Angiotensin II modulates CD40 expression in vascular smooth muscle cells

The signalling pathway CD40/CD40L (CD40 ligand) plays an important role in atherosclerotic plaque formation and rupture. AngII (angiotensin II), which induces oxidative stress and inflammation, is also implicated in the progression of atherosclerosis. In the present study, we tested the hypothesis that AngII increases CD40/CD40L activity in vascular cells and that ROS (reactive oxygen species) are part of the signalling cascade that controls CD40/CD40L expression. Human CASMCs (coronary artery smooth muscle cells) in culture exposed to IL (interleukin)-1 beta or TNF-alpha (tumour necrosis factor-a) had increased superoxide generation and enhanced CD40 expression, detected by EPR (electron paramagnetic resonance) and immunoblotting respectively. Both phenomena were abolished by previous incubation with membrane-permeant antioxidants or cell transfection with P22(phox) antisense. AngII (50-200 nmol/l) induced an early and sustained increase in CD40 mRNA and protein expression in CASMCs, which was blocked by treatment with antioxidants. Increased CD40 expression led to enhanced activity of the pathway, as AngII-treated cells stimulated with recombinant CD40L released higher amounts of IL-8 and had increased COX-2 (cyclo-oxygenase-2) expression. We conclude that AngII stimulation of vascular cells leads to a ROS-dependent increase in CD40/CD40L signalling pathway activity. This phenomenon may be an important mechanism modulating the arterial injury observed in atherosclerosis-related vasculopathy.

Quantitative detection of periodontopathic bacteria in atherosclerotic plaques from coronary arteries

Oral pathogens, including periodontopathic bacteria, are thought to be aetiological factors in the development of cardiovascular disease. In this study, the presence of Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum-periodonticum-simiae group, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Tannerella forsythia in atheromatous plaques from coronary arteries was determined by real-time PCR. Forty-four patients displaying cardiovascular disease were submitted to periodontal examination and endarterectomy of coronary arteries. Approximately 60-100 mg atherosclerotic tissue was removed surgically and DNA was obtained. Quantitative detection of periodontopathic bacteria was performed using universal and species-specific TaqMan probe/primer sets. Total bacterial and periodontopathic bacterial DNA were found in 94.9 and 92.3 %, respectively, of the atheromatous plaques from periodontitis patients, and in 80.0 and 20.0%, respectively, of atherosclerotic tissues from periodontally healthy subjects. All periodontal bacteria except for the F. nucleatum-periodonticum-simiae group were detected, and their DNA represented 47.3 % of the total bacterial DNA obtained from periodontitis; patients. Porphyromonas gingivalis, A. actinomycetemcomitans and Prevotella intermedia were detected most often. The presence of two or more periodontal species could be observed in 64.1 % of the samples. In addition, even in samples in which a single periodontal species was detected, additional unidentified microbial DNA could be observed. The significant number of periodontopathic bacterial DNA species in atherosclerotic tissue samples from patients with periodontitis suggests that the presence of these micro-organisms in coronary lesions is not coincidental and that they may in fact contribute to the development of vascular diseases.

Effect of Calcium Pre-rinse and Fluoride Dentifrice on Enamel and on Dental Plaque Formed In Situ

Purpose: The aim of this in situ double-blind randomised crossover study was to investigate the effect of calcium (Ca) pre-rinse on the composition of plaque and on enamel prior to the use of fluoride (F) dentifrice. Materials and Methods: During four phases (14 days each) of this study, 10 volunteers had agreed to wear dental appliances containing two healthy bovine enamel blocks. A fresh solution containing 20% weight/volume (w/v) sucrose was dripped on the enamel blocks ex vivo for 5 min three times a day. Subsequently, the appliances were replaced in the mouth, and the volunteers rinsed their mouth with 10 mL of a Ca (150 mmol/L) or a placebo rinse (1 min). In sequence, a slurry (1:3 w/v) of F (1030 ppm) or placebo dentifrice was dripped onto the blocks ex vivo for 1 min. During this time, the volunteers brushed their teeth with the respective dentifrice. The appliances were replaced in the mouth, and the volunteers rinsed their mouth with water. The plaque formed on the blocks was analysed for F and Ca. The enamel demineralisation as well as the incorporation of F on enamel was evaluated by cross-sectional microhardness and alkali-soluble F analysis, respectively. Data were tested using analysis of variance (P < 0.05). Results: The Ca pre-rinse prior to the use of the F dentifrice led to a three- and sixfold increase in the plaque F and Ca concentrations, respectively. It also did not have any additive effect on the F content on the enamel and the demineralisation of the enamel, in comparison with the use of F dentifrice alone. Conclusions: A Ca lactate rinse used prior to the F dentifrice was able to change the mineral content in the plaque, but it was unable to prevent enamel demineralisation.

Use of the DNA Checkerboard hybridization method for detection and quantitation of Candida species in oral microbiota

The DNA Checkerboard method enables the simultaneous identification of distinct microorganisms in a large number of samples and employs up to 45 whole genomic DNA probes to gram-negative and gram-positive bacterial species present in subgingival biofilms. Collectively, they account for 55%-60% of the bacteria in subgingival biofilms. In this study, we present the DNA Checkerboard hybridization as an alternative method for the detection and quantitation of Candida species in oral cavities. Our results reveal that DNA Checkerboard is sensitive enough and constitutes a powerful and appropriate method for detecting and quantifying Candida species found in the oral cavity.

The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method

Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA-DNA hybridization technique enables the simultaneous identification of distinct bacterial. species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA-DNA hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA-DNA hybridization technique. (c) 2007 Elsevier GmbH. All rights reserved.

The effect of a single episode of antimicrobial photodynamic therapy in the treatment of experimental periodontitis. Microbiological profile and cytokine pattern in the dog mandible

The purpose of this study was to evaluate the effect of a single application of antimicrobial photodynamic therapy (aPDT) on microbiological profile and cytokine pattern in dogs. Periodontal disease was induced by placing 3.0 silk ligatures around the mandibular pre-molars bilaterally during 8 weeks. The dogs were randomly treated with aPDT using a dye/laser system, scaling and root planning (SRP), or with the association of treatments (SRP + aPDT). Plaque samples were collected at baseline, 1, 3, and 4 weeks, and the mean counts of 40 species were determined using DNA-DNA hybridization. Gingival biopsies were removed and the expression of tumor necrosis factor alpha (TNF-alpha), receptor activator of NF-kB ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase (MMP-1), interleukin (IL) 6, IL-10 and total bacterial load by analysis of 16 S rRNA gene were evaluated through real-time PCR. The results shows that the levels of the majority of the species were reduced 1 week post-therapy for all treatments, however, an increase in counts of Prevotella intermedia (p = 0.00), Prevotella. nigrescens (p = 0.00) and Tannerella forsythia (p = 0.00) was observed for aPDT and SRP + aPDT. After 4 weeks, a regrowth of Porphyromonas gingivalis (p = 0.00) and Treponema denticola (p = 0.00), was observed for all treatments. Also, a strikingly reduction of counts on counts of Aggregatibacter actinomycetemcomitans was observed for the aPDT (p = 0.00). For the cytokine pattern, the results were similar for all treatments, and a reduction in the expression of cytokines and bacterial load was observed throughout the study. Our results suggest that SRP, aPDT in a single application, and SRP + aPDT affects different bacterial species and have similar effects on the expression of cytokines evaluated during the treatment of ligature-induced periodontitis.

Morphological differentiation between S. mutans and S. sobrinus on modified SB-20 culture medium

Due to the major role of Streptococcus mutans and Streptococcus sobrinus in the etiology of dental caries, it is important to use culture media that allow for differentiating these bacterial species. The aim of this study was to evaluate the suitability of a modified SB-20 culture medium (SB-20M) for the isolation and morphological differentiation of S. mutans and S. sobrinus, compared to biochemical identification (biotyping). Saliva samples were collected using the spatula method from 145 children, seeded on plates containing the SB-20M, in which sucrose was replaced by coarse granular cane sugar, and incubated in microaerophilia at 37 degrees C during 72 h. Identification of the microorganisms was performed under stereomicroscopy based on colony morphology of 4904 colonies. The morphological identification was examined by biochemical tests of 94 randomly selected colonies with the macroscopic characteristic of S. mutans and S. sobrinus using sugar fermentation, resistance to bacitracin and production of hydrogen peroxide. There was no statistically significant difference (p> 0.05) between morphological identification in the SB-20M medium and biochemical identification (biotyping). Biotyping confirmed that S. mutans and S. sobrinus colonies were correctly characterized in the SB-20M in 95.8% and 95.5% of the cases, respectively. Of the mutans streptococci detected in the children 98% were S. mutans and 2% S. sobrinus. The SB-20M medium is reliable for detection and direct morphological identification of S. mutans and S. sobrinus. (C) 2010 Elsevier GmbH. All rights reserved.

In vitro antibacterial action of Tetraclean, MTAD and five experimental irrigation solutions

P>Aim To investigate the antibacterial effect of Tetraclean, MTAD and five experimental irrigants using both direct exposure test with planktonic cultures and mixed-species in vitro biofilm model. Methodology Tetraclean, MTAD and five experimental solutions that were modifications of existing formulae including MTAD + 0.01% cetrimide (CTR), MTAD + 0.1% CTR, MTAC-1 (Tween 80 replaced by 0.01% CTR in MTAD), MTAC-2 (Tween 80 replaced by 0.1% CTR) and MTAD-D (MTAD without the Tween 80 and no CTR added) were used as disinfectants in the experiments. In the direct exposure test, a suspension of Enterococcus faecalis was mixed with each of the solutions. After 0.5, 1, 3 and 10 min, an inactivator was added and the number of surviving bacteria was calculated. A mixed-species biofilm from subgingival plaque bacteria was grown in brain heart infusion broth in anaerobic conditions on synthetic hydroxyapatite discs. Two-week-old biofilms were exposed to the solutions for 0.5, 1 and 3 min. The samples were observed by confocal laser scanning microscopy after bacterial viability staining. The scans were quantitatively analysed, and the volume of killed cells of all cells was calculated for each medicament. Results Tetraclean and MTAC-2 (0.1% CTR) killed planktonic E. faecalis in < 30 s. Complete killing of bacteria required 1 min by MTAC-1, 3 min by MTAD + 0.1% CTR and 10 min by MTAD, MTAD-D and MTAD + 0.01% CTR. In the biofilm test, there were significant differences in microbial killing between the different solutions and times of exposure (P < 0.005). MTAC-2 showed the best performance, killing 71% of the biofilm bacteria in 3 min, followed by MTAC-1 and Tetraclean. MTAD and the three MTAD modifications demonstrated the lowest antibacterial activity. Conclusion Tetraclean was more effective than MTAD against E. faecalis in planktonic culture and in mixed-species in vitro biofilm. CTR improved the antimicrobial properties of the solutions, whereas Tween 80 seemed to have a neutral or negative impact on their antimicrobial effectiveness.

Evaluation of three indices for biofilm accumulation on complete dentures

Objectives: The objective of this study was to evaluate the accuracy and reproducibility of three complete denture biofilm indices (Prosthesis Hygiene Index; Jeganathan et al. Index; Budtz-J circle divide rgensen Index) by means of a computerised comparison method. Background: Clinical studies into denture hygiene have employed a large number of biofilm indices among their outcome variables. However, the knowledge about the validity of these indices is still scarce. Materials and methods: Sixty-two complete denture wearers were selected. The internal surfaces of the upper complete dentures were stained (5% erythrosine) and photographed. The slides were projected on paper, and the biofilm indices were applied over the photos by means of a scoring method. For the computerised method, the areas (total and biofilm-covered) were measured by dedicated software (Image Tool). In addition, to compare the results of the computerised method and Prosthetic Hygiene Index, a new scoring scale (including four and five graded) was introduced. For the Jeganathan et al. and Budtz-J circle divide rgensen indices, the original scales were used. Values for each index were compared with the computerised method by the Friedman test. Their reproducibility was measured by means of weighed kappa. Significance for both tests was set at 0.05. Results: The indices tested provided similar mean measures but they tended to overestimate biofilm coverage when compared with the computerised method (p < 0.001). Agreement between the Prosthesis Hygiene Index and the computerised method was not significant, regardless of the scale used. Jeghanathan et al. Index showed weak agreement, and consistent results were found for Budtz-Jorgensen Index (kappa = 0.19 and 0.39 respectively). Conclusion: Assessment of accuracy for the biofilm indices showed instrument bias that was similar among the tested methods. Weak inter-instrument reproducibility was found for the indices, except for the Budtz-J circle divide rgensen Index. This should be the method of choice for clinical studies when more sophisticated approaches are not possible.

In vitro and clinical evaluation of specific dentifrices for complete denture hygiene

To study the physical properties of two experimental dentifrices for complete denture hygiene, their effect on denture biofilm removal and antimicrobial properties by means of a clinical trial. The experimental dentifrices comprised two compositions. One was based on the addition of 1% chloramine T (D1) and the other on the presence of 0.01% fluorosurfactant (D2). Measurements of density, pH, consistency, rheological features and abrasiveness were conducted. Sixty complete denture wearers were randomly assigned to three groups and were instructed to brush their dentures with a specific toothbrush: (1) Water (control); (2) D1; or (3) D2. Each method was used for 21 days. Denture biofilm was disclosed by a 1% neutral red solution and quantified by means of digital photos taken from the internal surface. Microbiological assessment was conducted to quantify Candida sp. and mutans streptococci. Data were evaluated by one-way anova and Tukey HSD, or Kruskal-Wallis (alpha = 0.05). Both dentifrices decreased biofilm coverage when compared with the control group. D1 was the most efficacious treatment to reduce mutans streptococci, whereas D2 showed an intermediate outcome (anova, p < 0.040). No treatment influenced Candida albicans or non-albicans species (Kruskal-Wallis, p = 0.163 and 0.746, respectively). It can be concluded that brushing complete dentures with the experimental dentifrices tested could be effective for the removal of denture biofilm.

Improved endothelial function with simvastatin but unchanged insulin sensitivity with simvastatin or ezetimibe

In addition to their expected effects on lipid profile, lipid-lowering agents may reduce cardiovascular events because of effects on nonclassic risk factors such as insulin resistance and inflammation. Ezetimibe specifically blocks the absorption of dietary and biliary cholesterol as well as plant sterols. Although it is known that an additional reduction of low-density lipoprotein cholesterol (LDL-C) levels can be induced by the combination of ezetimibe with statins, it is not known if this can enhance some pleiotropic effects, which may be useful in slowing the atherosclerotic process. This study assessed the effects of simvastatin and ezetimibe, in monotherapy or in combination, on markers of endothelial function and insulin sensitivity. Fifty prediabetic subjects with normo- or mild-to-moderate hypercholesterolemia were randomly allocated to 2 groups receiving either ezetimibe (10 mg/d) or simvastatin (20 mg/d) for 12 weeks, after which the drugs were combined for both groups for an additional 12-week period. Clinical and laboratory parameters were measured at baseline and after 12 and 24 weeks of therapy. Homeostasis model assessment of insulin resistance index and the area under the curve of insulin were calculated. As expected, both groups receiving drugs in isolation significantly reduced total cholesterol, LDL-C, apolipoprotein B, and triglyceride levels; and additional reductions were found after the combination period (P <.05). After 12 weeks of monotherapy, plasminogen activator inhibitor-1 levels and urinary albumin excretion were lower in the simvastatin than in the ezetimibe group. No change in homeostasis model assessment of insulin resistance index, area under the curve of insulin, and adiponectin levels was observed tiller either the monotherapies or the combined therapy. However, simvastatin combined with ezetimibe provoked significant reductions in E-selectin and intravascular cellular adhesion molecule-1 levels that were independent of LDL-C changes. Our findings support claims that simvastatin may be beneficial in preserving endothelial function in prediabetic subjects with normo- or mild-to-moderate hypercholesterolemia. Alternatively, a deleterious effect of ezetimibe on the endothelial function is suggested, considering the increase in intravascular cellular adhesion molecule I and E-selectin levels. Simvastatin and ezetimibe, in isolation or in combination, do not interfere with insulin sensitivity. (C) 2010 Elsevier Inc. All rights reserved.

A prospective 12-month study of the effect of smoking cessation on periodontal clinical parameters

P>Aim The aim of this 12-month prospective study was to assess the adjunctive effect of smoking cessation in non-surgical periodontal therapy of subjects with severe chronic periodontitis. Materials and methods Of the 201 subjects enrolled from a smoking cessation clinic, 93 were eligible and received non-surgical periodontal treatment and concurrent smoking cessation treatment. Periodontal maintenance was performed every 3 months. Full-mouth periodontal examination in six sites per tooth was performed by a calibrated examiner, blinded to smoking status, at baseline, 3, 6 and 12 months after non-surgical periodontal treatment. Furthermore, expired air carbon monoxide concentration measurements and interviews based on a structured questionnaire were performed in order to collect demographic and smoking data. Results Of the 93 eligible subjects, 52 remained in the study after 1 year. Of these, 17 quit smoking and 35 continued smoking or oscillated. After 1 year, only quitters presented significant clinical attachment gain (p=0.04). However, there were no differences between the groups regarding clinical attachment level, probing depth, bleeding on probing and plaque index after 1 year (p > 0.05). Conclusion Smoking cessation promoted clinical attachment gain in chronic periodontitis subjects from a smoking cessation clinic after 1 year of follow-up.

Apolipoprotein E genotype is related to nitric oxide production in platelets

The presence of the, 4 allele of apolipoprotein E (APOE) is considered a risk factor for sporadic Alzheimer`s disease (AD). Our recent data demonstrated that the systemic modulation of oxidative stress in platelets and erythrocytes is disrupted in aging and AD. In this study, the relationship between APOE genotype and oxidative stress markers, both in AD patients and controls, was evaluated. The AD group showed an increase in the content of thiobarbituric acid-reactive substances (TBARS) and in the activities of nitric oxide synthase (NOS) and Na, K-ATPase, when compared to controls. Both groups had a similar cGMP content and superoxide dismutase activity. APOE epsilon 4 allele carriers showed higher NOS activity than non-carriers. These results suggest a possible influence of APOE genotype on nitric oxide (NO) production that might enhance the effects of age-related specific factor(s) associated with neurodegenerative disorders. Copyright (C) 2008 John Wiley & Sons, Ltd.

Role of PPAR-gamma in the Modulation of CD36 and FcgammaRII induced by LDL with Low and High Degrees of Oxidation During the Differentiation of the Monocytic THP-1 Cell Line

Scavenger or Fc gamma receptors are important for capture and clearance of modified LDL particles by monocytes/macrophages. Uptake via scavenger receptors is not regulated by intracellular levels of cholesterol and in consequence, macrophages develop into foam cells in the arterial intima. The levels of scavenger receptor CD36 are increased in atherosclerotic lesions and there is evidence that some components of oxLDL auto-regulate the expression of this receptor. Fc gamma receptor expression is increased in cardiovascular diseases but it is not known weather their expression is regulated by oxLDL. The biological properties of oxLDLs vary depending on the degree of oxidation. In the present study we investigated the effect of LDL particles showing extensive or low oxidation (HoxLDL and LoxLDL) on the expression of CD36 and Fc gamma RII in a human monocytic cell line (THP-1), differentiated or not to macrophage, and the involvement of PPAR gamma. It was found that both forms of oxLDL are able to increase the expression of CD36 and Fc gamma RII and that this effect is dependent on the degree of oxidation and of the stage of cell differentiation ( monocyte or macrophage). We also showed that the increased expression of Fc gamma RII is dependent on PPAR. whereas that of the CD36 is independent of PPAR gamma. Copyright (c) 2008 S. Karger AG, Basel

Differential expression of cytokines, chemokines and chemokine receptors in patients with coronary artery disease

Monocytes/macrophages and lymphocytes have a key role in the pathogenesis of atherosclerosis through the production of inflammatory and anti-inflammatory cytokines. We evaluated mRNA expression and protein production of CCL2, CXCL8, CXCL9, CXCL10, IFN-gamma and IL-10 in vitro as well as the expression of the CCR2 and CXCR3 receptors in peripheral blood mononuclear cells (PBMCs) of patients with coronary artery disease (CAD) and healthy controls in the presence or absence of oxidized LDL (oxLDL). Patients with CAD showed higher constitutive expression of CCL2, CXCL8, CXCL9, CXCL10 and IFN-gamma mRNA and, after stimulation with oxLDL, higher expression of CCL2 and CXCL8 mRNA than the control group. We also detected higher levels of CCL2 and CXCL8 in supernatants of oxLDL-stimulated PBMCs from CAD patients than in corresponding supernatants from controls. Patients with CAD had a higher percentage of constitutive CCR2(+) and CXCR3(+) cells after stimulation with oxLDL. Among CAD patients, the main differences between the stable (SA) and unstable angina (UA) groups were lower IL-10 mRNA production in the latter group. Altogether, our data suggest that PBMCs from CAD patients are able to produce higher concentrations of chemokines and cytokines involved in the regulation of monocyte and lymphocyte migration and retention in atherosclerotic lesions. (C) 2008 Elsevier Ireland Ltd. All rights reserved.