AnEVs induce vascular permeability
Vascular permeability is one of the processes tightly associated to the anaphylactic reactions [28]. To analyze a plausible role of AnEVs in endothelial permeability, in vitro assays combined with immunofluorescence staining were carried. Images showed that an interaction between the PKH67-stained EVs and ECs exists. A clear perinuclear EVs localization revealed that HMVEC-Ls engulf and internalize these particles (Figure 5A-B ). Once confirmed that AnEVs and HMVEC-Ls interact with each other, the purpose was to evaluate the functional effect of these vesicles on the endothelial barrier. Vascular permeability assays showed that the incubation of EVs extracted from anaphylactic patients produced a decrease in the resistance of the endothelial monolayer (Figure 5C-D ). Importantly, this analysis including 16 patients demonstrated that AnEVs induced a greater increase of the cellular permeability compared to the BEVs (Figure 5D ). As control of the cellular system created, vasoactive agents were also evaluated showing the effect of thrombin and platelet-activating factor (PAF) as destabilizers of the monolayer and the AMPc as an endothelial stabilizer. In all cases the expected effect was observed (Supplemental Figure 1 ).