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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV110028	1/3	Homo sapiens Malassezia sympodialis	NAY	(d)(U)C DG IAF	Gehrmann U	2011	38%
Study summary Full title Nanovesicles from Malassezia sympodialis and host exosomes induce cytokine responses--novel mechanisms for host-microbe interactions in atopic eczema All authors Gehrmann U, Qazi KR, Johansson C, Hultenby K, Karlsson M, Lundeberg L, Gabrielsson S, Scheynius A Journal PLoS One Abstract BACKGROUND: Intercellular communication can occur via the release of membrane vesicles. Exosomes are (show more...)BACKGROUND: Intercellular communication can occur via the release of membrane vesicles. Exosomes are nanovesicles released from the endosomal compartment of cells. Depending on their cell of origin and their cargo they can exert different immunoregulatory functions. Recently, fungi were found to produce extracellular vesicles that can influence host-microbe interactions. The yeast Malassezia sympodialis which belongs to our normal cutaneous microbial flora elicits specific IgE- and T-cell reactivity in approximately 50% of adult patients with atopic eczema (AE). Whether exosomes or other vesicles contribute to the inflammation has not yet been investigated. OBJECTIVE: To investigate if M. sympodialis can release nanovesicles and whether they or endogenous exosomes can activate PBMC from AE patients sensitized to M. sympodialis. METHODS: Extracellular nanovesicles isolated from M. sympodialis, co-cultures of M. sympodialis and dendritic cells, and from plasma of patients with AE and healthy controls (HC) were characterised using flow cytometry, sucrose gradient centrifugation, Western blot and electron microscopy. Their ability to stimulate IL-4 and TNF-alpha responses in autologous CD14, CD34 depleted PBMC was determined using ELISPOT and ELISA, respectively. RESULTS: We show for the first time that M. sympodialis releases extracellular vesicles carrying allergen. These vesicles can induce IL-4 and TNF-? responses with a significantly higher IL-4 production in patients compared to HC. Exosomes from dendritic cell and M. sympodialis co-cultures induced IL-4 and TNF-? responses in autologous CD14, CD34 depleted PBMC of AE patients and HC while plasma exosomes induced TNF-? but not IL-4 in undepleted PBMC. CONCLUSIONS: Extracellular vesicles from M. sympodialis, dendritic cells and plasma can contribute to cytokine responses in CD14, CD34 depleted and undepleted PBMC of AE patients and HC. These novel observations have implications for understanding host-microbe interactions in the pathogenesis of AE. (hide) EV-METRIC 38% (79th 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 NAY Focus vesicles exosomes / "Nano(-sized) 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 DG IAF Protein markers EV: CD63/ MHC2 non-EV: Proteomics no EV density (g/ml) 1.1-1.2 Show all info Study aim Function Sample Species Homo sapiens / Malassezia sympodialis Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g 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 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Speed (g) 200000 Immunoaffinity capture Selected surface protein(s) MHC2 Western Blot Antibody details provided? No Detected EV-associated proteins MHC2/ MHC2 ELISA Antibody details provided? No Detected EV-associated proteins MHC2/ MHC2 Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein MHC2; M.sympodialis antigen Image type Close-up

	EV110028	2/3	Malassezia sympodialis	Fungus	(d)(U)C DG IAF	Gehrmann U	2011	33%
Study summary Full title Nanovesicles from Malassezia sympodialis and host exosomes induce cytokine responses--novel mechanisms for host-microbe interactions in atopic eczema All authors Gehrmann U, Qazi KR, Johansson C, Hultenby K, Karlsson M, Lundeberg L, Gabrielsson S, Scheynius A Journal PLoS One Abstract BACKGROUND: Intercellular communication can occur via the release of membrane vesicles. Exosomes are (show more...)BACKGROUND: Intercellular communication can occur via the release of membrane vesicles. Exosomes are nanovesicles released from the endosomal compartment of cells. Depending on their cell of origin and their cargo they can exert different immunoregulatory functions. Recently, fungi were found to produce extracellular vesicles that can influence host-microbe interactions. The yeast Malassezia sympodialis which belongs to our normal cutaneous microbial flora elicits specific IgE- and T-cell reactivity in approximately 50% of adult patients with atopic eczema (AE). Whether exosomes or other vesicles contribute to the inflammation has not yet been investigated. OBJECTIVE: To investigate if M. sympodialis can release nanovesicles and whether they or endogenous exosomes can activate PBMC from AE patients sensitized to M. sympodialis. METHODS: Extracellular nanovesicles isolated from M. sympodialis, co-cultures of M. sympodialis and dendritic cells, and from plasma of patients with AE and healthy controls (HC) were characterised using flow cytometry, sucrose gradient centrifugation, Western blot and electron microscopy. Their ability to stimulate IL-4 and TNF-alpha responses in autologous CD14, CD34 depleted PBMC was determined using ELISPOT and ELISA, respectively. RESULTS: We show for the first time that M. sympodialis releases extracellular vesicles carrying allergen. These vesicles can induce IL-4 and TNF-? responses with a significantly higher IL-4 production in patients compared to HC. Exosomes from dendritic cell and M. sympodialis co-cultures induced IL-4 and TNF-? responses in autologous CD14, CD34 depleted PBMC of AE patients and HC while plasma exosomes induced TNF-? but not IL-4 in undepleted PBMC. CONCLUSIONS: Extracellular vesicles from M. sympodialis, dendritic cells and plasma can contribute to cytokine responses in CD14, CD34 depleted and undepleted PBMC of AE patients and HC. These novel observations have implications for understanding host-microbe interactions in the pathogenesis of AE. (hide) EV-METRIC 33% (92nd 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 Fungus Sample origin NAY Focus vesicles exosomes / "Nano(-sized) 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 DG IAF Protein markers EV: M.sympodialis antigen/ MHC2 non-EV: Proteomics no EV density (g/ml) 1.1-1.2 Show all info Study aim Function Sample Species Malassezia sympodialis Sample Type Fungus Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Speed (g) 200000 Immunoaffinity capture Selected surface protein(s) MHC2 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins "MHC2/ MHC2/ M.sympodialis antigen" ELISA Antibody details provided? No Detected EV-associated proteins "MHC2/ MHC2/ M.sympodialis antigen" Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein MHC2; M.sympodialis antigen Image type Close-up

	EV110028	3/3	Homo sapiens	Blood plasma	(d)(U)C DG Filtration IAF	Gehrmann U	2011	33%
Study summary Full title Nanovesicles from Malassezia sympodialis and host exosomes induce cytokine responses--novel mechanisms for host-microbe interactions in atopic eczema All authors Gehrmann U, Qazi KR, Johansson C, Hultenby K, Karlsson M, Lundeberg L, Gabrielsson S, Scheynius A Journal PLoS One Abstract BACKGROUND: Intercellular communication can occur via the release of membrane vesicles. Exosomes are (show more...)BACKGROUND: Intercellular communication can occur via the release of membrane vesicles. Exosomes are nanovesicles released from the endosomal compartment of cells. Depending on their cell of origin and their cargo they can exert different immunoregulatory functions. Recently, fungi were found to produce extracellular vesicles that can influence host-microbe interactions. The yeast Malassezia sympodialis which belongs to our normal cutaneous microbial flora elicits specific IgE- and T-cell reactivity in approximately 50% of adult patients with atopic eczema (AE). Whether exosomes or other vesicles contribute to the inflammation has not yet been investigated. OBJECTIVE: To investigate if M. sympodialis can release nanovesicles and whether they or endogenous exosomes can activate PBMC from AE patients sensitized to M. sympodialis. METHODS: Extracellular nanovesicles isolated from M. sympodialis, co-cultures of M. sympodialis and dendritic cells, and from plasma of patients with AE and healthy controls (HC) were characterised using flow cytometry, sucrose gradient centrifugation, Western blot and electron microscopy. Their ability to stimulate IL-4 and TNF-alpha responses in autologous CD14, CD34 depleted PBMC was determined using ELISPOT and ELISA, respectively. RESULTS: We show for the first time that M. sympodialis releases extracellular vesicles carrying allergen. These vesicles can induce IL-4 and TNF-? responses with a significantly higher IL-4 production in patients compared to HC. Exosomes from dendritic cell and M. sympodialis co-cultures induced IL-4 and TNF-? responses in autologous CD14, CD34 depleted PBMC of AE patients and HC while plasma exosomes induced TNF-? but not IL-4 in undepleted PBMC. CONCLUSIONS: Extracellular vesicles from M. sympodialis, dendritic cells and plasma can contribute to cytokine responses in CD14, CD34 depleted and undepleted PBMC of AE patients and HC. These novel observations have implications for understanding host-microbe interactions in the pathogenesis of AE. (hide) EV-METRIC 33% (65th 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 Blood plasma Sample origin NAY Focus vesicles exosomes / "Nano(-sized) 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 DG Filtration IAF Protein markers EV: CD81/ CD63/ MHC2 non-EV: Proteomics no EV density (g/ml) 1.1-1.18 Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Speed (g) 200000 Filtration steps 0.22µm or 0.2µm Immunoaffinity capture Selected surface protein(s) MHC2 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ MHC2/ MHC2 ELISA Antibody details provided? No Detected EV-associated proteins MHC2/ MHC2 Characterization: Particle analysis None

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EV-TRACK ID	EV110028
species	Homo sapiens Malassezia sympodialis	Malassezia sympodialis	Homo sapiens
sample type	Cell culture	Fungus	Blood plasma
cell type	NAY	NA	NA
medium	EV Depleted
condition	NAY	NAY	NAY
separation protocol	(d)(U)C DG IAF	(d)(U)C DG IAF	(d)(U)C DG Filtration IAF
Exp. nr.	1	2	3
EV-METRIC %	38	33	33