Topical anti-inflammatory activity of yacon leaf extracts

Smallanthus sonchifolius (Poepp.) H. Rob. , Asteraceae, known as yacon, is an herb that is traditionally used for the treatment of diabetes in folk medicine. However, recent studies have demonstrated that this plant has other interesting properties such as anti-microbial and anti-inflammatory actions. Thus, the purpose of this study was to evaluate the topical anti-inflammatory property of different extracts prepared from yacon leaves and analyze the role of different chemical classes in this activity. Three yacon leaf extracts were obtained: aqueous extract, where chlorogenic acid derivatives and sesquiterpene lactones were detected; leaf rinse extract, rich in sesquiterpene lactones; and polar extract, rich in chlorogenic acid derivatives. All the extracts exhibited anti-edematogenic activity in vivo (aqueous extract: 25.9% edema inhibition at 0.50 mg/ear; polar extract: 42.7% inhibition at 0.25 mg/ear; and leaf rinse extract: 44.1% inhibition at 0.25 mg/ear). The leaf rinse extract furnished the best results regarding neutrophil migration inhibition, and NO, TNF-? and PGE2 inhibition. These data indicate that both sesquiterpene lactones and chlorogenic acid derivatives contribute to the anti-inflammatory action, although sesquiterpene lactones seem to have more pronounced effects. In conclusion, yacon leaf extracts, particularly the sesquiterpene lactone-rich extract, has potential use as topical anti-inflammatory agent.FAPESP #2007/00844-3CAPES #314/0

Inflammasome activation is critical to the protective immune response during chemically induced squamous cell carcinoma

Chronic inflammation affects most stages of tumorigenesis, including initiation, promotion, malignant differentiation, invasion and metastasis. Inflammasomes have been described as involved with persistent inflammation and are known to exert both pro and antitumour effects. We evaluated the influence of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase (CASP)-1 in the antitumor immune response using a multistage model of squamous cell carcinoma (SCC) development. Absence of ASC and CASP-1 resulted in an earlier incidence and increased number of papilloma. Loss of inflammassome function in mice resulted in decreased presence of natural killer (NK), dendritic (DC), CD4+, CD8+ and CD45RB+ T cells in the tumor lesions as well as in lymph nodes (LN) compared with WT mice. Increased percentage of CD4+CD25+Foxp3+ T cells was associated with association with inflammasome loss of function. Moreover, significant differences were also found with neutrophils and macrophage infiltrating the lesions. Myeloperoxidase (MPO), but not elastase (ELA), activity oscillated among the groups during the SCC development. Levels of proinflammatory cytokines IL-1β, IL-18, Tumor Necrosis Factor (TNF)-α and Interferon (IFN)-γ were decreased in the tumor microenvironment in the absence of inflammasome proteins. These observations suggest a link between inflammasome function and SCC tumorigenesis, indicating an important role for inflammasome activation in the control of SCC development.Fil: Gasparoto, Thais Helena. Universidad de São Paulo. Faculdade de Odontologia de Bauru. Departamento de Ciencias Biológicas; BrasilFil: Ervolino de Oliveira, Carine. Universidad de São Paulo. Faculdade de Odontologia de Bauru. Departamento de Ciencias Biológicas; BrasilFil: Thomazini de Freitas, Luisa. Universidad de São Paulo. Faculdade de Odontologia de Bauru. Departamento de Ciencias Biológicas; BrasilFil: Ramos Pinheiro, Claudia. Universidad de São Paulo. Faculdade de Odontologia de Bauru. Departamento de Ciencias Biológicas; BrasilFil: Issa Hori, Juliana. University of São Paulo. Faculdade de Medicina de Ribeirão Preto; BrasilFil: Pompermaier Garlet, Gustavo. Universidad de São Paulo. Faculdade de Odontologia de Bauru. Departamento de Ciencias Biológicas; BrasilFil: Cavassani, Karen Angélica. University Of Michigan; Estados UnidosFil: Schillaci, Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Santana Da Silva, Joao. University of São Paulo. Faculdade de Medicina de Ribeirão Preto; BrasilFil: Simmões Zamboni, Darío. University of São Paulo. Faculdade de Medicina de Ribeirão Preto; BrasilFil: Campanelli, Ana Paula. Universidad de São Paulo. Faculdade de Odontologia de Bauru. Departamento de Ciencias Biológicas; Brasi

Inflammatory events during murine squamous cell carcinoma development

Background: Squamous cell carcinoma (SCC) is one of the most common human cancers worldwide. In SCC, tumour development is accompanied by an immune response that leads to massive tumour infiltration by inflammatory cells, and consequently, local and systemic production of cytokines, chemokines and other mediators. Studies in both humans and animal models indicate that imbalances in these inflammatory mediators are associated with cancer development. Methods: We used a multistage model of SCC to examine the involvement of elastase (ELA), myeloperoxidase (MPO), nitric oxide (NO), cytokines (IL-6, IL-10, IL-13, IL-17, TGF-β and TNF-α), and neutrophils and macrophages in tumour development. ELA and MPO activity and NO, IL-10, IL −17, TNF-α and TGF-β levels were increased in the precancerous microenvironment. Results: ELA and MPO activity and NO, IL-10, IL −17, TNF-α and TGF-β levels were increased in the precancerous microenvironment. Significantly higher levels of IL-6 and lower levels of IL-10 were detected at 4 weeks following 7,12-Dimethylbenz(a)anthracene (DMBA) treatment. Similar levels of IL-13 were detected in the precancerous microenvironment compared with control tissue. We identified significant increases in the number of GR-1+ neutrophils and F4/80+/GR-1- infiltrating cells in tissues at 4 and 8 weeks following treatment and a higher percentage of tumour-associated macrophages (TAM) expressing both GR-1 and F4/80, an activated phenotype, at 16 weeks. We found a significant correlation between levels of IL-10, IL-17, ELA, and activated TAMs and the lesions. Additionally, neutrophil infiltrate was positively correlated with MPO and NO levels in the lesions. Conclusion: Our results indicate an imbalance of inflammatory mediators in precancerous SCC caused by neutrophils and macrophages and culminating in pro-tumour local tissue alterations.FAPESP [2011/03195-1; 2006/01617-8; 2009/14127-7; 2009/03471-9]Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Study of neutrophils from elderly in the defense mechanisms against Candida-related denture stomatitis

Os neutrófilos são as primeiras células a migrar para sítios de infecção e desempenham importante papel na defesa contra vários patógenos, especialmente Candida albicans. A função dos neutrófilos envolve fagocitose e destruição de leveduras e formas filamentosas do fungo, além de produção de citocinas e quimiocinas, capazes de ativar outras células envolvidas na eliminação de C. albicans. Recentemente, tem sido mostrado que estas células apresentam prejuízos com o avanço da idade e estes fenômenos fazem parte de uma área da Imunologia conhecida como Imunossenescência. A estomatite protética é a lesão oral mais verificada em idosos e a sua persistência é diretamente relacionada com a presença de espécies de Candida, principalmente C. albicans, nas lesões e na superfície interna de próteses, especialmente totais superiores (PTS). Com o objetivo de avaliar possíveis alterações em neutrófilos que poderiam predispor usuários de prótese total superior à estomatite protética, foram analisados neutrófilos de sangue e de saliva de idosos e jovens com estomatite protética associada à Candida (EPC), bem como de indivíduos controles. No primeiro capítulo foi avaliada a positividade de Candida e as diferentes espécies deste patógeno em lesões de estomatite protética e superfície interna de PTS. C. albicans foi a espécie mais detectada em lesões de EP e na PTS. No segundo capítulo, os neutrófilos do sangue dos indivíduos da pesquisa foram caracterizados quanto ao fenótipo ex vivo, função fagocítica e produção de citocinas e quimiocinas após desafio com C. albicans. Neutrófilos do sangue de indivíduos com EPC, bem como de idosos sem EPC, apresentaram características relacionadas com suscetibilidade às doenças causadas por C. albicans. No terceiro capítulo, os neutrófilos salivares dos indivíduos foram caracterizados quanto ao fenótipo ex vivo e função fagocítica após desafio com C. albicans. Além disso, níveis de citocinas e quimiocinas relacionadas com resposta imune de proteção ou suscetibilidade à infecções por C. albicans foram detectadas nas amostras de saliva. Os resultados indicaram diferenças nos neutrófilos salivares e componentes solúveis da saliva de pacientes com EPC. Também, alterações observadas nos neutrófilos resultantes da imunossenescência podem tornar os idosos mais suscetíveis a infecções por Candida e doenças associadas com a presença deste fungo.Neutrophils are the first line of immune cells to migrate into infection sites, playing an important role against pathogens, especially Candida albicans. These cells phagocytose and destroy yeasts or filamentous forms of this fungus, beyond secrete cytokines and chemokines that activate other immune cells to eliminate C. albicans. Nowadays, it has been shown that the ageing damages neutrophil function and such events are described as part of Imunosenescence. Denture stomatitis is the most oral lesion present in elderly, and its persistence is related to Candida species, especially C. albicans, in the lesions and prosthesis fitting surface, particularly maxillary prosthesis (MP). In order to evaluate possible neutrophil alterations predisposing denture wearers to Candida- related denture stomatitis (DS), blood and salivary neutrophils from elderly and younger individuals with DS, as well control ones, were analyzed. Thus, the first chapter addresses the presence of Candida species on the denture stomatitis lesions and MP fitting surface. C. albicans was the most species detected in DS lesions and MP. On the second chapter, blood neutrophils from the individuals were characterized about ex vivo phenotype, phagocytic function, and cytokines and chemokines production after challenged with C. albicans. Blood neutrophils from DS individuals, as well elderly without DS, presented characteristics of susceptibility to C. albicans diseases. On the third chapter, salivary neutrophils from the individuals were characterized about ex vivo phenotype and phagocytic function against C. albicans. In addition, salivary cytokines and chemokines involved with resistance or susceptibility to C. albicans infection were detected. Results indicated differences in salivary neutrophils and soluble components from patients with DS. In addition, alterations observed in neutrophils by immunosenescence could facilitate Candida infections and diseases related to the presence of this pathogen in elderly

The pattern recognition receptors expressed on neutrophils and the associated cytokine profile from different aged patients with Candida-related denture stomatitis

This study evaluated the expression of pattern recognition receptors (PRRs) and activation factors associated with salivary and blood neutrophils from different aged patients diagnosed with Candida-related denture stomatitis (DS). Expression of neutrophil PRRs was determined by flow cytometry and immunofluorescence, and the levels of selected cytokines that influence immune activation were determined by ELISA. The salivary (but not the serum derived) neutrophils of individuals with DS were found to have an increased expression of CD69 regardless of the age of the patient compared to patients without DS. However, these salivary neutrophils had a lower expression of CD66b and CD64. Expression of TLR2 was lower on the salivary-and serum-derived neutrophils from elderly individuals compared to the neutrophils of younger subjects, regardless of whether the individual had DS. Salivary interleukin (IL)-4 was elevated in both of the elderly subject groups (with or without DS). Only elderly DS patients were observed to have increased serum IL-4 levels and reduced salivary IL-12 levels. Younger DS patients showed an increase in salivary IL-10 levels, and both the saliva and the serum levels of IFN-gamma were increased in all of the younger subjects. Our data demonstrated that changes in both the oral immune cells and the protein components could be associated with DS. Furthermore, changes in the blood-derived factors were more associated with age than DS status. (C) 2012 Elsevier Inc. All rights reserved.FAPESPFAPESP [05/60668-9, 06/59612-1

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

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

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

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

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

The purpose of this study was to explore the potential of confocal laser scanning microscopy (CLSM) for in situ identification of live and dead Enterococcus faecalis in infected dentin. Eight cylindrical dentin specimens were infected with Enterococcus faecalis in BHI for 21 days. After the experimental period, the specimens were stained with fluorescein diacetate (FDA) and propidium iodide (PI) or acridine orange (0.01 %) and analyzed by CLSM. Two noninfected dentin specimens were used as negative controls. CLSM analysis shows that the discrimination between viable (green) and dead (red) bacteria in infected dentinal tubules could be observed after staining with FDA/PI. Acridine orange was able to show metabolic activity of the E. faecalis cells inside the dentinal tubules showed by its red fluorescence. The viability of bacteria in infected dentin can be determined in situ by CLSM. FDA/PI and acricline orange are useful for this technique.FAPESP[2007/01838-7

Isolation of Candida dubliniensis from denture wearers

Candida albicans is considered the most important Candida species able to cause oral infections in denture wearers. In recent years, Candida dubliniensis has emerged as a pathogenic yeast in humans. The close phenotypic similarities of C. albicans and C. dubliniensis have led to the misidentification of these species. In this work, our aim was to verify through PCR the presence of C. dubliniensis in palate and maxillary denture samples from 112 denture wearers presenting with or without denture-related stomatitis (DRS). C. dubliniensis was isolated at low rates from both palate (5.3% and 10.7%) and maxillary denture (5.3% and 8.9%) samples from wearers regardless of the presence of the disease. However, when C. dubliniensis was detected in individuals with DRS, it was always associated with C. albicans. In addition, our results showed that C. albicans was the most commonly identified candidal species in maxillary denture and hard palate samples from DRS patients (78.5% and 89.2%, respectively) as well as from controls (31.2% and 28.5%, respectively). In conclusion, C. dubliniensis was detected in the oral environment of denture wearers. The association of C. dubliniensis with C. albicans occurred in approximately 10% of the DRS cases.FAPESP[05/60668-9]FAPESP[06/59612-1

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

Objective: Aggregatibacter actinomycetemcomitans is an oral Gram-negative bacterium that contributes to periodontitis progression. Isolated antigens from A. actinomycetemcomitans could be activating innate immune cells through Toll-like receptors (TLRs). In this study, we evaluated the role of TLR4 in the control of A. actinomycetemcomitans infection. Material and Methods: We examined the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR4(-/-) mice. The production of cytokines was evaluated by ELISA. The bacterial load was determined by counting the number of colony-forming units per gram of tissue. Results: The results showed that TLR4-deficient mice developed less severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly lower bone loss and inflammatory cell migration to periodontal tissues. However, the absence of TLR4 facilitated the A. actinomycetemcomitans dissemination. Myeloperoxidase activity was diminished in the periodontal tissue of TLR4(-/-) mice. We observed a significant reduction in the production of tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1 beta in the periodontal tissue of TLR4(-/-) mice. Conclusion: The results of this study highlighted the role of TLR4 in controlling A. actinomycetemcomitans infection.State of Sao Paulo Research Foundation (FAPESP)[06/06072-0