Transcriptomic analysis of Porphyromonas gingivalis-infected head and neck cancer cells: Identification of PLAU as a candidate prognostic biomarker.

Periodontal disease is a risk factor for head and neck squamous cell carcinoma (HNSCC), and Porphyromonas gingivalis, a major periodontal pathogen, has been identified as a specific and potentially independent microbial factor that increases the risk of cancer mortality. Gene expression in HNSCC due to P. gingivalis infection and how changes in gene expression affect the prognosis of HNSCC patients are not clarified. When P. gingivalis was cultured with HNSCC cells, it efficiently adhered to these cells and enhanced their invasive ability. A transcriptome analysis of P. gingivalis -infected HNSCC cells showed that genes related to migration, including CCL20, CITED2, CTGF, C8orf44-SGK3, DUSP10, EGR3, FUZ, HBEGF, IL1B, IL24, JUN, PLAU, PTGS2, P2RY1, SEMA7A, SGK1 and SIX2, were highly up- or down-regulated. The expression of up-regulated genes was examined using the expression data of HNSCC patients obtained from The Cancer Genome Atlas (TCGA) database, and the expression of 5 genes, including PLAU, was found to be higher in cancer tissue than in solid normal tissue. An analysis of protein-protein interactions revealed that these 5 genes formed a dense network. A Cox regression analysis showed that high PLAU expression levels were associated with a poor prognosis in patients with TCGA-HNSCC. Furthermore, the prognostic impact correlated with tumour size and the presence or absence of lymph node metastasis. Collectively, these results suggest the potential of PLAU as a molecular prognostic marker in HNSCC patients. Further in vivo and in vitro studies are needed to verify the findings of this study.

Prevalencia de Porphyromonas gingivalis en fluido gingival y su relación con la periodontitis / Prevalence of Porphyromonas gingivalis in gingival fluid and its relationship with periodontitis

Introducción: la periodontitis es una enfermedad infecciosa multifactorial asociada a un biofilm de microorganismos patógenos. Objetivo: el objetivo del trabajo fue establecer la prevalencia de Porphyromonas gingivalis en pacientes con periodontitis y relacionarla con la severidad de la enfermedad. Material y métodos: participaron 45 pacientes, sistémicamente saludables, con edades entre 35 y 65 años. El grado de periodontitis se definió según los criterios de Papapanou y colaboradores. Como grupo control, se incluyeron 20 sujetos de ambos sexos sin periodontitis y sin enfermedades sistémicas. Se tomaron muestras de fluido gingival en dos sitios más profundos. Porphyromonas gingivalis se detectó por PCR (reacción en cadena de la polimerasa). Resultados: la frecuencia relativa de periodontitis fue de 13.3% grado I, 46.7% grado II y 40% grado III. El sexo masculino presentó periodontitis grado III 72.2% y grado II 52.3%. El grado I se registró con mayor frecuencia en el sexo femenino, 66.7%. La prevalencia de Porphyromonas gingivalis en la población con periodontitis fue de 44.4%. Se obtuvieron diferencias estadísticamente significativas entre los grados de severidad de periodontitis y la presencia de Porphyromonas gingivalis (p = 0.0002, α = 5%). Conclusión: la periodontitis predominó en el sexo masculino. La prevalencia de Porphyromonas gingivalis en la población con periodontitis crónica fue de 44.4% y su presencia está relacionada con la severidad (AU)

Introduction: periodontitis is a multifactorial infectious disease associated with a biofilm of pathogenic microorganisms. Objective: the objective of the work was to establish the prevalence of Porphyromonas gingivalis in patients with periodontitis and relate it to the severity of the disease. Material and methods: 45 systemically healthy patients, aged between 35 and 65 years old, participated. The degree of periodontitis was defined according to the criteria of Papapanou et al. As a control group, 20 patients of both sexes without periodontitis and without systemic diseases were included. Gingival fluid samples were taken from two deeper sites. Porphyromonas gingivalis was detected by PCR (polymerase chain reaction). Results: the relative frequency of periodontitis was 13.3% grade I, 46.7% grade II and 40% grade III. The male sex presented periodontitis grade III 72.2% and grade II 52.3%. Grade I was recorded more frequently in the female sex, 66.7%. The prevalence of Porphyromonas gingivalis in the population with periodontitis was 44.4%. Statistically significant differences were obtained between the degrees of severity of periodontitis and the presence of Porphyromonas gingivalis (p = 0.0002, α = 5%). Conclusion: periodontitis predominated in males. The prevalence of Porphyromonas gingivalis in the population with chronic periodontitis was 44.4% and its presence is related to severity (AU)

Prevalencia de Porphyromonas gingivalis en un grupo de pacientes con aparatología fija de ortodoncia / Prevalence of Porphyromonas gingivalis in a group of patients with fixed orthodontic appliances

Introducción: existen diversos patógenos que pueden afectar no sólo la salud periodontal, sino también la salud general de los pacientes. Objetivo: determinar la Porphyromonas gingivalis (PG) en el primer molar superior derecho de adolescentes, de entre 12 y 18 años, con al menos un mes de tratamiento de ortodoncia con aparatología fija. Material y métodos: se realizó un estudio observacional, descriptivo, transversal de casos en un grupo de 26 adolescentes con tratamiento de ortodoncia, compuesto de brackets metálicos, tubos o bandas, arcos NiTi termoactivos, módulos, cadenas o ligaduras; sin importar sexo, edad, tiempo de tratamiento o maloclusión. Se formaron dos pares de grupos 1 y 2 (15 mujeres y 11 hombres), A y B (13 mujeres y 13 hom- bres) comparando los resultados obtenidos entre los grupos. Resulta- dos: dentro del grupo 1 y 2 la detección molecular de microorganismos arroja que 80% fueron positivas a la PG, 58.33% presenta maloclusión y en promedio 89% de las pacientes son positivas a PG. La detección molecular del grupo A y B indica que 54.54% fueron positivos a PG, mientras que 83.3% presenta maloclusión y en promedio 47% son positivos a PG. Conclusión: la explicación de los eventos moleculares que se desencadenan en la cavidad oral y los sistemas afectados por PG contribuyen a la prevención de complicaciones al tener una mejor comprensión de los fenómenos infecciosos (AU)

Introduction: there are various pathogens that can affect not only periodontal health, but also the general health of patients. Objective: to determine Porphyromonas gingivalis (PG) in the upper right first molar of adolescents, between 12 and 18 years old, with at least one month of orthodontic treatment with fixed appliances. Material and methods: a cross-sectional descriptive observational study of cases was carried out in a group of 26 adolescents with orthodontic treatment, consisting of metal brackets, tubes or bands, thermoactive NiTi archwires, modules, chains or ligatures; regardless of sex, age, treatment time or malocclusion. Two pairs of groups 1 and 2 (15 women and 11 men), A and B (13 women and 13 men) were formed, comparing the results obtained between the groups. Results: within group 1 and 2, the molecular detection of microorganisms shows that 80% were positive for PG, 58.33% presented malocclusion and an average of 89% of patients were positive for PG. The molecular detection of group A and B indicates that 54.54% were positive for PG while 83.3% presented malocclusion and on average 47% were positive for PG. Conclusion: the explanation of the molecular events that are triggered in the oral cavity and the systems affected by PG contribute to the prevention of complications by having a better understanding of the infectious phenomena (AU)

Associations of Porphyromonas gingivalis Infection and Low Beclin1 Expression With Clinicopathological Parameters and Survival of Esophageal Squamous Cell Carcinoma Patients.

Purpose: The present study focused on exploring the associations of Porphyromonas gingivalis (P. gingivalis) infection and low Beclin1 expression with clinicopathological parameters and survival of esophageal squamous cell carcinoma (ESCC) patients, so as to illustrate its clinical significance and prognostic value. Methods: Immunohistochemistry (IHC) was used to detect P. gingivalis infection status and Beclin1 expression in 370 ESCC patients. The chi-square test was adopted to illustrate the relationship between categorical variables, and Cohen's kappa coefficient was used for correlation analysis. Kaplan-Meier survival curves with the log-rank test were used to analyse the correlation of P. gingivalis infection and low Beclin1 expression with survival time. The effects of P. gingivalis infection and Beclin1 downregulation on the proliferation, migration and antiapoptotic abilities of ESCC cells in vitro were detected by Cell Counting Kit-8, wound healing and flow cytometry assays. For P. gingivalis infection of ESCC cells, cell culture medium was replaced with antibiotic-free medium when the density of ESCC cells was 70-80%, cells were inoculated with P. gingivalis at a multiplicity of infection (MOI) of 10. Result: P. gingivalis infection was negatively correlated with Beclin1 expression in ESCC tissues, and P. gingivalis infection and low Beclin1 expression were associated with differentiation status, tumor invasion depth, lymph node metastasis, clinical stage and prognosis in ESCC patients. In vitro experiments confirmed that P. gingivalis infection and Beclin1 downregulation potentiate the proliferation, migration and antiapoptotic abilities of ESCC cells (KYSE150 and KYSE30). Our results provide evidence that P. gingivalis infection and low Beclin1 expression were associated with the development and progression of ESCC. Conclusion: Long-term smoking and alcohol consumption causes poor oral and esophageal microenvironments and ESCC patients with these features were more susceptible to P. gingivalis infection and persistent colonization, and exhibited lower Beclin1 expression, worse prognosis and more advanced clinicopathological features. Our findings indicate that effectively eliminating P. gingivalis colonization and restoring Beclin1 expression in ESCC patients may contribute to preventation and targeted treatment, and yield new insights into the aetiological research on ESCC.

The effects of periodontitis associated microbiota on the development of oral squamous cell carcinoma.

Epidemiological data have shown that periodontal bacterial infection, periodontitis, and oral squamous cell carcinoma have close relationship on the disease progress and risk. However, the specific role of periodontal microbes and their mechanism in the development of oral squamous cell carcinoma is not yet clear. In our previous work, metagenomic Illumina Mi-seq analysis was used to identify tstructure and abundance of periodontital microbiome. Accoding to the results, we used Porphyromonas.spp. and Fusobacterium.spp. as the periodontitis positive microbiota; Neisseria.spp and Corynebacterium.spp as periodontitis negative microbiota (their average relative abundance were >5%). These representative strains of the above genus were used to infect OSCC cells to explore their effect on tumor cell biology behavior, and detect the expression level of the gene in related to inflammation, migration, invasion and cell cycle. We find that periodontitis positive correlated microbiota had a promoting effect on the development of oral squamous cell carcinoma in vitro by regulating mRNA and protein expression of IL-6, IL-8, MMP-9 and Cyclin-D1. Periodontitis negative correlated microbiota had suppression effect on the development of oral squamous cell carcinoma in vitro analysis.

Frequencies of Porphyromonas gingivalis Detection in Oral-Digestive Tract Tumors.

Mounting evidence suggests a causal relationship between specific bacterial infections and the development of certain malignancies. In this study, we examined the presence of Porphyromonas gingivalis (P. gingivalis) in oral-digestive tract tumors by immunohistochemistry (IHC) and PCR and analyzed the correlation between P. gingivalis detection and clinicopathological characteristics and prognosis of oral and esophageal carcinoma. The IHC results showed that the positive rates of P. gingivalis were 60.00, 46.00, 20.00, 6.67, and 2.86% in oral, esophagus, cardiac, stomach, and colorectal cancer tissues, respectively. Likewise, PCR results showed rates of 56.00, 42.00, 16.67, 3.33, and 2.86%, respectively. The two methods were consistent, and the kappa value was 0.806, P < 0.001. In addition, P. gingivalis expression was significantly correlated with lymph node metastasis and the clinical stages of oral and esophageal cancer (P < 0.05). The overall survival rate of the P. gingivalis undetected group (86, 50%) was significantly higher than that of the P. gingivalis detected group (57, 14%) for oral and esophageal cancer, respectively. In conclusion, the detection rate of P. gingivalis showed a decreasing trend in oral-digestive tract tumors. Detection with P. gingivalis was associated with poor prognosis for oral and esophageal cancer.

Feasibility of investigating the association between bacterial pathogens and oral leukoplakia in low and middle income countries: A population-based pilot study in India.

BACKGROUND: Certain oral bacterial pathogens may play a role in oral carcinogenesis. We assessed the feasibility of conducting a population-based study in India to examine the distributions and levels of Porphyromonas gingivalis, Fusobacterium nucleatum and Prevotella intermedia in relation to oral leukoplakia (a potentially malignant disorder) and other participant characteristics. METHODS: This exploratory case-control study was nested within a large urban Indian cohort and the data included 22 men and women with oral leukoplakia (cases) and 69 leukoplakia-free controls. Each participant provided a salivary rinse sample, and a subset of 34 participants (9 cases; 25 controls) also provided a gingival swab sample from keratinized gingival surface for quantitative polymerase chain reaction (qPCR). RESULTS: Neither the distribution nor the levels of pathogens were associated with oral leukoplakia; however, individual pathogen levels were more strongly correlated with each other in cases compared to controls. Among controls, the median level of total pathogens was the highest (7.55×104 copies/ng DNA) among persons of low socioeconomic status. Salivary rinse provided better DNA concentration than gingival swab for qPCR analysis (mean concentration: 1.8 ng/µl vs. 0.2 ng/µl). CONCLUSIONS: This study confirms the feasibility of population studies evaluating oral microbiome in low-resource settings and identifies promising leads for future research.

Development and Optimization of a Rapid Colorimetric Membrane Immunoassay for Porphyromonas gingivalis.

Porphyromonas gingivalis (P. gingivalis) is a major bacterial pathogen that causes periodontitis, a chronic inflammatory disease of tissues around the teeth. Periodontitis is known to be related to other diseases, such as oral cancer, Alzheimer's disease, and rheumatism. Thus, a precise and sensitive test to detect P. gingivalis is necessary for the early diagnosis of periodontitis. The objective of this study was to optimize a rapid visual detection system for P. gingivalis. First, we performed a visual membrane immunoassay using 3,3',5,5'-tetramethylbenzidine (TMB; blue) and coating and detection antibodies that could bind to the host laboratory strain, ATCC 33277. Antibodies against the P. gingivalis surface adhesion molecules RgpB (arginine proteinase) and Kgp (lysine proteinase) were determined to be the most specific coating and detection antibodies, respectively. Using these two selected antibodies, the streptavidin-horseradish peroxidase (HRP) reaction was performed using a nitrocellulose membrane and visualized with a detection range of 103-105 bacterial cells/ml following incubation for 15 min. These selected conditions were applied to test other oral bacteria, and the results showed that P. gingivalis could be detected without crossreactivity to other bacteria, including Streptococcus mutans and Escherichia fergusonii. Furthermore, three clinical strains of P. gingivalis, KCOM 2880, KCOM 2803, and KCOM 3190, were also recognized using this optimized enzyme immunoassay (EIA) system. To conclude, we established optimized conditions for P. gingivalis detection with specificity, accuracy, and sensitivity. These results could be utilized to manufacture economical and rapid detection kits for P. gingivalis.

Preliminary functional analysis of the subgingival microbiota of cats with periodontitis and feline chronic gingivostomatitis.

The subgingival microbial communities of domestic cats remain incompletely characterized and it is unknown whether their functional profiles are associated with disease. In this study, we used a shotgun metagenomic approach to explore the functional potential of subgingival microbial communities in client-owned cats, comparing findings between periodontally healthy cats and cats with naturally occurring chronic periodontitis, aggressive periodontitis, and feline chronic gingivostomatitis. Subgingival samples were subjected to shotgun sequencing and the metagenomic datasets were analyzed using the MG-RAST metagenomic analysis server and STAMP v2.1.3 (Statistical Analysis of Metagenomic Profiles) software. The microbial composition was also described to better understand the predicted features of the communities. The Respiration category in the level 1 Subsystems database varied significantly among groups. In this category, the abundance of V-Type ATP-synthase and Biogenesis of cytochrome c oxidases were significantly enriched in the diseased and in the healthy groups, respectively. Both features have been previously described in periodontal studies in people and are in consonance with the microbial composition of feline subgingival sites. In addition, the narH (nitrate reductase) gene frequency, identified using the KEGG Orthology database, was significantly increased in the healthy group. The results of this study provide preliminary functional insights of the microbial communities associated with periodontitis in domestic cats and suggest that the ATP-synthase and nitrate-nitrite-NO pathways may represent appropriate targets for the treatment of this common disease.

Invasion of Gingival Epithelial Cells by Porphyromonas gingivalis.

Porphyromonas gingivalis is a major pathogen responsible for severe and chronic manifestations of periodontal disease, which is one of the most common infectious disorders of humans. Although human gingival epithelium prevents intrusions by periodontal bacteria, P. gingivalis is able to invade gingival epithelial cells. To study the dynamics and the fate of intracellular P. gingivalis, confocal laser scanning microscopy (CLSM) is a method of choice. Information gained with CLSM contains not only the number of P. gingivalis associated with gingival epithelial cells but also the bacterial localization on/inside the host cells, morphological change of host cells, and physical interaction between the bacteria and host organelle. In this chapter, we describe the protocols for microscopy techniques to morphologically study gingival epithelial cells infected by P. gingivalis.

Oral infectious bacteria in dental plaque and saliva as risk factors in patients with esophageal cancer.

BACKGROUND: High levels of periodontopathic bacteria as well as Streptococcus anginosus were detected in cancer tissue from patients with esophageal cancer. An association between oral infectious bacteria and esophageal cancer has been reported. METHODS: Characteristics of the oral microbiota and periodontal conditions were studied as clinicopathologic factors in patients with esophageal cancer. The study included 61 patients with esophageal cancer and 62 matched individuals without any cancers. Samples of subgingival dental plaque and unstimulated saliva were collected to evaluate the prevalence and abundance of the following oral bacteria using a real-time polymerase chain reaction assay: Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema denticola, and S. anginosus. RESULTS: In the cancer group, the prevalence of all bacteria, with the exception of F. nucleatum, in dental plaque; the prevalence of A. actinomycetemcomitans in saliva; the abundance of all bacteria, with the exception of F. nucleatum and P. intermedia, in dental plaque; and the abundance of A. actinomycetemcomitans and S. anginosus in saliva were significantly higher. Furthermore, a logistic regression analysis suggested that the prevalence of T. forsythia and S. anginosus in dental plaque and of A. actinomycetemcomitans in saliva, as well as a drinking habit, were associated with a high risk of esophageal cancer, with a high odds ratio. CONCLUSIONS: The current findings have potential implications for the early diagnosis of esophageal cancer.

Prevalencia de Prevotella spp y Porphyromona spp en periodontitis crónica / Prevalence of Prevotella spp and Porphyromona spp in chronic periodontitis

Las enfermedades del periodonto tienen una etiopatogenia compleja y puede considerarse multifactorial. El factor etiológico esencial en la patología inflamatoria periodontal es la biopelícula dental y cuando el desequilibrio entre el huésped y los microorganismos cambia la complejidad de la flora. Ciertas bacterias como Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Prevotella loescheii, Fusobacterium nucleatum, Tannerrella forsythia, Campylobacter rectus, Eikenella corrodens y Treponema spp., han sido comúnmente relacionadas con la periodontitis crónica y son consideradas como indicadores de riesgo para la progresión de dicha enfermedad. El objetivo de este trabajo fue establecer la prevalencia de Prevotella spp y Porphyromona spp en los distintos estadios de periodontitis crónicas. Material y métodos: Se estudiaron 48 pacientes sistémicamente saludables con diagnóstico de periodontitis crónica. Se completó el consentimiento informado, se realizó historia clínica y examen periodontal. El estado periodontal se clasificó en distintos grados de severidad: leve, moderada y severa. Se tomaron muestras de dos sitios con mayor profundidad de sondaje con conos de papel absorbente estériles y se transportaron en un medio prerreducido. Para el aislamiento de Prevotella spp se utilizó agar Brucella más sangre ovina al 5%, hemina, vitamina K al que se agregaron vancomicina y kanamicina; Porphyromonas sp se aisló en el mismo medio con el agregado de bacitracina y colistina. Se sembraron 10 µl de muestra entera y las placas fueron incubadas en jarras de anaerobiosis por 5 a 7 días a 37ºC. Resultados: los distintos grados de periodontitis correspondieron a un 17% periodontits leve, 57% moderada y 26% severa. En el total de pacientes se determinó la presencia de Prevotella spp en el 54% de los casos y un 12,5% de Porphyromona spp. Conclusión: De los pacientes estudiados con periodontits crónica, un 52% correspondió al sexo masculino, un 57% de los casos correspondieron a periodontitis moderada. Se aisló Prevotella sp en todos los estadios de periodontitis crónica y Porphyromonas sp sólo en periodontitis severas (AU)

Periodontal diseases have a complex etiopathogenesis and can be considered multifactorial. The essential etiological factor in periodontal inflammatory pathology is the dental biofilm and when the imbalance between the host and the microorganisms changes the complexity of the flora. Certain bacteria such as Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Prevotella loescheii, Fusobacterium nucleatum, Tannerrella forsythia, Campylobacter rectus, Eikenella corrodens and Treponema spp., Have been commonly related to chronic periodontitis and are considered as risk indicators for the progression of said disease. The objective of this work was to establish the prevalence of Prevotella spp and Porphyromonas spp in the different stages of chronic periodontitis. Forty eight systemically healthy patients diagnosed with chronic periodontitis were studied. Informed consent was completed, a medical history and periodontal examination was carried out. The periodontal state was classified into different degrees of severity: mild, moderate and severe. Samples were taken from two sites with greater depth of probing with sterile absorbent paper cones and transported in a prereduced medium. For the isolation of Prevotella spp, Brucella agar plus 5% sheep blood, hemin, vitamin K to which vancomycin and kanamycin were added. For Porphyromonas spp, the same medium was used and bacitracin and colistin were added. 10 µl of the whole sample was seeded and the plates were incubated in anaerobic jars for 5 to 7 days at 37 ° C. Different degrees of periodontitis corresponded to 17% mild periodontitis, 57% moderate and 26% severe. In the total number of patients, the presence of Prevotella spp was determined in 54% of the cases and 12.5% of Porphyromona spp. Of the patients studied with chronic periodontitis, 52% corresponded to the male sex, 57% of the cases corresponded to moderate periodontitis. Prevotella spp was isolated in all stages of chronic periodontitis and Porphyromonas sp only in severe periodontitis (AU)

Epigenetic regulation of inflammation in periodontitis: cellular mechanisms and therapeutic potential.

Epigenetic mechanisms, namely DNA and histone modifications, are critical regulators of immunity and inflammation which have emerged as potential targets for immunomodulating therapies. The prevalence and significant morbidity of periodontitis, in combination with accumulating evidence that genetic, environmental and lifestyle factors cannot fully explain the susceptibility of individuals to disease development, have driven interest in epigenetic regulation as an important factor in periodontitis pathogenesis. Aberrant promoter methylation profiles of genes involved in inflammatory activation, including TLR2, PTGS2, IFNG, IL6, IL8, and TNF, have been observed in the gingival tissue, peripheral blood or buccal mucosa from patients with periodontitis, correlating with changes in expression and disease severity. The expression of enzymes that regulate histone acetylation, in particular histone deacetylases (HDACs), is also dysregulated in periodontitis-affected gingival tissue. Infection of gingival epithelial cells, gingival fibroblasts and periodontal ligament cells with the oral pathogens Porphyromonas gingivalis or Treponema denticola induces alterations in expression and activity of chromatin-modifying enzymes, as well as site-specific and global changes in DNA methylation profiles and in histone acetylation and methylation marks. These epigenetic changes are associated with excessive production of inflammatory cytokines, chemokines, and matrix-degrading enzymes that can be suppressed by small molecule inhibitors of HDACs (HDACi) or DNA methyltransferases. HDACi and inhibitors of bromodomain-containing BET proteins ameliorate inflammation, osteoclastogenesis, and alveolar bone resorption in animal models of periodontitis, suggesting their clinical potential as host modulation therapeutic agents. However, broader application of epigenomic methods will be required to create a comprehensive map of epigenetic changes in periodontitis. The integration of functional studies with global analyses of the epigenetic landscape will provide critical information on the therapeutic and diagnostic potential of epigenetics in periodontal disease.

An evaluation of direct PCR assays for the detection and quantification of Porphyromonas gingivalis.

Porphyromonas gingivalis has been linked to the development and progression of oesophageal squamous cell carcinoma (ESCC), and is considered to be a high-risk factor for ESCC. Currently, the commonly used methods for P. gingivalis detection are culture or DNA extraction-based, which are either time and labour intensive especially for high-throughput applications. We aimed to establish and evaluate a rapid and sensitive direct quantitative polymerase chain reaction (qPCR) protocol for the detection of P. gingivalis without DNA extraction which is suitable for large-scale epidemiological studies. Paired gingival swab samples from 192 subjects undergoing general medical examinations were analysed using two direct and one extraction-based qPCR assays for P. gingivalis. Tris-EDTA buffer-based direct qPCR (TE-direct qPCR), lysis-based direct qPCR (lysis-direct qPCR) and DNA extraction-based qPCR (kit-qPCR) were used, respectively, in 192, 132 and 60 of these samples for quantification of P. gingivalis. The sensitivity and specificity of TE-direct qPCR was 95.24% and 100% compared with lysis-direct qPCR, which was 100% and 97.30% when compared with kit-qPCR; TE-direct qPCR had an almost perfect agreement with lysis-direct qPCR (κ = 0.954) and kit-qPCR (κ = 0.965). Moreover, the assay time used for TE-direct qPCR was 1.5 h. In conclusion, the TE-direct qPCR assay is a simple and efficient method for the quantification of oral P. gingivalis and showed high sensitivity and specificity compared with routine qPCR.

The effect of periodontal bacteria infection on incidence and prognosis of cancer: A systematic review and meta-analysis.

BACKGROUND: Periodontal bacteria is the major pathogens in the oral cavity and the main cause of adult chronic periodontitis, but their association with incidence and prognosis in cancer is controversial. The aim of this study was to evaluate the effect of periodontal bacteria infection on incidence and prognosis of cancer. METHODS: A systematic literature search of PubMed, Embase, Web of Science, and Cochrane Library databases was performed to obtain 39 studies comprising 7184 participants. The incidence of cancer was evaluated as odd ratios (OR) with a 95% confidence interval (95% CI) using Review Manager 5.2 software. Overall survival, cancer-specific survival and disease-free survival, which were measured as hazard ratios (HR) with a 95% CI using Review Manager 5.2 software. RESULTS: Our results indicated that periodontal bacteria infection increased the incidence of cancer (OR = 1.25; 95%CI: 1.03-1.52) and was associated with poor overall survival (HR = 1.75; 95% CI: 1.40-2.20), disease-free survival (HR = 2.18; 95%CI: 1.24-3.84) and cancer-specific survival (HR = 1.85, 95%CI: 1.44-2.39). Subgroup analysis indicted that the risk of cancer was associated with Porphyromonas gingivalis (Pg) infection (OR = 2.16; 95%CI: 1.34-3.47) and Prevotella intermedia (Pi) infection (OR = 1.28; 95%CI: 1.01-1.63) but not Tannerella forsythia (Tf) (OR = 1.06; 95%CI: 0.8-1.41), Treponema denticola (Td) (OR = 1.30; 95%CI: 0.99-1.72), Aggregatibacter actinomycetemcomitans (Aa) (OR = 1.00; 95%CI: 0.48-2.08) and Fusobacterium nucleatum (Fn) (OR = 0.61; 95%CI: 0.32-1.16). CONCLUSION: This meta-analysis revealed periodontal bacteria infection increased the incidence of cancer and predicted poor prognosis of cancer.

A peptide coating preventing the attachment of Porphyromonas gingivalis on the surfaces of dental implants.

OBJECTIVES: The aim of this study was to investigate whether a peptide-based coating can prevent the adhesion of Porphyromonas gingivalis, a key human pathogen associated with periodontitis and peri-implantitis. BACKGROUND: Nonsurgical and surgical interventions have been used for the treatment of peri-implantitis; however, the effectiveness of these approaches is usually unsatisfactory. The main reason is that dental plaque on the surface of the implant is difficult to remove due to its rough surface and thread design. Recently, a peptide-based coating for implant surfaces that can reject the adhesion of Escherichia coli and improve the attachment of host cells was developed. METHODS: A salivary pellicle was created on the surfaces of peptide-coated bare discs and verified with anti-human immunoglobulin G, A and M, and anti-fibrinogen. Early colonizers, Veillonella parvula and Streptococcus sobrinus, and the later colonizer, Porphyromonas gingivalis, were labelled with green and red fluorescent dyes, respectively, and seeded on the discs. Bacterial attachment was semi-quantified by fluorescence intensity. RESULTS: The salivary pellicle was evenly distributed on the discs, with or without the peptide coating, with an average thickness of 3.84 µm. A multi-species dental biofilm was created on the salivary pellicle. The peptide coating resulted in an approximate 25% reduction in the attachment of Veillonella parvula and Streptococcus sobrinus, and a 50% reduction in Porphyromonas gingivalis, when compared to control, uncoated implant discs. CONCLUSION: The novel peptide-based coating can inhibit the attachment of Porphyromonas gingivalis. It may have the potential to impede the development of peri-implantitis.

Substance P participates in periodontitis by upregulating HIF-1α and RANKL/OPG ratio.

BACKGROUND: Both substance P and hypoxia-inducible factor 1 alpha (HIF-1α) are involved in inflammation and angiogenesis. However, the relationship between substance P and HIF-1α in rat periodontitis is still unknown. METHODS: Ligation-induced rat periodontitis was established to observe the distribution and expression of substance P and HIF-1α by immunohistochemistry. Rat gingival fibroblasts were cultured and stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). Recombinant substance P was applied to elaborate the relationship between substance P and HIF-1α in gingival fibroblasts in vitro. Primary mouse bone marrow-derived macrophages (BMMs) were isolated and cultured to observe the effect of substance P on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis by TRAP staining. Western blotting was used to investigate the expression of HIF-1α, osteoprotegerin (OPG) and RANKL. RESULTS: Rat experimental periodontitis was successfully established 6 weeks after ligation. Gingival inflammatory infiltration and alveolar bone loss were observed. Positive expression of substance P was found in the infiltrating cells. Higher HIF-1α levels were observed in periodontitis compared to that of normal tissues. Substance P upregulated the level of HIF-1α in gingival fibroblasts with or without 1 µg/ml LPS in vitro (*P < 0.05). Substance P upregulated the expression of HIF-1α in RANKL-stimulated BMMs in vitro. Substance P also increased the RANKL/OPG ratio in gingival fibroblasts (*P < 0.05). Both 10 nM and 50 nM substance P promoted RANKL-induced osteoclast differentiation (*P < 0.05). CONCLUSION: Substance P participates in periodontitis by upregulating HIF-1α and the RANKL/OPG ratio.

Gingival crevicular fluid levels of SLIT3 are increased in periodontal disease.

This study aims to investigate the levels of SLIT3 in gingival crevicular fluid (GCF) of healthy and periodontal disease subjects, and their correlations to periodontal disease. A total of 45 periodontal patients and 45 periodontally healthy volunteers were enrolled. The clinical parameters, radiographic bone loss and the levels of SLIT3, receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) in GCF were measured. The prevalences of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia in subgingival plaque were also analyzed. The expression of SLIT3 and RANKL was detected in the periodontium of experimental periodontitis in rats and lipopolysaccharide (LPS)-induced mouse macrophage. The total amounts and concentrations of SLIT3 and RANKL were significantly higher in periodontitis than those in healthy, while the level of OPG was significantly lower (p < .05). Significant positive correlations were observed between the level of GCF SLIT3 and clinical attachment level and radiographic bone loss (p < .05). There existed a significant positive correlation between SLIT3 and RANKL (p < .05). Increased expression of SLIT3 and RANKL was observed in the periodontium of periodontal rats. SLIT3 expression was induced by LPS stimulation in macrophages. These results suggest that SLIT3 may act as a diagnostic indicator of periodontal disease and should be further investigated.

Clinical and microbiological evaluation of non-surgical periodontal therapy in obese and non-obese individuals with periodontitis: a 9-month prospective longitudinal study

Abstract Objective Obesity is a chronic disease that negatively affects an individual's general and oral health. The present study aimed to compare the clinical and microbiological effects of non-surgical periodontal therapy with the full mouth disinfection (FMD) protocol on obese and non-obese individuals at 9 months post-therapy. Methodology This clinical study was first submitted and approved by the Ethics Committee. Fifty-five obese patients and 39 non-obese patients with periodontitis were evaluated. The full-mouth periodontal clinical parameters, clinical attachment level (CAL), probing depth (PD), gingival index (GI), and plaque index (PI), were monitored at baseline, 3, 6, and 9 months after periodontal treatment with full mouth disinfection (FMD) protocol. The mean count of Tannerella forsythia , Porphyromonas gingivalis , Treponema Denticola , and Aggregatibacter actinomycetemcomitans was determined by quantitative polymerase chain reaction on subgingival biofilm samples. Demographic data were assessed by Chi-square test. For clinical and microbiological parameters, two-factor repeated-measures ANOVA was used. Results In both groups, periodontal therapy using the one-stage full-mouth disinfection protocol significantly improved CAL, PD, GI, and PI (p<0.05). Obese and non-obese patients equally responded to non-surgical periodontal therapy (p>0.05). Microbial count found no major differences (p>0.05) between obese and non-obese individuals who had undergone non-surgical periodontal therapy. Conclusions Obesity did not affect the clinical and microbiological outcomes of non-surgical periodontal therapy.

Microbiological study of the subgingival biofilm in HIV+/HAART patients at a specialized dental service / Estudio microbiológico del biofilm subgingival de pacientes VIH+ bajo TARGA en un servicio dental especializado

ABSTRACT The aim of this study was to describe the microbiological profile of HIV patients under highly active antiretroviral treatment (HAART). This crosssectional study comprised 32 HIV patients with periodontal disease (PD) who had been under HAART for more than 6 months. Information about the patients' medical history was obtained from clinical records. Clinical dental examination was performed by a calibrated researcher using standard dental instruments to determine probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP). A total 4,765 periodontal sites were evaluated, 125 of which were also studied microbiologically. Subgingival biofilm samples were obtained using sterile paper points; one set was used for microbiological culture studies and the other for endpoint PCR. Statistical analysis was performed using KruskalWallis and posthoc DunnBonferroni contrast tests. All participants were on HAART at the time of the study, and 90.6% had a viral load below 50 copies/mm3. Prevalence of periodontally active sites was low in the study population. Microbiological studies: Black pigmented anaerobic bacteria and fusiform CFU counts were significantly higher in samples from sites with BOP and PD ≥4mm (p 0.020 and p 0.005, respectively). Molecular Assays: Detection of Porphyromonas gingivalis (p 0.002), Tannerella forsythia (p 0.023) and Treponema denticola (p 0.015) was significantly more frequent at sites with BOP and PD ≥4mm. Conclusions: The patients living with HIV/AIDS under HAART studied here had low prevalence of clinical periodontal disease signs. However, significant detection of P. gingivalis, T. denticola, and T. forsythia in periodontal active sites, and the involvement of these microorganisms as potential HIV reactivators, show the importance of creating awareness among dental health professionals of the need for close dental and periodontal monitoring in HIV patients.

RESUMEN El objetivo de este estudio fue describir el perfil microbiológico del biofilm subgingival de los pacientes con VIH bajo tratamiento antirretroviral de alta actividad (TARGA). El estudio comprendió a 32 pacientes VIH seropositivos con enfermedad periodontal (EP) que se encontraran en tratamiento con TARGA por más de 6 meses. Los antecedentes médicos de los pacientes se obtuvieron de las historias clínicas. El examen clínico instrumental (profun didad de sondaje (PS), nivel de inserción clínico (NIC) y sangrado al sondaje (SS)) fue realizado con instrumental odontológico estándar por un investigador calibrado. De este modo, se evaluaron un total de 4.765 sitios periodontales de los cuales 125 fueron estudiados microbiológicamente. Las muestras de biope lícula subgingival se obtuvieron empleando conos de papel estéril. Las muestras se emplearon en estudios microbiológicos y moleculares por PCR de punto final. El análisis estadístico se realizó según KruskalWallis y pruebas de contrastes posthoc de DunnBonferroni. El 90,6% de la población en estudio presentó carga viral inferior a 50 copias/mm3. La prevalencia de sitios periodontales activos fue baja (1%). Los recuentos de bacterias anaerobias estrictas pigmentadas de negro y fusiformes fueron significativamente más altos en muestras de sitios periodontales con SS positivo y PS ≥4 mm (p 0.020 y p 0.005). La detección molecular de Porphyromonas gingivalis (p 0.002), Tannerella forsythia (p 0.023) y Treponema denticola (p 0.015) fue significativamente mayor en los sitios con SS y PS ≥4mm. La prevalencia del 1% de enfermedad periodontal en el grupo de pacientes estudiados fue menor a la esperada, sin embargo; la detección significativa de P. gingivalis, T. denticola y T. forsythia en sitios periodontales activos y su potencial participación como agentes reactivadores del VIH, nos alerta de la importancia de crear conciencia en los profesionales de la salud (médicos y odontólogos) acerca de la necesidad de un monitoreo minucioso del estado periodontal de pacientes con características semejantes a las descriptas en la muestra poblacional estudiada.