Saliva and serum biomarkers in periodontitis and coronary artery disease.

AIM: Matrix metalloproteinase (MMP)-8, MMP-9, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and myeloperoxidase (MPO) participate in extracellular matrix breakdown both in periodontium and atherosclerotic plaques. We investigated the diagnostic value of serum and saliva biomarkers in periodontitis and acute coronary syndrome (ACS). MATERIALS AND METHODS: The population was PAROGENE (n = 481), a random cohort of patients with an indication for coronary angiography. All patients underwent a clinical and radiographic oral examination. Groups consisting of periodontitis versus non-periodontitis, and ACS versus non-ACS patients were compared. RESULTS: Saliva MMP-8, MMP-9 and MPO provided significant area-under-curve (AUC) values for periodontitis, 0.69 (<0.001), 0.66 (<0.001) and 0.68 (<0.001), respectively. Serum MMP-8, MMP-9 and MPO levels distinguished ACS from non-ACS patients with AUCs of 0.73 (<0.001), 0.58 (0.03) and 0.68 (<0.001), respectively. Periodontitis confounded the use of serum MMP-9 in diagnostics of ACS. Cardiac status complicated the use of saliva TIMP-1 in periodontal diagnostics. Saliva biomarkers could not be used in ACS diagnosis, and serum biomarkers were not useful in diagnosis of periodontitis. CONCLUSIONS: MMP-8, MMP-9, TIMP-1and MPO are valuable biomarkers for both ACS and periodontitis, but the selection of sample material is crucial; serum is suitable for ACS and saliva for periodontal diagnostic aid.

Aggregatibacter actinomycetemcomitans serotypes associate with periodontal and coronary artery disease status.

AIM: We investigated the association between the Aggregatibacter actinomycetemcomitans serotypes, periodontal status and coronary artery disease (CAD). MATERIALS AND METHODS: The study population included 497 patients who underwent coronary angiography, and clinical oral examination. Quantitative polymerase chain reaction assays were designed to identify the serotypes from saliva samples. RESULTS: Aggregatibacter actinomycetemcomitans serotype frequencies were as follows: serotype "c" 35.7%, "b" 28.6%, "a" 26.2%, "e" 7.1%, "d" 2.4% and "f" 0%. The subjects with a detectable serotype had less teeth and higher bleeding on probing than those with no serotype. Serotypes "b" and "c" associated with periodontal probing depths and periodontal inflammatory burden. The saliva and subgingival bacterium quantities and serum antibody levels against A. actinomycetemcomitans were highest in patients harbouring serotype "c." Serotypes "b" and "c" were most frequent (59.3%) in patients with CAD (p = .040), and they associated with the risk of stable CAD with an odds ratio of 2.67 (95% confidence interval 1.06-7.44). Also, the severity of CAD (p = .018) associated with serotypes "b" and "c." CONCLUSIONS: Aggregatibacter actinomycetemcomitans serotypes "b" and "c" associate with both periodontal and CAD status. Detectable serotypes associate with the quantity and the serology of the bacterium emphasizing both local and systemic effect of the A. actinomycetemcomitans serotypes.

Lipopolysaccharide, a possible molecular mediator between periodontitis and coronary artery disease.

AIM: We aimed to study how lipopolysaccharide (LPS) in saliva and serum associates with each other, periodontal microbial burden, periodontitis and coronary artery disease (CAD). MATERIALS AND METHODS: The used Parogene cohort comprised N = 505 Finnish adults. Coronary diagnosis was acquired by coronary angiography, and the main outcomes were as follows: no significant CAD (n = 123), stable CAD (n = 184) and acute coronary syndrome (n = 169). Periodontitis was defined according to clinical and radiographic examinations. Levels for 75 strains of subgingival bacteria were determined by checkerboard DNA-DNA hybridization. Saliva and serum LPS activity was analysed by Limulus amebocyte lysate assay. RESULTS: The level of 11 bacterial strains, which were mainly oral and respiratory Gram-negative species, associated with salivary LPS levels in an age- and gender-adjusted linear regression. A total of 4.9% of the serum LPS, that is endotoxemia, variation was explainable by saliva LPS among patients with periodontitis (n = 247, R2  = .049, Pearson's r = .222, p < .001). Endotoxemia associated with stable CAD in a confounder adjusted multinomial logistic regression model (OR 1.99, 95% CI 1.04-3.81, p = .039, 3rd tertile). CONCLUSIONS: In particular in periodontitis patients, subgingival microbial burden contributes to endotoxemia. LPS is a possible molecular mediator between periodontitis and CAD.

Genetic variation on the BAT1-NFKBIL1-LTA region of major histocompatibility complex class III associates with periodontitis.

Periodontitis is a chronic inflammatory disease with a multifactorial etiology. We investigated whether human major histocompatibility complex (MHC) polymorphisms (6p21.3) are associated with periodontal parameters. Parogene 1 population samples (n = 169) were analyzed with 13,245 single nucleotide polymorphisms (SNPs) of the MHC region. Eighteen selected SNPs (P ≤ 0.001) were replicated in Parogene 2 population samples (n = 339) and the Health 2000 Survey (n = 1,420). All subjects had a detailed clinical and radiographic oral health examination. Serum lymphotoxin-α (LTA) concentrations were measured in the Parogene populations, and the protein was detected in inflamed periodontal tissue. In the Parogene 1 population, 10 SNPs were associated with periodontal parameters. The strongest associations emerged from the parameters bleeding on probing (BOP) and a probing pocket depth (PPD) of ≥6 mm with the genes BAT1, NFKBIL1, and LTA. Six SNPs, rs11796, rs3130059, rs2239527, rs2071591, rs909253, and rs1041981 (r(2), ≥0.92), constituted a risk haplotype. In the Parogene 1 population, the haplotype had the strongest association with the parameter BOP, a PPD of ≥6 mm, and severe periodontitis with odds ratios (95% confidence intervals) of 2.63 (2.21 to 3.20), 2.90 (2.37 to 3.52), and 3.10 (1.63 to 5.98), respectively. These results were replicated in the other two populations. High serum LTA concentrations in the Parogene population were associated with the periodontitis risk alleles of the LTA SNPs (rs909253 and rs1041981) of the haplotype. In addition, the protein was expressed in inflamed gingival connective tissue. We identified a novel BAT1-NFKBIL1-LTA haplotype as a significant contributor to the risk of periodontitis. The genetic polymorphisms in the MHC class III region may be functionally important in periodontitis susceptibility.

Salivary biomarkers of bacterial burden, inflammatory response, and tissue destruction in periodontitis.

AIM: Chronic periodontitis has an episodic and multifactorial character, with fluctuations in bacterial burden, inflammatory response, and tissue destruction. We investigated the association of selected salivary biomarkers with periodontal parameters and validated the use of a novel salivary diagnostic approach, the cumulative risk score (CRS), in detection of periodontitis in subjects with angiographically verified coronary artery disease diagnosis. MATERIALS AND METHODS: The concentrations of matrix metalloproteinase (MMP)-8, interleukin (IL)-1ß, and Porphyromonas gingivalis were analysed from saliva of 493 subjects. The subjects participated in a detailed clinical and radiographic oral examination. The CRS index, combining the three salivary biomarkers, was calculated for each subject. RESULTS: High salivary concentrations of MMP-8, IL-1ß, and P. gingivalis were associated with deepened periodontal pockets and alveolar bone loss, and MMP-8 and IL-1ß with bleeding on probing. The CRS index had a stronger association with moderate to severe periodontitis (OR 6.13; 95% CI 3.11-12.09) than any of the markers alone. CONCLUSIONS: Salivary concentrations of MMP-8, IL-1ß, and P. gingivalis are associated with various clinical and radiographic measures of periodontitis. The CRS index, combining the three salivary biomarkers, is associated with periodontitis more strongly than any of the markers alone regardless of the coronary artery disease status of the patients.

Subgingival Aggregatibacter actinomycetemcomitans associates with the risk of coronary artery disease.

AIM: We investigated the association between angiographically verified coronary artery disease (CAD) and subgingival Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. MATERIALS AND METHODS: The cross-sectional study population (n = 445) comprised 171 (38.4%) patients with Stable CAD, 158 (35.5%) with acute coronary syndrome (ACS) and 116 (26.1%) with no significant CAD (No CAD). All patients participated in clinical and radiological oral health examinations. Pooled subgingival bacterial samples were analysed by checkerboard DNA-DNA hybridization assays. RESULTS: In all study groups, the presence of P. gingivalis, T. forsythia and T. denticola indicated a significant (p ≤ 0.001) linear association with the extent of alveolar bone loss (ABL), but A. actinomycetemcomitans did not (p = 0.074). With a threshold level of bacterial cells 1 × 10(5) A. actinomycetemcomitans was significantly more prevalent in the Stable CAD group (42.1%) compared to the No CAD group (30.2%) (p = 0.040). In a multi-adjusted logistic regression analysis using this threshold, A. actinomycetemcomitans positivity associated with Stable CAD (OR 1.83, 95% CI 1.00-3.35, p = 0.049), but its level or levels of other bacteria did not. CONCLUSIONS: The presence of subgingival A. actinomycetemcomitans associates with an almost twofold risk of Stable CAD independently of alveolar bone loss.

Periodontitis is associated with angiographically verified coronary artery disease.

INTRODUCTION: We investigated the association of periodontitis and severity of coronary artery disease (CAD) as verified using coronary angiography. MATERIAL AND METHODS: Participants were recruited among those attending coronary angiography at Helsinki University Central Hospital, Finland, in 2007 and 2008. Detailed clinical periodontal examination [number of teeth, bleeding on probing, periodontal probing depth (PPD)] and oral panoramic radiographs [alveolar bone loss (ABL), angular bone defects] were performed. RESULTS: Of 506 patients, 123 (24.3%) had no significant CAD, whereas 184 (36.4%) had stable CAD and 169 (33.4%) acute coronary syndrome (ACS). Both stable CAD and ACS were associated with 8-17 missing teeth with ORs 4.33 (1.61-11.7, p = 0.020) and 5.24 (1.90-14.5, p = 0.014), and more than seven teeth with PPD ≥6 mm with ORs 2.44 (1.01-6.07, p = 0.049) and 2.75 (1.16-6.53, p = 0.022) respectively. Severe ABL was associated with ACS with an OR 5.39 (1.23-23.6, p = 0.025). Number of stenosed arteries was linearly associated with ABL (p for trend <0.001), number of missing teeth (p < 0.001), and pockets with probing depth ≥6 mm (p = 0.033). CONCLUSIONS: Compared with patients with no significant stenosis, poor periodontal health including missing teeth, periodontal inflammation, and bone loss is associated with angiographically verified coronary artery narrowing in patients with stable CAD or ACS.

Smoking confounds the periodontal diagnostics using saliva biomarkers.

BACKGROUND: Smoking is a risk factor for periodontal disease because of its complex impact on the inflammatory response in the periodontium. We investigated the effect of smoking on salivary periodontal biomarkers, matrix metalloproteinase (MMP)-8, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and myeloperoxidase (MPO). METHODS: Saliva biomarkers were analyzed in the Parogene population (n = 480) comprising a random cohort of patients that have undergone coronary angiography and oral examination. The effect of time since cessation and pack years of smoking on biomarkers were investigated. RESULTS: Saliva MMP-8, MMP-9, TIMP-1, and MPO concentrations distinguished periodontitis patients significantly from patients without periodontitis. When the time since cessation was considered, the area-under-the-curve values (p-value) for periodontitis were 0.76 (<0.001), 0.74 (<0.001), 0.70 (<0.001), and 0.76 (<0.001), respectively. Adding information about smoking habits in the models improved slightly the sensitivities of all biomarkers. In logistic regression model saliva, MMP-8 was mainly affected by pack years of smoking, whereas saliva MMP-9, TIMP-1, and MPO were mostly affected by time since cessation, especially if smoking currently or quit recently (<1 year ago). CONCLUSION: Smoking confounds the salivary diagnostics of periodontitis and should be considered when interpreting the results obtained by potential diagnostic tests.

Immunologic burden links periodontitis to acute coronary syndrome.

BACKGROUND AND AIMS: Periodontitis, a common polymicrobial inflammatory disease in the tooth supporting tissues, is a risk factor for coronary artery disease. One of the proposed underlying mechanisms is the systemic immune response to periodontal infection. We studied how serum antibodies against seven periodontal pathogens and their subgingival levels associate with each other, periodontitis, and coronary artery disease. METHODS: The Parogene cohort included 505 Finnish patients (mean age 63 y) who underwent coronary angiography, and clinical and radiographic oral examinations. Coronary diagnosis was defined as no significant coronary artery disease (<50% stenosis, n = 152), stable coronary artery disease (≥50% stenosis, n = 184) and acute coronary syndrome (n = 169). Levels of subgingival Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, Tannerella forsythia, Campylobacter rectus, and Fusobacterium nucleatum were determined by checkerboard DNA-DNA hybridization. Serum antibody (IgA/IgG) levels were analyzed with enzyme-linked immunosorbent assay (ELISA). Aggregate IgA/IgG burdens were calculated by summing and standardizing the serum antibody levels. RESULTS: Patients with active periodontitis were characterized by higher levels of subgingival bacteria and corresponding IgA/IgG response. Quartiles 2-4 of serum IgA/IgG burden indicated higher risk for acute coronary syndrome (OR 1.84, 95%CI 1.01-3.35 for IgA; OR 1.87, 95%CI 1.01-3.46 for IgG) independently of established cardiovascular risk factors, body mass index, number of teeth, subgingival bacterial levels and periodontal diagnosis. CONCLUSIONS: Our findings support the hypothesis that the association between periodontitis and cardiovascular diseases is partly mediated by the immunologic response for periodontal pathogens.

Salivary biomarkers in association with periodontal parameters and the periodontitis risk haplotype.

Genetic factors play a role in periodontitis. Here we examined whether the risk haplotype of MHC class III region BAT1-NFKBIL1-LTA and lymphotoxin-α polymorphisms associate with salivary biomarkers of periodontal disease. A total of 455 individuals with detailed clinical and radiographic periodontal health data were included in the study. A 610 K genotyping chip and a Sequenom platform were used in genotyping analyses. Phospholipid transfer protein activity, concentrations of lymphotoxin-α, IL-8 and myeloperoxidase, and a cumulative risk score (combining Porphyromonas gingivalis, IL-1ß and matrix metalloproteinase-8) were examined in saliva samples. Elevated IL-8 and myeloperoxidase concentrations and cumulative risk scores associated with advanced tooth loss, deepened periodontal pockets and signs of periodontal inflammation. In multiple logistic regression models adjusted for periodontal parameters and risk factors, myeloperoxidase concentration (odds ratio (OR); 1.37, P = 0.007) associated with increased odds for having the risk haplotype and lymphotoxin-α concentration with its genetic variants rs2857708, rs2009658 and rs2844482. In conclusion, salivary levels of IL-8, myeloperoxidase and cumulative risk scores associate with periodontal inflammation and tissue destruction, while those of myeloperoxidase and lymphotoxin-α associate with genetic factors as well.

Is periodontal infection behind the failure of antibiotics to prevent coronary events?

The use of antimicrobial agents to prevent coronary events is under debate. They have reduced cardiovascular events in some studies, but in others, their effect has not been distinguishable from that of placebo. In addition to Chlamydophila (Chlamydia) pneumoniae as a target pathogen, very few other microbes or infections have been targeted, although an association for instance between cardiovascular disease and periodontitis has been established. In our recent pilot study, long-term clarithromycin treatment reduces recurrent cardiovascular events in subjects without periodontitis, but in subjects with periodontitis, fails to show any effect. As a background infection, periodontitis may overpower the beneficial effects of antibiotics. This paper presents the hypothesis that periodontitis is behind the failure of antibiotics to prevent coronary events. We discuss the systemic effects of periodontal infection and consider studies to test our hypothesis, which offers a novel viewpoint for discussion of antibiotics in coronary-disease prevention.

Clarithromycin reduces recurrent cardiovascular events in subjects without periodontitis.

Inflammation leading to acute coronary syndrome may be triggered by bacteria causing periodontal infection. We investigated if recurrence of cardiovascular events in unstable coronary patients are associated with periodontitis or microbiological/serological markers of it. Periodontitis-related parameters of 141 patients with acute non-Q-wave infarction or unstable angina pectoris, who participated in a double-blind, placebo-controlled study with clarithromycin for 3 months, were adjusted to the occurrence of a recurrent cardiovascular event during a follow-up period (average 519 days). In the age group under 65 years the patients with periodontitis had a univariate odds ratios (OR) 95% confidence intervals (95% CI) of 5.0 (1.02-24.55) for a recurrent cardiovascular event in comparison with patients without periodontitis. Dental status correlated positively with serum lipopolysaccharide concentrations and combined IgG antibody response to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. The end point frequency did not differ between clarithromycin and placebo groups in bacterium-positive, seropositive, or periodontitis patients. Fewer end points in clarithromycin group were seen in bacterium-negative, seronegative, edentulous, and non-periodontitis patients. Periodontitis and edentulousness are associated with recurrent cardiovascular events, especially in younger patients. Long-term clarithromycin therapy seems to be beneficial in prevention of recurrent cardiovascular events in non-periodontitis but not in periodontitis patients.

Quantitative PCR analysis of salivary pathogen burden in periodontitis.

Our aim was to investigate the value of salivary concentrations of four major periodontal pathogens and their combination in diagnostics of periodontitis. The Parogene study included 462 dentate subjects (mean age 62.9 ± 9.2 years) with coronary artery disease (CAD) diagnosis who underwent an extensive clinical and radiographic oral examination. Salivary levels of four major periodontal bacteria were measured by quantitative real-time PCR (qPCR). Median salivary concentrations of Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia, as well as the sum of the concentrations of the four bacteria, were higher in subjects with moderate to severe periodontitis compared to subjects with no to mild periodontitis. Median salivary Aggregatibacter actinomycetemcomitans concentrations did not differ significantly between the subjects with no to mild periodontitis and subjects with moderate to severe periodontitis. In logistic regression analysis adjusted for age, gender, diabetes, and the number of teeth and implants, high salivary concentrations of P. gingivalis, T. forsythia, and P. intermedia were significantly associated with moderate to severe periodontitis. When looking at different clinical and radiographic parameters of periodontitis, high concentrations of P. gingivalis and T. forsythia were significantly associated with the number of 4-5 mm periodontal pockets, ≥6 mm pockets, and alveolar bone loss (ABL). High level of T. forsythia was associated also with bleeding on probing (BOP). The combination of the four bacteria, i.e., the bacterial burden index, was associated with moderate to severe periodontitis with an odds ratio (OR) of 2.40 (95% CI 1.39-4.13). When A. actinomycetemcomitans was excluded from the combination of the bacteria, the OR was improved to 2.61 (95% CI 1.51-4.52). The highest OR 3.59 (95% CI 1.94-6.63) was achieved when P. intermedia was further excluded from the combination and only the levels of P. gingivalis and T. forsythia were used. Salivary diagnostics of periodontitis has potential especially in large-scale population studies and health promotion. The cumulative strategy appears to be useful in the analysis of salivary bacteria as markers of periodontitis.

Subgingival bacterial burden in relation to clinical and radiographic periodontal parameters.

BACKGROUND: This cross-sectional study characterizes the association between subgingival bacterial profile and periodontal parameters in patients assigned to coronary angiography because of cardiologic problems, which may affect the oral microbiota. METHODS: Pooled subgingival bacterial samples were collected from 477 dentate individuals during the oral examinations, along with periodontal probing depth (PD) and assessments of bleeding on probing (BOP) and radiographic alveolar bone loss (ABL). The checkerboard DNA-DNA hybridization assay was used to determine the levels of 29 oral bacteria, which were divided into three bacterial complexes. RESULTS: All bacterial combinations from the etiologic bacterial group and each species from the red complex were significantly associated (P <0.001) with grade of ABL. The prevalence of the etiologic bacterial group and the level of each species were also associated strongly with the proportion of sites with PD 4 to 5 mm and ≥ 6 mm, BOP, and ABL, except Aggregatibacter actinomycetemcomitans. Levels of Gram-negative oral bacteria correlated significantly with those of Gram-positive species (r = 0.840, P <0.001). In multiple logistic regression analysis, the prevalence of the etiologic bacterial group, levels of Gram-negative bacteria and Treponema denticola, and the prevalence of Porphyromonas gingivalis and T. denticola associated significantly with ABL, whereas other bacterial complexes and levels of Gram-positive species did not. CONCLUSIONS: Although levels of Gram-negative and -positive species paralleled periodontal parameters, only the species considered etiologic were associated with ABL.

A common periodontal pathogen has an adverse association with both acute and stable coronary artery disease.

OBJECTIVE: The aim of this study was to investigate the association between angiographically verified coronary artery disease (CAD) and salivary levels of four major periodontal pathogens. METHODS: The study population (n = 492) was composed of 179 (36.4%) patients with stable CAD, 166 (33.7%) with acute coronary syndrome (ACS), and 119 (24.2%) showing no pathological findings by coronary angiography. All patients were subjected to a detailed oral health examination. The saliva samples were analyzed for lipopolysaccharide activity as well as for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia by quantitative PCR. Serum antibodies levels against A. actinomycetemcomitans were analyzed. RESULTS: The level of bacterial burden was linearly associated with alveolar bone loss (p < 0.001) and bleeding on probing (p = 0.015). The median salivary levels of A. actinomycetemcomitans in pathogen-positive patients were significantly higher in the "Stable CAD" (p = 0.014) and the "ACS" (p = 0.044) groups when compared to "No significant CAD" patients. In logistic regression models, a 10-fold increase in the salivary A. actinomycetemcomitans levels was associated with a risk for stable CAD and ACS with odds ratios (ORs) of 7.47 (95% confidence interval [CI]: 1.57-35.5, p = 0.012) and 4.31 (95% CI: 1.06-17.5, p = 0.041), respectively. The OR for the association of IgA-class antibody levels against A. actinomycetemcomitans with ACS risk was 3.13 (95% CI: 1.38-7.12, p = 0.006)/log(10) unit increase. CONCLUSIONS: High salivary levels of A. actinomycetemcomitans and systemic exposure to the bacterium were associated with increased risk for CAD. These findings emphasize the importance of oral microbiota in cardiovascular risk assessment and therapeutics.