Release of membrane vesicles, a process conserved in both prokaryotes and eukaryotes, represents an evolutionary link, and suggests essential functions of a dynamic extracellular vesicular compartment (including exosomes, microparticles or microvesicles and apoptotic bodies). Compelling evidence supports the significance of this compartment in a broad range of physiological and pathological processes. However, classification of membrane vesicles, protocols of their isolation and detection, molecular details of vesicular release, clearance and biological functions are still under intense investigation. Here, we give a comprehensive overview of extracellular vesicles. After discussing the technical pitfalls and potential artifacts of the rapidly emerging field, we compare results from meta-analyses of published proteomic studies on membrane vesicles. We also summarize clinical implications of membrane vesicles. Lessons from this compartment challenge current paradigms concerning the mechanisms of intercellular communication and immune regulation. Furthermore, its clinical implementation may open new perspectives in translational medicine both in diagnostics and therapy.

The discovery that submicron-sized extracellular vesicles (EVs) are generated by both prokaryotic and eukaryotic cells might have a profound effect on experimental and clinical sciences, and could pave the way for new strategies to combat various diseases. EVs are carriers of pathogen-associated and damage-associated molecular patterns, cytokines, autoantigens and tissue-degrading enzymes. In addition to a possible role in the pathogenesis of a number of inflammatory conditions, such as infections and autoimmune diseases, EVs, including microvesicles (also known as microparticles), exosomes and apoptotic vesicles, have therapeutic potential and might be used as biomarkers for inflammatory diseases. Therefore, molecular diagnostics and targeted therapy could benefit from expanding knowledge in the field. In this Review, we summarize important developments and propose that extracellular vesicles could be used as therapeutic vehicles and as targets for the treatment and prevention of inflammatory diseases.

BackgroundExosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described.AimTherefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC).Methods and ResultsExosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4°C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4°C, or UC performed at 37°C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin.ConclusionHere we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.

Numerous diseases, recently reported to associate with elevated microvesicle/ microparticle (MP) counts, have also long been known to be characterized by accelerated immune complex (IC) formation. The goal of this study was to investigate the potential overlap between parameters of protein complexes (eg, ICs or avidinbiotin complexes) and MPs, which might perturb detection and/or isolation of MPs. In this work, after comprehensive characterization of MPs by electron microscopy, atomic force microscopy, dynamic lightscattering analysis, and flow cytometry, for the first time, we drive attention to the fact that protein complexes, especially insoluble ICs, overlap in biophysical properties (size, light scattering, and sedimentation) with MPs. This, in turn, affects MP quantification by flow cytometry and purification by differential centrifugation, especially in diseases in which IC formation is common, including not only autoimmune diseases, but also hematologic disorders, infections, and cancer. These data may necessitate reevaluation of certain published data on patient-derived MPs and contribute to correct the clinical laboratory assessment of the presence and biologic functions of MPs in health and disease. (Blood. 2011;117(4):e39-e48) IntroductionMembrane vesicles are small subcellular structures surrounded by a phospholipid bilayer. Their release by various cell types is enhanced during activation and apoptosis. 1 They represent heterogeneous structures and can be classified into several groups depending on their size, antigenic features, and mechanism of cellular release. 1 The two best characterized categories include exosomes and microvesicles/microparticles (MPs). Both populations are characterized by the exposure of phosphatidylserine, which allows annexin-V (AX) to bind to these-lipid surfaces. 1 Exosomes are composed of small, 50-to 100-nm-sized structures released on exocytosis of multivesicular bodies. 1 The diameter of MPs, formed by membrane blebbing, is described to be 100 to 1000 nm 2 . However, precise definitions of MPs are still lacking. 1,2 MPs are found in various biologic fluids, including blood plasma, 3 urine, 4 and synovial fluid (SF). 5,6 Numerous flow cytometry (FC) studies using blood plasma have shown correlation of MP counts with human cardiovascular 7 and autoimmune diseases, 8 hematologic disorders, 9 and cancer. 10 Of particular interest, autoimmune diseases were reported to be characterized by elevated levels of MPs. 11 The assessment of exosomes and MPs is complicated by the presence of further known categories of membrane bound subcellular structures, such as apoptotic vesicles, exosome-like vesicles, membrane particles, and ectosomes. 1 There is a substantial size overlap among the aforementioned vesicle categories, and the size distribution of a given vesicle preparation may also be affected by the method used for their isolation. 3,12,13 Recently, attempts have been made to standardize isolation and detection protocols for membrane vesicles. 3,14 Up until now, no systemat...

Key Points• Neutrophilic granulocytes stimulated with opsonized particles produce microvesicles (MVs) that are able to impair bacterial growth.• Antibacterial effect correlates with number and size of aggregates between bacteria and MVs and depends on cytoskeletal reorganization of MVs.Cell-derived vesicles represent a recently discovered mechanism for intercellular communication. We investigated their potential role in interaction of microbes with host organisms. We provide evidence that different stimuli induced isolated neutrophilic granulocytes to release microvesicles with different biologic properties. Only opsonized particles initiated the formation of microvesicles that were able to impair bacterial growth. The antibacterial effect of neutrophil-derived microvesicles was independent of production of toxic oxygen metabolites and opsonization or engulfment of the microbes, but depended on ␤ 2 integrin function, continuous actin remodeling, and on the glucose supply. Neutrophil-derived microvesicles were detected in the serum of healthy donors, and their number was significantly increased in the serum of bacteremic patients. We propose a new extracellular mechanism to restrict bacterial growth and dissemination. IntroductionCell-derived vesicles (such as exosomes, ectosomes, microvesicles, shedding microvesicles, and microparticles) represent a recently discovered mechanism for cell-cell communication. [1][2][3] Exosomes are small (50-100 nm) vesicles released from multivesicular bodies. 4 They are involved in antigen presentation [5][6][7] and cell-tocell transfer of receptors 8 or RNA, 9,10 thereby influencing or reprogramming neighboring cells and often promoting tumorigenesis. 8,11 Exosomes also play a role in host defense against microorganisms: tracheobronchial epithelial cells produce exosome-like vesicles with antiviral activity, 12 and macrophage-derived exosomes are able to transfer pathogen-associated molecular patterns of opportunistic intracellular pathogens to uninfected cells. 13 Larger vesicles, called microvesicles (MVs) or microparticles express tissue factor on their surface that is capable of initiating coagulation. 14 Both exosomes and MVs of different cellular origin were detected in various body fluids and selective enrichment was related to specific diseases. [15][16][17][18][19] Neutrophilic granulocytes (PMNs) play a critical role in innate immune mechanisms by engulfing, killing, and degrading various microorganisms. PMNs produce larger vesicles (named by the authors alternatively as ectosomes, microparticles, or MVs) after incubation with various stimuli. [19][20][21][22] Microparticles obtained from PMNs stimulated by chemotactic agents or phorbol esters activated cytokine (IL-6) secretion from endothelial cells 23 and platelets, 24 thereby contributing to the procoagulant effect of leukocytederived microparticles. 25 Chemotactic peptide-induced PMNectosomes increase the secretion of the anti-inflammatory cytokine transforming growth factor ␤ 26 and interfere with the maturatio...