-derived peptide 14 (Pep14) or peptide 19 (Pep19), or a Western diet and immunization with Pep14 or Pep19 before challenge with Pep14 or Pep19. RESULTS: Atherosclerotic plaques were significantly smaller in mice that received a Western diet with Pep14 nasal immunization than in mice that received a Western diet and no Pep14 immunization with or without Pep14 challenge. An immunoblot profile failed to detect serum reactivity to Pep14 in any of the study groups. Stimulation by either Pep14 or Pep19 strongly promoted the induction of CD4+CD25+forkhead box P3 (FoxP3)+ human regulatory T cells (Tregs) in vitro. However, the expression of mouse splenic CD4+CD25+FoxP3+ Tregs was lower in the Pep14-immunized mice than in the Pep14-challenged or Pep19-immunized mice. Levels of serum interferon gamma (IFN-γ) and transforming growth factor beta were higher and levels of interleukin (IL) 10 were lower in the Pep14-immunized mice than in the other groups. Induction of CD25- IL-17+ T helper 17 (Th17) cells was attenuated in the Pep14-immunized mice. CONCLUSIONS: Nasal immunization with Pep14 may be a mechanism for attenuating atherogenesis by promoting the secretion of IFN-γ and/or suppressing Th17-mediated immunity.

Detection of association between periodontitis and polymorphisms of IL-1ß + 3954 and TNF-α -863 in the Korean population after controlling for confounding risk factors.

BACKGROUND AND OBJECTIVE: Interleukin (IL)-1 and tumor necrosis factor (TNF)-α are inflammatory cytokines that play an important role in periodontitis, and their genetic variations have been suggested to be associated with increased risk of periodontitis. Focusing on three single nucleotide polymorphisms (SNPs) of IL-1α + 4845, IL-1ß + 3954, and TNF-α -863, we aimed to investigate the relationship between periodontitis risk and the polymorphisms of IL-1 α/ß and TNF-α in Koreans. MATERIAL AND METHODS: Mouthwash samples from 548 subjects (135 controls without periodontitis, 387 generalized chronic periodontitis patients, and 26 generalized aggressive periodontitis patients) were collected for isolation of genomic DNA. Genotyping of selected SNPs was performed using real-time PCR. Univariable associations between the polymorphisms and periodontitis were assessed by chi-squared test or Fisher's exact test. To evaluate the association after controlling for confounding effects of various risk factors, we stratified the subjects according to the presence or absence of self-reported diseases and employed multiple logistic regression model to adjust for age, smoking status, and oral hygiene indices and behaviors. RESULTS: Significant association of IL-1ß + 3954 and TNF-α -863 polymorphisms with periodontitis was observed after adjusting for the confounding risk factors, but not in univariable association analysis. The significant association between genotype CT of IL-1ß + 3954 and increased risk of advanced periodontitis was consistently detected regardless of the status of self-reported diseases. In the polymorphism of TNF-α -863, the genotype with minor allele (CA + AA) was significantly associated with periodontitis susceptibility, which was observed only in the subjects with self-reported diseases. CONCLUSION: The results suggest that genetic variations of IL-1ß + 3954 and TNF-α -863 are associated with increased risk of periodontitis in Koreans. In addition, our findings underscore the importance of controlling for confounding risk factors to detect significant association between genetic factors and risk of periodontitis. A further well-designed large-scale study is needed to warrant our results.

Gastrin-Releasing Peptide (GRP) Stimulates Osteoclastogenesis in Periodontitis.

Periodontitis is a chronic inflammatory disease with alveolar bone resorption and subsequent tooth loss as its ultimate outcomes. Gastrin-releasing peptide (GRP) is a neuropeptide with growth-stimulatory and tumorigenic properties, and neuropeptides have previously been suggested to play a role in the complex cascade of chemical activity associated with periodontal inflammation. In this study, GRP treatment enhanced the differentiation of bone marrow-derived macrophages (BMMs) into osteoclasts, and gastrin-releasing peptide receptor (GRPR) antagonists suppressed the pro-osteoclastogenic effect of GRP. Grpr-siRNA knockdown resulted in a significantly lower number of osteoclasts formed as compared with the control. Interestingly, gene expression analysis indicated downregulation of Grp and Grpr expressions in BMMs during osteoclastogenesis. Moreover, ligature-induced periodontitis model in mice and gingival samples from patients with periodontitis displayed increased immunostaining of GRP in the oral epithelium. Subsequently, stimulation of mouse primary epithelial cells (ECs) and HaCaT cells, human epidermal keratinocytes, with lipopolysaccharides (LPS) of Porphyromonas gingivalis or live P. gingivalis upregulated Grp and Grpr expressions. Finally, coculture of P. gingivalis-stimulated ECs and BMMs using Transwell system revealed that the differentiation of BMMs was induced when subjected to paracrine activation by LPS- as well as live-P. gingivalis stimulated ECs. Taken together, our results demonstrate that the pro-osteoclastogenic properties of BMMs may be modulated by GRP produced by ECs in the periodontal microenvironment.

Assessing the effects of cyclosporine A on the osteoblastogenesis, osteoclastogenesis, and angiogenesis mediated by human periodontal ligament stem cells.

BACKGROUND: This study was performed to investigate the effects of cyclosporine A (CsA) on the osteogenic differentiation, osteoclastogenic-supporting ability, and angiogenic potential of human periodontal ligament stem cells (hPDLSCs). METHODS: hPDLSCs were isolated from the extracted teeth of orthodontic patients. Cell proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) and alizarin red (ARS) staining. Real-time polymerase chain reaction (PCR) was used to quantify transcripts. Tartrate-resistant acid phosphatase staining of bone marrow-derived macrophages (BMMs) and tube formation assays on human umbilical vein endothelial cells (HUVECs) were performed after treating cells with the conditioned media from CsA-exposed or non-exposed hPDLSCs. Signaling pathways mediating the angiogenic activity were investigated using western blotting. RESULTS: CsA suppressed the proliferation of hPDLSCs but enhanced osteogenic differentiation as determined by ALP and ARS staining and PCR of osteogenic transcripts. The expressions of osteoclastogenic transcripts in hPDLSCs and the differentiation of BMMs treated with conditioned medium from CsA-exposed hPDLSCs were unaffected by CsA. However, the expressions of angiogenic transcripts and the transcripts known to support angiogenesis-phosphorylation of extracellular signal p-regulated kinase (ERK) and p38, and c-fos-were inhibited. Conditioned medium from CsA-exposed hPDLSCs suppressed the tube forming abilities of HUVECs. CONCLUSIONS: CsA enhanced the osteogenic differentiation and reduced angiogenesis by blocking the ERK and p38/c-fos pathway in hPDLSCs. It is necessary to confirm whether this phenomenon is also observed in vivo in subsequent animal experiments.