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Angiotensin II (Ang II) plays a role in several inflammatory diseases and is capable of inducing the release of inflammatory mediators in several cell types. The objective was to investigate the potential of Ang II to induce mRNA expression of inflammatory mediators by cultured primary human periodontal cells. A synergistic effect of a co-treatment with Ang II and IL1β on mRNA expression of inflammatory mediators was also explored. Primary cultures of human fibroblast-like cells isolated from gingival and periodontal ligament tissues from 3 subjects were established using explant techniques. Immunophenotyping of STRO-1, AT1 and AT2 receptors was performed by flow cytometry analysis. Cell cultures were challenged with Ang II (1M) for 3, 6 and 24h with or without a co-treatment with IL1β (0.1ng/mL) for 24h. mRNA expression of inflammatory mediators was performed by real time quantitative polymerase chain reaction (RT-qPCR). We present, for the first time, a precise quantification for AT1 and AT2 receptors on human gingival and periodontal fibroblast-like cell types; the percentage of positive immunostaining compared to the total population of cells varied from 3.35% through 5.29% for AT1 and 2.97% through 4.57% for AT2. Ang II slightly upregulated mRNA expression of IL6 and CCL2/MCP1 in gingival cells and IL8 and PTGS2/COX2 in periodontal ligament cells. IL1β induced upregulation of IL8, IL6 CCL2/MCP1, PTGS2/COX2 and IL1β mRNA in both cell types. Co-treatment with Ang II and IL1β did not show a synergistic effect under these experimental conditions. Ang II shows a low potential to induce mRNA upregulation of inflammatory mediators in cultured primary human gingival and periodontal ligament cells, most likely due to the low percentage of Ang II receptors in such cells, and no synergistic effect with the co-treatment with IL1β.

Made available in DSpace on 2022-04-29T08:46:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-02-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. Faculdade de Ciências Universidade Estadual Paulista (Unesp), SP Faculdade de Medicina Universidade Federal de Mato Grosso do Sul (UFMS), Cidade Universitária, Unit 9, MS Departamento de Bioquimica e Imunologia Universidade de São Paulo, Escola de Medicina de Ribeirão Preto, SP Lauro de Souza Lima Institute, SP Faculdade de Odontologia de Bauru (FOB) Universidade de São Paulo (USP), SP Faculdade de Ciências Universidade Estadual Paulista (Unesp), SP FAPESP: # 2013/15513-3

Background and objective Several studies have demonstrated that mast cells are equipped with versatile tools to combat and kill bacteria. Additionally, mast cells produce and secrete a variety of mediators, which either regulate the host's immune system or directly attack bacteria. In this study, the intracellular microbicidal capacity of mast cells against Aggregatibacter actinomycetemcomitans was evaluated. Methods Murine mast cells were challenged in vitro with A actinomycetemcomitans for 3, 5, 10, and 24 hours. Subsequently, the colony-forming units were counted. Additionally, the production and release of nitric oxide and hydrogen peroxide were analyzed by DAF-FM diacetate, the Griess reaction, and the Amplex Red kit, respectively. Cell death was evaluated using FITC Annexin V and propidium iodide staining. Results Mast cells are able to efficiently eliminate periodontopathogen, with best results after 10 hours of intracellular challenge. The production/release of nitric oxide-and to a lesser extent of hydrogen peroxide-by mast cells was in agreement with its microbicidal capacity. Ninety percent of the mast cells maintained their cellular viability even after 24 hours of bacterial challenge. Conclusions This is-to the best of our knowledge-the first report to describe the intracellular microbicidal activity of mast cells against A actinomycetemcomitans, concerning the production and release of potentially bactericidal substances. Further, the low number of cell deaths confirms that the decreased number of colony-forming units was due to the higher antimicrobial activity of mast cells. The results highlight the importance of these cells in the defense mechanisms of biofilm-induced periodontal disease.

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.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 χsup2/suptests, ANOVA and the Tukey test or the Friedman and Kruskal-Wallis tests (α=0.05).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 (Plt;0.05), but this difference was maintained at follow-up only for the relined dentures (Plt;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 (Plt;0.05).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.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.

Made available in DSpace on 2022-04-29T08:46:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-05 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. Department of Basic Sciences São Paulo State University (UNESP) School of Dentistry Multicenter Postgraduate Program in Physiological Sciences Brazilian Society of Physiology São Paulo State University (UNESP) School of Dentistry Department of Stomatology Bauru School of Dentistry University of São Paulo (USP) Department of Biological Science Bauru School of Dentistry University of São Paulo (USP) Department of Basic Sciences São Paulo State University (UNESP) School of Dentistry Multicenter Postgraduate Program in Physiological Sciences Brazilian Society of Physiology São Paulo State University (UNESP) School of Dentistry

Made available in DSpace on 2019-10-06T16:46:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 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. Department of Microbiology and Immunology Institute of Biosciences São Paulo State University (UNESP), Rua Dr. Plinio Pinto e Silva, S/N, Distrito de Rubião Júnior Department of Chemistry and Biochemistry Institute of Biosciences São Paulo State University (UNESP) Department of Surgery Stomatology Pathology and Radiology Bauru School of Dentistry University of São Paulo (USP) Department of Microbiology and Immunology Institute of Biosciences São Paulo State University (UNESP), Rua Dr. Plinio Pinto e Silva, S/N, Distrito de Rubião Júnior Department of Chemistry and Biochemistry Institute of Biosciences São Paulo State University (UNESP)

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 &lt, 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 &lt, 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 &lt, 0.0001), except the FBCh group (p = 0.100). No statistical differences were found between the PMMA base materials (p &gt, 0.050), except in the FB group (p &lt, 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.

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 granatum Linné 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.

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.

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.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.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.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.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.

Made available in DSpace on 2021-06-25T11:03:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-01 Universidade Estadual Paulista School of Dentistry, University of Michigan 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-1β, 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. Programa Multicêntrico de Pós-graduação em Ciências Fisiológicas SBFis São Paulo State University (UNESP) School of Dentistry Department of Basic Sciences São Paulo State University (UNESP) School of Dentistry Department of Biological Science Bauru School of Dentistry University of São Paulo (USP) Department of Stomatology Bauru School of Dentistry University of São Paulo (USP) Programa Multicêntrico de Pós-graduação em Ciências Fisiológicas SBFis São Paulo State University (UNESP) School of Dentistry Department of Basic Sciences São Paulo State University (UNESP) School of Dentistry

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.

Made available in DSpace on 2020-12-10T20:03:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-22 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) 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 withEscherichia colilipopolysaccharide/1 mu g/mL(EcLPS)orEnterococcus faecalislipoteichoic acid/1 mu g/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 (R), respectively, for Interleukin (IL)-1 alpha, IL-1 beta, 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-alpha), 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-alpha), Interferon-gamma (IFN gamma), Vascular Endothelial Growth Factor (VEGF), Colony stimulating factor 1 (CSF-1) and Macrophage colony-stimulating factor (M-CSF). Results EcLPSincreased IL-1 alpha, IL-1 beta, IL-8, CCL2, CCL5, TNF-alpha and CSF-1 mRNA and protein levels whileEfLTAwas 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 withEcLPSandEfLTA. Univ Sao Paulo, Bauru Sch Dent, Dept Biol Sci, Bauru, SP, Brazil Univ Sao Paulo, Bauru Sch Dent, Dept Surg Stomatol Pathol & Radiol, Bauru, SP, Brazil Univ Sao Paulo, Bauru Sch Dent, Dept Pediat Dent Orthodont & Publ Hlth, Bauru, SP, Brazil Sao Paulo State Univ, Sch Dent Aracatuba, Dept Basic Sci, Aracatuba, SP, Brazil Jackson Lab, 600 Main St, Bar Harbor, ME 04609 USA Sao Paulo State Univ, Sch Dent Aracatuba, Dept Basic Sci, Aracatuba, SP, Brazil FAPESP: 2015/03965-2 FAPESP: 2016/11450-5 CNPq: 307986/2017-9 CAPES: 001

Made available in DSpace on 2020-12-12T01:34:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Aim: To investigate the immune response of disseminated Ryzopus oryzae infection in immunocompetent mice. Methods: C57Bl/6, BALB/c and Swiss wild-type mice were intravenously infected with R. oryzae; the parameters of infection and immune response were determined. Transcriptional signature of Th17 immune response and infection in Il17ra-/- mice were also evaluated. Results: All mouse strains showed an initial spread of R. oryzae in the target tissues; however, after 30 days, C57Bl/6 and BALB/c mice showed an effective fungal clearance associated with specific production of IL-17 and IL-2. We also observed that 60% of Il17ra-/- mice succumbed to infection within 16 days. Conclusion: This study has established an immunocompetent model for disseminated mucormycosis and highlighted the role of IL-17 signaling in immunity against R. oryzae. Universidade Estadual Paulista (Unesp) Faculdade de Ciências Universidade Estadual Paulista (Unesp) Instituto de Biociências Lauro de Souza Lima Institute Universidade de São Paulo (USP), Faculdade de Odontologia de Bauru (FOB) Universidade Federal de Mato Grosso do Sul (UFMS) Faculdade de Medicina Universidade Estadual Paulista (Unesp) Faculdade de Ciências Universidade Estadual Paulista (Unesp) Instituto de Biociências

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.

Made available in DSpace on 2022-04-29T08:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-07-20 Squamous cell carcinoma (SCC) is the second most common form of skin cancer and the mechanism(s) involved in the progression of this tumor are unknown. Increases in the expression of IL-33/ST2 axis components have been demonstrated to contribute to neoplastic transformation in several tumor models and interleukin-33 is correlated with poor prognosis of patients with squamous cell carcinoma of the tongue. Based on these observations, we sought to determine the role of the IL-33/ST2 pathway during the development of SCC. Our findings show that ST2- deficiency led to a marked decrease in the severity of skin lesions, suggesting that ST2 signaling contributed to tumor development. An analysis of tumor lesions in wild-type and ST2KO mice revealed that a lack of ST2 was associated with specific and significant reductions in the numbers of CD4+ T cells, CD8+ T cells, dendritic cells, and macrophages. In addition, NK cells that were isolated from ST2KO mice exhibited higher cytotoxic activity than cells isolated from wild-type mice. Notably, ST2 deficiency resulted in lower IFN-γ, TNF-α, IL-10, and IL-17 production in tumor samples. Our findings indicate that the IL-33/ST2 pathway contributes to the development of SCC by affecting leukocyte migration to tumor microenvironment and impairing NK cytotoxic activity. Department of Biological Sciences Bauru School of Dentistry University of São Paulo, Al. Dr. Octávio Pinheiro Brisolla Department of Microbiology and Immunology Institute of Biosciences of Botucatu São Paulo State University, R. Prof. Dr. Antônio Celso Wagner Zanin Department of Stomatology - Oral Pathology Bauru School of Dentistry University of São Paulo, Al. Dr. Octávio Pinheiro Brisolla Department of Biochemistry and Immunology School of Medicine of Ribeirão Preto University of São Paulo Department of Biomedical Sciences (Research Division of Immunology) and Medicine F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute Cedars-Sinai Medical Center Department of Medicine Division of Pulmonary and Critical Care Medicine Cedars-Sinai Medical Center Samuel Oschin Comprehensive Cancer Institute Cedars-Sinai Medical Center Department of Microbiology and Immunology Institute of Biosciences of Botucatu São Paulo State University, R. Prof. Dr. Antônio Celso Wagner Zanin

Background Tumour metastasis has been associated with cancer stem cells, a small population with stem-like cells properties, higher rate of migration and metastatic potential compared to cells from the tumour bulk. Our aim was to evaluate the immunoexpression of the putative cancer stem cell biomarkers ALDH1 and CD44 in primary tumour and corresponding metastatic lymph nodes. Methods Tumour tissue specimens (n = 50) and corresponding metastatic lymph nodes (n = 25) were surgically obtained from 50 patients with oral squamous cell carcinoma and submitted to immunohistochemistry. CD44 and ALDH1 were semi-quantitatively scored according to the proportion and intensity of positive cells within the invasive front and metastatic lymph nodes as a whole. A combined score was obtained by multiplying both parameters and later dichotomized into a final score classified as low (≤2) or high (>2) immunoexpression. Results ALDH1 immunoexpression and CD44 immunoexpression were detected in both tumour sites, although the means of ALDH1 (P = .0985) and CD44 (P = .4220) cells were higher in metastasis compared to primary tumours. ALDH1high was positively associated (P = .0184) with angiolymphatic invasion, while CD44high was positively associated (P = .0181) with metastasis (N+). At multivariate analysis, CD44 significantly increased the odds of lymph node metastasis, regardless of T stage (OR = 8.24; 1.64-65.64, P = .0088). Conclusions CD44 immunoexpression was a significant predictor of lymph node metastasis, while ALDH1high immunostaining was associated with angiolymphatic invasion. Altogether, it suggests that immunoexpression of CD44 and ALDH1 links the cancer stem cell phenotype with oral squamous cell carcinoma invasion and metastasis.

Due to the low incidence of the Ewing's Sarcoma (ES) family tumors, the available epidemiology is likely to be unreliable, and at present, there are no standard diagnostic or clinical guidelines outlining their management. This report describes a case of peripheral primitive neuroectodermal tumor (ES/pPNET) which initially mimicked cystic lesions, and describes a comparison between ES and ES/pPNET in the jaws by the World Health Organization classification. This review addressed 63 cases published in the English literature between 1950 and 2016. The majority of cases were ES. Both ES and ES/pPNET mimicked other benign entities such as traumatic, cystic and inflammatory lesions. The patients who died of their disease had a history of metastatic tumors, and primary tumor located in the mandible and maxilla for ES and ES/pPNET, respectively. The differentiation of the ES family tumors from other small blue-cell tumors may be difficult and requires familiarity with histological and immunohistochemical features.

Context: Equisetum giganteum L. (Equisetaceae) is an endemic plant of Central and South America used in traditional medicine. Natural drugs have been frequently used in the treatment of a myriad of diseases, proving to be an alternative to synthetic chemicals, and have been intensively studied in the prevention of sicknesses, including oral diseases. Objective: This study evaluated the in vitro antiadherent activity of E. giganteum extract against Candida albicans biofilms. Materials and methods: Crystal violet and colony-forming units assays were used to quantify the total biofilm biomass and biofilm living cells on a denture base acrylic resin pretreated with hydroethanolic extract of E. giganteum in different concentrations (50, 25, 16, 8, and 4 mg/mL), after 24 h of biofilm development. Results: Equisetum giganteum affected biofilms by reduction of biomass and living cells per area of acrylic specimens. The results revealed reduction of 15–44% of the biofilm mass and reduction of numbers of colony-forming units (CFUs) present in biofilms (79%) compared to the untreated control (CTRL/PBS). At all concentrations, it demonstrated important antiadherent activity on Candida albicans biofilms, the main microbe in denture stomatitis. Discussion and conclusion: The present findings show that E. giganteum antimicrobial effects may qualify the extract as a promising natural alternative for topical treatment or prevention of denture stomatitis. The usage of drugs made of natural products shows advantages in relation to synthetic drugs on the market, such as lower cost, lower toxicity, and in relation to the occurrence of microbial resistance.

The presence of Candida albicans in the biofilm underlying the dental prosthesis is related to denture stomatitis (DS), an inflammatory reaction of the oral mucosa. The oral epithelium, a component of the innate immune response, has the ability to react to fungal invasion. In this study, we evaluated the in vitro effect of viable C. albicans on the apoptosis, nitric oxide (NO) production, and &beta, defensin 2 (hBD-2) expression and production of human palate epithelial cells (HPECs). We further determined whether or not these effects were correlated with fungal invasion of epithelial cells. Interaction between HPEC primary culture and C. albicans was obtained through either direct or indirect cell&ndash, cell contact with a supernatant from a hyphal fungus. We found that the hyphae supernatants were sufficient to induce slight HPEC apoptosis, which occurred prior to the activation of the specific mechanisms of epithelial defense. The epithelial defense responses were found to occur via NO and antimicrobial peptide hBD-2 production only during direct contact between C. albicans and HPECs and coincided with the fungus&rsquo, s intraepithelial invasion. However, although the hBD-2 levels remained constant in the HPEC supernatants over time, the NO release and hBD-2 gene expression were reduced at a later time (10 h), indicating that the epithelial defense capacity against the fungal invasion was not maintained in later phases. This aspect of the immune response was associated with increased epithelial invasion and apoptosis maintenance.

ResumoA paracoccidioidomicose (PCM) é uma doença fúngica limitante com ocorrência em países latino‐americanos. Lesões secundárias em mucosa e pele são observadas em sua forma crónica. Um homem leucoderma de 60 anos, etilista, hipertenso, fumante e ex‐garimpeiro apresentou‐se cancerofóbico e com a queixa principal de dor do lado direito da boca. Ao exame intraoral, observou‐se uma úlcera extensa com bordas irregulares, endurecida e edemaciada. Foi realizada biópsia incisional e, por meio de exame histopatológico após coloração por hematoxina e eosina e Gomori‐Grocott, concluiu‐se que o referente caso clínico era de PCM. O paciente foi encaminhado a um infectologista e foi tratado com itraconazol 200mg. Assim, objetivamos relatar um caso clínico de PCM com sinais e sintomas intraorais.AbstractParacoccidioidomycosis (PCM) is a fungal disease with occurrence in Latin America. Secondary lesions in mucosa and skin are observed in chronic presentation. One man, caucasian, 60 years old, alcoholic, hypertensive, smoke and ex‐miner, cancer‐phobia and with main complain of pain in the right side of the mouth. At intraoral exam, was observed a large ulcer with irregular borders, hard and edematous. It was made a incisional biopsy and through histopathologic exam, after Hematoxin and Eosin and Gomori‐Groccot, the final diagnosis was PCM. The patient was referred to a infectologist and the treatment was Itraconazol 200mg. We aimed report a case of PCM with intraoral signs and symptoms.

Although some morphological investigations on aged human sublingual glands (HSG) found eventual phenomena identified as autolysis and mucous extravasation, the exact meaning of these findings has not been elucidated. Objective The aim of this work is to investigate whether acinar autolysis and mucous extravasation are related to the aging process in human sublingual glands. We also speculate if autolytic changes may assist forensic pathologists in determining time of death. Material and Methods 186 cadavers’ glands were allocated to age groups: I (0–30 years); II (31–60), and III (61–90). Time and mode of death were also recorded. Acinar autolysis and mucous extravasation were classified as present or absent. Ultrastructural analysis was performed using transmission electron microscopy (TEM). Data were compared using Mann-Whitney U, Spearman’s correlation coefficient, Kruskal-Wallis, and Dunn tests (p

Made available in DSpace on 2015-12-07T15:34:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2015. Added 1 bitstream(s) on 2015-12-07T15:52:49Z : No. of bitstreams: 1 PMC4531177.pdf: 2455935 bytes, checksum: f43a0355a37eee2d18dbbb1909e90828 (MD5) Equisetum giganteum L. (E. giganteum), Equisetaceae, commonly called giant horsetail, is an endemic plant of Central and South America and is used in traditional medicine as diuretic and hemostatic in urinary disorders and in inflammatory conditions among other applications. The chemical composition of the extract EtOH 70% of E. giganteum has shown a clear presence of phenolic compounds derived from caffeic and ferulic acids and flavonoid heterosides derived from quercitin and kaempferol, in addition to styrylpyrones. E. giganteum, mainly at the highest concentrations, showed antimicrobial activity against the relevant microorganisms tested: Escherichia coli, Staphylococcus aureus, and Candida albicans. It also demonstrated antiadherent activity on C. albicans biofilms in an experimental model that is similar to dentures. Moreover, all concentrations tested showed anti-inflammatory activity. The extract did not show cytotoxicity in contact with human cells. These properties might qualify E. giganteum extract to be a promising alternative for the topic treatment and prevention of oral candidiasis and denture stomatitis. Department of Stomatology (Pathology), Bauru School of Dentistry, University of São Paulo (USP), Alameda Doutor Octávio Pinheiro Brisolla 9-75, 17012-901 Bauru, SP, Brazil. Institute of Biosciences, São Paulo State University (UNESP), Botucatu, SP, Brazil. Department of Prosthodontics, Bauru School of Dentistry, University of São Paulo (USP), Alameda Doutor Octávio Pinheiro Brisolla 9-75, 17012-901 Bauru, SP, Brazil. Department of Biological Sciences, Faculty of Sciences, São Paulo State University (UNESP), Bauru, SP, Brazil. Institute of Biosciences, São Paulo State University (UNESP), Botucatu, SP, Brazil. Department of Biological Sciences, Faculty of Sciences, São Paulo State University (UNESP), Bauru, SP, Brazil.

Existem poucos estudos a respeito da presença de constituintes fúngicos na circulação sanguínea de indivíduos com estomatite por dentadura (ED) (AHMAD et al., 2002), considerada uma forma localizada de candidose; o que poderia caracterizar o poder de invasão sistêmica do fungo nesta condição local, bem como um prévio reconhecimento desses antígenos por células presentes na circulação sanguínea do hospedeiro, o que poderia explicar aspectos específicos da resposta imune localizada e sistêmica. Assim sendo, este trabalho teve por objetivo avaliar a presença do fungo Candida albicans (C. albicans) no palato, na superfície interna das próteses totais superiores e no sangue de pacientes com ED, em dois momentos distintos. A população de estudo foi composta por indivíduos usuários de prótese total superior (PTS), com e sem estomatite por dentadura, avaliados e selecionados nas clínicas de graduação e pós-graduação da Disciplina de Prótese, do Departamento de Prótese, da Faculdade de Odontologia de Bauru da Universidade de São Paulo (FOB USP). Indivíduos não usuários de próteses removíveis constituíram o grupo controle. Assim o trabalho foi constituído por três grupos, cada um com 14 pacientes. As lesões de estomatite por dentadura foram diagnosticadas clinicamente e por meio de confirmação microbiológica em CHROMAgar Candida, a partir de material biológico coletado da mucosa palatal e da superfície interna da PTS. A PCR foi realizada quando da ocorrência do crescimento de colônias verdes para diferenciação das espécies C. albicans e C. dubliniensis. As amostras de sangue foram analisadas para a detecção de fragmentos de DNA responsáveis pela codificação da proteína da parede da hifa1(Hwp1) de C. albicans, utilizando a técnica da PCR. Os resultados demonstraram que nem os usuários de PTS, independentemente da presença de ED, nem os voluntários não usuários apresentaram a proteína Hwp1 no sangue, em nenhuma das amostras coletadas. A presença de fungos do gênero Candida foi mais frequente (p 0,005) entre os usuários de PTS com ED quando comparado com os outros indivíduos. Além disso, pudemos constatar que os pacientes com diagnóstico clínico e microbiológico de ED não apresentaram distribuição sanguínea de C. albicans. There are few studies about the presence of yeast constituents in the bloodstream of patients with denture stomatitis (DE), a localizated kind of candidiasis; what could characterize the yeast systemic invasion power in this local condidition, and also previous acknowledgement of these antigens by cells of the entertainer bloodstream, and explain specific features of the immune located and systemic answer. So being, this work had as a goal to evaluate the presence of the yeast Candida albicans (C. albicans) at the palate, at the internal surface of the upper denture and in the blood of patients with DE, at two different moments. The population of study was composed by individuals both with and without upper denture, with and without stomatitis, assessed and selected in the clinics of graduation and postgraduation of the Discipline of Prosthesis, of the Department of Prosthesis, of the Faculdade de Odontologia de Bauru of the University of São Paulo (FOB USP). Individuals who are not users of removable dentures constituted the group control. So the work was constituted by three groups, each one with 14 patients. The injuries of stomatitis were diagnosed clinically and through microbiological confirmation in CHROMAgar Candida, from biological collected material of the palatal mucosa and of the internal surface of the upper denture. The PCR was carried out when the growth of green colonies for differentiation of the stain C. albicans and C. dubliniensis happened. The samples of blood were analyzed for the detection of fragments of DNA responsible for the codification of the hyphal wall protein (Hwp1) of C. albicans. The results demonstrated that not even the users of upper denture, independently of the presence of the DE, not even the volunteers who are not users presented the protein Hwp1 in the blood, in none of the collected samples. The yeast Candida presence was more frequent (p 0,005) in the group 1 when compared with the other groups. Morever, we can conclude that patients with clinic and microbiologic diagnostic have not presented bloodstream distribution of C. albicans.

Lipomas are very common benign slow-growing soft tissue neoplasms composed of mature adipose tissue mostly diagnosed in the fifth decade of life. These tumors rarely present in the oral cavity, representing less than approximately 5% of all benign mouth tumors. They are usually less than 2cm in size and etiology remains unclear. We report a young male patient presenting with a giant lipoma in the buccal mucosa. Histopathology revealed a large area of mature fat cells consistent with conventional lipoma and an area of the mucosal lining of the lesion suggestive of morsicatio buccarum. In the present article, we emphasize the clinicopathological features and differential diagnosis of the disease.

O objetivo deste trabalho foi avaliar, in vitro, se a incorporacao de extratos hidroalcoolicos de Equisetum giganteum (Eg) e de Punica granatum (Pg) a um adesivo protetico influencia no desenvolvimento do biofilme de Candida albicans sobre a superficie de resina acrilica termopolimerizavel. Apos identificacao dos compostos por HPLC-PAD, foi selecionada a fracao e a concentracao de interesse por meio da concentracao inibitoria minima (CIM). Os biofilmes de C. albicans foram induzidos durante 3, 6 ou 12 horas sobre corpos de prova de resina acrilica, previamente submetidos ao tratamento com o adesivo associado aos fitoterapicos (AD/Eg ou AD/Pg). Como controles, corpos de prova foram tratados apenas com adesivo (AD), ou com a associacao adesivo/nistatina (AD/Nt) ou nao recebam tratamento (PBS), n=2 por grupo, em cada experimento. A atividade antimicrobiana foi avaliada por meio das unidades formadoras de colonias (UFC/mL), e pelo percentual de reducao da atividade metabolica pelo ensaio colorimetrico - XTT. Os resultados foram expressos como media ± desvio padrao, e submetidos ao teste de Kruscal-Wallis e Mann-Whitney (UFC/mL); e ANOVA-2 seguido do teste de Tukey HSD e o teste de Dunnett (XTT), com significância quando p˂ 0,05. Foi possivel identificar compostos derivados de kaempferol e quercetina em Eg e punicalina, em Pg. A associacao de ambos os fitoterapicos ao adesivo (AD/Eg ou AD/Pg) reduziram significativamente o biofilme de C. albicans sobre a superficie da resina, em comparacao ao grupo AD. Em acordo, houve aumento do percentual de reducao da atividade metabolica do biofilme em todos os periodos, na presenca dos fitoterapicos. Por fim, sugerimos que a associacao destes fitoterapicos ao adesivo protetico podera constituir uma alternativa temporaria, viavel e inovadora para auxiliar no tratamento e/ou prevencao da Estomatite Protetica. Apoio Financeiro: FAPESP #2014/07012-7 Descritores: Estomatite sob Protese; Adesivos Teciduais; Fitoterapia.

Squamous cell carcinoma (SCC) is one of the most common human cancers worldwide. Recent studies show that regulatory T cells (Treg) have a critical role in the modulation of an antitumor immune response, and consequently the SCC development. Because the accumulation of Tregs at the tumor site is, in part, due to selective recruitment through CCR5- and CCR5-associated chemokines, we investigated the role of CCR5 in the SCC development. Our findings showed that CCR5-deficient mice (CCR5KO) were efficient in controlling papilloma's incidence when compared with wild-type mice. Analysis of tumor lesions in wild-type (WT) and CCR5KO mice revealed that lack of CCR5 lead to significant reduction in frequency of Tregs and increased of CD4 T cells into the tumors. Moreover, the adoptive transfer of naturally occurring Tregs CD4+CD25+CCR5+, CD4+CD25−CCR5+ or CD8+CCR5+ conventional T cells to CCR5KO mice resulted in an increased papilloma incidence. Interestingly, adoptive transfer of WT CD4+CD25+CCR5+ cells to CCR5KO mice induced more undifferentiated SCC lesions, characterized by higher infiltration of macrophages and dendritic cells. In this study, we also demonstrated that Treg migration to the tumor microenvironment is mediated by CCR5, and these cells are promoting tumor growth via inhibition of antitumor cells such as cytotoxic CD8+ T cells. Our findings reinforce the therapeutic potential of CCR5 inhibition for cancer treatment, and indicate an attractive approach for SCC treatment. Mol Cancer Ther; 16(12); 2871–80. ©2017 AACR.