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

Although our results do not allow a direct clinical application, we believe that caution should be warranted when intramuscular bupivacaine is used.

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.