Anti-Candida activity and biocompatibility of silver nanoparticles associated with denture glaze: a new approach to the management of denture stomatitis.

The association of silver nanoparticles (AgNps) to sealant agent Palaseal® can be a promising alternative for complete denture wearers who may develop denture stomatitis (DS). The study aimed to evaluate the anti-Candida and biocompatible potential of silver nanoparticles synthesized by three routes associated with denture glaze to prevent and/or treat oral candidiasis. Surface acrylic resin specimens were treated with different associations of glaze with AgNps (VER+AgUV, VER+AgTurk and VER+AgGm). As controls, specimens were treated with glaze+nystatin (VER+Nyst), glaze only (VER) or submerged in PBS (PBS). Afterwards, Candida albicans biofilm was developed for 24 h, 15 d and 30 d. Subsequently, the biofilm was quantified by CFU/mL, XTT assay and confocal laser scanning microscopy. Fibroblasts were submitted to conditioned medium with the same associations for 24, 48 and 72 h and LIVE/DEAD® viability test was carried out. Regardless of the period, there was a significant reduction (p < 0.01) of viable fungal cells load, as well as inhibition of fungal metabolic activity, in specimens treated with glaze+AgNps associations, compared to VER and PBS. The anti-Candida effects of the associations were similar to the VER+Nyst group, with emphasis on VER+AgGm, which showed the highest percentage values of non-viable fungal cells maintained over time. The associations did not prove toxicity to fibroblasts. The AgNps exerted antimicrobial activity against C. albicans biofilms and are biocompatible. The most effective results were achieved with the association of glaze+silver nanoparticles synthesized by the green chemistry method (AgGm), proving to be an innovative alternative in the management of DS.

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.

Silver nanoparticles in denture adhesive: An antimicrobial approach against Candida albicans.

OBJECTIVE: To evaluate the antimicrobial potential of silver nanoparticles (Ag NPs) synthesized using three different routes (ultraviolet light, Turkevich, and green chemistry method using Glycine max extract) associated with COREGA® denture powder adhesive. METHODS: Heat-cured acrylic resin specimens were treated with different Ag NPs associated with the adhesive (AD + Ag UV, AD + Ag Turk, and AD + Ag Gm groups). As controls, the specimens were treated with a combination of adhesive and nystatin (AD + Nyst group), only adhesive (AD group), or submerged on the surface of the specimens (PBS group). After the treatments, biofilms of C. albicans developed for 3, 6, and 12 h on the specimen surfaces. The biofilm was quantified using colony-forming units per milliliter, colorimetric assay, and confocal laser scanning microscopy. RESULTS: Regardless of the period, we observed an inhibition of fungal load and a reduction in metabolic activity and biofilm mass in the resin specimens treated with the combinations AD/Ag NPs, compared to AD and PBS. The antimicrobial action of the AD + Turk and AD + Ag Gm groups was similar than that for the AD + Nyst group in all periods and viability tests, except for the biofilm mass (12 h). CONCLUSIONS: The COREGA® adhesive with Ag NPs, mainly those synthesized using the Turkevich and Glycine max methods, showed excellent antimicrobial activity against C. albicans biofilms, maintained for up to 12 h. CLINICAL SIGNIFICANCE: The association of Ag NPs to the adhesive can add preventive or therapeutic effects against denture stomatitis, to this prosthetic material.

Resilient liner modified by antimicrobials for denture stomatitis treatment: A randomized controlled trial.

OBJECTIVES: This randomized clinical trial evaluated the effectiveness of an interim denture resilient liner (Trusoft) modified with minimum inhibitory concentrations (MICs) of antimicrobial agents for Candida albicans biofilm in the denture stomatitis (DS) treatment. METHODS: Forty participants with DS and maxillary complete denture (MCD) wearers were randomly assigned to one of the treatments for 14 days (n=10): nystatin oral suspension (Control-100,000IU/mL; 4 × /day), MCD relined with Trusoft either without (Tru) or with nystatin (Ny) or chlorhexidine diacetate (Chx) at MICs. Cytological smears and mycological quantitative cultures were taken from the palate and denture before treatment (baseline), at the end of treatment (day 14), and at follow-up (days 30, 45, and 60). Photographs of the palate were made at each visit. Data were analyzed by the Cochran and χ2 tests, ANOVA and the Tukey test or the Friedman and Kruskal-Wallis tests (α=0.05). RESULTS: Palatal smears of the Ny and Chx groups exhibited no mycelial Candida (0%) on day 14, and, at the 60-day follow-up, it was observed for only 1 participant from Chx group. MCD smears showed reduction in mycelial forms for all groups on day 14 (P<0.05), but this difference was maintained at follow-up only for the relined dentures (P<0.05). Unlike Tru and the control groups, Ny and Chx groups evidenced a significant reduction in CFU/mL values and DS severity throughout the trial (P<0.05). CONCLUSIONS: The addition of nystatin and chlorhexidine at MICs to an interim resilient liner is a promising treatment for DS, with better results than those for conventional therapy with nystatin suspension, including the follow-ups. CLINICAL SIGNIFICANCE: Compared with conventional topical antifungal therapy, modifying a denture reline material with antimicrobials provided a therapeutic option for denture stomatitis. This non-invasive, straightforward therapeutic approach can be easily adopted by dentists and has the advantage of not requiring patient compliance while maintaining therapeutic concentrations on the denture base.

Medication-Related Osteonecrosis of the Jaw Associated with Pazopanib Monotherapy

ABSTRACT: Pazopanib, an antiangiogenic agent, has shown promising results in controlling tumor growth and metastasis in patients with renal cell carcinoma. The use of pazopanib in the management of malignancies has increased over recent years, with more patients at risk of developing medication-related osteonecrosis of the jaw (MRONJ). This paper presents the first case report of MRONJ associated with pazopanib monotherapy. A 59-year-old man was referred to the dental clinic with complaints of dysphagia and dysgeusia. The patient was prescribed pazopanib (400 mg) daily following surgical treatment and chemotherapy for metastatic renal cell carcinoma. He had undergone extraction of the maxillary left second premolar nine weeks previously. Intraoral examination revealed exposed necrotic bone, which was treated effectively with leukocyte and platelet-rich fibrin (LPRF). The patient was followed up for 150 days after dental treatment with no signs of relapse.

RESUMEN: Pazopanib, un agente antiangiogénico, ha mostrado resultados prometedores en el control del crecimiento tumoral y las metástasis en pacientes con carcinoma de células renales. El uso de pazopanib en el tratamiento de las neoplasias malignas ha aumentado en los últimos años, con más pacientes en riesgo de desarrollar osteonecrosis de la mandíbula relacionada con la medicación (MRONJ). Este artículo presenta el primer reporte de caso de MRONJ asociado con la monoterapia con pazopanib. Un hombre de 59 años fue remitido a la clínica dental con quejas de disfagia y disgeusia. Al paciente se le prescribió pazopanib (400 mg) al día tras tratamiento quirúrgico y quimioterapia por carcinoma metastásico de células renales. Había sido sometido a extracción del segundo premolar superior izquierdo nueve semanas antes. El examen intraoral reveló hueso necrótico expuesto, que fue tratado eficazmente con leucocitos y fibrina rica en plaquetas (LPRF). El paciente fue seguido durante 150 días después del tratamiento dental sin signos de recidiva.

Ganoderma lucidum polysaccharides inhibit in vitro tumorigenesis, cancer stem cell properties and epithelial-mesenchymal transition in oral squamous cell carcinoma.

ETHNOPHARMACOLOGICAL RELEVANCE: The polysaccharides of the millenary mushroom Ganoderma lucidum (GL) have been shown for decades to present anti-tumor activities, but few studies evaluated its importance on cancer stem cells and EMT process. Cancer stem cells (CSC) drive the development of carcinoma and are also involved in cancer treatment failure, being a good target for treatment success. Also, the process of epithelial-mesenchymal transition (EMT) is involved in metastasis and cancer relapse. Besides that, the increasing incidence worldwide of oral squamous cell carcinoma (OSCC) became a public health issue with a high rate of metastasis and poor quality of life for patients during and after treatment. AIM OF THE STUDY: to evaluate G. lucidum polysaccharides (GLPS) in vitro effects on OSCC, focusing on hallmarks associated with tumorigenesis using the SCC-9, a squamous cells carcinoma lineage from the tongue. MATERIALS AND METHODS: SCC-9 cells were treated in vitro for 72h with different GLPS concentrations. The controls cells were maintained with culture media only and cisplatin was used as treatment control. After the treatment period, the cells were evaluated. RESULTS: GLPS treatment changed cell morphology and granularity, delayed migration, decreased colony, and impaired sphere formation, thereby leading to a non-invasive and less proliferative behavior of tumoral cells. Additionally, GLPS downregulated CSC, EMT, and drug sensitivity (ABC) markers. CONCLUSIONS: These results show that the natural product GLPS has the potential to be an important ally for tongue squamous cell carcinoma treatment, bringing the millenary compound to modern therapy, providing a basis for future studies and the improvement of life quality for OSCC patients.

IFN-γ Mediated Signaling Improves Fungal Clearance in Experimental Pulmonary Mucormycosis.

We established three immunocompetent murine models of pulmonary mucormycosis to determine the involvement of the adaptive immune response in host resistance in pulmonary mucormycosis, a rapidly fatal disease caused mainly by Rhizopus spp. Immunocompetent inbred (C57BL/6, BALB/c) and outbred (Swiss) strains of mice were inoculated with R. oryzae via the intratracheal route. The inoculation resulted in a disseminated infection that spread to the brain, spleen, kidney, and liver. After 7 and 30 days of R. oryzae infection, BALB/c mice showed the lowest fungal load and highest production of IFN-γ and IL-2 by splenocytes. Swiss mice showed a higher fungal load 30 days p.i. and was associated with a weak development of the Th-1 profile. To confirm our findings, R. oryzae-infected IFN-γ-/- mice were evaluated after 60 days, where the mice still showed viable fungi in the lungs. This study showed, for the first time, that pulmonary mucormycosis in three widely used mouse strains resulted in an acute fungal dissemination without immunosuppression whose outcome varies according to the genetic background of the mice. We also identified the partial role of IFN-γ in the efficient elimination of R. oryzae during pulmonary infection.

Telmisartan impairs the in vitro osteogenic differentiation of mesenchymal stromal cells from spontaneously hypertensive male rats.

Telmisartan (TELM) is an angiotensin II (Ang II) type 1 receptor (Agtr1) antagonist, with partial agonism for Pparg, and has been shown to affect bone metabolism. Therefore, the aim of this study was to investigate the effects of TELM in the in vitro osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSC) from spontaneously hypertensive rats (SHRs). BMSC were obtained from male SHR, and the osteogenic medium (OM) was added to the cells concomitantly with TELM (0.005, 0.05, and 0.5 µM). Undifferentiated BMSC, in control medium (CM), showed an increased viability, while the addition of OM reduced this parameter, and TELM did not show cytotoxicity in the concentrations used. BMSC in OM had an alkaline phosphatase (ALP) activity peak at d10, which decreased at d14 and d21, and TELM reduced ALP at d10 in a dose-dependent manner. Mineralization was observed in the OM at d14, which intensified at d21, but was inhibited by TELM. Agtr1b was increased in the OM, and TELM inhibited its expression. TELM reduced Opn, Ocn, and Bsp and increased Pparg expression, and at the higher concentration TELM also increased the expression of adipogenic markers, Fabp4 and Adipoq. In addition, TELM 0.5 µM increased Irs1 and Glut4, insulin and glucose metabolism markers, known to be regulated by Pparg and to be related to adipogenic phenotype. Our data shows that TELM inhibited the osteogenic differentiation and mineralization of SHR BMSC, by favoring an adipogenic prone phenotype due to Pparg upregulation.

Fcγ receptors on aging neutrophils.

OBJECTIVE: Neutrophils are key effector cells of the innate immune system. They recognize antigens through membrane receptors, which are expressed during their maturation and activation. Neutrophils express FcγRII (CD32), FcγRIII (CD16), and FcγRI (CD64) after being activated by different factors such as cytokines and bacterial products. These receptors are involved with phagocytosis of IgG-opsonized microbes and enhance defense mechanisms. Based on that, our study seeks to compare the expression of FcγRII, FcγRIII, FcγRI, and CD11b on neutrophils from elderly and young subjects and their expression after in vitro activation with cytokines and LPS. METHODOLOGY: Neutrophils were isolated from human peripheral blood and from mice bone marrow by density gradient. After isolation, FCγRs expression was immediately analyzed by flow cytometry or after in vitro stimulation. RESULTS: In freshly isolated cells, the percentage of FcγRIIIb+ and CD11b+ neutrophils were higher in samples from young individuals; FcγRIIIa expression was more prominent on aged neutrophils; FcγRIA expression was similar in all samples analyzed. Exposure to CXCL8 and LPS resulted in a higher percentage of FcγRIa+ neutrophils on elderly individuals' samples but lower when compared with neutrophils from young donors. We observed that LPS caused an increase in FcγRIIa expression on aging human neutrophils. In contrast, FcγRIIIb expression in response to CXCL8 and LPS stimulation was not altered in the four groups. CD11b expression was lower in neutrophils from elderly individuals even in response to LPS and CXCL8. In mice, we observed differences only regarding CD11b expression, which was increased on aged neutrophils. LPS exposure caused an increase in all FcγRs. CONCLUSIONS: Our results suggest that, in humans, the overall pattern of FcγR expression and integrin CD11b are altered during aging and immunosenescence might contribute to age-related infection.

Fibrin Biopolymer Incorporated with Antimicrobial Agents: A Proposal for Coating Denture Bases.

The characteristics of the denture base surface, in combination with the oral environment, promote the colonization and development of Candida albicans biofilm, which is the main cause of denture stomatitis. This study evaluated the effectiveness of fibrin biopolymer with digluconate chlorhexidine or Punica granatum alcoholic extract to prevent C. albicans biofilm. Conventional heat polymerized and pre-polymerized poly(methyl methacrylate) (PMMA) circular specimens (10 × 2 mm) were fabricated (n = 504) and randomly divided into groups: no treatment (control-CT), fibrin biopolymer coating (FB), fibrin biopolymer with P. granatum (FBPg), or digluconate of chlorhexidine (FBCh) coating. The specimens were inoculated with C. albicans SC5314 (1 × 107 cells/mL) and incubated for 24, 48, and 72 h. Crystal violet and colony-forming unit assays were used to quantify the total biofilm biomass and biofilm-living cells. A qualitative analysis was performed using confocal laser scanning microscopy. Data obtained are expressed as means and standard deviations and were statistically analyzed using a three-way analysis of variance (α = 0.05). The FBPg and FBCh groups inhibited the growth of C. albicans biofilm in both PMMA materials analyzed, with FBCh performing better in all periods evaluated (p < 0.0001). The colony forming unit (CFU) assay showed that the FB group favored the C. albicans biofilm growth at 24 h and 48 h (p < 0.0001), with no differences with CT group at 72 h (p = 0.790). All groups showed an enhancement in biofilm development up to 72 h (p < 0.0001), except the FBCh group (p = 0.100). No statistical differences were found between the PMMA base materials (p > 0.050), except in the FB group (p < 0.0001). Fibrin biopolymer, albeit a scaffold for the growth of C. albicans, when combined with chlorhexidine digluconate or P. granatum, demonstrated excellent performance as a drug delivery system, preventing and controlling the formation of denture biofilm.

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.

Late complications after root coverage with two types of subepithelial connective tissue grafts, clinical and histopathological evaluation: A prospective cohort study.

AIM: This prospective cohort study evaluated late complications (LC) on recipient sites comparing two types of connective tissue grafts (CTG). MATERIALS AND METHODS: Participants (n: 60) were treated with coronally advanced flap (CAF) plus CTG harvested by de-epithelialized technique (DE) (n:31) or two-parallel incision (PI) (n:29). Areas were evaluated to identify white discharge associated or not with gingival cul-de-sac. Patients were ordered in groups with (DE+and PI+) or without (DE- and PI-) LC. Biopsies for histopathological analysis in LC areas were proposed. RESULTS: Six cases exhibited LC, 5 in DE graft (DE+) and 1 in PI graft (PI+) group; 2 were diagnosed at 3 months postoperatively, 3 at 6 months and one at 12 months. The relative risk for LC was 1.7 times greater for DE graft (p: 0.01; CI: 1.10 to 2.72; RR>1). Differences were not observed for clinical outcomes after both types of CTGs (p > 0.05). Biopsies showed deep invagination of the epithelial lining suggesting cyst-like area/ cavity with keratin content and consolidated in fibrous connective tissue. After 24 months biopsied areas presented no recurrence of LC, in non-biopsied patients the clinical condition remained unchanged. CONCLUSIONS: Considering the limitations of this study, LC on recipient sites demonstrated no statistical difference between two types of CTG.

Beneficial effects of three natural products for the treatment of denture stomatitis: a randomized clinical trial / Efeitos benéficos de três produtos naturais para o tratamento da estomatite protética: um ensaio clínico randomizado

Aim: To evaluate the effect of three natural antifungal agents combined with routine denture care on the treatment of DS, using a quantitative mycological culture analysis. Methods: Thirty denture wearers with denture stomatitis DS were treated using five substances: sterile distilled water (G1), nystatin oral suspension (G2), 20% alcoholic extract propolis (G3), Punica granatumLinné gel (G4), and Uncaria tomentosa gel (G5). The substances were used 3 times a day for 14 days. Quantitative mycological culture analysis of samples collected from the palatal mucosa was performed at three stages: before treatment (T0), after 14 days of treatment (T1), and 30 days after treatment completion (T2). Data were evaluated using Kruskal-Wallis and Friedman tests (p < 0.05). Results: Palatal mucosa intragroup analysis showed a significant reduction of Candida CFU/mL values for all groups at T1 compared to T0 (p < 0.05). However, they did not present statistical differences when comparing T1 and T2 (p > 0.05). The intergroup analysis demonstrated that there are no statistical differences, regardless of the evaluation time (p > 0.05). Conclusion:The natural products tested showed a satisfactory result on DS treatment, which proved to be equivalent to conventional topical therapy with nystatin and to treatment using only regular oral hygiene procedures.

Objetivo: Avaliar o efeito de três antifúngicos naturais combinados com o cuidado rotineiro com próteses dentárias no tratamento da EP, por meio de uma análise quantitativa de cultura micológica. Métodos: Trinta usuários de próteses dentárias com EP foram tratados com cinco substâncias: água destilada estéril (G1), suspensão oral de nistatina (G2), extrato alcoólico de própolis 20% (G3), gel Punica granatum L. (G4) e gel Uncaria tomentosa (G5). As substâncias foram utilizadas 3 vezes ao dia durante 14 dias. A análise micológica quantitativa das amostras coletadas da mucosa palatina foi realizada em três etapas: antes do tratamento (T0), após 14 dias do tratamento (T1) e 30 dias após o término do tratamento (T2). Os dados foram avaliados pelos testes de Kruskal-Wallis e Friedman (p < 0,05). Resultados: A análise intragrupo da mucosa palatina mostrou uma redução significativa dos valores de Candida UFC/mL para todos os grupos em T1 em comparação com T0 (p < 0,05). No entanto, não apresentaram diferenças estatísticas na comparação de T1 e T2 (p > 0,05). A análise intergrupos demonstrou que não há diferenças estatísticas, independentemente do tempo de avaliação (p > 0,05). Conclusão: Os produtos naturais testados apresentaram resultado satisfatório no tratamento da EP, sendo equivalente à terapia tópica convencional com nistatina e ao tratamento apenas com procedimentos rotineiros de higiene bucal.

Fcγ receptors on aging neutrophils

Abstract Objective Neutrophils are key effector cells of the innate immune system. They recognize antigens through membrane receptors, which are expressed during their maturation and activation. Neutrophils express FcγRII (CD32), FcγRIII (CD16), and FcγRI (CD64) after being activated by different factors such as cytokines and bacterial products. These receptors are involved with phagocytosis of IgG-opsonized microbes and enhance defense mechanisms. Based on that, our study seeks to compare the expression of FcγRII, FcγRIII, FcγRI, and CD11b on neutrophils from elderly and young subjects and their expression after in vitro activation with cytokines and LPS. Methodology Neutrophils were isolated from human peripheral blood and from mice bone marrow by density gradient. After isolation, FCγRs expression was immediately analyzed by flow cytometry or after in vitro stimulation. Results In freshly isolated cells, the percentage of FcγRIIIb+ and CD11b+ neutrophils were higher in samples from young individuals; FcγRIIIa expression was more prominent on aged neutrophils; FcγRIA expression was similar in all samples analyzed. Exposure to CXCL8 and LPS resulted in a higher percentage of FcγRIa+ neutrophils on elderly individuals' samples but lower when compared with neutrophils from young donors. We observed that LPS caused an increase in FcγRIIa expression on aging human neutrophils. In contrast, FcγRIIIb expression in response to CXCL8 and LPS stimulation was not altered in the four groups. CD11b expression was lower in neutrophils from elderly individuals even in response to LPS and CXCL8. In mice, we observed differences only regarding CD11b expression, which was increased on aged neutrophils. LPS exposure caused an increase in all FcγRs. Conclusions Our results suggest that, in humans, the overall pattern of FcγR expression and integrin CD11b are altered during aging and immunosenescence might contribute to age-related infection.

Telmisartan Prevents Alveolar Bone Loss by Decreasing the Expression of Osteoclasts Markers in Hypertensive Rats With Periodontal Disease.

Periodontal disease (PD) is a prevalent inflammatory disease with the most severe consequence being the loss of the alveolar bone and teeth. We therefore aimed to evaluate the effects of telmisartan (TELM), an angiotensin II type 1 receptor (Agtr1) antagonist, on the PD-induced alveolar bone loss, in Wistar (W) and Spontaneous Hypertensive Rats (SHRs). PD was induced by ligating the lower first molars with silk, and 10 mg/kg TELM was concomitantly administered for 15 days. The hemimandibles were subjected to microtomography, ELISA was used for detecting tumor necrosis factor (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), CXCL3, and CCL2, while qRT-PCR was used for analyzing expression of components of renin-angiotensin system (RAS) (Agt, Ace, Agt1r, Agt2r, Ace2, and Masr), and bone markers (Runx2, Osx, Catnb, Alp, Col1a1, Opn, Ocn, Bsp, Bmp2, Trap, Rank, Rankl, CtsK, Mmp-2, Mmp-9, and osteoclast-associated receptor (Oscar)). The SHR + PD group showed greater alveolar bone loss than the W + PD group, what was significantly inhibited by treatment with TELM, especially in the SHR group. Additionally, TELM reduced the production of TNF-α, IL-1ß, and CXCL3 in the SHR group. The expression of Agt increased in the groups with PD, while Agtr2 reduced, and TELM reduced the expression of Agtr1 and increased the expression of Agtr2, in W and SHRs. PD did not induce major changes in the expression of bone formation markers, except for the expression of Alp, which decreased in the PD groups. The bone resorption markers expression, Mmp9, Ctsk, and Vtn, was higher in the SHR + PD group, compared to the respective control and W + PD group. However, TELM attenuated these changes and increased the expression of Runx2 and Alp. Our study suggested that TELM has a protective effect on the progression of PD, especially in hypertensive animals, as evaluated by the resorption of the lower alveolar bone. This can be partly explained by the modulation in the expression of Angiotensin II receptors (AT1R and AT2R), reduced production of inflammatory mediators, the reduced expression of resorption markers, and the increased expression of the bone formation markers.

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.

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.

Should mast cells be considered therapeutic targets in multiple sclerosis?

Mast cells are immune cells of the myeloid lineage that are found throughout the body, including the central nervous system. They perform many functions associated with innate and specific immunity, angiogenesis, and vascular homeostasis. Moreover, they have been implicated in a series of pathologies (e.g., hypersensitivity reactions, tumors, and inflammatory disorders). In this review, we propose that this cell could be a relevant therapeutic target in multiple sclerosis, which is a central nervous system degenerative disease. To support this proposition, we describe the general biological properties of mast cells, their contribution to innate and specific immunity, and the participation of mast cells in the various stages of multiple sclerosis and experimental autoimmune encephalomyelitis development. The final part of this review is dedicated to an overview of the available mast cells immunomodulatory drugs and their activity on multiple sclerosis and experimental autoimmune encephalomyelitis, including our own experience related to the effect of ketotifen fumarate on experimental autoimmune encephalomyelitis evolution.

Calming Down Mast Cells with Ketotifen: A Potential Strategy for Multiple Sclerosis Therapy?

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by extensive inflammation, demyelination, axonal loss and gliosis. Evidence indicates that mast cells contribute to immunopathogenesis of both MS and experimental autoimmune encephalomyelitis (EAE), which is the most employed animal model to study this disease. Considering the inflammatory potential of mast cells, their presence at the CNS and their stabilization by certain drugs, we investigated the effect of ketotifen fumarate (Ket) on EAE development. EAE was induced in C57BL/6 mice by immunization with MOG35-55 and the animals were injected daily with Ket from the seventh to the 17th day after disease induction. This early intervention with Ket significantly reduced disease prevalence and severity. The protective effect was concomitant with less NLRP3 inflammasome activation, rebalanced oxidative stress and also reduced T cell infiltration at the CNS. Even though Ket administration did not alter mast cell percentage at the CNS, it decreased the local CPA3 and CMA1 mRNA expression that are enzymes typically produced by these cells. Evaluation of the CNS-barrier permeability indicated that Ket clearly restored the permeability levels of this barrier. Ket also triggered an evident lymphadenomegaly due to accumulation of T cells that produced higher levels of encephalitogenic cytokines in response to in vitro stimulation with MOG. Altogether these findings reinforce the concept that mast cells are particularly relevant in MS immunopathogenesis and that Ket, a known stabilizer of their activity, has the potential to be used in MS control.

Aliskiren Attenuates the Inflammatory Response and Wound Healing Process in Diabetic Mice With Periodontal Disease.

The aim of this study was to characterize the role of local RAS (renin-angiotensin system) in the inflammatory response of normal (N) and diabetic (D) mice with periodontal disease (PD). Diabetes Mellitus (DM) was induced by peritoneal injection of streptozotocin in Balb/c mice. PD was induced by ligature around the first molar in both N and D, irrespective of whether they were treated with aliskiren (50 mg/kg, Alisk). Mandibles were harvested for histomorphometric analyses, and gingival tissue (GT) was collected to evaluate gene expression and extracellular matrix components (ECM). Immunohistochemical (IHC) analyses were used to localize RAS in GT. The production of C-reactive protein (CRP), IL-1ß, CXCL2, and CCL8 was evaluated by enzyme-linked immunosorbent assay (ELISA). Renin was found to exacerbate the inflammation and periodontal bone loss at 14 days after PD, and Alisk inhibited this process in GT of N and D. PD increased CRP, CXCL2, CCL8, and IL-1ß production in both animals. Alisk could inhibit CRP, CXCL2, and CCL8 primarily in D animals. However, only CCL8 was decreased in N animals after Alisk pretreatment. PD enhanced expression and production of AGT, ACE, AT1R, and AT2R in both N and D. AT1R expression was higher in D with PD, and AT2R expression was higher in N with PD. ACE2 and receptor Mas (MasR) expression and production was elevated in the control group of both animals. PD inhibited ACE2 in N but not in D. MasR expression was unaffected in both N and D with PD. Alisk reduced expression and production of all RAS components in GT of both animals, except for ACE2 in N. RAS staining was observed in all layers of epithelium, basal cell layer, and lamina propria and was higher in N with PD. Col1a1, Col1a2, Col3a1, and fibronectin (Fn1) were increased in both animals with PD. Alisk inhibited Col1a1 and Fn in both animals, Col1a2 was decreased only in D, while levels of Col3a1 remained unchanged in all animal groups. In conclusion, these data demonstrated the presence and functional role of local RAS in GT, exacerbating the inflammatory response, periodontal bone loss, and wound healing processes in both N and D animal groups. In addition, Alisk was able to significantly reduce gingival inflammation, excessive wound healing processes, and periodontal bone loss.