Background miRNAs are master regulators of signaling pathways critically involved in asthma and are transferred between cells in extracellular vesicles (EV). We aimed to investigate whether the miRNA content of EV secreted by primary normal human bronchial epithelial cells (NHBE) is altered upon asthma development. Methods NHBE cells were cultured at air‐liquid interface and treated with interleukin (IL)‐13 to induce an asthma‐like phenotype. EV isolations by precipitation from basal culture medium or apical surface wash were characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blot, and EV‐associated miRNAs were identified by a RT‐qPCR‐based profiling. Significant candidates were confirmed in EVs isolated by size‐exclusion chromatography from nasal lavages of children with mild‐to‐moderate (n = 8) or severe asthma (n = 9), and healthy controls (n = 9). Results NHBE cells secrete EVs to the apical and basal side. 47 miRNAs were expressed in EVs and 16 thereof were significantly altered in basal EV upon IL‐13 treatment. Expression of miRNAs could be confirmed in EVs from human nasal lavages. Of note, levels of miR‐92b, miR‐210, and miR‐34a significantly correlated with lung function parameters in children (FEV1FVC%pred and FEF25‐75%pred), thus lower sEV‐miRNA levels in nasal lavages associated with airway obstruction. Subsequent ingenuity pathway analysis predicted the miRNAs to regulate Th2 polarization and dendritic cell maturation. Conclusion Our data indicate that secretion of miRNAs in EVs from the airway epithelium, in particular miR‐34a, miR‐92b, and miR‐210, might be involved in the early development of a Th2 response in the airways and asthma.

Adipocyte-derived extracellular vesicles (AdEVs) are membranous nanoparticles that convey communication from adipose tissue to other organs. Here, to delineate their role as messengers with glucoregulatory nature, we paired fluorescence AdEV-tracing and SILAC-labeling with (phospho)proteomics, and revealed that AdEVs transfer functional insulinotropic protein cargo into pancreatic β-cells. Upon transfer, AdEV proteins were subjects for phosphorylation, augmented insulinotropic GPCR/cAMP/PKA signaling by increasing total protein abundances and phosphosite dynamics, and ultimately enhanced 1st-phase glucose-stimulated insulin secretion (GSIS) in murine islets. Notably, insulinotropic effects were restricted to AdEVs isolated from obese and insulin resistant, but not lean mice, which was consistent with differential protein loads and AdEV luminal morphologies. Likewise, in vivo pre-treatment with AdEVs from obese but not lean mice amplified insulin secretion and glucose tolerance in mice. This data suggests that secreted AdEVs can inform pancreatic β-cells about insulin resistance in adipose tissue in order to amplify GSIS in times of increased insulin demand.

Glioblastomas are among the most aggressive tumors, and with low survival rates. They are characterized by the ability to create a highly immunosuppressive tumor microenvironment. Exosomes, small extracellular vesicles (EVs), mediate intercellular communication in the tumor microenvironment by transporting various biomolecules (RNA, DNA, proteins, and lipids), therefore playing a prominent role in tumor proliferation, differentiation, metastasis, and resistance to chemotherapy or radiation. Exosomes are found in all body fluids and can cross the blood–brain barrier due to their nanoscale size. Recent studies have highlighted the multiple influences of tumor-derived exosomes on immune cells. Owing to their structural and functional properties, exosomes can be an important instrument for gaining a better molecular understanding of tumors. Furthermore, they qualify not only as diagnostic and prognostic markers, but also as tools in therapies specifically targeting aggressive tumor cells, like glioblastomas.

The determination of the somatic cell count of a milk sample is one of the most common methods to monitor udder health of a dairy cow. However, this procedure does not take into account the fact that cells in milk present a great variety of different cell types. The objective of our study was to establish a high-resolution differential cell count (HRDCC) by means of flow cytometry in blood and milk. We were able to detect ten subpopulations among the three main populations of immune cells and to determine their viability. Additionally, blood samples were analyzed for common laboratory biomarkers, i.e. differential blood counts, haptoglobin levels and several metabolic parameters. In this first feasibility study, we used three different vaccines to stimulate the immune system of five healthy cows each. Samples were collected shortly before, in between and after the vaccinations. Using multivariate statistical methods we saw a diagnostic benefit when HRDCCs were included compared to only the standard laboratory parameters. The impacts of all three vaccinations on the immune system were visible in blood HRDCCs as well as in milk HRDCCs. Cluster of Differentiation 8+ (CD8+) T cells, B cells and monocyte/macrophage subpopulations were among the most important and statistically relevant parameters for all treatments in both biofluids. Moreover, in one of the treatment groups intermediate monocytes showed a significant increase after both vaccinations. Although the use of HRDCC in blood or milk was shown to be highly relevant for early systemic diagnostic, to confirm these subpopulations further investigations in cows of different breed, lactation stage or health status are required.

Extracellular vesicles (EVs) have recently come into the spotlight as potential cancer biomarkers. Isolation of pure EVs is complex, so wider use requires reliable and time-efficient isolation methods. In the present study, galectin-based magnetic glycan recognition particles, EXÖBead ® were investigated for their practicality as a novel EV isolation technique, exemplified here for squamous cell carcinoma of the head and neck. Analysis of the isolation method showed a high concentration of pure EVs with detection of specific EV markers such as CD9, CD63, CD81 and TSG101. No apolipoprotein A1 was shown in the isolates, indicating low contamination of this isolation technique compared with size exclusion chromatography. In addition, common leukocyte antigen (CD45), three HNSCC [epithelial cell adhesion molecule (EpCAM), pan-cytokeratin and programmed death-ligand 1 (PD-L1)] and PanEV markers (premixed CD9, CD63 and CD81 anti-bodies) were measured by bead-based flow cytometry (BFC). BFC revealed that CD45 Neg PanEV + , EpCAM + PanEV + and PD-L1 + PanEV + were significantly higher in tumor patients compared with healthy control plasma. CD45 Neg PanEV + and CD45 + PanEV + carrying two or three HNSCC biomarkers were also significantly higher in tumor patients compared with healthy controls (BFC). Comparison of the functional immunosuppression effect of eluted tumor patient plasma EVs from EXÖBead ® and commercial polyethylene glycol isolation showed a significant tumor-dependent increase in concentration of EVs. A peripheral blood mononuclear cell activation assay also showed that the T-cell functionality of tumor patient plasma EVs isolated with EXÖBead ® was preserved in vitro . In conclusion, isolation using galectin-based magnetic glycan recognition particles is a novel method for isolating plasma EVs with low lipoprotein contamination. Bead-based flow cytometry provided an easy way to understand EV subpopulations. EXÖBead ® therefore showed great potential as a new isolation tool with high throughput capacity that could potentially be used in a clinical setting.