Background Ultrasound (US) has been widely used to improve the efficiency of nonviral vector transfection. The mechanism of plasmid uptake is usually attributed to sonoporation, although there is not clear evidence for this attribution. Based on our previous results, we hypothesized that other mechanisms, such as endocytosis, could be involved in this process.Methods NIH3T3 cells were transfected with plasmid vector pEGFP-N3 (4.7kb) using a therapeutic US without microbubbles. Bioeffects such as calcium influx, reactive oxygen species (ROS) generation and membrane potential alterations were accessed with fluorescent dyes in real-time by confocal microscopy after US insonation. Localization of labeled plasmid DNA in cells was also monitored with endocytosis markers using an immunofluorescence assay.Results US at 2 W/cm 2 with a duty-cycle of 20% for 30 s resulted in approximately 40% transfection efficiency but, at 1 W/cm 2 , resulted in a very low level of transfection. Both the production of ROS and calcium influx were augmented during the insonation, although they were stopped soon after turning off US, with the exception of calcium influx with 1 W/cm 2 . US also changed the cell membrane potential to the hyperpolarization state, which returned to the normal state soon after insonation. Labeled plasmids DNA could be co-localized with clathrin-mediated endocytosis marker but not with caveolin-1. ConclusionsThe present data indicate that plasmid DNA uptake promoted by US should occur via clathrin-mediated endocytosis. Figure 2. Representative images of NIH3T3 cells transfected by US. Cells were transfected with 20mg of pEGFP-N3 and US parameters were adjusted to 20% DC and 2 W/cm 2 for 30 s. Efficiency of transfection in other conditions can be seen in the Tables 1, 2 and 3. Fluorescent micrograph of cells expressing green fluorescent protein 48 h after of transfection (a). Micrograph without fluorescence of the cells in (b).Plasmid DNA endocytosis induced by ultrasound 395 using 20% DC for 30 s at 1 W/cm 2 (a) and 2 W/cm 2 (b). Graphs show calcium influx at different time points. The two dotted lines represent when the US was switched on. F t /F 0 is the ratio of total fluorescence to fluorescence at time zero. Plasmid DNA endocytosis induced by ultrasound 397 DAPI dye. The images represent the responses of at least three independent experiments.Plasmid DNA endocytosis induced by ultrasound 399

Paracoccidioides brasiliensis is a pathogenic, dimorphic fungus that causes paracoccidioidomycosis, a systemic human mycosis that is highly prevalent in Latin America. In this study, we demonstrated that P. brasiliensis yeasts induced interleukin (IL)-8 and IL-6 secretion by human lung epithelial A549 cells. However, tumor necrosis factor-α and interferon-γ were undetectable in these cultures. Moreover, P. brasiliensis yeasts induced activation of p38 mitogen-activated protein kinase (MAPK), c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in A549 cells, and IL-8 and IL-6 secretion promoted by this fungus was dependent on activation of p38 MAPK and ERK 1/2. In addition, IL-8 and IL-6 levels were significantly higher in culture supernatants of A549 cells that were incubated with formaldehyde-fixed P. brasiliensis compared to cultures of cells that were infected with live yeasts. Our results indicate that the observed cytokine level differences were due to protease expression, in live yeasts, that degraded these cytokines. Degradation of human recombinant IL-8 and IL-6 by live P. brasiliensis was inhibited by AEBSF and aprotinin, suggesting that these proteases belong to a family of serine proteases. This is the first report showing that P. brasiliensis may modulate host inflammation by expressing proteases that degrade proinflammatory cytokines.

In transplantation, parasite diseases are transmitted from the donor, or appear as de novo infections, or activate from a dormant insource as a consequence of immunosuppression. Clinical findings have shown that an intact immune system is crucial to prevent recurrence of Leishmania infection. We used BALB/c and C57BL/6 mice to evaluate the role of FTY720 in leishmaniasis. Mice inoculated with Leishmania (Leishmania) amazonensis were followed over 7 weeks for foot thickness measurements after initiation of FTY720 treatment. After 10 days of treatment, spleen, blood, and the foot were harvested for evaluation. BALB/c showed greater evident foot thickness than C57BL/6 mice. Oral treatment with FTY720 (1 mg/kg/d) over 10 days produced the same outcome. Increases in CD4(+) and CD8(+) T cells were observed after infection; FTY720 treatment was associated with a decrease in CD4(+) T cells only in BALB/c mice, whereas CD8(+) T cells were decreased in both mice strains. CD11b(+) expression decreased after infection with a discrete increase after FTY720 treatment. Lymphopenia was observed among all FTY720-treated mice. In conclusion, we observed that FTY720 produced no worse an outcome as monotherapy in established infections with L (L) amazonensis.

− (Chemical composition, acetylcholinesterase inhibitory and antifungal activities of Pera glabrata (Schott) Baill. (Euphorbiaceae)). Pera glabrata (Schott) Baill. was selected for this study after showing a preliminary positive result in a screening of Atlantic Forest plant species in the search for acetylcholinesterase inhibitors and antifungal compounds. The bioassays were conducted with crude ethanol extract of the leaves using direct bioautography method for acetylcholinesterase and antifungal activities. This extract was partitioned with hexane, chloroform and ethyl acetate solvents. The active chloroform fraction was submitted to silica gel chromatography column affording 12 groups. Caffeine, an alkaloid, which showed detection limits of 0.1 and 1.0 μg for anticholinesterasic and antifungal activities, respectively, was isolated from group nine. After microplate analyses, only groups four, nine, 10, 11 and 12 showed acetylcholinesterase inhibitory activity of 40% or higher. The group 12 was purifi ed by preparative layer chromatography affording four sub-fractions. Two sub-fractions from this group were analyzed by gas chromatography-mass spectrometry and gas chromatography-fl ame ionization detector. The fi rst sub-fraction showed anticholinesterasic activity and contained two major compounds: 9-hydroxy-4-megastigmen-3-one (84%) and caffeine (6%). The second sub-fraction presented fi ve major compounds identifi ed as 9-hydroxy-4-megastigmen-3-one, isololiolide, (-) loliolide, palmitic acid and lupeol and did not show activity. Key words -9-hydroxy-4-megastigmen-3-one, biological activities, caffeine, EuphorbiaceaeResumo -(Composição química, atividades inibidora da acetilcolinesterase e antifúngica de Pera glabrata (Schott) Baill. (Euphorbiaceae)). Pera glabrata (Schott) Baill. foi selecionada para este estudo a partir de uma triagem de espécies vegetais da Mata Atlântica na busca de substâncias com atividades anticolinesterásica e antifúngica. A técnica da bioautografi a direta foi utilizada para a detecção das atividades anticolinesterásica e antifúngica. O extrato etanólico bruto obtido das folhas foi particionado com hexano, clorofórmio e acetato de etila. A fração clorofórmica ativa foi fracionada por cromatografi a em coluna de sílica gel fornecendo 12 grupos. Do grupo nove foi isolado o alcalóide cafeína com limites de detecção de 0,1 e 1,0 μg para as atividades anticolinesterásica e antifúngica, respectivamente. Após bioensaio em microplaca, somente os grupos quatro, nove, 10, 11 e 12 apresentaram inibição da acetilcolinesterase maior ou igual a 40%. O grupo 12 foi purifi cado por cromatografi a em camada delgada preparativa de sílica gel fornecendo quatro sub-frações. Duas sub-frações deste grupo foram analisadas por cromatografi a a gás-espectrometria de massas e cromatografi a a gás com detector de ionização de chama. A primeira sub-fração contém dois compostos majoritários: 9-hidroxi-4-megastigmen-3-ona (78%) e cafeína (6%), e apresentou atividade anticolinesterásica. A segunda

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