BackgroundUnderstanding the pathogenic role of extracellular vesicles (EVs) in disease and their potential diagnostic and therapeutic utility is extremely reliant on in-depth quantification, measurement and identification of EV sub-populations. Quantification of EVs has presented several challenges, predominantly due to the small size of vesicles such as exosomes and the availability of various technologies to measure nanosized particles, each technology having its own limitations.Materials and MethodsA standardized methodology to measure the concentration of extracellular vesicles (EVs) has been developed and tested. The method is based on measuring the EV concentration as a function of a defined size range. Blood plasma EVs are isolated and purified using size exclusion columns (qEV) and consecutively measured with tunable resistive pulse sensing (TRPS). Six independent research groups measured liposome and EV samples with the aim to evaluate the developed methodology. Each group measured identical samples using up to 5 nanopores with 3 repeat measurements per pore. Descriptive statistics and unsupervised multivariate data analysis with principal component analysis (PCA) were used to evaluate reproducibility across the groups and to explore and visualise possible patterns and outliers in EV and liposome data sets.ResultsPCA revealed good reproducibility within and between laboratories, with few minor outlying samples. Measured mean liposome (not filtered with qEV) and EV (filtered with qEV) concentrations had coefficients of variance of 23.9% and 52.5%, respectively. The increased variance of the EV concentration measurements could be attributed to the use of qEVs and the polydisperse nature of EVs.ConclusionThe results of this study demonstrate the feasibility of this standardized methodology to facilitate comparable and reproducible EV concentration measurements.
IntroductionDendritic cells (DCs) are key regulators of adaptive immunity by selectively promoting or suppressing T-cell responses. 1 One of the suppressive mechanisms involves the expression of the enzyme indoleamine 2,3-dioxygenase (IDO) by DCs. 2 IDO degrades the essential amino acid tryptophan into kynurenine, which leads to tryptophan depletion resulting in suppression of T-cell proliferation 3-5 or induction of apoptosis in activated T cells both in vitro and in vivo, 6 and, consequently, the induction of tolerance. 7,8 IDO can be induced in DCs by a variety of stimuli, including ligation of CD40 or CD80/CD86 by, respectively, CD40L 3,9,10 or CTLA-4 11,12 on activated T cells, as well as soluble factors such as IFN-␥ and IL-1 (reviewed in Mellor and Munn 2 ). Some other factors, such as LPS, require additional signals such as IFN-␥ to effectively induce IDO in DCs. 3,13 Remarkably, the conditions resulting in the expression of anti-inflammatory IDO also result in the expression of proinflammatory cytokines.NF-B transcription factors are essential for the expression of proinflammatory cytokines in DCs 14 and have been implicated in IDO induction. 15 NF-B can be activated via 2 distinct signal transduction pathways. The canonical (also known as classical) NF-B pathway requires activation of the IKK complex, consisting of the catalytic subunits IKK␣ and IKK, and the regulatory subunit NEMO/IKK␥, and controls NF-B activation in response to proinflammatory stimuli such as LPS, TNF␣, and CD40L. [16][17][18][19] Activation of this pathway results predominantly in the activation, nuclear translocation, and DNA binding of the classical NF-B dimer p50-RelA. In this pathway, IKK is essential for NF-B activation, whereas IKK␣ is dispensable for the activation and induction of NF-B DNA-binding activity in most cell types. [19][20][21] In contrast, the noncanonical (also known as alternative) pathway is strictly dependent on IKK␣ homodimers and requires neither IKK nor NEMO/IKK␥. 22,23 The target for IKK␣ homodimers is NF-B2/p100, which upon activation of IKK␣ by NF-B-inducing kinase (NIK) is incompletely degraded into p52, resulting in the release and nuclear translocation of mainly p52-RelB dimers. This pathway can be triggered by the activation of members of the TNF-receptor superfamily such as the lymphotoxin  receptor, B-cell activating factor belonging to the TNF family (BAFF) receptor, and CD40 (which also induce canonical NF-B signaling), but not via pattern recognition receptors such as Toll-like receptor 4 (TLR4), the receptor for LPS. 24 It has been suggested that the canonical and noncanonical NF-B pathways play distinct roles in immunity (reviewed in Bonizzi and Karin 25 ). Recent literature proposes a role for the noncanonical pathway in the regulation of immune responses, as IKK␣ is implicated in the negative regulation of inflammation 26,27 and NIK has a role in the development of regulatory T cells (Tregs). 28 In addition, it has been demonstrated that IKK␣ has an important function in thymic organo...
IntroductionThe size of extracellular vesicles (EVs) can be determined with a tunable resistive pulse sensor (TRPS). Because the sensing pore diameter varies from pore to pore, the minimum detectable diameter also varies. The aim of this study is to determine and improve the reproducibility of TRPS measurements.MethodsExperiments were performed with the qNano system (Izon) using beads and a standard urine vesicle sample. With a combination of voltage and stretch that yields a high blockade height, we investigate whether the minimum detected diameter is more reproducible when we configure the instrument targeting (a) fixed stretch and voltage, or (b) fixed blockade height.ResultsDaily measurements with a fixed stretch and voltage (n=102) on a standard urine sample show a minimum detected vesicle diameter of 128±19 nm [mean±standard deviation; coefficient of variation (CV) 14.8%]. The vesicle concentration was 2.4·109±3.8·109 vesicles/mL (range 1.4·108–1.8·1010). When we compared setting a fixed stretch and voltage to setting a fixed blockade height on 3 different pores, we found a minimum detected vesicle diameter of 118 nm (CV 15.5%, stretch), and 123 nm (CV 4.5%, blockade height). The detected vesicle concentration was 3.2–8.2·108 vesicles/mL with fixed stretch and 6.4–7.8·108 vesicles/mL with fixed blockade height.Summary/conclusionPore-to-pore variability is the cause of the variation in minimum detected size when setting a fixed stretch and voltage. The reproducibility of the minimum detectable diameter is much improved by setting a fixed blockade height.
Dendritic cells (DC) are the only antigen-presenting cells for naive T cells and, therefore, they are crucial players in the initiation of immune responses. Because DC maturation and cytokine production are NF-jB dependent, we hypothesized that blocking NF-jB activity in DC by selectively targeting the inhibitor of jB (IjB) kinase (IKK) complex using the novel NF-jB inhibitor NEMO-binding domain (NBD) peptide could inhibit DC maturation and other functional characteristics, resulting in modulation of the immune response. We used human monocyte-derived DC to test the biological effects of the NBD peptide in vitro. NF-jB inhibition by the NBD peptide resulted in blockade of IKKmediated IjBa phosphorylation and subsequent nuclear translocation and DNA binding of NF-jB p65 in DC. In addition, IL-6, IL-12, and TNF-a production was dosedependently blocked and NBD peptide treatment also led to a strong reduction of LPSinduced maturation. Functional analysis of these DC showed marked inhibition of T cell proliferation in the allogeneic mixed lymphocyte reaction, accompanied by less Th1 and Th2 polarization. The current study reveals for the first time the unique properties of this novel, highly specific NF-jB inhibitor in DC. Also, these data indicate that the NBD peptide could be used as an elegant tool in DC based immunotherapy for unwanted cellular immune responses.
We investigated the contribution of the intracellular calcium (Ca transients (Indo-1 fluorescence) were recorded from single isolated rabbit SAN cells, whereas intracellular cAMP content was measured in SAN cell suspensions using a cAMP assay (LANCE Ò
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