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Introduction Recurrent pregnancy loss (RPL), defined as two or more consecutive pregnancy losses in the first trimester, affects around 5% of fertile women. The underlying causes remain unknown in up to 60% of patients; however, most studies point at an immunological pathology in unexplained RPL, and therefore, an effective treatment may be immunomodulatory. This study aims to evaluate the effect of intravenous immunoglobulin (IVIg) and prednisolone on reproductive outcome and the immune system in women with unexplained RPL undergoing assisted reproductive technology treatment.Methods and analysis This randomised, placebo-controlled trial with double-blinded randomisation to two parallel arms evaluate if immunomodulatory (active) treatment is superior to placebo in increasing the chance of ongoing pregnancy assessed at nuchal translucency scan in gestational weeks (GW) 11–13 after embryo transfer (ET) in 74 RPL patients with ≥2 pregnancy losses as its primary objective. The active treatment consists of IVIg (one infusion preferably 1–5 days before ET and in GW 5, 6 and 7) and prednisolone (5 mg/day from first day of menstrual bleeding until ET and 10 mg/day from ET to GW 8+0) while the comparator consists of intravenous human albumin (5%) and placebo tablets. Allocation is concealed for participants, caregivers, and investigators until trial termination and is performed in a 1:1 ratio. The secondary objective is to evaluate treatment safety, and the tertiary objective is exploration of the association between treatment, reproductive outcome after ET, and the lymphocyte subset distribution in peripheral blood collected before and after intravenous infusion(s). Excess biological material is stored in a biobank for future research.Ethics and dissemination The North Denmark Region Committee on Health Research Ethics (N-20200066) approved this trial. The results will be published in peer-reviewed scientific journals and presented to relevant patient associations, at relevant academic conferences and to key stakeholders.Trial registration number NCT04701034.

Abstract Plasma extracellular vesicle (EV) number and composition are altered following myocardial infarction (MI), but to properly understand the significance of these changes it is essential to appreciate how the different isolation methods affect EV characteristics, proteome and sphingolipidome. Here, we compared plasma EV isolated from platelet‐poor plasma from four healthy donors and six MI patients at presentation and 1‐month post‐MI using ultracentrifugation (UC), polyethylene glycol precipitation, acoustic trapping, size‐exclusion chromatography (SEC) and immunoaffinity capture. The isolated EV were evaluated by Nanoparticle Tracking Analysis (NTA), Western blot, transmission electron microscopy (TEM), an EV‐protein array, untargeted proteomics (LC‐MS/MS) and targeted sphingolipidomics (LC‐MS/MS). The application of the five different plasma EV isolation methods in patients presenting with MI showed that the choice of plasma EV isolation method influenced the ability to distinguish elevations in plasma EV concentration following MI, enrichment of EV‐cargo (EV‐proteins and sphingolipidomics) and associations with the size of the infarct determined by cardiac magnetic resonance imaging 6 months post‐MI. Despite the selection bias imposed by each method, a core of EV‐associated proteins and lipids was detectable using all approaches. However, this study highlights how each isolation method comes with its own idiosyncrasies and makes the comparison of data acquired by different techniques in clinical studies problematic.

Background and aims: OxLDL modulates innate and adaptive immunity, and extracellular vesicles (EVs) released from both non-immune and immune cells are proposed key players in atherosclerosis development. In the present study, we aimed to investigate EVs expressing markers related to adaptive immunity-driven inflammation and endothelial activation/dysfunction in hypercholesterolemic patients. Methods: EVs were phenotyped in thirty patients with familial hypercholesterolemia (FH) and twenty-three healthy controls using the Extracellular Vesicle (EV) Array with antibodies targeting proteins expressed on B and T cells, and endothelial cells. Results: FH patients had a higher atherosclerotic burden, as determined by the mean carotid intima-media thickness (IMT) (0.64 ± 0.12 mm vs. 0.58 ± 0.07 mm; p = 0.033), higher oxLDL levels (p

Background: Blood–barrier (BBB) breakdown and active inflammation are hallmarks of relapsing multiple sclerosis (RMS), but the molecular events contributing to the development of new lesions are not well explored. Leaky endothelial junctions are associated with increased production of endothelial-derived extracellular microvesicles (EVs) and result in the entry of circulating immune cells into the brain. MRI with intravenous gadolinium (Gd) can visualize acute blood–barrier disruption as the initial event of the evolution of new lesions. Methods: Here, weekly MRI with Gd was combined with proteomics, multiplex immunoassay, and endothelial stress-optimized EV array to identify early markers related to BBB disruption. Five patients with RMS with no disease-modifying treatment were monitored weekly using high-resolution 3T MRI scanning with intravenous gadolinium (Gd) for 8 weeks. Patients were then divided into three groups (low, medium, or high MRI activity) defined by the number of new, total, and maximally enhancing Gd-enhancing lesions and the number of new FLAIR lesions. Plasma samples taken at each MRI were analyzed for protein biomarkers of inflammation by quantitative proteomics, and cytokines using multiplex immunoassays. EVs were characterized with an optimized endothelial stress EV array based on exosome surface protein markers for the detection of soluble secreted EVs. Results: Proteomics analysis of plasma yielded quantitative information on 208 proteins at each patient time point (n = 40). We observed the highest number of unique dysregulated proteins (DEPs) and the highest functional enrichment in the low vs. high MRI activity comparison. Complement activation and complement/coagulation cascade were also strongly overrepresented in the low vs. high MRI activity comparison. Activation of the alternative complement pathway, pathways of blood coagulation, extracellular matrix organization, and the regulation of TLR and IGF transport were unique for the low vs. high MRI activity comparison as well, with these pathways being overrepresented in the patient with high MRI activity. Principal component analysis indicated the individuality of plasma profiles in patients. IL-17 was upregulated at all time points during 8 weeks in patients with high vs. low MRI activity. Hierarchical clustering of soluble markers in the plasma indicated that all four MRI outcomes clustered together with IL-17, IL-12p70, and IL-1β. MRI outcomes also showed clustering with EV markers CD62E/P, MIC A/B, ICAM-1, and CD42A. The combined cluster of these cytokines, EV markers, and MRI outcomes clustered also with IL-12p40 and IL-7. All four MRI outcomes correlated positively with levels of IL-17 (p < 0.001, respectively), and EV-ICAM-1 (p < 0.0003, respectively). IL-1β levels positively correlated with the number of new Gd-enhancing lesions (p < 0.01), new FLAIR lesions (p < 0.001), and total number of Gd-enhancing lesions (p < 0.05). IL-6 levels positively correlated with the number of new FLAIR lesions (p < 0.05). Random Forests and linear mixed models identified IL-17, CCL17/TARC, CCL3/MIP-1α, and TNF-α as composite biomarkers predicting new lesion evolution. Conclusions: Combination of serial frequent MRI with proteome, neuroinflammation markers, and protein array data of EVs enabled assessment of temporal changes in inflammation and endothelial dysfunction in RMS related to the evolution of new and enhancing lesions. Particularly, the Th17 pathway and IL-1β clustered and correlated with new lesions and Gd enhancement, indicating their importance in BBB disruption and initiating acute brain inflammation in MS. In addition to the Th17 pathway, abundant protein changes between MRI activity groups suggested the role of EVs and the coagulation system along with innate immune responses including acute phase proteins, complement components, and neutrophil degranulation.

Extracellular vesicles (EVs) have become a topic of interest within the field of diagnostic biomarkers; however, recent developments in the study of EVs have increased the need for simpler but still comprehensive methods for characterization. Here, we describe how to simultaneously measure several surface or surface-associated proteins on EVs using a multiparametric microarray-based analysis termed Extracellular Vesicle Array (EV Array), which is developed to catch and phenotypically characterize small EVs. Previously, this analysis has been limited to measuring only one fluorescent signal per analysis. The analysis relies on antibodies printed onto a solid surface, for catching the EVs carrying the specific surface or surface-associated proteins, and on the subsequent fluorescent detection. For the optimization of detection, two antibodies with attached Cy3 or Cy5 were added to various combinations of the EV surface or surface-associated proteins: CD9, CD63, CD81, flotillin-1, and HSP90. In this study, the EV surface or surface-associated proteins were analyzed in human plasma from six healthy subjects. Changes observed in signal intensities from Cy3 and Cy5 related specifically to these combinations and allowed for a comparison of the two different fluorescent signals. When comparing the results, it was observed that it is possible to measure the EV surface or surface-associated proteins at both 532 nm (Cy3) and 635 nm (Cy5) simultaneously without a significant change in signals from the detection molecules. This allows us to measure multiple EV marker proteins in a single analysis, thereby more quickly finding complex biomarker patterns in a sample.

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.

Potato late blight is one the most important crop diseases worldwide. Even though potato has been studied for many years, the potato disease late blight still has a vast negative effect on the potato production [1–3]. Late blight is caused by the pathogen Phytophthora infestans (P. infestans), which initiates infection through leaves. However, the biological activities during different stages of infection are poorly described, and could enable novel or improved ways of defeating late blight infection [4]. Therefore, we investigated the interactions between P. infestans (mixed strain culture) and potato (Solanum tuberosum). Three commercially available field potato cultivars of different resistance to late blight infection; Kuras (moderate), Sarpo Mira (highly resistant) and Bintje (very susceptable) were grown under controlled green house conditions and inoculated with a diversity of P. infestans populations.We used label-free quantitative proteomics to investigate the infection with P. infestans in a time-course study over 258 h. Several key issues limits proteome analysis of potato leaf tissue [5–7]. Firstly, the immense complexity of the plant proteome, which is further complicated by the presence of highly abundant proteins, such as ribulose bisphosphate carboxylase/oxygenase (RuBisCO). Secondly, plant leaf and potato, in particular, contain abundant levels amounts of phenols and polyphenols, which hinder or completely prevent a successful protein extraction. Hitherto, protein profiling of potato leaf tissues have been limited to few proteome studies and only 1484 proteins have been extracted and comprehensively described [5,8,9]. We here present the detailed methods and raw data by optimized gel-enhanced label free quantitative approach. The methodology enabled us to detect and quantify between 3248 and 3529 unique proteins from each cultivar, and up to 758 P. infestans derived proteins. The complete dataset is available via ProteomeXchange, with the identifier http://www.ebi.ac.uk/pride/archive/projects/PXD002767.

The majority of patients diagnosed with rheumatoid arthritis (RA) have developed autoantibodies against neoepitopes in proteins that have undergone post-translational modification, e.g., citrullination or carbamylation. There is growing evidence of their molecular relevance and their potential utility to improve diagnosis, patient stratification, and prognosis for precision medicine. Autoantibodies reacting to native proteins may also have a role in RA pathogenesis, however, their reactivity patterns remain much less studied. We hypothesized that a high-density protein array technology could shed light onto the normal and disease-related autoantibodies produced in healthy and RA patient subgroups. In an exploratory study, we investigated the global reactivity of autoantibodies in plasma pools from 15 anti-cyclic citrullinated peptide (CCP)-positive and 10 anti-CCP-negative RA patients and 10 healthy donors against more than 1600 native and unmodified human proteins using a high-density protein array. A total of 102 proteins recognized by IgG autoantibodies were identified, hereof 86 were recognized by antibodies from CCP-positive RA patients and 76 from anti-CCP-negative RA patients, but not by antibodies from healthy donors. Twenty-four of the identified autoantigens have previously been identified in synovial fluid. Multiple human proteins in their native conformation are recognized by autoantibodies from anti-CCP-positive as well as anti-CCP-negative RA patients.

Extracellular vesicles (EVs) and exosomes are difficult to enrich or purify from biofluids, hence quantification and phenotyping of these are tedious and inaccurate. The multiplexed, highly sensitive and high-throughput platform of the EV Array presented by Jørgensen et al., (J Extracell Vesicles, 2013; 2: 10) has been refined regarding the capabilities of the method for characterization and molecular profiling of EV surface markers. Here, we present an extended microarray platform to detect and phenotype plasma-derived EVs (optimized for exosomes) for up to 60 antigens without any enrichment or purification prior to analysis.

Extracellular vesicles (EVs) are involved in inter-cellular communication and their cargo may provide prognostic/diagnostic biomarkers. To discover EV-associated biomarkers for Multiple Sclerosis (MS), we used an immune marker array to identify surface proteins on circulating EVs that differ between MS patients and controls (n = 3 each). We identified toll-like receptor-3 (TLR3) as a potential target for further validation. We utilized prospectively collected serum from relapsing-remitting MS patients (n = 18) and controls (n = 16) and confirmed lower concentration of TLR3 and higher concentration of mechanistically related TLR4 in MS EVs compared to controls. Future studies may further evaluate the utility of EV-associated TLRs as MS biomarkers and uncover their mechanistic significance.

Appropriate and well-documented in vitro cell-culturing systems are necessary to study the activity and biological function of extracellular vesicles (EVs). The aim of this study was to describe an experimental system, in which dynamic, vesicle-based cell communication can be investigated. A commercially available cell-culturing system was applied to study contact-independent cell communication, which separated two cell populations using a membrane with a pore size of 0.4 μm. The EV exchange characteristics between the two compartments in the culture set-up was preliminarily investigated in a cell-free set-up, and analysed using the Extracellular Vesicle (EV) Array and Nanoparticle Tracking Analysis. The application of the cell-culturing set-up was demonstrated using co-cultures of human primary cells. The effects of the relative placement of the two cell populations on the phenotype of EVs found in the cell supernatant were investigated. The results indicate that this placement can be important for the biological hypothesis that is being investigated. These observations are relevant for short (

Extracellular vesicles (EVs) have a demonstrated involvement in modulating the immune system. It has been proposed that EVs could be used as biomarkers for detection of inflammatory and immunological disorders. Consequently, it is of great interest to investigate EVs in more detail with focus on immunological markers. In this study, five major leukocyte subpopulations and the corresponding leukocyte-derived EVs were phenotyped with focus on selected immunological lineage-specific markers and selected vesicle-related markers. The leukocyte-derived EVs displayed phenotypic differences in the 34 markers investigated. The majority of the lineage-specific markers used for identification of the parent cell types could not be detected on EVs released from monocultures of the associated cell types. In contrast, the vesicular presentation of CD9, CD63, and CD81 correlated to the cell surface expression of these markers, however, with few exceptions. Furthermore, the cellular expression of CD9, CD63, and CD81 varied between leukocytes present in whole blood and cultured leukocytes. In summary, these data demonstrate that the cellular and vesicular presentation of selected lineage-specific and vesicle-related markers may differ, supporting the accumulating observations that sorting of molecular cargo into EVs is tightly controlled.

Extracellular vesicles (EVs) are one of several tools that cells use to communicate with each other. This communication is facilitated by a number of surface-associated proteins and the cargo of the vesicles. For several cancer types, the amount of EVs is observed to be up-regulated in patients compared to healthy individuals, possibly signifying the presence of an aberrant process. The hypoxia-induced release of EVs from cancer cells has been hypothesized to cause the malignant transformation of healthy recipient cells. In this study, the phenotype of cells and EVs from the ovarian cancer cell lines, COV504, SKOV3, and Pt4, were quantified and analysed under normoxic and hypoxic conditions. It was shown that both cells and EVs express common markers and that the EV phenotype varies more than the cellular phenotype. Additionally, cells subjected to 24 hours of hypoxia compared to normoxia produced more EVs. The phenotyping of EVs from cancer cell lines provides information about their molecular composition. This information may be translated to knowledge regarding the functionality of EVs and lead to a better understanding of their role in cancer.

Appropriate and well-documented in vitro cell-culturing systems are necessary to study the activity and biological function of extracellular vesicles (EVs). The aim of this study was to describe an experimental system, in which dynamic, vesicle-based cell communication can be investi‐ gated. A commercially available cell-culturing system was applied to study contact-independent cell communication, which separated two cell populations using a membrane with a pore size of 0.4 μm. The EV exchange characteristics between the two compartments in the culture set-up was preliminarily investigated in a cell-free set-up, and ana‐ lysed using the Extracellular Vesicle (EV) Array and Nanoparticle Tracking Analysis. The application of the cell- culturing set-up was demonstrated using co-cultures of human primary cells. The effects of the relative placement of the two cell populations on the phenotype of EVs found in the cell supernatant were investigated. The results indicate that this placement can be important for the biological hypothesis that is being investigated. These observations are relevant for short (

Extracellular vesicles (EVs) are one of several tools that cells use to communicate with each other. This communication is facilitated by a number of surface-associated proteins and the cargo of the vesicles. For several cancer types, the amount of EVs is observed to be up-regulated in patients com‐ pared to healthy individuals, possibly signifying the presence of an aberrant process. The hypoxia-induced release of EVs from cancer cells has been hypothesized to cause the malignant transformation of healthy recipient cells. In this study, the phenotype of cells and EVs from the ovarian cancer cell lines, COV504, SKOV3, and Pt4, were quantified and analysed under normoxic and hypoxic conditions. It was shown that both cells and EVs express common markers and that the EV phenotype varies more than the cellular phenotype. Additionally, cells subjected to 24 hours of hypoxia compared to normoxia produced more EVs. The phenotyping of EVs from cancer cell lines provides information about their molecular composition. This information may be translated to knowledge regarding the functionality of EVs and lead to a better understanding of their role in cancer.