43 kDa Glycoprotein (gp43) from Paracoccidioides brasiliensis Induced IL-17A and PGE2 Production by Human Polymorphonuclear Neutrophils: Involvement of TLR2 and TLR4.

The glycoprotein gp43 is the major antigenic/diagnostic component of Paracoccidioides brasiliensis, one of the etiologic agents of paracoccidioidomycosis (PCM). Gp43 has protective roles in mice, but due to adhesive properties, this glycoprotein has also been associated with immune evasion mechanisms. The present study evaluated gp43 interaction in vitro with Toll-like receptors 2 and 4 (TLR2 and TLR4) present in polymorphonuclear neutrophils (PMNs) from healthy human individuals and the consequent modulation of the immune response through the expression and release of cytokines and eicosanoids. PMNs were incubated in the absence or presence of monoclonal antibodies anti-TLR2 and anti-TLR4 (individually or in combination) before gp43 stimulation. Then, PMNs were analyzed for the expression of both surface receptors and the detection of intracytoplasmic IL-17A and IL-4 using flow cytometry, while the production of PGE2, LTB4, IL-6, IL-10, IL-12, IFN-γ, and TNF-α was evaluated in the supernatants by enzyme-linked immunosorbent assay (ELISA). Our results showed that gp43 increased TLR2 and TLR4 expression by PMNs and induced PGE2 and IL-17A via TLR4 and TLR2, respectively. Thus, our data suggest that gp43 from P. brasiliensis might modulate host susceptibility to the fungal infection by affecting PGE2 and IL-17A production.

Causes for hospitalization of elderly individuals due to primary care sensitive conditions and its associated contextual factors.

OBJECTIVE: The objective of this study was to analyze the hospitalizations of the elderly for conditions sensitive to primary care (ICSAP) and associated contextual factors, referring to elderly people aged 60 and over, living in municipalities in the Northeast region. METHODS: Characterized as being an ecological study using data from the Hospital Information System (SIH) and the Basic Attention Information System (SIAB) referring to elderly people aged 60 and over. RESULTS: The total hospitalization rate was 527,524, with the highest number due to heart failure, followed by cerebrovascular diseases, and infectious gastroenteritis. Analyzing the ICSAP rates with the contextual factors, all were significant. Regarding the coverage of basic care, a similarity occurred between them, and for the rate of the number of consultations among the elderly, despite the greater number of these in the municipalities with higher hospitalization rates, there was no significant difference between them. CONCLUSION: We conclude that the contextual factors interfere in the conditions of this hospitalization, necessitating, besides the improvement of primary care, an improvement in the living conditions of the elderly population.

Causes for hospitalization of elderly individuals due to primary care sensitive conditions and its associated contextual factors

SUMMARY OBJECTIVE The objective of this study was to analyze the hospitalizations of the elderly for conditions sensitive to primary care (ICSAP) and associated contextual factors, referring to elderly people aged 60 and over, living in municipalities in the Northeast region. METHODS Characterized as being an ecological study using data from the Hospital Information System (SIH) and the Basic Attention Information System (SIAB) referring to elderly people aged 60 and over. RESULTS The total hospitalization rate was 527,524, with the highest number due to heart failure, followed by cerebrovascular diseases, and infectious gastroenteritis. Analyzing the ICSAP rates with the contextual factors, all were significant. Regarding the coverage of basic care, a similarity occurred between them, and for the rate of the number of consultations among the elderly, despite the greater number of these in the municipalities with higher hospitalization rates, there was no significant difference between them. CONCLUSION We conclude that the contextual factors interfere in the conditions of this hospitalization, necessitating, besides the improvement of primary care, an improvement in the living conditions of the elderly population.

RESUMO OBJETIVO Objetivou-se realizar uma análise das internações dos idosos por condições sensíveis à atenção primária (ICSAP) e fatores contextuais associados em idosos com 60 anos ou mais, residentes em municípios da Região Nordeste. MÉTODOS Caracterizou-se por ser um estudo ecológico utilizando dados do Sistema de Informação Hospitalar (SIH) e do Sistema de Informação da Atenção Básica (Siab) referentes a idosos com 60 anos ou mais. RESULTADOS A taxa total de internação foi de 527,524, sendo em maior quantidade aquelas por insuficiência cardíaca, seguidas das doenças cerebrovasculares e, em terceiro, as gastroenterites infecciosas. Analisando as taxas de ICSAP com os fatores contextuais, todas foram significativas. Em relação à cobertura da atenção básica, ocorreu uma similaridade entre eles, e para a taxa do número de consultas entre idosos, apesar do maior número destas nos municípios com maiores taxas de internação, não existiu diferença significativa entre eles. CONCLUSÃO Concluímos que os fatores contextuais interferem nas condições dessa internação, necessitando, além da melhoria da atenção primária, uma melhoria nas condições de vida da população idosa.

Concordância entre índices de necessidade de tratamento ortodôntico em estudo de base populacional com adolescentes escolares / Agreement between indexes of orthodontic treatment need in population based study with teenager students

Diversos índices analisam a prevalência das oclusopatias, sendo métodos de avaliar a necessidade e priorização do tratamento ortodôntico. Baseado nas possíveis divergências, este estudo avaliou a concordância entre o ICON (Índice de Complexidade, Efeito e Necessidade de Tratamento Ortodôntico), DHC (Componente de Saúde Dental) do IOTN (Índice de Necessidade de Tratamento Ortodôntico), AC (Componente de Estética Dental) do examinador e DAI (Índice de Estética Dental) ao diagnosticarem indivíduos com ou sem necessidade de tratamento para oclusopatias e em seus graus de severidade. Examinou-se 300 estudantes de 15 a 19 anos de Macaíba/RN. A concordância para a categorização com ou sem necessidade foi dada pelo Kappa, e para severidade através do coeficiente W de Kendall com significância de 5%. A prevalência de necessidade de tratamento foi de 76% no DAI, 31% no ICON, 73,7% no DHC e 29,3% no AC. O Kappa não foi significativo (k=0,054; p=0,351) ao comparar o DAI com DHC. Outras comparações foram significativas (p<0,001), com concordância fraca (k=0,148-0,158), exceção a do ICON com AC (k=0,866). A concordância no coeficiente W de Kendall foi fraca (w=0,365), embora significativa (p<0,001). Há classificação divergente para a necessidade de tratamento ortodôntico ao utilizar diferentes índices, com consequências ao diagnóstico e priorização do tratamento em nível populacional e individual.(AU)

everal indices analyse the prevalence of malocclusion as methods of evaluating and prioritizing orthodontic treatment need. Based on possible divergences, this study examined the correlation between ICON (Complexity Index, Effect and Need Orthodontic Treatment), DHC (Dental Health Component), IOTN (Index of Orthodontic Treatment Need), AC (Component Dental Aesthetics) of examiner and DAI (Dental Aesthetic Index) to diagnose individuals with or without treatment need for malocclusion and its severity degree. We examined 300 students from 15 to 19 years of Macaíba/RN. The agreement for the categorization with or without treatment need was given by Kappa coefficient and for severity by Kendall W coeficient with 5% significance. The prevalence of treatment need was 76% in the DAI, 31% in the ICON, 73.7% in the DHC and 29.3% in the AC. Kappa was not significant (k = 0.054, p = 0.351) when comparing the DAI with DHC. Other comparisons were significant (p <0.001), with weak agreement (k = 0.148 to 0.158), exception to the ICON with AC (k = 0.866). The coefficient correlation in W Kendall was weak (w = 0.365), although significant (p <0.001). There is divergent classification for orthodontic treatment need when using different indices, with implications for diagnosis and treatment priority in individual and population level.(AU)

Role of Dectin-1 receptor on cytokine production by human monocytes challenged with Paracoccidioides brasiliensis.

Fungal recognition by Dectin-1 receptor triggers a series of cellular mechanisms involved in a protective activation of the immune system. In this study, we aimed to evaluate the participation of Dectin-1 receptor in the induction of IL-8, TNF-α, IL-12, IL-10 and IL-17A secretion by human monocytes activated with different cytokines, and challenged in vitro with Paracoccidioides brasiliensis (P. brasiliensis). Our results show that monocytes challenged with P. brasiliensis (Pb265) are able to produce IL-12, IL-8, IL-17, IL-10 and TNF-α. Dectin-1 receptor blockage decreased the IL-12, IL-17, IL-10 and TNF-α levels indicating the participation of such receptor in the induction of these cytokines. Only IL-8 production was not affected by the blockage. Cells activation with different cytokines showed that GM-CSF was able to induce secretion of all cytokines and the receptor blockage prior to the challenge also decreased the cytokine secretion, except IL-8. Monocytes activated with TNF-α promoted IL-8, IL-10 and TNF-α production, whereas stimulation with IFN-γ promoted mainly IL-12 and TNF-α. Thus, these findings bring new and important knowledge about Dectin-1 participation in cytokines production by monocytes challenged with Pb265.

TLR9 stimulation induces increase in fungicidal activity of human dendritic cells challenged with Paracoccidioides brasiliensis.

Microorganisms killing by dendritic cells (DCs) is an important effector mechanism during innate immune response, as it can avoid dissemination of infection during migration of these cells toward draining lymph nodes. However, this function depends on pattern recognition receptors (PRRs) to which the microorganism will bind in these cells. Regarding this, TLR9 activation, by stimulating the oxidative metabolism, induces increase in microbicidal activity of these cells. Accordingly, we showed that DCs treatment with a TLR9 agonist results in an increase in fungicidal activity of these cells against the fungus Paracoccidioides brasiliensis (Pb), which however, was not associated to higher H2O2 levels.

Cryptococcus neoformans and gattii promote DNA damage in human peripheral blood mononuclear cells.

Cryptococcosis, a systemic mycosis capable of disseminating to the central nervous system with frequent lethal effects, is caused by the species Cryptococus neoformans and Cryptococcus gattii. Several infectious agents such as virus, bacteria, and parasites may be associated to DNA damage and carcinogenesis in humans. Products of the oxidative metabolism, such as NO, produced as a host defense mechanism to destroy these pathogens, have been implicated in this damage process, due to excessive production related to an established chronic inflammatory response. Here, we investigated whether C. neoformans and /or C. gattii can cause DNA damage in human peripheral blood mononuclear cells (PBMCs) and whether this process is related to NO levels produced by PBMCs. We found that both species are equally able to induce genotoxicity in PBMCs. However, an association between DNA damage and high NO levels was only detected in relation to C. gattii. The results point to the possibility that patients with cryptococcosis are more susceptible to the development of other diseases.

Internações em idosos e fatores contextuais associados no nordeste brasileiro / Hospitalizations in the elderly and associated contextual factors in northeastern Brazil

Esse estudo objetivou investigar as internações dos idosos por grupo de causa e por condições sensíveis à atenção primária (ICSAP), identificando sua relação com fatores contextuais na região Nordeste do Brasil. Realizou-se um estudo com delineamento ecológico, que utilizou dados secundários oriundos do Sistema de Informação Hospitalar (SIHSUS) para a descrição das taxas de internação por grupo de causas e por ICSAP em idosos a partir de 60 anos ou mais, nos anos de 2010 a 2015. Como variável dependente, utilizou-se a taxa de internação hospitalar nos idosos por grupo de causas e por ICSAP. Realizou-se a análise de cluster não-hierárquica (K-means) agrupando os municípios com base nos grupos de causas de internações gerais e aquelas sensíveis à atenção primária, sendo formado 3 Clusters, os quais foram classificados como altas taxas, intermediárias e baixas taxas de internação. Para a associação com as variáveis dependentes, foram selecionadas 11 variáveis independentes socioeconômicas contextuais, resumindo-as através da Análise Fatorial, tendo a extração dos fatores sido realizada pela Análise de Componentes Principais (ACP). Obteve-se, portanto, um componente denominado "Urbanização e seus reflexos", o segundo nominado de "Contexto socioeconômico favorável" e o terceiro intitulado "Pouca escolaridade e dependência do Estado". Além dessas variáveis, utilizou-se o Índices de GINI, IDH, FIRJAN, a média da cobertura da Atenção Básica dos municípios nos anos de 2010 a 2015 e a taxa do número de consultas em idosos na atenção básica nesse mesmo período. A taxa total de internação no período foi 1.212,60, sendo em maior quantidade aquelas por doenças do aparelho circulatório, seguidas de doenças do aparelho respiratório e das doenças infecciosas e parasitárias. Os clusters de taxas de internação diferiram significativamente em relação aos fatores contextuais, de modo que os municípios que possuem as maiores taxas de internações apresentaram os valores mais altos para o contexto socioeconômico favorável, sendo os maiores valores relativos à pouca escolaridade e dependência do estado os dos municípios de menores taxas de internação. Em relação às ICSAP, a taxa total de internação foi de 527,524 (43,5% do total das internações), sendo em maior quantidade aquelas por insuficiência cardíaca, seguidas das doenças cerebrovasculares e das gastroenterites infecciosas. Existiu diferença significativa entre os clusters e os fatores, onde o cluster que possui as maiores taxas de internações foi o que apresentou as maiores médias para o contexto socioeconômico favorável e para pouca escolaridade e dependência do estado. O cluster que possui menores taxas de internação possuiu as maiores médias para o fator "urbanização e seus reflexos". Na análise da cobertura da atenção básica e taxa de consultas de idosos na atenção básica, observou-se que os clusters não diferiram significativamente. Diante desse contexto, conclui-se que na população idosa do Nordeste há predomínio de internações por doenças do aparelho circulatório e das ICSAP por insuficiência cardíaca. Os fatores contextuais interferem de modo significativo nas internações da população idosa, sendo importante analisá-los na construção das políticas públicas de saúde, atentando para as vulnerabilidades dessa população e contribuindo com a equidade em saúde (AU).

This study aimed to investigate the hospitalizations of the elderly by cause group and conditions sensitive to primary care (ICSAP), identifying their relationship with contextual factors in the Northeast region of Brazil. A study with an ecological proposal was carried out by using secondary data from the Hospital Information System (SIH-SUS) to describe hospitalization rates by cause group and by ICSAP in the elderly aged 60 and over from the years 2010 to 2015. As a dependent variable, the hospital admission rate in the elderly was used by cause group and by ICSAP. Non-hierarchical cluster analysis (K-means) was performed by grouping the municipalities based on the groups of causes of general admissions and those sensitive to primary care, with 3 Clusters being formed, which were classified as high, intermediate and low hospitalization rates. For the association with the dependent variables, 11 independent socioeconomic contextual variables were selected, summarizing them through the Factor Analysis, with the extraction of the factors being performed by Principal Component Analysis (PCA). Therefore, a component called "Urbanization and its reflexes" was obtained, the second named "Favorable socioeconomic context" and the third entitled "Little education and necessity of the State". Other than these variables, the indexes of GINI, IDH, FIRJAN, the average coverage of primary care in the municipalities from the years 2010 to 2015, and the rate of the number of consultations in the elderly in basic care in the same period were used. The total hospitalization rate in the period was 1,212.60, with the greatest number being due to diseases of the circulatory system, followed by diseases of the respiratory system and infectious and parasitic diseases. The clusters of hospitalization rates differed significantly in relation to the contextual factors, so that the municipalities with the highest hospitalization rates presented the highest values for the favorable socioeconomic context, the highest values being related to the little education and state necessity of municipalities with lower hospitalization rates. In relation to the ICSAP, the total hospitalization rate was 527,524 (43.5% of all hospitalizations), with the highest number due to heart failure, followed by cerebrovascular diseases and infectious gastroenteritis. There was a significant difference between all the clusters and factors, where the cluster with the highest admission rates was the one that presented the highest averages for the favorable socioeconomic context and for little education and state necessity. The cluster with the lowest hospitalization rates had the highest averages for the "urbanization and its reflexes" factor. In the analysis of the coverage of primary care and the rate of consultations of the elderly in basic care, it was observed that the clusters did not differ significantly. Having this context in mind, one may conclude that in the elderly population of the Northeast there is a majority of hospitalizations due to diseases of the circulatory system and ICSAP due to heart failure. Contextual factors interfere significantly in hospitalizations of the elderly population, and it is important to analyze them in the construction of public health policies, taking into account the vulnerabilities of this population and contributing to health equity (AU).

Agaricus brasiliensis polysaccharides stimulate human monocytes to capture Candida albicans, express toll-like receptors 2 and 4, and produce pro-inflammatory cytokines.

BACKGROUND: Agaricus brasiliensis is a medicinal mushroom with immunomodulatory and antitumor activities attributed to the ß-glucans presented in the polysaccharide fraction of its fruiting body. Since ß-glucans enhance cellular immunoresponsiveness, in this study we aimed to evaluate the effect of an acid-treated polysaccharide-rich fraction (ATF) of A. brasiliensis on the ability of human monocytes to adhere/phagocyte C. albicans yeast cells, their expression of pattern recognition receptors and their ability to produce cytokines. METHODS: Adhesion/phagocytosis of FITC-labeled C. albicans was evaluated by flow cytometry. Cells were incubated with specific fluorochrome-labeled antibodies for TLR2 and 4, ßGR and MR and also evaluated by flow cytometry. Monocytes were cultured with ATF, and culture supernatants were collected for analysis of in vitro cytokine production by ELISA (TNF-α, IL-1ß, IL-12 and IL-10). RESULTS: ATF significantly increased the adherence/phagocytosis of C. albicans by monocytes and this was associated with enhanced expression of TLR2 and TLR4, while no effect was observed on ßGR or MR. Moreover, expression of TLR4 and TLR2 was associated with higher levels of in vitro production of TNF-α and IL-1, respectively. Production of IL-10 was also increased by ATF treatment, but we found no association between its production and the expression of Toll-like receptors. CONCLUSION: Our results provided us with evidence that A. brasiliensis polysaccharides affect human monocytes probably through the modulation of Toll-like receptors.

Agaricus brasiliensis polysaccharides stimulate human monocytes to capture Candida albicans, express toll-like receptors 2 and 4, and produce pro-inflammatory cytokines

Abstract Background Agaricus brasiliensis is a medicinal mushroom with immunomodulatory and antitumor activities attributed to the -glucans presented in the polysaccharide fraction of its fruiting body. Since -glucans enhance cellular immunoresponsiveness, in this study we aimed to evaluate the effect of an acid-treated polysaccharide-rich fraction (ATF) of A. brasiliensis on the ability of human monocytes to adhere/phagocyte C. albicans yeast cells, their expression of pattern recognition receptors and their ability to produce cytokines. Methods Adhesion/phagocytosis of FITC-labeled C. albicans was evaluated by flow cytometry. Cells were incubated with specific fluorochrome-labeled antibodies for TLR2 and 4, GR and MR and also evaluated by flow cytometry. Monocytes were cultured with ATF, and culture supernatants were collected for analysis of in vitro cytokine production by ELISA (TNF-, IL-1, IL-12 and IL-10). Results ATF significantly increased the adherence/phagocytosis of C. albicans by monocytes and this was associated with enhanced expression of TLR2 and TLR4, while no effect was observed on GR or MR. Moreover, expression of TLR4 and TLR2 was associated with higher levels of in vitro production of TNF- and IL-1, respectively. Production of IL-10 was also increased by ATF treatment, but we found no association between its production and the expression of Toll-like receptors. Conclusion Our results provided us with evidence that A. brasiliensis polysaccharides affect human monocytes probably through the modulation of Toll-like receptors.

Agaricus brasiliensis polysaccharides stimulate human monocytes to capture Candida albicans, express toll-like receptors 2 and 4, and produce pro-inflammatory cytokines

Agaricus brasiliensis é um cogumelo medicinal com atividades imunomoduladoras e antitumorais atribuídas aos ß-glucanos presentes na fração polissacarídica de seu corpo de frutificação. Uma vez que os ß-glucanos aumentam a imunorresponsividade celular, neste estudo objetivamos avaliar o efeito de uma fração rica em polissacarídeos tratados com ácido (ATF) de A. brasiliensis sobre a capacidade de monócitos humanos de aderir / fagocitar células de levedura C. albicans . expressão de receptores de reconhecimento de padrões e sua capacidade de produzir citocinas. Métodos: A adesão / fagocitose de C. albicans marcada com FITC foi avaliada por citometria de fluxo. As células foram incubadas com anticorpos marcados com fluorocromo específicos para TLR2 e 4, ßGR e MR e também avaliadas por citometria de fluxo. Os monócitos foram cultivados com ATF, e os sobrenadantes da cultura foram coletados para análise da produção de citocinas in vitro por ELISA (TNF-α, IL-1ß, IL-12 e IL-10). Resultados: ATF aumentou significativamente a aderência / fagocitose de C. albicans por monócitos e isso foi associado com expressão aumentada de TLR2 e TLR4, enquanto nenhum efeito foi observado em ßGR ou MR. Além disso, a expressão de TLR4 e TLR2 foi associada a níveis mais elevados de produção in vitro de TNF-α e IL-1, respectivamente. A produção de IL-10 também foi aumentada pelo tratamento com ATF, mas não encontramos associação entre sua produção e a expressão de receptores Toll-like. Conclusão: Nossos resultados nos forneceram evidências de que polissacarídeos de A. brasiliensis afetam monócitos humanos provavelmente através da modulação de receptores Toll-like.(AU)

Participation of dectin-1 receptor on NETs release against Paracoccidioides brasiliensis: Role on extracellular killing.

Paracoccidioides brasiliensis is a dimorphic fungus from the Paracoccidioides genus, which is the causative agent of paracoccidioidomycosis, a chronic, subacute or acute mycosis, with visceral and cutaneous involvement. This disease that is acquired through inhalation primarily attacks the lungs but, can spread to other organs. Phagocytic cells as neutrophils play an important role during innate immune response against this fungus, but studies on antifungal activities of these cells are scarce. In addition to their ability to eliminate pathogens by phagocytosis and antimicrobial secretions, neutrophils can trap and kill microorganisms by release of extracellular structures composed by DNA and antimicrobial proteins, called neutrophil extracellular traps (NETs). Here, we provide evidence that P. brasiliensis virulent strain (P. brasiliensis 18) induces NETs release. These structures were well evidenced by scanning electron microscopy, and specific NETs compounds such as histone, elastase and DNA were shown by confocal microscopy. In addition, we have shown that dectin-1 receptor is the main PRR to which fungus binds to induce NETS release. Fungi were ensnared by NETs, denoting the role of these structures in confining the fungus, avoiding dissemination. NETs were also shown to be involved in fungus killing, since fungicidal activity detected before and mainly after neutrophils activation with TNF-α, IFN-γ and GM-CSF was significantly inhibited by cocultures treatment with DNAse.

Neutrophil Extracellular Traps Identification in Tegumentary Lesions of Patients with Paracoccidioidomycosis and Different Patterns of NETs Generation In Vitro.

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil. It is caused by the thermo-dimorphic fungus of the genus Paracoccidioides (Paracoccidioides brasiliensis and Paracoccidioides lutzii). Innate immune response plays a crucial role in host defense against fungal infections, and neutrophils (PMNs) are able to combat microorganisms with three different mechanisms: phagocytosis, secretion of granular proteins, which have antimicrobial properties, and the most recent described mechanism called NETosis. This new process is characterized by the release of net-like structures called Neutrophil Extracellular Traps (NETs), which is composed of nuclear (decondensed DNA and histones) and granular material such as elastase. Several microorganisms have the ability of inducing NETs formation, including gram-positive and gram-negative bacteria, viruses and some fungi. We proposed to identify NETs in tegumentary lesions of patients with PCM and to analyze the interaction between two strains of P. brasiliensis and human PMNs by NETs formation in vitro. In this context, the presence of NETs in vivo was evidenced in tegumentary lesions of patients with PCM by confocal spectrum analyzer. Furthermore, we showed that the high virulent P. brasiliensis strain 18 (Pb18) and the lower virulent strain Pb265 are able to induce different patterns of NETs formation in vitro. The quantification of extracellular DNA corroborates the idea of the ability of P. brasiliensis in inducing NETs release. In conclusion, our data show for the first time the identification of NETs in lesions of patients with PCM and demonstrate distinct patterns of NETs in cultures challenged with fungi in vitro. The presence of NETs components both in vivo and in vitro open new possibilities for the detailed investigation of immunity in PCM.

Interleukin-18 increases TLR4 and mannose receptor expression and modulates cytokine production in human monocytes.

Interleukin-18 is a proinflammatory cytokine belonging to the interleukin-1 family of cytokines. This cytokine exerts many unique biological and immunological effects. To explore the role of IL-18 in inflammatory innate immune responses, we investigated its impact on expression of two toll-like receptors (TLR2 and TLR4) and mannose receptor (MR) by human peripheral blood monocytes and its effect on TNF-α, IL-12, IL-15, and IL-10 production. Monocytes from healthy donors were stimulated or not with IL-18 for 18 h, and then the TLR2, TLR4, and MR expression and intracellular TNF-α, IL-12, and IL-10 production were assessed by flow cytometry and the levels of TNF-α, IL-12, IL-15, and IL-10 in culture supernatants were measured by ELISA. IL-18 treatment was able to increase TLR4 and MR expression by monocytes. The production of TNF-α and IL-10 was also increased by cytokine treatment. However, IL-18 was unable to induce neither IL-12 nor IL-15 production by these cells. Taken together, these results show an important role of IL-18 on the early phase of inflammatory response by promoting the expression of some pattern recognition receptors (PRRs) that are important during the microbe recognition phase and by inducing some important cytokines such as TNF-α and IL-10.

The involvement of TLR2 and TLR4 in cytokine and nitric oxide production in visceral leishmaniasis patients before and after treatment with anti-leishmanial drugs.

Toll-like receptors (TLRs) have significant involvement in Leishmania infection, although little is known about the relationship between these receptors, cytokines and nitric oxide (NO) in patients with visceral leishmaniasis (VL) before or after treatment with anti-leishmanial drugs. The goal of this study was to evaluate the expression of TLR2 and TLR4 in CD3+ and CD14+ cells and the production of TNF-α, IFN-γ, IL-17, IL-10, TGF-ß and NO in peripheral blood mononuclear cells (PBMCs) from VL patients pre- and post-treatment with anti-leishmanial drugs. In addition, we investigated whether these receptors were involved in the production of these cytokines and NO. In the active VL patients, increased TLR2 and TLR4 expression in lymphocytes and monocytes, increased production of TNF-α, IL-10 and TGF-ß and decreased production of IFN-γ, IL-17 and NO were observed. After treatment, TLR2 and TLR4 were still expressed in lymphocytes and monocytes, the TNF-α and IL-10 levels were lower, the production of IFN-γ, IL-17 and NO was higher, and the TGF-ß level remained high. Before treatment, the production of TNF-α and NO was associated with TLR2 and TLR4 expression, while IL-10 production was only associated with TLR2 expression. After treatment, both receptors were associated with the production of TNF-α, IFN-γ, IL-10 and NO, while the production of IL-17 was associated only with TLR4 expression. The results presented in this study suggest that both TLR2 and TLR4 participate in the modulation of cytokine and NO production in VL patients, contributing to the pathogenesis of VL prior to treatment and the protective immune response after treatment.

Interferon-gamma production by human neutrophils upon stimulation by IL-12, IL-15 and IL-18 and challenge with Paracoccidioides brasiliensis.

Paracoccidiodomycosis is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb), which is endemic in Latin America. The host innate immune response against the fungus has been well characterized and several studies have shown the important role played by phagocytic cells. Our laboratory has studied the relationship between human neutrophils (PMNs)/Pb, focusing the effector mechanisms of these cells against the fungus. However, in last years, studies have shown that in addition to their phagocytic and killer functions, PMNs can modulate and instruct the immune response, since these cells have been shown to produce and release several cytokines. Thus, we evaluated whether PMNs stimulated with Pb can modulate the immune response to a Th1 phenotype through the production of IFN-γ, as well as the role of "pattern-recognition receptors" (PRRs) such as TLR2, TLR4 and Dectin-1 in this production. Furthermore, we asked whether activation of the cells with the cytokines IL-12, IL-15 and IL-18 could result in increased levels of this cytokine. Peripheral blood PMNs obtained from 20 healthy donors were nonactivated or activated with IL-12, IL-15 or IL-18 in different concentrations and challenged with strain 18 Pb (Pb18) for 2 h, 4 h, 12 h, 24 h and 48 h and evaluated for IFN-γ production, by ELISA. In other experiments, PMNs were treated with monoclonal antibodies anti-TLR2, TLR4 and Dectin-1, challenged with Pb and evaluated for IFN-γ production. We found that Pb induces human PMNs to produce IFN-γ, probably by binding to TLR4 and Dectin-1 receptors expressed by these cells. Moreover, IFN-γ levels were significantly increased when cells were activated with each of the tested cytokines or a combination of two of them, being the association IL-12 plus IL-15 the most effective. The results support our hypothesis that during infection by Pb, human PMNs modulate the adaptive immune response to a Th1 response pattern, via IFN-γ production.

[Reliability of functional vision assessment in children with low vision from 2 to 6 years old--searching for evidences]. / Confiabilidade da avaliação da visão funcional para crianças com baixa visão de dois a seis anos--em busca de evidências.

PURPOSE: The functional vision assessment (AVIF-2 to 6 years) for low vision children with ages from 2 to 6 years old was analyzed. The inter-rater, test-retest and the internal consistency were verified for reliability. METHODS: The test, composed by seven domains (visual fixation, visual following, visual field confrontation, eye-hand coordination and surrounding locomotion, contrast vision and color vision discrimination), was applied to 40 children, 20 with low vision and 20 without low vision. The inter-rater and test-retest reliability were analyzed with 12 children and the internal consistency with 40 children. RESULTS: Concerning test-retest reliability, six domains exhibited intra-class correlation coefficients ranging from 0.5361 to 1.000. For the movement within surroundings category, this coefficient was 0.37. For inter-rater reliability, only the category of confrontation visual field was below expectations (0.3901). The weighted kappa coefficient exhibited values ranged from -0.087 to 1.000. Cronbach's alfa ranged from 0.584 (movement within surroundings) to 0.973 (contrast sensitivity). CONCLUSION: AVIF-2 to 6 years presented good reliability indexes, however a few items within the domains need minor adjustments in order to improve scores' consistency of some items.

Confiabilidade da avaliação da visão funcional para crianças com baixa visão de dois a seis anos: em busca de evidências / Reliability of functional vision assessment in children with low vision from 2 to 6 years old: searching for evidences

OBJETIVOS: Avaliar a confiabilidade interexaminadores, fazer o teste-reteste e verificar a consistência interna da avaliação da visão funcional para crianças com baixa visão de dois a seis anos de idade (AVIF-2 a 6 anos). MÉTODOS: Foram testados sete domínios: fixação visual, seguimento visual, campo visual de confrontação, coordenação olho-mão, visão de contraste, deslocamento no ambiente e visão de cores, em 40 crianças, 20 com baixa visão e 20 sem alterações visuais. A consistência interna foi examinada para o teste de todas as crianças e a confiabilidade interexaminadores e teste-reteste para 12 delas. RESULTADOS: Seis domínios apresentaram ao teste-reteste coeficiente de correlação intraclasse com valores de 0,5361 a 1,000. Para o domínio deslocamento no ambiente esse coeficiente foi de 0,37. Na confiabilidade interexaminadores, somente o campo visual de confrontação apresentou valor abaixo do esperado (0,3901). O coeficiente kappa ponderado obteve resultados que variaram de -0,087 a 1,000. O valor do alfa de Cronbach variou de 0,584 (no deslocamento no ambiente) a 0,973 (na visão de contraste). CONCLUSÃO: A AVIF-2 a 6 anos apresentou bons índices de confiabilidade, mas a confiabilidade dos domínios, quando analisados isoladamente, precisa ser aprimorada.

PURPOSE: The functional vision assessment (AVIF-2 to 6 years) for low vision children with ages from 2 to 6 years old was analyzed. The inter-rater, test-retest and the internal consistency were verified for reliability. METHODS: The test, composed by seven domains (visual fixation, visual following, visual field confrontation, eye-hand coordination and surrounding locomotion, contrast vision and color vision discrimination), was applied to 40 children, 20 with low vision and 20 without low vision. The inter-rater and test-retest reliability were analyzed with 12 children and the internal consistency with 40 children. RESULTS: Concerning test-retest reliability, six domains exhibited intra-class correlation coefficients ranging from 0.5361 to 1.000. For the movement within surroundings category, this coefficient was 0.37. For inter-rater reliability, only the category of confrontation visual field was bellow expectations (0.3901). The weighted kappa coefficient exhibited values ranged from -0.087 to 1.000. Cronbach's alfa ranged from 0.584 (movement within surroundings) to 0.973 (contrast sensitivity). CONCLUSION: AVIF-2 to 6 years presented good reliability indexes, however a few items within the domains need minor adjustments in order to improve scores' consistency of some items.

High expression of human monocyte iNOS mRNA induced by Paracoccidioides brasiliensis is not associated with increase in NO production.

In this study we investigated the role of nitric oxide (NO) in monocyte fungicidal activity against Paracoccidioides brasiliensis. We found that cells primed with IFN-γ, TNF-α or GM-CSF and challenged with a high-(Pb18) or low-virulence (Pb265) strain of the fungus increase their fungicidal activity. Expression of iNOS mRNA was increased after priming cells with each cytokine, and tended to be inhibited by Pb18. Despite up-regulation of iNOS mRNA expression by Pb265, an equivalent increase in NO production was not detected, as metabolite levels were similar in all cultures. The results indicated that high expression of human monocyte iNOS mRNA induced by P. brasiliensis is not correlated with NO concentrations produced.

Production of leukotriene B4 by Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent deep mycosis in Latin America. The production of eicosanoids during fungal infection has been associated with the biology of these microorganisms and modulation of host immune response. The aim of our study was to evaluate whether P. brasiliensis strains with high or low virulence produce leukotriene B4 (LTB4), using endogenous and/or exogenous sources of arachidonic acid (AA). Moreover, we assessed whether this fungus might use the same metabolic pathway, described for mammalian cells, that involves the lipoxygenase (LOX) enzyme. The association between the production of this eicosanoid and fungus survival and growth was also evaluated. Our results showed that P. brasiliensis, irrespective of its virulence, produces high levels of LTB4 using endogenous AA. In addition, in cultures treated with exogenous AA, LTB4 levels were significantly higher, showing that this fungus also uses exogenous sources of fatty acids. Treatment with MK886, which blocks the activity of lipoxygenase, by inhibiting five-lipoxygenase-activating protein (FLAP) or with nordihydroguaiaretic acid (NDGA), a non-selective lipoxygenase inhibitor, resulted in a significant reduction in LTB4 levels, indicating that the fungus produces this eicosanoid by using the LOX pathway or an enzyme with biochemically similar function. The significant reduction in viability detected in cultures treated with these inhibitors was, however, restored by adding exogenous LTB4 , confirming the role of this eicosanoid in fungus survival. Moreover, the addition of LTB4 to cultures capable of producing LTs induces fungal growth. These results provide a foundation for additional studies on the contributions of LTB4 in P. brasiliensis virulence.