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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170031	1/1	Homo sapiens	Serum	(d)(U)C Filtration	Ramanathan S	2019	44%
Study summary Full title Exosome microRNA signatures in patients with complex regional pain syndrome undergoing plasma exchange. All authors Ramanathan S, Douglas SR, Alexander GM, Shenoda BB, Barrett JE, Aradillas E, Sacan A, Ajit SK Journal J Transl Med Abstract BACKGROUND: Therapeutic plasma exchange (PE) or plasmapheresis is an extracorporeal procedure employ (show more...)BACKGROUND: Therapeutic plasma exchange (PE) or plasmapheresis is an extracorporeal procedure employed to treat immunological disorders. Exosomes, nanosized vesicles of endosomal origin, mediate intercellular communication by transferring cargo proteins and nucleic acids and regulate many pathophysiological processes. Exosomal miRNAs are potential biomarkers due to their stability and dysregulation in diseases including complex regional pain syndrome (CRPS), a chronic pain disorder with persistent inflammation. A previous study showed that a subset of CRPS patients responded to PE. METHODS: As a proof-of-concept, we investigated the PE-induced exosomal miRNA changes in six CRPS patients. Plasma cytokine levels were measured by HPLC and correlated with miRNA expression. Luciferase assay following co-transfection of HEK293 cells with target 3'UTR constructs and miRNA mimics was used to evaluate miRNA mediated gene regulation of target mRNA. Transient transfection of THP-1 cells with miRNA mimics followed by estimation of target gene and protein expression was used to validate the findings. RESULTS: Comparison of miRNAs in exosomes from the serum of three responders and three poor-responders showed that 17 miRNAs differed significantly before and after therapy. Of these, poor responders had lower exosomal hsa-miR-338-5p. We show that miR-338-5p can bind to the interleukin 6 (IL-6) 3' untranslated region and can regulate IL-6 mRNA and protein levels in vitro. PE resulted in a significant reduction of IL-6 in CRPS patients. CONCLUSIONS: We propose that lower pretreatment levels of miR-338-5p in poor responders are linked to IL-6 levels and inflammation in CRPS. Our data suggests the feasibility of exploring exosomal miRNAs as a strategy in patient stratification for maximizing therapeutic outcome of PE. (hide) EV-METRIC 44% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Complex regional pain syndrome Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 142.9 (pelleting) / 142.9 (washing) Protein markers EV: CD81/ CD63 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 142.9 Wash: time (min) 70 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 110000 Wash: adjusted k-factor 142.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD63 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 85.7 EV concentration Yes Particle yield 394000000000 EM EM-type Transmission-EM/ Immune-EM EM protein CD81 Image type Close-up, Wide-field

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EV-TRACK ID	EV170031
species	Homo sapiens
sample type	Serum
condition	Complex regional pain syndrome
separation protocol	(d)(U)C Filtration
Exp. nr.	1
EV-METRIC %	44