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Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field. We and others have shown that single vesicles can be detected as light diffraction limited fluorescent spots using standard confocal and widefield fluorescence microscopes. Advancing this simple strategy into a process for routine EV quantitation, we developed 'EVAnalyzer', an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated, quantitative single vesicle analysis from imaging data. Using EVAnalyzer, we established a robust protocol for capture, (immuno‐)labelling and fluorescent imaging of EVs. To exemplify the application scope, the process was optimised and systematically tested for (i) quantification of EV subpopulations, (ii) validation of EV labelling reagents, (iii) in situ determination of antibody specificity, sensitivity and species cross‐reactivity for EV markers and (iv) optimisation of genetic EV engineering. Additionally, we show that the process can be applied to synthetic nanoparticles, allowing to determine siRNA encapsulation efficiencies of lipid‐based nanoparticles (LNPs) and protein loading of SiO2 nanoparticles. EVAnalyzer further provides a pipeline for automated quantification of cell uptake at the single cell–single vesicle level, thereby enabling high content EV cell uptake assays and plate‐based screens. Notably, the entire procedure from sample preparation to the final data output is entirely based on standard reagents, materials, laboratory equipment and open access software. In summary, we show that EVAnalyzer enables rigorous characterisation of EVs with generally accessible tools. Since we further provide the plugin as open‐source code, we expect EVAnalyzer to not only be a resource of immediate impact, but an open innovation platform for the EV and nanoparticle research communities. [ABSTRACT FROM AUTHOR]

Nanosafety assessment has experienced an intense era of research during the past decades driven by a vivid interest of regulators, industry, and society. Toxicological assays based on in vitro cellular models have undergone an evolution from experimentation using nanoparticulate systems on singular epithelial cell models to employing advanced complex models more realistically mimicking the respective body barriers for analyzing their capacity to alter the immune state of exposed individuals. During this phase, a number of lessons were learned. We have thus arrived at a state where the next chapters have to be opened, pursuing the following objectives: (1) to elucidate underlying mechanisms, (2) to address effects on vulnerable groups, (3) to test material mixtures, and (4) to use realistic doses on (5) sophisticated models. Moreover, data reproducibility has become a significant demand. In this context, we studied the emerging concept of adverse outcome pathways (AOPs) from the perspective of immune activation and modulation resulting in pro‐inflammatory versus tolerogenic responses. When considering the interaction of nanomaterials with biological systems, protein corona formation represents the relevant molecular initiating event (e.g., by potential alterations of nanomaterial‐adsorbed proteins). Using this as an example, we illustrate how integrated experimental–computational workflows combining in vitro assays with in silico models aid in data enrichment and upon comprehensive ontology‐annotated (meta)data upload to online repositories assure FAIRness (Findability, Accessibility, Interoperability, Reusability). Such digital twinning may, in future, assist in early‐stage decision‐making during therapeutic development, and hence, promote safe‐by‐design innovation in nanomedicine. Moreover, it may, in combination with in silico‐based exposure‐relevant dose‐finding, serve for risk monitoring in particularly loaded areas, for example, workplaces, taking into account pre‐existing health conditions. This article is categorized under:Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials [ABSTRACT FROM AUTHOR]

Background: Transforming growth factor β1 (TGFβ1) is a cytokine that exerts immunosuppressive functions, as reflected by its ability to induce regulatory T (Treg) cell differentiation and inhibit Th1 and Th2 responses. Hence, peptides that mimic the active core domain of TGFβ1 may be promising candidates for modulation of the allergic response. This study aimed to investigate a synthetic TGFβ1 mimetic peptide (TGFβ1‐mim) for its ability to modulate the immune response during allergic sensitization to grass pollen allergens. Methods: The in vitro action of TGFβ1‐mim was evaluated in human lung epithelial cells, Jurkat cells, and rat basophilic leukemia cells. The in vivo action was evaluated in a murine model of Phl p 5 allergic sensitization. Additionally, the Th2 modulatory response was evaluated in IL‐4 reporter mice. Results: In vitro, TGFβ1‐mim downregulated TNF‐α production, IL‐8 gene expression, and cytokine secretion, upregulated IL‐10 secretion, and inhibited Phl p 5‐induced basophil degranulation. During Phl p 5 sensitization in mice, TGFβ1‐mim downregulated IL‐2, IL‐4, IL‐5, IL‐13, and IFN‐γ, upregulated IL‐10, and induced Treg cell production. Furthermore, mice treated with TGFβ1‐mim had lower levels of IgE, IgG1, IgG2a and higher levels of IgA antibodies than control mice. In a reporter mouse, the mimetic inhibited Th2 polarization. Conclusion: The TGFβ1‐mim efficiently modulated various important events that exacerbate the allergic microenvironment, including the production of main cytokines that promote Th1 and Th2 differentiation, and the induction of allergen‐specific regulatory T cells, highlighting its potential use in therapeutic approaches to modulate the immune response toward environmental allergens. [ABSTRACT FROM AUTHOR]