| 1. |
- Portelius, Erik, 1977, et al.
(author)
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Characterization of tau in cerebrospinal fluid using mass spectrometry.
- 2008
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In: Journal of proteome research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:5, s. 2114-20
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Journal article (peer-reviewed)abstract
- The neurodegenerative disorder Alzheimer's disease (AD) is the most common cause of dementia in the elderly. The presence of neurofibrillary tangles, consisting of hyperphosphorylated tau protein, is one of the major neuropathologic characteristics of the disease, making this protein an attractive biomarker for AD and a possible target for therapy. Here, we describe an optimized immunoprecipitation mass spectrometry method that enables, for the first time, detailed characterization of tau in human cerebrospinal fluid. The identities of putative tau fragments were confirmed using nanoflow liquid chromatography and tandem mass spectrometry. Nineteen tryptic fragments of tau were detected, of which 16 are found in all tau isoforms while 3 represented unique tau isoforms. These results pave the way for clinical CSF studies on the tauopathies.
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| 2. |
- Folkesson Hansson, Sara, 1976, et al.
(author)
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Enteroviruses and the pathogenesis of type 1 diabetes revisited: cross-reactivity of enterovirus capsid protein (VP1) antibodies with human mitochondrial proteins
- 2013
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In: Journal of Pathology. - : Wiley. - 0022-3417 .- 1096-9896. ; 229:5, s. 719-728
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Journal article (peer-reviewed)abstract
- Current or recent enteroviral infections show an association with type 1 diabetes. However, evidence for this has mainly been generated using a particular mouse monoclonal antibody (clone 5-D8/1) which binds the viral capsid protein VP1. Difficulty in confirming these findings using other independent methods has led to the concern that this might be artefactual. To address this, we examined the potential cross-reactivity of clone 5-D8/1 with normal islet proteins. Western blotting, two-dimensional gel electrophoresis, and mass spectrometry were used to identify human islet proteins bound by the clone 5-D8/1. We found a distinct reactivity with two mitochondrial proteins, creatine kinase B-type and ATP synthase beta subunit. Immunohistochemistry using the clone 5-D8/1 revealed a granular cytoplasmic staining pattern in mitochondria-rich cells, ie hepatocytes, ductal epithelial cells, vascular endothelial cells, skeletal muscle cells, and the neoplastic salivary gland oncocytoma cells, whereas connective tissue and infiltrating immune cells were negative. Staining on islets of Langerhans from subjects with recent-onset type 1 diabetes, but not on isolated human islets infected in vitro with enteroviruses, could be blocked after mixing the clone 5-D8/1 with the mitochondrial proteins. Collectively, our data show that the clone 5-D8/1 detects two human mitochondrial enzymes in addition to enteroviral VP1. The notion that the previously reported VP1 positivity in islets of recent-onset type 1 diabetes patients could reflect cross-reactivity to native islet proteins and not the presence of EV is supported by difficulties in demonstrating EV infection by independent techniques such as PCR or in situ hybridization. These findings call for revisiting the presence of enteroviruses in pancreatic islets of patients with type 1 diabetes. Copyright © 2013 Pathological Society of Great Britain and Ireland
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| 3. |
- Folkesson Hansson, Sara, 1976
(author)
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Proteomic strategies for analysis of cerebrospinal fluid in neurodegenerative disorders
- 2008
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Doctoral thesis (other academic/artistic)abstract
- ABSTRACT There is a great need for biomarkers to diagnose neurodegenerative disorders, such as the cognitive disorders Alzheimer?s disease (AD) and frontotemporal dementia (FTD). Cerebrospinal fluid (CSF) is in contact with the extracellular fluid of the brain and is consequently a valuable medium for identifying biomarkers for neurological disorders. Biomarkers can be used for early identification of disease, to facilitate homogenous classification and to extend our basic knowledge of disease pathogenesis. Proteomics, an approach for biomarker discovery, generally combines various separation techniques with mass spectrometry (MS) and bioinformatics to identify and characterize proteins, reflecting a defined state at a specific time point. The aim of this thesis was to develop and evaluate proteomic strategies for analysis of CSF proteins to reveal disease mechanisms and identify potential biomarkers to distinguish AD from FTD. Two approaches to improve the detection of CSF proteins by two-dimensional gel electrophoresis (2-DGE) were used. First, to enrich the proteins, CSF was prefractionated using liquid phase isoelectric focusing followed by 2-DGE profiling. Secondly, zoom 2D gels increased protein separation directly in the gels. These studies showed that in the CSF proteome of AD and FTD patients several proteins were differentially expressed, suggesting that different mechanisms are involved in the pathogenesis of these disorders. To validate some of the findings from the 2-DGE studies, beta-trace, transthyretin (TTR), alpha-1-antitrypsin and cystatin C (CysC) were quantified in CSF. The concentrations of all these proteins, previously shown to bind amyloid-beta (Abeta) peptides, were reduced in AD CSF, while only CysC and beta-trace were reduced in FTD. Furthermore, we found a strong positive correlation between beta-trace, TTR and CysC, and levels of Abeta peptides specifically in the AD group, suggesting that a lack of proteins binding to Abeta peptides in AD CSF might cause increased extracellular Abeta aggregation, a major pathological hallmark in the AD brain. Additionally, we showed that incorrect storage conditions can influence the isoform levels of some CSF proteins. Thus, standardization of CSF sample handling is important in avoiding ambiguous results. Furthermore, very low-abundant neuron specific tau protein isoforms, were for the first time characterized in CSF using a targeted immunoprecipitation-MS approach, opening up new possibilities for further differentiation of tauopathies, including AD and FTD.
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| 4. |
- Folkesson Hansson, Sara, 1976, et al.
(author)
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Validation of a prefractionation method followed by two-dimensional electrophoresis - Applied to cerebrospinal fluid proteins from frontotemporal dementia patients.
- 2004
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In: Proteome science. - : Springer Science and Business Media LLC. - 1477-5956. ; 2:1
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Journal article (peer-reviewed)abstract
- BACKGROUND: The aim of this study was firstly, to improve and validate a cerebrospinal fluid (CSF) prefractionation method followed by two-dimensional electrophoresis (2-DE) and secondly, using this strategy to investigate differences between the CSF proteome of frontotemporal dementia (FTD) patients and controls. From each subject three ml of CSF was prefractionated using liquid phase isoelectric focusing prior to 2-DE. RESULTS: With respect to protein recovery and purification potential, ethanol precipitation of the prefractionated CSF sample was found superior, after testing several sample preparation methods.The reproducibility of prefractionated CSF analyzed on 2-D gels was comparable to direct 2-DE analysis of CSF. The protein spots on the prefractionated 2-D gels had an increased intensity, indicating a higher protein concentration, compared to direct 2-D gels. Prefractionated 2-DE analysis of FTD and control CSF showed that 26 protein spots were changed at least two fold. Using mass spectrometry, 13 of these protein spots were identified, including retinol-binding protein, Zn-alpha-2-glycoprotein, proapolipoproteinA1, beta-2-microglobulin, transthyretin, albumin and alloalbumin. CONCLUSION: The results suggest that the prefractionated 2-DE method can be useful for enrichment of CSF proteins and may provide a new tool to investigate the pathology of neurodegenerative diseases. This study confirmed reduced levels of retinol-binding protein and revealed some new biomarker candidates for FTD.
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| 5. |
- Lässer, Cecilia, 1981, et al.
(author)
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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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In: Journal of Translational Medicine. - : Springer Science and Business Media LLC. - 1479-5876. ; 14:1
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Journal article (peer-reviewed)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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