Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer

Ryanodine receptors: physiological function and deregulation in Alzheimer disease

BackgroundHealth-care professionals have a responsibility to be attentive to patients’ adherence behavior but it could be difficult to identify poor adherence in the context of clinical practice. Assessment of personality could be used to identify individuals who are in need for support with their adherence behavior. To our knowledge, existing adherence questionnaires are not based on individuals reflecting asthmatics in the general population and there is limited research describing adherence with asthma medication in relation to personal goals with the treatment. The aim was to develop and validate an adherence questionnaire in adult individuals with asthma from the general population and to assess adherence in relation to personality traits and goals with the asthma medication using the developed questionnaire.MethodsThe study was conducted in three phases: 1. A preliminary postal 46-item questionnaire was refined after psychometric testing (n = 157). 2. The questionnaire was validated (n = 104). 3. The developed adherence questionnaire was analyzed in relation to personality traits and achieved goals with the asthma medication. Adult respondents with physician diagnosed asthma using asthma medications were selected from the population-based West Sweden Asthma Study. The respondents completed the Neuroticism, Extraversion and Openness to Experience Five-Factor Inventory and the Medication Adherence Report Scale and stated their goals with the asthma medication. Data were analyzed using t-tests, correlations, multiple regression and principal component analysis.ResultsA final questionnaire was developed consisting of ten items organized in three subscales - “medication routines”, “self-adjusting the medication” and “concerns about side-effects”. Two of the subscales - “medication routines” and “self-adjusting the medication” – were associated with the Medication Adherence Report Scale. The subscale “medication routines” was associated with the personality traits – Conscientiousness and Neuroticism and unachieved goals with the asthma medication.ConclusionsThe developed questionnaire appears to be useful for measuring adherence to asthma medication in adult individuals with asthma. The study suggests that both individual differences and personal treatment goals need to be addressed in efforts to promote adherence to asthma medication treatment

Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum

Extracellular vesicles (EVs), including the nano-sized exosomes, have the capacity to transfer multiple functional molecules between cells. In cell culture experiments, fetal bovine serum (FBS) is often used to supplement cell culture medium as a nutrient, but it is important to know that the FBS also contain significant quantities of EVs. The aim of the current study was to determine whether the FBS EVs can influence cultured cell phenotype, and secondly to determine the efficiency of FBS-EV elimination protocols. Firstly, FBS that had not been depleted of EVs induced a migratory phenotype in a lung cancer epithelial cell line (A549 cells), an effect that could be mimicked by isolated FBS EVs alone. FBS-derived EVs also contained RNA, which was protected from consecutive proteinase K and RNase A treatment. Comparison of common isolation protocols suggested that an 18-hour centrifugation period eliminates approximately 95% of RNA-containing FBS EVs, whereas a 1.5-hour protocol is insufficient. In conclusion, this study shows that FBS EVs substantially influence cultured cell behaviour, but also that they can be virtually removed by an 18-hour ultracentrifugation protocol

Rhinitis phenotypes correlate with different symptom presentation and risk factor patterns of asthma

SummaryBackgroundAsthma and rhinitis frequently coexist, but no population study has previously determined the relationship between nasal comorbidities and symptom expression and risk factors of asthma.MethodsIn 2008, a postal questionnaire on respiratory health was sent to 30 000 randomly selected subjects aged 16–75 years in West Sweden; 29218 could be traced and 18 087 (62%) responded. The questionnaire included questions on asthma, rhinitis, chronic rhinosinusitis, respiratory symptoms and possible determinants.ResultsPrevalence of allergic rhinitis in asthma was 63.9% and of asthma in allergic rhinitis 19.8%. Prevalence of chronic rhinosinusitis in asthma was 8.4% and of asthma in chronic rhinosinusitis 24.4%. Asthma subjects with chronic rhinitis, or chronic rhinosinusitis, had more symptoms of asthma and bronchitis than those without rhinitis (p < 0.001). There was an obvious trend of higher ORs for various environmental exposures including occupational exposure to dust, gases and fumes (OR 2.32 vs. OR 1.44), visible mould at home (OR 1.72 vs. OR 1.27) and water damage at home (OR 1.82 vs. OR 1.06) for asthma with chronic rhinosinusitis than for asthma with allergic rhinitis. Family history of allergy yielded a higher OR for asthma with allergic rhinitis than with asthma with chronic rhinosinusitis (OR 7.15 vs. OR 4.48).ConclusionConsiderable overlap between asthma and nasal comorbidities was documented, confirming a close relationship between nasal disease and asthma. Allergic rhinitis, chronic rhinitis and chronic rhinosinusitis were associated with different risk factor patterns and symptom expression of asthma. Thus, different nasal comorbidities may reflect different phenotypes of asthma

Short Course in Extracellular Vesicles – The Transition from Tissue to Liquid Biopsies

Extracellular vesicles (EVs), including exosomes and microvesicles, carry a variety of bio-macromolecules, including mRNA, microRNA, other non-coding RNAs, proteins and lipids. EVs have emerged as a promising, minimally invasive (liquid biopsies) and novel source of material for molecular diagnostics, and may provide a surrogate to tissue biopsy-based biomarkers for a variety of diseases. Although EVs can be easily identified and collected from biological fluids using commercial kits, further research and proper validation is needed in order for them to be useful in the clinical setting. Currently, several EV-based research and diagnostic companies have developed research-based kits and are in the process of working with clinical laboratories to develop and validate EV-based assays for a variety of diseases. The successful clinical application of EV-based diagnostic assays will require close collaboration between industry, academia, regulatory agencies and access to patient samples. We expect that international, integrative and interdisciplinary translational research teams, along with the emergence of FDA-approved platforms, will set the framework for EV-based diagnostics. We recognize that the EV field offers new promise for personalized/precision medicine and targeted treatment in a variety of diseases. A short course was held as a four-session webinar series in September and October 2014, presented by pioneers and experts in the EV domain, covering a broad range of topics from an overview of the field to its applications, and the current state and challenges of the commercialization of EVs for research and an introduction to the clinic. It was concluded with a panel discussion on the regulatory aspects and funding opportunities in this field. A summary of the short course is presented as a meeting dispatch

Immune response modifiers in the treatment of asthma: A PRACTALL document of the American Academy of Allergy, Asthma & Immunology and the European Academy of Allergy and Clinical Immunology

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Extracellular vesicle DNA from human melanoma tissues contains cancer-specific mutations

Liquid biopsies are promising tools for early diagnosis and residual disease monitoring in patients with cancer, and circulating tumor DNA isolated from plasma has been extensively studied as it has been shown to contain tumor-specific mutations. Extracellular vesicles (EVs) present in tumor tissues carry tumor-derived molecules such as proteins and nucleic acids, and thus EVs can potentially represent a source of cancer-specific DNA. Here we identified the presence of tumor-specific DNA mutations in EVs isolated from six human melanoma metastatic tissues and compared the results with tumor tissue DNA and plasma DNA. Tumor tissue EVs were isolated using enzymatic treatment followed by ultracentrifugation and iodixanol density cushion isolation. A panel of 34 melanoma-related genes was investigated using ultra-sensitive sequencing (SiMSen-seq). We detected mutations in six genes in the EVs (BRAF, NRAS, CDKN2A, STK19, PPP6C, and RAC), and at least one mutation was detected in all melanoma EV samples. Interestingly, the mutant allele frequency was higher in DNA isolated from tumor-derived EVs compared to total DNA extracted directly from plasma DNA, supporting the potential role of tumor EVs as future biomarkers in melanom

Spin and Statistics and First Principles

It was shown in the early Seventies that, in Local Quantum Theory (that is the most general formulation of Quantum Field Theory, if we leave out only the unknown scenario of Quantum Gravity) the notion of Statistics can be grounded solely on the local observable quantities (without assuming neither the commutation relations nor even the existence of unobservable charged field operators); one finds that only the well known (para)statistics of Bose/Fermi type are allowed by the key principle of local commutativity of observables. In this frame it was possible to formulate and prove the Spin and Statistics Theorem purely on the basis of First Principles. In a subsequent stage it has been possible to prove the existence of a unique, canonical algebra of local field operators obeying ordinary Bose/Fermi commutation relations at spacelike separations. In this general guise the Spin - Statistics Theorem applies to Theories (on the four dimensional Minkowski space) where only massive particles with finite mass degeneracy can occur. Here we describe the underlying simple basic ideas, and briefly mention the subsequent generalisations; eventually we comment on the possible validity of the Spin - Statistics Theorem in presence of massless particles, or of violations of locality as expected in Quantum Gravity.Comment: Survey based on a talk given at the Meeting on "Theoretical and experimental aspects of the spin - statistics connection and related symmetries", Trieste, Italy - October 21-25, 200

Network analysis of quantitative proteomics on asthmatic bronchi: effects of inhaled glucocorticoid treatment

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>Endobronchial biopsies were taken from untreated asthmatic patients (<it>n </it>= 12) and healthy controls (<it>n </it>= 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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p> <p>Trial registration</p> <p>ClinicalTrials.gov registration <a href="http://www.clinicaltrials.gov/ct2/show/NCT01378039">NCT01378039</a></p