Development of nanobiosilicate, tricalcium phosphate and chlorhexidine materials for biomineralization with crystallographic similarity to hydroxyapatite and biomodified collagen.

OBJECTIVES: The aim of this work is to test experimental cements, doped with a silicate based bioactive nanoparticle (NanoBiosilicate). Methods, we synthesized a glass nanoparticle by Sol-Gel Stöber method, used to be incorporated in a dental material for endodontic uses. MATERIALS AND METHODS: We assess the mineralizing properties and biocompatibility. Besides the crystallography characterization of the resultant new crystals. Results, After analysis, and comparison with commercial materials, the material tested was similar in mechanical properties required by ISO, The ion release was effective after 2 hr. of setting and the novel material was cell compatible accepted by ISO. RESULTS: We found new formed Calcium Phosphate peaks in the spectroscopic analysis (FTIR), remarkably the crystals formed were comparable to hydroxyapatite when analyzed with a Selected Area Electron Diffractometer, with rings of 2.84 Å for 002, and the 2.77 Å is also visible for 210. The 6.83 Å and 6.88 Å, for respective 222 and 004. The incorporation of Chlorhexidine was not detrimental for this property, Significance, the features mentioned represented a progress in biomineralization field that was associated to an improved mineral structure formation with increased crystallographic similarity to natural hydroxyapatite. When chlorhexidine was added a favorable biomodification of the remaining collagen in dentinal walls and antimicrobial activity potential were also observed.

Aggregatibacter actinomycetemcomitans Outer Membrane Proteins 29 and 29 Paralogue Induce Evasion of Immune Response.

Aggregatibacter actinomycetemcomitans (Aa) is abundant within the microbial dysbiotic community of some patients with periodontitis. Aa outer membrane protein 29 (OMP29), a member of the OMPA family, mediates the invasion of Aa to gingival epithelial cells (GECs). This study evaluated the effect of OMP29 and its paralogue OMP29par on the response of GECs to Aa. The omp29 or/and omp29 par deletion mutants AaΔ29, AaΔ29P, and AaΔ29Δ29P were constructed, and recombinant Aa OMP29His was obtained. Microarray analysis and the evaluation of cxcl-8 gene expression were performed to examine the response of GECs line OBA-09 to Aa and its mutants. The expression of cxcl-8 and its product CXCL-8 was examined in LPS-stimulated OBA-09 cells with Aa OMP29His. Proteomics analysis showed that the deletion of omp29 led to overexpression of both OMP29par and another membrane protein OMP39, the expression of which was further increased in AaΔ29Δ29P. OBA-09 cells challenged with AaΔ29Δ29P exhibited a higher expression of cxcl-8 in comparison to wildtype Aa strain AaD7S or single-deletion mutants AaΔ29 or AaΔ29P. LPS-stimulated OBA-09 cells challenged with Aa OMP29His showed reduced expressions of cxcl-8 and its product CXCL-8. OBA-09 cells challenged with AaΔ29Δ29P in comparison to Aa strain AaD7S resulted in higher expressions of genes involved in apoptosis and inflammatory response such as bcl2, birc3, casp3, c3, ep300, fas, fosb, grb2, il-1α, il-1ß, il-6, cxcl-8, nr3c1, prkcq, socs3, and tnfrsf1ß and reduced expressions of cd74, crp, faslg, tlr1, and vcam1. The results suggested a novel strategy of Aa, mediated by OMP29 and OMP29par, to evade host immune response by inhibiting CXCL-8 expression and modulating the genes involved in apoptosis and inflammatory response in GECs. Pending further confirmation, the strategy might interfere with the recruitment of neutrophils and dampen the host inflammatory response, leading to a more permissive subgingival niche for bacterial growth.

Anandenanthera colubrina (Vell.) Brenan as an inhibitor of HIV-1 BaL infection.

We reported the in vitro anti-HIV-1 activity, cytotoxicity, cytokines expression and chemical profile of Anadenanthera colubrina. Cytotoxicity was evaluated on TZM-bl, HL2/3 cells and macrophages. Anti-HIV-1 activity was determined by Luciferase assay (TZM-bl cells) and by HIV-p24 quantification (macrophages) assessed by ELISA. TZM-bl and HL2/3 cells were used to determine cell-cell fusion inhibition. Cytokines expression was assessed by ELISA. Chemical composition was determined by Gas Chromatography Coupled to Mass Spectrometry. At 66.6 µg/mL, the extract maintained the cell viability above 90%. At 33.28 µg/mL, the extract reduced 82.8% of HIV-1 infection (TZM-bl cells) and HIV-p24 expression (macrophages). The extract inhibited approximately 70% of TZM-bl and HL2/3 cells fusion. Extract did't induce inflammatory response. Phytochemical analysis showed presence of flavonoid, phenolic acids, fatty acids and sugars. This is the first study presenting the anti-HIV effect of A. colubrina, showing low cytotoxicity and no inflammatory stimuli, important requirements for a microbicide development.

Yeast-Host Interactions: Anadenanthera colubrina Modulates Virulence Factors of C. albicans and Inflammatory Response In Vitro.

Oral candidiasis is one of the most common fungal infections in humans. Its incidence has increased widely, as well as the antifungal resistance, demanding for the search for novel antifungal therapeutic agents. Anadenanthera colubrina (Vell.) Brenan is a plant species that has been proven to possess pharmacological effects, including antifungal and anti-inflammatory activities. This study evaluated in vitro the effects of standardized A. colubrina extract on virulence factors of Candida albicans and its regulation on immune response through C. albicans-host interaction. Antifungal activity was evaluated by Broth Microdilution Method against reference Candida strains (C. albicans, C. glabrata, C. tropicalis; C. dubliniensis). Anti-biofilm effect was performed on C. albicans mature biofilm and quantified by CFU/mL/g of biofilm dry weight. Proleotlytic enzymatic activities of proteinase and phospholipase were assessed by Azocasein and Phosphatidylcholine assays, respectively. Cytotoxicity effect was determined by Cell Titer Blue Viability Assay on Human Gingival Fibroblasts. Co-cultured model was used to analyze C. albicans coexisting with HGF by Scanning Electron Microscopy and fluorescence microscopies; gene expression was assessed by RT-PCR of C. albicans enzymes (SAP-1, PLB-1) and of host inflammatory cytokines (IL-6, IL-8, IL-1ß, IL-10). Cytokines secretion was analysed by Luminex. The extract presented antifungal effect with MIC<15.62 µg/ml against Candida strains. Biofilm and proteolytic activity were significant reduced at 312.4 µg/ml (20 × 15.62 µg/ml) extract concentration. Cell viability was maintained higher than 70% in concentrations up to 250 µg/ml (LD50 = 423.3 µg/ml). Co-culture microscopies demonstrated a substantial decreased in C. albicans growth and minimal toxicity against host cells. Gene expressions of SAP-1/PLB-1 were significantly down-regulated and host immune response was modulated by a significant decreased on IL-6 and IL-8 cytokines secretion. A. colubrina had antifungal activity on Candida strains, antibiofilm, and anti-proteolytic enzyme effects against C. albicans. Presented low cytotoxicity to the host cells and modulatory effects on the host immune response.

Malva sylvestris derivatives as inhibitors of HIV-1 BaL infection.

The emphasis of the present study is to evaluate a natural product and the potential microbicide activity using a dual chamber infection method. Malva sylvestris extracts and fractions were screened for anti-HIV activity by measuring the virus-antibody neutralization. Plant extracts with strong antiviral activity working in nanomolar or picomolar range can be used to enhance the activity of synthetic compounds and work as anti-HIV agents. The aqueous fraction (AF) of M. sylvestris demonstrated antiviral activity in a model with epithelial and blood cell lines. The AF showed an effective antiviral potential on the TZM-bl cells with reduction scores higher than 60% of infectivity. Quantification of p24 in the supernatant of the co-culture model demonstrated a reduction in the number of viral particles after AF treatment (p < 0.05). Cytokines were quantified and all signaling inflammatory markers; IL1-alpha, IL-beta, IL-6, IL-8 and GM-CSF (p < 0.05) were modulated by positive control and AF treatments. In particular, IL-6 had lower levels of expression in Malva groups when compared to the Zidovudine positive control group. Natural occurring derivatives of M. sylvestris demonstrated to work inhibiting reverse transcriptase enzyme action. M. sylvestris contains highly potential anti-HIV-1 BaL components and may be considered a potential source for new formulations in the development of topical microbicides.

Effects of Monolaurin on Oral Microbe-Host Transcriptome and Metabolome.

The aim of this in vitro study was to evaluate the effects of monolaurin against Aggregatibacter actinomycetemcomitans (Aa) and determine their effects on the host transcriptome and metabolome, using an oral cell/bacteria co-culture dual-chamber model to mimic the human periodontium. For this, the Aa, was applied to cross the monolayer of epithelial keratinocytes (OBA-9) to reach the fibroblasts layer (HGF-1) in the basal chamber. The Monolaurin treatments (25 or 50 µM) were added immediately after the inoculation of the dual-chamber with Aa. After 24 h, the transcriptional factors and metabolites produced were quantified in the remaining cell layers (insert and basal chamber) and in supernatant released from the cells. The genes IL-1α, IL-6, IL-18, and TNF analyzed in HGF-1 concentrations showed a decreased expression when treated with both concentration of Monolaurin. In keratinocytes, the genes IL-6, IL-18, and TNF presented a higher expression and the expression of IL-1α decreased when treated with the two cited concentrations. The production of glycerol and pyruvic acid increased, and the 2-deoxytetronic acid NIST, 4-aminobutyric acid, pinitol and glyceric acid, presented lower concentrations because of the treatment with 25 and/or 50 µM of Monolaurin. Use of monolaurin modulated the immune response and metabolite production when administered for 24 h in a dual-chamber model inoculated with A. actinomycetemcomitans. In summary, this study indicates that monolaurin had antimicrobial activity and modulated the host immune response and metabolite production when administered for 24 h in a dual-chamber model inoculated with A. actinomycetemcomitans.

Fungal-Host Interaction: Curcumin Modulates Proteolytic Enzyme Activity of Candida albicans and Inflammatory Host Response In Vitro.

Current treatments for Candida albicans infection are limited due to the limited number of antifungal drugs available and the increase in antifungal resistance. Curcumin is used as a spice, food preservative, flavoring, and coloring agent that has been shown to have many pharmacological activities. Thus, this study evaluated the modulatory effects of curcumin on major virulence factors associated with the pathogenicity of C. albicans. The minimum inhibitory concentration (MIC) of curcumin against C. albicans (SC5314) was determined. Biofilm formation was quantified and the proteinase and phospholipase secretion was measured. The cytotoxicity was tested in oral fibroblast cells. A cocultured model was used to analyze the gene expression of proinflammatory cytokines (IL-1ß, IL-1α, and IL-6) from host cells, as well SAP-1 and PLB-1 by RT-PCR. The MIC was between 6.25 and 12.5 µM, and the activity of proteinase enzyme was significantly decreased in biofilms treated with curcumin. However, proteinase gene expression was not downregulated after curcumin treatment. Furthermore, gene expressions of host inflammatory response, IL-1ß and IL-1α, were significantly downregulated after exposure to curcumin. In conclusion, curcumin exhibited antifungal activity against C. albicans and modulated the proteolytic enzyme activities without downregulating the gene expression. In host inflammatory response, curcumin downregulated IL-1ß and IL-1α gene expression.

Anti-HIV-1 activity of flavonoid myricetin on HIV-1 infection in a dual-chamber in vitro model.

HIV infection by sexual transmission remains an enormous global health concern. More than 1 million new infections among women occur annually. Microbicides represent a promising prevention strategy that women can easily control. Among emerging therapies, natural small molecules such as flavonoids are an important source of new active substances. In this study we report the in vitro cytotoxicity and anti-HIV-1 and microbicide activity of the following flavonoids: Myricetin, Quercetin and Pinocembrin. Cytotoxicity tests were conducted on TZM-bl, HeLa, PBMC, and H9 cell cultures using 0.01-100 µM concentrations. Myricetin presented the lowest toxic effect, with Quercetin and Pinocembrin relatively more toxic. The anti-HIV-1 activity was tested with TZM-bl cell plus HIV-1 BaL (R5 tropic), H9 and PBMC cells plus HIV-1 MN (X4 tropic), and the dual tropic (X4R5) HIV-1 89.6. All flavonoids showed anti-HIV activity, although Myricetin was more effective than Quercetin or Pinocembrin. In TZM-bl cells, Myricetin inhibited ≥90% of HIV-1 BaL infection. The results were confirmed by quantification of HIV-1 p24 antigen in supernatant from H9 and PBMC cells following flavonoid treatment. In H9 and PBMC cells infected by HIV-1 MN and HIV-1 89.6, Myricetin showed more than 80% anti-HIV activity. Quercetin and Pinocembrin presented modest anti-HIV activity in all experiments. Myricetin activity was tested against HIV-RT and inhibited the enzyme by 49%. Microbicide activities were evaluated using a dual-chamber female genital tract model. In the in vitro microbicide activity model, Myricetin showed promising results against different strains of HIV-1 while also showing insignificant cytotoxic effects. Further studies of Myricetin should be performed to identify its molecular targets in order to provide a solid biological foundation for translational research.

TLR2 and TLR4 gene promoter methylation status during chronic periodontitis.

AIM: The objective of this study was to analyse the status of DNA methylation in the promoter region of the toll-like receptor (TLR)2 and TLR4 genes in gingival tissue samples from healthy subjects, smokers and non-smokers affected by chronic periodontitis. MATERIAL AND METHODS: Genomic DNA and total RNA were purified from gingival tissue using the TRIZOL reagent protocol. Genomic DNA was then digested by methylation-sensitive restriction enzymes, amplified by polymerase chain reaction (PCR), electrophoresed on a 10% polyacrylamide gel and stained using SYBR Gold. Real-time PCR was also performed to verify the transcript levels. RESULTS: The CpG dinucleotides analysed were observed to be unmethylated in the majority of DNA samples of the three groups and statistical differences were not found among groups (p>0.05). However, a trend towards methylation was observed in the TLR2 HhaI site in the samples of the periodontitis non-smoker groups. In fact, the analysis of all CpG sites together shows which complete methylation is observed in the shortest level in the samples of periodontitis non-smoker group. The analysis of transcript levels demonstrated no difference among groups (p>0.05). CONCLUSION: The results demonstrated major unmethylation of the TLR4 gene promoter in all groups. However, the results for the TLR2 gene promoter are inconclusive; this gene was found as a mosaic of methylated and unmethylated DNA in the majority of samples of the three groups and we also observed a trend towards the DNA methylation of CpG sites recognized by the HhaI enzyme.

Latex use as an occlusive membrane for guided bone regeneration.

Latex extracted from Hevea brasiliensis was used as an occlusive membrane for guided bone regeneration. Twenty-four rabbits were divided in two groups: treated and control group. Critical size bone defects (2 cm × 1 cm) were surgically made in the rabbit calvarium. Two latex membranes were implanted in each animal of the treated group, whereas the control defect was filled only with autogenous blood clot. After 15, 30, 60, and 120 days, animals from each group were euthanized, and the samples with regenerated bone were removed. No signs of allergy or rejection were noticed around the calvarial bone defect of the treated group. In the histological analysis, no foreign body inflammatory reaction was observed in the adjacent tissues in contact with the membranes demonstrating that latex can be used at injured sites as an aid in the healing process. Histological analysis, digital radiography, and electron spin resonance were used to evaluate the progress of bone repair. The results show significant differences between groups (p < 0.05) suggesting that latex membranes accelerates healing in critical bone defects.

Antiinflammatory activity of ricinus communis derived polymer

Aim: The present study aimed to evaluate the antiinflammatory activity of the polymer derived from Ricinus communis and its mechanism of action. Methods: The antiinflammatory activity was investigated in chronic and acute animal models and the mechanism of action involved in the antiinflammatory activity was determined by the in vitro phospholipase A2 (PLA2) enzyme assay. Results: In mouse ear edema (10.0 mg/ear) and granulomatous tissue formation (500 mg/kg) models, the polymer inhibited the inflammatory response in 75.08 ± 1.80% and 61.70 ± 1.80% of the cases, respectively (p<0.001). Oral administration of the Ricinus communis polymer (500 mg/kg) inhibited 72.00 ± 1.20% of formalin-induced inflammation. Topical administration of the polymer on oral lesions of mice showed that the oral mucosa was recovered in 60.00 ± 1.40% (p<0.05) of the cases. In in vitro assay, the phospholipase A2 enzyme was inhibited by the Ricinus communis polymer (5.0 mg/mL) in a dose-dependent manner (84.60 ± 1.41%). Conclusion: the polymer derived from Ricinus communis showed a significant antiinflammatory activity, confirming that the pharmacological mechanism involved in this antiinflammatory action was related to the inhibition of the PLA2 enzyme.

Effects of maternal ethanol intake on immunoexpression of epidermal growth factor in developing rat mandibular molar.

OBJECTIVE: A polyclonal antibody was used to investigate the effects of ethanol ingestion before and during pregnancy, in the expression of EGF on dentinogenesis and amelogenesis of rat mandibular first molar. DESIGN: Ethanol was administered to drinking water (treated group) starting at concentrations of 1% and increasing weekly to 5, 10, 15, 20 and 25% (v/v). During week 7, these rats were mated and continued to receive the 25% alcoholic solution, up to delivery. The control group received tap water. On postnatal days 0, 4 and 9, two offspring of each litter were killed, their hemimandibles removed and prepared for paraffin processing and immunohistochemistry. RESULTS: At postnatal day 0 the EGF immunoreactivity of the inner enamel epithelium and presecretory ameloblasts was weak when compared to controls. At postnatal day 4 EGF immunoreactivity of the secretory ameloblasts and odontoblasts was only moderate compared to controls. At postnatal day 9 EGF staining of the ameloblasts was weak when compared to controls. CONCLUSIONS: These results suggest that, maternal alcoholism interferes with EGF expression during initial dentinogenesis and amelogenesis and in the secretion and maturation of the dentin and enamel, therefore, which may cause a reduction of dentin and enamel formation.