| 1. |
- Lässer, Cecilia, 1981, et al.
(författare)
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Exosomes in the nose induce immune cell trafficking and harbour an altered protein cargo in chronic airway inflammation.
- 2016
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Ingår i: Journal of Translational Medicine. - : Springer Science and Business Media LLC. - 1479-5876. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Exosomes are nano-sized extracellular vesicles participating in cell-to-cell communication both in health and disease. However, the knowledge about the functions and molecular composition of exosomes in the upper airways is limited. The aim of the current study was therefore to determine whether nasal exosomes can influence inflammatory cells and to establish the proteome of nasal lavage fluid-derived exosomes in healthy subjects, as well as its alterations in individuals with chronic airway inflammatory diseases [asthma and chronic rhinosinusitis (CRS)]. METHODS: Nasal lavage fluid was collected from 14 healthy subjects, 15 subjects with asthma and 13 subjects with asthma/CRS. Exosomes were isolated with differential centrifugation and the proteome was analysed by LC-MS/MS with the application of two exclusion lists as well as using quantitative proteomics. Ingenuity Pathways Analysis and GO Term finder was used to predict the functions associated with the exosomal proteome and a migration assay was used to analyse the effect on immune cells by nasal exosomes. RESULTS: Firstly, we demonstrate that nasal exosomes can induce migration of several immune cells, such as monocytes, neutrophils and NK cells in vitro. Secondly, a mass spectrometry approach, with the application of exclusion lists, was utilised to generate a comprehensive protein inventory of the exosomes from healthy subjects. The use of exclusion lists resulted in the identification of ~15 % additional proteins, and increased the confidence in ~20 % of identified proteins. In total, 604 proteins were identified in nasal exosomes and the nasal exosomal proteome showed strong associations with immune-related functions, such as immune cell trafficking. Thirdly, a quantitative proteomics approach was used to determine alterations in the exosome proteome as a result of airway inflammatory disease. Serum-associated proteins and mucins were more abundant in the exosomes from subjects with respiratory diseases compared to healthy controls while proteins with antimicrobial functions and barrier-related proteins had decreased expression. CONCLUSIONS: Nasal exosomes were shown to induce the migration of innate immune cells, which may be important as the airway epithelium is the first line of defence against pathogens and allergens. The decreased expression in barrier and antimicrobial exosomal proteins in subjects with airway diseases, could possibly contribute to an increased susceptibility to infections, which have important clinical implications in disease progression.
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| 2. |
- Lässer, Cecilia, 1981, et al.
(författare)
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RNA-containing exosomes in human nasal secretions.
- 2011
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Ingår i: American journal of rhinology & allergy. - : SAGE Publications. - 1945-8932 .- 1945-8924. ; 25:2, s. 89-93
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Exosomes are nanovesicles of endocytic origin released by cells and present in human body fluids such as plasma, breast milk, andbronchoalveolar lavage fluid. These vesicles take part in communication between cells. Recently, it was shown that exosomes contain both mRNA andmicroRNA. This RNA can be shuttled between cells (exosomal shuttle RNA), which is a new route of communication between cells. The aim of this study wasto determine whether nasal secretions harbor exosomes and furthermore, whether these exosomes contain RNA.METHODS: Human nasal lavage fluid (NLF) underwent centrifugation and filtration to discard cells and debris, followed by a final ultracentrifugation at 120,000 X g to pellet the exosomes. Exosomes were detected using electron microscopy (EM), flow cytometry, and Western blot. RNA was extracted and analyzed using a Bioanalyzer.RESULTS: Exosomes were visualized as 40-80 nm, CD63+ vesicles using EM. Flow cytometry of exosomes using anti-major histocompatibility complex classII beads revealed exosomes positive for the tetraspanins CD9, CD63, and CD81. Western blot confirmed the presence of exosomal protein and absence ofproteins from the endoplasmic reticulum (ER), because the exosomes were positive for Tsg101, but negative for the ER marker, calnexin. Bioanalyzer analysis revealed that, these exosomes contain RNA.CONCLUSION: This study shows for the first time that NLF contains exosomes and that these exosomes contain RNA. Further characterization of the exosomalRNA and proteins may provide important information about communication in the nose and potentially provide a source of biomarkers for upper airwaydiseases.
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| 3. |
- Park, Kyong-Su, et al.
(författare)
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Detoxified synthetic bacterial membrane vesicles as a vaccine platform against bacteria and SARS-CoV-2
- 2023
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Ingår i: Journal of Nanobiotechnology. ; 21:1
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Tidskriftsartikel (refereegranskat)abstract
- The development of vaccines based on outer membrane vesicles (OMV) that naturally bud off from bacteria is an evolving field in infectious diseases. However, the inherent inflammatory nature of OMV limits their use as human vaccines. This study employed an engineered vesicle technology to develop synthetic bacterial vesicles (SyBV) that activate the immune system without the severe immunotoxicity of OMV. SyBV were generated from bacterial membranes through treatment with detergent and ionic stress. SyBV induced less inflammatory responses in macrophages and in mice compared to natural OMV. Immunization with SyBV or OMV induced comparable antigen-specific adaptive immunity. Specifically, immunization with Pseudomonas aeruginosa-derived SyBV protected mice against bacterial challenge, and this was accompanied by significant reduction in lung cell infiltration and inflammatory cytokines. Further, immunization with Escherichia coli-derived SyBV protected mice against E. coli sepsis, comparable to OMV-immunized group. The protective activity of SyBV was driven by the stimulation of B-cell and T-cell immunity. Also, SyBV were engineered to display the SARS-CoV-2 S1 protein on their surface, and these vesicles induced specific S1 protein antibody and T-cell responses. Collectively, these results demonstrate that SyBV may be a safe and efficient vaccine platform for the prevention of bacterial and viral infections
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| 4. |
- Park, Kyong-Su, et al.
(författare)
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Mesenchymal stromal cell-derived nanovesicles ameliorate bacterial outer membrane vesicle-induced sepsis via IL-10.
- 2019
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Ingår i: Stem cell research & therapy. - : Springer Science and Business Media LLC. - 1757-6512. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Sepsis remains a source of high mortality in hospitalized patients despite proper antibiotic approaches. Encouragingly, mesenchymal stromal cells (MSCs) and their produced extracellular vesicles (EVs) have been shown to elicit anti-inflammatory effects in multiple inflammatory conditions including sepsis. However, EVs are generally released from mammalian cells in relatively low amounts, and high-yield isolation of EVs is still challenging due to a complicated procedure. To get over these limitations, vesicles very similar to EVs can be produced by serial extrusions of cells, after which they are called nanovesicles (NVs). We hypothesized that MSC-derived NVs can attenuate the cytokine storm induced by bacterial outer membrane vesicles (OMVs) in mice, and we aimed to elucidate the mechanism involved.NVs were produced from MSCs by the breakdown of cells through serial extrusions and were subsequently floated in a density gradient. Morphology and the number of NVs were analyzed by transmission electron microscopy and nanoparticle tracking analysis. Mice were intraperitoneally injected with Escherichia coli-derived OMVs to establish sepsis, and then injected with 2×109 NVs. Innate inflammation was assessed in peritoneal fluid and blood through investigation of infiltration of cells and cytokine production. The biodistribution of NVs labeled with Cy7 dye was analyzed using near-infrared imaging.Electron microscopy showed that NVs have a nanometer-size spherical shape and harbor classical EV marker proteins. In mice, NVs inhibited eye exudates and hypothermia, signs of a systemic cytokine storm, induced by intraperitoneal injection of OMVs. Moreover, NVs significantly suppressed cytokine release into the systemic circulation, as well as neutrophil and monocyte infiltration in the peritoneum. The protective effect of NVs was significantly reduced by prior treatment with anti-interleukin (IL)-10 monoclonal antibody. In biodistribution study, NVs spread to the whole mouse body and localized in the lung, liver, and kidney at 6h.Taken together, these data indicate that MSC-derived NVs have beneficial effects in a mouse model of sepsis by upregulating the IL-10 production, suggesting that artificial NVs may be novel EV-mimetics clinically applicable to septic patients.
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| 5. |
- Park, Kyong-Su, et al.
(författare)
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Synthetic bacterial vesicles combined with tumour extracellular vesicles as cancer immunotherapy
- 2021
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Ingår i: Journal of Extracellular Vesicles. - : Wiley. - 2001-3078. ; 10:9
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial outer membrane vesicles (OMV) have gained attention as a promising new cancer vaccine platform for efficiently provoking immune responses. However, OMV induce severe toxicity by activating the innate immune system. In this study, we applied a simple isolation approach to produce artificial OMV that we have named Synthetic Bacterial Vesicles (SyBV) that do not induce a severe toxic response. We also explored the potential of SyBV as an immunotherapy combined with tumour extracellular vesicles to induce anti-tumour immunity. Bacterial SyBV were produced with high yield by a protocol including lysozyme and high pH treatment, resulting in pure vesicles with very few cytosolic components and no RNA or DNA. These SyBV did not cause systemic pro-inflammatory cytokine responses in mice compared to naturally released OMV. However, SyBV and OMV were similarly effective in activation of mouse bone marrow-derived dendritic cells. Co-immunization with SyBV and melanoma extracellular vesicles elicited tumour regression in melanoma-bearing mice through Th-1 type T cell immunity and balanced antibody production. Also, the immunotherapeutic effect of SyBV was synergistically enhanced by anti-PD-1 inhibitor. Moreover, SyBV displayed significantly greater adjuvant activity than other classical adjuvants. Taken together, these results demonstrate a safe and efficient strategy for eliciting specific anti-tumour responses using immunotherapeutic bacterial SyBV.
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| 6. |
- Rupert, Deborah, 1986, et al.
(författare)
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Determination of exosome concentration in solution using surface plasmon resonance spectroscopy.
- 2014
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 86:12, s. 5929-5936
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Tidskriftsartikel (refereegranskat)abstract
- Exosomes are cell-secreted nanometer-sized extracellular vesicles that have been reported to play an important role in intercellular communication. They are also considered potential diagnostic markers for various health disorders, and intense investigations are presently directed towards their use as carriers in drug-delivery and gene-therapy applications. This has generated a growing need for sensitive methods capable of accurately and specifically determining the concentration of exosomes in complex biological fluids. Here, we explore the use of label-free surface-based sensing with surface plasmon resonance (SPR) read-out to determine the concentration of exosomes in solution. Human mast cell secreted exosomes carrying the tetraspanin membrane protein CD63 were analyzed by measuring their diffusion-limited binding rate to an SPR sensor surface functionalized with anti-CD63 antibodies. The concentration of suspended exosomes was determined by first converting the SPR response into surface-bound mass. The increase in mass uptake over time was then related to the exosome concentration in solution using a formalism describing diffusion-limited binding under controlled flow conditions. The proposed quantification method is based on a calibration and control measurements performed with proteins and synthetic lipid vesicles and takes into account i) the influence of the broad size distribution of the exosomes on the surface coverage, ii) the fact that their size is comparable to the ~150 nm probing depth of SPR, and iii) possible deformation of exosomes upon adsorption. Under those considerations, the accuracy of the concentration determination was estimated to be better than ±50% and significantly better if exosome deformation is negligible.
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| 7. |
- Rupert, Deborah, 1986, et al.
(författare)
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Dual-Wavelength Surface Plasmon Resonance for Determining the Size and Concentration of Sub-Populations of Extracellular Vesicles
- 2016
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 88:20, s. 9980-9988
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Tidskriftsartikel (refereegranskat)abstract
- Accurate concentration determination of subpopulations of extracellular vesicles (EVs), such as exosomes, is of importance both in the context of understanding their fundamental biological role and of potentially using them as disease biomarkers. In principle, this can be achieved by measuring the rate of diffusion-limited mass uptake to a sensor surface modified with a receptor designed to only bind the subpopulation of interest. However, a significant error is introduced if the targeted EV subpopulation has a size, and thus hydrodynamic diffusion coefficient, that differs from the mean size and diffusion coefficient of the whole EV population and/or if the EVs become deformed upon binding to the surface. We here demonstrate a new approach to determine the mean size (or effective film thickness) of bound nanoparticles, in general, and EV subpopulation carrying a marker of interest, in particular. The method is based on operating surface plasmon resonance simultaneously at two wavelengths with different sensing depths and using the ratio of the corresponding responses to extract the particle size on the surface. By estimating in this way the degree of deformation of adsorbed EVs, we markedly improved their bulk concentration determination and showed that EVs carrying the exosomal marker CD63 correspond to not more than around 10% of the EV sample.
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| 8. |
- Rupert, Deborah, 1986, et al.
(författare)
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Methods for the physical characterization and quantification of extracellular vesicles in biological samples
- 2017
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Ingår i: Biochimica et Biophysica Acta - General Subjects. - : Elsevier BV. - 1872-8006 .- 0304-4165. ; Epub ahead of print:1, s. 3164-3179
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Forskningsöversikt (refereegranskat)abstract
- BACKGROUND:Our body fluids contain a multitude of cell-derived vesicles, secreted by most cell types, commonly referred to as extracellular vesicles. They have attracted considerable attention for their function as intercellular communication vehicles in a broad range of physiological processes and pathological conditions. Extracellular vesicles and especially the smallest type, exosomes, have also generated a lot of excitement in view of their potential as disease biomarkers or as carriers for drug delivery. In this context, state-of-the-art techniques capable of comprehensively characterizing vesicles in biological fluids are urgently needed.SCOPE OF REVIEW:This review presents the arsenal of techniques available for quantification and characterization of physical properties of extracellular vesicles, summarizes their working principles, discusses their advantages and limitations and further illustrates their implementation in vesicle research.MAJOR CONCLUSIONS:The small size and physicochemical heterogeneity of extracellular vesicles make their physical characterization and quantification an extremely challenging task. Currently, structure, size, buoyant density, optical properties and zeta potential have most commonly been studied. The concentration of vesicles in suspension can be expressed in terms of biomolecular or particle content depending on the method at hand. In addition, common quantification methods may either provide a direct quantitative measurement of vesicle concentration or solely allow for relative comparison between samples.GENERAL SIGNIFICANCE:The combination of complementary methods capable of detecting, characterizing and quantifying extracellular vesicles at a single particle level promises to provide new exciting insights into their modes of action and to reveal the existence of vesicle subpopulations fulfilling key biological tasks.
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| 9. |
- Svennerholm, Kristina, 1981, et al.
(författare)
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Escherichia coli outer membrane vesicles can contribute to sepsis induced cardiac dysfunction.
- 2017
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Sepsis induced cardiac dysfunction (SIC) is a severe complication to sepsis which significantly worsens patient outcomes. It is known that bacteria have the capacity to release outer membrane vesicles (OMVs), which are nano-sized bilayered vesicles composed of lipids and proteins, that can induce a fatal inflammatory response. The aim of this study was to determine whether OMVs from a uropathogenic Escherichia coli strain can induce cardiac dysfunction, and to elucidate any mechanisms involved. OMVs induced irregular Ca2+ oscillations with a decreased frequency in cardiomyocytes through recordings of intracellular Ca2+ dynamics. Mice were intraperitoneally injected with bacteria-free OMVs, which resulted in increased concentration of pro-inflammatory cytokine levels in blood. Cytokines were increased in heart lysates, and OMVs could be detected in the heart after OMVs injection. Troponin T was significantly increased in blood, and echocardiography showed increased heart wall thickness as well as increased heart rate. This study shows that E. coli OMVs induce cardiac injury in vitro and in vivo, in the absence of bacteria, and may be a causative microbial signal in SIC. The role of OMVs in clinical disease warrant further studies, as bacterial OMVs in addition to live bacteria may be good therapeutic targets to control sepsis.
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| 10. |
- Zhang, Guo-Qiang, et al.
(författare)
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Exogenous female sex steroid hormones and new-onset asthma in women: a matched case-control study
- 2023
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Ingår i: BMC medicine. - 1741-7015. ; 21:1
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Tidskriftsartikel (refereegranskat)abstract
- Evidence on the role of exogenous female sex steroid hormones in asthma development in women remains conflicting. We sought to quantify the potential causal role of hormonal contraceptives and menopausal hormone therapy (MHT) in the development of asthma in women.We conducted a matched case-control study based on the West Sweden Asthma Study, nested in a representative cohort of 15,003 women aged 16-75years, with 8-year follow-up (2008-2016). Data were analyzed using Frequentist and Bayesian conditional logistic regression models.We included 114 cases and 717 controls. In Frequentist analysis, the odds ratio (OR) for new-onset asthma with ever use of hormonal contraceptives was 2.13 (95% confidence interval [CI] 1.03-4.38). Subgroup analyses showed that the OR increased consistently with older baseline age. The OR for new-onset asthma with ever MHT use among menopausal women was 1.17 (95% CI 0.49-2.82). In Bayesian analysis, the ORs for ever use of hormonal contraceptives and MHT were, respectively, 1.11 (95% posterior interval [PI] 0.79-1.55) and 1.18 (95% PI 0.92-1.52). The respective probability of each OR being larger than 1 was 72.3% and 90.6%.Although use of hormonal contraceptives was associated with an increased risk of asthma, this may be explained by selection of women by baseline asthma status, given the upward trend in the effect estimate with older age. This indicates that use of hormonal contraceptives may in fact decrease asthma risk in women. Use of MHT may increase asthma risk in menopausal women.
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