Gabriella Pocsfalvi

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

EVpedia: a community web portal for extracellular vesicles research

MOTIVATION Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. AVAILABILITY AND IMPLEMENTATION The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.

Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine

Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.

A multiplex quantitative proteomics strategy for protein biomarker studies in urinary exosomes

Urinary exosomes have received considerable attention as a potential biomarker source for the diagnosis of renal diseases. Notwithstanding, their use in protein biomarker research is hampered by the lack of efficient methods for vesicle isolation, lysis, and protein quantification. Here we report an improved ultracentrifugation-based method that facilitates the solubilization and removal of major impurities associated with urinary exosomes. A double-cushion sucrose/D(2)O centrifugation step was used after a two-step differential centrifugation to separate exosomes from the heavier vesicles. After the removal of uromodulin, 378 and 79 unique proteins were identified, respectively, in low- and high-density fractions. Comparison of our data with two previously published data sets helped to define proteins commonly found in urinary exosomes. Lysis, protein extraction, and in-solution digestion of exosomes were then optimized for MudPIT application. More than a hundred exosomal proteins were quantified by four-plex iTRAQ analysis of single and pooled samples from two different age groups. For healthy men, six proteins (TSN1, PODXL, IDHC, PPAP, ACBP, and ANXA5) showed significant expression differences between exosome pools of those aged 25-50 and 50-70 years old. Thus, exosomes isolated by our method provide the basis for the development of robust quantitative methods for protein biomarker research.

Proteomic analysis of exoproteins expressed by enterotoxigenic Staphylococcus aureus strains

Pathogenic bacteria excrete a variety of virulence factors into extracellular medium and to the cell surface which have essential roles in the colonization and insurrection of the host cells, and thus reflect the degree of bacterial pathogenicity. For the exploration of virulence factors expressed in the secreted proteome fraction, different Staphylococcus aureus strains were analyzed using gel‐based bottom‐up proteomic approach. A total of 119 distinct proteins were identified for the enterotoxin gene cluster (egc) negative and seb gene positive S. aureus American Type Culture Collection (ATCC) 14458 strain by the use of one‐ and 2‐DE based proteomics. Detailed analysis of enterotoxin region of the 2‐D map confirmed, beside the highly expressed staphylococcal enterotoxin B (SEB), the presence of enterotoxin‐like proteins SElK and SElQ previously predicted by genotyping (Sergeev et al.., J. Clin. Microbiol. 2004, 42, 2134–2143). Exoprotein patterns at the late‐exponential (7 h) and stationary (24 h) phases of cellular growth show a high‐level similarity in this region. Comparative analysis of enterotoxin region of five S. aureus strains including two clinical isolates (RIMD 31092 and A900322), a food derived strain (AB‐8802) with highly prevalent egc positive operon and a nonenterotoxigenic reference strain (ROS) revealed the presence of different known enterotoxins and other virulence factors along with a number of core exoproteins. In addition, production of SElL (RIMD 31092) and SElP (A900322) was demonstrated for the first time at the protein level. Under the experimental conditions applied none of the enterotoxins encoded by the genes of egc operon was identified.

Mass spectrometric and linear discriminant analysis of N-glycans of human serum alpha-1-acid glycoprotein in cancer patients and healthy individuals

N-glycan oligosaccharides of human serum alpha(1)-acid glycoprotein (AGP) samples isolated from 43 individuals (healthy individuals and patients with lymphoma and with ovarian tumor) were analyzed by MALDI-TOF mass spectrometry and a multivariate statistical method (linear discriminant analysis, LDA). 34 different glycan structures have been identified. From the glycosylation pattern determined by mass spectrometry fucosylation and branching indices have been calculated. These parameters show only small differences between the patient groups studied, but these differences are not sufficiently large to use as a potential biomarker. LDA analysis, on the other hand shows a very good separation between the three groups (with a classification of 88%). Cross-validation indicates that the method has predictive power: Identifying cancerous vs. healthy individuals shows 96% selectivity and 93% specificity; identification of lymphoma vs. the mixed group of healthy and ovarian tumor cases is also promising (72% selectivity and 84% specificity). The pilot study presented here demonstrates that mass spectrometry combined with linear discriminant analysis (LDA) may provide valuable data for identifying and studying the pathophysiology of malignant diseases.

Phosphorylation of B14.5a Subunit from Bovine Heart Complex I Identified by Titanium Dioxide Selective Enrichment and Shotgun Proteomics

Shotgun proteomics was used to study the steady phosphorylation state of NADH:ubiquinone oxidoreductase (complex I) subunits from bovine heart mitochondria. A total tryptic digestion of enzymatically active complex I was performed, and the resulting peptide mixture was subjected to phosphopeptide enrichment by the use of titanium dioxide (TiO2). The phosphopeptide-enriched fraction was separated and analyzed with nanoscale reverse-phase HPLC-ESI-MS/MS in single information-dependent acquisition. Hence two phosphorylated complex I subunits were detected: 42 kDa and B14.5a. Phosphorylation of 42-kDa subunit at Ser-59 has already been determined with fluorescent phosphoprotein-specific gel staining and mass spectrometry (Schilling, B., Aggeler, R., Schulenberg, B., Murray, J., Row, R. H., Capaldi, R. A., and Gibson, B. W. (2005) Mass spectrometric identification of novel phosphorylation site in subunit NDUFA10 of bovine mitochondrial complex I. FEBS Lett. 579, 2485–2490). In our work, this finding was confirmed using a non-gel-based approach. In addition, we report novel phosphorylation on B14.5a nuclear encoded subunit. We demonstrated evidence of the phosphorylation site at Ser-95 residue by collision-induced dissociation experiments on three different molecular ions of two tryptic phosphopeptides of B14.5a.

Coupling Immunomagnetic Separation on Magnetic Beads with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Detection of Staphylococcal Enterotoxin B

ABSTRACT The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low-nanogram levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB antibody on the surface of para-toluene-sulfonyl-functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly “on beads” by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, “off beads” after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of Staphylococcus aureus strains and raw milk samples.

Mass spectrometry of extracellular vesicles

The review briefly summaries main features of extracellular vesicles, a joint terminology for exosomes, microvesicles, and apoptotic vesicles. These vesicles are in the center of interest in biology and medical sciences, and form a very active field of research. Mass spectrometry (MS), with its specificity and sensitivity, has the potential to identify and characterize molecular composition of these vesicles; but as yet there are only a limited, but fast-growing, number of publications that use MS workflows in this field. MS is the major tool to assess protein composition of extracellular vesicles: qualitative and quantitative proteomics approaches are both reviewed. Beside proteins, lipid and metabolite composition of vesicles might also be best assessed by MS techniques; however there are few applications as yet in this respect. The role of alternative analytical approaches, like gel-based proteomics and antibody-based immunoassays, are also mentioned. The objective of the review is to give an overview of this fast-growing field to help orient MS-based research on extracellular vesicles.

Observation of Non-covalent Interactions Between Beauvericin and Oligonucleotides Using Electrospray Ionization Mass Spectrometry

Electrospray ionization mass spectrometry has been used to study the possible non-covalent interaction between oligonucleotides and beauvericin (B) mycotoxin. Beauvericin-oligonucleotide adduct formation was observed even at low mycotoxin concentration (25 pmol/microL). Adducts were found with different numbers of B ligands attached. The selectivity of binding of B ligands to two different oligonucleotides has been shown to be similar indicating that beauvericin does not have a strongly preferred base sequence or base site in the DNA. In a competitive complexation reaction, beauvericin forms specific adducts with oligonucleotide while another mycotoxin, nigeromicin, which causes apoptosis without fragmentation of DNA, does not.