Efficacy of local phytotherapy in the nonsurgical treatment of periodontal disease: A systematic review.

Herbal drugs are commonly used in the treatment of several diseases, including periodontitis. So far, no systematic review had evaluated the evidence regarding the efficacy of these agents in the treatment of periodontal disease. Therefore, the purpose of this review was to evaluate the effect of local application of phytotherapic agents as adjuncts to scaling and root planing (SRP), compared to SRP alone, on clinical parameters of chronic periodontal patients. Only randomized controlled trials of at least 3 months follow-up, of SRP alone in association with local phytotherapic agents were included. MEDLINE (PubMed), Google Scholar and LILACS databases were searched for articles published up to October 2016. Random-effects meta-analyses were conducted for clinical attachment level and probing pocket depth (PPD) change after treatment. Of 1861 papers potentially relevant, 7 were included. All studies showed that periodontal treatment in association with local phytotherapic delivery promotes a significant PPD reduction and the majority of them showed clinical attachment level gain. The local use of phytotherapy as an adjunct to SRP may promote additional benefits in PPD reduction and clinical attachment level gain. However, these results must be interpreted with caution due to the small sample size, high risk of bias and heterogeneity of the studies.

Inactivation of the spxA1 or spxA2 gene of Streptococcus mutans decreases virulence in the rat caries model.

In oral biofilms, the major environmental challenges encountered by Streptococcus mutans are acid and oxidative stresses. Previously, we showed that the transcriptional regulators SpxA1 and SpxA2 are involved in general stress survival of S. mutans with SpxA1 playing a primary role in activation of antioxidant and detoxification strategies whereas SpxA2 serves as a back up activator of oxidative stress genes. We have also found that spxA1 mutant strains (∆spxA1 and ∆spxA1∆spxA2) are outcompeted by peroxigenic oral streptococci in vitro and have impaired abilities to colonize the teeth of rats fed a highly cariogenic diet. Here, we show that the Spx proteins can also exert regulatory roles in the expression of additional virulence attributes of S. mutans. Competence activation is significantly impaired in Δspx strains and the production of mutacin IV and V is virtually abolished in ΔspxA1 strains. Unexpectedly, the ∆spxA2 strain showed increased production of glucans from sucrose, without affecting the total amount of bacteria within biofilms when compared with the parent strain. By using the rat caries model, we showed that the capacity of the ΔspxA1 and ΔspxA2 strains to cause caries on smooth tooth surfaces is significantly impaired. The ∆spxA2 strain also formed fewer lesions on sulcal surfaces. This report reveals that global regulation via Spx contributes to the cariogenic potential of S. mutans and highlights that animal models are essential in the characterization of bacterial traits implicated in virulence.

The effect of an inhibitor of gut serotonin (LP533401) during the induction of periodontal disease.

BACKGROUND AND OBJECTIVE: LP533401 is an inhibitor of tryptophan hydroxylase 1, which regulates serotonin production in the gut. Previous work indicates that LP533401 has an anabolic effect in bone. Thus, we hypothesized that inhibition of gut serotonin production may modulate the host response in periodontal disease. In this study, we aimed to analyze the effects of LP533401 in a rat periodontitis model to evaluate the role of gut serotonin in periodontitis pathophysiology. MATERIAL AND METHODS: Twenty-four rats were divided into three groups: treated group (T: ligature-induced periodontal disease and LP533401, 25 mg/kg/d) by gavage; ligature group (L: ligature-induced periodontal disease only); and control group (C: without ligature-induced periodontal disease). After 28 d, radiographic alveolar bone support was measured on digital radiographs, and alveolar bone volume fraction, tissue mineral density and trabeculae characteristics were quantified by microcomputed tomography in the right hemi-mandible. Left hemi-mandibles were decalcified and alveolar bone loss, attachment loss and area of collagen in the gingiva were histologically analyzed. RESULTS: Significant difference between the L and C groups was found, confirming that periodontal disease was induced. We observed no difference between the T and L groups regarding alveolar bone destruction and area of collagen. CONCLUSION: LP533401 (25 mg/kg/d) for 28 d does not prevent bone loss and does not modulate host response in a rat model of induced periodontal disease.

Dependence of cytokine levels on the sex of experimental animals: a pilot study on the effect of oestrogen in the temporomandibular joint synovial tissues.

The aim of this study was to evaluate the effects of 17ß-oestradiol (E2) on cartilage thickness and cytokine levels in the temporomandibular joint (TMJ). Thirty rats (15 female, 15 male) were orchidectomized (ORX), ovariectomized (OVX), or sham-operated. After 21 days, animals were assigned to six groups: (1) sham-ORX; (2) ORX; (3) ORX+E2; (4) sham-OVX; (5) OVX; and (6) OVX+E2. Treatments were administered daily for 21 days. The thickness of cartilage layers (fibrous, proliferative, maturation, and hypertrophic) and cytokine levels (interleukins IL-1α, IL-1ß, IL-6, and tumour necrosis factor alpha (TNF-α)) were measured by histomorphometry and ELISA, respectively. Kruskal-Wallis/Dunn's tests were used (alpha=5%). Sham-ORX showed thicker layers than ORX+E2, but not thicker than ORX. All layers, except the hypertrophic layer, were thicker in sham-OVX than OVX or OVX+E2. Although IL-1ß levels were higher in castrated animals, E2 did not affect the level of this cytokine. IL-1α levels were higher in both ORX (P=0.0010) and ORX+E2 (P=0.0053) than in sham-ORX. However, E2 decreased IL-1α levels in OVX (P=0.0129). When compared to sham-ORX/OVX, IL-6 levels were not affected by E2 in males but were reduced in OVX (P=0.0079) and increased in OVX+E2 (P=0.0434). Levels of TNF-α were reduced by E2 in both ORX+E2 and OVX+E2. E2 treatment caused gender- and layer-dependent changes in the cartilage. Castration increased all cytokine levels, except for IL-6, without respect to gender.

Catecholamines promote the expression of virulence and oxidative stress genes in Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Stress has been identified as an important risk factor in the development of many infectious diseases, including periodontitis. Porphyromonas gingivalis, a gram-negative oral anaerobic bacterium, is considered an important pathogen in chronic periodontitis. Microorganisms, including P. gingivalis, that participate in infectious diseases have been shown to respond to catecholamines released during stress processes by modifying their growth and virulence. Therefore, the purpose of this study was to evaluate the effects of adrenaline and noradrenaline on the growth, antimicrobial susceptibility and gene expression in P. gingivalis. MATERIAL AND METHODS: P. gingivalis was incubated in the presence of adrenaline and noradrenaline (100 µm) for different time-periods in rich (Tryptic soy broth supplemented with 0.2% yeast extract, 5 µg/mL of hemin and 1 µg/mL of menadione) and poor (serum-SAPI minimal medium and serum-SAPI minimal medium supplemented with 5 µg/mL of hemin and 1 µg/mL of menadione) media, and growth was evaluated based on absorbance at 660 nm. Bacterial susceptibility to metronidazole was examined after exposure to adrenaline and noradrenaline. The expression of genes involved in iron acquisition, stress oxidative protection and virulence were also evaluated using RT-quantitative PCR. RESULTS: Catecholamines did not interfere with the growth of P. gingivalis, regardless of nutritional or hemin conditions. In addition, bacterial susceptibility to metronidazole was not modified by exposure to adrenaline or noradrenaline. However, the expression of genes related to iron acquisition (hmuR), oxidative stress (tpx, oxyR, dps, sodB and aphC) and pathogenesis (hem, hagA and ragA) were stimulated upon exposure to adrenaline and/or noradrenaline. CONCLUSION: Adrenaline and noradrenaline can induce changes in gene expression related to oxidative stress and virulence factors in P. gingivalis. The present study is, in part, a step toward understanding the stress-pathogen interactions that may occur in periodontal disease.

Proteomic analysis of Porphyromonas gingivalis exposed to nicotine and cotinine.

BACKGROUND AND OBJECTIVE: Smokers are more predisposed than nonsmokers to infection with Porphyromonas gingivalis, one of the most important pathogens involved in the onset and development of periodontitis. It has also been observed that tobacco, and tobacco derivatives such as nicotine and cotinine, can induce modifications to P. gingivalis virulence. However, the effect of the major compounds derived from cigarettes on expression of protein by P. gingivalis is poorly understood. Therefore, this study aimed to evaluate and compare the effects of nicotine and cotinine on the P. gingivalis proteomic profile. MATERIAL AND METHODS: Total proteins of P. gingivalis exposed to nicotine and cotinine were extracted and separated by two-dimensional electrophoresis. Proteins differentially expressed were successfully identified through liquid chromatography-mass spectrometry and primary sequence databases using MASCOT search engine, and gene ontology was carried out using DAVID tools. RESULTS: Of the approximately 410 protein spots that were reproducibly detected on each gel, 23 were differentially expressed in at least one of the treatments. A particular increase was seen in proteins involved in metabolism, virulence and acquisition of peptides, protein synthesis and folding, transcription and oxidative stress. Few proteins showed significant decreases in expression; those that did are involved in cell envelope biosynthesis and proteolysis and also in metabolism. CONCLUSION: Our results characterized the changes in the proteome of P. gingivalis following exposure to nicotine and cotinine, suggesting that these substances may modulate, with minor changes, protein expression. The present study is, in part, a step toward understanding the potential smoke-pathogen interaction that may occur in smokers with periodontitis.

Aggregatibacter actinomycetemcomitans serotypes infections and periodontal conditions: a two-way assessment.

This study investigated a large population of individuals positive for A. actinomycetemcomitans and performed a two way analysis assessing the relation between the different serotypes of the bacterium and periodontal conditions. The serotypes analysis (serotypes a, b, c, d, e, f) showed that out of the 204 selected individuals positive for A. actinomycetemcomitans, 152 were positive for a single serotype, 27 showed a variable mixed infection and 25 individuals were not positive for any of the serotypes tested. Serotypes a, b and c were largely found (98%), and serotype c was the most prevalent. Serotypes d, e, and f were either not detected or relatively rare. It was also verified that in non-periodontitis individuals, serotypes a and c were more prevalent (p<0.05); in individuals with mild or moderate/severe chronic periodontitis serotype c was also more common (p<0.05); and aggressive periodontitis subjects showed high prevalence of both serotypes b and c (p<0.05). In conclusion, our study showed that serotype c was the most prevalent in both diseased and healthy subjects. Aggressive periodontitis subjects were not exclusively associated with A. actinomycetemcomitans serotype b. Non-typeable strains were either not detected or were relatively infrequent, and serotypes d and f were not detected in the examined Brazilian population.

Prevalence of periodontopathogens in a black Brazilian secluded community matched with a black urban population.

OBJECTIVE: To evaluate the prevalence of periodontopathogens according to periodontal profile in a black Brazilian secluded community matched with an urban black population. PARTICIPANTS: A total of 84 subjects were selected, 42 (mean age 25.7 sd 18.0 years) from a secluded community called Santo Antonio do Guapore (SAG) and 42 (mean age 25.4 sd 18.1 years) from an urban area of Sao Paulo State (SPT). METHODS: Participants received clinical examinations as follows: periodontal pocket depth; clinical attachment loss; plaque and gingival indexes. After examination, the secluded population was classified as periodontal health (13), gingivitis (15) or periodontitis (14). Then, 182 urban volunteers were screened and 42 subjects were selected matched for the variables: periodontal diagnosis, age (+/- 2 years) and gender. Samples were taken for microbial analysis. Genomic DNA for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Campylobacter rectus, Tannerella forsythia and Prevotella intermedia was provided by polymerase chain reaction. RESULTS: Except for C. rectus, all pathogens were present in both groups with no statistically significant difference. In particular, C. rectus was more prevalent only in gingivitis subjects from the SPT group (p<0.05). A high frequency of periodontopathogens was related to the severity of periodontal disease. CONCLUSION: In general, the prevalence of the examined periodontopathogens in this study did not differ between a secluded black Brazilian population and an urban black population.

Protease-activated receptor-2 (PAR(2)) in human periodontitis.

No evidence for the role of protease-activated receptor-2 (PAR(2)) in human periodontal disease has been demonstrated so far. Thus, we sought to investigate the expression of PAR(2) mRNA in chronic periodontitis, and to examine whether its expression is related to the presence of PAR(2) potential activators. Microbiological and gingival crevicular fluid samples were collected from individuals with chronic periodontitis and control individuals, and the presence of neutrophil serine proteinase 3 (P3) and Porphyromonas gingivalis was evaluated. PAR(2) mRNA expression was higher (p < 0.001) in those with chronic periodontitis compared with control individuals, and it was statistically decreased (p = 0.0006) after periodontal treatment. Furthermore, those with chronic periodontitis presented higher (p < 0.05) levels of IL-1alpha, IL-6, IL-8, and TNF-alpha, total proteolytic activity, P. gingivalis prevalence, and P3mRNA expression compared with control individuals. We conclude that PAR(2) mRNA expression and its potential activators are elevated in human chronic periodontitis, therefore suggesting that PAR(2) may play a role in periodontal inflammation.

A PK/PD approach on the effects of clarithromycin against oral and nasal microbiota of healthy volunteers.

OBJECTIVE: To assess the pharmacokinetics of clarithromycin (CLR) and its effects on oral and nasal microbiota in healthy volunteers in an open, randomized, two-period crossover design. METHODS: A single 500 mg oral dose of CLR (Group 1: Merck; Group 2: Klaricid) was administered observing a 1-week interval between doses. Blood samples were collected from pre-dose to 24 h. Plasmatic concentrations of CLR were quantified by the LC-MS-MS method. Saliva and nasal mucosa swabs were obtained previously and after 1.33, 2, 6 and 12 h of drug administration. Pharmacokinetics and PK/PD (t > MIC, %t > MIC and AUC0-24/MIC ratio) parameters were estimated. The microorganism counts were obtained on different culture media. RESULTS: No statistically significant differences were observed between the two formulations (p > 0.05) regarding the pharmacokinetic parameters. Total microorganisms, staphylococci and streptococci counts did not show statistical differences (p > 0.05) between the two groups during each sampling time. Considering the microorganisms of each group, no statistically significant differences were found after drug administration, but all differed from pre-dose counts (p < 0.05). The observed t > MIC ranged from 14.45 h (+/- 1.69) to 1.19 h (+/- 2.17) considering MICs of 0.25 microg/ml and 2.0 microg/ml, respectively. There was no correlation between any t > MIC, %t > MIC or AUC0-24 and bacterial reduction (between 0- and 12-h periods). However, the profile of reduction of microorganisms in both saliva and nasal samples were compatible with high values of t > MIC verified for both clarithromycin formulations. CONCLUSION: Both formulations of clarithromycin had similar pharmacokinetics and efficacy.

Plasma and salivary amoxicillin concentrations and effect against oral microorganisms.

Plasma and salivary amoxicillin (AMO) concentrations were quantified following a single oral dose (875 mg) of two formulations of AMO (Amoxicillin-EMS Sigma Pharma and Amoxil BD 875 mg). In addition, the effect of amoxicillin against oral microorganisms was accessed. The open, randomized, two-period crossover study was carried out in 20 volunteers. Saliva and blood samples were collected at 0, 0.5, 1, 2, 4, 8 and 12 h after drug administration, and quantified using HPLC-ESI-MS and HPLC, respectively. Streptococci counts, anaerobe counts and total microorganism counts were obtained. No differences were observed between formulations (p > 0.05) in the plasma and salivary AMO concentrations and the pharmacokinetic parameters (C(max), t(max), AUC(0-8), and AUC(0-infinity)) also showed no statistically significant differences between formulations (p > 0.05). Microorganism counts for the two formulations at all sampling times did not differ (p > 0.05) but all microorganism counts at 60 min post-dose showed a significant decrease (p < 0.05). Amoxicillin was effective in reducing oral microorganism levels up to 12 h post-dose.

Comparative bioavailability of clarithromycin formulations in healthy brazilian volunteers.

OBJECTIVE: To compare the bioavailability of clarithromycin 500 mg tablets (Merck S.A Industrias Quimicas, Sao Paulo, SP, Brazil, used as test formulation) and Klaricid (Abbott Laboratórios do Brasil Ltda, Sao Paulo, SP, Brazil, used as reference formulation) in 24 healthy volunteers. MATERIAL AND METHODS: The study was conducted using an open, randomized, two-period crossover design with one-week interval between doses. Blood samples were collected at pre-dose, 0.33, 0.66, 1, 1.33, 1.66, 2, 2.5, 3, 4, 6, 8, 10, 12, 16, 20 and 24 hours after the administration. AUC was calculated by the trapezoidal rule extrapolation method. Cmax and tmax were compiled from the plasmatic concentration-time data. Analysis of variance was carried out using logarithmically transformed AUC(0-inf), AUC(0-24 h), Cmax and untransformed tmax. RESULTS: Intraindividual coefficient of variation (CV%) values were 14.25% and 12.62%, respectively for Cmax and AUC(0-24 h). The geometric mean values (+/- SD) for AUC(0-24 h) (microg x h/ml), AUC(0-inf) (microg x h/ml), and Cmax (microg/ml) for test medication were 18.56 (+/- 6.87), 18.8 (+/- 5.70) and 2.45 (+/- 0.88); the obtained values for reference medication were 18.29 (+/- 5.39), 19.10 (+/- 7.21) and 2.5 (+/- 0.69). 90% Cl for clarithromycin geometric mean of AUC(0-24 h), AUC(0-inf) and Cmax ratios (test/reference) were: 93.6-105.9%, 93.8-106.2% and 89- 103.2%. CCONCLUSION The test medication was considered bioequivalent to the reference medication based on the rate and extent of absorption.

Comparative bioavailability of 875 mg amoxicillin tablets in healthy human volunteers.

OBJECTIVE: To compare the bioavailability of amoxicillin 875 mg tablets (EMS Sigma Pharma used as test formulation) and Amoxil BD 875 mg tablets (GlaxoSmithKline used as reference formulation) in 26 healthy volunteers. MATERIAL AND METHODS: 26 healthy volunteers (13 males and 13 females) received each formulation in an open, 2 x 2 crossover, randomized study with seven days of washout period between doses. Plasma samples were obtained over a 12-hour interval after administration. Plasmatic amoxicillin concentrations were obtained by combined reversed-phase liquid chromatography and mass spectrometry with positive ion electrospray ionization using the select ion monitoring method. AUC was calculated by the trapezoidal rule extrapolation method. Cmax and tmax were compiled from the plasmatic concentration-time data. Analysis of variance was carried out using logarithmically transformed AUC0-inf, AUC0-12 h, Cmax and untransformed tmax. RESULTS: The mean values (+/- SD) for AUC0-12 h (microg x h x ml(-1)), AUC0-inf (microg x h x ml(-1)), Cmax (microg x ml(-1)), t1/2 (h) and tmax (h), were, respectively: 55.42 (+/- 16.85), 55.42 (+/- 16.85), 18.59 (+/- 6.3), 1.49 (+/- 1.57) and 2.04 (+/- 0.75) concerning the test formulation, and 51.11 (+/- 18.9), 51.29 (+/- 19.12), 17.83 (+/- 5.86), 1.52 (+/- 1.31) and 2.02 (+/- 0.87) concerning the reference formulation. Confidence intervals (90%) of amoxicillin means of AUC0-12 h and Cmax ratios (test/reference) were: 0.961-1.149 and 0.914-1.142, respectively, agreeing with the bioequivalence criteria established by the Brazilian National Health Surveillance Agency. CONCLUSION: Both formulations were bioequivalent based on both the rate and extent of absorption.

The contribution of mild thiamine deficiency and ethanol consumption to central cholinergic parameter dysfunction and rats' open-field performance impairment.

We studied at the biochemical, morphological, and behavioral levels the effect of chronic ethanol consumption, associated or not with a mild thiamine deficiency episode. We found that (i) thiamine deficiency induced a significant decrease of the acetylcholinesterase (AChE) activity both in cortex and hippocampus; (ii) chronic ethanol treatment has no effect on cortical AChE activity, but induced a significant decrease of hippocampal enzyme activity; (iii) the reduction in cortical and hippocampal AChE activity induced by chronic ethanol treatment associated with a 1-week thiamine deficiency was also significant and was greater than that induced by ethanol alone. Furthermore, either chronic ethanol or thiamine deficiency induced a significant decrease in the release of acetylcholine (ACh) in the stimulated condition using high potassium concentration; and when both treatments were associated the decrease was even greater. In the unstimulated condition, the reduction in the release of ACh was greater for ethanol treatment than for thiamine deficiency. Open-field tests showed that only in the "sniffing" category were there significant differences among the experimental groups. No morphological change was detected by optical microscopy, suggesting that the injury process was in its initial stages in which only functional and behavioral changes are displayed. In addition, our biochemical results indicate that cortical cholinergic susceptibilities to ethanol and thiamine deficiency are significantly different.

Uso de um sistema de ELISA para detecçäo de anticorpos contra herpesvírus eqüino 1 (HVE-1) em éguas gestantes assintomáticas e potros recém-nascidos / Use of an ELISA system for detection of equine herpesvirus 1 (EHV-1) antibodies in non-symptomatic pregnant mares and neonatal foals

Um sistema de ELISA foi utilizado para se estudar a prevalência do herpesvirus eqüino 1 (HVE-1) em plantéis de cavalos brasileiros vacinados e näo vacinados. Amostras de soros foram coletadas de éguas gestantes assintomáticas, antes e após o parto e de seus respectivos potros recém-nascidos. O antígeno foi preparado de uma fraçäo viral purificada (HVE-1 amostra Ab1). O ponto de corte (absorvância de 0,3516) foi determinado considerando o limite máximo para os valores negativos. Esse valor determinou sensibilidade de 100 por cento e especificidade de 94,7 por cento, utilizando um banco de soros de éguas gestantes e fetos. No estudo observou-se alta correlaçäo entre o ELISA e a soro-neutralizaçäo para detecçäo de anticorpos específicos contra o EHV-1

Chronic ethanol consumption impairs spatial remote memory in rats but does not affect cortical cholinergic parameters.

We have studied learning, memory and cortical cholinergic parameters after oral administration of 20% v/v ethanol solution to male Fisher rats for 6 months. A group of rats were trained to behave efficiently in an eight-arm radial maze and after that split into two subgroups submitted to ethanol or control treatment. Ethanol-treated rats had more difficulty in relearning the same task 1 year later, compared to ethanol-untreated rats (control). Differences in working memory performance were found, but only in the first 10 training sessions. Another group of rats, which had not been pretrained, was also split into two subgroups submitted to ethanol or control treatment. After that, these rats were trained in the radial maze task for the first time. No significant difference was found between the reference memory performance of the untreated subgroup and the treated one. These two subgroups did not significantly differ in their working memory performance either. Moreover, there were no significant differences between treated and control subjects in the following biochemical brain cortical parameters: in vitro acetylcholinesterase (AChE) activity, and stimulated acetylcholine (ACh) release. This work presents an experimental design that allows assessment of remote memory performance after ethanol chronic consumption and shows that the experimental subject is able to retain the behaviors learned 1 year before. It was concluded that chronic ethanol treatment may cause retrograde amnesia, which does not seem to be linked with a cortical cholinergic deficit.