Despite the strong evidence for the immunomodulatory activity of mesenchymal stromal cells (MSCs), clinical trials have so far failed to clearly show benefit, likely reflecting methodological shortcomings and lack of standardization. MSC-mediated tissue repair is commonly believed to occur in a paracrine manner, and it has been stated that extracellular vesicles (EVs) secreted by MSCs (EVMSC) are able to recapitulate the immunosuppressive properties of parental cells. As a next step, clinical trials to corroborate preclinical studies should be performed. However, effective dose in large mammals, including humans, is quite high and EVs industrial production is hindered by the proliferative senescence that affects MSCs during massive cell expansion. We generated a genetically modified MSC cell line overexpressing hypoxia-inducible factor 1-alpha and telomerase to increase the therapeutic potency of EVMSC and facilitate their large-scale production. We also developed a cytokine-based preconditioning culture medium to prime the immunomodulatory response of secreted EVs (EV_MSC-T-HIF^c). We tested the efficacy of this system in vitro and in a delayed-type hypersensitivity mouse model. MSC-T with an HIF-1α-GFP lentiviral vector (MSC-T-HIF) can be effectively expanded to obtain large amounts of EVs without major changes in cell phenotype and EVs composition. EV_MSC-T-HIF^c suppressed the proliferation of activated T-cells more effectively than did EVs from unmodified MSC in vitro, and significantly blunted the ear-swelling response in vivo by inhibiting cell infiltration and improving tissue integrity. We have developed a long-lived EV source that secretes high quantities of immunosuppressive EVs, facilitating a more standard and cost-effective therapeutic product.

Helminth Extracellular Vesicles (EVs) have emerged as important mediators in host-parasite communications, participating in the parasite survival and its pathogenic effects. In the last decade, a growing amount of information reporting the isolation and characterization of EVs from different helminth species has appeared, but unfortunately, few reports have focused on functional studies of helminth EVs in different cell lines, organoids or animal models. We here review these in vitro and in vivo studies, which clearly demonstrate that helminths secrete EVs, which affect their environment. Helminth EVs are actively internalized by different cell lines, modulating cellular functions important for host-parasite communication. We discuss how these lines of investigation should provide potential new biomarkers of infection, and since helminth EVs can modulate the host immune response, we also discuss how they can provide a new landscape for the development of new vaccine tools against helminthiases as well as immunotherapy.

Seminal plasma contains many morphologically heterogeneous extracellular vesicles (sEVs). These are sequentially released by cells of the testis, epididymis, and accessory sex glands and involved in male and female reproductive processes. This study aimed to define in depth sEV subsets isolated by ultrafiltration and size exclusion chromatography, decode their proteomic profiles using liquid chromatography-tandem mass spectrometry, and quantify identified proteins using sequential window acquisition of all theoretical mass spectra. The sEV subsets were defined as large (L-EVs) or small (S-EVs) by their protein concentration, morphology, size distribution, and EV-specific protein markers and purity. Liquid chromatography-tandem mass spectrometry identified a total of 1034 proteins, 737 of them quantified by SWATH in S-EVs, L-EVs, and non-EVs-enriched samples (18-20 size exclusion chromatography-eluted fractions). The differential expression analysis revealed 197 differentially abundant proteins between both EV subsets, S-EVs and L-EVs, and 37 and 199 between S-EVs and L-EVs versus non-EVs-enriched samples, respectively. The gene ontology enrichment analysis of differentially abundant proteins suggested, based on the type of protein detected, that S-EVs could be mainly released through an apocrine blebbing pathway and be involved in modulating the immune environment of the female reproductive tract as well as during sperm-oocyte interaction. In contrast, L-EVs could be released by fusion of multivesicular bodies with the plasma membrane becoming involved in sperm physiological processes, such as capacitation and avoidance of oxidative stress. In conclusion, this study provides a procedure capable of isolating subsets of EVs from pig seminal plasma with a high degree of purity and shows differences in the proteomic profile between EV subsets, indicating different sources and biological functions for the sEVs.

There is increasing evidence that food is an important factor that influences the composition of the gut microbiota. Usually, all the attention has been focused on nutrients such as lipids, proteins, vitamins, or polyphenols. However, a pivotal role in these processes has been linked to dietary-derived exosome-like nanoparticles (DELNs). While food macro- and micronutrient composition are largely well established, there is considerable interest in these DELNs and their cargoes. In this sense, traditionally, all the attention was focused on the proteins or miRNAs contained in these vesicles. However, it has been shown that DELNs would also carry other bioactive molecules with a key role in regulating biochemical pathways and/or interactions with the host’s gut microbiome affecting intracellular communication. Due to the scarce literature, it is necessary to compile the current knowledge about the antimicrobial capacity of DELNs and its possible molecular mechanisms that will serve as a starting point. For this reason, in this review, we highlight the impact of DENLs on different bacteria species modulating the host gut microbiota or antibacterial properties. It could be concluded that DELNs, isolated from both plant and animal foods, exert gut microbiota modulation. However, the presence of miRNA in the vesicle cargoes is not the only one responsible for this effect. Lipids present in the DELNs membrane or small molecules packed in may also be responsible for apoptosis signaling, inhibition, or growth promoters.

Background: Because of their specific and biologically relevant cargo, urine extracellular vesicles (EVs) constitute a valuable source of potential non-invasive biomarkers that could support the clinical decision-making to improve the management of prostate cancer (PCa) patients. Different EV isolation methods differ in terms of complexity and yield, conditioning, as consequence, the analytical result. Methods: The aim of this study was to compare three different isolation methods for urine EVs: ultracentrifugation (UC), size exclusion chromatography (SEC), and a commercial kit (Exolute® Urine Kit). Urine samples were collected from 6 PCa patients and 4 healthy donors. After filtered through 0.22 µm filters, urine was divided in 3 equal volumes to perform EVs isolation with each of the three approaches. Isolated EVs were characterized by spectrophotometric protein quantification, nanoparticle tracking analysis, transmission electron microscopy, AlphaScreen Technology, and whole miRNA Transcriptome. Results: Our results showed that UC and SEC provided better results in terms of EVs yield and purity than Exolute®, non-significant differences were observed in terms of EV-size. Interestingly, luminescent AlphaScreen assay demonstrated a significant enrichment of CD9 and CD63 positive microvesicles in SEC and UC methods compared with Exolute®. This heterogeneity was also demonstrated in terms of miRNA content indicating that the best correlation was observed between UC and SEC. Conclusions: Our study highlights the importance of standardizing the urine EV isolation methods to guaranty the analytical reproducibility necessary for their implementation in a clinical setting.

Plant-derived nanovesicles (PDNVs) are promising therapeutic agents, valued for their bioactive compound content and as drug delivery systems. However, the effects of industrial food processing techniques on PDNVs remain underexplored. This study evaluated the impact of lyophilization and pasteurization on the properties and uptake efficiency of pomegranate-derived nanovesicles (PgNVs). PgNVs were isolated from pomegranate juice using tangential flow filtration and size exclusion chromatography. Treated PgNVs were analyzed via transmission electron microscopy, nanoparticle tracking analysis, and LC-MS/MS, assessing their functional properties and cellular uptake. High-purity PgNVs were obtained, although both treatments reduced their yield. Pre-treated PgNVs displayed diminished anti-inflammatory and wound-healing capacities, though antioxidant activity remained unaffected. Treated PgNVs also exhibited lower internalization by human macrophages compared to untreated PgNVs. Proteomic analyses revealed that the damage of membrane proteins, such as Tetraspanin-8, might be responsible for these effects. Our findings emphasize the need of optimizing processing to preserve PDNV therapeutic potential.