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BackgroundExosomes 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)].MethodsNasal 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.ResultsFirstly, 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.ConclusionsNasal 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0927-4) contains supplementary material, which is available to authorized users.

In a prospective study 101 children aged less than 2 years (median age 10 months), were examined the first time they were admitted to a paediatric ward for asthmatic symptoms. Two-thirds were boys and 58 had parents or siblings with allergic symptoms. During winter-spring, respiratory syncytial (RS) virus was verified in 50% of children. Other viral agents were adenovirus, parainfluenza 3, coxsackie B 2, ECHO 6 and rotavirus. At the acute stage, 54% of the children displayed changes on pulmonary X-ray. The total IgE value was greater than or equal to +2 SD score units in 14 children. At reinvestigation after 3-4.5 years, when the children were aged 3.3-6.3 years, 53% were free from asthmatic symptoms; the median age for the last episode was 2 years. A total of 33% had mild asthma, 8% moderate and 6% severe asthma. The factors which correlated significantly with persistent asthma were: (1) The need for daily medication for at least 6 months. (2) A young age in conjunction with the first wheezing episode and on the first admission to a paediatric ward because of asthmatic symptoms. (3) Other past or present atopic symptoms. Heredity, tobacco smoking at home, having a furry pet, RS virus infection, or high total IgE at the time of the first admission did not correlate significantly with the persistence of asthma 3-4.5 years later. The results emphasize the good overall prognosis of wheezing in early childhood, even when the wheezing is severe enough to lead to inpatient treatment.

Amyloid-beta(Abeta) aggregation is a major hallmark of Alzheimer's disease (AD). Previous studies have suggested that only unbound Abeta can take part in the aggregation process. Therefore, endogenous Abeta-binding proteins may have an important role in preventing AD. Here, we analyzed cerebrospinal fluid (CSF) samples from 35 subjects with AD, 18 subjects with frontotemporal dementia (FTD) and 29 non-demented controls to test if reduced Abeta-binding capacity in CSF is a specific feature of AD. A panel of known Abeta-binding CSF proteins, including beta-trace/prostaglandin D2 synthase (beta-trace), transthyretin (TTR), cystatin C (CysC) and alpha(1)-antitrypsin (AAT), were quantified and related to diagnosis and CSF levels of Abeta(1-38), Abeta(1-40) and Abeta(1-42). AD patients displayed a mild reduction in the CSF levels of beta-trace (p=0.020), CysC (p=0.017), AAT (p=0.019) and TTR (p=0.012) compared with controls. While the reductions in AAT and TTR were AD-specific, the levels of beta-trace and CysC were also reduced in FTD. As expected, CSF Abeta(1-42) was reduced in AD compared with controls (p=0.00005) and with FTD patients (p=0.015). Positive correlations between Abeta(1-42) and beta-trace, CysC and TTR, respectively, were seen only in the AD group, suggesting that deficient Abeta-binding capacity in CSF may contribute to the amyloidogenic process in AD.

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.

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.

Objective: A recent study using surface-enhanced laser desorption/ionization time-of-flight analysis of cerebrospinal fluid identified a 12.5kDa truncated isoform of cystatin C (CysC) as a specific biomarker for multiple sclerosis (MS). Methods:Surface-enhanced laser desorption/ionization time-of-flight analysis of cerebrospinal fluid samples from 43 MS patients and 46 healthy control subjects. Results: Full-length CysC (13.4kDa) concentration was similar in MS and control samples. The 12.5kDa CysC protein was produced from full-length CysC by N-terminal cleavage during storage at Ϫ20°C. Interpretation: The 12.5kDa CysC isoform is a storagerelated artifact and is not useful as a diagnostic marker for MS.Ann Neurol 2007;62:193-196 A recent study that used surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry on cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) identified a unique 12.5kDa protein peak that distinguished MS from other neurological disorders with 100% specificity.

Despite improvements in pre-clinical drug testing models, predictability of clinical outcomes continues to be inadequate and costly due to poor evidence of drug metabolism. Humanized miniature organs integrating decellularized rodent organs with tissue specific cells are translational models that can provide further physiological understanding and evidence. Here, we evaluated 4-Flow cannulated rat hearts as the fundamental humanized organ model for cardiovascular drug validation. Results show clearance of cellular components in all chambers in 4-Flow hearts with efficient perfusion into both coronary arteries and cardiac veins. Furthermore, material characterization depicts preserved organization and content of important matrix proteins such as collagens, laminin, and elastin. With access to the complete vascular network, different human cell types were delivered to show spatial distribution and integration into the matrix under perfusion for up to three weeks. The feature of 4-Flow cannulation is the preservation of whole heart conformity enabling ventricular pacing via the pulmonary vein as demonstrated by noninvasive monitoring with fluid pressure and ultrasound imaging. Consequently, 4-Flow hearts surmounting organ mimicry challenges with intact complexity in vasculature and mechanical compliance of the whole organ providing an ideal platform for improving pre-clinical drug validation in addition to understanding cardiovascular diseases.