Detection and quantification of prostaglandin E2 in saliva by liquid chromatography-tandem mass spectrometry using microextraction by packed sorbent.

The detection of eicosanoids in saliva samples can assist pharmacokinetic/pharmacodynamic studies due to the facility of obtaining samples, minimal discomfort and high adherence of volunteers to the study. The present study enabled determine prostaglandin E2 concentrations in saliva samples, using a microextraction by packed sorbent methodology and subsequent detection in liquid chromatography-tandem mass spectrometry. Twelve volunteers underwent scaling and coronary-radicular polishing of the upper molars and sequential saliva collections: 0.25-96 h after ingestion of a 600 mg ibuprofen tablet, to quantify prostaglandin E2 concentrations. There was an increase in the level of prostaglandin E2 with a significant difference after the dental procedure (0.25 h) compared to 11, 24, 48 and 72 h (*p < 0.05). After taking the drug, these levels begin to decrease up to 5 h, returning to normal in the subsequent hours. The method was developed and validated with linearity between 2.4 and 1250 ng/mL and r2 above 0.9932. The limit of quantitation was about 2.4 ng/mL. The coefficients of variation and the relative standard errors of the accuracy and precision analyzes were < 15%. The proposed extraction and analysis methodology proved to be efficient, fast and promising for pharmacokinetic/pharmacodynamic assays after using anti-inflammatory drugs.

Could the photobiomodulation therapy induce angiogenic growth factors expression from dental pulp cells?

This study aimed to evaluate the effect of different photobiomodulation (PBM) radiant exposures on the viability, proliferation, and gene expression of pulp fibroblasts from human primary teeth (HPF) involved in the pulp tissue repair. HPF were irradiated with Laser InGaAlP (Twin Flex Evolution, MMOptics®) at 660-nm wavelength (red); single time, continuous mode, 0.04-cm2 laser tip area, and 0.225-cm laser tip diameter, keeping the distance of 1 mm between the laser beam and the cell culture. The doses used were between 1.2 and 6.2 J/cm2 and were evaluated at the 6 h, 12 h, and 24 h after PBM. MTT and crystal violet assays evaluated the cell viability and proliferation. RT-PCR verified VEGF and FGF-2 mRNA expression. A blinded examiner analyzed the data through two-way ANOVA followed by Tukey test (p < 0.05). The groups with higher powers (10 mW, 15 mW, 20 mW, and 25 mW), shortest application periods (10 s), and radiant exposures between 2.5 and 6.2 J/cm2 exhibited statistically higher viability than that of the groups with small power (5 mW), longer application period (50 s), and radiant exposure of 6.2 J/cm2 (p < 0.05). VEGF and FGF-2 mRNA expression were observed at the three evaluated periods (6 h, 12 h, and 24 h) and the highest expression was in the shortest period (p < 0.05). All radiant exposures maintained HPF viable. The period of 6 h after irradiation showed statistically greater gene expression for both growth factors than other periods. VEGF mRNA had no differences among the dosimetries studied. The best radiant exposures for FGF-2 gene expression were 2.5 J/cm2 and 3.7 J/cm2.

What is the response profile of deciduous pulp fibroblasts stimulated with E. coli LPS and E. faecalis LTA?

BACKGROUND: Oral fibroblast immunological responses to bacterial stimuli are well known. However, there are few studies about pulp fibroblasts from deciduous teeth (HDPF) responses, which are important for the treatment of pulp infections in children. The aim of this study was to evaluate expression and production of inflammatory cytokines and chemokines by HDPF when challenged with bacterial antigens normally present in advanced caries lesions. METHODS: Triplicate HDPF from 4 children (n = 4; 2 boys and 2 girls) were cultured by explant technique and challenged or not with Escherichia coli lipopolysaccharide/1 µg/mL (EcLPS) or Enterococcus faecalis lipoteichoic acid/1 µg/mL (EfLTA) for 6 and 24 h. Most of published studies employed immortalized cells, i.e., without checking possible gender and genetic variables. mRNA expression and protein production were evaluated by RT-qPCR and ELISA MILLIPLEX®, respectively, for Interleukin (IL)-1α, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, Chemokine C-C motif ligand 2/monocyte chemoattractant protein 1 (CCL2/MCP-1), Chemokine C-C motif ligand 3/macrophage inflammatory protein 1-alpha (CCL3/MIP1-α), Chemokine C-C motif ligand 5/ regulated on activation, normal T cell expressed and secreted (CCL5/RANTES), C-X-C motif chemokine 12/ stromal cell-derived factor 1 (CXCL12/SDF-1), Tumor Necrosis Factor-alpha (TNF-α), Interferon-gamma (IFN γ), Vascular Endothelial Growth Factor (VEGF), Colony stimulating factor 1 (CSF-1) and Macrophage colony-stimulating factor (M-CSF). RESULTS: EcLPS increased IL-1α, IL-1ß, IL-8, CCL2, CCL5, TNF-α and CSF-1 mRNA and protein levels while EfLTA was only able to positively regulate gene expression and protein production of IL-8. CONCLUSION: The results of the present study confirmed our hypothesis, since pulp fibroblasts from deciduous teeth are capable of increasing gene expression and protein production after being stimulated with EcLPS and EfLTA.

A Biobank of Stem Cells of Human Exfoliated Deciduous Teeth: Overview of Applications and Developments in Brazil.

A biobank is an organized collection of biological human material and its associated information stored for research according to regulations under institutional responsibility, without commercial purposes, being a mandatory and strategical activity for research, regenerative medicine, and innovation. Stem cells have largely been employed in research and frequently stored in biobanks, which have been used as an essential source of biological materials. Stem cells of human exfoliated deciduous teeth (SHED) are stem cells which have a high multipotency and can be easily obtained. Besides, this extremely accessible tissue has advantages with respect to storage, as the SHED obtained in childhood can be used in later life, which implies the necessity for the creation and regulation of biobanks. The proper planning for the creation of a biobank includes knowledge of the material types to be stored, requirements regarding handling and storage conditions, storage time, and room for the number of samples. Thus, this study aimed to establish an overview of the development of a SHED biobank. Ethical and legal standardization, current applications, specific orientations, and challenges for the implementation of a SHED biobank were discussed. Through this overview, we hope to encourage further studies to use SHED biobanks.

Antifungal activity of Punicalagin-nystatin combinations against Candida albicans.

OBJECTIVES: Oral candidiasis is the most common opportunistic fungal infection of oral mucosa and results from an overgrowth of Candida, especially Candida albicans. The potential anti-C. albicans and cytotoxicity of punicalagin (PCG), isolated from Punica granatum, alone or with nystatin (NYS) were evaluated. METHODS: Activity of compounds alone or in combinations was determined against two C. albicans strains (ATCC 90028 and SC5314). Minimal inhibitory concentration (MIC)-50 and Minimum Fungicidal Concentration (MFC) were assessed by XTT assay and CFU counts, respectively. For combinations, determination of fractional inhibitory concentration index was performed. Ergosterol pathway was investigated as a possible PCG antifungal mechanism. Cytotoxicity assays were undertaken on human primary oral keratinocytes and gingival fibroblasts incubated with antifungal concentrations of PCG and/or NYS for 24 hr. RESULTS: Combination of NYS and PCG increased antifungal efficacy, compared with compounds tested alone. Combinations 4 (PCG-6.25 µg/ml; NYS-3.9 µg/ml) and 5 (PCG-12.5 µg/ml; NYS-1.95 µg/ml) were more effective since they reduced the MIC-50 of PCG (50 µg/ml) by 8 and 4 times, respectively, increased the candidal inhibition and nullified the PCG cytotoxicity for keratinocytes. PCG antifungal mechanism did not involve ergosterol biosynthesis pathway. CONCLUSIONS: The favorable outcomes for combination of PCG and NYS encourage further testing this therapeutic strategy against C. albicans.

Angiogenic protein synthesis after photobiomodulation therapy on SHED: a preliminary study.

This study evaluated the viability, proliferation, and protein expression after photobiomodulation (PBM) of stem cell from human exfoliated deciduous teeth (SHED). The groups were the following: G1 (2.5 J/cm2), G2 (3.7 J/cm2), and control (not irradiated). According to the groups, cells were irradiated with InGaAlP diode laser at 660 nm wavelength, continuous mode, and single time application. After 6 h, 12 h, and 24 h from irradiation, the cell viability and proliferation, and the protein expression were analyzed by MTT, crystal violet, and ELISA multiplex assay, respectively. Twenty-four hours after PBM, SHED showed better proliferation. Over time in the supernatant, all groups had an increase at the levels of VEGF-C, VEGF-A, and PLGF. In the lysate, the control and G2 exhibited a decrease of the VEGF-A, PECAM-1, and PLGF expression, while control and G3 decreased VEGF-C, VEGF-A, and PDGF expression. The dosimetries of 2.5 J/cm2 and 3.7 J/cm2 maintained viability, improved proliferation, and synthesis of the angiogenic proteins in the supernatant in the studied periods on SHED.

Photobiomodulation effect on angiogenic proteins produced and released by dental pulp cells.

OBJECTIVE: To verify the photobiomodulation effect on angiogenic proteins produced and released by dental human pulpal fibroblasts (HPFs). MATERIAL AND METHODS: HPFs were irradiated with 660-nm low-level laser at fluences of 2.5 J/cm2 and 3.7 J/cm2. The control group was not irradiated. MTT, crystal violet, and ELISA assays respectively verified viability, proliferation, and angiogenic protein (supernatant/lysate) at 6 h, 12 h, and 24 h after photobiomodulation. Capillary-like structure formation assay verified functional role. Two-way ANOVA/Tukey's test and ANOVA/Bonferroni's multiple comparisons test respectively verified cell viability/proliferation and intragroup and intergroup comparisons of protein synthesis (p < 0.05). RESULTS: Irradiated and non-irradiated HPFs showed statistically similar cell viability and proliferation pattern. Intragroup comparisons showed similar patterns of protein synthesis for all groups: VEGF-A, VEGF-C, and vascular endothelial growth factor receptor 1 (VEGFR1) increased significantly in the supernatant, while FGF-2 and VEGF-A increased significantly in the lysate. The lower fluence significantly increased BMP-9 (6 h) in the supernatant and VEGFR1 (6 h and 12 h) and VEGF-D (24 h) in the lysate, while the higher fluence significantly increased BMP-9 (6 h) in the supernatant and VEGFR1 (12 h) in the lysate. Regardless of the time, both fluences statistically downregulated placental growth factor (PLGF) and PDGF secretion. Both fluences statistically decreased VEGF-A secretion (24 h) and PLGF production (6 h). CONCLUSION: Photobiomodulation produced stimulatory effects on angiogenic protein secretion by pulp fibroblasts. In terms of photobiomodulation, over time, both fluences significantly increased the secretion of VEGF-A, VEGF-C, and VEGFR1 and significantly upregulated BMP-9 (6 h) in the supernatant; for capillary-like structure formation, the fluence of 2.5 J/cm2 was better than the fluence of 3.7 J/cm2. CLINICAL RELEVANCE: This study results addressed effective photobiomodulation parameters tailored for pulp angiogenesis.

Recognition of Candida albicans by gingival fibroblasts: The role of TLR2, TLR4/CD14, and MyD88.

Recent evidence indicates that nonprofessional immune cells such as epithelial cells, endothelial cells, and fibroblasts also contribute to innate immunity via secretion of cytokines. Fibroblasts are the principal type of cell found in the periodontal connective tissues and they are involved in the immune response during periodontal disease. The role of fibroblasts in the recognition of pathogens via Toll-like receptors (TLRs) has been established; however, few studies have been conducted concerning the involvement of innate immune receptors in the recognition of Candida albicans by gingival fibroblast. In the current study, we investigate the functional activity of TLR2, cluster of differentiation 14 (CD14), and myeloid differentiation primary response gene 88 (MyD88) molecules in the recognition of C. albicans by gingival fibroblast. First, we identified that gingival fibroblasts expressed TLR2, TLR3, and TLR4. Our results showed that TLR agonists had no effect on these receptors' expression by TLR2, MyD88, and CD14-deficient cells. Notably, C. albicans and a synthetic triacylated lipoprotein (Pam3CSK4) induced a remarkable increase of TLR3 expression on MyD88-deficient gingival fibroblasts. TLR4 expression levels were lower than TLR2 and TLR3 levels and remained unchanged after TLR agonist stimulation. Gingival fibroblasts presented morphological similarities; however, TLR2 deficiency on these cells leads to a lower proliferative response, whereas the deficiency on CD14 expression resulted in lower levels of type I collagen by these cells. In addition, the recognition of C. albicans by gingival fibroblasts had an effect on the secretion of cytokines and it was dependent on a specific recognition molecule. Specifically, tumor necrosis factor-α (TNF-α) production after the recognition of C. albicans was dependent on MyD88, CD14, and TLR2 molecules, whereas the production of interleukin-1ß (IL-1ß) and IL-13 was dependent on TLR2. These findings are the first to describe a role of gingival fibroblast in the recognition of C. albicans and the pathways involved in this process. An understanding of these pathways may lead to alternative treatments for patients with periodontal disease.

Effects of PBM in different energy densities and irradiance on maintaining cell viability and proliferation of pulp fibroblasts from human primary teeth.

This study aimed to compare the effects of photobiomodulation (PBM) in different energy densities and irradiances on maintaining cell viability, and proliferation of pulp fibroblasts from human primary teeth (HPF) were cultured in DMEM and used between the fourth and eighth passages. Then, HPF were irradiated with the following different energy densities: 1.25 J/cm2 (a), 2.50 J/cm2 (b), 3.75 J/cm2 (c), 5.00 J/cm2 (d), and 6.25 J/cm2 (e); but varying either the time of irradiation (groups 1a-1e) or the output power (groups 2a-2e). Positive (groups 1f and 2f) and negative controls (groups 1g and 2g), respectively, comprised non-irradiated cells grown in regular nutritional conditions (10% fetal bovine serum [FBS]) and under nutritional deficit (1% FBS). Cell viability and proliferation were respectively assessed through MTT and crystal violet (CV) assays at 24, 48, and 72 h after irradiation. Statistical analysis was performed by two-way ANOVA, followed by Tukey test (P < 0.05). The negative controls showed significantly lower viability in relation to most of the corresponding subgroups, both for MTT and CV assays. For both assays, the intragroup comparison showed that the periods of 24 h exhibited lower viability than the periods of 48 and 72 h for most of the subgroups, except the negative controls with lower viability. The different irradiation protocols (equal energy densities applied with different irradiances) showed no statistically significant differences on cell viability and proliferation at the evaluated periods. The proposed PBM in different energy densities and irradiance did not affect the viability and proliferation of pulp fibroblasts from human primary teeth.

Expression of Secreted Aspartyl Proteinases in an Experimental Model of Candida albicans-Associated Denture Stomatitis.

PURPOSE: Candida albicans is known to produce secreted aspartyl proteinases (SAPs) to aid adhesion, invasion, and host tissue destruction. SAPs may contribute to denture stomatitis (DS) pathogenesis. The aim of this study was to develop an in vivo experimental model for Candida-associated DS that allows the analysis of SAP2, SAP5, and SAP9 expression by C. albicans from biofilm induced on the denture surface. MATERIALS AND METHODS: Thirty-five male Wistar rats were divided into three groups: control, denture, and denture/Candida group. The last two groups remained with dentures for 2, 4, and 6 days, with or without induced biofilm. SAP expression was concomitant with leukocyte counts as well as clinical and histological changes shown by animal palate. RESULTS: The signs observed at 4 days in the denture/Candida group were clinically closer to the Candida-associated DS, showing a significant increase of neutrophils and decrease of lymphocytes in peripheral blood, presence of inflammation signs on the palate similar to DS Newton type I, and fungal invasion in the epithelial layer. Accordingly, the denture/Candida group at 4 days presented the highest relative expression of all SAPs studied. CONCLUSION: The results showed a coincidence between SAP expression and clinical, microscopic, and blood data. Finally, the molecular findings were consistent with the virulence capacities of C. albicans from biofilm formed on the denture resin, which possibly allowed epithelial invasion by the fungus.

Palatal mucosa derived fibroblasts present an adaptive behavior regarding cytokine secretion when grafted onto the gingival margin.

BACKGROUND: Considering that grafted gingival tissue might have to be adapted to the receptor area and that fibroblasts have the ability to respond to bacterial stimuli through the release of various cytokines, this study investigated whether fibroblasts from the palatal mucosa behave differently when grafted onto the gingival margin regarding cytokine secretion. METHODS: Biopsies from the palatal mucosa were collected at the time of free gingival graft surgery, and after four months re-collection was performed upon surgery for root coverage. Fibroblasts were isolated by the explant technique, cultured and stimulated with Porphyromonas gingivalis (Pg) and Escherichia coli (Ec) LPS for 24 or 48 h for comparative evaluation of the secretion of cytokines and chemokines, such as IL-6, IL-8/CXCL8, MIP-1α/CCL3, TGF-ß, VEGF and CXCL16. Unstimulated cells were used as the control group. Cells were tested for viability through MTT assay, and secretion of cytokines and chemokines was evaluated in the cell supernatants by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: Fibroblasts from the palatal mucosa maintained the same secretion pattern of IL-6 when grafted onto the gingival margin. On the contrary, fibroblasts from the marginal gingival graft showed increased secretion of IL-8/CXCL8 even in the absence of stimulation. Interestingly, MIP-1α/CCL3 secretion by fibroblasts from the marginal gingival graft was significantly increased after 48 hours of stimulation with Pg LPS and after 24 h with Ec LPS. Only fibroblasts from the marginal gingival graft showed secretion of TGF-ß. VEGF and CXCL16 secretion were not detected by both subsets of fibroblasts. CONCLUSION: Fibroblasts from the palatal mucosa seem to be adapted to local conditions of the site microenvironment when grafted onto the gingival marginal area. This evidence supports the effective participation of fibroblasts in the homeostasis of the marginal periodontium through secretion modulation of important inflammatory mediators.

Losartan Plays a Fungistatic and Fungicidal Activity Against Candida albicans Biofilms: Drug Repurposing for Localized Candidosis.

Candidosis is one of the most frequent opportunistic infections and exhibits variable clinical presentations, including oral localized forms. Drugs affecting the renin-angiotensin system targets inhibit secreted aspartic proteases from Candida albicans. The objective of the study was to evaluate whether losartan has antimicrobial action against C. albicans biofilms. Biofilms were treated with losartan or aliskiren (for comparison) for 24 h. Metabolic activity of viable cells and growth inhibition of C. albicans biofilms were assessed using XTT [2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-5-[(Phenyl-Amino)Carbonyl]-2H-Tetrazolium Hydroxide] and colony-forming unit assays, respectively. In addition, the cytotoxicity of the drugs on human cells was evaluated using the AlamarBlue assay. Both drugs decreased fungal viability at all concentrations. In addition, all concentrations of losartan inhibited the growth of C. albicans biofilm, ranging from 47% to 88.5%, whereas aliskiren showed inhibition from 1 to 10 mg/mL, which ranged from 16% to 97.6%. Furthermore, at certain concentrations, these drugs maintained the viability of human cells. Losartan and aliskiren have fungistatic and fungicidal action against C. albicans biofilms and are compatible with human cells. Therefore, these antihypertensive drugs can be repurposed to interfere with the metabolism and development of Candida biofilms, which are widely associated with clinical forms of candidosis, including oral localized forms such as denture stomatitis.

The use of chronic gingivitis as reference status increases the power and odds of periodontitis genetic studies: a proposal based in the exposure concept and clearer resistance and susceptibility phenotypes definition.

AIM: Current literature on chronic periodontitis genetics encompasses numerous single nucleotide polymorphisms-focused case-control studies with inconsistent and controversial results, which typically disregards the exposure concept embraced by case-control definition. Herein, we propose a case-control design reappraisal by clear phenotype selection, where chronic gingivitis represents a genetically resistant phenotype/genotype opposing the susceptible cohort. MATERIAL AND METHODS: The hypothesis was tested in healthy, chronic periodontitis and gingivitis groups through Real-time PCR-based allelic discrimination of classic variants IL1B-3954, IL6-174, TNFA-308, IL10-592 and TLR4-299. RESULTS: Observed allele/genotype frequencies characterize the healthy group with an intermediate genetic profile between periodontitis and gingivitis cohorts. When comparing genotype/allele frequencies in periodontitis versus healthy and periodontitis versus gingivitis scenarios, the number of positive associations (2-4) and the degree of association (p and odds ratio values) were significantly increased by the new approach proposed (periodontitis versus gingivitis), suggesting the association of IL1B-3954, TNFA-308, IL10-592 and TLR4-299 with periodontitis risk. Power study was also significantly improved by the new study design proposed when compared to the traditional approach. CONCLUSIONS: The data presented herein support the use of new case-control study design based on the case-control definition and clear resistance/susceptibility phenotypes selection, which can significantly impact the study power and odds of identification of genetic factors involved in PD.

Simultaneous separation of naproxen and 6-O-desmethylnaproxen metabolite in saliva samples by liquid chromatography-tandem mass spectrometry: Pharmacokinetic study of naproxen alone and associated with esomeprazol-Results.

After performing liquid-liquid extraction with ethyl acetate and HCl, samples from 12 volunteers who performed sequential collections after taking a tablet of naproxen alone (n = 6) or associated with esomeprazole (n = 6) were analyzed in a triple quadrupole mass spectrometer 8040 LC MS/MS Shimadzu. Separation of naproxen and its main metabolite 6-O-desmethylnaproxen was performed in a Shim-Pack XR-ODS 75Lx2.0 column and C18 pre-column at 40°C using a mixture of methanol and ammonium acetate 10 mM (70:30, v/v) with an injection rate of 0.3 ml/min. The total analytical run time for each sample was 5 min. The association of naproxen with esomeprazole take considerably longer time to reach the maximum concentration [Tmax 0.17 h (interquartile range, 0.13-1.95) for naproxen alone and 13.18*h (interquartile range, 10.12-27.15) for naproxen with esomeprazole, p = 0.002], also to be eliminated [T1/2 0.12 h (interquartile range, 0.09-1.35) for naproxen alone and 9.16*h (interquartile range, 7.16-41.40) for naproxen with esomeprazole, p = 0.002] and lower maximum concentrations (Cmax 4.6 ± 2.5 ug/mL for naproxen alone and 2.04 ± 0.78* µg/mL, p = 0.038). The association of naproxen with esomeprazole showed increased values of AUC0-t [82.06* h*µg/mL (interquartile range, 51.90-157.00) with esomeprazole and 2.97 h*µg/mL (interquartile range, 1.82-7.84) naproxen alone, p = 0.002] in drug concentrations in relation to the naproxen tablet alone, probably, such differences are due to the delay in the absorption of naproxen when it is associated with the drug proton pump inhibitor, esomeprazole. As well as reduced values of full clearance when naproxen is combined with esomeprazole (0.07* µg/h (interquartile range, 0.005-0.01) with esomeprazole and 7.29 µg/h (interquartile range, 3.17-16.23) in naproxen alone, p = 0.002). Both naproxen and 6-O-desmethylnaproxen in saliva samples can be effectively quantified using LC-MS/MS, this methodology proved to be rapid, sensitive, accurate and selective for each drug and allows for the analysis of their pharmacokinetic parameters, in both situations.

Pharmacogenetic and Pharmacokinetic Assays from Saliva Samples Can Guarantee Personalized Drug Prescription.

Saliva is widely used for clinical and laboratory analysis. This study proposed to use DNA extracted from saliva for genotyping and pharmacokinetics of piroxicam. A fast and efficient genotyping method was used to determine relevant allelic variants of CYP2C9 (*2 and *3), since genetic factors can influence in non-steroidal anti-inflammatory drugs (NSAIDs) metabolization. DNA Extract All Reagents Kit® was used for DNA extraction and genotyping was performed using TaqMan® GTXpress™ Master Mix, SNP genotyping assays and a Viia7 Real-Time PCR system. Volunteers performed sequential collections of saliva samples before and after taking a single dose of piroxicam (0.25 to 72 h) which were used for pharmacokinetics assays. Piroxicam concentrations were analyzed using LC-MS/MS. Sixty-six percent of volunteers were ancestral homozygous (CYP2C9*1/*1), and 34% showed one or both polymorphisms. Of these 34%, 22 individuals showed CYP2C9*2 polymorphism, 8 CYP2C9*3, and 4 CYP2C9*2/*3. Piroxicam pharmacokinetics were performed in 5 subjects. Areas under the curve (AUC0-t(h*ng/mL)) for CYP2C9*1/*1, *1/*2 and *1/*3 were, respectively, 194.33±70.93, 166 and 303. Maximum concentrations (Cmax(ng/mL)) for these genotypes were respectively 6.46±2.56, 4.3 and 10.2. Saliva sampling was a very effective matrix for both pharmacogenetic and pharmacokinetic tests, ensuring the speed of the procedure and the well-being and agreement of the participants. Once having the knowledge about the slow and fast metabolizers, it is possible to make an adequate prescription in order to avoid the adverse effects of the medication and to guarantee greater analgesic comfort to the patients respectively.

Mast cells contribute to alveolar bone loss in Spontaneously Hypertensive Rats with periodontal disease regulating cytokines production.

Mast cells (MCs) play a pivotal role in inflammatory responses and had been studied in inflammatory bone disorders, however, their role in alveolar bone loss induced by periodontal disease (PD) is not yet fully understood. We, therefore, aimed to evaluate the effects of MCs depletion in the PD-induced alveolar bone loss in Wistar (W) and Spontaneously Hypertensive Rats (SHRs). PD was induced by ligating the lower first molars with silk thread one day after the MCs depletion, by the pre-treatment with compound 48/80 for 4 days. After 15 days of PD induction, the hemi-mandibles were surgically collected for qRT-PCR, histological analyses, immunostaining, and ELISA. Systolic blood pressure (SBP) was verified by tail plethysmography to confirm the hypertensive status, and SHR presented SBP >150 mmHg, and previous MC depletion alone or associated with PD did not alter this parameter. SHRs showed a more severe alveolar bone loss compared to W, and MC depletion significantly inhibited this response in both strains, with a more significant response in SHRs. MCs were less abundant in 48/80+PD groups, thus validating the previous MCs depletion in our model. PD increased the number of MC in the gingival tissue of SHR. Cytokine production (TNF-α, IL-6, IL-1ß, and CXCL3) was constitutively higher in SHR and increased further after PD, which was also significantly reduced in the MCs-depleted animals. PD led to an increased expression of Opn, Rankl, Rank, Vtn, Itga5, Itgb5, Trap, and Ctsk in the mandible of W and SHRs, which was reversed in MCs-depleted animals. These results suggest that MCs significantly contributes to the PD-induced alveolar bone resorption, especially in the SHR, which is associated with a more severe PD progression compared to Wistar, partly explained by these cells contribution to the inflammatory status and mediator production, stimulating osteoclast-related response markers, which were reduced after MC depletion in our experimental model.

The essential role of toll like receptor-4 in the control of Aggregatibacter actinomycetemcomitans infection in mice.

OBJECTIVE: Aggregatibacter actinomycetemcomitans is an oral Gram-negative bacterium that contributes to periodontitis progression. Isolated antigens from A. actinomycetemcomitans could be activating innate immune cells through Toll-like receptors (TLRs). In this study, we evaluated the role of TLR4 in the control of A. actinomycetemcomitans infection. MATERIAL AND METHODS: We examined the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR4(-/-) mice. The production of cytokines was evaluated by ELISA. The bacterial load was determined by counting the number of colony-forming units per gram of tissue. RESULTS: The results showed that TLR4-deficient mice developed less severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly lower bone loss and inflammatory cell migration to periodontal tissues. However, the absence of TLR4 facilitated the A. actinomycetemcomitans dissemination. Myeloperoxidase activity was diminished in the periodontal tissue of TLR4(-/-) mice. We observed a significant reduction in the production of tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta in the periodontal tissue of TLR4(-/-) mice. CONCLUSION: The results of this study highlighted the role of TLR4 in controlling A. actinomycetemcomitans infection.

Influence of dental plaque on human enamel erosion: in situ / ex vivo study.

PURPOSE: The objective of the present in situ study was to evaluate the influence of dental plaque on human enamel erosion. MATERIALS AND METHODS: Thirteen volunteers wore acrylic palatal devices with four enamel specimens that were prepared from freshly extracted impacted human third permanent molars (4 x 4 mm), randomly selected and distributed into two vertical rows, corresponding to the following groups: GI, erosion of dental plaque-free samples, and GII, erosion of dental plaque-covered samples. For the formation of dental plaque, the specimens were placed 1 mm below the level of the appliance and covered with a plastic mesh to allow the accumulation of dental plaque. The palatal device was continuously worn by the volunteers for 14 consecutive days and then immersed in a soft drink (Coca-Cola, 150 ml) for 5 min, three times a day. Half of the surfaces of specimens were coated with nail varnish for profilometry tests. The study variables included the depth of enamel surface wear (profilometer, vertical ranges in lm) and the percentage of superficial microhardness change (%SMHC). Data were analysed using the t test (P < 0.05). RESULTS: The %SMHC and depth of enamel surface wear were significantly higher for GI (-87.82% +/- 3.66 and 4.70 lm +/- 1.65) than for GII (-13.79% +/- 4.22 and 0.14 lm +/- 0.03). CONCLUSIONS: It was concluded that the dental plaque formed in situ was able to protect the enamel surface against erosion by a cola soft drink, thus reducing the depth of enamel surface wear and the %SMHC.

Angiotensin II Regulates Proliferation and Function of Stem Cells of Apical Papilla.

INTRODUCTION: Stem cells of apical papilla (SCAP) may be affected by inflammatory mediators released by activation with lipopolysaccharide (LPS) from infected pulpal cavities of necrotic immature teeth. Therefore, this study aimed to investigate the presence of a local renin-angiotensin system (RAS) and the role of angiotensin II (Ang II) on the modulation of SCAP in vitro. METHODS: Primary cultures of SCAP were incubated with LPS (0.1-10 µg/mL) for cell viability and quantification of the chemokine CCL2. Components of RAS were searched by gene expression of angiotensinogen (AGTN), angiotensin converting enzyme (ACE), renin, angiotensin receptor 1 (AT1) and 2 (AT2), and Mas receptor. Ang II was investigated in SCAP supernatants. Immunofluorescence was used to detect AGTN and AT1. Next, cells were treated with Ang II for viability/proliferation assessment, quantification of CCL2 and interleukin 6, and mineralization assay. Data were evaluated by analysis of variance using Tukey post hoc comparisons or the Student t test. P values <.05 were considered to be significant. RESULTS: LPS increased CCL2 production at 1 and 10 µg/mL. The gene expression of AGTN, renin, ACE, and AT1 was detected, but only ACE was increased by LPS. Ang II peptide was found in SCAP supernatants but unaltered by LPS. Both AGTN and AT1 proteins were detected by immunostaining. Ang II significantly induced SCAP proliferation, increased CCL2 production, down-regulated IL-6 release, and reduced the SCAP mineralization rate. CONCLUSIONS: A local RAS was found at the apical papilla, and Ang II was able to modulate SCAP function in vitro.

Simultaneous separation of naproxen and 6-O-desmethylnaproxen metabolite in saliva samples by liquid chromatography-tandem mass spectrometry: Pharmacokinetic study of naproxen alone and associated with esomeprazole.

Naproxen is a widely used non-steroidal anti-inflammatory drug for the control of postoperative inflammatory signs and symptoms in dentistry. Its association with esomeprazole has been widely studied and has yielded good results for the control of acute pain, even with the delayed absorption of naproxen owing to the presence of esomeprazole. To further understand the absorption, distribution, and metabolism of this drug alone and in combination with esomeprazole, we will analyze the pharmacokinetic parameters of naproxen and its major metabolite, 6-O-desmethylnaproxen, in saliva samples. A rapid, sensitive, and selective liquid chromatography-tandem mass spectrometric method for the simultaneous determination of naproxen and 6-O-desmethylnaproxen in saliva will be developed and validated. Sequential saliva samples from six patients will be analyzed before and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6 8, 11, 24, 48, 72, and 96 h after the ingestion of one naproxen tablet (500 mg) and esomeprazole-associated naproxen tablets (500 + 20 mg), at two different times. After liquid-liquid extraction with ethyl acetate and HCl, the samples will be analyzed using an 8040 Triple Quadrupole Mass Spectrometer (Shimadzu, Kyoto, Japan). Separation of naproxen and its major metabolic products will be performed using a Shim-Pack XR-ODS 75Lx2.0 column and C18 pre-column (Shimadzu, Kyoto, Japan) at 40°C using a mixture of methanol and 10 mM ammonium acetate (70:30, v/v) with an injection flow of 0.3 mL/min. The total analytical run time will be 5 min. The detection and quantification of naproxen and its metabolite will be validated, which elucidate the pharmacokinetics of this drug, thereby contributing to its proper prescription for the medical and dental interventions that cause acute pain.