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.

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.

Intravenous immunoglobulin and prednisolone to women with unexplained recurrent pregnancy loss after assisted reproductive technology treatment: a protocol for a randomised, double-blind, placebo-controlled trial.

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.

[Myocarditis and development of dilated cardiomyopathy].

Inflammation is increasingly recognised as a causal factor in the development and progression of cardiovascular disease. With the introduction of immune checkpoint inhibitors in oncology and the ongoing COVID-19 pandemic the role of the immune system in myocardial inflammation (myocarditis) and subsequent inflammatory cardiomyopathy has once again regained attention. In this review, we want to bring myocardial inflammation to the clinician's attention and provide up-to-date knowledge on its diagnostic workup, prognostication, and current management recommendations.

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.

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.

Protein array-based companion diagnostics in precision medicine.

INTRODUCTION: The development of companion diagnostics (CDx) will increase efficacy and cost-benefit markedly, compared to the currently prevailing trial-and-error approach for treatment. Recent improvements in high-throughput protein technology have resulted in large amounts of predictive biomarkers that are potentially useful components of future CDx assays. Current high multiplex protein arrays are suitable for discovery-based approaches, while low-density and more simple arrays are suitable for use in point-of-care facilities. AREA COVERED: This review discusses the technical platforms available for protein array focused CDx, explains the technical details of the platforms and provide examples of clinical use, ranging from multiplex arrays to low-density clinically applicable arrays. We thereafter highlight recent predictive biomarkers within different disease areas, such as oncology and autoimmune diseases. Lastly, we discuss some of the challenges connected to the implementation of CDx assays as point-of-care tests. EXPERT OPINION: Recent advances in the field of protein arrays have enabled high-density arrays permitting large biomarker discovery studies, which are beneficial for future CDx assays. The density of protein arrays range from a single protein to proteome-wide arrays, allowing the discovery of protein signatures that may correlate with drug response. Protein arrays will undoubtedly play a key role in future CDx assays.

Extracellular vesicle-associated proteins as potential biomarkers.

Every cell in the body secretes extracellular vesicles (EVs) possibly as cellular signaling components and these cell-derivatives can be found in multiple numbers in biological fluids. EVs have in the scientific field received great attention in relation to pathophysiology and disease diagnostics. Altered protein expressions associated with circulating EVs in diseased individuals can serve as biomarkers for different disease states. This capacity paves the way for non-invasive screening tools and early diagnostic markers. However, no isolation method of EVs has been acknowledged as the "golden standard," thus reproducibility of the studies remains inadequate. Increasing interest in EV proteins as disease biomarkers could give rise to more scientific knowledge with diagnostic applicability. In this chapter, studies of proteins believed to be associated with EVs within cancer, autoimmunity, metabolic and neurodegenerative diseases have been outlined.

Identification of Novel Native Autoantigens in Rheumatoid Arthritis.

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.

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.

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.

Induction of a Regulatory Phenotype in CD3+ CD4+ HLA-DR+ T Cells after Allogeneic Mixed Lymphocyte Culture; Indications of Both Contact-Dependent and -Independent Activation.

Although the observation of major histocompatibility complex II (MHCII) receptors on T cells is longstanding, the explanation for this occurrence remains enigmatic. Reports of an inducible, endogenous expression exist, as do studies demonstrating a protein acquisition from other cells by mechanisms including vesicle transfer. Irrespective of origin, the presence of the human MHCII isotype, human leukocyte antigen DR (HLA-DR), potentially identifies a regulatory T cell population. Using an allogeneic mixed lymphocyte culture (MLC) to induce an antigen-specific immune response, the role of antigen-presenting cells (APCs) for the presence of HLA-DR on cluster of differentiation 3(CD3)+ CD4+ T cells was evaluated. Moreover, a functional phenotype was established for these T cells. It was demonstrated that APCs were essential for HLA-DR on CD3+ CD4+ T cells. Additionally, a regulatory T cell phenotype was induced in CD3+ CD4+ HLA-DR+ responder T cells with an expression of CD25, CTLA-4, CD62L, PD-1, and TNFRII. This phenotype was induced both with and without physical T cell:APC contact, which could reveal novel indications about its functionality. To further investigate contact-independent communication, a phenotype of the small cell-derived vesicles from the MLCs was determined. Yet heterogeneous, this vesicle phenotype displayed contact-dependent differences, providing clues about their intended function in cellular communication.

Multiplexed Phenotyping of Small Extracellular Vesicles Using Protein Microarray (EV Array).

The Extracellular Vesicle (EV) Array is based on the technology of protein microarray and provides the opportunity to detect and phenotype small EVs from unpurified starting material in a high-throughput manner (Jørgensen et al., J Extracell vesicles 2:1-9, 2013). The technology was established to perform multiplexed phenotyping of EVs in an open platform. This protocol outlines the microarray printing procedure followed by the steps of capture and detection of small extracellular vesicles from plasma/serum or cell culture supernatants. The principles of data treatment and analysis are thoroughly described as well.