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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170023	1/5	Haemophilus influenzae	non-typeable Haemophilus influenzae	(d)(U)C Filtration SEC UF	Volgers C	2017	14%
Study summary Full title Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection. All authors Volgers C, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM Journal Scientific Reports Abstract Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations (show more...)Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations and it is estimated that up to 50% of these exacerbations are associated with bacterial infections. The mainstay treatment for these infection-related exacerbations constitutes the administration of glucocorticoids, alone or in combination with antibiotics. A recent line of evidence demonstrates that many hormones including the steroid beclomethasone can also directly affect bacterial growth, virulence, and antibiotic resistance. The effect of these regimens on the release of potentially virulent and toxic membrane vesicles (MVs) is at present unclear. In this study, we determined the effect of several pharmacological agents on MVs release by and bacterial growth of common respiratory pathogens. We found that neither the release of MVs nor the bacterial growth was affected by the glucocorticoids budesonide and fluticasone. The macrolide antibiotic azithromycin only inhibited the growth of Moraxella catarrhalis but no effects were observed on bacterial MV release at a concentration that is achieved locally in the epithelial lining on administration. The macrophage pro-inflammatory response to MVs was significantly reduced after treatment with budesonide and fluticasone but not by azithromycin treatment. Our findings suggest that these glucocorticoids may have a positive effect on infection-related inflammation although the bacterial growth and MV release remained unaffected. (hide) EV-METRIC 14% (44th 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 membrane vesicles 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 SEC UF Protein markers EV: non-typeable Haemophilus influenzae antigen non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Haemophilus influenzae Sample Type Cell culture supernatant EV-producing cells non-typeable Haemophilus influenzae EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) non-typeable Haemophilus influenzae antigen Characterization: Lipid analysis No Characterization: Particle analysis TRPS EV concentration Yes

	EV170023	2/5	Homo sapiens	THP1	(d)(U)C Filtration SEC UF	Volgers C	2017	14%
Study summary Full title Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection. All authors Volgers C, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM Journal Scientific Reports Abstract Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations (show more...)Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations and it is estimated that up to 50% of these exacerbations are associated with bacterial infections. The mainstay treatment for these infection-related exacerbations constitutes the administration of glucocorticoids, alone or in combination with antibiotics. A recent line of evidence demonstrates that many hormones including the steroid beclomethasone can also directly affect bacterial growth, virulence, and antibiotic resistance. The effect of these regimens on the release of potentially virulent and toxic membrane vesicles (MVs) is at present unclear. In this study, we determined the effect of several pharmacological agents on MVs release by and bacterial growth of common respiratory pathogens. We found that neither the release of MVs nor the bacterial growth was affected by the glucocorticoids budesonide and fluticasone. The macrolide antibiotic azithromycin only inhibited the growth of Moraxella catarrhalis but no effects were observed on bacterial MV release at a concentration that is achieved locally in the epithelial lining on administration. The macrophage pro-inflammatory response to MVs was significantly reduced after treatment with budesonide and fluticasone but not by azithromycin treatment. Our findings suggest that these glucocorticoids may have a positive effect on infection-related inflammation although the bacterial growth and MV release remained unaffected. (hide) EV-METRIC 14% (44th 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 membrane vesicles 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 SEC UF Protein markers EV: CD81/ CD63 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP1 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD63 Characterization: Lipid analysis No Characterization: Particle analysis TRPS EV concentration Yes

	EV170023	3/5	Streptococcus pneumoniae	Streptococcus pneumoniae	(d)(U)C Filtration SEC UF	Volgers C	2017	14%
Study summary Full title Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection. All authors Volgers C, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM Journal Scientific Reports Abstract Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations (show more...)Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations and it is estimated that up to 50% of these exacerbations are associated with bacterial infections. The mainstay treatment for these infection-related exacerbations constitutes the administration of glucocorticoids, alone or in combination with antibiotics. A recent line of evidence demonstrates that many hormones including the steroid beclomethasone can also directly affect bacterial growth, virulence, and antibiotic resistance. The effect of these regimens on the release of potentially virulent and toxic membrane vesicles (MVs) is at present unclear. In this study, we determined the effect of several pharmacological agents on MVs release by and bacterial growth of common respiratory pathogens. We found that neither the release of MVs nor the bacterial growth was affected by the glucocorticoids budesonide and fluticasone. The macrolide antibiotic azithromycin only inhibited the growth of Moraxella catarrhalis but no effects were observed on bacterial MV release at a concentration that is achieved locally in the epithelial lining on administration. The macrophage pro-inflammatory response to MVs was significantly reduced after treatment with budesonide and fluticasone but not by azithromycin treatment. Our findings suggest that these glucocorticoids may have a positive effect on infection-related inflammation although the bacterial growth and MV release remained unaffected. (hide) EV-METRIC 14% (44th 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 membrane vesicles 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 SEC UF Protein markers EV: Streptococcus pneumoniae antigen non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Streptococcus pneumoniae Sample Type Cell culture supernatant EV-producing cells Streptococcus pneumoniae EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Streptococcus pneumoniae antigen Characterization: Lipid analysis No Characterization: Particle analysis TRPS EV concentration Yes

	EV170023	4/5	Pseudomonas aeruginosa	Pseudomonas aeruginosa	(d)(U)C Filtration SEC UF	Volgers C	2017	14%
Study summary Full title Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection. All authors Volgers C, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM Journal Scientific Reports Abstract Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations (show more...)Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations and it is estimated that up to 50% of these exacerbations are associated with bacterial infections. The mainstay treatment for these infection-related exacerbations constitutes the administration of glucocorticoids, alone or in combination with antibiotics. A recent line of evidence demonstrates that many hormones including the steroid beclomethasone can also directly affect bacterial growth, virulence, and antibiotic resistance. The effect of these regimens on the release of potentially virulent and toxic membrane vesicles (MVs) is at present unclear. In this study, we determined the effect of several pharmacological agents on MVs release by and bacterial growth of common respiratory pathogens. We found that neither the release of MVs nor the bacterial growth was affected by the glucocorticoids budesonide and fluticasone. The macrolide antibiotic azithromycin only inhibited the growth of Moraxella catarrhalis but no effects were observed on bacterial MV release at a concentration that is achieved locally in the epithelial lining on administration. The macrophage pro-inflammatory response to MVs was significantly reduced after treatment with budesonide and fluticasone but not by azithromycin treatment. Our findings suggest that these glucocorticoids may have a positive effect on infection-related inflammation although the bacterial growth and MV release remained unaffected. (hide) EV-METRIC 14% (44th 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 membrane vesicles 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 SEC UF Protein markers EV: Pseudomonas aeruginosa antigen non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Pseudomonas aeruginosa Sample Type Cell culture supernatant EV-producing cells Pseudomonas aeruginosa EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Pseudomonas aeruginosa antigen Characterization: Lipid analysis No Characterization: Particle analysis TRPS EV concentration Yes

	EV170023	5/5	Moraxella catarrhalis	Moraxella catarrhalis	(d)(U)C Filtration SEC UF	Volgers C	2017	14%
Study summary Full title Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection. All authors Volgers C, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM Journal Scientific Reports Abstract Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations (show more...)Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations and it is estimated that up to 50% of these exacerbations are associated with bacterial infections. The mainstay treatment for these infection-related exacerbations constitutes the administration of glucocorticoids, alone or in combination with antibiotics. A recent line of evidence demonstrates that many hormones including the steroid beclomethasone can also directly affect bacterial growth, virulence, and antibiotic resistance. The effect of these regimens on the release of potentially virulent and toxic membrane vesicles (MVs) is at present unclear. In this study, we determined the effect of several pharmacological agents on MVs release by and bacterial growth of common respiratory pathogens. We found that neither the release of MVs nor the bacterial growth was affected by the glucocorticoids budesonide and fluticasone. The macrolide antibiotic azithromycin only inhibited the growth of Moraxella catarrhalis but no effects were observed on bacterial MV release at a concentration that is achieved locally in the epithelial lining on administration. The macrophage pro-inflammatory response to MVs was significantly reduced after treatment with budesonide and fluticasone but not by azithromycin treatment. Our findings suggest that these glucocorticoids may have a positive effect on infection-related inflammation although the bacterial growth and MV release remained unaffected. (hide) EV-METRIC 14% (44th 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 membrane vesicles 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 SEC UF Protein markers EV: Moraxella catarrhalis antigen non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Moraxella catarrhalis Sample Type Cell culture supernatant EV-producing cells Moraxella catarrhalis EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Moraxella catarrhalis antigen Characterization: Lipid analysis No Characterization: Particle analysis TRPS EV concentration Yes

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EV-TRACK ID	EV170023
species	Haemophilus influenzae	Homo sapiens	Streptococcus pneumoniae	Pseudomonas aeruginosa	Moraxella catarrhalis
sample type	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture
cell type	non-typeable Haemophilus influenzae	THP1	Streptococcus pneumoniae	Pseudomonas aeruginosa	Moraxella catarrhalis
condition	Control condition	Control condition	Control condition	Control condition	Control condition
separation protocol	(d)(U)C Filtration SEC UF	(d)(U)C Filtration SEC UF	(d)(U)C Filtration SEC UF	(d)(U)C Filtration SEC UF	(d)(U)C Filtration SEC UF
Exp. nr.	1	2	3	4	5
EV-METRIC %	14	14	14	14	14