74 research outputs found
Novel insights into the isolation of extracellular vesicles by anion exchange chromatography
Extracellular vesicles (EVs) are membrane structures enclosed by a lipid bilayer that are released into the extracellular space by all types of cells. EVs are involved in many physiological processes by transporting biologically active substances. Interest in EVs for diagnostic biomarker research and therapeutic drug delivery applications has increased in recent years. The realization of the full therapeutic potential of EVs is currently hampered by the lack of a suitable technology for the isolation and purification of EVs for downstream pharmaceutical applications. Anion Exchange Chromatography (AEX) is an established method in which specific charges on the AEX matrix can exploit charges on the surface of EVs and their interactions to provide a productive and scalable separation and purification method. The established AEX method using Eshmuno® Q, a strong tentacle anion exchange resin, was used to demonstrate the principal feasibility of AEX-based isolation and gain insight into isolated EV properties. Using several EV analysis techniques to provide a more detailed insight into EV populations during AEX isolation, we demonstrated that although the composition of CD9/63/81 remained constant for tetraspanin positive EVs, the size distribution and purity changed during elution. Higher salt concentrations eluted larger tetraspanin negative vesicles
Validation of extracellular miRNA quantification in blood samples using RT‐qPCR
Extracellular microRNAs (miRs) have been proposed as important blood‐based biomarkers for several diseases. Contrary to proteins and other RNA classes, miRs are stable and easily detectable in body fluids. In this respect, miRs represent a perfect candidate for minimal invasive biomarkers which can hopefully become a complement for invasive histological examinations of tumor tissue. Despite the high number of miR biomarker studies, the specificity and reproducibility of these studies is missing. Therefore, the standardization of pre‐analytical and analytical methods is urgently needed. Here, we validated miR analysis for RNA isolation and miR quantification by quantitative polymerase chain reaction (RT‐qPCR) based on good laboratory practice (GLP). Validation was carried out exemplarily on four miRs, which had already been described as potential biomarkers in previous studies. As basis for RNA analysis using RT‐qPCR, the Minimum Information for Publication of Quantitative Real‐Time PCR Experiments were applied and adapted on the analysis of circulating miRs from human plasma. In our study, we identified and solved several pitfalls from handling to normalization strategy in the analysis of extracellular miRs that lead to inconsistent and non‐repeatable data. Principles of GLP set a framework of experimental design, performance and monitoring to ensure high quality and reliable data. Within this study, we appointed first acceptance criteria for circulating miR quantification during validation which set standards for future miR quantification in blood samples
Novel insights into the isolation of extracellular vesicles by anion exchange chromatography
Extracellular vesicles (EVs) are membrane structures enclosed by a lipid bilayer that are released into the extracellular space by all types of cells. EVs are involved in many physiological processes by transporting biologically active substances. Interest in EVs for diagnostic biomarker research and therapeutic drug delivery applications has increased in recent years. The realization of the full therapeutic potential of EVs is currently hampered by the lack of a suitable technology for the isolation and purification of EVs for downstream pharmaceutical applications. Anion Exchange Chromatography (AEX) is an established method in which specific charges on the AEX matrix can exploit charges on the surface of EVs and their interactions to provide a productive and scalable separation and purification method. The established AEX method using Eshmuno® Q, a strong tentacle anion exchange resin, was used to demonstrate the principal feasibility of AEX-based isolation and gain insight into isolated EV properties. Using several EV analysis techniques to provide a more detailed insight into EV populations during AEX isolation, we demonstrated that although the composition of CD9/63/81 remained constant for tetraspanin positive EVs, the size distribution and purity changed during elution. Higher salt concentrations eluted larger tetraspanin negative vesicles
Proteomics-Based Characterization of miR-574-5p Decoy to CUGBP1 Suggests Specificity for mPGES-1 Regulation in Human Lung Cancer Cells
MicroRNAs (miRs) are one of the most important post-transcriptional repressors of gene expression. However, miR-574-5p has recently been shown to positively regulate the expression of microsomal prostaglandin E-synthase-1 (mPGES-1), a key enzyme in the prostaglandin E2 (PGE2) biosynthesis, by acting as decoy to the RNA-binding protein CUG-RNA binding protein 1 (CUGBP1) in human lung cancer. miR-574-5p exhibits oncogenic properties and promotes lung tumor growth in vivo via induction of mPGES-1-derived PGE2 synthesis. In a mass spectrometry-based proteomics study, we now attempted to characterize this decoy mechanism in A549 lung cancer cells at a cellular level. Besides the identification of novel CUGBP1 targets, we identified that the interaction between miR-574-5p and CUGBP1 specifically regulates mPGES-1 expression. This is supported by the fact that CUGBP1 and miR-574-5p are located in the nucleus, where CUGBP1 regulates alternative splicing. Further, in a bioinformatical approach we showed that the decoy-dependent mPGES-1 splicing pattern is unique. The specificity of miR-574-5p/CUGBP1 regulation on mPGES-1 expression supports the therapeutic strategy of pharmacological inhibition of PGE2 formation, which may provide significant therapeutic value for NSCLC patients with high miR-574-5p levels
Detailed Characterization of Small Extracellular Vesicles from Different Cell Types Based on Tetraspanin Composition by ExoView R100 Platform
Small extracellular vesicles (sEV) hold enormous potential as biomarkers, drug carriers, and therapeutic agents. However, due to previous limitations in the phenotypic characterization of sEV at the single vesicle level, knowledge of cell type-specific sEV signatures remains sparse. With the introduction of next-generation sEV analysis devices, such as the single-particle interferometric reflectance imaging sensor (SP-IRIS)-based ExoView R100 platform, single sEV analyses are now possible. While the tetraspanins CD9, CD63, and CD81 were generally considered pan-sEV markers, it became clear that sEV of different cell types contain several combinations and amounts of these proteins on their surfaces. To gain better insight into the complexity and heterogeneity of sEV, we used the ExoView R100 platform to analyze the CD9/CD63/CD81 phenotype of sEV released by different cell types at a single sEV level. We demonstrated that these surface markers are sufficient to distinguish cell-type-specific sEV phenotypes. Furthermore, we recognized that tetraspanin composition in some sEV populations does not follow a random pattern. Notably, the tetraspanin distribution of sEV derived from mesenchymal stem cells (MSCs) alters depending on cell culture conditions. Overall, our data provide an overview of the cell-specific characteristics of sEV populations, which will increase the understanding of sEV physiology and improve the development of new sEV-based therapeutic approaches
The multiple functions of miR-574-5p in the neuroblastoma tumor microenvironment
Neuroblastoma is the most common extracranial solid tumor in childhood and arises from neural crest cells of the developing sympathetic nervous system. Prostaglandin E2 (PGE2) has been identified as a key pro-inflammatory mediator of the tumor microenvironment (TME) that promotes neuroblastoma progression. We report that the interaction between the microRNA miR-574-5p and CUG-binding protein 1 (CUGBP1) induces the expression of microsomal prostaglandin E2 synthase 1 (mPGES-1) in neuroblastoma cells, which contributes to PGE2 biosynthesis. PGE2 in turn specifically induces the sorting of miR-574-5p into small extracellular vesicles (sEV) in neuroblastoma cell lines. sEV are one of the major players in intercellular communication in the TME. We found that sEV-derived miR-574-5p has a paracrine function in neuroblastoma. It acts as a direct Toll-like receptor 7/8 (TLR7/8) ligand and induces α-smooth muscle actin (α-SMA) expression in fibroblasts, contributing to fibroblast differentiation. This is particularly noteworthy as it has an opposite function to that in the TME of lung carcinoma, another PGE2 dependent tumor type. Here, sEV-derived miR-574-5p has an autokrine function that inhibits PGE2 biosynthesis in lung cancer cells. We report that the tetraspanin composition on the surface of sEV is associated with the function of sEV-derived miR-574-5p. This suggests that the vesicles do not only transport miRs, but also appear to influence their mode of action
Plasma lipid profiles discriminate bacterial from viral infection in febrile children
Fever is the most common reason that children present to Emergency Departments. Clinical signs and symptoms suggestive of bacterial infection are often non-specific, and there is no definitive test for the accurate diagnosis of infection. The 'omics' approaches to identifying biomarkers from the host-response to bacterial infection are promising. In this study, lipidomic analysis was carried out with plasma samples obtained from febrile children with confirmed bacterial infection (n = 20) and confirmed viral infection (n = 20). We show for the first time that bacterial and viral infection produces distinct profile in the host lipidome. Some species of glycerophosphoinositol, sphingomyelin, lysophosphatidylcholine and cholesterol sulfate were higher in the confirmed virus infected group, while some species of fatty acids, glycerophosphocholine, glycerophosphoserine, lactosylceramide and bilirubin were lower in the confirmed virus infected group when compared with confirmed bacterial infected group. A combination of three lipids achieved an area under the receiver operating characteristic (ROC) curve of 0.911 (95% CI 0.81 to 0.98). This pilot study demonstrates the potential of metabolic biomarkers to assist clinicians in distinguishing bacterial from viral infection in febrile children, to facilitate effective clinical management and to the limit inappropriate use of antibiotics
Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines
The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points
Standardisierte mikroRNA-Analytik — die große Chance für Biomarkerstudien?
Circulating microRNAs (miRs) represent promising diagnostic and prognostic biomarkers for various diseases. Despite the high number of biomarker studies, the results of these studies are hardly reproducible. This makes it difficult to transfer the results into clinical application. In this context, acceptance criteria for the quantification of miRs by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) may help to standardize miR analysis and improve reproducibility
- …