Seerupblanchard5313

Collectively, our findings reveal the effect of hESC-sEVs in reversing BM-MSCs senescence and age-related osteogenic dysfunction, thereby preventing age-related bone loss. Because hESC-sEVs could alleviate senescence of tissue-resident stem cells, they might be promising therapeutic candidates for age-related diseases.Extracellular vesicles (EVs) are naturally occurring nano-sized carriers that are secreted by cells and facilitate cell-to-cell communication by their unique ability to transfer biologically active cargo. Despite the pronounced increase in our understanding of EVs over the last decade, from disease pathophysiology to therapeutic drug delivery, improved molecular tools to track their therapeutic delivery are still needed. Unfortunately, the present catalogue of tools utilised for EV labelling lacks sensitivity or are not sufficiently specific. Here, we have explored the bioluminescent labelling of EVs using different luciferase enzymes tethered to CD63 to achieve a highly sensitive system for in vitro and in vivo tracking of EVs. Using tetraspanin fusions to either NanoLuc or ThermoLuc permits performing highly sensitive in vivo quantification of EVs or real-time imaging, respectively, at low cost and in a semi-high throughput manner. We find that the in vivo distribution pattern of EVs is determined by the route of injection, but that different EV subpopulations display differences in biodistribution patterns. By applying this technology for real-time non-invasive in vivo imaging of EVs, we show that their distribution to different internal organs occurs just minutes after administration.Dendritic cells (DC) have the unique capacity to activate naïve T cells by presenting T cell receptor specific peptides from exogenously acquired antigens bound to Major Histocompatibility Complex (MHC) molecules. MHC molecules are displayed on the DC plasma membrane as well as on extracellular vesicles (EV) that are released by DC, and both have antigen-presenting capacities. However, the physiological role of antigen presentation by EV is still unclear. We here demonstrate that the release of small EV by activated DC is strongly stimulated by phagocytic events. We show that, concomitant with the enhanced release of EV, a significant proportion of phagocytosed bacteria was expulsed back into the medium. High-resolution fluorescence microscopic images revealed that bacteria in phagosomes were surrounded by EV marker-proteins. Moreover, expulsed bacteria were often found associated with clustered HLA II and CD63. Together, these observations suggest that exosomes may be formed by the inward budding into phagosomes, whereupon they are secreted together with the phagosomal content. These findings may have important implications for selective loading of peptides derived from phagocytosed pathogens onto exosome associated HLA molecules, and have important implications for vaccine design.Severe COVID-19 infection results in bilateral interstitial pneumonia, often leading to acute respiratory distress syndrome (ARDS) and pulmonary fibrosis in survivors. Most patients with severe COVID-19 infections who died had developed ARDS. Currently, ARDS is treated with supportive measures, but regenerative medicine approaches including extracellular vesicle (EV)-based therapies have shown promise. Herein, we aimed to analyse whether EV-based therapies could be effective in treating severe pulmonary conditions that affect COVID-19 patients and to understand their relevance for an eventual therapeutic application to human patients. Using a defined search strategy, we conducted a systematic review of the literature and found 39 articles (2014-2020) that reported effects of EVs, mainly derived from stem cells, in lung injury models (one large animal study, none in human). EV treatment resulted in (1) attenuation of inflammation (reduction of pro-inflammatory cytokines and neutrophil infiltration, M2 macrophage polarization); (2) regeneration of alveolar epithelium (decreased apoptosis and stimulation of surfactant production); (3) repair of microvascular permeability (increased endothelial cell junction proteins); (4) prevention of fibrosis (reduced fibrin production). These effects were mediated by the release of EV cargo and identified factors including miRs-126, -30b-3p, -145, -27a-3p, syndecan-1, hepatocyte growth factor and angiopoietin-1. This review indicates that EV-based therapies hold great potential for COVID-19 related lung injuries as they target multiple pathways and enhance tissue regeneration. However, before translating EV therapies into human clinical trials, efforts should be directed at developing good manufacturing practice solutions for EVs and testing optimal dosage and administration route in large animal models.An immune reaction is a protector of our body but a target to be overcome for all non-self-derived medicine. Extracellular Vesicles (EVs), noted as a primary alternative to cell therapy products that exhibit immune rejection due to mismatching-major histocompatibility complex (MHC), were discovered to have excellent curative effects through the delivery of various biologically active substances. Although EVs are sure to incur immune reaction by immunogenicity due to alloantigens from their parental cells, their immune rejection is rarely known. Hence, to develop cell lines and EVs as medicines with no immune rejection, we noted the immune tolerance where the foetus, as semi-allograft, is perfectly protected from the maternal immune system. We designed the ex-vivo culture systems to simulate in-vivo environmental factors inducing extravillous trophoblast (EVT)-specific Human Leukocyte Antigen-G (HLA-G) expression and secretion of HLA-G-bearing EVs at the mother-foetus interface. Using them, we confirmed that iility complex; MSC mesenchymal stem cells; NK natural killer cells; NTA nanoparticle tracking analysis; PBMC peripheral blood mononuclear cells; PHA phytohemagglutinin; SP-IRIS single particle interferometric reflectance imaging sensing; STB syncytiotrophoblast.Chronic bone degenerative diseases represent a major threat to the health and well-being of the population, particularly those with advanced age. This study isolated exosomes (EXO), natural nano-particles, from dendritic cells, the "directors" of the immune response, to examine the immunobiology of DC EXO in mice, and their ability to reprogram immune cells responsible for experimental alveolar bone loss in vivo. Distinct DC EXO subtypes including immune-regulatory (regDC EXO), loaded with TGFB1 and IL10 after purification, along with immune stimulatory (stimDC EXO) and immune "null" immature (iDCs EXO) unmodified after purification, were delivered via I.V. route or locally into the soft tissues overlying the alveolar bone. Locally administrated regDC EXO showed high affinity for inflamed sites, and were taken up by both DCs and T cells in situ. RegDC EXO-encapsulated immunoregulatory cargo (TGFB1 and IL10) was protected from proteolytic degradation. Moreover, maturation of recipient DCs and induction of Th17 effectors was suppressed by regDC EXO, while T-regulatory cell recruitment was promoted, resulting in inhibition of bone resorptive cytokines and reduction in osteoclastic bone loss. This work is the first demonstration of DC exosome-based therapy for a degenerative alveolar bone disease and provides the basis for a novel treatment strategy.Innate immunity is a first line of defence against danger. Exogenous pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) trigger innate immune responses through well-understood cellular pathways. In contrast, endogenous damage-associated molecular patterns (DAMPs) convey "danger signals" via their (mis)localization or modification. Both MAMPs and DAMPs are often communicated on or within extracellular vesicles (EVs). Despite growing evidence for the importance of EVs and their cargo in modulating innate immune responses, in some cases, it is unclear how EV-transported molecules are sensed as abnormal. In particular, EVs constitutively carry RNA, which is also abundant in the cytoplasm. How, then, would RNA convey a danger signal as a cargo of EVs? In this Perspective, we offer some thoughts on how EV-associated RNAs might raise the alarm for innate immune responses - or silence them.Probiotics offer various health benefits. Lactobacillus plantarum has been used for decades to enhance human intestinal mucosal immunity and improve skin barrier integrity. Extracellular vesicles (EVs) derived from eukaryotic or prokaryotic cells have been recognized as efficient carriers for delivery of biomolecules to recipient cells, and to efficiently regulate human pathophysiology. However, the mechanism underlying the beneficial effects of probiotic bacteria-derived EVs on human skin is unclear. Herein, we investigated how L. plantarum-derived EVs (LEVs) exert beneficial effects on human skin by examining the effect of LEVs on cutaneous immunity, particularly on macrophage polarization. LEVs promoted differentiation of human monocytic THP1 cells towards an anti-inflammatory M2 phenotype, especially M2b, by inducing biased expression of cell-surface markers and cytokines associated with M2 macrophages. Pre- or post-treatment with LEVs under inflammatory M1 macrophage-favouring conditions, induced by LPS and interferon-γ, inhibited M1-associated surface marker, HLA-DRα expression. Moreover, LEV treatment significantly induced expression of macrophage-characteristic cytokines, IL-1β, GM-CSF and the representative anti-inflammatory cytokine, IL-10, in human skin organ cultures. Hence, LEVs can trigger M2 macrophage polarization in vitro, and induce an anti-inflammatory phenomenon in the human skin, and may be a potent anti-inflammatory strategy to alleviate hyperinflammatory skin conditions.The in vivo detection of dead cells remains a major challenge due to technical hurdles. Here, we present a novel method, where injection of fluorescent milk fat globule-EGF factor 8 protein (MFG-E8) in vivo combined with imaging flow cytometry and deep learning allows the identification of dead cells based on their surface exposure of phosphatidylserine (PS) and other image parameters. A convolutional autoencoder (CAE) was trained on defined pictures and successfully used to identify apoptotic cells in vivo. However, unexpectedly, these analyses also revealed that the great majority of PS+ cells were not apoptotic, but rather live cells associated with PS+ extracellular vesicles (EVs). During acute viral infection apoptotic cells increased slightly, while up to 30% of lymphocytes were decorated with PS+ EVs of antigen-presenting cell (APC) exosomal origin. The combination of recombinant fluorescent MFG-E8 and the CAE-method will greatly facilitate analyses of cell death and EVs in vivo.Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellular communication, and their presence in biofluids can be indicative for (patho)physiological conditions. Studies aiming to resolve functionalities of EV or to discover EV-associated biomarkers for disease in liquid biopsies are hampered by limitations of current protocols to isolate EV from biofluids or cell culture medium. EV isolation is complicated by the >105-fold numerical excess of other types of particles, including lipoproteins and protein complexes. In addition to persisting contaminants, currently available EV isolation methods may suffer from inefficient EV recovery, bias for EV subtypes, interference with the integrity of EV membranes, and loss of EV functionality. In this study, we established a novel three-step non-selective method to isolate EV from blood or cell culture media with both high yield and purity, resulting in 71% recovery and near to complete elimination of unrelated (lipo)proteins. This EV isolation procedure is independent of ill-defined commercial kits, and apart from an ultracentrifuge, does not require specialised expensive equipment.