Journal of Extracellular Vesicles最新文献

Extracellular vesicles (EVs) are valuable targets for liquid biopsy. However, attempts to introduce EV-based biomarkers into clinical practice have not been successful to the extent expected. One of the reasons for this failure is the lack of reliable methods for EV baseline purification from complex biofluids, such as cell-free plasma or serum. Because available one-step approaches for EV isolation are insufficient to purify EVs, the majority of studies on clinical samples were performed either on a mixture of EVs and lipoproteins, whilst the real number of EVs and their individual specific biomarker content remained elusive, or on a low number of samples of sufficient volume to allow elaborate 2-step EV separation by size and density, resulting in a high purity but utmost low recovery. Here we introduce Fast Protein Liquid Chromatography (FPLC) using Superose 6 as a matrix to obtain small EVs from biofluids that are almost free of soluble proteins and lipoproteins. Along with the estimation of a realistic number of small EVs in human samples, we show temporal resolution of the effect of the duration of postprandial phase on the proportion of lipoproteins in purified EVs, suggesting acceptable time frames additionally to the recommendation to use fasting samples for human studies. Furthermore, we assessed a potential value of pure EVs for liquid biopsy, exemplarily examining EV- and tumour-biomarkers in pure FPLC-derived fractions isolated from the serum of patients with pancreatic cancer. Consistent among different techniques, showed the presence of diseases-associated biomarkers in pure EVs, supporting the feasibility of using single-vesicle analysis for liquid biopsy.

Escherichia coli A0 34/86 (EcO83) is a probiotic strain used in newborns to prevent nosocomial infections and diarrhoea. This bacterium stimulates both pro- and anti-inflammatory cytokine production and its intranasal administration reduces allergic airway inflammation in mice. Despite its benefits, there are concerns about the use of live probiotic bacteria due to potential systemic infections and gene transfer. Extracellular vesicles (EVs) derived from EcO83 (EcO83-EVs) might offer a safer alternative to live bacteria. This study characterizes EcO83-EVs and investigates their interaction with host cells, highlighting their potential as postbiotic therapeutics. EcO83-EVs were isolated, purified, and characterised following the Minimal Information of Studies of Extracellular Vesicles (MISEV) guidelines. Ex vivo studies conducted in human nasal epithelial cells showed that EcO83-EVs increased the expression of proteins linked to oxidative stress and inflammation, indicating an effective interaction between EVs and the host cells. Further in vivo studies in mice demonstrated that EcO83-EVs interact with nasal-associated lymphoid tissue, are internalised by airway macrophages, and stimulate neutrophil recruitment in the lung. Mechanistically, EcO83-EVs activate the NF-κΒ signalling pathway, resulting in the nitric oxide production. EcO83-EVs demonstrate significant potential as a postbiotic alternative to live bacteria, offering a safer option for therapeutic applications. Further research is required to explore their clinical use, particularly in mucosal vaccination and targeted immunotherapy strategies.

Extracellular vesicles (EVs) are highly heterogeneous, and different EV subpopulations from various origins mediate different biological effects. The separation of different subpopulations of EVs from mixtures is critical but challenging. Epididymosomes are secreted by the epididymal epithelium and play a key role in sperm maturation and function. However, limited access to human epididymal tissue and epididymal fluid has hampered further study of epididymosomes and their potential clinical applications. Here, we established a novel strategy based on flow cytometry sorting to isolate human CD63-positive epididymosomes from ejaculate. We identified CD52, a membrane-located protein expressed exclusively in the epididymis, as the sorting marker for human epididymosomes. Then, CD63-positive epididymosomes were isolated from human semen using a flow cytometry sorting instrument and concentrated. Additionally, we observed that isolated CD63-positive epididymosomes improved sperm function more than other CD63-positive seminal EV subpopulations did, demonstrating the successful isolation of a subpopulation of epididymosomes from human semen and their potential application in the clinic.

Atherosclerotic lesions mainly form in arterial areas exposed to low shear stress (LSS), where endothelial cells express a senescent and inflammatory phenotype. Conversely, areas exposed to high shear stress (HSS) are protected from plaque development. Endothelial extracellular vesicles (EVs) have been shown to regulate inflammation and senescence, and therefore play a crucial role in vascular homeostasis. Whilst previous studies have shown links between hemodynamic forces and EV release, the effects of shear stress on the release and uptake of endothelial EVs remains elusive. We aim to decipher the interplay between these processes in endothelial cells exposed to atheroprone or atheroprotective shear stress. Confluent HUVECs were exposed to LSS or HSS for 24 h. Large and small EVs were isolated from conditioned medium by centrifugation and size exclusion chromatography. They were characterised by TEM, Western blot, tunable resistive pulse sensing, flow cytometry and proteomics. Uptake experiments were performed using fluorescently-labelled EVs and differences between groups were assessed by flow cytometry and confocal microscopy. We found that levels of large and small EVs in conditioned media were fifty and five times higher in HSS than in LSS conditions, respectively. In vivo and in vitro uptake experiments revealed greater EV incorporation by cells exposed to LSS conditions. Additionally, endothelial LSS-EVs have a greater affinity for HUVECs than HSS-EVs or EVs derived from platelets, erythrocytes and leukocytes. Proteomic analysis revealed that LSS-EVs were enriched in adhesion proteins (PECAM1, MCAM), participating in EV uptake by endothelial cells. LSS-EVs also carried mitochondrial material, which may be implicated in elevating ROS levels in recipient cells. These findings suggest that shear stress influences EV biogenesis and uptake. Given the major role of EVs and shear stress in vascular health, deciphering the relation between these processes may yield innovative strategies for the early detection and treatment of endothelial dysfunction.

Immunofluorescence analysis of individual extracellular vesicles (EVs) in common fluorescence microscopes is gaining popularity due to its accessibility and high fluorescence sensitivity; however, EV number and size are only measurable using fluorescent stains requiring extensive sample manipulations. Here we introduce highly sensitive label-free EV size photometry (SP) based on interferometric scattering (iSCAT) imaging of immersed EVs immobilized on a glass coverslip. We implement SP on a common inverted epifluorescence microscope with LED illumination and a simple 50:50 beamsplitter, permitting seamless integration of SP with fluorescence imaging (SPFI). We present a high-throughput SPFI workflow recording >10,000 EVs in 7 min over ten 88 × 88 µm² fields of view, pre- and post-incubation imaging to suppress background, along with automated image alignment, aberration correction, spot detection and EV sizing. We achieve an EV sizing range from 37 to ∼220 nm in diameter with a dual 440 and 740 nm SP illumination scheme, and suggest that this range can be extended by more advanced image analysis or additional hardware customization. We benchmark SP to flow cytometry using calibrated silica nanoparticles and demonstrate superior, label-free sensitivity. We showcase SPFI's potential for EV analysis by experimentally distinguishing surface and volumetric EV dyes, observing the deformation of EVs adsorbed to a surface, and by uncovering distinct subpopulations in <100 nm-in-diameter EVs with fluorescently tagged membrane proteins.

Bacterial infections, especially those caused by multidrug-resistant pathogens, pose a significant threat to public health. Vaccines are a crucial tool in fighting these infections; however, no clinically available vaccine exists for the most common bacterial infections, such as those caused by Pseudomonas aeruginosa. Herein, a multiantigenic antibacterial nanovaccine (AuNP@HMV@SPs) is reported to combat P. aeruginosa infections. This nanovaccine utilizes the hybrid membrane vesicles (HMVs) created by fusing macrophage membrane vesicles (MMVs) with bacterial outer membrane vesicles (OMVs). The HMVs mitigate the toxic effects of both OMVs and bacterial secreted toxins (SP) adsorbed on the surface of MMVs, while preserving their stimulating properties. Gold nanoparticles (AuNPs) are utilized as adjuvant to enhance immune response without comprising safety. The nanovaccine AuNP@HMV@SPs induces robust humoral and cellular immune responses, leading to destruction of bacterial cells and neutralization of their secreted toxins. In murine models of septicemia and pneumonia caused by P. aeruginosa, AuNP@HMV@SPs exhibits superior prophylactic efficacy compared to control groups including OMVs, or MMVs@SPs and HMV@SPs, achieving 100% survival in septicemia and > 99.9% reduction in lung bacterial load in pneumonia. This study highlights AuNP@HMV@SPs as a safe and effective antibacterial nanovaccine, targeting both bacteria and their secreted toxins, and offers a promising platform for developing multiantigenic antibacterial vaccines against multidrug-resistant pathogens.

Extracellular vesicles (EVs) are lipid nanoparticles and play an important role in cell-cell communications, making them potential therapeutic agents and allowing to engineer for targeted drug delivery. The expanding applications of EVs in next generation medicine is still limited by existing tools for scaling standardized EV production, single EV tracing and analytics, and thus provide only a snapshot of tissue-specific EV cargo information. Here, we present the Snorkel-tag, for which we have genetically fused the EV surface marker protein CD81, to a series of tags with an additional transmembrane domain to be displayed on the EV surface, resembling a snorkel. This system enables the affinity purification of EVs from complex matrices in a non-destructive form while maintaining EV characteristics in terms of surface protein profiles, associated miRNA patterns and uptake into a model cell line. Therefore, we consider the Snorkel-tag to be a widely applicable tool in EV research, allowing for efficient preparation of EV standards and reference materials, or dissecting EVs with different surface markers when fusing to other tetraspanins in vitro or in vivo.

Adult T cell leukaemia (ATL), caused by infection with human T- lymphotropic virus type 1 (HTLV-1), is often complicated by hypercalcemia and osteolytic lesions. Therefore, we studied the communication between patient-derived ATL cells (ATL-PDX) and HTLV-1 immortalized CD4+ T cell lines (HTLV/T) with osteoclasts and their effects on bone mass in mice. Intratibial inoculation of some HTLV/T leads to a profound local decrease in bone mass similar to marrow-replacing ATL-PDX, despite the fact that few HTLV/T cells persisted in the bone. To study the direct effect of HTLV/T and ATL-PDX on osteoclasts, supernatants were added to murine and human osteoclast precursors. ATL-PDX supernatants from hypercalcemic patients promoted the formation of mature osteoclasts, while those from HTLV/T were variably stimulatory, but had largely consistent effects between human and murine cultures. Interestingly, this osteoclastic activity did not correlate with expression of osteoclastogenic cytokine receptor activator of nuclear factor kappa-B ligand (RANKL), suggesting an alternative mechanism. HTLV/T and ATL-PDX produce small extracellular vesicles (sEV), known to facilitate HTLV-1 infection. We hypothesized that these sEV also mediate bone loss by targeting osteoclasts. We isolated sEV from both HTLV/T and ATL-PDX, and found they carried most of the activity found in supernatants. In contrast, sEV from uninfected activated T cells had little effect. Analysis of sEV (both active and inactive) by mass spectrometry and electron microscopy confirmed absence of RANKL and intact virus. Viral proteins Tax and Env were only present in sEV from the active, osteoclast-stimulatory group, along with increased representation of proteins involved in osteoclastogenesis and bone resorption. sEV from osteoclast-active HTLV/T injected over mouse calvaria in the presence of low-dose RANKL caused more osteolysis than osteoclast-inactive sEV or RANKL alone. Thus, HTLV-1 infection of T cells can cause release of sEV with strong osteolytic potential, providing a mechanism beyond RANKL production that modifies the bone microenvironment, even in the absence of overt leukaemia.

The morphogenesis of higher plants requires communication among distant organs throughout vascular tissues (xylem and phloem). Numerous investigations have demonstrated that phloem also act as a distribution route for signalling molecules being observed that different macromolecules translocated by the sap, including nucleic acids and proteins, change under stress situations. The participation of extracellular vesicles (EVs) in this communication has been suggested, although little is known about their role. In fact, in the last decade, the presence of EVs in plants has originated a great controversy, where major concerns arose from their origin, isolation methods, and even the appropriate nomenclature for plant nanovesicles. Phloem sap exudates from melon plants, either aphid-free or infested with Aphis gossypii, were collected by stem incision. After sap concentration (Amicon), phloem EVs (PhlEVs) were isolated by size exclusion chromatography. PhlEVs were characterised using Nanoparticle Tracking Analysis, Transmission electron microscopy and proteomic analysis. Here we confirm the presence of EVs in phloem sap in vivo and the detection of changes in the particles/protein ratio and composition of PhlEVs in response to insect feeding, revealing the presence of typical defence proteins in their cargo as well as components of the proteasome complex. PhlEVs from infested plants showed lower particles/protein ratio and almost two times more proteolytic activity than PhlEVs from aphid-free plants. In both cases, such activity was inhibited in a dose-dependent manner by the proteasome inhibitor MG132. Our results suggest that plants may use this mechanism to prepare themselves to receive infectious agents and open up the possibility of an evolutionary conserved mechanism of defence against pathogens/stresses in eukaryotic organisms.

Bacterial extracellular vesicles (BEVs) have emerged as pivotal mediators between bacteria and host. In addition to being crucial players in host homeostasis, they have recently been implicated in disease pathologies such as cancer. Hence, the study of BEVs represents an intriguing and rapidly evolving field with substantial translational potential. In this review, we briefly introduce the fundamentals of BEV characteristics, cargo and biogenesis. We emphatically summarize the current relationship between BEVs across various cancer types, illustrating their role in tumorigenesis, treatment responses and patient survival. We further discuss the inherent advantages of BEVs, such as stability, abundance and specific cargo profiles, that make them attractive candidates for non-invasive diagnostic and prognostic approaches. The review also explores the potential of BEVs as a strategy for cancer therapy, considering their ability to deliver therapeutic agents, modulate the tumour microenvironment (TME) and elicit immunomodulatory responses. Understanding the clinical significance of BEVs may lead to the development of better-targeted and personalized treatment strategies. This comprehensive review evaluates the current progress surrounding BEVs and poses questions to encourage further research in this emerging field to harness the benefits of BEVs for their full potential in clinical applications against cancer.