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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200070	1/2	Homo sapiens	primary CD14+ cells	(d)(U)C	Russell, Ashley	2022	67%
Study summary Full title Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication All authors Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication Journal Toxicol Lett. Abstract Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contribu (show more...)Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contributor to many health problems. Cigarette smoking can reduce immune system functioning while concurrently increasing inflammation. Dendritic cells in the lung exposed to cigarette smoke become stimulated and go on to activate T-cells. Extracellular vesicles (EVs) are nano-sized particles released by cells. They participate in intercellular communication by transferring functional proteins and nucleic acids to recipient cells and have been implicated in immune responses. For example, they can display MHC-peptide complexes to activate T-cells. In the current study, we sought to understand the role of cigarette smoke extract (CSE) on dendritic cell-derived EVs and their capacity to activate and differentiate T-cells. Primary human dendritic cells (iDCs) were exposed to CSE and EVs were separated and characterized. We exposed autologous primary CD4 + T-cells to iDC-EVs and observed T helper cell populations skewing towards Th1 and Th17 phenotypes. As HIV + individuals are disproportionately likely to be current smokers, we also examined the effects of iDC-EVs on acutely infected T-cells as well as on a cell model of HIV latency (ACH-2). We found that in most cases, iDC-CSE EVs tended to reduce p24 release from the acutely infected primary T-cells, albeit with great variability. We did not observe large effects of iDC-EVs or direct CSE exposure on p24 release from the ACH-2 cell line. Together, these data suggest that iDC-CSE EVs have the capacity to modulate the immune responses, in part by pushing T-cells towards Th1 and Th17 phenotypes. (hide) EV-METRIC 67% (94th 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 Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle 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 Protein markers EV: CD81/ TSG101/ CD63/ Syntenin non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary CD14+ cells EV-harvesting Medium Serum free medium 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) 90 Pelleting: rotor type AH-629 (36 ml) Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Not detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Calnexin/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 245.6 nm EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 6.89E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200070	2/2	Homo sapiens	primary CD14+ cells	(d)(U)C	Russell, Ashley	2022	67%
Study summary Full title Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication All authors Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication Journal Toxicol Lett. Abstract Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contribu (show more...)Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contributor to many health problems. Cigarette smoking can reduce immune system functioning while concurrently increasing inflammation. Dendritic cells in the lung exposed to cigarette smoke become stimulated and go on to activate T-cells. Extracellular vesicles (EVs) are nano-sized particles released by cells. They participate in intercellular communication by transferring functional proteins and nucleic acids to recipient cells and have been implicated in immune responses. For example, they can display MHC-peptide complexes to activate T-cells. In the current study, we sought to understand the role of cigarette smoke extract (CSE) on dendritic cell-derived EVs and their capacity to activate and differentiate T-cells. Primary human dendritic cells (iDCs) were exposed to CSE and EVs were separated and characterized. We exposed autologous primary CD4 + T-cells to iDC-EVs and observed T helper cell populations skewing towards Th1 and Th17 phenotypes. As HIV + individuals are disproportionately likely to be current smokers, we also examined the effects of iDC-EVs on acutely infected T-cells as well as on a cell model of HIV latency (ACH-2). We found that in most cases, iDC-CSE EVs tended to reduce p24 release from the acutely infected primary T-cells, albeit with great variability. We did not observe large effects of iDC-EVs or direct CSE exposure on p24 release from the ACH-2 cell line. Together, these data suggest that iDC-CSE EVs have the capacity to modulate the immune responses, in part by pushing T-cells towards Th1 and Th17 phenotypes. (hide) EV-METRIC 67% (94th 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 Cell culture supernatant Sample origin cells exposed to cigarette smoke extract Focus vesicles extracellular vesicle 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 Protein markers EV: TSG101/ CD81/ CD63/ Syntenin non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary CD14+ cells EV-harvesting Medium Serum free medium 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) 90 Pelleting: rotor type AH-629 (36 ml) Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ Syntenin/ CD81 Not detected EV-associated proteins TSG101 Detected contaminants Calnexin/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 252.45 nm EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 8.51E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV200070
species	Homo sapiens
sample type	Cell culture
cell type	primary CD14+ cells
condition	Control condition	cells exposed to cigarette smoke extract
separation protocol	(d)(U)C	(d)(U)C
Exp. nr.	1	2
EV-METRIC %	67	67