Nanoparticles as drug delivery system against tuberculosis in zebrafish embryos: direct visualization and treatment.

Nanoparticles (NPs) enclosing antibiotics have provided promising therapy against Mycobacterium tuberculosis (Mtb) in different mammalian models. However, the NPs were not visualized in any of these animal studies. Here, we introduce the transparent zebrafish embryo as a system for noninvasive, simultaneous imaging of fluorescent NPs and the fish tuberculosis (TB) agent Mycobacterium marinum (Mm). The study was facilitated by the use of transgenic lines of macrophages, neutrophils, and endothelial cells expressing fluorescent markers readily visible in the live vertebrate. Intravenous injection of Mm led to phagocytosis by blood macrophages. These remained within the vasculature until 3 days postinfection where they started to extravasate and form aggregates of infected cells. Correlative light/electron microscopy revealed that these granuloma-like structures had significant access to the vasculature. Injection of NPs induced rapid uptake by both infected and uninfected macrophages, the latter being actively recruited to the site of infection, thereby providing an efficient targeting into granulomas. Rifampicin-loaded NPs significantly improved embryo survival and lowered bacterial load, as shown by quantitative fluorescence analysis. Our results argue that zebrafish embryos offer a powerful system for monitoring NPs in vivo and rationalize why NP therapy was so effective against Mtb in earlier studies; bacteria and NPs share the same cellular niche.

A two-tier Golgi-based control of organelle size underpins the functional plasticity of endothelial cells.

Weibel-Palade bodies (WPBs), endothelial-specific secretory granules that are central to primary hemostasis and inflammation, occur in dimensions ranging between 0.5 and 5 µm. How their size is determined and whether it has a functional relevance are at present unknown. Here, we provide evidence for a dual role of the Golgi apparatus in controlling the size of these secretory carriers. At the ministack level, cisternae constrain the size of nanostructures ("quanta") of von Willebrand factor (vWF), the main WPB cargo. The ribbon architecture of the Golgi then allows copackaging of a variable number of vWF quanta within the continuous lumen of the trans-Golgi network, thereby generating organelles of different sizes. Reducing the WPB size abates endothelial cell hemostatic function by drastically diminishing platelet recruitment, but, strikingly, the inflammatory response (the endothelial capacity to engage leukocytes) is unaltered. Size can thus confer functional plasticity to an organelle by differentially affecting its activities.

Poly(lactide-co-glycolide)-rifampicin nanoparticles efficiently clear Mycobacterium bovis BCG infection in macrophages and remain membrane-bound in phago-lysosomes.

Nanoparticles (NPs) are increasingly used as biodegradable vehicles to selectively deliver therapeutic agents such as drugs or antigens to cells. The most widely used vehicle for this purpose is based on copolymers of lactic acid and glycolic acid (PLGA) and has been extensively used in experiments aimed at delivering antibiotics against Mycobacterium tuberculosis in animal models of tuberculosis. Here, we describe fabrication of PLGA NPs containing either a high concentration of rifampicin or detectable levels of the green fluorescent dye, coumarin-6. Our goal here was twofold: first to resolve the controversial issue of whether, after phagocytic uptake, PLGA NPs remain membrane-bound or whether they escape into the cytoplasm, as has been widely claimed. Second, we sought to make NPs that enclosed sufficient rifampicin to efficiently clear macrophages of infection with Mycobacterium bovis BCG. Using fluorescence microscopy and immuno-electron microscopy, in combination with markers for lysosomes, we show that BCG bacteria, as expected, localized to early phagosomes, but that at least 90% of PLGA particles were targeted to, and remained in, low pH, hydrolase-rich phago-lysosomes. Our data collectively argue that PLGA NPs remain membrane-enclosed in macrophages for at least 13 days and degrade slowly. Importantly, provided that the NPs are fabricated with sufficient antibiotic, one dose given after infection is sufficient to efficiently clear the BCG infection after 9-12 days of treatment, as shown by estimates of the number of bacterial colonies in vitro.

Physiological responses to the Coriolis illusion: effects of head position and vision.

INTRODUCTION: Changes in sympathetic outflow during Type II spatial disorientation are well documented. In this study we investigated the influences of head position and eye state (open or closed) on sympathetic activation. METHODS: There were 11 naive subjects (6 men, 5 women) who were tested in a General Aviation Trainer that accelerated at a subthreshold rate for 60 s until a constant angular velocity of 90 degrees x s(-1) was reached. Approximately 40 s later, subjects were instructed to tilt their heads along either the pitch or roll axis, stimulating a Coriolis illusion. Subjects reported the perceived intensity and duration of disorientation. Heart rate, heart rate variability, and electrodermal responses were recorded before, during, and after the period of disorientation. Each subject completed four trials, which were crossed combinations of head position and eye state. RESULTS: There were significant increases in heart rate and the electrodermal response during disorientation, but no significant change in heart rate variability. Head position had no significant effect on any physiological parameters or on the perceived intensity of disorientation; subjects reported a shorter duration of disorientation when the head was tilted into the roll versus the pitch axis. Eye state had no effect on heart rate, heart rate variability, or the intensity of disorientation, but the electrodermal response was somewhat greater, and the duration of disorientation shorter when eyes were open. CONCLUSIONS: The results suggest that head position and eye state (open or closed) do not need to be included as factors when investigating sympathetic outflow during a mild Coriolis illusion.

Polymerization rate and mechanism of ultrasonically initiated emulsion polymerization of n-butyl acrylate.

The factors affecting the induction period and polymerization rate in ultrasonically initiated emulsion polymerization of n-butyl acrylate (BA) were investigated. The induction period takes only an instant in ultrasonically initiated emulsion polymerization of BA without any added initiator by enhancing the N2 flow rate. Increasing temperature, power output and SDS concentration, decreasing the monomer concentration results in further decreasing induction period and enhanced polymerization rate. Under optimized reaction conditions the conversion of BA reaches 92% in 11 min. The polymerization rate can be controlled by varying reaction parameters. The apparatus of ultrasonically initiated semi-continuous and continuous emulsion polymerization were set up and the feasibility was first studied. Based on the experimental results, a free radical polymerization mechanism for ultrasonically initiated emulsion polymerization was proposed, including the sources of the radicals, the process of radical formation, the locus of polymerization and the polymerization process. Compared with conventional emulsion polymerization, where the radicals come from thermal decomposition of a chemical initiator, ultrasonically initiated emulsion polymerization has attractive features such as no need for a chemical initiator, lower reaction temperature, faster polymerization rate, and higher molecular weight of the polymer prepared.