Extracellular Vesicles from Scedosporium apiospermum Mycelial Cells: Implication for Fungal-Host Interplays.

The release of extracellular vesicles (EVs) has been implicated as an alternative transport mechanism for the passage of macromolecules through the fungal cell wall, a phenomenon widely reported in yeasts but poorly explored in mycelial cells. In the present work, we have purified and characterized the EVs released by mycelia of the emerging, opportunistic, widespread and multidrug-resistant filamentous fungus Scedosporium apiospermum. Transmission electron microscopy images and light scattering measurements revealed the fungal EVs, which were observed individually or grouped with heterogeneous morphology, size and electron density. The mean diameter of the EVs, evaluated by the light scattering technique, was 179.7 nm. Overall, the structural stability of S. apiospermum EVs was preserved during incubation under various storage conditions. The lipid, carbohydrate and protein contents were quantified, and the EVs' protein profile was evidenced by SDS-PAGE, revealing proteins with molecular masses ranging from 20 to 118 kDa. Through immunoblotting, ELISA and immunocytochemistry assays, antigenic molecules were evidenced in EVs using a polyclonal serum (called anti-secreted molecules) from a rabbit inoculated with conditioned cell-free supernatant obtained from S. apiospermum mycelial cells. By Western blotting, several antigenic proteins were identified. The ELISA assay confirmed that the anti-secreted molecules exhibited a positive reaction up to a serum dilution of 1:3200. Despite transporting immunogenic molecules, S. apiospermum EVs slightly induced an in vitro cytotoxicity effect after 48 h of contact with either macrophages or lung epithelial cells. Interestingly, the pretreatment of both mammalian cells with purified EVs significantly increased the association index with S. apiospermum conidia. Furthermore, EVs were highly toxic to Galleria mellonella, leading to larval death in a typically dose- and time-dependent manner. Collectively, the results represent the first report of detecting EVs in the S. apiospermum filamentous form, highlighting a possible implication in fungal pathogenesis.

Ultrastructural viewpoints on the interaction events of Scedosporium apiospermum conidia with lung and macrophage cells.

BACKGROUND: Scedosporium apiospermum is a ubiquitous, emerging and multidrug-resistant fungal pathogen with still rather unknown virulence mechanisms. OBJECTIVES/METHODS: The cellular basis of the in vitro interaction between fungi and host cells/tissues is the determinant factor for the development of a successful in vivo infection. Herein, we evaluated the interaction of S. apiospermum conidia with lung epithelial (A549), lung fibroblast (MRC-5) and RAW 264.7 macrophages by light and scanning/transmission electron microscopy. FINDINGS: After 4 h of fungi-host cell contact, the percentage of infected mammalian cells and the number of fungi per infected cell was measured by light microscopy, and the following association indexes were calculated for A549, MRC-5 and macrophage cells: 73.2 ± 25.9, 69.7 ± 22.5 and 59.7 ± 11.1, respectively. Both conidia and germinated conidia were regularly observed interacting with the evaluated cells, with a higher prevalence of non-germinated conidia. Interestingly, nests of germinated conidia were evidenced at the surface of lung cells by scanning electron microscopy. Some germination projections and hyphae were seen penetrating/evading the mammalian cells. Furthermore, internalised conidia were seen within vacuoles as visualised by transmission electron microscopy. MAIN CONCLUSIONS: The present study contributes to a better understanding of S. apiospermum pathogenesis by demonstrating the first steps of the infection process of this opportunistic fungus.

Ultrastructural viewpoints on the interaction events of Scedosporium apiospermum conidia with lung and macrophage cells

BACKGROUND Scedosporium apiospermum is a ubiquitous, emerging and multidrug-resistant fungal pathogen with still rather unknown virulence mechanisms. OBJECTIVES/METHODS The cellular basis of the in vitro interaction between fungi and host cells/tissues is the determinant factor for the development of a successful in vivo infection. Herein, we evaluated the interaction of S. apiospermum conidia with lung epithelial (A549), lung fibroblast (MRC-5) and RAW 264.7 macrophages by light and scanning/transmission electron microscopy. FINDINGS After 4 h of fungi-host cell contact, the percentage of infected mammalian cells and the number of fungi per infected cell was measured by light microscopy, and the following association indexes were calculated for A549, MRC-5 and macrophage cells: 73.2 ± 25.9, 69.7 ± 22.5 and 59.7 ± 11.1, respectively. Both conidia and germinated conidia were regularly observed interacting with the evaluated cells, with a higher prevalence of non-germinated conidia. Interestingly, nests of germinated conidia were evidenced at the surface of lung cells by scanning electron microscopy. Some germination projections and hyphae were seen penetrating/evading the mammalian cells. Furthermore, internalised conidia were seen within vacuoles as visualised by transmission electron microscopy. MAIN CONCLUSIONS The present study contributes to a better understanding of S. apiospermum pathogenesis by demonstrating the first steps of the infection process of this opportunistic fungus.

Detection of avian paramyxoviruses in migratory and resident birds in the state of Rio de Janeiro, Brazil.

Paramyxoviruses and avian influenza viruses are present worldwide, and wild birds are known natural reservoirs of these viruses. This study monitored the circulation of these viruses in migratory and resident coastal birds captured in the state of Rio de Janeiro, Brazil. In total, 494 birds were trapped, and their fecal samples were collected and inoculated into embryonated chicken eggs. The allantoic fluids were evaluated using a hemagglutination test and PCR amplification of the genes of the M and L proteins of influenza A virus and paramyxovirus, respectively. Avian paramyxovirus was detected in 5 (1.01%) of the birds. The majority of these viruses were isolated from migratory birds classified into the order Charadriiformes (families Scolopacidae and Charadriidae). Four samples were characterized as avian paramyxovirus serotype-2 (APMV-2) by a hemagglutination inhibition test. These results reinforce the importance of continuous surveillance of wild species in Brazil.

Influence of the relative composition of trace elements and vitamins in physicochemical stability of total parenteral nutrition formulations for neonatal use.

OBJECTIVE: The present study aimed to evaluate the influence of the relative composition of trace elements and vitamins in physicochemical stability of neonatal parenteral nutrition. MATERIAL AND METHODS: Three formulations for neonatal administration were selected; the main variable was the presence of trace elements and vitamins. The analyses where carried out immediately after preparation and at 24 h, 48 h, 72 h and 7 days after preparation. Three methods were selected to determine globule size: light obscuration, dynamic light scattering and optical microscopy. Complementary evaluation including visual inspection, determination of pH and osmolarity, peroxide levels and measurements of zeta potential were also performed. RESULTS: There was an observable alteration in color and phase separation in the PN stored at 25°C and 40°C. Neither globule size pattern, nor any other physicochemical characteristic evaluated appeared to be considerably altered in any of the analyzed formulations even after 7 days of storage at 5°C. Globule size in all the PN studied was consistent with the established limit, below 500 nm by DLS measurement, and PFAT5 was below 0.05% under all storage temperatures. CONCLUSION: Concomitant presence of trace elements and vitamins in the same neonatal formulation did not alter the evaluated aspects of stability.

Detection of paramyxoviruses in Magellanic penguins (Spheniscus magellanicus) on the Brazilian tropical coast.

Aquatic migratory birds are a major vectors by which influenza viruses and paramyxoviruses are spread in nature. Magellanic penguins (Spheniscus magellanicus) are usually present on the southern shores of South America and can swim as far as the southern coast of Brazil in winter. In 2008, however, several Magellanic penguins were observed on the northeastern coast of Brazil. Paramyxoviruses were isolated from Magellanic penguins on the Espírito Santo state coast, approximately 4000 km from their breeding colonies, although influenza viruses were not detected. Among the paramyxoviruses, five Avulavirus isolates belonging to serotype APMV-2 and the serotype APMV-10, which was proposed by Miller et al. (2010), were identified. These results highlight the risks associated with the spread of paramyxoviruses between natural to non-natural habitats by birds exhibiting unusual migration patterns, and they document for the first time the presence of the APMV-2 and APMV-10 serotypes on penguins in Brazil. The local avifauna may become infected with these viruses through close contact between migratory and resident birds. Continued surveillance of virus incidence in these migratory populations of penguins is necessary to detect and prevent the potential risks associated with these unusual migration patterns.

O uso da homeopatia para prevenir sintomas da gripe humana e infecção respiratória aguda: um estudo clínico duplo cego, randomizado, placebo controlado com 600 crianças do Serviço Público de Sáude do Brasil / The use of homeopathy to prevent symptoms of human flu and acute respiratory infections: a double-blind, randomized, placebo-controlled clinical trial with 600 children from Brazilian Public Health Service

In Brazil, homeopathy was implemented in the Public Health Service through the National Policy on Complementary and Integrative Practices of the Health Ministry, published in 2006. Homeopathy appears as a very interesting therapy to be used in the Public Health Services since its medicines are compounded at a very low cost. Considering this interesting scenario to develop research in the Public Health, the Family Health Program (FHP) in Petropolis and the Faculty of Pharmacy at UFRJ started a partnership with the Roberto Costa Institute.(AU)

No Brasil, a homeopatia foi implementada no Serviço Público de Saúde, através da Política Nacional de Práticas Integrativas e Complementares do Ministério da Saúde, publicada em 2006. A homeopatia surge como uma terapia muito interessante para ser usada no serviço público de saúde uma vez que seus medicamentos tem um custo muito baixo. Considerando este cenário interessante para desenvolver uma pesquisa em Saúde Pública, o Programa de Saúde da Família (PSF) de Petrópolis e a Faculdade de Farmácia da UFRJ iniciaram uma parceria com o Instituto Roberto Costa. (AU)

Crude ethanol extract from babassu (Orbignya speciosa): cytotoxicity on tumoral and non-tumoral cell lines.

Plant-derived substances have been considered as important sources of drugs, including antineoplasic agents. Babassu mesocarp is popularly used in Brazil as a food additive, and in popular medicine against several conditions, such as inflammations, menstrual pains and leukaemia. From babassu Orbignya speciosa (Mart.) Barb. Rodr. [Arecaceae (Palmae)] epicarp/mesocarp, an ethanol extract was prepared and named OSEME, which was tested on the viability,morphology and metabolism of several cell lines, such as the leukaemic cell lines, HL-60, K562 and the latter multidrug resistant counterpart K562-Lucena 1, the human breast cancer cell line MCF-7, the mouse fibroblast cell line 3T3-L1 and fresh human lymphocytes. OSEME promoted a dose-dependent decrease on the viability of all cells. This effect was much more pronounced on the tumoral cell lines than on non-tumoral cells, a phenomenon revealed by the dose of OSEME which promotes half of maximal effect (ID50). The decrease on viability was followed by shrinkage of cells, alteration on their morphology, and a markedly nuclear condensation. Curiously, stimulation of 6-phosphofructokinase activity (6.6-times) was observed on HL-60 cells, treated with OSEME, when compared to control treated with ethanol (vehicle). These results support evidences to suggest OSEME as a promising source of novel antineoplasic agents.

Crude ethanol extract from babassu (Orbignya speciosa): cytotoxicity on tumoral and non-tumoral cell lines

Plant-derived substances have been considered as important sources of drugs, including antineoplasic agents. Babassu mesocarp is popularly used in Brazil as a food additive, and in popular medicine against several conditions, such as inflammations, menstrual pains and leukaemia. From babassu Orbignya speciosa (Mart.) Barb. Rodr. [Arecaceae (Palmae)] epicarp/mesocarp, an ethanol extract was prepared and named OSEME, which was tested on the viability,morphology and metabolism of several cell lines, such as the leukaemic cell lines, HL-60, K562 and the latter multidrug resistant counterpart K562-Lucena 1, the human breast cancer cell line MCF-7, the mouse fibroblast cell line 3T3-L1 and fresh human lymphocytes. OSEME promoted a dose-dependent decrease on the viability of all cells. This effect was much more pronounced on the tumoral cell lines than on non-tumoral cells, a phenomenon revealed by the dose of OSEME which promotes half of maximal effect (ID50). The decrease on viability was followed by shrinkage of cells, alteration on their morphology, and a markedly nuclear condensation. Curiously, stimulation of 6-phosphofructokinase activity (6.6-times) was observed on HL-60 cells, treated with OSEME, when compared to control treated with ethanol (vehicle). These results support evidences to suggest OSEME as a promising source of novel antineoplasic agents.

Substâncias derivadas de plantas têm sido usadas como importante fonte de agentes antineoplásicos. O mesocarpo do babaçu é popularmente usado no Brasil como suplemento alimentar e na medicina popular para o tratamento de várias afecções, tais como: inflamações, cólicas menstruais e leucemia. A partir do epicarpo/mesocarpo do babaçu Orbignya speciosa (Mart.) Barb. Rodr. [Arecaceae (Palmae)] foi preparado um extrato etanólico, denominado OSEME, o qual foi incubado com as seguintes linhagens humanas leucêmicas: HL-60, K562 e a sua derivada resistente a múltiplas drogas, K562-Lucena 1; além destas, foram testadas a linhagem humana de câncer de mama, MCF-7; a linhagem de fibroblastos de camundongo, 3T3-L1 e linfócitos humanos de sangue periférico. OSEME promoveu diminuição da viabilidade em todas as linhagens celulares testadas de maneira dose-dependente. Este efeito foi mais pronunciado sobre as linhagens celulares tumorais quando comparado às não tumorais, o que foi revelado pela dose de OSEME capaz de promover metade do efeito máximo (ID50). A diminuição da viabilidade foi acompanhada por danos sobre a morfologia celular com pronunciada condensação citoplasmática e nuclear. Curiosamente, quando a linhagem HL-60 foi tratada com OSEME, foi detectado um aumento de 6,6 vezes da atividade da enzima 6-fosfofrutoquinase, quando comparado ao grupo controle (células tratadas com o veículo etanol). Esses resultados sugerem que OSEME pode ser uma promissora fonte de novos agentes antineoplásicos.

Clotrimazole decreases human breast cancer cells viability through alterations in cytoskeleton-associated glycolytic enzymes.

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. Glycolysis is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. Clotrimazole is an anti-fungal azole derivative recently recognized as a calmodulin antagonist with promising anti-cancer effect. Here, we show that clotrimazole induced morphological and functional alterations on human breast cancer derived cell line, MCF-7. The drug decreased cell viability in a dose- and time-dependent manner, exhibiting an IC50 of 88.6+/-5.3 microM and a t0.5 of 89.7+/-7.2 min, with 50 microM clotrimazole. Morphological changes were evident as observed by scanning electron microscopy, which revealed the completely loss of protrusion responsible for cell adhesion after a 180 min of treatment with 50 microM clotrimazole. Giemsa stained cells observed by optical microscopy show morphological alterations and a marked nuclear condensation. These changes occurred in parallel to the detachment of the glycolytic enzymes, 6-phosphofructo-1-kinase and aldolase, from cytoskeleton. After a 45 min treatment with 50 microM clotrimazole, the remaining activities in a cytoskeleton enriched fraction was 16.4+/-3.6% and 41.0+/-15.6% of control for 6-phosphofructo-1-kinase and aldolase, respectively. Immunocytochemistry experiments revealed a decrease in the co-localization of 6-phosphofructo-1-kinase and F-actin after clotrimazole treatment, suggesting the site of detachment of the enzymes. Altogether, our results support evidence for apoptotic events that might be started by clotrimazole involving inhibition of glycolytic flux in MCF-7 cells and makes this drug a promising agent in the fight against human breast cancer.

Exposure of human leukemic cells to direct electric current: generation of toxic compounds inducing cell death by different mechanisms.

Treatment with direct electric current (DC) influences the growth of several cancer cells. In this work, we evaluated the effects of DC treatment on the human leukemic cell line HL60. Human cells were separately treated in the presence of the cathode or the anode or without contact with the electrodes. In all systems, DC-treated cells presented an impaired ability to proliferate. Growth inhibition was dependent on the generation of soluble products of electrolysis. Cathodic treatment of HL60 cells predominantly induced lysis, whereas treatment without contact with electrodes did not induce alterations in cell viability. In contrast, cell stimulation by the anode resulted in irreversible membrane damage, as demonstrated by trypan blue and 7-aminoactinomycin staining. Analysis of these cells by transmission electron microscopy indicated that necrosis is a major mechanism inducing cell death. In addition, apoptotic-like cells were observed under light microscopy after anodic treatment. Accordingly, DNA from anodic-treated cells presented a typical pattern of apoptosis. Apoptotic cell death was only generated after the treatment of HL60 cells in conditions in which the generation of chloride-derived compounds was favored. These results indicate that the nature of the products from cathodic or anodic reactions differently influences the mechanisms of cell death induced by DC-derived toxic compounds.