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Peptide gpMBP72–85, containing amino acids 72–85 of guinea pig myelin basic protein is commonly used to induce experimental autoimmune encephalomyelitis in Lewis rats. The N-terminal glutamine in this peptide can cyclize to pyroglutamic acid, leading to loss of the first MHC anchor for binding to MHC class II. Acetylation of the peptide N-terminus prevents pyroglutamic acid formation and ensures a constant quality. An increased MHC binding affinity after N-terminal acetylation was observed. This modification also enhanced T cell proliferation of a gpMBP reactive T cell clone. The encephalitogenicity of peptide gpMBP72–85 was unaffected by acetylation. It is concluded that acetylation improves the chemical stability of gpMBP72–85, and is not detrimental but rather favorable for its biochemical and immunological, in vitro, and in vivo behavior. [Copyright &y& Elsevier]

The major histocompatibility complex (MHC) class II binding requirements for solvent-exposed peptide residues were systematically studied using amino acid and peptoid substitutions. In a peptoid residue, the side chain is present on the backbone nitrogen atom as opposed to the α-carbon atom in an amino acid residue. To investigate the effect of this side chain shifting on MHC binding, three amino acids in the central part of the peptide sticking out of the binding groove were replaced by corresponding peptoid residues. Two peptoid–peptide hybrids showed large affinity decreases in the MHC–peptide binding assay. To investigate this affinity loss, the individual contributions to MHC binding affinity of the side chain (position), the putative hydrogen bond, and the flexibility were dissected. We conclude that the side chain position as well as the backbone nitrogen atom hydrogen bonding features of solvent-exposed residues in the peptide can be important for MHC binding affinity. [Copyright &y& Elsevier]

Recent progress in molecular medicine has provided important tools to identify antigen-specific T cells. In most cases, the approach is based on oligomeric combinations of recombinant major histocompatibility complex-peptide complexes fixed to various rigid supports available for binding by the T-cell receptor. These tools have greatly increased our insight into mechanisms of immune responses mediated by CD8[sup +] T cells. Examples of the diverse fields of application for this technology include immunization, viral infections and oral tolerance induction. [ABSTRACT FROM AUTHOR]

[Display omitted] Communication between cells located in different parts of an organism is often mediated by membrane-enveloped nanoparticles, such as extracellular vesicles (EVs). EV binding and cell uptake mechanisms depend on the heterogeneous composition of the EV membrane. From a colloidal perspective, the EV membrane interacts with other biological interfaces via both specific and non-specific interactions, where the latter include long-ranged electrostatic and van der Waals forces, and short-ranged repulsive "steric-hydration" forces. While electrostatic forces are generally exploited in most EV immobilization protocols, the roles played by various colloidal forces in controlling EV adsorption on surfaces have not yet been thoroughly addressed. In the present work, we study the adsorption of EVs onto supported lipid bilayers (SLBs) carrying different surface charge densities using a combination of quartz crystal microbalance with dissipation monitoring (QCM-D) and confocal laser scanning microscopy (CLSM). We demonstrate that EV adsorption onto lipid membranes can be controlled by varying the strength of electrostatic forces and we theoretically describe the observed phenomena within the framework of nonlinear Poisson-Boltzmann theory. Our modelling results confirm the experimental observations and highlight the crucial role played by attractive electrostatics in EV adsorption onto lipid membranes. They furthermore show that simplified theories developed for model lipid systems can be successfully applied to the study of their biological analogues and provide new fundamental insights into EV-membrane interactions with potential use in developing novel EV separation and immobilization strategies. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) in blood plasma are recognized as potential biomarkers for disease. Although blood plasma is easily obtainable, analysis of EVs at the single particle level is still challenging due to the biological complexity of this body fluid. Besides EVs, plasma contains different types of lipoproteins particles (LPPs), that outnumber EVs by orders of magnitude and which partially overlap in biophysical properties such as size, density and molecular makeup. Consequently, during EV isolation LPPs are often co-isolated. Furthermore, physical EV-LPP complexes have been observed in purified EV preparations. Since co-isolation or association of LPPs can impact EV-based analysis and biomarker profiling, we investigated the presence and formation of EV-LPP complexes in biological samples by using labelfree atomic force microscopy, cryo-electron tomography and synchronous Rayleigh and Raman scattering analysis of optically trapped particles and fluorescence-based high sensitivity single particle flow cytometry. Furthermore, we evaluated the impact on flow cytometric analysis in the presence of LPPs using in vitro spike-in experiments of purified tumour cell line-derived EVs in different classes of purified human LPPs. Based on orthogonal single-particle analysis techniques we demonstrate that EV-LPP complexes can form under physiological conditions. Furthermore, we show that in fluorescence-based flow cytometric EV analysis staining of LPPs, as well as EV-LPP associations, can influence quantitative and qualitative EV analysis. Lastly, we demonstrate that the colloidal matrix of the biofluid in which EVs reside impacts their buoyant density, size and/or refractive index (RI),which may have consequences for down-stream EV analysis and EV biomarker profiling. [ABSTRACT FROM AUTHOR]

Cell-derived extracellular vesicles (EVs) are currently in the limelight as potential disease biomarkers. The promise of EV-based liquid biopsy resides in the identification of specific disease-associated EV signatures. Knowing the reference EV profile of a body fluid can facilitate the identification of such disease-associated EV-biomarkers. With this aim, we purified EVs from paired human milk and serum samples and used the MACSPlex bead-based flow-cytometry assay to capture EVs on bead-bound antibodies specific for a certain surface protein, followed by EV detection by the tetraspanins CD9, CD63, and CD81. Using this approach we identified body fluid-specific EV signatures, e.g. breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, as well as body fluid-specific markers associated to immune cells and stem cells. Interestingly, comparison of pan-tetraspanin detection (simultaneous CD9, CD63 and CD81 detection) and single tetraspanin detection (detection by CD9, CD63 or CD81) also unveiled body fluid-specific tetraspanin distributions on EVs. Moreover, certain EV surface proteins were associated with a specific tetraspanin distribution, which could be indicative of the biogenesis route of this EV subset. Altogether, the identified body fluid-specific EV profiles can contribute to study EV profile deviations in these fluids during disease processes. [ABSTRACT FROM AUTHOR]

Spondyloarthritis (SpA) patients suffer from joint inflammation resulting in tissue damage, characterized by the presence of numerous neutrophils in the synovium and synovial fluid (SF). As it is yet unclear to what extent neutrophils contribute to the pathogenesis of SpA, we set out to study SF neutrophils in more detail. We analyzed the functionality of SF neutrophils of 20 SpA patients and 7 disease controls, determining ROS production and degranulation in response to various stimuli. In addition, the effect of SF on neutrophil function was determined. Surprisingly, our data show that SF neutrophils in SpA patients have an inactive phenotype, despite the presence of many neutrophil-activating stimuli such as GM-CSF and TNF in SF. This was not due to exhaustion as SF neutrophils readily responded to stimulation. Therefore, this finding suggests that one or more inhibitors of neutrophil activation may be present in SF. Indeed, when blood neutrophils from healthy donors were activated in the presence of increasing concentrations of SF from SpA patients, degranulation and ROS production were dose-dependently inhibited. This effect was independent of diagnosis, gender, age, and medication in the patients from which the SF was isolated. Treatment of SF with the enzyme hyaluronidase strongly reduced the inhibitory effect of SF on neutrophil activation, indicating that hyaluronic acid that is present in SF may be an important factor in preventing SF neutrophil activation. This finding provides novel insights into the role of soluble factors in SF regulating neutrophil function and may lead to the development of novel therapeutics targeting neutrophil activation via hyaluronic acid or associated pathways. [ABSTRACT FROM AUTHOR]

Flow cytometry (FCM) offers a multiparametric technology capable of characterizing single extracellular vesicles (EVs). However, most flow cytometers are designed to detect cells, which are larger than EVs. Whereas cells exceed the background noise, signals originating from EVs partly overlap with the background noise, thereby making EVs more difficult to detect than cells. This technical mismatch together with complexity of EV‐containing fluids causes limitations and challenges with conducting, interpreting and reproducing EV FCM experiments. To address and overcome these challenges, researchers from the International Society for Extracellular Vesicles (ISEV), International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH) joined forces and initiated the EV FCM working group. To improve the interpretation, reporting, and reproducibility of future EV FCM data, the EV FCM working group published an ISEV position manuscript outlining a framework of minimum information that should be reported about an FCM experiment on single EVs (MIFlowCyt‐EV). However, the framework contains limited background information. Therefore, the goal of this compendium is to provide the background information necessary to design and conduct reproducible EV FCM experiments. This compendium contains background information on EVs, the interaction between light and EVs, FCM hardware, experimental design and preanalytical procedures, sample preparation, assay controls, instrument data acquisition and calibration, EV characterization, and data reporting. Although this compendium focuses on EVs, many concepts and explanations could also be applied to FCM detection of other particles within the EV size range, such as bacteria, lipoprotein particles, milk fat globules, and viruses. [ABSTRACT FROM AUTHOR]

The molecular signature of cell-derived extracellular vesicles (EVs) from synovial fluid (SF) offers insights into the cells and molecular processes associated with joint disorders and can be exploited to define biomarkers. The EV-signature is determined by cargo molecules and the lesser-studied lipid bilayer. We here investigated the lipidome of SF-EVs in inflamed joints derived from Rheumatoid Arthritis (RA) and Spondyloarthritis (SpA) patients, two autoimmune-driven joint diseases, and compared these signatures to the lipid profile of equine SF-EVs obtained during induced acute synovitis. Since neutrophils are primary SF-infiltrating cells during these inflammatory joint diseases, we also analyzed how inflammatory stimuli alter the lipidomic profile of human and equine neutrophil-derived EVs (nEVs) in vitro and how these signatures relate to the lipidome signatures of SF-EVs from inflamed joints. We identified neutrophil stimulation intensity-dependent changes in the lipidomic profile of nEVs with elevated presence of dihexosylceramide (lactosylceramide), phosphatidylserine, and phosphatidylethanolamine ether-linked lipid classes in human nEVs upon full neutrophil activation. In horses, levels of monohexosylceramide (glucosylceramide) increased instead of dihexosylceramide, indicating species-specific differences. The lipid profiles of RA and SpA SF-EVs were relatively similar and showed a relative resemblance with stimulated human nEVs. Similarly, the lipidome of equine synovitis-derived SF-EVs closer resembled the one of stimulated equine nEVs. Hence, lipidome profiling can provide insights into the contribution of nEVs to the heterogeneous pool of SF-EVs, deepening our understanding of inflammatory joint diseases and revealing molecular changes in joint homeostasis, which can lead to the development of more precise disease diagnosis and treatment strategies. • Neutrophil activation alters the lipid composition of neutrophil-derived EVs. • Human and equine neutrophils and the derived EVs show species-specific lipid shifts. • Neutrophil activation alters the EV lipids, not the overall cellular lipid profile. • Synovial fluid EVs from RA and SpA patients have similar lipid profiles. [ABSTRACT FROM AUTHOR]

Abstract: Small-sized (less than 150nm) long-circulating liposomes (LCL) may be useful as drug-targeting vehicles for anti-inflammatory agents in arthritis, since they selectively home at inflamed joints after i.v. administration. Previously it was shown in experimental arthritis that encapsulation of glucocorticoids (GC) as water-soluble phosphate esters in PEG–liposomes resulted in a strong improvement of the anti-inflammatory effect as compared to the free drug. In the present study, we compared the therapeutic activity and adverse effects induced by 3 different GC encapsulated in LCL in an attempt to further optimize the therapeutic index of liposomal GC in arthritis. Our data showed that with GC (dexamethasone, budesonide) of higher potency than prednisolone, the therapeutic activity of liposomal GC can be increased. However, side effects at the level of body weight and hyperglycemia were noted, related to the sustained free GC level observed after injection of the liposomal GC. An inverse relationship with the clearance rate of the free GC in question was shown. This study stresses the importance of a high clearance rate of the GC to be encapsulated for achieving a maximal therapeutic index with liposomal GC. Therefore high-clearance GC, which until now are only applied in local treatment approaches, may be very useful for the development of novel, highly effective anti-inflammatory preparations for systemic treatment of inflammatory disorders. [Copyright &y& Elsevier]

Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis development and therapy resistance. In response to hypoxia, tumour cells secrete pro‐angiogenic factors to induce blood vessel formation and restore oxygen supply to hypoxic regions. Extracellular vesicles (EVs) are emerging as mediators of intercellular communication in the tumour microenvironment. Here we demonstrate that increased expression of the LC3/GABARAP protein family member GABARAPL1, is required for endosomal maturation, sorting of cargo to endosomes and the secretion of EVs. Silencing GABARAPL1 results in a block in the early endosomal pathway and impaired secretion of EVs with pro‐angiogenic properties. Tumour xenografts of doxycycline inducible GABARAPL1 knockdown cells display impaired vascularisation that results in decreased tumour growth, elevated tumour necrosis and increased therapy efficacy. Moreover, our data show that GABARAPL1 is expressed on the EV surface and targeting GABARAPL1+EVs with GABARAPL1 targeting antibodies results in blockade of pro‐angiogenic effects in vitro. In summary, we reveal that GABARAPL1 is required for EV cargo loading and secretion. GABARAPL1+EVs are detectable and targetable and are therefore interesting to pursue as a therapeutic target. [ABSTRACT FROM AUTHOR]

Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients' cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers.Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk.Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression.Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108-2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4-23.3 ± 10.0 mg/μL in processed milk, P < 0.05).Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. [ABSTRACT FROM AUTHOR]

Maternal milk is nature's first functional food. It plays a crucial role in the development of the infant's gastrointestinal (GI) tract and the immune system. Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer enclosed vesicles released by cells for intercellular communication and are a component of milk. Recently, we discovered that human milk EVs contain a unique proteome compared to other milk components. Here, we show that physiological concentrations of milk EVs support epithelial barrier function by increasing cell migration via the p38 MAPK pathway. Additionally, milk EVs inhibit agonist‐induced activation of endosomal Toll like receptors TLR3 and TLR9. Furthermore, milk EVs directly inhibit activation of CD4+ T cells by temporarily suppressing T cell activation without inducing tolerance. We show that milk EV proteins target key hotspots of signalling networks that can modulate cellular processes in various cell types of the GI tract. [ABSTRACT FROM AUTHOR]

Announcing the ISEV2020 special achievement award recipients: Andrew Hill and Edit Buzás; and the recipient of the ISEV2020 special education award: Carolina Soekmadji The International Society for Extracellular Vesicles (ISEV) is pleased to announce two Special Achievement Awards that were presented at the virtual ISEV2020 annual meeting, 20-22 July 2020. ISEV Special Achievement Awards are given each year to one or more individuals who have made outstanding contributions to the field of extracellular vesicle (EV) research or have performed extraordinary service to ISEV (Witwer, Hill, & Tahara, 2019). For her major contributions to the EV field and to ISEV, Professor Edit I. Buzás was a 2020 recipient of the ISEV Special Achievement Award. [Extracted from the article]

Announcing the ISEV2020 special achievement award recipients: Andrew Hill and Edit Buzás; and the recipient of the ISEV2020 special education award: Carolina Soekmadji The International Society for Extracellular Vesicles (ISEV) is pleased to announce two Special Achievement Awards that were presented at the virtual ISEV2020 annual meeting, 20-22 July 2020. ISEV Special Achievement Awards are given each year to one or more individuals who have made outstanding contributions to the field of extracellular vesicle (EV) research or have performed extraordinary service to ISEV (Witwer, Hill, & Tahara, 2019). For her major contributions to the EV field and to ISEV, Professor Edit I. Buzás was a 2020 recipient of the ISEV Special Achievement Award. [Extracted from the article]

There is an increasing interest in exploring clinically relevant information that is present in body fluids, and extracellular vesicles (EVs) are intrinsic components of body fluids ("liquid biopsies"). In this report, we will focus on blood. Blood contains not only EVs but also cells, and non-EV particles including lipoproteins. Due to the high concentration of soluble proteins and lipoproteins, blood, plasma and serum have a high viscosity and density, which hampers the concentration, isolation and detection of EVs. Because most if not all studies on EVs are single-centre studies, their clinical relevance remains limited. Therefore, there is an urgent need to improve standardization and reproducibility of EV research. As a first step, the International Society on Extracellular Vesicles organized a biomarker workshop in Birmingham (UK) in November 2017, and during that workshop several working groups were created to focus on a particular body fluid. This report is the first output of the blood EV work group and is based on responses by work group members to a questionnaire in order to discover the contours of a roadmap. From the answers it is clear that most respondents are in favour of evidence-based research, education, quality control procedures, and physical models to improve our understanding and comparison of concentration, isolation and detection methods. Since blood is such a complex body fluid, we assume that the outcome of the survey may also be valuable for exploring body fluids other than blood. [ABSTRACT FROM AUTHOR]

Features like small size, low refractive index and polydispersity pose challenges to the currently available detection methods for Extracellular Vesicles (EVs). In addition, the lack of appropriate standards to set up the experimental conditions makes it difficult to compare analyses obtained by different technical approaches. By modifying synthetic nanovesicles with recombinant antigenic regions of EV-enriched tetraspanins, we aimed to construct an EV-mimetic that can be used as a suitable standard for EV analyses. To this end, the sequences of the large extracellular loops of the tetraspanins CD9, CD63 and CD81 were tagged with a target sequence for the biotin ligase BirA, and co-transformed with a BirA expression plasmid into Escherichia coli. GST fusion proteins were then isolated by affinity chromatography and released using thrombin. Biotinylated recombinant tetraspanin-loops were then coupled to (strept)avidin-coated synthetic nanovesicles and analysed and characterised by Dot-blot, Western-blot, Nanoparticle Tracking Analysis, Flow Cytometry and Transmission Electron Microscopy. With this method, we were able to efficiently produce tetraspanin-domain decorated nanovesicles that share biophysical properties with natural EVs, can be detected using specific antibodies against common EV markers such as tetraspanins, and can be used as robust reference materials for detection techniques that are often used in the EV field. [ABSTRACT FROM AUTHOR]

• GABARAPL1 is essential for metastasis development. • GABARAPL1 increases cancer cell migration. • Extracellular vesicles increase cancer cell invasiveness. • GABARAPL1 is essential for extracellular vesicle composition. • GABARAPL1 does not affect extracellular vesicle induced invasion. Hypoxia is a common feature of tumours, associated with poor prognosis due to increased resistance to radio- and chemotherapy and enhanced metastasis development. Previously we demonstrated that GABARAPL1 is required for the secretion of extracellular vesicles (EV) with pro-angiogenic properties during hypoxia. Here, we explored the role of GABARAPL1+ EV in the metastatic cascade. GABARAPL1 deficient or control MDA-MB-231 cells were injected in murine mammary fat pads. Lungs were dissected and analysed for human cytokeratin 18. EV from control and GABARAPL1 deficient cells exposed to normoxia (21% O 2) or hypoxia (O 2 < 0.02%) were isolated and analysed by immunoblot, nanoparticle tracking analysis, high resolution flow cytometry, mass spectrometry and next-generation sequencing. Cellular migration and invasion were analysed using scratch assays and transwell-invasion assays, respectively. The number of pulmonary metastases derived from GABARAPL1 deficient tumours decreased by 84%. GABARAPL1 deficient cells migrate slower but display a comparable invasive capacity. Both normoxic and hypoxic EV contain proteins and miRNAs associated with metastasis development and, in line, increase cancer cell invasiveness. Although GABARAPL1 deficiency alters EV content, it does not alter the EV-induced increase in cancer cell invasiveness. GABARAPL1 is essential for metastasis development. This is unrelated to changes in migration and invasion and suggests that GABARAPL1 or GABARAPL1+ EV are essential in other processes related to the metastatic cascade. [ABSTRACT FROM AUTHOR]

The discovery that extracellular vesicles (EVs) can transfer functional extracellular RNAs (exRNAs) between cells opened new avenues into the study of EVs in health and disease. Growing interest in EV RNAs and other forms of exRNA has given rise to research programmes including but not limited to the Extracellular RNA Communication Consortium (ERCC) of the US National Institutes of Health. In 2017, the International Society for Extracellular Vesicles (ISEV) administered a survey focusing on EVs and exRNA to canvass-related views and perceived needs of the EV research community. Here, we report the results of this survey. Overall, respondents emphasized opportunities for technical developments, unraveling of molecular mechanisms and standardization of methodologies to increase understanding of the important roles of exRNAs in the broader context of EV science. In conclusion, although exRNA biology is a relatively recent emphasis in the EV field, it has driven considerable interest and resource commitment. The ISEV community looks forward to continuing developments in the science of exRNA and EVs, but without excluding other important molecular constituents of EVs. [ABSTRACT FROM AUTHOR]

Several naked virus species, including members of the Picornaviridae family, have recently been described to escape their host cells and spread infection via enclosure in extracellular vesicles (EV). EV are 50–300 nm sized lipid membrane-enclosed particles produced by all cells that are broadly recognized for playing regulatory roles in numerous (patho)physiological processes, including viral infection. Both pro- and antiviral functions have been ascribed to EV released by virus-infected cells. It is currently not known whether this reported functional diversity is a result of the release of multiple virus-containing and non-virus containing EV subpopulations that differ in composition and function. Using encephalomyocarditis virus infection (EMCV, Picornaviridae family), we here provide evidence that EV populations released by infected cells are highly heterogeneous. Virus was contained in two distinct EV populations that differed in physical characteristics, such as sedimentation properties, and in enrichment for proteins indicative of different EV biogenesis pathways, such as the plasma membrane resident proteins Flotillin-1 and CD9, and the autophagy regulatory protein LC3. Additional levels of EV heterogeneity were identified using high-resolution flow cytometric analysis of single EV. Importantly, we demonstrate that EV subsets released during EMCV infection varied largely in potency of transferring virus infection and in their kinetics of release from infected cells. These data support the notion that heterogeneous EV populations released by virus-infected cells can exert diverse functions at distinct time points during infection. Unraveling the compositional, temporal and functional heterogeneity of these EV populations using single EV analysis technologies, as employed in this study, is vital to understanding the role of EV in virus dissemination and antiviral host responses. [ABSTRACT FROM AUTHOR]

The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of intercellular communication. The molecular composition of EV, and thereby their signaling function to target cells, is regulated by cellular activation and differentiation stimuli. EV are regarded as snapshots of cells and are, therefore, in the limelight as biomarkers for disease. Although research on EV-associated RNA has predominantly focused on microRNAs, the transcriptome of EV consists of multiple classes of small non-coding RNAs with potential gene-regulatory functions. It is not known whether environmental cues imposed on cells induce specific changes in a broad range of EV-associated RNA classes. Here, we investigated whether immune-activating or -suppressing stimuli imposed on primary dendritic cells affected the release of various small non-coding RNAs via EV. The small RNA transcriptomes of highly pure EV populations free from ribonucleoprotein particles were analyzed by RNA sequencing and RT-qPCR. Immune stimulus-specific changes were found in the miRNA, snoRNA, and Y-RNA content of EV from dendritic cells, whereas tRNA and snRNA levels were much less affected. Only part of the changes in EV-RNA content reflected changes in cellular RNA, which urges caution in interpreting EV as snapshots of cells. By comprehensive analysis of RNA obtained from highly purified EV, we demonstrate that multiple RNA classes contribute to genetic messages conveyed via EV. The identification of multiple RNA classes that display cell stimulation-dependent association with EV is the prelude to unraveling the function and biomarker potential of these EV-RNAs. [ABSTRACT FROM AUTHOR]

Innate immune cells are recognized for their rapid and critical contribution to the body’s first line of defense against invading pathogens and harmful agents. These actions can be further amplified by specific adaptive immune responses adapted to the activating stimulus. Recently, the awareness has grown that virtually all innate immune cells, i.e., mast cells, neutrophils, macrophages, eosinophils, basophils, and NK cells, are able to communicate with dendritic cells (DCs) and/or T and B cells, and thereby significantly contribute to the orchestration of adaptive immune responses. The means of communication that are thus far primarily associated with this function are cell-cell contacts and the release of a broad range of soluble mediators. Moreover, the possible contribution of innate immune cell-derived extracellular vesicles (EVs) to the modulation of adaptive immunity will be outlined in this review. EVs are submicron particles composed of a lipid bilayer, proteins, and nucleic acids released by cells in a regulated fashion. EVs are involved in intercellular communication between multiple cell types, including those of the immune system. A good understanding of the mechanisms by which innate immune cell-derived EVs influence adaptive immune responses, or vice versa, may reveal novel insights in the regulation of the immune system and can open up new possibilities for EVs (or their components) in controlling immune responses, either as a therapy, target, or as an adjuvant in future immune modulating treatments. [ABSTRACT FROM AUTHOR]

Inflammation is the hallmark of most joint disorders. However, the precise regulation of induction, perpetuation, and resolution of joint inflammation is not entirely understood. Since extracellular vesicles (EVs) are critical for intercellular communication, we aim to unveil their role in these processes. Here, we investigated the EVs' dynamics and phospholipidome profile from synovial fluid (SF) of healthy equine joints and from horses with lipopolysaccharide (LPS)-induced synovitis. LPS injection triggered a sharp increase of SF-EVs at 5-8 h post-injection, which started to decline at 24 h post-injection. Importantly, we identified significant changes in the lipid profile of SF-EVs after synovitis induction. Compared to healthy joint-derived SF-EVs (0 h), SF-EVs collected at 5, 24, and 48 h post-LPS injection were strongly increased in hexosylceramides. At the same time, phosphatidylserine, phosphatidylcholine, and sphingomyelin were decreased in SF-EVs at 5 h and 24 h post-LPS injection. Based on the lipid changes during acute inflammation, we composed specific lipid profiles associated with healthy and inflammatory state-derived SF-EVs. The sharp increase in SF-EVs during acute synovitis and the correlation of specific lipids with either healthy or inflamed states-derived SF-EVs are findings of potential interest for unveiling the role of SF-EVs in joint inflammation, as well as for the identification of EV-biomarkers of joint inflammation. • Acute joint inflammation induces a rapid increase in the number of synovial fluid extracellular vesicles • Induced synovitis results in changes of the phospholipid composition of synovial fluid-derived extracellular vesicles • The phospholipidome profile of extracellular vesicles is dynamic • Extracellular vesicles in healthy and inflammatory state synovial fluid have distinct phospholipid profiles [ABSTRACT FROM AUTHOR]

The discovery that extracellular vesicles (EVs) can transfer functional extracellular RNAs (exRNAs) between cells opened new avenues into the study of EVs in health and disease. Growing interest in EV RNAs and other forms of exRNA has given rise to research programmes including but not limited to the Extracellular RNA Communication Consortium (ERCC) of the US National Institutes of Health. In 2017, the International Society for Extracellular Vesicles (ISEV) administered a survey focusing on EVs and exRNA to canvass-related views and perceived needs of the EV research community. Here, we report the results of this survey. Overall, respondents emphasized opportunities for technical developments, unraveling of molecular mechanisms and standardization of methodologies to increase understanding of the important roles of exRNAs in the broader context of EV science. In conclusion, although exRNA biology is a relatively recent emphasis in the EV field, it has driven considerable interest and resource commitment. The ISEV community looks forward to continuing developments in the science of exRNA and EVs, but without excluding other important molecular constituents of EVs. [ABSTRACT FROM AUTHOR]

Background: To address the lack of standard terminology to describe extracellular RNA (exRNA) data/metadata, we have launched an inter-community effort to extend the Gene Ontology (GO) with subcellular structure concepts relevant to the exRNA domain. By extending GO in this manner, the exRNA data/metadata will be more easily annotated and queried because it will be based on a shared set of terms and relationships relevant to extracellular research. Methods: By following a consensus-building process, we have worked with several academic societies/consortia, including ERCC, ISEV, and ASEMV, to identify and approve a set of exRNA and extracellular vesicle-related terms and relationships that have been incorporated into GO. In addition, we have initiated an ongoing process of extractions of gene product annotations associated with these terms from Vesiclepedia and ExoCarta, conversion of the extracted annotations to Gene Association File (GAF) format for batch submission to GO, and curation of the submitted annotations by the GO Consortium. As a use case, we have incorporated some of the GO terms into annotations of samples from the exRNA Atlas and implemented a faceted search interface based on such annotations. Results: We have added 7 new terms and modified 9 existing terms (along with their synonyms and relationships) to GO. Additionally, 18,695 unique coding gene products (mRNAs and proteins) and 963 unique non-coding gene products (ncRNAs) which are associated with the terms: "extracellular vesicle", "extracellular exosome", "apoptotic body", and "microvesicle" were extracted from ExoCarta and Vesiclepedia. These annotations are currently being processed for submission to GO. Conclusions: As an inter-community effort, we have made a substantial update to GO in the exRNA context. We have also demonstrated the utility of some of the new GO terms for sample annotation and metadata search. [ABSTRACT FROM AUTHOR]

Cell-derived extracellular vesicles (EVs), present in synovial fluid and cartilage extracellular matrix (ECM), are involved in joint development and in the regulation of joint homeostasis. Although the exact function of EVs in these processes remains incompletely defined, the knowledge already acquired in this field suggests a role for these EVs as biomarkers of joint disease, and as a new tool to restore joint homeostasis and enhance articular tissue regeneration. In addition to direct injection of therapeutic EVs into the target site, surface coating of scaffolds and embedding of EVs in hydrogels might also lead to novel therapeutic possibilities. Based on the existing literature of EVs in synovial fluid and articular tissues, and investigation of the molecular factors (including microRNAs) active in joint homeostasis (or during its disturbance), we postulate novel perspectives for the implementation of EVs as a regenerative medicine approach in joint repair. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) represent an important mode of intercellular communication. Research in this field has grown rapidly in the last few years, and there is a plethora of techniques for the isolation and characterization of EVs, many of which are poorly standardized. EVs are heterogeneous in size, origin and molecular constituents, with considerable overlap in size and phenotype between different populations of EVs. Little is known about current practices for the isolation, purification and characterization of EVs. We report here the first large, detailed survey of current worldwide practices for the isolation and characterization of EVs. Conditioned cell culture media was the most widely used material (83%). Ultracentrifugation remains the most commonly used isolation method (81%) with 59% of respondents use a combination of methods. Only 9% of respondents used only 1 characterization method, with others using 2 or more methods. Sample volume, sample type and downstream application all influenced the isolation and characterization techniques employed. [ABSTRACT FROM AUTHOR]

Nano-sized extracelullar vesicles (EVs) released by various cell types play important roles in a plethora of (patho)physiological processes and are increasingly recognized as biomarkers for disease. In addition, engineered EV and EV-inspired liposomes hold great potential as drug delivery systems. Major technologies developed for high-throughput analysis of individual EV include nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (tRPS) and high-resolution flow cytometry (hFC). Currently, there is a need for comparative studies on the available technologies to improve standardization of vesicle analysis in diagnostic or therapeutic settings. We investigated the possibilities, limitations and comparability of NTA, tRPS and hFC for analysis of tumor cell-derived EVs and synthetic mimics (i.e. differently sized liposomes). NTA and tRPS instrument settings were identified that significantly affected the quantification of these particles. Furthermore, we detailed the differences in absolute quantification of EVs and liposomes using the three technologies. This study increases our understanding of possibilities and pitfalls of NTA, tRPS and hFC, which will benefit standardized and large-scale clinical application of (engineered) EVs and EV-mimics in the future. [ABSTRACT FROM AUTHOR]

Secreted membrane‐enclosed vesicles, collectively called extracellular vesicles (EVs), which include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets, encompass a very rapidly growing scientific field in biology and medicine. Importantly, it is currently technically challenging to obtain a totally pure EV fraction free from non‐vesicular components for functional studies, and therefore there is a need to establish guidelines for analyses of these vesicles and reporting of scientific studies on EV biology. Here, the International Society for Extracellular Vesicles (ISEV) provides researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or functions to EVs. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EV) in breast milk carry immune relevant proteins and could play an important role in the instruction of the neonatal immune system. To further analyze these EV and to elucidate their function it is important that native populations of EV can be recovered from (stored) breast milk samples in a reproducible fashion. However, the impact of isolation and storage procedures on recovery of breast milk EV has remained underexposed. Here, we aimed to define parameters important for EV recovery from fresh and stored breast milk. To compare various protocols across different donors, breast milk was spiked with a well‐defined murine EV population. We found that centrifugation of EV down into density gradients largely improved density‐based separation and isolation of EV, compared to floatation up into gradients after high‐force pelleting of EV. Using cryo‐electron microscopy, we identified different subpopulations of human breast milk EV and a not previously described population of lipid tubules. Additionally, the impact of cold storage on breast milk EV was investigated. We determined that storing unprocessed breast milk at −80°C or 4°C caused death of cells present in breast milk, leading to contamination of the breast milk EV population with storage‐induced EV. Here, an alternative method is proposed to store breast milk samples for EV analysis at later time points. The proposed adaptations to the breast milk storage and EV isolation procedures can be applied for EV‐based biomarker profiling of breast milk and functional analysis of the role of breast milk EV in the development of the neonatal immune system. [ABSTRACT FROM AUTHOR]

Abstract: Seminal plasma contains various types of extracellular vesicles, including ‘prostasomes’. Prostasomes are small vesicles secreted by prostatic epithelial cells that can be recruited by and fuse with sperm cells in response of progesterone that is released by oocyte surrounding cumulus cells. This delivers Ca2+ signaling tools that allow the sperm cell to gain hypermotility and undergo the acrosome reaction. Conditions for binding of prostasomes to sperm cells are however unclear. We found that classically used prostasome markers are in fact heterogeneously expressed on distinct populations of small and large vesicles in seminal plasma. To study interactions between prostasomes and spermatozoa we used the stallion as a model organism. A homogeneous population of ~60nm prostasomes was first separated from larger vesicles and labeled with biotin. Binding of biotinylated prostasomes to individual live spermatozoa was then monitored by flow cytometry. Contrary to assumptions in the literature, we found that such highly purified prostasomes bound to live sperm only after capacitation had been initiated, and specifically at pH ≥7.5. Using fluorescence microscopy, we observed that prostasomes bound primarily to the head of live sperm. We propose that in vivo, prostasomes may bind to sperm cells in the uterus, to be carried in association with sperm cells into oviduct and to fuse with the sperm cell only during the final approach of the oocyte. This article is part of a Special Issue entitled: An Updated Secretome. [Copyright &y& Elsevier]

The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the "ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)", 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments. [ABSTRACT FROM AUTHOR]

Professional antigen-presenting cells secrete major histocompatibility complex class II (MHC II) carrying exosomes with unclear physiological function(s). Exosomes are first generated as the intraluminal vesicles (ILVs) of a specific type of multivesicular body, and are then secreted by fusion of this compartment with the plasma membrane. We have previously shown that in contrast to the sorting of MHC II at lysosomally targeted multivesicular bodies, sorting of MHC II into exosomes does not rely on MHC II ubiquitination. In search for proteins that drive the incorporation of MHC II into exosomes or functionally discriminate exosomal from plasma membrane MHC II, we first analyzed the total proteome of highly purified B cell-derived exosomes using sensitive and accurate mass spectrometry (MS), and identified 539 proteins, including known and not previously identified constituents. Using quantitative MS, we then identified a small subset of proteins that were specifically co-immunoprecipitated with MHC II from detergent-solubilized exosomes. These include HSC71, HSP90, 14-3-3ɛ, CD20 and pyruvate kinase type M2 (PKM2), and we speculate on the functionality of their interaction with exosomal MHC II. [ABSTRACT FROM AUTHOR]

In dendritic cells (DC), newly synthesized MHCII is directed to endosomes by its associated invariant chain (Ii). Here, Ii is degraded after which MHCII is loaded with peptides. In immature DC, ubiquitination of peptide-loaded MHCII drives its sorting to lysosomes for degradation. Ubiquitination of MHCII is strongly reduced in response to inflammatory stimuli, resulting in increased expression of MHCII at the plasma membrane. Whether surface exposure of MHCII is also regulated during DC maturation by changing the rate of Ii degradation remained unresolved by conflicting results in the literature. We here pinpoint experimental problems that have contributed to these controversies and demonstrate that immature and mature DC degrade Ii equally efficient at proper culture conditions. Only when DC were cultured in glutamine containing media, endosome acidification and Ii degradation were restricted in immature DC and enhanced in response to lipopolysaccharide (LPS). These effects are caused by ammonia, a glutamine decomposition product. This artificial behavior could be prevented by culturing DC in media containing a stable dipeptide as glutamine source. We conclude that Ii degradation is a prerequisite for but not a rate limiting step in MHCII processing. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) express major histocompatibility complex class II (MHC II) to present peptide antigens to T cells. In immature DCs, which bear low cell surface levels of MHC II, peptide-loaded MHC II is ubiquitinated. Ubiquitination drives the endocytosis and sorting of MHC II to the luminal vesicles of multivesicular bodies (MVBs) for lysosomal degradation. Ubiquitination of MHC II is abrogated in activated DCs, resulting in an increased cell surface expression. We here provide evidence for an alternative MVB sorting mechanism for MHC II in antigen-loaded DCs, which is triggered by cognately interacting antigen-specific CD4+ T cells. At these conditions, DCs generate MVBs with MHC II and CD9 carrying luminal vesicles that are secreted as exosomes and transferred to the interacting T cells. Sorting of MHC II into exosomes was, in contrast to lysosomal targeting, independent of MHC II ubiquitination but rather correlated with its incorporation into CD9 containing detergent-resistant membranes. Together, these data indicate two distinct MVB pathways: one for lysosomal targeting and the other for exosome secretion. [ABSTRACT FROM AUTHOR]

Abstract: Therapeutic intervention in experimental autoimmune diseases by modulation of the T cell mediated autoimmune response has been accomplished in the past using altered peptide ligands (APLs). These peptides are usually created by applying alterations to the T cell epitope recognized by the autoaggressive T cells. In this study, we investigated whether it was possible to design APLs in a rational way, using knowledge of molecular interaction in the MHC–peptide–T cell receptor (TCR) complex, for the therapeutic intervention in experimental autoimmune encephalomyelitis (EAE). Additionally, the value of peptidomimetic modification and alterations based on posttranslational modifications for the design of APLs was examined. Based on a molecular model of the MHC-peptide complex, the T cell receptor contact residues were identified and selected alterations were applied. The designed APLs were tested for MHC binding capacity, T cell recognition, blocking of the autoreactive T cell response, immunogenicity, encephalitogenicity, and therapeutic activity. Based on the results of the in vitro assays, it was expected that some of our APLs would be able to modulate EAE. Nevertheless, none of these APLs displayed clear therapeutic activity in vivo. Thus, rational design of modified peptides for immunotherapy has to await further insights into the relationships between structure and peptide/peptidomimetic induced T cell activation, and until that, there is no possibility to take advantage of the tailor made origin of peptidomimetics. [Copyright &y& Elsevier]

Abstract: The balance between specific and degenerate T cell recognition of MHC class II bound peptides is crucial for T cell repertoire selection, and holds important implications for protective immunity versus autoimmunity. To investigate the degree of degeneracy in T cell recognition, we applied selected modifications to T cell receptor (TCR) contact residue amino acids in the MHC class II bound epitope gpMBP72-85. By using glycosylated amino acids, as an example of a posttranslational modification, large alterations were applied. Small modifications were accomplished by exchanging an arginine residue for a citrulline or an ornithine residue. Finally, the unmodified TCR contact residue side chains were shifted one atom position to the left, using peptoid residues. Both these large and subtle changes in the wild type (WT) peptide caused lack of recognition by WT peptide specific monoclonal and polyclonal T cells. Furthermore, T cells specific for the modified peptides did not cross recognize the WT peptide. Using a set of additional compounds, we investigated the specificity of these T cell populations into detail. Our data reveal a strongly limited plasticity in T cell recognition, and a high specificity for TCR contact residue side chains. [Copyright &y& Elsevier]

Heat shock proteins (hsp) are highly conserved, immune-dominant micro-bial proteins, whose expression is increased at sites of inflammation. In the experi-mental model of adjuvant arthritis (AA) immune responses to hsp determine the out-come of disease. AA can be transferred with a single T cell clone specific for a sequence of mycobacterial hsp65 (Mhsp65). Immunization with whole Mhsp65 on the other hand, protects in virtually all forms of experimental arthritis, including AA. This protective effect seems the consequence of the induction of a T cell response di-rected against self-hsp60. A similar protective effect of self-hsp60-specific T cells seems present in patients with a spontaneous remitting form of juvenile idiopathic ar-thritis. Next to hsp60, other hsp have similar protective effects in arthritis, while oth-er conserved microbial proteins lack such capacity. Nasal administration of hsp60 peptides induces IL-10-driven regulatory T cells that are highly effective in sup-pressing arthritis. Thus hsp60, or peptides derived from hsp60, are suitable candi-dates for immune therapy in chronic arthritis. [ABSTRACT FROM AUTHOR]

Due to the central role of T cells in the pathogenesis of inflammatory diseases of the peripheral nervous system like the Guillain–Barre´ syndrome, specific immunotherapies aim at modifying T cell responses. Use of truncated mutants of the neuritogenic peptide of myelin basic protein (MBP) has been shown to anergize autoreactive T cells and to reverse experimental autoimmune encephalitis (EAE). To establish a rationale basis for the use of altered peptide ligands (APLs) in the treatment of autoimmune diseases we designed a set of N- and C-terminally truncated mutants of the minimal experimental autoimmune neuritis (EAN) inducing bovine P2 (bP2) (60–70) peptide and compared them for the ability to induce immune responses and T cell receptor (TCR) cell signaling. Truncated peptides bound to MHC class II molecules and induced TCR internalization and expression of interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) with decreasing potency. None of the shortened mutants elicited a proliferative response in P2-specific T cells. Stimulation of these antigen-specific T cells with peptide bP2(62–69) using antigen presenting cells (APCs) prepulsed with bP2(60–70) resulted in a significant decrease of the proliferative response. In agreement with the observed effects on T cell activation, analysis of TCR signaling demonstrated a lack of CD3&epsiv; phosphorylation and MAPK activation. Moreover, repeated injection of bP2(62–69) significantly slowed progression of adoptive transfer EAN (AT-EAN). Taken together, these findings strongly suggest that peptide bP2(62–69) can favorably modulate the antigen-induced response of neuritogenic T cells. [Copyright &y& Elsevier]

Adjuvant arthritis (AA) is a T cell mediated disease which can be induced in genetically susceptible rats by immunization with heat-killed Mycobacterium tuberculosis ( Mt ) suspended in incomplete Freund's adjuvant. The critical mycobacterial T cell epitope for the induction of AA was previously identified as residues 178-186 of the mycobacterial 65 kDa heat shock protein ( Mt. hsp65 178-186 ). It was suggested that the development of AA was due to molecular mimicry between a mycobacterial epitope and a cartilage-associated self-antigen. However, until now such cartilage-associated mimicry epitope has not been identified. In this study we designed a computer search profile to predict mimicry self-epitopes, and investigated whether one or more of these self-epitopes could serve as mimicry epitopes in AA. Although several of these self-epitopes were recognized by arthritogenic T cells, no cross-reactivity was found between T cells specific for these self-epitopes and Mt. hsp65 178-186 specific T cells. [ABSTRACT FROM AUTHOR]

The highly conserved 60 kD endogenous heat shock protein (hsp60) has been suggested to be a target for T cell recognition in autoimmune diseases such as type I diabetes. We previously reported cross-recognition of both mycobacterial hsp60 (Mt60) and self hsp60 (m60) by Mt60 immunized NOD mice. To identify the epitopes involved, we generated T cell lines against m60 or its mycobacterial counterpart and tested these lines for recognition of complete sets of overlapping peptides spanning either hsp60 sequence. T cell lines responded to identical regions in the hsp60 proteins, regardless of their degree of conservation or I-Ag7 binding-affinity. Additionally, we determined whether a protective genetic background would affect the presence of hsp60 cross-reactive T cells in the peripheral repertoire by comparing epitope recognition in I-Ag7 expressing NOD, NOD-asp and Biozzi AB/H mice. Two out of five immunodominant murine peptides were able to induce proliferation in NOD and NOD-asp Mt60 T cell lines, but not in Biozzi AB/H T cell lines. Our results point out that Mt60 immunization not necessarily leads to proliferative T cells responding to endogenous hsp60 peptides in the context of diabetes-predisposing I-Ag7. Moreover, the capacity of T cells to respond to self hsp60 is not influenced by the presence of protective I-Ag7asp. Yet, proliferation of hsp60 autoreactive T cells is solely measured in combination with insulitis and as such serves as a surrogate marker for islet inflammation. [Copyright &y& Elsevier]

Neutrophils are abundantly present in the synovium and synovial fluid of patients suffering from arthritis. Neutrophils can be activated by a multitude of stimuli and the current dogma states that this is a two-step process, consisting of a priming step followed by an activation step. Considering that neutrophil activation occurs in an inflammatory environment, where multiple stimuli are present, we argue that a two-step process is highly unlikely. Here, we indeed demonstrate that neutrophils require simultaneous ligation of two different receptors for efficient activation. We isolated human peripheral blood neutrophils and cultured them with various combinations of stimuli (GM-CSF, fMLF, TNF, and LPS). Next, we evaluated essential neutrophil functions, including degranulation and ROS production using flow cytometry, mediator release using ELISA, NETosis by a live cell imaging method, phagocytosis by imaging flow cytometry, and extracellular vesicle (EV) release quantified by high-resolution flow cytometry. Exposure of neutrophils to any combination of stimuli, but not to single stimuli, resulted in significant degranulation, and mediator and EV release. Furthermore, ROS production increased substantially by dual stimulation, yet appeared to be more dependent on the type of stimulation than on dual stimulation. Phagocytosis was induced to its maximum capacity by a single stimulus, while NETosis was not induced by any of the used physiological stimuli. Our data indicate that neutrophil activation is tightly regulated and requires activation by two simultaneous stimuli, which is largely independent of the combination of stimuli. [ABSTRACT FROM AUTHOR]

Adjuvant arthritis (AA) in Lewis rats if T-cell mediated and seems to depend on T cells recognising the 180-188 epitope of mycobacterial heat-shock protein (hsp)60. Analysis of arthritogenic T-cell clone A2b has revealed a mimicry of this particular epitope with an articular cartilage-associated target T-cell epitope. Nasal administration of synthetic peptides covering this 180-188 sequence led to epitope-specific tolerance and resistance to AA. Since this tolerisation protocol also inhibited avridine arthritis, one may conclude that this form of epitope-specific tolerance had effectuated a spreading tolerisation at the level of target antigens that included a diverse set of possible arthritis-associated antigens. In vitro anergised T cells exhibited suppressive activity in a co-culture system. As in this case--depending on the presence of the antigen of the anergic T cell--such T cells suppressed responder T cells of a different antigenic specificity, we postulated that anergic T cells may be responsible for spreading of tolerance. It seemed that such spreading of tolerance was channeled through the antigen-presenting cells (APC) and was dependent on direct cell-cell contact. This and additional forms of spreading of tolerance could be responsible for specific nasal tolerance, causing inhibition of the development of an arthritogenic inflammatory response. This can be similarly the case for the arthritis protection that resulted from immunisation with hsps. Analysis of T-cell responses following hsp immunisations revealed that the arthirits inhibitory activity resided in T cells with specificity for a conserved part of microbial hsp60. The same T cells cross-responded to rat self-hsp60. Low level expression of the latter molecule... [ABSTRACT FROM AUTHOR]

In the past years, extracellular vesicles (EVs) have become an important field of research since EVs have been found to play a central role in biological processes. In pathogens, EVs are involved in several events during the host–pathogen interaction, including invasion, immunomodulation, and pathology as well as parasite–parasite communication. In this report, we summarised the role of EVs in infections caused by viruses, bacteria, fungi, protozoa, and helminths based on the talks and discussions carried out during the International Society for Extracellular Vesicles (ISEV) workshop held in São Paulo (November, 2016), Brazil, entitled Cross-organism Communication by Extracellular Vesicles: Hosts, Microbes and Parasites. [ABSTRACT FROM PUBLISHER]