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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210184	2/4	Homo sapiens	Blood plasma	DG (d)(U)C Filtration	Pillay, Preenan	2020	50%
Study summary Full title Exosomal Th1/Th2 cytokines in preeclampsia and HIV-positive preeclamptic women on highly active anti-retroviral therapy All authors Preenan Pillay, Kogi Moodley, Manu Vatish, Jagidesa Moodley, Raquel Duarte, Irene Mackraj Journal Cytokine Abstract Preeclampsia (PE) is a hypertensive disorder of pregnancy which is a leading cause of maternal and f (show more...)Preeclampsia (PE) is a hypertensive disorder of pregnancy which is a leading cause of maternal and foetal morbidity and mortality. Furthermore, HIV/Highly Active Anti-Retroviral Treatment has been associated with the increased risk of preeclampsia due to maternal immune reconstitution, which complicates the clinical diagnosis of PE in these patients. It is therefore necessary to identify biomarkers involved in the pathology of both disorders with the intent to diagnose. Exosomal cytokines represent ideal biomarkers of PE and inflammatory conditions due to their immunomodulatory role in pregnancy. We therefore quantified exosomal Th1 (IL-2 and TNF-α) and Th2 cytokines (IL-10) in maternal circulation. A significant dysregulation in total exosomes, placental-derived exosomes and exosomal cytokines in PE and HIV-positive PE pregnant woman on Highly Active Antiretroviral Treatment (HAART) was observed (p < 0.01). Additionally, we observed a significant shift towards Th1 immunity in PE which becomes amplified in HIV-positive PE pregnant woman on HAART (p < 0.01). Moreover, we show the potential application of exosomal Tumor necrosis factor alpha (TNF-α) as a biomarker of PE and PE in HIV-positive pregnant women on HAART (CI: 95%, LHR > 10, sensitivity of 100% and specificity of 90%). These findings are in support of exosome release and exosome cytokine encapsulation as a tightly regulated process in favour of maintaining the immune microenvironment, which can orchestrate either normal pregnancy, or the pathogenesis of preeclampsia and preeclampsia in HIV/HAART pregnancies. (hide) EV-METRIC 50% (83rd 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 Normotensive pregnant with HIV 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 DG (d)(U)C Filtration Protein markers EV: PLAP/ CD63/ IL-2/ IL-10/ TNF-? non-EV: None Proteomics no EV density (g/ml) Not specified Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type MLA-55 Pelleting: speed (g) 110000 Density gradient Type Continuous Lowest density fraction 6% Highest density fraction 18% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Not specified Rotor type MLA-55 Speed (g) 200000 Duration (min) 120 Fraction volume (mL) 12 Fraction processing Not specified Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? No Detected EV-associated proteins CD63 ELISA Antibody details provided? No Detected EV-associated proteins CD63/ PLAP Other 1 Multiplex cytokine assay Detected EV-associated proteins IL-2/ IL-10/ TNF-? Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 20-130nm EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 9.96e9 (< 33 gest weeks), 1.22e10 (>34 gest weeks) EM EM-type Transmission-EM Image type Wide-field Report size (nm) 20-130nm

	EV210184	1/4	Homo sapiens	Blood plasma	DG (d)(U)C Filtration	Pillay, Preenan	2020	38%
Study summary Full title Exosomal Th1/Th2 cytokines in preeclampsia and HIV-positive preeclamptic women on highly active anti-retroviral therapy All authors Preenan Pillay, Kogi Moodley, Manu Vatish, Jagidesa Moodley, Raquel Duarte, Irene Mackraj Journal Cytokine Abstract Preeclampsia (PE) is a hypertensive disorder of pregnancy which is a leading cause of maternal and f (show more...)Preeclampsia (PE) is a hypertensive disorder of pregnancy which is a leading cause of maternal and foetal morbidity and mortality. Furthermore, HIV/Highly Active Anti-Retroviral Treatment has been associated with the increased risk of preeclampsia due to maternal immune reconstitution, which complicates the clinical diagnosis of PE in these patients. It is therefore necessary to identify biomarkers involved in the pathology of both disorders with the intent to diagnose. Exosomal cytokines represent ideal biomarkers of PE and inflammatory conditions due to their immunomodulatory role in pregnancy. We therefore quantified exosomal Th1 (IL-2 and TNF-α) and Th2 cytokines (IL-10) in maternal circulation. A significant dysregulation in total exosomes, placental-derived exosomes and exosomal cytokines in PE and HIV-positive PE pregnant woman on Highly Active Antiretroviral Treatment (HAART) was observed (p < 0.01). Additionally, we observed a significant shift towards Th1 immunity in PE which becomes amplified in HIV-positive PE pregnant woman on HAART (p < 0.01). Moreover, we show the potential application of exosomal Tumor necrosis factor alpha (TNF-α) as a biomarker of PE and PE in HIV-positive pregnant women on HAART (CI: 95%, LHR > 10, sensitivity of 100% and specificity of 90%). These findings are in support of exosome release and exosome cytokine encapsulation as a tightly regulated process in favour of maintaining the immune microenvironment, which can orchestrate either normal pregnancy, or the pathogenesis of preeclampsia and preeclampsia in HIV/HAART pregnancies. (hide) EV-METRIC 38% (72nd 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 Healthy pregnant 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 DG (d)(U)C Filtration Protein markers EV: PLAP/ CD63/ IL-2/ IL-10/ TNF-? non-EV: None Proteomics no EV density (g/ml) Not specified Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type MLA-55 Pelleting: speed (g) 110000 Density gradient Type Continuous Lowest density fraction 6% Highest density fraction 18% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Not specified Rotor type MLA-55 Speed (g) 200000 Duration (min) 120 Fraction volume (mL) 12 Fraction processing Not specified Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Lowry-based assay ELISA Antibody details provided? No Detected EV-associated proteins CD63/ PLAP Other 1 Multiplex cytokine assay Detected EV-associated proteins IL-2/ IL-10/ TNF-? Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 20-130nm EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 6.16E9 (< 33 gest weeks), 8.45E9 (>34 gest weeks)

	EV210184	3/4	Homo sapiens	Blood plasma	DG (d)(U)C Filtration	Pillay, Preenan	2020	38%
Study summary Full title Exosomal Th1/Th2 cytokines in preeclampsia and HIV-positive preeclamptic women on highly active anti-retroviral therapy All authors Preenan Pillay, Kogi Moodley, Manu Vatish, Jagidesa Moodley, Raquel Duarte, Irene Mackraj Journal Cytokine Abstract Preeclampsia (PE) is a hypertensive disorder of pregnancy which is a leading cause of maternal and f (show more...)Preeclampsia (PE) is a hypertensive disorder of pregnancy which is a leading cause of maternal and foetal morbidity and mortality. Furthermore, HIV/Highly Active Anti-Retroviral Treatment has been associated with the increased risk of preeclampsia due to maternal immune reconstitution, which complicates the clinical diagnosis of PE in these patients. It is therefore necessary to identify biomarkers involved in the pathology of both disorders with the intent to diagnose. Exosomal cytokines represent ideal biomarkers of PE and inflammatory conditions due to their immunomodulatory role in pregnancy. We therefore quantified exosomal Th1 (IL-2 and TNF-α) and Th2 cytokines (IL-10) in maternal circulation. A significant dysregulation in total exosomes, placental-derived exosomes and exosomal cytokines in PE and HIV-positive PE pregnant woman on Highly Active Antiretroviral Treatment (HAART) was observed (p < 0.01). Additionally, we observed a significant shift towards Th1 immunity in PE which becomes amplified in HIV-positive PE pregnant woman on HAART (p < 0.01). Moreover, we show the potential application of exosomal Tumor necrosis factor alpha (TNF-α) as a biomarker of PE and PE in HIV-positive pregnant women on HAART (CI: 95%, LHR > 10, sensitivity of 100% and specificity of 90%). These findings are in support of exosome release and exosome cytokine encapsulation as a tightly regulated process in favour of maintaining the immune microenvironment, which can orchestrate either normal pregnancy, or the pathogenesis of preeclampsia and preeclampsia in HIV/HAART pregnancies. (hide) EV-METRIC 38% (72nd 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 Preeclamptic pregnancy 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 DG (d)(U)C Filtration Protein markers EV: PLAP/ CD63/ IL-2/ IL-10/ TNF-? non-EV: None Proteomics no EV density (g/ml) Not specified Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type MLA-55 Pelleting: speed (g) 110000 Density gradient Type Continuous Lowest density fraction 6% Highest density fraction 18% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Not specified Rotor type MLA-55 Speed (g) 200000 Duration (min) 120 Fraction volume (mL) 12 Fraction processing Not specified Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Lowry-based assay ELISA Antibody details provided? No Detected EV-associated proteins CD63/ PLAP Other 1 Multiplex cytokine assay Detected EV-associated proteins IL-2/ IL-10/ TNF-? Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 20-130nm EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.73e10 (early onset PE), 1.71e10 (late onset PE)

	EV210184	4/4	Homo sapiens	Blood plasma	DG (d)(U)C Filtration	Pillay, Preenan	2020	38%
Study summary Full title Exosomal Th1/Th2 cytokines in preeclampsia and HIV-positive preeclamptic women on highly active anti-retroviral therapy All authors Preenan Pillay, Kogi Moodley, Manu Vatish, Jagidesa Moodley, Raquel Duarte, Irene Mackraj Journal Cytokine Abstract Preeclampsia (PE) is a hypertensive disorder of pregnancy which is a leading cause of maternal and f (show more...)Preeclampsia (PE) is a hypertensive disorder of pregnancy which is a leading cause of maternal and foetal morbidity and mortality. Furthermore, HIV/Highly Active Anti-Retroviral Treatment has been associated with the increased risk of preeclampsia due to maternal immune reconstitution, which complicates the clinical diagnosis of PE in these patients. It is therefore necessary to identify biomarkers involved in the pathology of both disorders with the intent to diagnose. Exosomal cytokines represent ideal biomarkers of PE and inflammatory conditions due to their immunomodulatory role in pregnancy. We therefore quantified exosomal Th1 (IL-2 and TNF-α) and Th2 cytokines (IL-10) in maternal circulation. A significant dysregulation in total exosomes, placental-derived exosomes and exosomal cytokines in PE and HIV-positive PE pregnant woman on Highly Active Antiretroviral Treatment (HAART) was observed (p < 0.01). Additionally, we observed a significant shift towards Th1 immunity in PE which becomes amplified in HIV-positive PE pregnant woman on HAART (p < 0.01). Moreover, we show the potential application of exosomal Tumor necrosis factor alpha (TNF-α) as a biomarker of PE and PE in HIV-positive pregnant women on HAART (CI: 95%, LHR > 10, sensitivity of 100% and specificity of 90%). These findings are in support of exosome release and exosome cytokine encapsulation as a tightly regulated process in favour of maintaining the immune microenvironment, which can orchestrate either normal pregnancy, or the pathogenesis of preeclampsia and preeclampsia in HIV/HAART pregnancies. (hide) EV-METRIC 38% (72nd 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 Preeclamptic pregnancy with HIV 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 DG (d)(U)C Filtration Protein markers EV: PLAP/ CD63/ IL-2/ IL-10/ TNF-? non-EV: None Proteomics no EV density (g/ml) Not specified Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type MLA-55 Pelleting: speed (g) 110000 Density gradient Type Continuous Lowest density fraction 6% Highest density fraction 18% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Not specified Rotor type MLA-55 Speed (g) 200000 Duration (min) 120 Fraction volume (mL) 12 Fraction processing Not specified Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Lowry-based assay ELISA Antibody details provided? No Detected EV-associated proteins CD63/ PLAP Other 1 Multiplex cytokine assay Detected EV-associated proteins IL-2/ IL-10/ TNF-? Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 20-130nm EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 3.71e10 (early onset PE), 2.15e10 (late onset PE)

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EV-TRACK ID	EV210184
species	Homo sapiens
sample type	Blood plasma
condition	Normotensive pregnant with HIV	Healthy pregnant	Preeclamptic pregnancy	Preeclamptic pregnancy with HIV
separation protocol	DG (d)(U)C Filtration	DG (d)(U)C Filtration	DG (d)(U)C Filtration	DG (d)(U)C Filtration
Exp. nr.	2	1	3	4
EV-METRIC %	50	38	38	38