Cryptococcus neoformans in Association with Dermatophagoides pteronyssinus has Pro- (IL-6/STAT3 Overproduction) and Anti-inflammatory (CCL2/ERK1/2 Downregulation) Effects on Human Bronchial Epithelial Cells.

Cryptococcosis (caused, for example, by Cryptococcus neoformans) and allergic asthma (caused, for example, by Dermatophagoides pteronyssinus) target the respiratory tract (the lung and bronchial epithelium). C. neoformans and D. pteronyssinus can coexist in the same indoor environment, and exposure to both can cause alterations in the local airway inflammatory milieu and exacerbation of airway inflammatory diseases. Here, we evaluated the effects of the association between C. neoformans and D. pteronyssinus in the modulation of airway inflammatory responses in an in vitro experimental model using human bronchial epithelial cells. BEAS-2B cells were cultivated and stimulated with D. pteronyssinus (10 µg/mL) and/or C. neoformans (MOI 100) for 24 h. No cytotoxic effect was observed in cells stimulated by C. neoformans and/or D. pteronyssinus. The production of IL-8, IL-6, and/or CCL2, but not IL-10, as well as the activation of NF-kB, STAT3, STAT6, and/or ERK1/2 were increased in cells stimulated by C. neoformans or D. pteronyssinus compared to controls. C. neoformans in association with D. pteronyssinus inhibited the CCL2­ERK1/2 signaling pathway in cells treated with both pathogens compared to cells stimulated by D. pteronyssinus alone. In addition, their association induced an additive effect on the IL-6/STAT3 signaling pathway in cells compared to cells stimulated with D. pteronyssinus or C. neoformans only. D. pteronyssinus increased the internalization and growth of C. neoformans in BEAS-2B cells. D. pteronyssinus in association with C. neoformans promoted pro- and anti-inflammatory responses, which can modulate cryptococcal infection and asthmaticus status.

Agaphelin modulates the activation of human bronchial epithelial cells induced by lipopolysaccharide and IL-4.

Sand fly saliva presents molecules with potential to development of compounds for treatment of inflammatory diseases. Agaphelin, isolated from the saliva of the mosquito Anopheles gambiae, demonstrates anti-inflammatory properties such as neutrophils chemotaxis inhibition. Here, we extend these results and evaluated the role of agaphelin (0.1-100 nM) in an in vitro model consisting in the activation of human bronchial epithelial cells (BEAS-2B) by IL-4 (50 ng/mL) or lipopolysaccharide (LPS; 10 ng/mL). Agaphelin is non-cytotoxic for BEAS-2B cells. Notably, agaphelin markedly reduces CCL2 and IL-8 production induced by IL-4 or LPS, without altering the IL-10 production. The TLR4 expression and STAT1 phosphorylation induced by LPS were inhibited by agaphlin. In addition, agaphelin decreased the phosphorylation of STAT6 induce by IL-4, whose effect was independent of IL-4-binding activity. Taken together, these findings identify agaphelin as a potential anti-inflammatory therapeutic agent for airway inflammations.

Toxoplasma gondii: effects of neuwiedase, a metalloproteinase from Bothrops neuwiedi snake venom, on the invasion and replication of human fibroblasts in vitro.

The major aim to the present study was to determine the effects of neuwiedase, a metalloproteinase isolated from Bothrops neuwiedi snake venom, on invasion and replication of Toxoplasma gondii in human fibroblasts in vitro. Neuwiedase treatment was done on host cells previously infected with T. gondii or on parasite before fibroblast infection. When treatments were done after or before infection, infection rates were inhibited in 71% and 61%, respectively. Considering that therapy protocols currently used in T. gondii infection cause considerable side effects, particularly in immunocompromised individuals and pregnant women, the results of neuwiedase treatment described herein could be taken into account for the development of new synthetic therapeutic agents, mainly due to the capacity of this enzyme to degrade extracellular matrix components, such as laminin, fibronectin and type I collagen, which is important to interfere in T. gondii host cell invasion.

[Susceptibility to antifungal agents among Cryptococcus neoformans varieties isolated from patients at a university hospital]. / Suscetibilidade a antifúngicos de variedades de Cryptococcus neoformans isoladas de pacientes em hospital universitário.

This study identified Cryptococcus neoformans varieties isolated from 35 patients at teaching hospital of the Federal University of the Triângulo Mineiro and evaluated the susceptibility to antifungal agents among these samples using the protocol M27-A2 from the National Committee for Clinical Laboratory Standards. The gattii variety was identified in 11.4% of the cases (n = 4). The minimum inhibitory concentration (mg/ml) of Cryptococcus neoformans neoformans isolates ranged from 0.062 to 2.000 (amphotericin B), 0.250 to 8.000 (fluconazole), 0.062 to 1.000 (itraconazole) and 0.125 to 1.000 (ketoconazole). The gattii variety presented a minimum inhibitory concentration range of 0.125 to 2.000 (amphotericin B), 0.250 to 16.00 (fluconazole), 0.062 to 1.000 (itraconazole) and 0.125 to 4.000 (ketoconazole). Two isolates resistant to itraconazole and two resistant to amphotericin B (one isolate of each variety per antifungal agent) were found. These data show the importance of determining the variety and minimum inhibitory concentration of Cryptococcus neoformans isolates, in order to monitor resistance development and enable better treatment for cryptococcosis.

AT-RvD1 modulates CCL-2 and CXCL-8 production and NF-κB, STAT-6, SOCS1, and SOCS3 expression on bronchial epithelial cells stimulated with IL-4.

Bronchial epithelial cells represent the first line of defense against microorganisms and allergens in the airways and play an important role in chronic inflammatory processes such as asthma. In an experimental model, both RvD1 and AT-RvD1, lipid mediators of inflammation resolution, ameliorated some of the most important phenotypes of experimental asthma. Here, we extend these results and demonstrate the effect of AT-RvD1 on bronchial epithelial cells (BEAS-2B) stimulated with IL-4. AT-RvD1 (100 nM) decreased both CCL2 and CXCL-8 production, in part by decreasing STAT6 and NF-κB pathways. Furthermore, the effects of AT-RvD1 were ALX/FRP2 receptor dependent, as the antagonist of this receptor (BOC1) reversed the inhibition of these chemokines by AT-RvD1. In addition, AT-RvD1 decreased SOCS1 and increased SOCS3 expression, which play important roles in Th1 and Th17 modulation, respectively. In conclusion, AT-RvD1 demonstrated significant effects on the IL-4-induced activation of bronchial epithelial cells and consequently the potential to modulate neutrophilic and eosinophilic airway inflammation in asthma. Taken together, these findings identify AT-RvD1 as a potential proresolving therapeutic agent for allergic responses in the airways.

Interferon-gamma signaling is downregulated in peripheral blood mononuclear cells from asthma patients / A sinalização de interferon-gama é diminuída em células mononucleares do sangue periférico de pacientes asmáticos

Introduction: Interferon-gamma (IFN-g) signaling is mediated by crosstalk of receptors, such as IFN-g receptor 1 (IFN-g R1), transcription factors, such as signal transducer and activator of transcription 1 (STAT1) and suppressors of cytokine signaling 1 (SOCS1). Here, we evaluated the role of IFN-g signaling in peripheral blood mononuclear cells (PBMCs) from asthma patients and control individuals. Methods: PBMCs from adult healthy nonasthmatic controls (n = 12; male and female, 18-60 years old) and patients diagnosed with asthma (n = 18; male and female, 18-60 years old) were stimulated with IFN-g (0.25, 0.5 and/or 1.0 ng/mL) and, after 24h, the production of CXC motif chemokine 10 (CXCL10) was evaluated (by enzyme linked immunosorbent assay) as well as the expression of IFN-g R1, STAT1 (both by flow cytometry assay) and SOCS1 (by real-time qPCR assay). Results: CXCL10 production was reduced in a dose-dependent manner in PBMCs from asthma patients stimulated with IFN-g when compared to control individuals. While IFN-g induced an increase in IFN-g R1 expression and phosphorylated STAT1 (pSTAT1) activation in PBMCs from the control group, a reduction in both IFN-g R1 and pSTAT1 was observed in PBMCs from asthma patients. IFN-g increased SOCS1 mRNA expression in PBMCs from asthma patients when compared to IFN-g-stimulated cells from control individuals. Conclusion: Taken together, our results demonstrated that IFN-g signaling is downregulated in asthma patients.

Introdução: A sinalização de interferon-gama (IFN-g) é mediada por receptores, como o receptor 1 de IFN-gama (IFN-gR1), fatores de transcrição, como o transdutor de sinal e o ativador de transcrição 1 (STAT1) e supressores de sinalização de citocina 1 (SOCS1). Neste trabalho, avaliamos o papel da sinalização de IFN-g em células mononucleares do sangue periférico (PBMCs) de indivíduos com asma e controle. Métodos: Células mononucleares do sangue periférico (PBMCs) de adultos saudáveis e não asmáticos (n = 12, homens e mulheres, 18-60 anos) e pacientes diagnosticados com asma (n = 18, homens e mulheres, 18-60 anos) foram estimuladas com IFN-g (0,25, 0,5 e/ou 1,0 ng/mL) e após 24 horas a produção de CXCL10 foi avaliada por ensaio de imunoabsorção enzimática (ELISA), bem como o receptor 1 de IFN-g (IFN-g R1). Também foram avaliadas as expressões do transdutor de sinal e ativador da transcrição 1 (STAT1) (por citometria de fluxo) e supressor de expressão de sinalização de citocinas 1 (SOCS1) (por ensaio qPCR em tempo real). Resultados: A produção de CXCL10, uma quimiocina induzida por IFNg, foi reduzida de maneira dependente da dose em PBMCs de pacientes com asma estimulados com IFN-g (0,25-1,0 ng/mL) quando comparado ao grupo controle. Enquanto IFN-g induziu um aumento da expressão de IFN-g R1 e ativação da fosforilação de STAT1 (pSTAT1) em PBMCs do grupo controle, uma redução de ambas (IFN-g R1 e pSTAT1) foi observada em PBMCs de pacientes com asma. O IFN-g aumentou as PBMCs de expressão do mRNA de SOCS1 de pacientes com asma quando comparado às células estimuladas por IFN-g do controle. Conclusão: Em conjunto, nossos resultados demonstraram que a sinalização de IFN-g é sub-regulada em pacientes com asma.

Suscetibilidade a antifúngicos de variedades de Cryptococcus neoformans isoladas de pacientes em hospital universitário / Susceptibility to antifungal agents among Cryptococcus neoformans varieties isolated from patients at a university hospital

Este trabalho identificou variedades de Cryptococcus neoformans e avaliou a suscetibilidade a antifúngicos pelo protocolo M27-A2 do National Committee for Clinical Laboratory Standards em isolados de 35 pacientes do Hospital Escola da Universidade Federal do Triângulo Mineiro. A variedade gatti foi identificada em 11.4 por cento (nº = 4) dos casos. A concentração inibitória mínima (mg/ml) dos isolados de Cryptococcus neoformans neoformans variou de 0,062 - 2,000 (anfotericina B), 0,250 - 8,000 (fluconazol), 0,062 - 1,000 (itraconazol) e 0,125 - 1,000 (cetoconazol). A variedade gattii apresentou concentração inibitória mínima de 0,125 - 2,000 (anfotericina B), 0,250 - 16,00 (fluconazol), 0,062 - 1,000 (itraconazol) e 0,125 - 4,000 (cetoconazol). Detectaram-se 2 isolados resistentes ao itraconazol e 2 a anfotericina B (1 isolado de cada variedade por antifúngico). Os dados mostram a importância da determinação da variedade e da concentração inibitória mínima de isolados de Cryptococcus neoformans para monitorar o desenvolvimento de resistência e possibilitar uma terapia mais adequada na criptococose.

This study identified Cryptococcus neoformans varieties isolated from 35 patients at teaching hospital of the Federal University of the Triângulo Mineiro and evaluated the susceptibility to antifungal agents among these samples using the protocol M27-A2 from the National Committee for Clinical Laboratory Standards. The gattii variety was identified in 11.4 percent of the cases (n = 4). The minimum inhibitory concentration (mg/ml) of Cryptococcus neoformans neoformans isolates ranged from 0.062 to 2.000 (amphotericin B), 0.250 to 8.000 (fluconazole), 0.062 to 1.000 (itraconazole) and 0.125 to 1.000 (ketoconazole). The gattii variety presented a minimum inhibitory concentration range of 0.125 to 2.000 (amphotericin B), 0.250 to 16.00 (fluconazole), 0.062 to 1.000 (itraconazole) and 0.125 to 4.000 (ketoconazole). Two isolates resistant to itraconazole and two resistant to amphotericin B (one isolate of each variety per antifungal agent) were found. These data show the importance of determining the variety and minimum inhibitory concentration of Cryptococcus neoformans isolates, in order to monitor resistance development and enable better treatment for cryptococcosis.

Valores de referência de parâmetros bioquímicos no sangue de duas linhagens de camundongos / Reference values for blood-based biochemical parameters in BALB/C and C57BL/6 wild-type mice

INTRODUÇÃO: Conhecer os parâmetros bioquímicos individuais de animais de laboratório utilizados na experimentação é importante, pois eles servirão como parâmetros para avaliar alterações funcionais em órgãos e como base para estabelecer valores de referência. OBJETIVO:Estabelecer valores de referência bioquímicos do sangue em camundongos das linhagens BALB/c e C57BL/6 selvagens do Biotério da Disciplina de Biologia Celular da Universidade Federal do Triângulo Mineiro (UFTM). Materiais e métodos: Foram utilizados 30 camundongos (BALB/c e C57BL/6 selvagem). Os exames realizados foram glicose, triglicérides, colesterol, proteínas totais, albumina, amilase, ácido úrico, uréia, fosfatase alcalina (kits Wiener), e as determinações foram realizadas no equipamento BIOPLUS-2000. RESULTADOS:Entre os nove analitos observou-se que quatro (albumina, glicose, proteínas totais e colesterol) apresentaram diferenças estatisticamente significativas entre as linhagens. Padronizamos como valores de referência para os camundongos os valores do intervalo de confiança (IC). Nos analitos em que houve diferença significativa entre as linhagens (p < 0,05) adotamos os valores do IC de cada linhagem; para os que não apresentaram diferenças foram utilizados os valores mínimos e máximos do IC entre as duas linhagens. CONCLUSÃO:Ao final da análise, acreditamos que os resultados obtidos sugerem a padronização de intervalos de referência próprios de cada biotério, pois refletem a condição da população para os quais os testes serão aplicados no dia-a-dia.

INTRODUCTION: Identifying individual biochemical parameters of laboratory animal species is important inasmuch as they may be used in the evaluation of functional changes in organs and in the establishment of reference values. OBJECTIVE: To establish biochemical reference values for blood tests in BALB/c and C57BL/6 wild-type mice from the Vivarium of the Department of Cellular Biology at the Federal University of "Triângulo Mineiro". MATERIALS AND METHODS: Thirty wild-type mice of the lineages BALB/c and C57BL/6 were used to evaluate the serum levels of glucose, triglycerides, cholesterol, total protein, albumin, amylase, uric acid, urea and alkaline phosphatase. The determinations were performed in a BIOPLUS-2000 analyzer. Results: Four out of the nine analytes (albumin, glucose, total proteins and cholesterol) showed significant statistical differences between the strains. Confidence interval (CI) values were standardized as reference values. In those analytes in which there was significant difference between strains (p < 0.05), confidence interval values of each lineage were adopted, whereas in those ones in which there were no differences, the minimum and maximum values of confidence interval from both lineages were applied. CONCLUSION: The results show the need for reference interval standardization of each Vivarium inasmuch as it reflects the conditions of the population in which the tests will be routinely performed.