Absence of functional TLR4 impairs response of macrophages after Candida albicans infection

Candida albicans is recognized by phagocytic cells through a set of recognition receptors patterns. Recently, we showed the importance of TLR2 in the regulation of neutrophil survival after C. albicans infection. In the present work, we analyzed the involvement of TLR4 in the recognition of C. albicans by neutrophils and macrophages. Our results show that the absence of functional TLR4 resulted in lower chemotaxis of neutrophils to the site of infection, lower levels of TNF-alpha, CXCL1 and nitric oxide, and dissemination and persistence of the pathogen in lymph nodes and spleen. In vitro, the phagocytic activity, nitric oxide production and myeloperoxidase activity, CXCL1, IL-1 beta production by neutrophils from TLR4-defective mice were not changed. In contrast, macrophages from TLR4-defective mice demonstrated lower phagocytosis and lower levels of CXCL1, IL-1 beta and TNF-alpha. Together, these data demonstrate that TLR4 signals are important for the recognition of C. albicans by macrophages and their absence allows persistence of the infection.

Patients with oral squamous cell carcinoma are characterized by increased frequency of suppressive regulatory T cells in the blood and tumor microenvironment

Oral squamous cell carcinoma (OSCC) is a cancerous lesion with high incidence worldwide. The immunoregulatory events leading to OSCC persistence remain to be elucidated. Our hypothesis is that regulatory T cells (Tregs) are important to obstruct antitumor immune responses in patients with OSCC. In the present study, we investigated the frequency, phenotype, and activity of Tregs from blood and lesions of patients with OSCC. Our data showed that > 80% of CD4(+)CD25(+) T cells isolated from PBMC and tumor sites express FoxP3. Also, these cells express surface Treg markers, such as GITR, CD45RO, CD69, LAP, CTLA-4, CCR4, and IL-10. Purified CD4(+)CD25(+) T cells exhibited stronger suppressive activity inhibiting allogeneic T-cell proliferation and IFN-gamma production when compared with CD4(+)CD25(+) T cells isolated from healthy individuals. Interestingly, approximately 25% of CD4(+)CD25(-) T cells of PBMC from patients also expressed FoxP3 and, although these cells weakly suppress allogeneic T cells proliferative response, they inhibited IFN-gamma and induced IL-10 and TGF-beta secretion in these co-cultures. Thus, our data show that Treg cells are present in OSCC lesions and PBMC, and these cells appear to suppress immune responses both systemically and in the tumor microenvironment.

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)-1 beta in the periodontal tissue of TLR4(-/-) mice. Conclusion: The results of this study highlighted the role of TLR4 in controlling A. actinomycetemcomitans infection.

Candida albicans and Denture Stomatitis: Evaluation of Its Presence in the Lesion, Prosthesis, and Blood

Denture stomatitis is a primarily oral disease that affects denture wearers. The presence of Candida albicans in the palatal mucosa, on the internal surface of the maxillary denture, and in the blood of patients with denture stomatitis was evaluated. Although the results did not show C albicans in the bloodstream of patients, a strong relationship between denture stomatitis and Candida sp was confirmed for both the palatal mucosa and maxillary denture. Int J Prosthodont 2010;23:158-159.

Understanding the impact of divalent cation substitution on hydroxyapatite: An in vitro multiparametric study on biocompatibility

Hydroxyapatite (HA), a stable and biocompatible material for bone tissue therapy, may present a variable stoichiometry and accept a large number of cationic substitutions. Such substitutions may modify the chemical activity of HA surface, with possible impact on biocompatibility. In this work, we assessed the effects of calcium substitution with diverse divalent cations (Pb(2+), Sr(2+), Co(2+), Zn(2+), Fe(2+), Cu(2+), or Mg(2+)) on the biological behavior of HA. Physicochemical analyses revealed that apatite characteristics related to crystallinity and calcium dissolution/uptake rates are very sensitive to the nature of cationic substitution. Cytocompatibility was evaluated by mitochondrial activity, membrane integrity, cell density, proapoptotic potential, and adhesion tests. With the exception of Zn-HA, all the substituted HAs induced some level of apoptosis. The highest apoptosis levels were observed for Mg-HA and Co-HA. Cu-HA was the only material to impair simultaneously mitochondrial activity, membrane integrity, and cell density. The highest relative cell densities after exposure to the modified HAs were observed for Mg-HA and Zn-HA, while Co-HA significantly improved cell adhesion onto HA surface. These results show that changes on surface dissolution caused by cationic substitution, as well as the increase of metal species released to biological media, were the main responsible factors related to alterations on HA biocompatibility. (C) 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 98A: 351-358, 2011.

Absence of TLR2 influences survival of neutrophils after infection with Candida albicans

Candida albicans is an opportunistic pathogen, which causes local and/or disseminated diseases in immunosuppressed humans. Phagocytic cells play a critical role in the immune response against C. albicans. Toll like receptors (TLR) are important in the identification of invading microorganisms and in the regulation of neutrophil survival. TLR2 has been shown to participate in the response against pathogenic yeasts and to increase the functional life span of neutrophils. In view of these observations, we studied the involvement of TLR2 in neutrophil function after C. albicans infection. The absence of TLR2 resulted in lower chemotaxis of neutrophils to the site of infection. This in turn was associated with lower levels of chemokines from neutrophils, facilitating the dissemination of the pathogen to the lymph nodes and spleen. A high frequency of apoptotic neutrophils and macrophages in the inflammatory exudates from TLR2(-/-) mice was found. In addition, the phagocytic activity of neutrophils and macrophages, nitric oxide production and myeloperoxidase, activity were diminished in cells from TLR2(-/-) mice. Together, these data demonstrate the importance of TLR2 signals for neutrophils activation and survival after C albicans infection.

The Role of Toll-Like Receptor 2 in the Recognition of Aggregatibacter actinomycetemcomitans

Background: Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) is a Gram-negative bacterium present in the oral cavity and is usually associated with localized aggressive periodontitis. Isolated antigens from A. actinomycetemcomitans can activate innate immune cells through Toll-like receptors (TLRs), which are molecules that recognize structural components conserved among microorganisms. In this study, we evaluate the role of TLR2 in the recognition of A. actinomycetemcomitans. Methods: Macrophages and neutrophils from knockout mice with targeted disruption of TLR2 (TLR2(-/-) mice) and wild-type mice were collected and used for the subsequent assays. The production of cytokines and chemokines was evaluated by enzyme-linked immunosorbent assay (ELISA), and the presence of apoptotic cells was determined by flow cytometry. In addition, the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR2(-/-) mice were examined. Results: The results show that TLR2-deficient mice developed more severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly higher bone loss and inflammatory cell migration to periodontal tissues. The inflammatory cell influx into the peritoneal cavities of TLR2(-/-) mice was three-fold lower than that observed for the littermate controls. A significantly diminished production of the cytokines tumor necrosis factor-alpha and interleukin-1 beta as well as the chemokine CC-ligand-5 in the peritoneal cavities of TLR2(-/-) mice was observed. In addition, a high frequency of apoptotic cells in the inflammatory exudates from TLR2(-/-) mice was observed. Phagocytosis and nitric oxide production was diminished in cells from TLR2(-/-) mice, facilitating the dissemination of the pathogen to the spleen. Conclusion: The results of this study highlight the involvement of TLR2 in recognizing A. actinomycetemcomitans and its essential role in controlling A. actinomycetemcomitans infection. J Periodontot 2009,80:2070-2019.

Isolation of Candida dubliniensis from denture wearers

Candida albicans is considered the most important Candida species able to cause oral infections in denture wearers. In recent years, Candida dubliniensis has emerged as a pathogenic yeast in humans. The close phenotypic similarities of C. albicans and C. dubliniensis have led to the misidentification of these species. In this work, our aim was to verify through PCR the presence of C. dubliniensis in palate and maxillary denture samples from 112 denture wearers presenting with or without denture-related stomatitis (DRS). C. dubliniensis was isolated at low rates from both palate (5.3% and 10.7%) and maxillary denture (5.3% and 8.9%) samples from wearers regardless of the presence of the disease. However, when C. dubliniensis was detected in individuals with DRS, it was always associated with C. albicans. In addition, our results showed that C. albicans was the most commonly identified candidal species in maxillary denture and hard palate samples from DRS patients (78.5% and 89.2%, respectively) as well as from controls (31.2% and 28.5%, respectively). In conclusion, C. dubliniensis was detected in the oral environment of denture wearers. The association of C. dubliniensis with C. albicans occurred in approximately 10% of the DRS cases.

Enhanced programmed death 1 (PD-1) and PD-1 ligand (PD-L1) expression in patients with actinic cheilitis and oral squamous cell carcinoma

PD-1 and PD-L1 can be involved in tumor escape, and little is known about the role of these molecules in oral tumors or pre-malignant lesions. In the present study, we investigated the expression of PD-1 and PD-L1 in the blood and lesion samples of patients with actinic cheilitis (AC) and oral squamous cell carcinoma (OSCC). Our results showed that lymphocytes from peripheral blood and tissue samples exhibited high expression of PD-1 in both groups analyzed. Patients with AC presented higher percentage as well as the absolute numbers of CD4(+)PD-1(+) and CD8(+)PD-1(+) lymphocytes in peripheral blood mononuclear cells (PBMC) than healthy individuals, while patients with OSCC presented an increased frequency of CD8(+)PD1(+) in PBMC when compared with controls. On the other hand, increased frequency of CD4(+) and CD8(+) T cells expressing PD-1(+) accumulate in samples from OSCC, and the expression of PD-L1 was intense in OSCC and moderate in AC lesion sites. Lower levels of IFN-gamma and higher levels of TGF-beta were detected in OSCC samples. Our data demonstrate that PD-1 and PD-L1 molecules are present in blood and samples of AC and OSCC patients. Further studies are required to understand the significance of PD-1 and PD-L1 in oral tumors microenvironment.

Confocal laser scanning microscopy is appropriate to detect viability of Enterococcus faecalis in infected dentin

The purpose of this study was to explore the potential of confocal laser scanning microscopy (CLSM) for in situ identification of live and dead Enterococcus faecalis in infected dentin. Eight cylindrical dentin specimens were infected with Enterococcus faecalis in BHI for 21 days. After the experimental period, the specimens were stained with fluorescein diacetate (FDA) and propidium iodide (PI) or acridine orange (0.01 %) and analyzed by CLSM. Two noninfected dentin specimens were used as negative controls. CLSM analysis shows that the discrimination between viable (green) and dead (red) bacteria in infected dentinal tubules could be observed after staining with FDA/PI. Acridine orange was able to show metabolic activity of the E. faecalis cells inside the dentinal tubules showed by its red fluorescence. The viability of bacteria in infected dentin can be determined in situ by CLSM. FDA/PI and acricline orange are useful for this technique.

The Drop Technique: a Method to Control the Amount of Fluoride Dentifrice Used by Young Children

Purpose: The purpose of this study was to evaluate the amount of dentifrice applied to the toothbrush by school children using a liquid dentifrice (drop technique), when compared to toothpaste. Materials and Methods: A total of 178 school children (4-8 years old) from two cities in Brazil (Bauru and Bariri) participated in the present two-part crossover study. Children from Bauru received training regarding tooth-brushing techniques and use of dentifrice before data collection. In each phase, the amount of toothpaste or liquid dentifrice applied by the children to the toothbrush was measured, using a portable analytical balance (+/- 0.01 g). Data were tested by analysis of covariance (Ancova) and linear regression (p < 0.05). Results: The mean (+/- standard deviation) amounts of toothpaste and liquid dentifrice applied to the toothbrushes for children from Bauru were 0.41 +/- 0.20 g and 0.15 +/- 0.06 g, respectively. For children from Bariri, the amounts applied were and 0.48 +/- 0.24 g and 0.14 +/- 0.05 g, respectively. The amount of toothpaste applied was significantly larger than the amount of liquid dentifrice for both cities. Children from Bariri applied a significantly larger amount of toothpaste, when compared to those from Bauru. However, for the liquid dentifrice, there was no statistically significant difference between the cities. A significant correlation between the amount of toothpaste applied and the age of the children was verified, but the same was not found for the liquid dentifrice. Conclusion: The use of the drop technique reduced and standardised the amount of dentifrice applied to the toothbrush, which could reduce the risk of dental fluorosis for young children.

Dental caries in adolescents from public schools in Maputo, Mozambique

Aim: To assess dental caries prevalence in adolescents at urban and sub-urban areas of Maputo-City, Mozambique and to identify its relationship with dental fluorosis, dental plaque, nutritional status, frequency of sugar consumption and the concentration of fluoride in public water supply. Methods: Subjects (n=601) were randomly selected from five urban schools and five sub-urban schools. Clinical examinations were performed under standardised conditions by a trained examiner using DMFT index, SiC index, fluorosis index, PHP, BMI, a sugar consumption questionnaire and water supply analysis. The bivariate analysis and Pearson correlation was used (p<0.05). Results: The mean (DMFT) was 0.9 (+/- 1.65 SD). Children in urban schools showed less dental caries (0.8 +/- 1.49SD) than children in sub-urban schools (1.1 +/- 1.80SD, p=0.03). Only 8.15% had very mild to moderate fluorosis but most presented poor oral hygiene. Cases of malnutrition were found in more sub-urban schools (n = 109; 36.22%) than in urban schools (n = 66; 22.00%) (p=0.03). The frequency of sugar consumption was higher among urban children compared to suburban schools (p<0.00). The level of fluoride in water consumption in urban schools was 0.4 ppmF, above the level of fluoride in sub-urban schools, 0.2 ppmF. Conclusion: Dental caries should not be considered a major oral health problem in Maputo at the moment. However the data suggest the implementation of a population strategy to reduce dental caries rates, in children of both urban and sub-urban areas, in Maputo.

Tooth-wear patterns in adolescents with normal occlusion and Class II Division 2 malocclusion

Introduction: In this study, we investigated tooth-wear patterns in adolescents with either normal occlusion or Class II Division 2 malocclusion. Methods: The sample consisted of dental casts from 165 subjects that were divided into 2 groups: 115 normal occlusion subjects (mean age, 14.3 years) and 50 complete Class II Division 2 subjects (mean age, 13.9 years). Dental wear was assessed by using a modified version of the tooth wear index. The 2 groups were compared with the Mann-Whitney test for the frequency and severity of wear on each surface of each group of teeth. The level of statistical significance was set at 5%. Results: The normal occlusion group statistically had greater tooth wear on the incisal surfaces of the maxillary lateral incisors and the incisal surfaces of the maxillary canines than did the Class II Division 2 malocclusion group. The malocclusion group showed statistically greater tooth wear on the labial surfaces of the mandibular lateral incisors, the occlusal surfaces of the maxillary premolars and first molars, the occlusal surfaces of the mandibular premolars, the palatal surfaces of the maxillary second premolars, and the buccal surfaces of the mandibular premolars and first molars than did the normal occlusion group. Conclusions: Subjects with normal occlusion and those with complete Class II Division 2 malocclusions have different tooth-wear patterns. Tooth wear on the malocclusion subjects should not be considered pathologic but, rather, the consequence of different interocclusal arrangements. (Am J Orthod Dentofacial Orthop 2010;137:730.e1-730.e5)

Tooth-wear patterns in subjects with Class II Division 1 malocclusion and normal occlusion

Introduction: The aim of this study was to investigate the prevalence of tooth wear in adolescents with Class II malocclusion, compared with those with normal occlusion. Methods: The sample consisted of dental casts obtained from 310 subjects, divided into 3 groups: group 1, 110 subjects with normal occlusion (mean age, 13.51 years); group 2, 100 complete Class II Division 1 patients (mean age, 13.44 years); and group 3, 100 half-cusp Class II Division 1 patients (mean age, 13.17 years). Dental wear was assessed by using a modified version of the tooth-wear index. The 3 groups were compared by means of the Kruskal-Wallis and Dunn tests, considering the frequency and the severity of wear on each surface of each group of teeth. The level of statistical significance was set at 5%. Results: The normal occlusion group had statistically greater tooth wear on the palatal surfaces of the maxillary central incisors and the incisal surfaces of the maxillary canines than the corresponding surfaces in both Class II malocclusion groups. The complete and half-cusp Class II Division 1 malocclusion groups had statistically greater tooth wear on the occlusal surfaces of the maxillary second premolar and first molar, the occlusal surfaces of the mandibular premolars, and the buccal surfaces of the mandibular posterior teeth compared with the normal occlusion group. The half-cusp Class II Division 1 malocclusion group had significantly greater tooth wear on the incisal surfaces of the mandibular incisors compared with the complete Class II Division 1 malocclusion group. Conclusions: Subjects with normal occlusion and complete or half-cusp Class II Division 1 malocclusions have different tooth-wear patterns. Tooth wear on the malocclusion subjects should not be considered pathologic but rather consequent to the different interocclusal tooth arrangement. (Am J Orthod Dentofacial Orthop 2010; 137: 14. e1-14.e7)

Preventive effect of an iron varnish on bovine enamel erosion in vitro

Objective: The aim of this study was to evaluate, in vitro, the effect of an experimental varnish containing iron on the dissolution of bovine enamel by carbonated beverage. Methods: Eighty specimens were randomly allocated to four groups (n = 20 per group), according to the following treatments: Fe varnish (FeV, 10 mmoL/L Fe), F varnish (FV, 2.71% F), placebo varnish (PV) and control (not treated, NT). The varnishes were applied in a thin layer and removed after 6 h. Then, the samples were submitted to six cycles, alternating re- and demineralisation (only 1 day). Demineralisation was performed with the beverage Coca-Cola (R) (10 min, 30 mL/block) and remineralisation with artificial saliva for I h. In order to determine the amount of enamel dissolved, the wear was analysed by profilometry. Data were analysed by ANOVA and Tukey`s test (p < 0.05). Results: The mean wear (+/- S.E.) was significantly lesser for the FeV (0.451 +/- 0.018 mu m) when compared to the other treatments. The FV caused significantly less wear (0.554 +/- 0.022 mu m) when compared to PV (0.991 +/- 0.039 mu m) and NT (1.014 +/- 0.033), which did not significantly differ from each other. Conclusions: The results suggest that the iron varnish can interfere with the dissolution of dental enamel in the presence of acidic beverages. (C) 2008 Elsevier Ltd. All rights reserved.