New Enhancing MRI Lesions Associate with IL-17, Neutrophil Degranulation and Integrin Microparticles: Multi-Omics Combined with Frequent MRI in Multiple Sclerosis.

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-1ß. 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-1ß 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-1ß 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.

Increased extracellular vesicles (EVs) related to T cell-mediated inflammation and vascular function in familial hypercholesterolemia.

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 < 0.0001), and showed increased levels of EV-specific markers: CD9 (p = 0.017), CD63 (p = 0.045), CD81 (p = 0.003), Annexin V (p = 0.018), and EV markers related to adaptive/lymphocyte immunity: CD28 (p = 0.034), CD4 (p = 0.049), CD152 (p = 0.029), LFA-1 (p = 0.024), and endothelial function: CD62E (p = 0.032), CD144 (p = 0.018), tPA (p = 0.017), CD31 (p = 0.024). Linear regression revealed a positive relationship between carotid IMT and several of the increased markers observed within the FH group, including CD9 (ß = 0.33; p = 0.022), CD63 (ß = 0.35; p 225 = 0.026), CD28 (ß = 0.37; p = 0.026), CD4 (ß = 0.40; p = 0.025), CD152 (ß = 0.41; p = 0.017), LFA-1 (ß = 0.42; p = 0.014) and CD62E (ß = 0.38; p = 0.024). Conclusion: EVs associated with adaptive immunity and endothelial dysfunction are elevated in FH patients, and several markers related to a higher atherosclerotic burden.

Hepatocyte-derived biomarkers predict liver-related events at 2 years in Child-Pugh class A alcohol-related cirrhosis.

BACKGROUND & AIMS: In patients with compensated alcohol-related cirrhosis, reliable prognostic biomarkers are lacking. Keratin-18 and hepatocyte-derived large extracellular vesicle (lEV) concentrations reflect disease activity, but their ability to predict liver-related events is unknown. METHODS: We measured plasma keratin-18 and hepatocyte lEV concentrations in 500 patients with Child-Pugh class A alcohol-related cirrhosis. The ability of these hepatocyte-derived biomarkers, alone or combined with model for end-stage liver disease (MELD) and FibroTest scores, to predict liver-related events at 2 years was analyzed, taking into account the alcohol consumption at inclusion and during follow-up. RESULTS: Keratin-18 and hepatocyte lEV concentrations increased with alcohol consumption. In patients without active alcohol consumption at enrollment (n = 419), keratin-18 concentration predicted liver-related events at 2 years, independently of FibroTest and MELD. Patients with both keratin-18 concentrations >285 U/L and FibroTest >0.74 had a 24% cumulative incidence of liver-related events at 2 years, vs. 5% to 14% in other groups of patients. Similar results were obtained when combining keratin-18 concentrations >285 U/L with MELD >10. In patients with active alcohol consumption at enrollment (n = 81), hepatocyte lEVs predicted liver-related events at 2 years, independently of FibroTest and MELD. Patients with both hepatocyte lEV concentrations >50 U/L and FibroTest >0.74 had a 62% cumulative incidence of liver-related events at 2 years, vs. 8% to 13% in other groups of patients. Combining hepatocyte lEV concentrations >50 U/L with MELD >10 had a lower discriminative ability. Similar results were obtained when using decompensation of cirrhosis, defined according to Baveno VII criteria, as an endpoint. CONCLUSION: In patients with Child-Pugh class A alcohol-related cirrhosis, combining hepatocyte-derived biomarkers with FibroTest or MELD scores identifies patients at high risk of liver-related events, and could be used for risk stratification and patient selection in clinical trials. IMPACT AND IMPLICATIONS: In patients with compensated alcohol-related cirrhosis, reliable predictors of outcome are lacking. In patients with Child-Pugh class A alcohol-related cirrhosis, combining hepatocyte-derived biomarkers (keratin-18 and hepatocyte-large extracellular vesicles) with FibroTest or MELD scores identifies those at high risk of liver-related events at 2 years. The identified patients at high risk of liver-related events are the target-of-choice population for intensive surveillance (e.g., referral to tertiary care centers; intensive control of risk factors) and inclusion in clinical trials.

Surface proteome of plasma extracellular vesicles as mechanistic and clinical biomarkers for malaria.

PURPOSE: Malaria is a life-threatening mosquito-borne disease caused by Plasmodium parasites, mainly in tropical and subtropical countries. Plasmodium falciparum (P. falciparum) is the most prevalent cause on the African continent and responsible for most malaria-related deaths globally. Important medical needs are biomarkers for disease severity or disease outcome. A potential source of easily accessible biomarkers are blood-borne small extracellular vesicles (sEVs). METHODS: We performed an EV Array to find proteins on plasma sEVs that are differentially expressed in malaria patients. Plasma samples from 21 healthy subjects and 15 malaria patients were analyzed. The EV array contained 40 antibodies to capture sEVs, which were then visualized with a cocktail of biotin-conjugated CD9, CD63, and CD81 antibodies. RESULTS: We detected significant differences in the protein decoration of sEVs between healthy subjects and malaria patients. We found CD106 to be the best discrimination marker based on receiver operating characteristic (ROC) analysis with an area under the curve of > 0.974. Additional ensemble feature selection revealed CD106, Osteopontin, CD81, major histocompatibility complex class II DR (HLA-DR), and heparin binding EGF like growth factor (HBEGF) together with thrombocytes to be a feature panel for discrimination between healthy and malaria. TNF-R-II correlated with HLA-A/B/C as well as CD9 with CD81, whereas Osteopontin negatively correlated with CD81 and CD9. Pathway analysis linked the herein identified proteins to IFN-γ signaling. CONCLUSION: sEV-associated proteins can discriminate between healthy individuals and malaria patients and are candidates for future predictive biomarkers. TRIAL REGISTRATION: The trial was registered in the Deutsches Register Klinischer Studien (DRKS-ID: DRKS00012518).

Rapid neutrophil mobilization by VCAM-1+ endothelial cell-derived extracellular vesicles.

AIMS: Acute myocardial infarction rapidly increases blood neutrophils (<2 h). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 h after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 min) release of extracellular vesicles (EVs), which have emerged as an important means of cell-cell signalling and are thus a potential mechanism for communicating with remote tissues. METHODS AND RESULTS: Here, we show that injury to the myocardium rapidly mobilizes neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma-EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilized splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing, we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilization of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. CONCLUSIONS: Our findings show a novel EV-dependent mechanism for the rapid mobilization of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.

The Prognostic Value of Plasma Small Extracellular Vesicles' Phenotype in Patients With Gastrointestinal Stromal Tumor.

BACKGROUND/AIM: For patients with local gastrointestinal stromal tumor (GIST), risk stratification is used to assess the prognosis and identify patients to offer adjuvant treatment. For patients with advanced or metastatic GIST, no such risk stratification exists. This study aimed to investigate the prognostic value of 31 different plasma small extracellular vesicles' (SEVs) surface proteins in GIST patients. MATERIALS AND METHODS: GIST patients from the two sarcoma centers in Denmark were included. Patients were divided into three groups; group 1: patients undergoing radical surgery; group 2: patients with local, locally advanced, or metastatic GIST; and group 3: patients without evidence of disease after radical surgery. Protein microarray technology was used for the analysis of plasma SEVs. The median plasma SEV marker level was used when comparing groups of patients. The primary endpoint was the progression of GIST. Iterative statistical modeling was used to identify a SEV marker profile/model with a prognostic value. RESULTS: A total of 157 patients were included, with a median follow-up time of 2.05 years. In group 2, a high level of carcinoembryonic antigen (CEA) and a low level of glucose transporter 1 (GLUT-1) were found to be poor prognostic factors [univariate analysis; GLUT-1: hazard ratio (HR)=0.47, 95% confidence interval (CI)=0.22-0.98; CEA: HR=2.12, 95%CI=1.02-4.44]. Composing a model consisting of CEA and GLUT-1 adjusted for age at inclusion was found to have a prognostic value (HR=4.93, 95%CI=2.30-10.57, p<0.0001). CONCLUSION: Plasma SEVs in GIST showed that CEA and GLUT-1 might be of prognostic value. However, external validation is needed.

Photometric method for dual targeting of surface and surface-associated proteins on extracellular vesicles in the multiparametric test.

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.

Profiling Blood Serum Extracellular Vesicles in Plaque Psoriasis and Psoriatic Arthritis Patients Reveals Potential Disease Biomarkers.

Psoriasis vulgaris (PsV) and psoriatic arthritis (PsA) are inflammatory diseases with unresolved pathophysiological aspects. Extracellular vesicles (EVs) play an important role in intercellular communication. We compared the miRNA contents and surface proteome of the EVs in the blood serum of PsV and PsA patients to healthy controls. Size-exclusion chromatography was used to isolate EVs from the blood serum of 12 PsV patients, 12 PsA patients and 12 healthy control subjects. EV samples were characterized and RNA sequencing was used to identify differentially enriched EV-bound miRNAs. We found 212 differentially enriched EV-bound miRNAs present in both PsV and PsA groups-a total of 13 miRNAs at FDR ≤ 0.05. The predicted target genes of these miRNAs were significantly related to lesser known but potentially disease-relevant pathways. The EV array revealed that PsV patient EV samples were significantly enriched with CD9 EV-marker compared to controls. Analysis of EV-bound miRNAs suggests that signaling via EVs in the blood serum could play a role in the pathophysiological processes of PsV and PsA. EVs may be able to fill the void in clinically applicable diagnostic and prognostic biomarkers for PsV and PsA.

The Role of Plasma Extracellular Vesicles in Remote Ischemic Conditioning and Exercise-Induced Ischemic Tolerance.

Ischemic conditioning and exercise have been suggested for protecting against brain ischemia-reperfusion injury. However, the endogenous protective mechanisms stimulated by these interventions remain unclear. Here, in a comprehensive translational study, we investigated the protective role of extracellular vesicles (EVs) released after remote ischemic conditioning (RIC), blood flow restricted resistance exercise (BFRRE), or high-load resistance exercise (HLRE). Blood samples were collected from human participants before and at serial time points after intervention. RIC and BFRRE plasma EVs released early after stimulation improved viability of endothelial cells subjected to oxygen-glucose deprivation. Furthermore, post-RIC EVs accumulated in the ischemic area of a stroke mouse model, and a mean decrease in infarct volume was observed for post-RIC EVs, although not reaching statistical significance. Thus, circulating EVs induced by RIC and BFRRE can mediate protection, but the in vivo and translational effects of conditioned EVs require further experimental verification.

Comparative and integrated analysis of plasma extracellular vesicle isolation methods in healthy volunteers and patients following myocardial infarction.

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.

Extracellular Vesicles: An Important Biomarker in Recurrent Pregnancy Loss?

Recurrent pregnancy loss (RPL) has an estimated incidence of 1-3% of all couples. The etiology is considered to be multifactorial. Extracellular vesicles (EVs) take part in numerous different physiological processes and their contents show the originating cell and pathophysiological states in different diseases. In pregnancy disorders, changes can be seen in the composition, bioactivity and concentration of placental and non-placental EVs. RPL patients have an increased risk of pregnancy complications. The aim of this prospective study was to examine whether measuring different specific EV markers in plasma before and during pregnancy could be used as predictors of pregnancy loss (PL) in women with RPL. Thirty-one RPL patients were included in this study; 25 had a live birth (LB group) and six had a new PL (PL group). Five blood samples were obtained, one before achieved pregnancy and the others in gestational week 6, 8, 10 and 16. Moreover, some of the patients received intravenous immunoglobulin (IVIG) infusions as part of treatment, and it was also examined whether this treatment influenced the EV levels. Seventeen EV markers specific for the immune system, coagulation, placenta and hypoxia were analyzed in the samples with EV Array, a method able to capture small EVs by using an antibody panel targeting membrane proteins. Comparing the LB and PL groups, one EV marker, CD9, showed a significant increase from before pregnancy to gestational week 6 in the PL group. The changes in the other 16 markers were nonsignificant. One case of late-onset PL showed steeply increasing levels, with sudden decrease after gestational week 10 in nine of 17 markers. Moreover, there was an overall increase of all 17 markers after IVIG treatment in the LB group, which was significant in 15 of the markers. Whether increases in EVs positive for CD9 characterize RPL patients who subsequently miscarry should be investigated in future larger studies.

Optimization of High-Throughput Multiplexed Phenotyping of Extracellular Vesicles Performed in 96-Well Microtiter Plates.

Extracellular vesicles (EVs) are promising biomarkers for several diseases, however, no simple and robust methods exist to characterize EVs in a clinical setting. The EV Array analysis is based on a protein microarray platform, where antibodies are printed onto a solid surface that enables the capture of small EVs (sEVs) by their surface or surface-associated proteins. The EV Array analysis was transferred to an easily handled microtiter plate (MTP) format and a range of optimization experiments were performed within this study. The optimization was performed in a comprehensive analytical setup where the focus was on the selection of additives added to spotting-, blocking-, and incubation buffers as well as the storage of printed antibody arrays under different temperatures from one day to 12 weeks. After ending the analysis, the stability of the fluorescent signal was investigated at different storage conditions for up to eight weeks. The various parameters and conditions tested within this study were shown to have a high influence on each other. The reactivity of the spots was found to be preserved for up to 12 weeks when stored at room temperature and using blocking procedure IV in combination with trehalose in the spotting buffer. Similar preservation could be obtained using glycerol or sciSPOT D1 in the spotting buffers, but only if stored at 4 °C after blocking procedure I. Conclusively, it was found that immediate scanning of the MTPs after analysis was not critical if stored dried, in the dark, and at room temperature. The findings in this study highlight the necessity of performing optimization experiments when transferring an established analysis to a new technological platform.

Cardioprotection by remote ischemic conditioning is transferable by plasma and mediated by extracellular vesicles.

BACKGROUND: Remote ischemic conditioning (RIC) by brief periods of limb ischemia and reperfusion protects against ischemia-reperfusion injury. We studied the cardioprotective role of extracellular vesicles (EV)s released into the circulation after RIC and EV accumulation in injured myocardium. METHODS: We used plasma from healthy human volunteers before and after RIC (pre-PLA and post-PLA) to evaluate the transferability of RIC. Pre- and post-RIC plasma samples were separated into an EV enriched fraction (pre-EV + and post-EV +) and an EV poor fraction (pre-EV- and post-EV-) by size exclusion chromatography. Small non-coding RNAs from pre-EV + and post-EV + were purified and profiled by NanoString Technology. Infarct size was compared in Sprague-Dawley rat hearts perfused with isolated plasma and fractions in a Langendorff model. In addition, fluorescently labeled EVs were used to assess homing in an in vivo rat model. (ClinicalTrials.gov, number: NCT03380663) RESULTS: Post-PLA reduced infarct size by 15% points compared with Pre-PLA (55 ± 4% (n = 7) vs 70 ± 6% (n = 8), p = 0.03). Post-EV + reduced infarct size by 16% points compared with pre-EV + (53 ± 15% (n = 13) vs 68 ± 12% (n = 14), p = 0.03). Post-EV- did not affect infarct size compared to pre-EV- (64 ± 3% (n = 15) and 68 ± 10% (n = 16), p > 0.99). Three miRNAs (miR-16-5p, miR-144-3p and miR-451a) that target the mTOR pathway were significantly up-regulated in the post-EV + group. Labelled EVs accumulated more intensely in the infarct area than in sham hearts. CONCLUSION: Cardioprotection by RIC can be mediated by circulating EVs that accumulate in injured myocardium. The underlying mechanism involves modulation of EV miRNA that may promote cell survival during reperfusion.

Molecular characteristics of porcine alpha-synuclein splicing variants.

Alpha-synuclein (α-syn) is a 140 amino acid, intrinsically disordered protein with a potential role in neurotransmitter vesicle release. The protein is natively unfolded under physiological conditions, and is expressed predominantly in neural tissue. α-syn is associated with neuropathological conditions in Parkinson's disease, where the protein misfolds into oligomers and fibrils resulting in aggregates in Lewy bodies. Here we report the molecular cloning of SNCA cDNA encoding porcine α-syn and transcript variants hereof. Six transcripts coding for porcine α-syn are presented in the report, of which three result from exon skipping, generating in-frame splicing of coding exons 3 and 5. The splicing pattern of these alternative spliced variants is conserved between human and pig. All the observed in-frame deletions yield significantly shorter α-syn proteins compared with the 140 amino acid full-length protein. Expression analysis performed by real-time quantitative RT-PCR revealed a differential expression of the six transcript splicing variants in different pig organs and tissues. Common for all splicing variants, a very high transcript expression was detected in brain tissues and in spinal cord and very low or no expression outside the central nervous system. The porcine α-syn protein demonstrated markedly different biophysical characteristics compared with its human counterpart. No fibrillation of porcine α-syn was observed with the pig wild-type α-syn and A30P α-syn, and both variants show significantly reduced ability to bind to lipid vesicles. Overexpression of mutated porcine α-syn might recapitulate the human PD pathogenesis and lead to the identification of genetic modifiers of the disease.

Treatment with intravenous immunoglobulin increases the level of small EVs in plasma of pregnant women with recurrent pregnancy loss.

Extracellular vesicles (EVs), which are small cell-derived compartments, take part in numerous different physiological processes. The contents of EVs reveal the cell of origin and indicates pathophysiological states in different diseases. In pregnancy disorders, changes have been reported in the composition, bioactivity and concentration of placental and non-placental EVs. The purpose of this study was to monitor the effects on EVs in patients receiving intravenous immunoglobulin (IVIG) or placebo (albumin) treatment due to recurrent pregnancy loss (RPL). In a placebo-controlled trial study of IVIG treatment, plasma collected from 39 women with RPL were investigated using the Extracellular Vesicle Array (EV Array). Plasma was sampled consecutively (from gestational week (GW) 5) and the protein phenotypes of the smaller EVs (sEVs) were analyzed for the presence of 34 markers. The levels of sEVs or changes in their levels in early pregnancy were correlated with treatment. There was statistically significant increased levels of sEVs in patients who received IVIG versus placebo. In conclusion, the treatment with high-doses of IVIG clearly boosted the production and release of sEVs to the circulation; however, the biological role of this boost remains to be clarified in further studies.

Blood flow-restricted resistance exercise alters the surface profile, miRNA cargo and functional impact of circulating extracellular vesicles.

Ischemic exercise conducted as low-load blood flow restricted resistance exercise (BFRE) can lead to muscle remodelling and promote muscle growth, possibly through activation of muscle precursor cells. Cell activation can be triggered by blood borne extracellular vesicles (EVs) as these nano-sized particles are involved in long distance signalling. In this study, EVs isolated from plasma of healthy human subjects performing a single bout of BFRE were investigated for their change in EV surface profiles and miRNA cargos as well as their impact on skeletal muscle precursor cell proliferation. We found that after BFRE, five EV surface markers and 12 miRNAs were significantly altered. Furthermore, target prediction and functional enrichment analysis of the miRNAs revealed several target genes that are associated to biological pathways involved in skeletal muscle protein turnover. Interestingly, EVs from BFRE plasma increased the proliferation of muscle precursor cells. In addition, alterations in surface markers and miRNAs indicated that the combination of exercise and ischemic conditioning during BFRE can stimulate blood cells to release EVs. These results support that BFRE promotes EV release to engage in muscle remodelling and/or growth processes.

Individually cultured bovine embryos produce extracellular vesicles that have the potential to be used as non-invasive embryo quality markers.

Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 µl droplets of culture media, and 50 µl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 108/ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 108/ml, p < 0.05). Moreover, at day 8, the concentration of EVs isolated from media conditioned by degenerating embryos (7.17 × 108/ml) was higher compared to that of blastocysts (5.68 × 108/ml, p < 0.05). Furthermore, at day 8, the mean diameter of EVs isolated from media conditioned by degenerating embryos (153.7 nm) was smaller than EVs from media conditioned by blastocysts (163.5 nm, p < 0.05). In conclusion, individually cultured preimplantation bovine embryos secrete EVs in the culture media and their concentration and size are influenced by embryo quality and may indicate their prospective development potential.

Surface Proteome of Plasma Extracellular Vesicles as Biomarkers for Pneumonia and Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

BACKGROUND: Community-acquired pneumonia (CAP) and acute exacerbation of chronic obstructive pulmonary disease (AECOPD) represent a major burden of disease and death and their differential diagnosis is critical. A potential source of relevant accessible biomarkers are blood-borne small extracellular vesicles (sEVs). METHODS: We performed an extracellular vesicle array to find proteins on plasma sEVs that are differentially expressed and possibly allow the differential diagnosis between CAP and AECOPD. Plasma samples were analyzed from 21 healthy controls, 24 patients with CAP, and 10 with AECOPD . The array contained 40 antibodies to capture sEVs, which were then visualized with a cocktail of biotin-conjugated CD9, CD63, and CD81 antibodies. RESULTS: We detected significant differences in the protein decoration of sEVs between healthy controls and patients with CAP or AECOPD. We found CD45 and CD28 to be the best discrimination markers between CAP and AECOPD in receiver operating characteristic analyses, with an area under the curve >0.92. Additional ensemble feature selection revealed the possibility to distinguish between CAP and AECOPD even if the patient with CAP had COPD, with a panel of CD45, CD28, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), tumor necrosis factor-R-II, and CD16. CONCLUSION: The discrimination of sEV-associated proteins is a minimally invasive method with potential to discriminate between CAP and AECOPD.

Altered Levels of Toll-Like Receptors in Circulating Extracellular Vesicles in Multiple Sclerosis.

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.

Platelets, endothelial cells and leukocytes contribute to the exercise-triggered release of extracellular vesicles into the circulation.

Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physical health. Recent work demonstrated that exercise triggers the release of extracellular vesicles (EVs) into the circulation, possibly contributing to exercise-associated adaptive systemic signalling. Circulating EVs comprise a heterogeneous collection of different EV-subclasses released from various cell types. So far, a comprehensive picture of the parental and target cell types, EV-subpopulation diversity and functional properties of EVs released during exercise (ExerVs) is lacking. Here, we performed a detailed EV-phenotyping analysis to explore the cellular origin and potential subtypes of ExerVs. Healthy male athletes were subjected to an incremental cycling test until exhaustion and blood was drawn before, during, and immediately after the test. Analysis of total blood plasma by EV Array suggested endothelial and leukocyte characteristics of ExerVs. We further purified ExerVs from plasma by size exclusion chromatography as well as CD9-, CD63- or CD81-immunobead isolation to examine ExerV-subclass dynamics. EV-marker analysis demonstrated increasing EV-levels during cycling exercise, with highest levels at peak exercise in all EV-subclasses analysed. Phenotyping of ExerVs using a multiplexed flow-cytometry platform revealed a pattern of cell surface markers associated with ExerVs and identified lymphocytes (CD4, CD8), monocytes (CD14), platelets (CD41, CD42, CD62P), endothelial cells (CD105, CD146) and antigen presenting cells (MHC-II) as ExerV-parental cells. We conclude that multiple cell types associated with the circulatory system contribute to a pool of heterogeneous ExerVs, which may be involved in exercise-related signalling mechanisms and tissue crosstalk.