Proteomic characterisation of leech microglia extracellular vesicles (EVs): comparison between differential ultracentrifugation and Optiprep™ density gradient isolation.

In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.

Clinical effects of beta-adrenergic blockade in chronic heart failure: a meta-analysis of double-blind, placebo-controlled, randomized trials.

BACKGROUND: beta-Blockers have improved symptoms and reduced the risk of cardiovascular events in studies of patients with heart failure, but it is unclear which end points are most sensitive to the therapeutic effects of these drugs. METHODS AND RESULTS: We combined the results of all 18 published double-blind, placebo-controlled, parallel-group trials of beta-blockers in heart failure. From this combined database of 3023 patients, we evaluated the strength of evidence supporting an effect of treatment on left ventricular ejection fraction, NYHA functional class, hospitalizations for heart failure, and death. beta-Blockers exerted their most persuasive effects on ejection fraction and on the combined risk of death and hospitalization for heart failure. beta-Blockade increased the ejection fraction by 29% (P<10(-9)) and reduced the combined risk of death or hospitalization for heart failure by 37% (P<0.001). Both effects remained significant even if >90% of the trials were eliminated from the analysis or if a large number of trials with a neutral result were added to the analysis. In contrast, the effect of beta-blockade on NYHA functional class was of borderline significance (P=0.04) and disappeared with the addition or removal of only 1 moderate-size study. Although beta -blockade reduced all-cause mortality by 32% (P=0.003), this effect was only moderately robust and varied according to the type of ss-blocker tested, ie, the reduction of mortality risk was greater for nonselective beta-blockers than for beta1-selective agents (49% versus 18%, P=0.049). However, selective and nonselective beta-blockers did not differ in their effects on other measures of clinical efficacy. CONCLUSIONS: These analyses indicate that there is persuasive evidence supporting a favorable effect of beta-blockade on ejection fraction and the combined risk of death and hospitalization for heart failure. In contrast, the effect of these drugs on other end points requires additional study.