A Potential Role of Phospholipase 2 Group IIA (PLA2-IIA) in P. gingivalis-Induced Oral Dysbiosis.

Porphyromonas gingivalis is an oral pathogen with the ability to induce oral dysbiosis and periodontal disease. Nevertheless, the mechanisms by which P. gingivalis could abrogate the host-microbe symbiotic relationship leading to oral dysbiosis remain unclear. We have recently demonstrated that P. gingivalis specifically increased the antimicrobial properties of oral epithelial cells, through a strong induction of the expression of PLA2-IIA in a mechanism that involves activation of the Notch-1 receptor. Moreover, gingival expression of PLA2-IIA was significantly increased during initiation and progression of periodontal disease in non-human primates and interestingly, those PLA2-IIA expression changes were concurrent with oral dysbiosis. In this chapter, we present an innovative hypothesis of a potential mechanism involved in P. gingivalis-induced oral dysbiosis and inflammation based on our previous observations and a robust body of literature that supports the antimicrobial and proinflammatory properties of PLA2-IIA as well as its role in other chronic inflammatory diseases.

On the diagnostic discrimination ability of mouthrinse and salivary aMMP-8 point-of-care testing regarding periodontal health and disease.

This study investigated the diagnostic utility of mouthrinse and saliva in aMMP-8 measurements to analyze patients' risk for active periodontal tissue destruction and progression of periodontal disease among 47 adolescents. Results show that measurements from mouthrinse produce better discrimination and should be used instead of saliva measurements.

The Ability of Quantitative, Specific, and Sensitive Point-of-Care/Chair-Side Oral Fluid Immunotests for aMMP-8 to Detect Periodontal and Peri-Implant Diseases.

The analysis of the disease-specific oral and systemic biomarkers in saliva and oral fluids (i.e., mouth rinse, gingival crevicular fluid (GCF), and peri-implantitis fluid (PISF)) is demanding. Several hosts and microbial factors may influence their expression, release, and levels. The type of saliva/oral fluids utilized for the diagnostics affects the analysis. High sensitivity and specificities together with sophisticated methods and techniques are essential for valuable outcome. We describe here recently developed practical, convenient, inexpensive, noninvasive, and quantitative mouth rinse and PISF/GCF/chair-side/point-of-care (PoC) lateral-flow aMMP-8 immunoassays (PerioSafe and ImplantSafe/ORALyser) to detect, predict, and monitor successfully the course, treatment, and prevention of periodontitis and peri-implantitis, respectively. The tests have been independently and successfully validated to differentiate periodontal and peri-implant health and disease in Finland, Germany, Netherland, Sweden, Turkey, Nigeria, Malawi, and USA. The clinical use of salivary/oral fluid biomarkers to identify oral and systemic conditions requires additional studies utilizing these noninvasive screening, diagnostic, and preventive aMMP-8 PoC/chair-side technologies.

aMMP-8 in correlation to caries and periodontal condition in adolescents-results of the epidemiologic LIFE child study.

OBJECTIVES: The suitability of a chairside aMMP-8 test in determination of periodontal inflammation and caries in adolescents was assessed. Secondly, the influence of orthodontic treatment on aMMP-8 test result was analyzed. MATERIALS AND METHODS: Within the LIFE Child study, 434 adolescents (10 to 18 years) were included. Clinical dental examinations comprised caries experience (DMF/T-Index), signs of periodontal inflammation (probing pocket depth, PPD; community periodontal index of treatment needs; CPITN) at six index teeth and oral hygiene (OH). Information about orthodontic appliances (OA) and socioeconomic status (SES) were obtained by validated questionnaires. Test's sensitivity and specificity to detect periodontal inflammation and carious lesions were evaluated. The influence of OA on the test result was analyzed (multivariate model). RESULTS: No associations between age, gender, SES or OH, and test outcome were found (p > 0.05). Positive test results correlated to periodontal findings (CPITN, mean PPD; p < 0.001). However, for the detection of ≥ 1 site(s) with PPD ≥ 4 mm, the test's sensitivity and specificity were found to be 61 and 69%, respectively. Multivariate analysis revealed a higher probability for a positive test result in cases of fixed OA (odds ratio 5.02, 95% confidence interval 1.90-13.19). The test had no diagnostic value considering carious lesions. CONCLUSIONS: The chairside aMMP-8 test does not reliably identify adolescents with periodontal inflammation. Positive test results were more frequent in case of OA. CLINICAL RELEVANCE: The chairside aMMP-8 test is no appropriate tool to screen children and adolescents neither for periodontal inflammation nor for carious lesions.

Detection of Biomarkers of Periodontal Disease in Human Saliva Using Stabilized, Vertical Flow Immunoassays.

We report methods for stabilizing cellulose-based immunoassays and using this platform to analyze human saliva. Stabilization treatments of immunoassays for matrix metalloproteinases (MMP)-8 and -9, biomarkers of periodontal disease, were conducted and compared, revealing that anti-MMP-8 and -9 capture antibodies could be stabilized with the addition of a 5% trehalose solution to the test zones, followed by drying in a vacuum oven. After stabilization, the paper devices retained equivalent binding activity to that of freshly prepared tests for 14 days-a time frame that enables US-based clinical testing of this diagnostic assay. A saliva pretreatment method was developed to remove viscous elements without reducing the concentration or binding activity of dissolved proteins. Immunoassays were stored in ziplock bags containing desiccant, and used to detect nanomolar concentrations of MMP-9 in human saliva across the relevant clinical concentration range. These methods and findings facilitate rapid, affordable validation studies of this and other biomarkers that are found in saliva using vertical flow immunoassays.

Evaluation of Metalloproteinase-8 Levels in Crevicular Fluid of Patients with Healthy Implants or Periodontitis.

Evaluation of periodontal and peri-implant tissue condition is mainly based on clinical examination and imaging diagnostics. Some data imply that Metalloproteinase-8 (MMP-8) level examination in peri-implant sulcular fluid (PISF) might be useful for evaluating the condition of peri-implant tissues and monitoring a development of peri-implant inflammation, including both mucositis and peri-implantitis. Hence, in this study, we decided to evaluate the level of MMP-8 in PISF obtained from patients without clinical symptoms of mucositis or peri-implantitis and compare it with MMP-8 level in gingival crevicular fluid (GCF) obtained from patients with healthy periodontium and those with varying severity of periodontitis. A total of 189 subjects were included in the study, and GCF/PISF samples were analysed for MMP-8 level by ELISA test. We documented that MMP-8 level in PISF obtained from patients without symptoms of mucositis or peri-implantitis was significantly higher not only than in GCF of periodontally healthy patients but also, which seems to be very interesting, than in GCF of patients with varying degrees of periodontal inflammation, consistent with earlier studies. Our observation might imply that monitoring of MMP-8 level in PISF could help to diagnose mucositis/peri-implantitis in an early stage, prior to clinical manifestations, which may allow for quick start of appropriate therapy.

Matrix Metalloproteinases as Regulators of Periodontal Inflammation.

Periodontitis are infectious diseases characterized by immune-mediated destruction of periodontal supporting tissues and tooth loss. Matrix metalloproteinases (MMPs) are key proteases involved in destructive periodontal diseases. The study and interest in MMP has been fuelled by emerging evidence demonstrating the broad spectrum of molecules that can be cleaved by them and the myriad of biological processes that they can potentially regulate. The huge complexity of MMP functions within the 'protease web' is crucial for many physiologic and pathologic processes, including immunity, inflammation, bone resorption, and wound healing. Evidence points out that MMPs assemble in activation cascades and besides their classical extracellular matrix substrates, they cleave several signalling molecules-such as cytokines, chemokines, and growth factors, among others-regulating their biological functions and/or bioavailability during periodontal diseases. In this review, we provide an overview of emerging evidence of MMPs as regulators of periodontal inflammation.

Periodontal status and serum creatine kinase levels among young soccer players: A preliminary report.

OBJECTIVES: It is hypothesized that soccer players with periodontal disease exhibit raised serum creatine kinase (CK) levels as compared to those without periodontal disease. We assessed the clinical gingival status and serum CK levels among young soccer players. MATERIALS AND METHODS: Demographic data were collected through a structured questionnaire. Full mouth bleeding on probing (BOP) and probing pocket depth (PPD) were assessed. Blood samples (4 mL) were collected for measurement of serum CK levels. All blood samples were collected from a vein in the antecubital region. Total CK activities in serum were determined with an optimized spectrophotometric method. Statistical analysis was performed using one-way analysis of variance, and P< 0.05 was considered statistically significant. RESULTS: Twenty-seven male soccer players volunteered to participate in the present study. The mean age of the participants in Groups 1 (n = 14) and 2 (n = 13) were 18.2 ± 2.3 years and 19.1 ± 0.6 years, respectively. Mean scores of BOP were significantly higher among individuals in Group 2 (56.8%) compared with individuals in Group 1 (19.4%) (P < 0.001). Mean scores of PPD ≥4 mm were significantly higher among subjects in Group 2 (12.1%) as compared to individuals in Group 1 (0.8%) (P < 0.001). Levels of CK were significantly higher among individuals in Group 2 (292.7 U/L) as compared to those in Group 1 (52.3 U/L) (P < 0.01). CONCLUSION: Increased BOP and PPD are associated with increased serum CK levels in young soccer players.

Rapid assessment of salivary MMP-8 and periodontal disease using lateral flow immunoassay.

OBJECTIVE: The objective of this study was to determine the efficacy of a novel point-of-care immunoflow device (POCID) for detecting matrix metalloproteinase (MMP)-8 concentrations in oral fluids in comparison with a gold standard laboratory-based immunoassay. METHODS: Oral rinse fluid and whole expectorated saliva samples were collected from 41 participants clinically classified as periodontally healthy or diseased. Samples were analyzed for MMP-8 by Luminex immunoassay and POCID. Photographed POCID results were assessed by optical scan and visually by two examiners. Data were analyzed by Pearson's correlation and receiver-operating characteristics. RESULTS: MMP-8 was readily detected by the POCID, and concentrations correlated well with Luminex for both saliva and rinse fluids (r = 0.57-0.93). Thresholds that distinguished periodontitis from health were delineated from both the optical scans and visual reads of the POCID (sensitivity: 0.7-0.9, specificity: 0.5-0.7; P < 0.05). CONCLUSIONS: Performance of this POCID for detecting MMP-8 in oral rinse fluid or saliva was excellent. These findings help demonstrate the utility of salivary biomarkers for distinguishing periodontal disease from health using a rapid point-of-care approach.

Potential Role of Reversion-Inducing Cysteine-Rich Protein with Kazal Motifs (RECK) in Regulation of Matrix Metalloproteinases (MMPs) Expression in Periodontal Diseases.

Periodontal diseases are characterized by pathological destruction of extracellular matrix (ECM) of periodontal tissues. Matrix metalloproteinases (MMPs) are a significant part of the degradation of ECM. However, the regulation of MMPs expression level in periodontal diseases is as yet undetermined. RECK (reversion-inducing cysteine-rich protein with Kazal motifs), a novel membrane-anchored inhibitor of MMPs, could regulate the expressions of MMP-2, 9 and MT1-MMP as a cell surface-signaling molecule. Thus, we propose that RECK may play an important role in regulating MMPs in the ECM degradation of periodontal diseases. The RECK/MMPs signaling pathway could provide a new approach for prevention and treatment of RECK in periodontal diseases by blocking MMPs.

Marcadores de remodelamiento óseo en pacientes con enfermedad periodontal / Bone remodeling biomarkers in patients with periodontal disease

La Fosfatasa Alcalina Ósea (FAO) es una isoforma de la Fosfatasa Alcalina (FAL). La medición de su actividad en saliva es una medida indirecta del proceso de formación ósea, más sensible y específica que la FAL. La catepsina K es la principal colagenasa del proceso de resorción ósea, es capaz de degradar al colágeno tipo I en varios sitios dando lugar a pequeños péptidos N- y C- terminales. El telopéptido C-terminal (CTx) es el marcador más sensible y específico en el aumento de la resorción ósea, ya que el colágeno tipo I constituye más del 90 por ciento de la matriz orgánica del hueso...

Analysis of matrix metalloproteinases, especially MMP-8, in gingival creviclular fluid, mouthrinse and saliva for monitoring periodontal diseases.

Matrix metalloproteinase-8 is a promising candidate biomarker for oral fluid (gingival crevicular fluid, peri-implant sulcular fluid and saliva) and mouthrinse chair-side/point-of-care diagnostics to predict, diagnose and determine the progressive phases of episodic periodontitis and peri-implantitis, as well as to monitor the treatments and medications. Matrix metalloproteinase-8 can be used alone or together with interleukin-1beta and Porphyromonas gingivalis to calculate cumulative risk score at the subject level as a successful diagnostic tool, especially in large-scale public health surveys, in which a thorough periodontal examination is not feasible.

Periodontal microbiota and phospholipases: the Oral Infections and Vascular Disease Epidemiology Study (INVEST).

OBJECTIVE: Periodontal infections have been linked to cardiovascular disease, including atherosclerosis, and systemic inflammation has been proposed as a possible mediator. Secretory phospholipase A2 (s-PLA2) and Lipoprotein-associated PLA2 (Lp-PLA2) are inflammatory enzymes associated with atherosclerosis. No data are available on the association between oral microbiota and PLA2s. We studied whether a relationship exists between periodontal microbiota and the activities of these enzymes. METHODS: The Oral Infection and Vascular Disease Epidemiology Study (INVEST) collected subgingival biofilms and serum samples from 593 dentate men and women (age 68.7 ± 8.6 years). 4561 biofilm samples were collected in the two most posterior teeth of each quadrant (average 7/participant) for quantitative assessment of 11 bacterial species using DNA-DNA checkerboard hybridization. Mean concentration of s-PLA2 and activities of s-PLA2 and Lp-PLA2 were regressed on tertiles of etiologic dominance (ED). ED is defined as the level of presumed periodontopathic species/combined level of all eleven species measured, and represents the relative abundance of periodontopathic organisms. Analyses were adjusted for age, sex, race/ethnicity, education, smoking, BMI, diabetes, LDL cholesterol and HDL cholesterol, and systolic blood pressure. RESULTS: Higher levels of s-PLA2 activity were observed across increasing tertiles of etiologic dominance (0.66 ± 0.04 nmol ml(-1) min(-1), 0.73 ± 0.04 nmol ml(-1) min(-1), 0.89 ± 0.04 nmol ml-1 min-1; p < 0.001), with also a trend of association between Lp-PLA2 activity and ED (p = 0.07), while s-PLA2 concentration was unrelated to ED. CONCLUSION: Increasingly greater s-PLA2 activity at higher tertiles of etiologic dominance may provide a mechanistic explanatory link of the relationship between periodontal microbiota and vascular diseases. Additional studies investigating the role of s-PLA2 are needed.

Do salivary and serum collagenases have a role in an association between obstructive sleep apnea syndrome and periodontal disease? A preliminary case-control study.

OBJECTIVES: Despite increasing evidence for an association of obstructive sleep apnea syndrome (OSAS) and periodontal disease, the pathophysiological linking mechanisms remain unclear. This study aims to evaluate the salivary and serum matrix metalloproteinase-2, -8, -9 (MMP-2, -8, -9), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), myeloperoxidase (MPO), neutrophil elastase (NE), neutrophil gelatinase-associated lipocalin (NGAL), as well as degree of activation of MMP-2, -9 of patients with and without OSAS. DESIGN: A total of 50 individuals were included in the study. There were 13, 17 and 20 individuals, respectively in the control (non-OSAS) group, mild-to-moderate OSAS and severe OSAS groups. Saliva, serum samples and clinical periodontal parameters were collected. Biofluid samples were analysed by immunofluorometric assay (IFMA), enzyme-linked immunosorbent assay (ELISA), western immunoblotting and gelatine zymography. Statistical analyses were performed using D'Agostino-Pearson omnibus normality test, Kruskal-Wallis test and Spearman rho rank correlation analysis. RESULTS: There were no statistically significant differences in clinical periodontal parameters between the study groups. Salivary NE and proMMP-2 levels were significantly lower in the OSAS groups than the control group (p<0.05). Serum proMMP-9 concentration and the degree of MMP-9 activation in saliva were significantly lower in the severe OSAS group than the control group (p<0.05). There were significant correlations between salivary and serum proMMP-9 and -2 concentrations (p<0.05). Serum proMMP-2, NE and salivary proMMP-9 and -2 negatively correlated with indicators of OSAS severity (p<0.05). CONCLUSIONS: The present findings do not support a pathophysiological link between the severity of OSAS and clinical periodontal status via neutrophil enzymes or MMPs.

Cyclooxygenase-2 and matrix metalloproteinase-9 expressions correlate with tissue inflammation degree in periodontal disease.

INTRODUCTION: Cyclooxygenase-2 (Cox-2) and matrix metalloproteinase-9 (MMP-9) have synergistic effects in the degradation of the extra-cellular matrix. OBJECTIVE: The aim of our study was to correlate the intensity of inflammation with MMP-9 and Cox-2 expression in the periodontal tissue of patients with chronic inflammatory disease (gingivitis and chronic periodontitis) in order to determine the role of these two biomarkers in the progression of periodontal disease. MATERIALS AND METHODS: To conduct this study we analyzed the gingival biopsies taken from patients clinically divided into three study groups: Group I (control): Patients free of periodontal disease (seven biopsies); Group II: Patients with gingivitis (10 biopsies); Group III: Patients with chronic periodontitis (10 biopsies). In these three groups, we graded the intensity of inflammation in the lamina propria and the immunohistochemical expression of MMP-9 and Cox-2. RESULTS: The presence of a large number of inflammatory cells in the lamina propria in patients with gingivitis or chronic periodontitis (Groups II and III) correlated with the clinically diagnosed inflammation of the gingival tissue. The expression of MMP-9 was higher in patients with chronic periodontitis than in those with gingivitis, showing a trend towards statistical significance (p=0.07, Mann-Whitney U-test). The expression of Cox-2 in periodontitis was also higher compared to gingivitis (p=0.05, Mann-Whitney U-test) and to controls (p=0.001, Mann-Whitney U-test).The inflammation score could be positively correlated to the MMP-9 and Cox-2 expression scores at the overall study group, but not separately on gingivitis and periodontitis patients. CONCLUSIONS: The presence of an intensive inflammatory infiltrate is characteristic both for periodontitis and gingivitis. MMP-9 and Cox-2 show higher expression in periodontitis, than in gingivitis and healthy controls, but MMP-9 and Cox-2 expression scores cannot be directly correlated to the grade of inflammatory infiltrate in the two different disease entities. As biomarkers of chronic inflammation activity, angiogenesis, and degradation of the extracellular matrix, MMP-9 and Cox-2 can be used in clinical practice for the detection of patients with chronic periodontitis risk, at whom treatment with Cox-2 and MMP-9 inhibitors may be considered.

Serum γ-glutamyltransferase level is associated with periodontal disease independent of drinking habits in Japanese adults.

Background Non-alcoholic fatty liver disease is considered a hepatic manifestation of metabolic syndrome. Periodontal disease is a mild chronic inflammatory disease with systemic effects, and many studies have indicated an association between metabolic syndrome and periodontitis. In the present study, we investigated the relationship between periodontitis and liver biochemical parameters according to alcohol drinking habits through a cross-sectional study based on data from Japanese people in occupational settings. Material and Methods The subjects were 1510 employees (1218 males, 292 females, mean age 50.4 years) who underwent dental and medical checkups in 2012. Associations between the presence of periodontal pockets and serum levels of liver biochemical parameters were assessed. Results Alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT) levels were higher in subjects with than without periodontal pockets. Multiple logistic regression analysis (adjusting for age, gender, cigarette smoking, and alcohol drinking habits, and components of metabolic syndrome) with GGT or ALT as the dependent variable revealed that there was a significant association between periodontal pockets and GGT (odds ratio, OR=1.48), but not ALT. Similar associations were observed when an analysis was performed according to the presence or absence of alcohol drinking habits; the OR was higher in subjects without (OR=1.84) than with drinking habits (OR=1.41). Conclusions The presence of periodontal pockets was associated with serum levels of GGT, a liver biochemical parameter, in Japanese adults with no drinking habit, suggesting that periodontal disease is associated with liver function, independent of alcohol ingestion.

Structural significance of the ß1K396 residue found in the Porphyromonas gingivalis sialidase ß-propeller domain: a computational study with implications for novel therapeutics against periodontal disease.

Porphyromonas gingivalis sialidase activity is associated with virulence and initiated by sialic acid (SA) binding to the ß-propeller domain (BPD). Sialidase BPD is structurally conserved in various bacterial species and the protein binding interfaces have the tendency to form salt bridges, whereas uncommitted charged residues may affect binding and protein structure. However, it is not clear whether the sialidase BPD of varying strains of the same bacterial species differ, particularly with regards to salt bridge formation. Here, we determined the P. gingivalis ATCC 33277 and W50 sialidase homology models and sialidase activities, while the putative salt bridge residues found in the sialidase BPDs were compared. We established that both ATCC 33277 and W50 have different sialidase homology models and activities, whereas, the BPD (ß1-6) is structurally conserved with most salt bridge-forming residues following a common orientation. Moreover, ß2D444-ß6K338 distance measurement in ATCC 33277 (5.99 Å) and W50 (3.09 Å) differ, while ß1K396A substitution alters the ß2D444-ß6K338 distance measurements in ATCC 33277 (3.09 Å) and W50 (3.01 Å) consequentially affecting each model. P. gingivalis plays a major role in periodontitis induction and its virulence is greatly influenced by the sialidase enzyme wherein the sialidase BPD is highly conserved. Our results suggest that alterations in the salt bridge formation within the BPD interface may affect the P. gingivalis sialidase structure. This would imply that disrupting the salt bridge formation within the P. gingivalis sialidase BPD could serve as a potential therapeutic strategy for the treatment of P. gingivalis-related periodontitis.

Determination of lysosomal exoglycosidases in human saliva.

BACKGROUND: Currently we observe a growing interest in human saliva as a non-invasive material for diagnosis and monitoring of general and oral diseases. METHODS: The aim of our study was adaptation of the Marciniak et al. (Marciniak J, Zalewska A, Popko J, Zwierz K, 2006, Clin Chem Lab Med 44: 933-937) method for determination of HEX and GLU activity in synovial fluid, and for determination of: HEX and GLU, as well as MAN, GAL, and FUC activity in human saliva. RESULTS: Under optimal conditions, 10 µl of saliva for HEX, and 30 µl for GLU, MAN, GAL and FUC, were sufficient for determination of human salivary exoglycosidases activity with variation coefficient ranging from 0.89 for GLU to 0.99 for GAL. CONCLUSION: The adapted method for exoglycosidases activity determination in human saliva is sufficiently sensitive and precise to use in clinical diagnosis.

The plasminogen activation system in periodontal tissue (Review).

The plasminogen activation system (PAS) plays an essential role in tissue proteolysis in physiological and pathological processes. Periodontitis is a chronic infection associated with increased proteolysis driven by plasminogen activation. In this comprehensive review, we summarise the effects of PAS in wound healing, tissue remodelling, inflammation, bacterial infection, and in the initiation and progression of periodontal disease. Specifically, we discuss the role of plasminogen activators (PAs), including urokinase PA (uPA), tissue-type PA (tPA), PA inhibitor type 1 (PAI-1) and 2 (PAI-2) and activated plasminogen in periodontal tissue, where their concentrations can reach much higher values than those found in other parts of the body. We also discuss whether PA deficiencies can have effects on periodontal tissue. We conclude that in periodontal disease, PAS is unbalanced and equalizing its function can improve the clinical periodontal tissue condition.

Matrix metalloproteinases and periodontal diseases.

Periodontitis is a chronic inflammatory disorder characterized a complex interaction between periodontopathic bacteria and the host inflammatory response resulting in release of pro-inflammatory cytokines leading to the destruction of periodontal tissues and alveolar bone. One of the important host factors involved in periodontal diseases is matrix metalloproteinases (MMPs), which is responsible for collagen and extracellular matrix (ECM) degradation of the periodontal tissues. MMPs comprise a family of around 25 members broadly categorized into six groups, which are involved in various physiological and pathological conditions. The activities of MMP are generally balanced by endogenous inhibitors such as tissue inhibitors of metalloproteinase (TIMP), and any imbalance between MMP and TIMP levels plays an important role in the disease progression. Assessment of MMP in tissues, GCF, and saliva may serve as an important biomarker in diagnosis of periodontal diseases and also for prognostic follow-up. Targeted therapy aimed at reducing effects of MMP may serve as a useful adjunct for treatment of periodontitis. This review provides an overview of MMP and its role in various physiological and pathological conditions with emphasis on its association with periodontal diseases. A note on its inhibitors and therapeutic importance is also provided.