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1.  Quantitative Stratification of Diffuse Parenchymal Lung Diseases 
PLoS ONE  2014;9(3):e93229.
Diffuse parenchymal lung diseases (DPLDs) are characterized by widespread pathological changes within the pulmonary tissue that impair the elasticity and gas exchange properties of the lungs. Clinical-radiological diagnosis of these diseases remains challenging and their clinical course is characterized by variable disease progression. These challenges have hindered the introduction of robust objective biomarkers for patient-specific prediction based on specific phenotypes in clinical practice for patients with DPLD. Therefore, strategies facilitating individualized clinical management, staging and identification of specific phenotypes linked to clinical disease outcomes or therapeutic responses are urgently needed. A classification schema consistently reflecting the radiological, clinical (lung function and clinical outcomes) and pathological features of a disease represents a critical need in modern pulmonary medicine. Herein, we report a quantitative stratification paradigm to identify subsets of DPLD patients with characteristic radiologic patterns in an unsupervised manner and demonstrate significant correlation of these self-organized disease groups with clinically accepted surrogate endpoints. The proposed consistent and reproducible technique could potentially transform diagnostic staging, clinical management and prognostication of DPLD patients as well as facilitate patient selection for clinical trials beyond the ability of current radiological tools. In addition, the sequential quantitative stratification of the type and extent of parenchymal process may allow standardized and objective monitoring of disease, early assessment of treatment response and mortality prediction for DPLD patients.
doi:10.1371/journal.pone.0093229
PMCID: PMC3968138  PMID: 24676019
2.  Lower Corticosteroid Skin Blanching Response Is Associated with Severe COPD 
PLoS ONE  2014;9(3):e91788.
Background
Chronic obstructive pulmonary disease (COPD) is characterized by chronic airflow limitation caused by ongoing inflammatory and remodeling processes of the airways and lung tissue. Inflammation can be targeted by corticosteroids. However, airway inflammation is generally less responsive to steroids in COPD than in asthma. The underlying mechanisms are yet unclear. This study aimed to assess whether skin corticosteroid insensitivity is associated with COPD and COPD severity using the corticosteroid skin blanching test.
Methods
COPD patients GOLD stage I–IV (n = 27, 24, 22, and 16 respectively) and healthy never-smokers and smokers (n = 28 and 56 respectively) were included. Corticosteroid sensitivity was assessed by the corticosteroid skin blanching test. Budesonide was applied in 8 logarithmically increasing concentrations (0–100 μg/ml) on subject's forearm. Assessment of blanching was performed after 7 hours using a 7-point scale (normal skin to intense blanching). All subjects performed spirometry and body plethysmography.
Results
Both GOLD III and GOLD IV COPD patients showed significantly lower skin blanching responses than healthy never-smokers and smokers, GOLD I, and GOLD II patients. Their area under the dose-response curve values of the skin blanching response were 586 and 243 vs. 1560, 1154, 1380, and 1309 respectively, p<0.05. Lower FEV1 levels and higher RV/TLC ratios were significantly associated with lower skin blanching responses (p = 0.001 and p = 0.004 respectively). GOLD stage I, II, III and IV patients had similar age and packyears.
Conclusions
In this study, severe and very severe COPD patients had lower skin corticosteroid sensitivity than mild and moderate COPD patients and non-COPD controls with comparable age and packyears. Our findings together suggest that the reduced skin blanching response fits with a subgroup of COPD patients that has an early-onset COPD phenotype.
doi:10.1371/journal.pone.0091788
PMCID: PMC3951419  PMID: 24622644
3.  Osteosarcoma Microenvironment: Whole-Slide Imaging and Optimized Antigen Detection Overcome Major Limitations in Immunohistochemical Quantification 
PLoS ONE  2014;9(3):e90727.
Background
In osteosarcoma survival rates could not be improved over the last 30 years. Novel biomarkers are warranted to allow risk stratification of patients for more individual treatment following initial diagnosis. Although previous studies of the tumor microenvironment have identified promising candidates, novel biomarkers have not been translated into routine histopathology. Substantial difficulties regarding immunohistochemical detection and quantification of antigens in decalcified and heterogeneous osteosarcoma might largely explain this translational short-coming. Furthermore, we hypothesized that conventional hot spot analysis is often not representative for the whole section when applied to heterogeneous tissues like osteosarcoma. We aimed to overcome these difficulties for major biomarkers of the immunovascular microenvironment.
Methods
Immunohistochemistry was systematically optimized for cell surface (CD31, CD8) and intracellular antigens (FOXP3) including evaluation of 200 different antigen retrieval conditions. Distribution patterns of these antigens were analyzed in formalin-fixed and paraffin-embedded samples from 120 high-grade central osteosarcoma biopsies and computer-assisted whole-slide analysis was compared with conventional quantification methods including hot spot analysis.
Results
More than 96% of osteosarcoma samples were positive for all antigens after optimization of immunohistochemistry. In contrast, standard immunohistochemistry retrieved false negative results in 35–65% of decalcified osteosarcoma specimens. Standard hot spot analysis was applicable for homogeneous distributed FOXP3+ and CD8+ cells. However, heterogeneous distribution of vascular CD31 did not allow reliable quantification with hot spot analysis in 85% of all samples. Computer-assisted whole-slide analysis of total CD31- immunoreactive area proved as the most appropriate quantification method.
Conclusion
Standard staining and quantification procedures are not applicable in decalcified formalin-fixed and paraffin-embedded samples for major parameters of the immunovascular microenvironment in osteosarcoma. Whole-slide imaging and optimized antigen retrieval overcome these limitations.
doi:10.1371/journal.pone.0090727
PMCID: PMC3940945  PMID: 24594971
4.  T Helper Cell Subsets Specific for Pseudomonas aeruginosa in Healthy Individuals and Patients with Cystic Fibrosis 
PLoS ONE  2014;9(2):e90263.
Background
We set out to determine the magnitude of antigen-specific memory T helper cell responses to Pseudomonas aeruginosa in healthy humans and patients with cystic fibrosis.
Methods
Peripheral blood human memory CD4+ T cells were co-cultured with dendritic cells that had been infected with different strains of Pseudomonas aeruginosa. The T helper response was determined by measuring proliferation, immunoassay of cytokine output, and immunostaining of intracellular cytokines.
Results
Healthy individuals and patients with cystic fibrosis had robust antigen-specific memory CD4+ T cell responses to Pseudomonas aeruginosa that not only contained a Th1 and Th17 component but also Th22 cells. In contrast to previous descriptions of human Th22 cells, these Pseudomonal-specific Th22 cells lacked the skin homing markers CCR4 or CCR10, although were CCR6+. Healthy individuals and patients with cystic fibrosis had similar levels of Th22 cells, but the patient group had significantly fewer Th17 cells in peripheral blood.
Conclusions
Th22 cells specific to Pseudomonas aeruginosa are induced in both healthy individuals and patients with cystic fibrosis. Along with Th17 cells, they may play an important role in the pulmonary response to this microbe in patients with cystic fibrosis and other conditions.
doi:10.1371/journal.pone.0090263
PMCID: PMC3937364  PMID: 24587305
5.  Airway, but not serum or urinary, levels of YKL-40 reflect inflammation in early cystic fibrosis lung disease 
Background
Cystic fibrosis (CF) lung disease begins in early life and is progressive with the major risk factor being an exaggerated inflammatory response. Currently, assessment of neutrophilic inflammation in early cystic fibrosis (CF) lung disease relies on bronchoalveolar lavage (BAL). The chitinase-like protein YKL-40 is raised in sputum and serum of adults with CF. We investigated YKL-40 in BAL, serum and urine to determine whether this reflected inflammation and infection in young children with CF.
Methods
YKL-40 was measured in matched samples of BAL, serum and urine obtained from 36 infants and young children with CF participating in an early surveillance program. Levels were compared to clinical data and markers of inflammation detected in the lung.
Results
YKL-40 in BAL correlated with pulmonary infection [β=1.30 (SE 0.34), p < 0.001] and BAL markers of inflammation [macrophage number: r2 = 0.34, p < 0.001; neutrophil number: r2 = 0.74, p < 0.001; neutrophil elastase: r2 = 0.47, p < 0.001; CXCL8: r2 = 0.45, p < 0.001; IL-β: r2 = 0.62, p < 0.001]. YKL-40 was detectable in serum but levels did not correlate with BAL levels in the same individuals (r2 = 0.04, p = 0.14) or with inflammatory markers. YKL-40 was below the limit of detection in urine (30 pg/ml).
Conclusions
This study demonstrates that levels of the chitinase-like protein YKL-40 reflect airway inflammation and infection in early CF lung disease. The lack of increased YKL-40 in serum in the absence of systemic inflammation limits the benefit of this potential biomarker in early disease.
doi:10.1186/1471-2466-14-28
PMCID: PMC3946043  PMID: 24576297
Cystic fibrosis; YKL-40; Biomarker; Lung disease
6.  Plasma sCD14 as a Biomarker to Predict Pulmonary Exacerbations in Cystic Fibrosis 
PLoS ONE  2014;9(2):e89341.
Background
One in four cystic fibrosis (CF) patients diagnosed with a pulmonary exacerbation will not recover their baseline lung function despite standard treatment. This highlights the importance of preventing such events. Clinical decision-making can be improved through a simple blood test that predicts individuals at elevated short-term risk of an exacerbation.
Methods
We obtained plasma samples from 30 stable CF patients from the St. Paul’s Hospital Adult CF Clinic (Vancouver, Canada). For 15 patients, an additional plasma sample was obtained during an exacerbation. Soluble CD14 (sCD14) and C-reactive protein (CRP) were quantified using ELISA kits. Myeloperoxidase (MPO), interleukin(IL)-6, IL-1β, monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and granulocyte colony-stimulating factor (G-CSF) were quantified using Luminex™ immunoassays. Stable state biomarker levels were examined in their ability to predict individuals that would experience a pulmonary exacerbation requiring intravenous (IV) antibiotics within 4 months. Paired stable and exacerbation plasma biomarker levels were also compared.
Results
sCD14 levels were significantly higher in patients that experienced a pulmonary exacerbation requiring IV antibiotics within 4 months (p = 0.001). sCD14 cut-off value of 1450 ng/mL was associated with an area under the curve of 0.91 (95% CI 0.83–0.99) for predicting an exacerbation within 4 months of a stable visit, with a sensitivity of 100% and specificity of 82%. Plasma sCD14 levels were significantly higher during exacerbations than during periods of clinical stability (p = 0.03).
Conclusions
Plasma sCD14 is a promising biomarker for identifying CF patients who will exacerbate within 4 months of a stable visit but requires further study in larger, independent cohorts.
doi:10.1371/journal.pone.0089341
PMCID: PMC3930718  PMID: 24586701
7.  Improvement of Chloride Transport Defect by Gonadotropin-Releasing Hormone (GnRH) in Cystic Fibrosis Epithelial Cells 
PLoS ONE  2014;9(2):e88964.
Cystic fibrosis (CF), the most common autosomal recessive disease in Caucasians, is due to mutations in the CFTR gene. F508del, the most frequent mutation in patients, impairs CFTR protein folding and biosynthesis. The F508del-CFTR protein is retained in the endoplasmic reticulum (ER) and its traffic to the plasma membrane is altered. Nevertheless, if it reaches the cell surface, it exhibits a Cl− channel function despite a short half-life. Pharmacological treatments may target the F508del-CFTR defect directly by binding to the mutant protein or indirectly by altering cellular proteostasis, and promote its plasma membrane targeting and stability. We previously showed that annexine A5 (AnxA5) directly binds to F508del-CFTR and, when overexpressed, promotes its membrane stability, leading to the restoration of some Cl− channel function in cells. Because Gonadotropin-Releasing Hormone (GnRH) increases AnxA5 expression in some cells, we tested it in CF cells. We showed that human epithelial cells express GnRH-receptors (GnRH-R) and that GnRH induces an AnxA5 overexpression and an increased Cl− channel function in F508del-CFTR cells, due to an increased stability of the protein in the membranes. Beside the numerous physiological implications of the GnRH-R expression in epithelial cells, we propose that a topical use of GnRH is a potential treatment in CF.
doi:10.1371/journal.pone.0088964
PMCID: PMC3929654  PMID: 24586461
8.  Angiopoietin-1 Upregulates De Novo Expression of Il-1β and Il1-Ra, and the Exclusive Release of Il1-Ra from Human Neutrophils 
PLoS ONE  2014;9(2):e88980.
The expression of the angiopoietin (Ang) receptor, Tie2, on both endothelial and inflammatory cells supports the idea that Ang signaling may play a fundamental role in initiating and maintaining the inflammatory response. We have previously shown that Ang1 and/or Ang2 alter the innate immune response by enhancing human neutrophil survival, chemotaxis and production of inflammatory cytokine interleukin-8 (IL-8) in vitro. Thus, we hypothesized that Ang1 and Ang2 could modulate other inflammatory signals in neutrophils, a possibility we explored through a gene-based assay looking at changes in the mRNA expression of 84 inflammatory cytokines and their receptors. We observed that Ang1 (10−8 M), but not Ang2, increased mRNA expression of prominent pro-inflammatory cytokine IL-1β and its natural antagonist IL-1RA, by up to 32.6- and 10.0-fold respectively, compared to PBS-control. The effects of Ang1 extended to the proteins, as Ang1 increased intracellular levels of precursor and mature IL-1β, and extracellular levels of IL-1RA proteins, by up to 4.2-, 5.0- and 4.4-fold respectively, compared to PBS-control. Interestingly, Ang1 failed at inducing IL-1β protein release or at increasing intracellular IL-1RA, but the ratio of IL-1RA to mature IL-1β remained above 100-fold molar excess inside and outside the cells. The above-noted effects of Ang1 were mediated by MAP kinases, whereby inhibiting MEK1/2 lead to up to 70% effect reduction, whereas the blockade of p38MAPK activity doubled Ang1's effect. These findings suggest that Ang1 selectively alters the balance of neutrophil-derived inflammatory cytokines, favoring the blockade of IL-1 activity, a consideration for future therapies of inflammatory diseases.
doi:10.1371/journal.pone.0088980
PMCID: PMC3923077  PMID: 24563688
9.  Molecular Analysis of Serum and Bronchoalveolar Lavage in a Mouse Model of Influenza Reveals Markers of Disease Severity That Can Be Clinically Useful in Humans 
PLoS ONE  2014;9(2):e86912.
Background
Management of influenza, a major contributor to the worldwide disease burden, is complicated by lack of reliable methods for early identification of susceptible individuals. Identification of molecular markers that can augment existing diagnostic tools for prediction of severity can be expected to greatly improve disease management capabilities.
Methodology/Principal Findings
We have analyzed cytokines, proteome flux and protein adducts in bronchoalveolar lavage (BAL) and sera from mice infected with influenza A virus (PR8 strain) using a previously established non-lethal model of influenza infection. Through detailed cytokine and protein adduct measurements of murine BAL, we first established the temporal profile of innate and adaptive responses as well as macrophage and neutrophil activities in response to influenza infection. A similar analysis was also performed with sera from a longitudinal cohort of influenza patients. We then used an iTRAQ-based, comparative serum proteome analysis to catalog the proteome flux in the murine BAL during the stages correlating with “peak viremia,” “inflammatory damage,” as well as the “recovery phase.” In addition to activation of acute phase responses, a distinct class of lung proteins including surfactant proteins was found to be depleted from the BAL coincident with their “appearance” in the serum, presumably due to leakage of the protein following loss of the integrity of the lung/epithelial barrier. Serum levels of at least two of these proteins were elevated in influenza patients during the febrile phase of infection compared to healthy controls or to the same patients at convalescence.
Conclusions/Significance
The findings from this study provide a molecular description of disease progression in a mouse model of influenza and demonstrate its potential for translation into a novel class of markers for measurement of acute lung injury and improved case management.
doi:10.1371/journal.pone.0086912
PMCID: PMC3914809  PMID: 24505273
10.  Smoking Dysregulates the Human Airway Basal Cell Transcriptome at COPD Risk Locus 19q13.2 
PLoS ONE  2014;9(2):e88051.
Genome-wide association studies (GWAS) and candidate gene studies have identified a number of risk loci associated with the smoking-related disease COPD, a disorder that originates in the airway epithelium. Since airway basal cell (BC) stem/progenitor cells exhibit the earliest abnormalities associated with smoking (hyperplasia, squamous metaplasia), we hypothesized that smoker BC have a dysregulated transcriptome, enriched, in part, at known GWAS/candidate gene loci. Massive parallel RNA sequencing was used to compare the transcriptome of BC purified from the airway epithelium of healthy nonsmokers (n = 10) and healthy smokers (n = 7). The chromosomal location of the differentially expressed genes was compared to loci identified by GWAS to confer risk for COPD. Smoker BC have 676 genes differentially expressed compared to nonsmoker BC, dominated by smoking up-regulation. Strikingly, 166 (25%) of these genes are located on chromosome 19, with 13 localized to 19q13.2 (p<10−4 compared to chance), including 4 genes (NFKBIB, LTBP4, EGLN2 and TGFB1) associated with risk for COPD. These observations provide the first direct connection between known genetic risks for smoking-related lung disease and airway BC, the population of lung cells that undergo the earliest changes associated with smoking.
doi:10.1371/journal.pone.0088051
PMCID: PMC3912203  PMID: 24498427
11.  Influenza Virus-Induced Lung Inflammation Was Modulated by Cigarette Smoke Exposure in Mice 
PLoS ONE  2014;9(1):e86166.
Although smokers have increased susceptibility and severity of seasonal influenza virus infection, there is no report about the risk of 2009 pandemic H1N1 (pdmH1N1) or avian H9N2 (H9N2/G1) virus infection in smokers. In our study, we used mouse model to investigate the effect of cigarette smoke on pdmH1N1 or H9N2 virus infection. Mice were exposed to cigarette smoke for 21 days and then infected with pdmH1N1 or H9N2 virus. Control mice were exposed to air in parallel. We found that cigarette smoke exposure alone significantly upregulated the lung inflammation. Such prior cigarette smoke exposure significantly reduced the disease severity of subsequent pdmH1N1 or H9N2 virus infection. For pdmH1N1 infection, cigarette smoke exposed mice had significantly lower mortality than the control mice, possibly due to the significantly decreased production of inflammatory cytokines and chemokines. Similarly, after H9N2 infection, cigarette smoke exposed mice displayed significantly less weight loss, which might be attributed to lower cytokines and chemokines production, less macrophages, neutrophils, CD4+ and CD8+ T cells infiltration and reduced lung damage compared to the control mice. To further investigate the underlying mechanism, we used nicotine to mimic the effect of cigarette smoke both in vitro and in vivo. Pre-treating the primary human macrophages with nicotine for 72 h significantly decreased their expression of cytokines and chemokines after pdmH1N1 or H9N2 infection. The mice subcutaneously and continuously treated with nicotine displayed significantly less weight loss and lower inflammatory response than the control mice upon pdmH1N1 or H9N2 infection. Moreover, α7 nicotinic acetylcholine receptor knockout mice had more body weight loss than wild-type mice after cigarette smoke exposure and H9N2 infection. Our study provided the first evidence that the pathogenicity of both pdmH1N1 and H9N2 viruses was alleviated in cigarette smoke exposed mice, which might partially be attributed to the immunosuppressive effect of nicotine.
doi:10.1371/journal.pone.0086166
PMCID: PMC3897646  PMID: 24465940
12.  Influenza A(H1N1)pdm09 and Cystic Fibrosis Lung Disease: A Systematic Meta-Analysis 
PLoS ONE  2014;9(1):e78583.
Background
To systematically assess the literature published on the clinical impact of Influenza A(H1N1)pdm09 on cystic fibrosis (CF) patients.
Methods
An online search in PUBMED database was conducted. Original articles on CF patients with Influenza A(H1N1)pdm09 infection were included. We analyzed incidence, symptoms, clinical course and treatment.
Results
Four surveys with a total of 202 CF patients infected by Influenza A(H1N1)pdm09 were included. The meta-analysis showed that hospitalisation rates were higher in CF patients compared to the general population. While general disease symptoms were comparable, the clinical course was more severe and case fatality rate (CFR) was higher in CF patients compared to asthmatics and the general population.
Conclusions
Evidence so far suggests that CF patients infected with Influenza A(H1N1)pdm09 show increased morbidity and a higher CFR compared to patients with other chronic respiratory diseases and healthy controls. Particularly, CF patients with advanced stage disease seem to be more susceptible to severe lung disease. Accordingly, early antiviral and antibiotic treatment strategies are essential in CF patients. Preventive measures, including vaccination as well as hygiene measures during the influenza season, should be reinforced and improved in CF patients.
doi:10.1371/journal.pone.0078583
PMCID: PMC3888399  PMID: 24427261
13.  Diabetes in Cystic Fibrosis: Multicenter Screening Results Based on Current Guidelines 
PLoS ONE  2013;8(12):e81545.
Background
Published estimates on age-dependent frequency of diabetes in cystic fibrosis (CF) vary widely, and are based mostly on older data. However, CF treatment and prevention of comorbidities changed over recent years. In many studies, definition of cystic fibrosis-related diabetes (CFRD) is not in line with current guideline recommendations. Therefore, we evaluated age-dependent occurrence of glucose abnormalities and associated risk factors in CF patients who participated in a multicenter screening program using oral glucose tolerance tests (OGTT).
Methods
Between 2001 and 2010, 43 specialized CF centers from Germany and Austria serially performed 5,179 standardized OGTTs in 1,658 clinically stable, non-pregnant CF patients with no prior steroid medication or lung transplantation. Age-dependent occurrence of impaired fasting glucose (IFG), impaired glucose tolerance (IGT), IFG+IGT, one (DGT) or two consecutive (CFRD) diabetic OGTTs was analyzed, using Kaplan Meier curves. Cox proportional-hazards models were created to elucidate the influence of sex or underweight.
Results
At baseline/last OGTT, median age was 15.9 years/18.2 years and 30.6%/31.8% of patients were underweight. 25% of patients showed IFG at age 14.3 years; IGT at age 16.3 years; IFG+IGT combined at age 17.7 years. DGT was observed in 25% of patients at age 22.6 years; CFRD at age 34.5 years. Females had a 3.54 [95% CI 1.23–10.18] times higher risk for CFRD; risk for DGT was 2.21 [1.22–3.98] times higher. Underweight was a risk factor for IGT (HR [95% CI]: 1.38 [1.11–1.71]) and IFG+IGT (1.43 [1.11–1.83]), and in males also for DGT (1.49 [1.09–2.04]).
Conclusions/Significance
If confirmation of diabetes by a second test is required, as recommended in current guidelines, age at CFRD diagnosis was higher compared to most previous studies. However, known risk factors for glucose abnormalities in CF were confirmed. Confirmation of diabetic OGT by a repeat test is important for a consistent diagnosis of CFRD.
doi:10.1371/journal.pone.0081545
PMCID: PMC3855692  PMID: 24324701
14.  Roscovitine-induced Apoptosis in Neutrophils and Neutrophil Progenitors Is Regulated by the Bcl-2-Family Members Bim, Puma, Noxa and Mcl-1 
PLoS ONE  2013;8(11):e79352.
Neutrophil granulocyte (neutrophil) apoptosis plays a key role in determining inflammation in infectious and non-infectious settings. Recent work has shown that inhibitors of cyclin-dependent kinases (cdk) such as roscovitine can potently induce neutrophil apoptosis and reduce inflammation. Using a conditional Hoxb8-expression system we tested the participation of Bcl-2-family proteins to roscovitine-induced apoptosis in mouse neutrophils and in neutrophil progenitor cells. Bcl-2 strongly protected against roscovitine-induced apoptosis in neutrophils. The isolated loss of either Bim or noxa provided significant, partial protection while protection through combined loss of Bim and noxa or Bim and Puma was only slightly greater than this individual loss. The only substantial change in protein levels observed was the loss of Mcl-1, which was not transcriptional and was inhibited by proteasome blockade. In progenitor cells there was no protection by the loss of Bim alone but substantial protection by the loss of both Bim and Puma; surprisingly, strongest protection was seen by the isolated loss of noxa. The pattern of protein expression and Mcl-1-regulation in progenitor cells was very similar to the one observed in differentiated neutrophils. In addition, roscovitine strongly inhibited proliferation in progenitor cells, associated with an accumulation of cells in G2/M-phase.
doi:10.1371/journal.pone.0079352
PMCID: PMC3815126  PMID: 24223929
15.  Sex Differences in the Relationship between Asthma and Overweight in Dutch Children: a Survey Study 
PLoS ONE  2013;8(10):e77574.
Objective
Obesity has been identified as a risk factor for asthma in children. However, in the Netherlands, the obesity prevalence is rising while the asthma prevalence in children is stabilising. The aim of this study is to clarify the association between asthma and Body Mass Index (BMI) in children and whether this association is influenced by sex.
Study Design
Parents of 39,316 children (6-16 years) in the south of the Netherlands were invited to complete an online questionnaire on respiratory symptoms, anthropometric variables and several potential confounding factors for asthma and obesity (including sex, birth weight and breastfeeding). Data was analysed by multivariable logistic regression models and an ordinal regression model.
Results
The response rate was 24% (n boys= 4,743, n girls= 4,529). The prevalence of asthma, overweight and obesity was 8%, 15% and 2% respectively. Body mass index - standard deviation Score (BMI-SDS) was related to current asthma (adjusted OR: 1.29; 95%CI: 1.14-1.45, p≤0.001). When stratified for sex, asthma and BMI-SDS were only related in girls (Girls: adjusted OR: 1.31; 95%CI: 1.13-1.51, p≤0.001. Boys: adjusted OR: 1.01; 95%CI: 0.91-1.14, p=0.72).
Conclusions
The positive association between BMI-SDS and asthma is only present in girls, not boys. Future studies into obesity and asthma should correct for sex in their analyses.
doi:10.1371/journal.pone.0077574
PMCID: PMC3804131  PMID: 24204876
16.  The Blood Neutrophil to Lymphocyte Ratio Correlates with Clinical Status in Children with Cystic Fibrosis: A Retrospective Study 
PLoS ONE  2013;8(10):e77420.
Purpose
The blood neutrophil to lymphocyte ratio (NLR) has been identified as a potentially useful marker of clinical outcome in disease states with an inflammatory component. The objective of this study was to evaluate the relationship between NLR and clinical status in children with cystic fibrosis.
Methods
This was a retrospective chart review. Data collected included NLR, body mass index, and forced expiratory volume in 1 second (FEV1) while asymptomatic, and during hospitalizations for pulmonary exacerbation. An NLR breakpoint of 3 was used for comparisons of body mass index and FEV1.
Results
A total of 159 charts were reviewed. An NLR ≥ 3 was significantly associated with lower body mass index and lower FEV1. NLR during hospitalization was significantly higher than NLR while asymptomatic. NLR measured during the first 3 months of life was negatively correlated with FEV1 at age 12.
Conclusion
NLR correlates with clinical status in children with cystic fibrosis and may be a useful biomarker in this population.
doi:10.1371/journal.pone.0077420
PMCID: PMC3788810  PMID: 24098587
17.  Tumor Necrosis Factor Induces Tumor Promoting and Anti-Tumoral Effects on Pancreatic Cancer via TNFR1 
PLoS ONE  2013;8(9):e75737.
Multiple activities are ascribed to the cytokine tumor necrosis factor (TNF) in health and disease. In particular, TNF was shown to affect carcinogenesis in multiple ways. This cytokine acts via the activation of two cell surface receptors, TNFR1, which is associated with inflammation, and TNFR2, which was shown to cause anti-inflammatory signaling. We assessed the effects of TNF and its two receptors on the progression of pancreatic cancer by in vivo bioluminescence imaging in a syngeneic orthotopic tumor mouse model with Panc02 cells. Mice deficient for TNFR1 were unable to spontaneously reject Panc02 tumors and furthermore displayed enhanced tumor progression. In contrast, a fraction of wild type (37.5%), TNF deficient (12.5%), and TNFR2 deficient mice (22.2%) were able to fully reject the tumor within two weeks. Pancreatic tumors in TNFR1 deficient mice displayed increased vascular density, enhanced infiltration of CD4+ T cells and CD4+ forkhead box P3 (FoxP3)+ regulatory T cells (Treg) but reduced numbers of CD8+ T cells. These alterations were further accompanied by transcriptional upregulation of IL4. Thus, TNF and TNFR1 are required in pancreatic ductal carcinoma to ensure optimal CD8+ T cell-mediated immunosurveillance and tumor rejection. Exogenous systemic administration of human TNF, however, which only interacts with murine TNFR1, accelerated tumor progression. This suggests that TNFR1 has basically the capability in the Panc02 model to trigger pro-and anti-tumoral effects but the spatiotemporal availability of TNF seems to determine finally the overall outcome.
doi:10.1371/journal.pone.0075737
PMCID: PMC3787053  PMID: 24098720
18.  Characterization and Prevention of the Adsorption of Surfactant Protein D to Polypropylene 
PLoS ONE  2013;8(9):e73467.
Surfactant Protein D (SP-D) is a multifunctional protein present in the lung and in respiratory secretions. In the process of developing new experimental approaches to examine SP-D function, we observed that SP-D adsorbs to polypropylene tubes to a great extent, thereby depleting SP-D from the solution. Although it is well known that proteins adsorb nonspecifically to plastic, this effect is usually diminished by treatments to make the plastic “low-retention” or “low-binding”. However, these treatments actually increased the binding of SP-D to the plastic. In addition, this adsorption affected the results of several assays, including proteolytic cleavage assays. In order to block SP-D from adsorbing to polypropylene and the effects caused by this adsorption, we coated the tubes with bovine serum albumin (BSA), as is commonly performed for ELISAs. This coating greatly diminished the amount of SP-D sticking to the plastic, providing an inexpensive and effective method for preventing adsorption and the artifacts resulting from this adsorption.
doi:10.1371/journal.pone.0073467
PMCID: PMC3770593  PMID: 24039953
19.  TLR2 Regulates Neutrophil Recruitment and Cytokine Production with Minor Contributions from TLR9 during Hypersensitivity Pneumonitis 
PLoS ONE  2013;8(8):e73143.
Hypersensitivity pneumonitis (HP) is an interstitial lung disease that develops following repeated exposure to environmental antigens. The disease results in alveolitis, granuloma formation and may progress to a fibrotic chronic form, which is associated with significant morbidity and mortality. The severity of the disease correlates with a neutrophil rich influx and an IL-17 response. We used the Saccharopolysporarectivirgula (SR) model of HP to determine whether Toll-like receptors (TLR) 2 and 9 cooperate in neutrophil recruitment and IL-17-associated cytokine production during the development of HP. Stimulation of bone marrow derived macrophages (BMDMs) from C57BL/6, MyD88-/- and TLR2/9-/- mice with SR demonstrate that SR is a strong inducer of neutrophil chemokines and growth factors. The cytokines induced by SR were MyD88-dependent and, of those, most were partially or completely dependent on TLRs 2 and 9. Following in vivo exposure to SR, CXCL2 production and neutrophil recruitment were reduced in TLR2-/- and TLR2/9-/- mice suggesting that the response was largely dependent on TLR2; however the reduction was greatest in the TLR2/9-/- double knockout mice indicating TLR9 may also contribute to the response. There was a reduction in the levels of pro-inflammatory cytokines TNFα and IL-6 as well as CCL3 and CCL4 in the BALF from TLR2/9-/- mice compared to WT and single knockout (SKO) mice exposed one time to SR. The decrease in neutrophil recruitment and TNFα production in the TLR2/9-/- mice was maintained throughout 3 weeks of SR exposures in comparison to WT and SKO mice. Both TLRs 2 and 9 contributed to the Th17 response; there was a decrease in Th17 cells and IL-17 mRNA in the TLR2/9-/- mice in comparison to the WT and SKO mice. Despite the effects on neutrophil recruitment and the IL-17 response, TLR2/9-/- mice developed granuloma formation similarly to WT and SKO mice suggesting that there are additional mediators and pattern recognition receptors involved in the disease.
doi:10.1371/journal.pone.0073143
PMCID: PMC3758260  PMID: 24023674
20.  CCDC65 Mutation Causes Primary Ciliary Dyskinesia with Normal Ultrastructure and Hyperkinetic Cilia 
PLoS ONE  2013;8(8):e72299.
Background
Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating.
Methods
Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion.
Results
A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells.
Conclusion
Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis.
doi:10.1371/journal.pone.0072299
PMCID: PMC3753302  PMID: 23991085
21.  Down Regulation of T Cell Receptor Expression in COPD Pulmonary CD8 Cells 
PLoS ONE  2013;8(8):e71629.
CD8 cells may contribute towards an autoimmune process in COPD. Down regulation of T cell receptor (TCR) signalling molecules occurs in autoimmune diseases with consequent T cell dysfunction. We hypothesise that TCR signalling is abnormal in COPD pulmonary CD8 cells. Micro-array gene expression analysis of blood and pulmonary COPD CD8 samples was performed and compared to pulmonary CD8 cells from smoker controls (S). We focused on the TCR signalling pathway, with validation of key findings using polymerase chain reaction and immunofluorescence. TCR signalling molecules in COPD pulmonary CD8 cells were down regulated compared to blood CD8 cells (CD247: fold change (FC) −2.43, Q = 0.001; LCK: FC −2.25, Q = 0.01). Micro-array analysis revealed no significant differences between COPD and S pulmonary CD8 cells. However, PCR revealed significantly lower gene expression levels of CD247 (FC −1.79, p = 0.04) and LCK (FC −1.77, p = 0.01) in COPD compared to S pulmonary CD8 cells. CD247 down regulation in COPD CD8 cells was confirmed by immunofluorescent staining of bronchoalveolar lavage cells: Significantly fewer COPD CD8 cells co-expressed CD247 compared to healthy non-smoker CD8 cells (mean 88.9 vs 75.2%, p<0.05) There is down regulation of TCR signalling molecules in COPD pulmonary CD8 cells. This may cause T cell dysfunction.
doi:10.1371/journal.pone.0071629
PMCID: PMC3747211  PMID: 23977094
22.  Indacaterol for Chronic Obstructive Pulmonary Disease: Systematic Review and Meta-Analysis 
PLoS ONE  2013;8(8):e70784.
Background
Inhaled bronchodilators are the first-line therapy for COPD. Indacaterol is a novel addition to existing long-acting bronchodilators.
Objectives
Systematic review of randomized controlled trials (RCT) on efficacy and safety of indacaterol as compared: 1) with placebo at different dosages, 2) with existing bronchodilators; (3) as add-on treatment to tiotropium.
Methods
We searched 13 electronic databases, including MEDLINE, EMBASE and CENTRAL, and contacted the manufacturer for unpublished data. Primary outcome was mean FEV1 change at 12th week, secondary outcomes included changes in SGRQ, TDI and BODE index at 6 months, exacerbation at 1 year, and worsening of symptoms.
Results
Twelve eligible RCTs of moderate risk of bias included data from 10,977 patients. Compared to placebo, indacaterol improved FEV1 by a weighted mean difference (WMD) of 0.16 L (95%CI: 0.15, 0.18 L, p<0.001), homogeneously above the minimally important difference of 0.10 L. It offered clinically relevant improvement in all secondary outcomes except exacerbation. Magnitude of benefit did not differ significantly by dosage, but one treatment related death was reported at 300 ug. Efficacy of Indacaterol was similar to formoterol and salmeterol (FEV1 WMD = 0.04L, 95%CI: 0.01L, 0.07 L, p = 0.02); and tiotropium (FEV1 WMD = 0.01L, 95%CI: −0.01, 0.03L, p = 0.61). The use of indacaterol on top of tiotropium yielded additional improvement on FEV1 (WMD = 0.07 L, 95%CI: 0.05L, 0.10 L, p<0.001).
Conclusion
Indacaterol is safe and beneficial for patients with COPD at dosage ≤150 ug. It may serve as a good alternative to existing bronchodilators, or as an add-on to tiotropium for unresponsive patients. Use of higher dosage requires further justification.
doi:10.1371/journal.pone.0070784
PMCID: PMC3743831  PMID: 23967106
23.  Hypersensitivity pneumonitis: lessons for diagnosis and treatment of a rare entity in children 
Hypersensitivity pneumonitis (HP) also called exogenous allergic alveolitis = extrinsic allergic alveolitis in children is an uncommon condition and may not be recognized and treated appropriately.
To assess current means of diagnosis and therapy and compare this to recommendations, we used the Surveillance Unit for Rare Paediatric Disorders (ESPED) to identify incident cases of HP in Germany during 2005/6. In addition, cases of HP reported for reference from all over Germany to our center in the consecutive year were included.
Twenty-three children with confirmed pediatric HP were identified. All (age 9.4 y (4.4-15.1) presented with dyspnoea at rest or with exercise, mean FVC was 39% of predicted, seven of the 23 children already had a chronic disease state at presentation. IgG against bird was elevated in 20, and against fungi in 15. Bronchoalveolar lavage was done in 18 subjects (41% lymphocytes, CD4/CD8 1.99), and lung biopsy in 6. Except 2, all children were treated with prolonged courses of systemic steroids. Outcome was not favourable in all cases.
Late diagnosis in up to a quarter of the children with HP and inappropriate steroid treatment must be overcome to improve management of HP. Inclusion of children with HP into international, web-based registry studies will help to study and follow up such rare lung diseases.
doi:10.1186/1750-1172-8-121
PMCID: PMC3751081  PMID: 23924322
Biopsy; Bronchoalveolar lavage; Children; Diffuse parenchymal lung diseases; Exogenous allergic alveolitis = extrinsic allergic alveolitis; Precipitins; Steroid treatment
24.  Absence of the Common Gamma Chain (γc), a Critical Component of the Type I IL-4 Receptor, Increases the Severity of Allergic Lung Inflammation 
PLoS ONE  2013;8(8):e71344.
The TH2 cytokines, IL-4 and IL-13, play critical roles in inducing allergic lung inflammation and drive the alternative activation of macrophages (AAM). Although both cytokines share receptor subunits, IL-4 and IL-13 have differential roles in asthma pathogenesis: IL-4 regulates TH2 cell differentiation, while IL-13 regulates airway hyperreactivity and mucus production. Aside from controlling TH2 differentiation, the unique contribution of IL-4 signaling via the Type I receptor in airway inflammation remains unclear. Therefore, we analyzed responses in mice deficient in gamma c (γc) to elucidate the role of the Type I IL-4 receptor. OVA primed CD4+ OT-II T cells were adoptively transferred into RAG2−/− and γc−/− mice and allergic lung disease was induced. Both γc−/− and γcxRAG2−/− mice developed increased pulmonary inflammation and eosinophilia upon OVA challenge, compared to RAG2−/− mice. Characteristic AAM proteins FIZZ1 and YM1 were expressed in lung epithelial cells in both mouse strains, but greater numbers of FIZZ1+ or YM1+ airways were present in γc−/− mice. Absence of γc in macrophages, however, resulted in reduced YM1 expression. We observed higher TH2 cytokine levels in the BAL and an altered DC phenotype in the γc−/− recipient mice suggesting the potential for dysregulated T cell and dendritic cell (DC) activation in the γc-deficient environment. These results demonstrate that in absence of the Type I IL-4R, the Type II R can mediate allergic responses in the presence of TH2 effectors. However, the Type I R regulates AAM protein expression in macrophages.
doi:10.1371/journal.pone.0071344
PMCID: PMC3734063  PMID: 23940740
25.  Competence-Independent Activity of Pneumococcal Enda Mediates Degradation of Extracellular DNA and Nets and Is Important for Virulence 
PLoS ONE  2013;8(7):e70363.
Membrane surface localized endonuclease EndA of the pulmonary pathogen Streptococcus pneumoniae (pneumococcus) is required for both genetic transformation and virulence. Pneumococcus expresses EndA during growth. However, it has been reported that EndA has no access to external DNA when pneumococcal cells are not competent for genetic transformation, and thus, unable to degrade extracellular DNA. Here, by using both biochemical and genetic methods, we demonstrate the existence of EndA-mediated nucleolytic activity independent of the competence state of pneumococcal cells. Pneumococcal mutants that are genetically deficient in competence development and genetic transformation have extracellular nuclease activity comparable to their parental wild type, including their ability to degrade neutrophil extracellular traps (NETs). The autolysis deficient ΔlytA mutant and its isogenic choline-treated parental wild-type strain D39 degrade extracellular DNA readily, suggesting that partial cell autolysis is not required for DNA degradation. We show that EndA molecules are secreted into the culture medium during the growth of pneumococcal cells, and contribute substantially to competence-independent nucleolytic activity. The competence-independent activity of EndA is responsible for the rapid degradation of DNA and NETs, and is required for the full virulence of Streptococcus pneumoniae during lung infection.
doi:10.1371/journal.pone.0070363
PMCID: PMC3729463  PMID: 23936195

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