Kati Hyvärinen

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)-1β, 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-1β, and P. gingivalis were associated with deepened periodontal pockets and alveolar bone loss, and MMP-8 and IL-1β 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-1β, 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.

Salivary interleukin-1β concentration and the presence of multiple pathogens in periodontitis

AIM This study aimed to find salivary enzymes and/or cytokines that would reflect periodontitis, alone or in combination with salivary microbial markers. MATERIAL AND METHODS The salivary concentrations of elastase, lactate dehydrogenase, interleukin-1beta (IL-1beta), interleukin-6, and tumour necrosis factor-alpha, and the presence of five periodontal pathogens, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, and Treponema denticola, were analysed from salivary specimens of 165 subjects, a subpopulation of Health 2000 Health Examination Survey in Finland; 84 of the subjects had probing pocket depth (PPD) of > or =4 mm at 14 or more teeth (the advanced periodontitis group), while 81 subjects had no teeth with PPD of > or =4 mm (the control group). All subjects had at least 20 teeth and no systemic diseases. RESULTS Among the salivary cytokines and enzymes tested, IL-1beta was the only biomarker associated with periodontitis. An association was also found with the presence of multiple periodontal pathogens. Salivary IL-1beta and the presence of multiple periodontal pathogens were associated with periodontitis at the same magnitude, when they were in the logistic regression model individually or together. CONCLUSION We suggest that salivary IL-1beta and the presence of multiple periodontal pathogens in saliva should be studied more thoroughly as markers of periodontitis.

Detection and quantification of five major periodontal pathogens by single copy gene-based real-time PCR

Periodontitis is a common chronic multibacterial infection in the tooth-supporting tissues. It has been shown that periodontitis patients carry higher number of disease-associated bacteria than healthy ones. The aim of this study was to generate a novel, single copy gene-based quantitative real-time PCR (qPCR) assay for five major periodontal pathogens — Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, and Tannerella forsythia. The primer/probe sets were designed for conservative lipopolysaccharide-coding gene regions. They proved to be sensitive and able to detect strains representing different serotypes of the target bacteria. The specificity of designed primers was tested using 49 selected bacterial species and no false positive or negative results were observed. We validated the assay with a case-control population, including 165 saliva samples, and proved the diagnostic accuracy by Receiver Operating Characteristic (ROC) curves. All quantified pathogens alone were able to distinguish significantly between the subjects with and without periodontitis, and provided areas under the ROC curve larger than 0.5. The total pathogen burden comprising all five species associated with periodontitis with an area of 0.821 (95% CI, 0.758—0.885, P50.001). Our prominently sensitive and specific assay may have major importance in the diagnosis, prevention, and treatment of periodontitis.

Use of host- and bacteria-derived salivary markers in detection of periodontitis: a cumulative approach.

In the present study, we propose a novel diagnostic approach, using 3 different salivary markers, representing periodontal pathogen burden, inflammation, and tissue degradation, for detecting periodontitis. The salivary concentrations of Porphyromonas gingivalis, interleukin-1β, and matrix metalloproteinase-8, available from salivary specimens of 165 subjects (84 subjects with advanced periodontitis and 81 controls), were calculated together to obtain a cumulative risk score (CRS). In the calculation of CRS, the concentrations of each marker were divided into tertiles, and cumulative sub-score per each subject were calculated by the multiplication of the tertile values. Three CRS groups, indicating the lowest, medium, or highest risk, were formed with the cumulative sub-scores. Logistic regression analysis and ROC curves were performed to study the association of CRS with periodontitis. The results indicate that CRS, calculated from the 3 salivary biomarkers, is associated with advanced periodontitis more strongly than any of the markers individually. CRS offers a novel, non-invasive model for advanced periodontitis risk categorization that is especially useful in large population surveys where a periodontal examination is not feasible.

Periodontal pathogen carriage, rather than periodontitis, determines the serum antibody levels

AIM We investigated in a nationally representative sample, how periodontitis modifies the association between the carriage of periodontal pathogens and serology. MATERIALS AND METHODS The population comprised 1586 dentate subjects who participated in an interview, clinical and radiological oral health examination, and saliva collection. Serum immunoglobulin A (IgA)- and IgG-class antibody levels against Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis and their salivary occurrence were determined in the whole population. The quantity of the pathogens was measured in a subpopulation. RESULTS In the univariate analyses, the corresponding antibody levels were higher in the pathogen carriers compared with the non-carriers, and clearly higher in the carriers with periodontal pockets compared with the carriers without. In the multi-variate analyses, however, all antibody levels associated strongly with age (p<0.001) and the carriage of the corresponding pathogen (p<0.001), but only weakly with the presence or number of teeth with periodontal pockets. In the subpopulation, the antibody levels and the numbers of corresponding bacteria in saliva had a positive association, which was not affected by the disease. CONCLUSIONS The carriage of A. actinomycetemcomitans and P. gingivalis is the strongest determinant of the systemic antibody response to these pathogens, and the extent of periodontitis has at most a modest modifying effect.

Chlamydial and Periodontal Pathogens Induce Hepatic Inflammation and Fatty Acid Imbalance in Apolipoprotein E-Deficient Mice

ABSTRACT Periodontitis and Chlamydia pneumoniae infection are independent risk factors for cardiovascular diseases. The aim of this study was to investigate the effect of C. pneumoniae and Aggregatibacter actinomycetemcomitans infection on hepatic inflammation and lipid homeostasis of apolipoprotein E-deficient mice. Mice were infected with viable C. pneumoniae intranasally three times for chronic infection or once for acute infection. Viable A. actinomycetemcomitans was administered 10 times intravenously alone or in concert with C. pneumoniae. Hepatic alterations were assessed by histochemistry, lipid quantification, and fatty acid profile analysis. The RNA expression levels and the presence of pathogens in the livers and lungs were detected by quantitative real-time PCR. Both pathogens were detected in the livers of the infected animals. Chronic C. pneumoniae infection induced marked changes in hepatic lipid homeostasis. A. actinomycetemcomitans infection resulted in inflammatory cell infiltration into the liver, accompanied by elevated hepatic RNA expression levels of inflammation-related genes and higher serum amyloid A and lipopolysaccharide concentrations. Our results indicate that proatherogenic pathogens infect the liver, causing proinflammatory alterations and lipid disturbances. This infection may maintain chronic systemic inflammation attributable to atherogenesis.

Combining Salivary Pathogen and Serum Antibody Levels Improves Their Diagnostic Ability in Detection of Periodontitis

BACKGROUND Initiation and progression of periodontitis correlates with increased quantities of periodontitis-associated bacteria in periodontal biofilms. In the present study, the aim is to measure Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis amounts in saliva and their antibody (immunoglobulin [Ig]A and IgG) levels in serum and evaluate their diagnostic abilities, together or alone, in chronic periodontitis. METHODS The study population comprised 230 Finnish dentate adults: 84 with generalized chronic periodontitis (GCP), 65 with localized chronic periodontitis (LCP), and 81 controls without periodontitis. General and oral health information was obtained by questionnaires, interviews, and clinical and radiographic examinations. Salivary and serum samples were analyzed by quantitative single copy gene-based real-time polymerase chain reaction and multiserotype enzyme-linked immunosorbent assay, respectively. RESULTS Pathogen carriers suffered mostly from GCP and seldom from LCP. A. actinomycetemcomitans and P. gingivalis quantities in saliva were strongly associated with corresponding serum IgA and IgG values (P <0.001) and with severity of disease (P <0.001). P. gingivalis exhibited more straightforward associations among salivary bacterial burdens, corresponding antibody formation, and periodontitis severity than A. actinomycetemcomitans. The combination of information on age, sex, smoking, and P. gingivalis results provided an area under the curve of 0.817 (95% confidence interval 0.76 to 0.87, P <0.001) for GCP. CONCLUSION The combination of saliva P. gingivalis quantity with pathogen-specific host response may be used to diagnose periodontitis with high accuracy.

Very low density lipoproteins derived from periodontitis patients facilitate macrophage activation via lipopolysaccharide function.

OBJECTIVE Periodontitis, a chronic oral infection caused mainly by gram-negative bacteria, induces endotoxemia and associates with the risk for atherosclerosis. We investigated the effect of periodontal treatment on proatherogenic properties of very low density lipoproteins (VLDL). METHODS VLDL were isolated from 30 systemically healthy periodontitis patients before (pre-treatment) and 3 months after treatment (post-treatment). The mass compositions were analyzed, and VLDL-induced changes in cellular cholesterol content and expression of selected genes of human THP-1 macrophages were measured. RESULTS Periodontal treatment decreased the local inflammation in the periodontium, but did not have a significant effect on C-reactive protein (CRP) levels, VLDL composition, or VLDL potential to induce cholesterol uptake or gene expression by the macrophages. Incubation of macrophages in the presence of VLDL resulted in more than twofold increase in their cellular cholesterol content. Uptake of VLDL with ensuing macrophage cholesterol accumulation correlated positively with VLDL-associated lipopolysaccharide (LPS) activity (r=0.436, P=.016) and apolipoprotein E content (r=0.374, P=.046). Pre-treatment VLDL derived from the patients with high CRP levels displayed higher LPS activity than that of VLDL derived from patients with low CRP (above vs. below median, P=.007). In addition, pre-treatment VLDL isolated from patients with high systemic inflammation induced higher relative mRNA expression of CD14, TNF-α, MCP-1, and IL-6 in the macrophages. CONCLUSION Inflammation and endotoxemia induced by severe periodontitis may increase VLDL-dependent macrophage activation and cellular cholesterol accumulation, and thereby atherogenesis.

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.

The effect of proatherogenic pathogens on adipose tissue transcriptome and fatty acid distribution in apolipoprotein E-deficient mice

BackgroundChronic infections have been demonstrated to maintain low-grade systemic inflammation and associate with atherosclerosis. We studied the inflammation- and lipid homeostasis-related effects of Aggregatibacter actinomycetemcomitans (Aa) and Chlamydia pneumoniae (Cpn) infections on the epididymal and inguinal adipose tissue (AT) transcriptomes and fatty acid distribution in apolipoprotein (apo) E-deficient mice. Chow-fed apoE-deficient mice were exposed to 1) chronic intranasal infection with C. pneumoniae (Cpn group), 2) recurrent intravenous infection with A. actinomycetemcomitans (Aa group), 3) a combination of both types of infection (Cpn + Aa group), or 4) infection with the vehicle (control group). Epididymal and inguinal AT gene expression was analyzed using an Illumina Mouse WG-6 v2.0 platform and quantitative PCR (QPCR). Microarray data were analyzed using Gene Ontology enrichment analysis. AT fatty acid analysis was performed using gas–liquid chromatography.ResultsThe transcriptomics data revealed significant enrichment in inflammation-associated biological pathways in both AT depots derived from the Aa and Cpn + Aa treated mice compared with the control group. The proportion of saturated fatty acids was higher in the inguinal AT in Aa (p = 0.027) and Cpn + Aa (p = 0.009) groups and in the epididymal AT in Aa group (p = 0.003). The proportion of polyunsaturated fatty acids was significantly lower among all Aa-infected groups in both depots. Chronic Cpn infection displayed only minor effects on transcriptomics and fatty acids of the AT depots.ConclusionsSystemic infection with A. actinomycetemcomitans activates inflammation-related biological pathways and modulates cellular lipid homeostasis. The adverse changes in adipose tissues during chronic infection may promote atherosclerosis.