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Robert A Star - One of the best experts on this subject based on the ideXlab platform.
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Isolation and purification of Exosomes in urine.
Methods of Molecular Biology, 2010Co-Authors: Patricia A. Gonzales, Trairak Pisitkun, Robert A Star, Mark A Knepper, Hua Zhou, Nam Sun Wang, Peter S.t. YuenAbstract:Exosomes represent an important and readily isolated subset of the urinary proteome that has the potential to shed much insight on the health status of the kidney. Each segment of the nephron sheds Exosomes into the urine. Exosomes are rich in potential biomarkers, especially membrane proteins such as transporters and receptors that may be up- or downregulated during disease states. Two differential centrifugation methods are available for simple purification of Exosomes: one uses ultracentrifugation, and the other uses a nanomembrane concentrator. Validation methods include western blots of pan-exosome markers and segment-specific exosome markers, and negative staining electron microscopy.
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large scale proteomics and phosphoproteomics of urinary Exosomes
Journal of The American Society of Nephrology, 2009Co-Authors: Patricia A. Gonzales, Trairak Pisitkun, Robert A Star, Nam Sun Wang, Jason D Hoffert, Dmitry Tchapyjnikov, Robert Kleta, Mark A KnepperAbstract:Normal human urine contains large numbers of Exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary Exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of Exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.
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collection storage preservation and normalization of human urinary Exosomes for biomarker discovery
Kidney International, 2006Co-Authors: Hua Zhou, Trairak Pisitkun, James W. Dear, H Yasuda, Mark A Knepper, Patricia A. Gonzales, Peter S.t. Yuen, Peter Gross, Robert A StarAbstract:Urinary Exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80°C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20°C caused a major loss in Exosomes compared to fresh urine. In contrast, recovery after freezing at -80°C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80°C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80°C with extensive vortexing after thawing maximizes the recovery of urinary Exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary Exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.
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Collection, storage, preservation, and normalization of human urinary Exosomes for biomarker discovery
Kidney International, 2006Co-Authors: P. S.t. Yuen, P. A. Gonzales, Trairak Pisitkun, James W. Dear, Petra Groß, H Yasuda, Mark A Knepper, Robert A StarAbstract:Urinary Exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80°C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20°C caused a major loss in Exosomes compared to fresh urine. In contrast, recovery after freezing at -80°C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80°C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80°C with extensive vortexing after thawing maximizes the recovery of urinary Exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary Exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts. © 2006 International Society of Nephrology.
Mark A Knepper - One of the best experts on this subject based on the ideXlab platform.
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Isolation and purification of Exosomes in urine.
Methods of Molecular Biology, 2010Co-Authors: Patricia A. Gonzales, Trairak Pisitkun, Robert A Star, Mark A Knepper, Hua Zhou, Nam Sun Wang, Peter S.t. YuenAbstract:Exosomes represent an important and readily isolated subset of the urinary proteome that has the potential to shed much insight on the health status of the kidney. Each segment of the nephron sheds Exosomes into the urine. Exosomes are rich in potential biomarkers, especially membrane proteins such as transporters and receptors that may be up- or downregulated during disease states. Two differential centrifugation methods are available for simple purification of Exosomes: one uses ultracentrifugation, and the other uses a nanomembrane concentrator. Validation methods include western blots of pan-exosome markers and segment-specific exosome markers, and negative staining electron microscopy.
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large scale proteomics and phosphoproteomics of urinary Exosomes
Journal of The American Society of Nephrology, 2009Co-Authors: Patricia A. Gonzales, Trairak Pisitkun, Robert A Star, Nam Sun Wang, Jason D Hoffert, Dmitry Tchapyjnikov, Robert Kleta, Mark A KnepperAbstract:Normal human urine contains large numbers of Exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary Exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of Exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.
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Urinary Exosomes: is there a future?
Nephrology Dialysis Transplantation, 2008Co-Authors: Patricia A. Gonzales, Trairak Pisitkun, Mark A KnepperAbstract:Tiny vesicles called ‘Exosomes’, recently discovered in normal urine [1], provide a non-invasive means of acquiring unique information about the physiological or pathophysiological state of their renal cells of origin. Exosomes are delivered to the urine from all renal epithelial cell types. Consequently, analysis of urinary Exosomes may provide a source of protein biomarkers for diseases involving glomerular podocytes, the various renal tubule segments or the transitional epithelium lining the urinary drainage tract [1]. Here, we discuss possible applications of urinary exosome analysis, as well as barriers to the development of practical clinical tools for exosome analysis. Exosomes originate as the internal vesicles of multivesicular bodies (MVBs) in cells (Figure 1). They were first described as products of circulating blood cells [2], such as erythrocytes and lymphocytes, and are probably formed by most cell types throughout the body. In the kidney, Exosomes are released to the urine by fusion of the outer membrane of the MVBs with the apical plasma membrane (Figure 1). Proteomic analysis of urinary Exosomes using tandem mass spectrometry approaches [1] revealed membrane proteins from each cell type facing the urinary space. In addition, the lumens of Exosomes contain many cytosolic proteins that are entrained when the Exosomes are formed in the MVBs. Thus, urinary Exosomes can provide the investigator with a sampling of membrane and cytosolic proteins from each renal epithelial cell type. Through the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS), more than a thousand proteins have been detected (Gonzales, Pisitkun and Knepper, unpublished data). In urine from normal subjects, urinary Exosomes account for ∼3% of the total urinary protein [3]. Hence, when Exosomes are isolated, their constituent proteins are enriched >30-fold, enhancing the detectability of rare proteins that may have diagnostic value.
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collection storage preservation and normalization of human urinary Exosomes for biomarker discovery
Kidney International, 2006Co-Authors: Hua Zhou, Trairak Pisitkun, James W. Dear, H Yasuda, Mark A Knepper, Patricia A. Gonzales, Peter S.t. Yuen, Peter Gross, Robert A StarAbstract:Urinary Exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80°C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20°C caused a major loss in Exosomes compared to fresh urine. In contrast, recovery after freezing at -80°C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80°C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80°C with extensive vortexing after thawing maximizes the recovery of urinary Exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary Exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.
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Collection, storage, preservation, and normalization of human urinary Exosomes for biomarker discovery
Kidney International, 2006Co-Authors: P. S.t. Yuen, P. A. Gonzales, Trairak Pisitkun, James W. Dear, Petra Groß, H Yasuda, Mark A Knepper, Robert A StarAbstract:Urinary Exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80°C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20°C caused a major loss in Exosomes compared to fresh urine. In contrast, recovery after freezing at -80°C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80°C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80°C with extensive vortexing after thawing maximizes the recovery of urinary Exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary Exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts. © 2006 International Society of Nephrology.
Trairak Pisitkun - One of the best experts on this subject based on the ideXlab platform.
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Isolation and purification of Exosomes in urine.
Methods of Molecular Biology, 2010Co-Authors: Patricia A. Gonzales, Trairak Pisitkun, Robert A Star, Mark A Knepper, Hua Zhou, Nam Sun Wang, Peter S.t. YuenAbstract:Exosomes represent an important and readily isolated subset of the urinary proteome that has the potential to shed much insight on the health status of the kidney. Each segment of the nephron sheds Exosomes into the urine. Exosomes are rich in potential biomarkers, especially membrane proteins such as transporters and receptors that may be up- or downregulated during disease states. Two differential centrifugation methods are available for simple purification of Exosomes: one uses ultracentrifugation, and the other uses a nanomembrane concentrator. Validation methods include western blots of pan-exosome markers and segment-specific exosome markers, and negative staining electron microscopy.
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large scale proteomics and phosphoproteomics of urinary Exosomes
Journal of The American Society of Nephrology, 2009Co-Authors: Patricia A. Gonzales, Trairak Pisitkun, Robert A Star, Nam Sun Wang, Jason D Hoffert, Dmitry Tchapyjnikov, Robert Kleta, Mark A KnepperAbstract:Normal human urine contains large numbers of Exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary Exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of Exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.
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Urinary Exosomes: is there a future?
Nephrology Dialysis Transplantation, 2008Co-Authors: Patricia A. Gonzales, Trairak Pisitkun, Mark A KnepperAbstract:Tiny vesicles called ‘Exosomes’, recently discovered in normal urine [1], provide a non-invasive means of acquiring unique information about the physiological or pathophysiological state of their renal cells of origin. Exosomes are delivered to the urine from all renal epithelial cell types. Consequently, analysis of urinary Exosomes may provide a source of protein biomarkers for diseases involving glomerular podocytes, the various renal tubule segments or the transitional epithelium lining the urinary drainage tract [1]. Here, we discuss possible applications of urinary exosome analysis, as well as barriers to the development of practical clinical tools for exosome analysis. Exosomes originate as the internal vesicles of multivesicular bodies (MVBs) in cells (Figure 1). They were first described as products of circulating blood cells [2], such as erythrocytes and lymphocytes, and are probably formed by most cell types throughout the body. In the kidney, Exosomes are released to the urine by fusion of the outer membrane of the MVBs with the apical plasma membrane (Figure 1). Proteomic analysis of urinary Exosomes using tandem mass spectrometry approaches [1] revealed membrane proteins from each cell type facing the urinary space. In addition, the lumens of Exosomes contain many cytosolic proteins that are entrained when the Exosomes are formed in the MVBs. Thus, urinary Exosomes can provide the investigator with a sampling of membrane and cytosolic proteins from each renal epithelial cell type. Through the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS), more than a thousand proteins have been detected (Gonzales, Pisitkun and Knepper, unpublished data). In urine from normal subjects, urinary Exosomes account for ∼3% of the total urinary protein [3]. Hence, when Exosomes are isolated, their constituent proteins are enriched >30-fold, enhancing the detectability of rare proteins that may have diagnostic value.
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collection storage preservation and normalization of human urinary Exosomes for biomarker discovery
Kidney International, 2006Co-Authors: Hua Zhou, Trairak Pisitkun, James W. Dear, H Yasuda, Mark A Knepper, Patricia A. Gonzales, Peter S.t. Yuen, Peter Gross, Robert A StarAbstract:Urinary Exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80°C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20°C caused a major loss in Exosomes compared to fresh urine. In contrast, recovery after freezing at -80°C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80°C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80°C with extensive vortexing after thawing maximizes the recovery of urinary Exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary Exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.
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Collection, storage, preservation, and normalization of human urinary Exosomes for biomarker discovery
Kidney International, 2006Co-Authors: P. S.t. Yuen, P. A. Gonzales, Trairak Pisitkun, James W. Dear, Petra Groß, H Yasuda, Mark A Knepper, Robert A StarAbstract:Urinary Exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80°C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20°C caused a major loss in Exosomes compared to fresh urine. In contrast, recovery after freezing at -80°C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80°C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80°C with extensive vortexing after thawing maximizes the recovery of urinary Exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary Exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts. © 2006 International Society of Nephrology.
Aled Clayton - One of the best experts on this subject based on the ideXlab platform.
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proteomics analysis of bladder cancer Exosomes
Molecular & Cellular Proteomics, 2010Co-Authors: Joanne L Welton, Sanjay Khanna, Peter Giles, Paul Brennan, Ian Andrew Brewis, John Staffurth, Malcolm David Mason, Aled ClaytonAbstract:Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of Exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell Exosomes. Using ultracentrifugation on a sucrose cushion, Exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various Exosomes with particular overlap with Exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary Exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function.
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human tumor derived Exosomes selectively impair lymphocyte responses to interleukin 2
Cancer Research, 2007Co-Authors: Aled Clayton, Malcolm David Mason, Paul J Mitchell, Jacquelyn Court, Zsuzsanna TabiAbstract:Exosomes are nanometer-sized vesicles, secreted by normal and neoplastic cells. The outcome following interaction between the cellular immune system and cancer-derived Exosomes is not well understood. Interleukin-2 (IL-2) is a key factor supporting expansion and differentiation of CTL and natural killer (NK) cells but can also support regulatory T cells and their suppressive functions. Our study examined whether tumor-derived Exosomes could modify lymphocyte IL-2 responses. Proliferation of healthy donor peripheral blood lymphocytes in response to IL-2 was inhibited by tumor Exosomes. In unfractionated lymphocytes, this effect was seen in all cell subsets. Separating CD4 + T cells, CD8 + T cells, and NK cells revealed that CD8 + T-cell proliferation was not inhibited in the absence of CD4 + T cells and that NK cell proliferation was only slightly impaired. Other exosome effects included selective impairment of IL-2–mediated CD25 up-regulation, affecting all but the CD3 + CD8 − T-cell subset. IL-2–induced Foxp3 expression by CD4 + CD25 + cells was not inhibited by tumor Exosomes, and the suppressive function of CD4 + CD25 + T cells was enhanced by Exosomes. In contrast, Exosomes directly inhibited NK cell killing function in a T-cell–independent manner. Analysis of tumor Exosomes revealed membrane-associated transforming growth factor β 1 (TGFβ 1 ), which contributed to the antiproliferative effects, shown by using neutralizing TGFβ 1 -specific antibody. The data show an exosome-mediated mechanism of skewing IL-2 responsiveness in favor of regulatory T cells and away from cytotoxic cells. This coordinated “double hit” to cellular immunity strongly implicates the role of Exosomes in tumor immune evasion. [Cancer Res 2007;67(15):7458–66]
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isolation and characterization of Exosomes from cell culture supernatants and biological fluids
Current protocols in pharmacology, 2006Co-Authors: Clotilde Thery, Graca Raposo, Sebastian Amigorena, Aled ClaytonAbstract:Exosomes are small membrane vesicles found in cell culture supernatants and in different biological fluids. Exosomes form in a particular population of endosomes, called multivesicular bodies (MVBs), by inward budding into the lumen of the compartment. Upon fusion of MVBs with the plasma membrane, these internal vesicles are secreted. Exosomes possess a defined set of membrane and cytosolic proteins. The physiological function of Exosomes is still a matter of debate, but increasing results in various experimental systems suggest their involvement in multiple biological processes. Because both cell-culture supernatants and biological fluids contain different types of lipid membranes, it is critical to perform high-quality exosome purification. This unit describes different approaches for exosome purification from various sources, and discusses methods to evaluate the purity and homogeneity of the purified exosome preparations.
Patricia A. Gonzales - One of the best experts on this subject based on the ideXlab platform.
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Isolation and purification of Exosomes in urine.
Methods of Molecular Biology, 2010Co-Authors: Patricia A. Gonzales, Trairak Pisitkun, Robert A Star, Mark A Knepper, Hua Zhou, Nam Sun Wang, Peter S.t. YuenAbstract:Exosomes represent an important and readily isolated subset of the urinary proteome that has the potential to shed much insight on the health status of the kidney. Each segment of the nephron sheds Exosomes into the urine. Exosomes are rich in potential biomarkers, especially membrane proteins such as transporters and receptors that may be up- or downregulated during disease states. Two differential centrifugation methods are available for simple purification of Exosomes: one uses ultracentrifugation, and the other uses a nanomembrane concentrator. Validation methods include western blots of pan-exosome markers and segment-specific exosome markers, and negative staining electron microscopy.
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large scale proteomics and phosphoproteomics of urinary Exosomes
Journal of The American Society of Nephrology, 2009Co-Authors: Patricia A. Gonzales, Trairak Pisitkun, Robert A Star, Nam Sun Wang, Jason D Hoffert, Dmitry Tchapyjnikov, Robert Kleta, Mark A KnepperAbstract:Normal human urine contains large numbers of Exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary Exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of Exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.
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Urinary Exosomes: is there a future?
Nephrology Dialysis Transplantation, 2008Co-Authors: Patricia A. Gonzales, Trairak Pisitkun, Mark A KnepperAbstract:Tiny vesicles called ‘Exosomes’, recently discovered in normal urine [1], provide a non-invasive means of acquiring unique information about the physiological or pathophysiological state of their renal cells of origin. Exosomes are delivered to the urine from all renal epithelial cell types. Consequently, analysis of urinary Exosomes may provide a source of protein biomarkers for diseases involving glomerular podocytes, the various renal tubule segments or the transitional epithelium lining the urinary drainage tract [1]. Here, we discuss possible applications of urinary exosome analysis, as well as barriers to the development of practical clinical tools for exosome analysis. Exosomes originate as the internal vesicles of multivesicular bodies (MVBs) in cells (Figure 1). They were first described as products of circulating blood cells [2], such as erythrocytes and lymphocytes, and are probably formed by most cell types throughout the body. In the kidney, Exosomes are released to the urine by fusion of the outer membrane of the MVBs with the apical plasma membrane (Figure 1). Proteomic analysis of urinary Exosomes using tandem mass spectrometry approaches [1] revealed membrane proteins from each cell type facing the urinary space. In addition, the lumens of Exosomes contain many cytosolic proteins that are entrained when the Exosomes are formed in the MVBs. Thus, urinary Exosomes can provide the investigator with a sampling of membrane and cytosolic proteins from each renal epithelial cell type. Through the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS), more than a thousand proteins have been detected (Gonzales, Pisitkun and Knepper, unpublished data). In urine from normal subjects, urinary Exosomes account for ∼3% of the total urinary protein [3]. Hence, when Exosomes are isolated, their constituent proteins are enriched >30-fold, enhancing the detectability of rare proteins that may have diagnostic value.
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collection storage preservation and normalization of human urinary Exosomes for biomarker discovery
Kidney International, 2006Co-Authors: Hua Zhou, Trairak Pisitkun, James W. Dear, H Yasuda, Mark A Knepper, Patricia A. Gonzales, Peter S.t. Yuen, Peter Gross, Robert A StarAbstract:Urinary Exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80°C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20°C caused a major loss in Exosomes compared to fresh urine. In contrast, recovery after freezing at -80°C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80°C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80°C with extensive vortexing after thawing maximizes the recovery of urinary Exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary Exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.