Zebrafish as a new model to study effects of periodontal pathogens on cardiovascular diseases.

Porphyromonas gingivalis (Pg) is a keystone pathogen in the aetiology of chronic periodontitis. However, recent evidence suggests that the bacterium is also able to enter the bloodstream, interact with host cells and tissues, and ultimately contribute to the pathogenesis of cardiovascular disease (CVD). Here we established a novel zebrafish larvae systemic infection model showing that Pg rapidly adheres to and penetrates the zebrafish vascular endothelium causing a dose- and time-dependent mortality with associated development of pericardial oedemas and cardiac damage. The in vivo model was then used to probe the role of Pg expressed gingipain proteases using systemically delivered gingipain-deficient Pg mutants, which displayed significantly reduced zebrafish morbidity and mortality compared to wild-type bacteria. In addition, we used the zebrafish model to show efficacy of a gingipain inhibitor (KYT) on Pg-mediated systemic disease, suggesting its potential use therapeutically. Our data reveal the first real-time in vivo evidence of intracellular Pg within the endothelium of an infection model and establishes that gingipains are crucially linked to systemic disease and potentially contribute to CVD

Benzyl Isothiocyanate, a Major Component from the Roots of Salvadora Persica Is Highly Active against Gram-Negative Bacteria

Plants produce a number of antimicrobial substances and the roots of the shrub Salvadora persica have been demonstrated to possess antimicrobial activity. Sticks from the roots of S. persica, Miswak sticks, have been used for centuries as a traditional method of cleaning teeth. Diverging reports on the chemical nature and antimicrobial repertoire of the chewing sticks from S. persica led us to explore its antibacterial properties against a panel of pathogenic or commensal bacteria and to identify the antibacterial component/s by methodical chemical characterization. S. persica root essential oil was prepared by steam distillation and solid-phase microextraction was used to sample volatiles released from fresh root. The active compound was identified by gas chromatography-mass spectrometry and antibacterial assays. The antibacterial compound was isolated using medium-pressure liquid chromatography. Transmission electron microscopy was used to visualize the effect on bacterial cells. The main antibacterial component of both S. persica root extracts and volatiles was benzyl isothiocyanate. Root extracts as well as commercial synthetic benzyl isothiocyanate exhibited rapid and strong bactericidal effect against oral pathogens involved in periodontal disease as well as against other Gram-negative bacteria, while Gram-positive bacteria mainly displayed growth inhibition or remained unaffected. The short exposure needed to obtain bactericidal effect implies that the chewing sticks and the essential oil may have a specific role in treatment of periodontal disease in reducing Gram-negative periodontal pathogens. Our results indicate the need for further investigation into the mechanism of the specific killing of Gram-negative bacteria by S. persica root stick extracts and its active component benzyl isothiocyanate

Usefulness of real time PCR for the differentiation and quantification of 652 and JP2 Actinobacillus actinomycetemcomitans genotypes in dental plaque and saliva

BACKGROUND: The aim of our study is to describe a fast molecular method, able to distinguish and quantize the two different genotypes (652 and JP2) of an important periodontal pathogen: Actinobacillus actinomycetemcomitans. The two genotypes show differences in the expression of an important pathogenic factor: the leukotoxin (ltx). In order to evidence this, we performed a real time PCR procedure on the ltx operon, able to recognize Aa clinical isolates with different leukotoxic potentials. METHODS: The specificity of the method was confirmed in subgingival plaque and saliva specimens collected from eighty-one Italian (Sardinian) subjects with a mean age of 43.9, fifty five (68 %) of whom had various clinical forms of periodontal disease. RESULTS: This procedure showed a good sensitivity and a high linear dynamic range of quantization (10(7)-10(2 )cells/ml) for all genotypes and a good correlation factor (R2 = 0.97–0.98). Compared with traditional cultural methods, this real time PCR procedure is more sensitive; in fact in two subgingival plaque and two positive saliva specimens Aa was only detected with the molecular method. CONCLUSION: A low number of Sardinian patients was found positive for Aa infections in the oral cavity, (just 10 positive periodontal cases out of 81 and two of these were also saliva positive). The highly leukotoxic JP2 strain was the most representative (60 % of the positive specimens); the samples from periodontal pockets and from saliva showed some ltx genotype for the same patient. Our experience suggests that this approach is suitable for a rapid and complete laboratory diagnosis for Aa infection

Platelet-activating factor levels of serum and gingival crevicular fluid in nonsmoking patients with periodontitis and/or coronary heart disease

The purpose of the present study was to investigate systemic and local levels of platelet-activating factor (PAF), a potent proinflammatory mediator implicated in cardiovascular pathophysiology in adult nonsmoking patients with periodontitis with or without coronary heart disease (CHD). Eighty-seven volunteers, 25 periodontitis patients, 19 periodontitis with CHD patients, 19 CHD patients, and 24 healthy controls were included, and periodontal conditions were assessed. Gingival crevicular fluid (GCF) and venous blood were collected, and PAF levels were measured by enzyme-linked immunosorbent assay. PAF levels in serum (303.3 ± 204 pg/ml) and in GCF (26.3 ± 6 pg/μl) of the periodontitis group with CHD, the periodontitis group (serum, 302.4 ± 241 pg/ml and GCF, 26.3 ± 8 pg/μl) and the CHD group (serum, 284.7 ± 192 pg/ml and GCF, 20.8 ± 6 pg/μl) were significantly higher than the healthy control group (serum, 65.4 ± 35 pg/ml and GCF, 7.7 ± 3 pg/μl; p < 0.05). In summary, the present study could demonstrate that in patients with periodontitis, the inflammatory mediator PAF is released into serum at least in the same range as for patients with coronary heart disease. However, no additive effects were seen when both conditions were present

Self-reported dental hygiene, obesity, and systemic inflammation in a pediatric rural community cohort

Background A growing body of epidemiologic evidence links oral health, obesity, and cardiovascular health, though few studies have reported on these relationships in children. While underlying mechanisms are unclear, adult studies have suggested sub-acute systemic inflammation, also implicated in the etiology of both obesity and cardiovascular disease. This study investigated associations between self-reported dental hygiene, obesity, and systemic inflammation in children. Methods 128 children \u3c 19 years of age from rural counties in West Virginia participated in a community-based health screening that included anthropometric assessments, blood collection, and a questionnaire about dental hygiene and self-assessed oral health. Results Participants ranged from 3.0-18.7 years. Univariate analysis demonstrated an association between parent-reported dental hygiene, including frequency of preventive dental care and parent-assessed overall dental health, and markers of systemic inflammation but not obesity. In multivariable regression, parent-assessed overall dental health and obesity were independent predictors of systemic inflammation, after adjustment for age, gender, and parent education. Conclusions This is the first known study of the association between dental hygiene, obesity, and systemic inflammation in children. These results highlight the importance of preventive dental care in overall, systemic health in children and are consistent with previous reports in adults

Oral microbe-host interactions: influence of β-glucans on gene expression of inflammatory cytokines and metabolome profile

Background: The aim of this study was to evaluate the effects of β-glucan on the expression of inflammatory mediators and metabolomic profile of oral cells [keratinocytes (OBA-9) and fibroblasts (HGF-1) in a dual-chamber model] infected by Aggregatibacter actinomycetemcomitans. The periodontopathogen was applied and allowed to cross the top layer of cells (OBA-9) to reach the bottom layer of cells (HGF-1) and induce the synthesis of immune factors and cytokines in the host cells. β-glucan (10 μg/mL or 20 μg/mL) were added, and the transcriptional factors and metabolites produced were quantified in the remaining cell layers and supernatant. Results: The relative expression of interleukin (IL)-1-α and IL-18 genes in HGF-1 decreased with 10 μg/mL or 20 μg/mL of β-glucan, where as the expression of PTGS-2 decreased only with 10 μg/mL. The expression of IL-1-α increased with 20 μg/mL and that of IL-18 increased with 10 μg/mL in OBA-9; the expression of BCL 2, EP 300, and PTGS-2 decreased with the higher dose of β-glucan. The production of the metabolite 4-aminobutyric acid presented lower concentrations under 20 μg/mL, whereas the concentrations of 2-deoxytetronic acid NIST and oxalic acid decreased at both concentrations used. Acetophenone, benzoic acid, and pinitol presented reduced concentrations only when treated with 10 μg/mL of β-glucan. Conclusions: Treatment with β-glucans positively modulated the immune response and production of metabolites

Biological foundation for periodontitis as a potential risk factor for atherosclerosis

Links between periodontal diseases and systemic diseases have been well documented by epidemiological studies. Recently, research has shifted to elucidating the biologic mechanism for a causal relationship. One focus of interest is atherosclerosis, the underlying event of cardiovascular diseases due to its serious health impact. However, it is still not clear whether periodontopathic pathogens are truly etiologic agents or ubiquitous bystanders. This article reviews the current understanding about the molecular biological interactions between periodontal disease and atherosclerosis and the biological plausibility of periodontitis as a potential risk factor for cardiovascular disease. Materials and methods:  The current literature regarding periodontal diseases and atherosclerosis and coronary vascular disease was searched using the Medline and PubMed databases. Results:  In vitro experiments and animal models are appropriate tools to investigate the biological interactions between periodontal disease and atherosclerosis at the cell molecular level. The concepts linking both pathologies refer to inflammatory response, immune responses, and hemostasis. In particular, Porphyromonas gingivalis appears to have unique, versatile pathogenic properties. Whether or not these findings from isolated cells or animal models are applicable in humans with genetic and environmental variations is yet to be determined. Likewise, the benefit from periodontal therapy on the development of atherosclerosis is unclear. Approaches targeting inflammatory and immune responses of periodontitis and atherosclerosis simultaneously are very intriguing. Conclusion:  An emerging concept suggests that a pathogenic burden from different sources might overcome an individual threshold culminating in clinical sequela. P. gingivalis contributes directly and indirectly to atherosclerosis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66109/1/j.1600-0765.2004.00771.x.pd

Pyrosequencing as a tool for better understanding of human microbiomes

Next-generation sequencing technologies have revolutionized the analysis of microbial communities in diverse environments, including the human body. This article reviews several aspects of one of these technologies, the pyrosequencing technique, including its principles, applications, and significant contribution to the study of the human microbiome, with especial emphasis on the oral microbiome. The results brought about by pyrosequencing studies have significantly contributed to refining and augmenting the knowledge of the community membership and structure in and on the human body in healthy and diseased conditions. Because most oral infectious diseases are currently regarded as biofilm-related polymicrobial infections, high-throughput sequencing technologies have the potential to disclose specific patterns related to health or disease. Further advances in technology hold the perspective to have important implications in terms of accurate diagnosis and more effective preventive and therapeutic measures for common oral diseases