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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV150103	7/7	Homo sapiens	Cell culture supernatant	dUC	Dieudé M	2015	55%
Study summary Full title The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection. All authors Dieudé M, Bell C, Turgeon J, Beillevaire D, Pomerleau L, Yang B, Hamelin K, Qi S, Pallet N, Béland C, Dhahri W, Cailhier JF, Rousseau M, Duchez AC, Lévesque T, Lau A, Rondeau C, Gingras D, Muruve D, Rivard A, Cardinal H, Perreault C, Desjardins M, Boilard É, Thibault P, Hébert MJ Journal J Transl Med Abstract Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rej (show more...)Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation. (hide) EV-METRIC 55% (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 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 Apoptosis Focus vesicles apoptotic exosome-like vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 127.9 (pelleting) Protein markers EV: LG3/ Fibronectin/ proteasome-alpha3/ Syntenin/ TCTP non-EV: Tubulin/ GM130 Proteomics yes Show all info Study aim Function, Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Apoptosis EV-producing cells HUVEC EV-harvesting Medium Serum free medium Cell viability 75 Separation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 1080 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 127.9 Characterization: Protein analysis Protein Concentration Method microBCA Protein Concentration 300 Western Blot Detected EV-associated proteins Syntenin, Fibronectin, TCTP, proteasome-alpha3, LG3 Not detected contaminants GM130, Tubulin Proteomics database Yes Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BDCantoII Special Order Research Product Hardware adjustment This high sensitivity Flow cytometer (hsFCM) is equipped with a small particle option. The forward scatter (FSC) on this dedicated equipment is coupled to a photomultiplier tube (PMT) with a 488 nm solid state;100mW output blue laser (rather than the conventional 20 mW);and includes a 633nmHeNe;20mW output red laser and a 405 nm solid state diode;50mW output violet laser. The hsFCM includes a FSC-PMT and a Fourier optical transformation unit;which reduces the background noise and increases the angle of diffusion;therby enhancing the detection of small-diameter particles. Calibration bead size 0.09,0.45,0.84,1,3.2 Report type Median Reported size (nm) 100-200 EV concentration Yes Particle yield 3.50E+07 particles/million cells EM EM-type Transmission-EM/ Immune-EM Proteïns LG3;proteasome-alpha3 Image type Close-up, Wide-field

	EV150103	5/7	Homo sapiens	Cell culture supernatant	dUC	Dieudé M	2015	44%
Study summary Full title The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection. All authors Dieudé M, Bell C, Turgeon J, Beillevaire D, Pomerleau L, Yang B, Hamelin K, Qi S, Pallet N, Béland C, Dhahri W, Cailhier JF, Rousseau M, Duchez AC, Lévesque T, Lau A, Rondeau C, Gingras D, Muruve D, Rivard A, Cardinal H, Perreault C, Desjardins M, Boilard É, Thibault P, Hébert MJ Journal J Transl Med Abstract Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rej (show more...)Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation. (hide) EV-METRIC 44% (80th 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 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 Apoptosis Focus vesicles apoptotic body Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 127.9 (pelleting) Protein markers EV: Tubulin/ TCTP/ Fibronectin/ Syntenin/ LG3/ GM130 non-EV: None Proteomics yes Show all info Study aim Function, Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Apoptosis EV-producing cells HUVEC EV-harvesting Medium Serum free medium Cell viability 75 Separation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 1080 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 127.9 Characterization: Protein analysis Protein Concentration Method microBCA Protein Concentration 2000 Western Blot Detected EV-associated proteins Syntenin, Fibronectin, TCTP, GM130, Tubulin, LG3 Proteomics database Yes Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BDCantoII Special Order Research Product Hardware adjustment This high sensitivity Flow cytometer (hsFCM) is equipped with a small particle option. The forward scatter (FSC) on this dedicated equipment is coupled to a photomultiplier tube (PMT) with a 488 nm solid state;100mW output blue laser (rather than the conventional 20 mW);and includes a 633nmHeNe;20mW output red laser and a 405 nm solid state diode;50mW output violet laser. The hsFCM includes a FSC-PMT and a Fourier optical transformation unit;which reduces the background noise and increases the angle of diffusion;therby enhancing the detection of small-diameter particles. Calibration bead size 0.09,0.45,0.84,1,3.2 Report type Median Reported size (nm) 100-200 EV concentration Yes Particle yield 3.50E+07 particles/million cells EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV150103	4/7	Mus musculus	Serum	dUC	Dieudé M	2015	22%
Study summary Full title The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection. All authors Dieudé M, Bell C, Turgeon J, Beillevaire D, Pomerleau L, Yang B, Hamelin K, Qi S, Pallet N, Béland C, Dhahri W, Cailhier JF, Rousseau M, Duchez AC, Lévesque T, Lau A, Rondeau C, Gingras D, Muruve D, Rivard A, Cardinal H, Perreault C, Desjardins M, Boilard É, Thibault P, Hébert MJ Journal J Transl Med Abstract Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rej (show more...)Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation. (hide) EV-METRIC 22% (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 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 Acute kidney injury model Focus vesicles exosome-like vesicle, membrane vesicle, nanovesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 127.9 (pelleting) Protein markers EV: LG3/ proteasome-alpha3 non-EV: None Proteomics no Show all info Study aim Function, Biomarker, Identification of content (omics approaches) Sample Species Mus musculus Sample Type Serum Sample Condition Acute kidney injury model Separation Method Differential ultracentrifugation dUC: centrifugation steps Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 1080 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 127.9 Characterization: Protein analysis Western Blot Detected EV-associated proteins LG3 Characterization: Particle analysis EM EM-type Transmission-EM/ Immune-EM Proteïns proteasome-alpha3 Image type Close-up, Wide-field

	EV150103	1/7	Mus musculus	Cell culture supernatant	dUC	Dieudé M	2015	11%
Study summary Full title The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection. All authors Dieudé M, Bell C, Turgeon J, Beillevaire D, Pomerleau L, Yang B, Hamelin K, Qi S, Pallet N, Béland C, Dhahri W, Cailhier JF, Rousseau M, Duchez AC, Lévesque T, Lau A, Rondeau C, Gingras D, Muruve D, Rivard A, Cardinal H, Perreault C, Desjardins M, Boilard É, Thibault P, Hébert MJ Journal J Transl Med Abstract Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rej (show more...)Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation. (hide) EV-METRIC 11% (26th 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 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 Apoptosis Focus vesicles apoptotic exosome-like vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 127.9 (pelleting) Protein markers EV: LG3 non-EV: None Proteomics no Show all info Study aim Function, Biomarker, Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant Sample Condition Apoptosis EV-producing cells primary aorta-derived endothelial cells EV-harvesting Medium Serum free medium Cell viability 80 Separation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 1080 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 127.9 Characterization: Protein analysis Western Blot Detected EV-associated proteins LG3 Characterization: Particle analysis EM

	EV150103	2/7	Mus musculus	Cell culture supernatant	dUC	Dieudé M	2015	11%
Study summary Full title The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection. All authors Dieudé M, Bell C, Turgeon J, Beillevaire D, Pomerleau L, Yang B, Hamelin K, Qi S, Pallet N, Béland C, Dhahri W, Cailhier JF, Rousseau M, Duchez AC, Lévesque T, Lau A, Rondeau C, Gingras D, Muruve D, Rivard A, Cardinal H, Perreault C, Desjardins M, Boilard É, Thibault P, Hébert MJ Journal J Transl Med Abstract Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rej (show more...)Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation. (hide) EV-METRIC 11% (26th 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 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 Apoptosis Focus vesicles apoptotic body Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 127.9 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function, Biomarker, Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant Sample Condition Apoptosis EV-producing cells primary aorta-derived endothelial cells EV-harvesting Medium Serum free medium Cell viability 80 Separation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 1080 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 127.9 Characterization: Protein analysis Characterization: Particle analysis EM

	EV150103	3/7	Mus musculus	Serum	dUC	Dieudé M	2015	11%
Study summary Full title The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection. All authors Dieudé M, Bell C, Turgeon J, Beillevaire D, Pomerleau L, Yang B, Hamelin K, Qi S, Pallet N, Béland C, Dhahri W, Cailhier JF, Rousseau M, Duchez AC, Lévesque T, Lau A, Rondeau C, Gingras D, Muruve D, Rivard A, Cardinal H, Perreault C, Desjardins M, Boilard É, Thibault P, Hébert MJ Journal J Transl Med Abstract Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rej (show more...)Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation. (hide) EV-METRIC 11% (45th 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 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 Control condition Focus vesicles exosome-like vesicle, membrane vesicle, nanovesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 127.9 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function, Biomarker, Identification of content (omics approaches) Sample Species Mus musculus Sample Type Serum Sample Condition Control condition Separation Method Differential ultracentrifugation dUC: centrifugation steps Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 1080 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 127.9 Characterization: Protein analysis Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up

	EV150103	6/7	Mus musculus	Serum	dUC	Dieudé M	2015	11%
Study summary Full title The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection. All authors Dieudé M, Bell C, Turgeon J, Beillevaire D, Pomerleau L, Yang B, Hamelin K, Qi S, Pallet N, Béland C, Dhahri W, Cailhier JF, Rousseau M, Duchez AC, Lévesque T, Lau A, Rondeau C, Gingras D, Muruve D, Rivard A, Cardinal H, Perreault C, Desjardins M, Boilard É, Thibault P, Hébert MJ Journal J Transl Med Abstract Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rej (show more...)Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation. (hide) EV-METRIC 11% (45th 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 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 Ischemic hindlimb model Focus vesicles exosome-like vesicle, nanovesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 127.9 (pelleting) Protein markers EV: LG3 non-EV: None Proteomics no Show all info Study aim Function, Biomarker, Identification of content (omics approaches) Sample Species Mus musculus Sample Type Serum Sample Condition Ischemic hindlimb model Separation Method Differential ultracentrifugation dUC: centrifugation steps Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 1080 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 127.9 Characterization: Protein analysis Western Blot Detected EV-associated proteins LG3

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EV-TRACK ID	EV150103
species	Homo sapiens	Homo sapiens	Mus musculus	Mus musculus	Mus musculus	Mus musculus	Mus musculus
sample type	Cell culture	Cell culture	Serum	Cell culture	Cell culture	Serum	Serum
cell type	HUVEC	HUVEC	NA	primary aorta-derived endothelial cells	primary aorta-derived endothelial cells	NA	NA
medium	Serum free medium	Serum free medium	NA	Serum free medium	Serum free medium	NA	NA
condition	Apoptosis	Apoptosis	Acute kidney injury model	Apoptosis	Apoptosis	Control condition	Ischemic hindlimb model
separation protocol	dUC	dUC	dUC	dUC	dUC	dUC	dUC
EV subtype	No	No	NA	No	No	NA	NA
vesicle related term	apoptotic exosome-like vesicle	apoptotic body	exosome-like vesicle membrane vesicle nanovesicle	apoptotic exosome-like vesicle	apoptotic body	exosome-like vesicle membrane vesicle nanovesicle	exosome-like vesicle nanovesicle
Exp. nr.	7	5	4	1	2	3	6
EV-METRIC %	55	44	22	11	11	11	11

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