| 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. |
- Lu, You, 1982, et al.
(författare)
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Expansion of CD4 + CD25 + and CD25- T-Bet, GATA-3, Foxp3 and RORγt Cells in Allergic Inflammation, Local Lung Distribution and Chemokine Gene Expression.
- 2011
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 6:5
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Tidskriftsartikel (refereegranskat)abstract
- Allergic asthma is associated with airway eosinophilia, which is regulated by different T-effector cells. T cells express transcription factors T-bet, GATA-3, RORγt and Foxp3, representing Th1, Th2, Th17 and Treg cells respectively. No study has directly determined the relative presence of each of these T cell subsets concomitantly in a model of allergic airway inflammation. In this study we determined the degree of expansion of these T cell subsets, in the lungs of allergen challenged mice. Cell proliferation was determined by incorporation of 5-bromo-2'-deoxyuridine (BrdU) together with 7-aminoactnomycin (7-AAD). The immunohistochemical localisation of T cells in the lung microenvironments was also quantified. Local expression of cytokines, chemokines and receptor genes was measured using real-time RT-PCR array analysis in tissue sections isolated by laser microdissection and pressure catapulting technology. Allergen exposure increased the numbers of T-bet(+), GATA-3(+), RORγt(+) and Foxp3(+) cells in CD4(+)CD25(+) and CD4(+)CD25(-) T cells, with the greatest expansion of GATA-3(+) cells. The majority of CD4(+)CD25(+) T-bet(+), GATA-3(+), RORγt(+) and Foxp3(+) cells had incorporated BrdU and underwent proliferation during allergen exposure. Allergen exposure led to the accumulation of T-bet(+), GATA-3(+) and Foxp3(+) cells in peribronchial and alveolar tissue, GATA-3(+) and Foxp3(+) cells in perivascular tissue, and RORγt(+) cells in alveolar tissue. A total of 28 cytokines, chemokines and receptor genes were altered more than 3 fold upon allergen exposure, with expression of half of the genes claimed in all three microenvironments. Our study shows that allergen exposure affects all T effector cells in lung, with a dominant of Th2 cells, but with different local cell distribution, probably due to a distinguished local inflammatory milieu.
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| 4. |
- O'Neil, Serena, 1981, et al.
(författare)
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Clinical proteomics stretch goals: EuPA 2012 roundtable report
- 2013
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 88, s. 37-40
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Tidskriftsartikel (refereegranskat)abstract
- The field of clinical proteomics is faced with multiple challenges which need to be overcome in order to improve our understanding of human diseases and provide management solutions. Researchers interested in clinical proteomics assembled for a roundtable discussion at the European Association for Proteomics (EuPA) conference held in Glasgow in July 2012, to discuss these challenges and highlight the key areas for successful clinical proteomic studies. This report shares topics of discussion and the resulting stretch goals of clinical proteomics for researchers to strive towards. This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].
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| 5. |
- O'Neil, Serena, 1981, et al.
(författare)
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Network analysis of quantitative proteomics on asthmatic bronchi: effects of inhaled glucocorticoid treatment
- 2011
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Ingår i: Respiratory Research. - : Springer Science and Business Media LLC. - 1465-9921 .- 1465-993X. ; 12:124
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Proteomic studies of respiratory disorders have the potential to identify protein biomarkers for diagnosis and disease monitoring. Utilisation of sensitive quantitative proteomic methods creates opportunities to determine individual patient proteomes. The aim of the current study was to determine if quantitative proteomics of bronchial biopsies from asthmatics can distinguish relevant biological functions and whether inhaled glucocorticoid treatment affects these functions. METHODS: Endobronchial biopsies were taken from untreated asthmatic patients (n = 12) and healthy controls (n = 3). Asthmatic patients were randomised to double blind treatment with either placebo or budesonide (800 μg daily for 3 months) and new biopsies were obtained. Proteins extracted from the biopsies were digested and analysed using isobaric tags for relative and absolute quantitation combined with a nanoLC-LTQ Orbitrap mass spectrometer. Spectra obtained were used to identify and quantify proteins. Pathways analysis was performed using Ingenuity Pathway Analysis to identify significant biological pathways in asthma and determine how the expression of these pathways was changed by treatment. RESULTS: More than 1800 proteins were identified and quantified in the bronchial biopsies of subjects. The pathway analysis revealed acute phase response signalling, cell-to-cell signalling and tissue development associations with proteins expressed in asthmatics compared to controls. The functions and pathways associated with placebo and budesonide treatment showed distinct differences, including the decreased association with acute phase proteins as a result of budesonide treatment compared to placebo. CONCLUSIONS: Proteomic analysis of bronchial biopsy material can be used to identify and quantify proteins using highly sensitive technologies, without the need for pooling of samples from several patients. Distinct pathophysiological features of asthma can be identified using this approach and the expression of these features is changed by inhaled glucocorticoid treatment. Quantitative proteomics may be applied to identify mechanisms of disease that may assist in the accurate and timely diagnosis of asthma
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| 6. |
- O'Neil, Serena, 1981, et al.
(författare)
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Proteomics in asthma and COPD phenotypes and endotypes for biomarker discovery and improved understanding of disease entities
- 2011
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 75:1, s. 192-201
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Tidskriftsartikel (refereegranskat)abstract
- The application of proteomics to respiratory diseases, such as asthma and COPD, has been limited compared to other fields, like cancer. Both asthma and COPD are recognised to be multi-factorial and complex diseases, both consisting of clusters of multiple disease phenotypes. The complexity of these diseases combined with the inaccessibility and invasiveness of disease relevant samples have provided a hurdle to the progress of respiratory proteomics. Advances in proteomic instrumentation and methodology have led to the possibility to identify proteomes in much smaller quantities of biological material. This review focuses on the efforts in respiratory proteomics in relation to asthma and COPD, and the importance of identifying subgroups of disease entities to establish appropriate biomarkers, and to enhance the understanding of underlying mechanisms in each subgroup. Careful phenotype characterisation of patient subpopulations is required to make improvement in the field of heterogeneous diseases such as asthma and COPD, and the clusters of phenotypes are likely to encompass subgroups of disease with distinct molecular mechanisms; endotypes. The utilisation of modern advanced proteomics in endotypes of asthma and COPD will likely contribute to the increased understanding of disease mechanisms, establishment of biomarkers for these endotypes and improved patient care.
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| 7. |
- O'Neil, Serena, 1981, et al.
(författare)
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Quantitative expression of osteopontin in nasal mucosa of patients with allergic rhinitis: effects of pollen exposure and nasal glucocorticoid treatment
- 2010
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Ingår i: Allergy, Asthma & Clinical Immunology. - : Springer Science and Business Media LLC. - 1710-1492. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Abstract Background Osteopontin (OPN) is a multifunctional cytokine that has been primarily investigated in Th1 diseases. Recently, it has also been implicated in Th2-mediated allergic diseases, such as asthma. The expression of OPN in allergic rhinitis (AR) is currently unknown, as is the effect of intranasal glucocorticosteroids (GCs) on that expression. Methods Subjects with AR were randomised to receive treatment with fluticasone propionate (FP) (n = 12) or a placebo (n = 16) over the grass pollen season and nasal biopsies were taken prior to, and during the season. OPN expression in the nasal mucosa was examined with immunohistochemistry. Healthy non-AR controls (n = 5) were used as a comparator. Results OPN expression was detected in epithelial cells, subepithelial infiltrating/inflammatory cells and cells lining the vessels and glands of all subjects. Comparison of the pre- and peak-pollen season biopsy sections in placebo treated patients revealed no increase in OPN expression during the grass pollen season (5.7% vs 6.4%). Treatment with a local glucocorticosteroid did not alter the expression of OPN during pollen exposure (6.2% vs 6.7%). Conclusion OPN has been increasingly associated with the pathogenesis of various Th2-mediated diseases. However, our finding that the OPN expression in the nasal mucosa of AR patients is not significantly affected by allergen exposure and is comparable to that of the healthy controls, suggests that intracellular OPN is not directly involved in the pathogenesis of allergic rhinitis.
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| 8. |
- Papadopoulos, Nikolaos G, et al.
(författare)
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Research needs in allergy: an EAACI position paper, in collaboration with EFA.
- 2012
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Ingår i: Clinical and translational allergy. - : Wiley. - 2045-7022. ; 2:1
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Tidskriftsartikel (refereegranskat)abstract
- ABSTRACT: In less than half a century, allergy, originally perceived as a rare disease, has become a major public health threat, today affecting the lives of more than 60 million people in Europe, and probably close to one billion worldwide, thereby heavily impacting the budgets of public health systems. More disturbingly, its prevalence and impact are on the rise, a development that has been associated with environmental and lifestyle changes accompanying the continuous process of urbanization and globalization. Therefore, there is an urgent need to prioritize and concert research efforts in the field of allergy, in order to achieve sustainable results on prevention, diagnosis and treatment of this most prevalent chronic disease of the 21st century.The European Academy of Allergy and Clinical Immunology (EAACI) is the leading professional organization in the field of allergy, promoting excellence in clinical care, education, training and basic and translational research, all with the ultimate goal of improving the health of allergic patients. The European Federation of Allergy and Airways Diseases Patients' Associations (EFA) is a non-profit network of allergy, asthma and Chronic Obstructive Pulmonary Disorder (COPD) patients' organizations. In support of their missions, the present EAACI Position Paper, in collaboration with EFA, highlights the most important research needs in the field of allergy to serve as key recommendations for future research funding at the national and European levels.Although allergies may involve almost every organ of the body and an array of diverse external factors act as triggers, there are several common themes that need to be prioritized in research efforts. As in many other chronic diseases, effective prevention, curative treatment and accurate, rapid diagnosis represent major unmet needs. Detailed phenotyping/endotyping stands out as widely required in order to arrange or re-categorize clinical syndromes into more coherent, uniform and treatment-responsive groups. Research efforts to unveil the basic pathophysiologic pathways and mechanisms, thus leading to the comprehension and resolution of the pathophysiologic complexity of allergies will allow for the design of novel patient-oriented diagnostic and treatment protocols. Several allergic diseases require well-controlled epidemiological description and surveillance, using disease registries, pharmacoeconomic evaluation, as well as large biobanks. Additionally, there is a need for extensive studies to bring promising new biotechnological innovations, such as biological agents, vaccines of modified allergen molecules and engineered components for allergy diagnosis, closer to clinical practice. Finally, particular attention should be paid to the difficult-to-manage, precarious and costly severe disease forms and/or exacerbations. Nonetheless, currently arising treatments, mainly in the fields of immunotherapy and biologicals, hold great promise for targeted and causal management of allergic conditions. Active involvement of all stakeholders, including Patient Organizations and policy makers are necessary to achieve the aims emphasized herein.
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