Ion mobility-tandem mass spectrometry of mucin-type O-glycans

The dense O-glycosylation of mucins plays an important role in the defensive properties of the mucus hydrogel. Aberrant glycosylation is often correlated with inflammation and pathology such as COPD, cancer, and Crohn’s disease. The inherent complexity of glycans and the diversity in the O-core structure constitute fundamental challenges for the analysis of mucin-type O-glycans. Due to coexistence of multiple isomers, multidimensional workflows such as LC-MS are required. To separate the highly polar carbohydrates, porous graphitized carbon is often used as a stationary phase. However, LC-MS workflows are time-consuming and lack reproducibility. Here we present a rapid alternative for separating and identifying O-glycans released from mucins based on trapped ion mobility mass spectrometry. Compared to established LC-MS, the acquisition time is reduced from an hour to two minutes. To test the validity, the developed workflow was applied to sputum samples from cystic fibrosis patients to map O-glycosylation features associated with disease

The ENaC-overexpressing mouse as a model of cystic fibrosis lung disease

AbstractChronic lung disease remains the major cause of morbidity and mortality of cystic fibrosis (CF) patients. Cftr mutant mice developed severe intestinal obstruction, but did not exhibit the characteristic CF ion transport defects (i.e. deficient cAMP-dependent Cl− secretion and increased Na+ absorption) in the lower airways, and failed to develop CF-like lung disease. These observations led to the generation of transgenic mice with airway-specific overexpression of the epithelial Na+ channel (ENaC) as an alternative approach to mimic CF ion transport pathophysiology in the lung. Studies of the phenotype of βENaC-transgenic mice demonstrated that increased airway Na+ absorption causes airway surface liquid (ASL) depletion, reduced mucus transport and a spontaneous CF-like lung disease with airway mucus obstruction and chronic airway inflammation. Here, we summarize approaches that can be applied for studies of the complex in vivo pathogenesis and preclinical evaluation of novel therapeutic strategies in this model of CF lung disease

Effects of lumacaftor—ivacaftor therapy on cystic fibrosis transmembrane conductance regulator function in F508del homozygous patients with cystic fibrosis aged 2–11 years

Rationale: Lumacaftor/ivacaftor was approved for the treatment of patients with cystic fibrosis who are homozygous for F508del aged 2 years and older following positive results from phase three trials. However, the improvement in CFTR function associated with lumacaftor/ivacaftor has only been studied in patients over 12 years of age, while the rescue potential in younger children is unknown.Methods: In a prospective study, we aimed to evaluate the effect of lumacaftor/ivacaftor on the CFTR biomarkers sweat chloride concentration and intestinal current measurement as well as clinical outcome parameters in F508del homozygous CF patients 2–11 years before and 8–16 weeks after treatment initiation.Results: A total of 13 children with CF homozygous for F508del aged 2–11 years were enrolled and 12 patients were analyzed. Lumacaftor/ivacaftor treatment reduced sweat chloride concentration by 26.8 mmol/L (p = 0.0006) and showed a mean improvement in CFTR activity, as assessed by intestinal current measurement in the rectal epithelium, of 30.5% compared to normal (p = 0.0015), exceeding previous findings of 17.7% of normal in CF patients homozygous for F508del aged 12 years and older.Conclusion: Lumacaftor/ivacaftor partially restores F508del CFTR function in children with CF who are homozygous for F508del, aged 2–11 years, to a level of CFTR activity seen in patients with CFTR variants with residual function. These results are consistent with the partial short-term improvement in clinical parameters

Hypertonic Saline Is Effective in the Prevention and Treatment of Mucus Obstruction, but Not Airway Inflammation, in Mice with Chronic Obstructive Lung Disease

Recent evidence suggests that inadequate hydration of airway surfaces is a common mechanism in the pathogenesis of airway mucus obstruction. Inhaled hypertonic saline (HS) induces osmotic water flux, improving hydration of airway surfaces. However, trials in patients with obstructive lung diseases are limited. The aim of this study was to investigate effects of HS on mucus obstruction and airway inflammation in the prevention and treatment of obstructive lung disease in vivo. We, therefore, used the β-epithelial Na+ channel (βENaC)–overexpressing mouse as a model of chronic obstructive lung disease and determined effects of preventive and late therapy with 3% HS and 7% HS on pulmonary mortality, airway mucus obstruction, and inflammation. We found that preventive treatment with 3% HS and 7% HS improved growth, reduced mortality, and reduced mucus obstruction in neonatal βENaC-overexpressing mice. In adult βENaC-overexpressing mice with chronic lung disease, mucus obstruction was significantly reduced by 7% HS, but not by 3% HS. Treatment with HS triggered airway inflammation with elevated keratinocyte chemoattractant levels and neutrophils in airways from wild-type mice, but reduced keratinocyte chemoattractant in chronic neutrophilic inflammation in adult βENaC-overexpressing mice. Our data demonstrate that airway surface rehydration with HS provides an effective preventive and late therapy of mucus obstruction with no consistent effects on inflammation in chronic lung disease. These results suggest that, through mucokinetic effects, HS may be beneficial for patients with a spectrum of obstructive lung diseases, and that additional strategies are required for effective treatment of associated airway inflammation

TRACK-CF prospective cohort study: Understanding early cystic fibrosis lung disease.

BACKGROUND Lung disease as major cause for morbidity in patients with cystic fibrosis (CF) starts early in life. Its large phenotypic heterogeneity is partially explained by the genotype but other contributing factors are not well delineated. The close relationship between mucus, inflammation and infection, drives morpho-functional alterations already early in pediatric CF disease, The TRACK-CF cohort has been established to gain insight to disease onset and progression, assessed by lung function testing and imaging to capture morpho-functional changes and to associate these with risk and protective factors, which contribute to the variation of the CF lung disease progression. METHODS AND DESIGN TRACK-CF is a prospective, longitudinal, observational cohort study following patients with CF from newborn screening or clinical diagnosis throughout childhood. The study protocol includes monthly telephone interviews, quarterly visits with microbiological sampling and multiple-breath washout and as well as a yearly chest magnetic resonance imaging. A parallel biobank has been set up to enable the translation from the deeply phenotyped cohort to the validation of relevant biomarkers. The main goal is to determine influencing factors by the combined analysis of clinical information and biomaterials. Primary endpoints are the lung clearance index by multiple breath washout and semi-quantitative magnetic resonance imaging scores. The frequency of pulmonary exacerbations, infection with pro-inflammatory pathogens and anthropometric data are defined as secondary endpoints. DISCUSSION This extensive cohort includes children after diagnosis with comprehensive monitoring throughout childhood. The unique composition and the use of validated, sensitive methods with the attached biobank bears the potential to decisively advance the understanding of early CF lung disease. ETHICS AND TRIAL REGISTRATION The study protocol was approved by the Ethics Committees of the University of Heidelberg (approval S-211/2011) and each participating site and is registered at clinicaltrials.gov (NCT02270476)

TRACK-CF prospective cohort study: Understanding early cystic fibrosis lung disease

BackgroundLung disease as major cause for morbidity in patients with cystic fibrosis (CF) starts early in life. Its large phenotypic heterogeneity is partially explained by the genotype but other contributing factors are not well delineated. The close relationship between mucus, inflammation and infection, drives morpho-functional alterations already early in pediatric CF disease, The TRACK-CF cohort has been established to gain insight to disease onset and progression, assessed by lung function testing and imaging to capture morpho-functional changes and to associate these with risk and protective factors, which contribute to the variation of the CF lung disease progression.Methods and designTRACK-CF is a prospective, longitudinal, observational cohort study following patients with CF from newborn screening or clinical diagnosis throughout childhood. The study protocol includes monthly telephone interviews, quarterly visits with microbiological sampling and multiple-breath washout and as well as a yearly chest magnetic resonance imaging. A parallel biobank has been set up to enable the translation from the deeply phenotyped cohort to the validation of relevant biomarkers. The main goal is to determine influencing factors by the combined analysis of clinical information and biomaterials. Primary endpoints are the lung clearance index by multiple breath washout and semi-quantitative magnetic resonance imaging scores. The frequency of pulmonary exacerbations, infection with pro-inflammatory pathogens and anthropometric data are defined as secondary endpoints.DiscussionThis extensive cohort includes children after diagnosis with comprehensive monitoring throughout childhood. The unique composition and the use of validated, sensitive methods with the attached biobank bears the potential to decisively advance the understanding of early CF lung disease.Ethics and trial registrationThe study protocol was approved by the Ethics Committees of the University of Heidelberg (approval S-211/2011) and each participating site and is registered at clinicaltrials.gov (NCT02270476)

Toxic Diatom Aldehydes Affect Defence Gene Networks in Sea Urchins.

Marine organisms possess a series of cellular strategies to counteract the negative effects of toxic compounds, including the massive reorganization of gene expression networks. Here we report the modulated dose-dependent response of activated genes by diatom polyunsaturated aldehydes (PUAs) in the sea urchin Paracentrotus lividus. PUAs are secondary metabolites deriving from the oxidation of fatty acids, inducing deleterious effects on the reproduction and development of planktonic and benthic organisms that feed on these unicellular algae and with anti-cancer activity. Our previous results showed that PUAs target several genes, implicated in different functional processes in this sea urchin. Using interactomic Ingenuity Pathway Analysis we now show that the genes targeted by PUAs are correlated with four HUB genes, NF-κB, p53, δ-2-catenin and HIF1A, which have not been previously reported for P. lividus. We propose a working model describing hypothetical pathways potentially involved in toxic aldehyde stress response in sea urchins. This represents the first report on gene networks affected by PUAs, opening new perspectives in understanding the cellular mechanisms underlying the response of benthic organisms to diatom exposure

Circulating microRNAs in sera correlate with soluble biomarkers of immune activation but do not predict mortality in ART treated individuals with HIV-1 infection: A case control study

Introduction: The use of anti-retroviral therapy (ART) has dramatically reduced HIV-1 associated morbidity and mortality. However, HIV-1 infected individuals have increased rates of morbidity and mortality compared to the non-HIV-1 infected population and this appears to be related to end-organ diseases collectively referred to as Serious Non-AIDS Events (SNAEs). Circulating miRNAs are reported as promising biomarkers for a number of human disease conditions including those that constitute SNAEs. Our study sought to investigate the potential of selected miRNAs in predicting mortality in HIV-1 infected ART treated individuals. Materials and Methods: A set of miRNAs was chosen based on published associations with human disease conditions that constitute SNAEs. This case: control study compared 126 cases (individuals who died whilst on therapy), and 247 matched controls (individuals who remained alive). Cases and controls were ART treated participants of two pivotal HIV-1 trials. The relative abundance of each miRNA in serum was measured, by RTqPCR. Associations with mortality (all-cause, cardiovascular and malignancy) were assessed by logistic regression analysis. Correlations between miRNAs and CD4+ T cell count, hs-CRP, IL-6 and D-dimer were also assessed. Results: None of the selected miRNAs was associated with all-cause, cardiovascular or malignancy mortality. The levels of three miRNAs (miRs -21, -122 and -200a) correlated with IL-6 while miR-21 also correlated with D-dimer. Additionally, the abundance of miRs -31, -150 and -223, correlated with baseline CD4+ T cell count while the same three miRNAs plus miR- 145 correlated with nadir CD4+ T cell count. Discussion: No associations with mortality were found with any circulating miRNA studied. These results cast doubt onto the effectiveness of circulating miRNA as early predictors of mortality or the major underlying diseases that contribute to mortality in participants treated for HIV-1 infection