Michael John Novak

Apoptotic Genes are Differentially Expressed in Aged Gingival Tissue

Cellular and molecular changes of the periodontium associated with a higher prevalence of oral diseases (e.g., chronic periodontitis) in aged populations have received little attention. Since impaired apoptosis during aging appears to be related to chronic inflammatory disorders, we hypothesized that the expression of genes associated with apoptotic processes are altered in aged healthy and periodontitis-affected gingival tissue. Ontology analysis of 88 genes related to apoptotic pathways was performed in gingival biopsies of healthy and periodontitis sites from young, adult, and aged non-human primates (Macaca mulatta), using the GeneChip® Rhesus Macaque Genome Array. Lower expression of anti-apoptotic and higher expression of pro-apoptotic genes were associated with healthy gingival tissue from young compared with aged animals. Few differences in gene expression were observed in healthy gingival tissue between adult and aged animals. Comparison between healthy and periodontitis gingival tissues showed that the up- or down-regulated apoptotic genes in diseased gingival tissue are different in adults compared with aged animals. These results suggest that apoptotic events normally occurring in gingival tissues could be reduced in aging,and unique aspects of apoptotic pathways are potentially involved in the pathophysiology of perio-dontal disease in adult vs. aged gingival tissues.

Change in periodontitis during pregnancy and the risk of pre-term birth and low birthweight.

AIM Determine whether periodontitis progression during pregnancy is associated with adverse birth outcomes. METHODS We used clinical data and birth outcomes from the Obstetrics and Periodontal Therapy Study, in which randomly selected women received periodontal treatment before 21 weeks of gestation (N=413) or after delivery (410). Birth outcomes were available for 812 women and follow-up periodontal data for 722, including 75 whose pregnancies ended <37 weeks. Periodontitis progression was defined as >or=3 mm loss of clinical attachment. Birth outcomes were compared between non-progressing and progressing groups using the log rank and t tests, separately in all women and in untreated controls. RESULTS The distribution of gestational age at the end of pregnancy (p>0.1) and mean birthweight (3295 versus 3184 g, p=0.11) did not differ significantly between women with and without disease progression. Gestational age and birthweight were not associated with change from baseline in percentage of tooth sites with bleeding on probing or between those who did versus did not progress according to a published definition of disease progression (p>0.05). CONCLUSIONS In these women with periodontitis and within this studys limitations, disease progression was not associated with an increased risk for delivering a pre-term or a low birthweight infant.

Caloric restriction and chronic inflammatory diseases

A reduction in calorie intake [caloric restriction (CR)] appears to consistently decrease the biological rate of aging in a variety of organisms as well as protect against age-associated diseases including chronic inflammatory disorders such as cardiovascular disease and diabetes. Although the mechanisms behind this observation are not fully understood, identification of the main metabolic pathways affected by CR has generated interest in finding molecular targets that could be modulated by CR mimetics. This review describes the general concepts of CR and CR mimetics as well as discusses evidence related to their effects on inflammation and chronic inflammatory disorders. Additionally, emerging evidence related to the effects of CR on periodontal disease in non-human primates is presented. While the implementation of this type of dietary intervention appears to be challenging in our modern society where obesity is a major public health problem, CR mimetics could offer a promising alternative to control and perhaps prevent several chronic inflammatory disorders including periodontal disease.

Extracorporeal Shock Wave Therapy Induces Alveolar Bone Regeneration

Periodontal inflammation with alveolar bone resorption is a hallmark of periodontitis. We hypothesized that extracorporeal shock wave therapy (ESWT) could promote the regeneration of alveolar bone following Porphyromonas gingivalis-induced periodontitis in rats. Rats were infected with P. gingivalis for 10 wks, which caused alveolar bone resorption. The rats were then treated with a single episode of 100, 300, or 1000 impulses of shock wave on both cheeks at energy levels 0.1 mJ/mm2. Alveolar bone levels were determined at 0, 3, 6, and 12 wks following ESWT and compared with those in untreated controls. Infected rats treated with 300 and 1000 impulses demonstrated significantly improved alveolar bone levels at 3 wks compared with untreated controls, and the improved levels remained for at least 6 wks in most rats. The results demonstrated effective regeneration of alveolar bone by ESWT and suggested that ESWT should be evaluated as an adjunct in the regeneration of periodontal tissues following periodontal disease. Abbreviations: ESWT, extracorporeal shock wave therapy; PCR, polymerase chain-reaction.

Optimizing qPCR for the Quantification of Periodontal Pathogens in a Complex Plaque Biofilm

Quantitative PCR (qPCR) has recently been used to quantify microorganisms in complex communities, including dental plaque biofilms. However, there is variability in the qPCR protocols being used. This study was designed to evaluate the validity of two of these variables with the intent of developing a more standardized qPCR protocol. The two variables evaluated were (1) the use of DNA content versus actual cell counts to estimate bacterial numbers in mixed plaque samples and (2) the effectiveness of three different universal primers versus species specific primers in amplifying specific target pathogens in these samples. Results lead to the development of a standardized protocol that was shown to be highly reproducible as demonstrated by low coefficients of variation. The results also confirmed that this standardized qPCR protocol can be used as a sensitive method for quantifying specific bacterial species in human plaque samples.

Differential gender effects of a reduced-calorie diet on systemic inflammatory and immune parameters in nonhuman primates

BACKGROUND AND OBJECTIVE Dietary manipulation, including caloric restriction, has been shown to impact host response capabilities significantly, particularly in association with aging. This investigation compared systemic inflammatory and immune-response molecules in rhesus monkeys (Macaca mulatta). MATERIAL AND METHODS Monkeys on continuous long-term calorie-restricted diets and a matched group of animals on a control ad libitum diet, were examined for systemic response profiles including the effects of both gender and aging. RESULTS The results demonstrated that haptoglobin and alpha1-antiglycoprotein levels were elevated in the serum of male monkeys. Serum IgG responses to Campylobacter rectus, Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis were significantly elevated in female monkeys. While only the antibody to Fusobacterium nucleatum was significantly affected by the calorie-restricted diet in female monkeys, antibody levels to Prevotella intermedia, C. rectus and Treponema denticola demonstrated a similar trend. CONCLUSION In this investigation, only certain serum antibody levels were influenced by the age of male animals, which was seemingly related to increasing clinical disease in this gender. More generally, analytes were modulated by gender and/or diet in this oral model system of mucosal microbial challenge.

Utility of Salivary Biomarkers for Demonstrating Acute Myocardial Infarction

The comparative utility of serum and saliva as diagnostic fluids for identifying biomarkers of acute myocardial infarction (AMI) was investigated. The goal was to determine if salivary biomarkers could facilitate a screening diagnosis of AMI, especially in cases of non-ST elevation MI (NSTEMI), since these cases are not readily identified by electrocardiogram (ECG). Serum and unstimulated whole saliva (UWS) collected from 92 AMI patients within 48 hours of chest pain onset and 105 asymptomatic healthy control individuals were assayed for 13 proteins relevant to cardiovascular disease, by Beadlyte technology (Luminex®) and enzyme immunoassays. Data were analyzed with concentration cut-points, ECG findings, logistic regression (LR) (adjusted for matching for age, gender, race, smoking, number of teeth, and oral health status), and classification and regression tree (CART) analysis. A sensitivity analysis was conducted by repetition of the CART analysis in 58 cases and 58 controls, each matched by age and gender. Serum biomarkers demonstrated AMI sensitivity and specificity superior to that of saliva, as determined by LR and CART. The predominant discriminators in serum by LR were troponin I (TnI), B-type natriuretic peptide (BNP), and creatine kinase-MB (CK-MB), and TnI and BNP by CART. In saliva, LR identified C-reactive protein (CRP) as the biomarker most predictive of AMI. A combination of smoking tobacco, UWS CRP, CK-MB, sCD40 ligand, gender, and number of teeth identified AMI in the CART decision trees. When ECG findings, salivary biomarkers, and confounders were included, AMI was predicted with 80.0% sensitivity and 100% specificity. These analyses support the potential utility of salivary biomarker measurements used with ECG for the identification of AMI. Thus, saliva-based tests may provide additional diagnostic screening information in the clinical course for patients suspected of having an AMI.

Effects of Low-energy Shock Waves on Oral Bacteria

We have recently demonstrated that extracorporeal shock-wave therapy (ESWT) is effective in promoting the healing of dermal wounds and in regenerating alveolar bone lost through periodontal disease. The objective of the present study was to determine any antibacterial effect of ESWT on oral bacteria. Monoculture suspensions of 6 bacterial species were treated with 100 to 500 pulses of ESWT at energy flux densities (EFD) of 0.12 mJ/mm2, 0.22 mJ/mm2, and 0.3 mJ/mm2. Following treatment, aliquots were plated for viability determination and compared with untreated controls. ESWT showed a significant microbicidal effect for Streptococcus mutans and an unencapsulated strain of Porphyromonas gingivalis following as few as 100 pulses at 0.3 mJ/mm2 (p ≤ 0.001). In addition, a significant disruption of bacterial aggregates was observed at lower EFDs. No significant reduction in viability was observed for all other bacteria at EFDs and pulses tested (p > 0.05). These findings suggest that low-energy ESWT may be bactericidal for selected oral bacteria.

Comparative analysis of gingival tissue antigen presentation pathways in ageing and periodontitis

AIM Gingival tissues of periodontitis lesions contribute to local elevations in mediators, including both specific T cell and antibody immune responses to oral bacterial antigens. Thus, antigen processing and presentation activities must exist in these tissues to link antigen-presenting cells with adaptive immunity. We hypothesized that alterations in the transcriptome of antigen processing and presentation genes occur in ageing gingival tissues and that periodontitis enhances these differences reflecting tissues less capable of immune resistance to oral pathogens. MATERIALS AND METHODS Rhesus monkeys (n = 34) from 3 to 23 years of age were examined. A buccal gingival sample from healthy or periodontitis sites was obtained, total RNA isolated, and microarray analysis was used to describe the transcriptome. RESULTS The results demonstrated increased transcription of genes related to the MHC class II and negative regulation of NK cells with ageing in healthy gingival tissues. In contrast, both adult and ageing periodontitis tissues showed decreased transcription of genes for MHC class II antigens, coincident with up-regulation of MHC class I-associated genes. CONCLUSION These transcriptional changes suggest a response of healthy ageing tissues through the class II pathway (i.e. endocytosed antigens) and altered responses in periodontitis that could reflect host-associated self-antigens or targeting cytosolic intracellular microbial pathogens.

Systemic endotoxin levels in chronic indolent periodontal infections.

BACKGROUND AND OBJECTIVE Periodontal disease has been linked with an increased risk of various systemic diseases. A plausible biologic explanation for this link includes the opportunity for oral pathogens to translocate to the circulation as a result of breakdown in integrity of the oral epithelium. This study refined a methodology used to detect endotoxin activity in the serum of subjects with indolent periodontal infections. MATERIAL AND METHODS The QCL Kinetic Chromogenic Assay (Cambrex) is a kinetic measure of endotoxin activity. Sera from 211 pregnant women with periodontitis enrolled in the Obstetrics and Periodontal Therapy Trial were used to develop the assay further and to evaluate the detection of endotoxin activity that might accompany a low-level bacteremia in chronic periodontitis. RESULTS We optimized the system to increase the sensitivity and reproducibility of the assay. The refined system was able to detect endotoxin activity in serum at > 0.0125 EU/mL. At baseline (13-16 wk of gestation), 35.5% of the women were positive for endotoxin activity (1.62 +/- 2.21; range: 0.38-15 EU/mL). CONCLUSION This report describes a sensitive measure of endotoxin activity in serum. The procedure allowed us to document levels of this microbial virulence factor in serum of individuals with indolent infections such as periodontal disease.