| 1. |
- Broman, Axel, et al.
(författare)
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Acoustic trapping based high throughput isolation and characterization of pathogen activated platelet derived extracellular vesicles from plasma
- 2023
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Konferensbidrag (refereegranskat)abstract
- We present the use of a high capacity and high throughput acoustic trapping platform for phenotypic characterization and functional studies of extracellular vesicles (EVs) from pathogen activated platelets. Platelet rich plasma was stimulated with bacterial M1 protein isolated from S. Pyogenes, which is known to activate platelets. The subsequently released platelet EVs were isolated from 400 μL plasma by acoustic trapping at a flowrate of 500 μL/min. We have previously reported on the acoustic trapping platform, which can process milliliter sized samples in minutes1. The EVs were then compared to EVs released by platelets stimulated with endogenous platelet activator (Thrombin) and negative control (HEPES buffer).A schematic of the sample processing can be seen in Fig. 1. Human plasma from healthy donors was incubated with HEPES buffer, Thrombin or M1 protein to stimulate platelets and induce EV release. The platelets were then removed by centrifugation, leaving EVs in plasma. The EVs were isolated and enriched by acoustic trapping and the protein content was analyzed using mass spectrometry. The EVs were also analyzed by immunoblotting, as well as immunogold labelling against CD42b and M1 protein and imaged by transmission electron microscopy (TEM). Additionally, isolated EVs were incubated with whole blood from healthy donors to investigate functional immunomodulatory effects compared to known platelet agonists (Thrombin, M1).The mass spectrometry data showed a clear distinction between isolated vesicles and plasma, Fig. 2A, with one protein cluster enriched for vesicles and one enriched for plasma samples. There was also a clear distinction between EVs from activated platelets (Thrombin, M1) and resting platelets (HEPES), Fig. 2B. Interestingly, the bacterial M1 protein was enriched in the vesicle fraction, Fig. 3A, suggesting that M1 protein binds to platelet EVs.To confirm that M1 binds to EVs, trapped samples and centrifuged samples (both pellet and supernatant) were analyzed with immunoblot against M1 protein, Fig. 3B. Clear bands are present around 54 kDa, in accordance with M1 mass, in samples containing enriched EVs. This further confirms that M1 protein binds to EVs. The TEM images showed isolated EVs for all samples, Fig. 3C. Vesicles from platelets stimulated with thrombin were found positive for CD42b. Pathogen activated platelet EVs were found positive for both CD42b and M1 protein, showing that the bacterial protein binds to platelet EVs. Although no CD42b positive EVs were found in HEPES stimulated samples in the TEM analysis, we observed a wealth of them in cytometry data.The whole blood assay showed that isolated platelet EVs stimulated platelet-neutrophil complex formation, compared to resting state, Fig. 4A. Additionally, platelet EVs stimulated IL-8 cytokine release from monocytes, Fig. 4B, suggesting functionally intact vesicles.We have demonstrated rapid isolation and enrichment of platelet EVs from plasma samples by acoustic trapping. The isolated vesicles were functionally intact, and it was possible to perform several downstream analyses, including whole blood stimulation. We found that bacterial M1 protein from S. Pyogenes binds to platelet EVs and is transported with them, a mechanism which could contribute to the rapid infectious progress in sepsis.
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| 2. |
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| 3. |
- Li, Yanhong, et al.
(författare)
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Urinary aquaporin-2 excretion during ibuprofen or indomethacin treatment in preterm infants with patent ductus arteriosus
- 2011
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Ingår i: Acta Paediatrica. - : Wiley-Blackwell. - 0803-5253 .- 1651-2227. ; 100:1, s. 59-66
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Tidskriftsartikel (refereegranskat)abstract
- Aim: Water channel AQP2 is the target for vasopressin (AVP) and a major determinant of urinary concentrating capacity. In mature kidneys, prostaglandins counteract the effect of AVP on AQP2 expression at functional sites. We investigated whether disturbances in water homeostasis in infants with patent ductus arteriosus (PDA) treated with prostaglandin inhibitors can be attributed to activation of AQP2. Methods: In 53 infants with symptomatic PDA (gestational age 24-33 weeks), 30 receiving ibuprofen and 23 indomethacin starting at 2-15 days of life, clinical and biochemical data were collected before treatment and after each dose of the drugs. Urinary AQP2 was determined by dot immunoblotting. Results: Urinary AQP2 level and osmolality were decreased in both groups. Urinary osmolality was overall low and correlated inversely with fluid uptake. In ibuprofen group, there was no correlation of AQP2 level with urinary osmolality. Conclusion: There was no AQP2 upregulation in the infants. The low urinary osmolality and dissociation between urinary osmolality and urinary AQP2 level indicate that the fluid retention sometimes observed in PDA infants treated with prostaglandin inhibitors is not caused by increased levels of functional AQP2. Thus, knowledge about the renal physiology of the adult cannot always be transferred to the infant kidney.
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| 4. |
- Marcoux, Genevieve, et al.
(författare)
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Platelet EVs contain an active proteasome involved in protein processing for antigen presentation via MHC-I molecules
- 2021
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 138:25, s. 2607-2620
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Tidskriftsartikel (refereegranskat)abstract
- In addition to their hemostatic role, platelets play a significant role in immunity. Once activated, platelets release extracellular vesicles (EVs) formed by the budding of their cytoplasmic membranes. Because of their heterogeneity, platelet EVs (PEVs) are thought to perform diverse functions. It is unknown, however, whether the proteasome is transferred from platelets to PEVs or whether its function is retained. We hypothesized that functional protein processing and antigen presentation machinery are transferred to PEVs by activated platelets. Using molecular and functional assays, we found that the active 20S proteasome was enriched in PEVs, along with major histocompatibility complex class I (MHC-I) and lymphocyte costimulatory molecules (CD40L and OX40L). Proteasome-containing PEVs were identified in healthy donor blood, but did not increase in platelet concentrates that caused adverse transfusion reactions. They were augmented, however, after immune complex injections in mice. The complete biodistribution of murine PEVs after injection into mice revealed that they principally reached lymphoid organs, such as spleen and lymph nodes, in addition to the bone marrow, and to a lesser extent, liver and lungs. The PEV proteasome processed exogenous ovalbumin (OVA) and loaded its antigenic peptide onto MHC-I molecules, which promoted OVA-specific CD8+ T-lymphocyte proliferation. These results suggest that PEVs contribute to adaptive immunity through cross-presentation of antigens and have privileged access to immune cells through the lymphatic system, a tissue location that is inaccessible to platelets.
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| 5. |
- Palm, Frida, et al.
(författare)
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Phenotypic characterization of acoustically enriched extracellular vesicles from pathogen-activated platelets
- 2023
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Ingår i: Journal of Innate Immunity. - : S. Karger. - 1662-811X .- 1662-8128. ; 15:1, s. 599-613
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) are derived from the membrane of platelets and released in the circulation upon activation or injury. Analogous to the parent cell, platelet derived EVs play an important role in hemostasis and immune responses by transfer of bioactive cargo from the parent cells. Platelet activation and release of EVs increases in several pathological inflammatory diseases, such as sepsis. We have previously reported that the M1 protein released from the bacterial pathogen Streptococcus pyogenes directly mediates platelet activation. In this study, EVs were isolated from these pathogen-activated platelets using acoustic trapping and their inflammation phenotype was characterized using quantitative mass spectrometry-based proteomics and cell-based models of inflammation. We determined that M1 protein mediated release of platelet derived EVs that contained the M1 protein. The isolated EVs derived from pathogen-activated platelets contained a similar protein cargo to those from physiologically activated platelets (thrombin), and included platelet membrane proteins, granule proteins and cytoskeletal proteins, coagulation factors and immune mediators. Immunomodulatory cargo, complement proteins and IgG3, were significantly enriched in EVs isolated from M1 protein-stimulated platelets. Acoustically enriched EVs were functionally intact and exhibited proinflammatory effects on addition to blood, including platelet-neutrophil complex formation, neutrophil activation, and cytokine release. Collectively, our findings reveal novel aspects of pathogen-mediated platelet activation during invasive streptococcal infection.
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