Yaping Pan

Porphorymonas gingivalis induces intracellular adhesion molecule-1 expression in endothelial cells through the nuclear factor-kappaB pathway, but not through the p38 MAPK pathway

BACKGROUND AND OBJECTIVE Porphyromonas gingivalis is a major pathogen in the development and progression of periodontal disease. The aim of this study was to investigate whether endothelial intracellular adhesion molecule-1 (ICAM-1), an inflammation biomarker for periodontitis, could be modified by infection with either of two strains of P. gingivalis with different virulence capacities: avirulent ATCC 33277 and virulent W83. MATERIAL AND METHODS We examined the expression of ICAM-1, IκBα, phospho-p38 MAPK and nuclear factor-kappaB (NF-κB) p65 in an umbilical vein endothelial cell line (ECV-304) treated with ATCC 33277 and W83, with or without the NF-κB antagonist MG132 and/or a specific p38 inhibitor (SB203580), by real-time PCR, western blotting and immunofluorescence. RESULTS Both strains could induce ICAM-1 expression; additionally W83 was able to increase ICAM-1 expression more significantly than ATCC 33277. In P. gingivalis-infected endothelial cells, both p38 MAPK and NF-κB signaling pathways were triggered by a rapid increase of p38 MAPK phosphorylation and a more delayed degradation of IκBα, followed by the nuclear translocation of NF-κB. It was found that ICAM-1 production in endothelial cells was abrogated by inhibition of the NF-κB pathway, but not by inhibition of the p38 MAPK pathway, using the inhibitors of the latter two molecules. CONCLUSION The induction of ICAM-1 by infection of umbilical vein endothelial cells with P. gingivalis might be mediated through the NF-κB pathway, but not by the p38 MAPK pathway.

16S rDNA‐based metagenomic analysis of dental plaque and lung bacteria in patients with severe acute exacerbations of chronic obstructive pulmonary disease

BACKGROUND AND OBJECTIVE Acute exacerbations of chronic obstructive pulmonary disease (AE-COPD) are leading causes of mortality in hospital intensive care units. We sought to determine whether dental plaque biofilms might harbor pathogenic bacteria that can eventually cause lung infections in patients with severe AE-COPD. MATERIAL AND METHODS Paired samples of subgingival plaque biofilm and tracheal aspirate were collected from 53 patients with severe AE-COPD. Total bacterial DNA was extracted from each sample individually for polymerase chain reaction amplification and/or generation of bacterial 16S rDNA sequences and cDNA libraries. We used a metagenomic approach, based on bacterial 16S rDNA sequences, to compare the distribution of species present in dental plaque and lung. RESULTS Analysis of 1060 sequences (20 clones per patient) revealed a wide range of aerobic, anaerobic, pathogenic, opportunistic, novel and uncultivable bacterial species. Species indistinguishable between the paired subgingival plaque and tracheal aspirate samples (97-100% similarity in 16S rDNA sequence) were dental plaque pathogens (Aggregatibacter actinomycetemcomitans, Capnocytophaga sputigena, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola) and lung pathogens (Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Streptococcus pneumoniae). Real-time polymerase chain reaction of 16S rDNA indicated lower levels of Pseudomonas aeruginosa and Porphyromonas gingivalis colonizing the dental plaques compared with the paired tracheal aspirate samples. CONCLUSION These results support the hypothesis that dental bacteria may contribute to the pathology of severe AE-COPD.

Virulence genes of Porphyromonas gingivalis W83 in chronic periodontitis

Objective. To identify virulence genes found in highly virulent strains of Porphyromonas gingivalis (P. gingivalis) among Chinese patients with chronic periodontitis and to evaluate the association of these virulence genes with clinical parameters and with periodontal tissue destruction. Material and methods.Suppression subtractive hybridization was applied to acquire short gene fragments harbored only in virulent strains of P. gingivalis W83. Eighteen genes, which were present in P. gingivalis W83 but absent from P. gingivalis ATCC 33277, were labeled with Cy5 and used as probes in DNA microarray hybridization to analyze DNA of P. gingivalis isolated from chronic periodontitis patients. Results. Spearman correlation analysis revealed 10 genes correlated with probing depth, clinical attachment loss, and tooth mobility (p<0.05). Conclusion. These genes may provide an important clue towards our understanding the mechanism of occurrence and the development of periodontal disease.

The effects of Porphyromonas gingivalis on the cell cycle progression of human gingival epithelial cells

OBJECTIVE Porphyromonas gingivalis is a major pathogen in the development and progression of periodontal disease. The interactions or cross-talk between bacteria and gingival epithelial cells drive bacteria to manipulate the cell cycle to favor bacterial survival and virulence expression within the host. This study aims to dissect the effects of P. gingivalis on the cell cycle in human gingival epithelial cells. MATERIALS AND METHODS We established a model of P. gingivalis invading IHGE cells. The cell cycle distribution of human gingival epithelial cells was analyzed by flow cytometry. Cyclin D and cyclin E mRNA and protein were detected by real-time PCR and Western blot, respectively. RESULTS Porphyromonas gingivalis-induced facilitation of cell growth was correlated with the acceleration of G1 phase of cell cycle. Cyclin D1 mRNA levels were significantly upregulated from 6 to 12 h after infection. Cyclin E protein and mRNA levels were elevated at 10 and 12 h after invasion. CONCLUSIONS We confirmed that P. gingivalis significantly enhances IHGE cell proliferation by promoting the G1/S transition, involving the up-regulation of cyclin D and cyclin E.

The Sociodemographic Characteristics, Periodontal Health Status, and Subgingival Microbiota of Patients With Chronic Periodontitis and Type 2 Diabetes Mellitus: A Case-Control Study in a Chinese Population

BACKGROUND In China, chronic periodontitis (CP) is common in patients with type 2 diabetes mellitus (T2DM). The purpose of this study is to identify the sociodemographic characteristics associated with such patients and to assess the periodontal health status and subgingival microbiota of patients with CP and T2DM (T2DMCP) in the Chinese population. METHODS A total of 150 patients with T2DMCP and 306 patients with CP without any systemic disease completed questionnaires, underwent clinical periodontal examinations and participated in diabetes-related parameter examinations. Subgingival plaques were obtained to determine the prevalence and amounts of selected oral bacterial species using polymerase chain reaction (PCR) and real-time PCR, respectively. RESULTS The income level and mean body mass index (BMI) of the patients with T2DMCP were significantly higher than those of the patients with CP. Additionally, the patients with T2DMCP were more likely to be urban residents, and they had significantly more severe periodontitis than did the patients with CP. In the patients with T2DMCP, the prevalence and amounts of Treponema denticola and Tannerella forsythia were significantly higher than those in the patients with CP. Finally, compared with the patients with CP, the patients with T2DMCP had a significantly lower prevalence and amount of Prevotella intermedia. CONCLUSIONS Compared with the patients with CP, the patients with T2DMCP were more likely to be urban residents and generally had higher incomes, higher mean BMI, and poorer periodontal health status. Higher levels of T. denticola and T. forsythia and lower levels of P. intermedia were identified in the subgingival plaque of the patients with T2DMCP.

Persistent Exposure to Porphyromonas gingivalis Promotes Proliferative and Invasion Capabilities, and Tumorigenic Properties of Human Immortalized Oral Epithelial Cells

Recent epidemiological studies revealed a significant association between oral squamous cell carcinoma (OSCC) and Porphyromonas gingivalis, a major pathogen of periodontal disease. As a keystone pathogen of periodontitis, P. gingivalis is known not only to damage local periodontal tissues, but also to evade the host immune system and eventually affect systemic health. However, its role in OSCC has yet to be defined. To explore the underlying effect of chronic P. gingivalis infection on OSCC and to identify relevant biomarkers as promising targets for therapy and prevention, we established a novel model by exposing human immortalized oral epithelial cells (HIOECs) to P. gingivalis at a low multiplicity of infection (MOI) for 5–23 weeks. The P. gingivalis infected HIOECs were monitored for tumor biological alteration by proliferation, wound healing, transwell invasion, and gelatin zymography assays. Microarray and proteomic analyses were performed on HIOECs infected with P. gingivalis for 15 weeks, and some selected data were validated by quantitative real-time PCR and (or) western blot on cells infected for 15 and 23 weeks. Persistent exposure to P. gingivalis caused cell morphological changes, increased proliferation ability with higher S phase fraction in the cell cycle, and promoted cell migratory and invasive properties. In combining results of bioinformatics analyses and validation assays, tumor-related genes such as NNMT, FLI1, GAS6, lncRNA CCAT1, PDCD1LG2, and CD274 may be considered as the key regulators in tumor-like transformation in response to long-time exposure of P. gingivalis. In addition, some useful clinical biomarkers and novel proteins were also presented. In conclusion, P. gingivalis could promote tumorigenic properties of HIOECs, indicating that chronic P. gingivalis infection may be considered as a potential risk factor for oral cancer. The key regulators detected from the present model might be used in monitoring the development of OSCC with chronic periodontal infection.

The Influences of Periodontal Status and Periodontal Pathogen Quantity on Salivary 8-Hydroxydeoxyguanosine and Interleukin 17 Levels

BACKGROUND Periodontitis is a biofilm-initiated disease that is characterized by elevated inflammatory status. 8-Hydroxydeoxyguanosine (8-OHdG) and interleukin (IL)-17 are highly associated with inflammation and bone resorption and therefore are regarded as potential biomarkers for periodontitis. In this study, the associations between salivary 8-OHdG and IL-17 levels and clinical and microbial parameters before and after non-surgical treatment are investigated. METHODS Forty-five patients with chronic periodontitis (CP) and 47 periodontally healthy volunteers were recruited for the study. Clinical parameters, including the probing depth (PD), clinical attachment level (CAL), sulcular bleeding index, and simplified oral hygiene index (OHI-S), were examined for each participant. Microbial parameters including the quantities of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola in the subgingival plaque and saliva were determined by real-time polymerase chain reaction at baseline and 1 and 3 months after the non-surgical treatment. Salivary 8-OHdG and IL-17 levels were detected by enzyme-linked immunosorbent assays. RESULTS Compared with healthy volunteers, CP group patients had significantly higher salivary 8-OHdG and IL-17 levels at baseline. Baseline salivary 8-OHdG and IL-17 levels were positively correlated with all clinical parameters as well as the quantities of T. forsythia and T. denticola. After non-surgical treatment, baseline levels of salivary 8-OHdG and IL-17 were reduced significantly at both the 1- and 3-month follow-ups. The hierarchical linear model revealed that variations in the PD, CAL, and OHI-S had significant positive effects on variation in the salivary 8-OHdG level. However, variations in the PD; quantity of T. forsythia in the subgingival plaque; and quantities of P. gingivalis, T. forsythia, and T. denticola in saliva were associated significantly with variation in the salivary IL-17 levels. CONCLUSIONS There was a strong association between salivary 8-OHdG and IL-17 levels and periodontitis. Variation in the salivary 8-OHdG level was correlated with variations in the clinical parameters, whereas variation in the IL-17 level was correlated with variation in the microbial parameters.

The relationship between root concavities in first premolars and chronic periodontitis.

OBJECTIVE To evaluate the significance of first premolar root concavity on clinical indices of chronic periodontal disease and alveolar bone defects. METHODS Three-dimensional reconstruction by cone beam computed tomography was used to observe root surface anatomy and the type of alveolar bone defect seen in the mesial and distal sites of 272 first premolars from 99 patients who had presented with chronic periodontitis. Periodontal clinical indicators at each site were measured using a Florida Probe Corporation (Gainesville, FL, USA). RESULTS The incidence of mesial and distal root concavities of the maxillary first premolars was 100% and 39.3% respectively, and in the mandibular, the incidence was 42.5% and 31.3% respectively. The distributions of the different types of concavities in terms of both age and gender of the patients were not statistically significant. The mean probing depth and clinical attachment loss of the first premolars with root concavities were significantly higher than those without concavity (p < 0.05). Plaque accumulation was significantly different in the premolars with/without root concavities (p < 0.001). The type of alveolar bone defects with concavities was significantly different from those without concavities (p < 0.05). Ramp shape bone defects were dominant for teeth without concavities, while crater shape was seen for teeth with concavities. CONCLUSION Root concavities of the first premolars were associated with periodontal disease, and the type of interproximal alveolar bone defect. Root concavities may be important in contributing to local periodontal disease of the first premolars.

Assessment of Alveolar Bone Status in Middle Aged Chinese (40-59 Years) with Chronic Periodontitis--Using CBCT.

Objective This study used con-beam computed tomography (CBCT) to investigate the prevalence and severity of alveolar bone loss in middle-aged (40–59 years) Chinese with chronic periodontitis. Materials and Methods The study group comprised 145 dentate individuals aged 40 to 59 years residing in China who suffered from chronic periodontitis. CBCT and the application of NNT software were used to examine the level and location of alveolar bone loss. Results The study revealed that 40–59 year old patients with chronic periodontitis had severe bone loss. At 5,286 sites (34.7%), alveolar bone loss was mild; severe alveolar bone loss was found at 5,978 sites (39.2%). A comparison of bone loss in different jaws revealed that the area with the highest degree of bone loss was on the lingual side of the maxillary molar (56.3 ± 7.2%), and that the area with the lowest degree was primarily on the lingual side of the mandibular canine (27.5 ± 6.3%). There was a lower degree of alveolar bone loss in males than females. Differences were observed when comparing the incidence of bone loss between males and females (P < 0.05). Menopause in females and smoking in both genders may affect the level of bone loss. Male smokers experienced a greater degree of bone loss (41.67 ± 5.76%) than male non-smokers (32.95 ± 4.31%). A 42.23 ± 6.34% bone loss was found in menopausal females versus 31.35 ± 3.62% in non-menopausal females. Conclusions The study revealed that different sites and teeth exhibited a diverse degree of bone loss. In middle-aged patients with chronic periodontitis, the highest degrees of bone loss in the incisors, premolars, and molars were on the lingual side, mesial side and lingual side, respectively. Menopause in females and smoking may affect the level of bone loss.

Sialidase Deficiency in Porphyromonas gingivalis Increases IL-12 Secretion in Stimulated Macrophages Through Regulation of CR3, IncRNA GAS5 and miR-21

Porphyromonas gingivalis (P. gingivalis) is a major periodontal pathogen that can induce an immune response leading to a destructive inflammatory process. During the inflammatory process, interleukin-12 (IL-12) is secreted, correlating with bacterial clearance by macrophages. Bacterial sialidase has recently been shown to influence the synthesis and modification of the macromolecules on its surface, and is associated with the interaction between bacteria and host cells. We have previously constructed a P. gingivalis sialidase gene mutant strain in P. gingivalis W83 (ΔPG0352) and found that ΔPG0352 showed less pathogenicity than the wild-type strain. In this study, U937-differentiated macrophages were stimulated by P. gingivalis W83, ΔPG0352, or PG0352 complemented strain (comΔPG0352). Transmission electron microscopy showed that P. gingivalis caused a loss of membrane integrity in macrophages and the intracellular bacteria were enclosed within endocytic vacuoles. The expression of both IL-12p35 and IL-12p40 genes and the levels of IL-12p70 were significantly higher in U937 stimulated by ΔPG0352 than in those with P. gingivalis W83 and comΔPG0352. In order to explain why ΔPG0352 induced more IL-12 in macrophages, immunofluorescence assays, PCR arrays, and gene silence or overexpression experiments were carried out. Immunofluorescence assays showed that ΔPG0352 induced lower expression of CR3 in macrophages. After CR3 was suppressed, there were no significant differences in the IL-12p70 levels between macrophages stimulated by P. gingivalis W83, ΔPG0352 or comΔPG0352. PCR array experiments showed that miR-21 and lncRNA GAS5 were differentially expressed between macrophages stimulated by P. gingivalis W83 and ΔPG0352, which had been identified by real-time PCR. The results of CR3 blocking and lncRNA GAS5 gene silence or overexpression showed that the difference in IL-12 levels between P. gingivalis W83 and ΔPG0352 groups was associated with CR3, lncRNA GAS5 and miR-21. Thus it can be concluded that the sialidase-deficient strain is more easily cleared by attenuating CR3 activation, reducing the inhibition of lncRNA GAS5, inducing less miR-21 and more IL-12 in macrophages. These results indicate that inhibiting the activity of sialidase in P. gingivalis will cause rapid clearing by macrophages.