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Netsanet A Negewo,1 Peter G Gibson,2– 4 Jodie L Simpson,1 Vanessa M McDonald,2– 5 Katherine J Baines1 1Immune Health Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia; 2Centre of Excellence in Treatable Traits, University of Newcastle, New Lambton Heights, NSW, Australia; 3Department of Respiratory and Sleep Medicine, John Hunter Hospital, Newcastle, NSW, Australia; 4Asthma and Breathing Research Centre, Hunter Medical Research Centre, New Lambton Heights, NSW, Australia; 5School of Nursing and Midwifery, The University of Newcastle, Callaghan, NSW, AustraliaCorrespondence: Katherine J Baines, Hunter Medical Research Institute, Level 2 East Wing, Locked Bag 1000, New Lambton Heights, NSW, 2305, Australia, Tel +61 2 40420090, Fax +61 2 40420046, Email katherine.baines@newcastle.edu.auPurpose: This study sought to characterize transcriptional phenotypes of COPD through unsupervised clustering of sputum gene expression profiles, and further investigate mechanisms underlying the characteristics of these clusters.Patients and methods: Induced sputum samples were collected from patients with stable COPD (n = 72) and healthy controls (n = 15). Induced sputum was collected for inflammatory cell counts, and RNA extracted. Transcriptional profiles were generated (Illumina Humanref-8 V2) and analyzed by GeneSpring GX14.9.1. Unsupervised hierarchical clustering and differential gene expression analysis were performed, and gene alterations validated in the ECLIPSE dataset (GSE22148).Results: We identified 2 main clusters (Cluster 1 [n = 35] and Cluster 2 [n = 37]), which further divided into 4 sub-clusters (Sub-clusters 1.1 [n = 14], 1.2 [n = 21], 2.1 [n = 20] and 2.2 [n = 17]). Compared with Cluster 1, Cluster 2 was associated with significantly lower lung function (p = 0.014), more severe disease (p = 0.009) and breathlessness (p = 0.035), and increased sputum neutrophils (p = 0.031). Sub-cluster 1.1 had significantly higher proportion of people with comorbid cardiovascular disease compared to the other 3 sub-clusters (92.5% vs 57.1%, 50% and 52.9%, p < 0.013). Through supervised analysis we determined that degree of airflow limitation (GOLD stage) was the predominant factor driving gene expression differences in our transcriptional clusters. There were 452 genes (adjusted p < 0.05 and ≥ 2 fold) altered in GOLD stage 3 and 4 versus 1 and 2, of which 281 (62%) were also found to be significantly expressed between these GOLD stages in the ECLIPSE data set (GSE22148). Differentially expressed genes were largely downregulated in GOLD stages 3 and 4 and connected in 5 networks relating to lipoprotein and cholesterol metabolism; metabolic processes in oxidation/reduction and mitochondrial function; antigen processing and presentation; regulation of complement activation and innate immune responses; and immune and metabolic processes.Conclusion: Severity of lung function drives 2 distinct transcriptional phenotypes of COPD and relates to immune and metabolic processes.Keywords: COPD, gene expression, inflammation, sputum

Katherine J Baines,1 Netsanet A Negewo,1 Peter G Gibson,1,2 Juan-Juan Fu,3 Jodie L Simpson,1 Peter AB Wark,1,2 Michael Fricker,1 Vanessa M McDonald1,2,4 1Priority Research Centre for Healthy Lungs, Hunter Medical Research Institute, The University of Newcastle, Callaghan, NSW, Australia; 2Department of Respiratory and Sleep Medicine, John Hunter Hospital, Newcastle, NSW, Australia; 3Respiratory Group, Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan Province, People’s Republic of China; 4School of Nursing and Midwifery, Faculty of Health and Medicine, The University of Newcastle, Callaghan, NSW, AustraliaCorrespondence: Katherine J BainesHunter Medical Research Institute, Level 2 West Wing, Locked Bag 1000, New Lambton, NSW 2305, AustraliaEmail katherine.baines@newcastle.edu.auBackground: The 6 gene expression signature (6GS) predicts inflammatory phenotype, exacerbation risk, and corticosteroid responsiveness in asthma. In COPD, patterns of airway inflammation are similar, suggesting the 6GS may be useful. This study determines the diagnostic and prognostic ability of 6GS in predicting inflammatory phenotypes and exacerbation risk in COPD.Methods: We performed 2 studies: a cross-sectional phenotype prediction study in stable COPD (total N=132; n=34 eosinophilic (E)-COPD, n=42 neutrophilic (N)-COPD, n=39 paucigranulocytic (PG)-COPD, n=17 mixed-granulocytic (MG)-COPD) that assessed 6GS ability to discriminate phenotypes (eosinophilia≥ 3%; neutrophilia≥ 61%); and a prospective cohort study (total n=54, n=8 E-COPD; n=18 N-COPD; n=20 PG-COPD; n=8 MG-COPD, n=21 exacerbation prone (≥ 2/year)) that investigated phenotype and exacerbation prediction utility. 6GS was measured by qPCR and evaluated using multiple logistic regression and area under the curve (AUC). Short-term reproducibility (intra-class correlation) and phenotyping method agreement (κ statistic) were assessed.Results: In the phenotype prediction study, 6GS could accurately identify and discriminate patients with E-COPD from N-COPD (AUC=96.4%; p< 0.0001), PG-COPD (AUC=88.2%; p< 0.0001) or MG-COPD (AUC=86.2%; p=0.0001), as well as N-COPD from PG-COPD (AUC=83.6%; p< 0.0001) or MG-COPD (AUC=87.4%; p< 0.0001) and was reproducible. In the prospective cohort study, 6GS had substantial agreement for neutrophilic inflammation (82%, κ=0.63, p< 0.001) and moderate agreement for eosinophilic inflammation (78%, κ=0.42, p< 0.001). 6GS could significantly discriminate exacerbation prone patients (AUC=77.2%; p=0.034). Higher IL1B levels were associated with poorer lung function and increased COPD severity.Conclusion: 6GS can significantly and reproducibly discriminate COPD inflammatory phenotypes and predict exacerbation prone patients and may become a useful molecular diagnostic tool assisting COPD management.Keywords: COPD, airway markers, inflammation, molecular biology, eosinophil

Ravi Lokwani,1–3 Peter AB Wark,1–3 Katherine J Baines,1,3 Michael Fricker,1,3 Daniel Barker,3 Jodie L Simpson1–3 1Priority Research Centre for Healthy Lungs, Hunter Medical Research Institute, University of Newcastle, Callaghan, NSW 2308, Australia; 2Department of Respiratory and Sleep Medicine, John Hunter Hospital, New Lambton Heights, NSW 2305, Australia; 3School of Medicine and Public Health, Faculty of Health and Medicine, University of Newcastle, Callaghan, NSW 2308, AustraliaCorrespondence: Jodie L SimpsonLevel 2, East Wing, Hunter Medical Research Institute, Locked Bag, New Lambton 1000, NSW 2305, AustraliaTel +61 422220992Email jodie.simpson@newcastle.edu.auPurpose: To characterize neutrophils in obstructive airway disease by measuring their surface adhesion molecules and oxidative burst along with characterizing them into different subsets as per their adhesion molecule expression.Patients and methods: Peripheral blood from adults with COPD (n=17), asthma (n=20), and healthy participants (n=19) was examined for expression of CD16, CD62L, CD11b, CD11c, and CD54, and analyzed by flow cytometry. For oxidative burst and CD62L shedding analysis, CD16 and CD62L stained leukocytes were loaded with Dihydrorhodamine-123 (DHR-123) and stimulated with N-Formylmethionine-leucyl-phenylalanine (fMLF). Neutrophil subsets were characterized based on CD16 and CD62L expression. Marker surface expression was recorded on CD16+ neutrophils as median fluorescence intensity (MFI).Results: Neutrophil surface expression of CD62L was significantly reduced in COPD (median (IQR) MFI: 1156 (904, 1365)) compared with asthma (1865 (1157, 2408)) and healthy controls (2079 (1054, 2960)); p=0.028. COPD neutrophils also demonstrated a significant reduction in CD62L expression with and without fMLF stimulation. Asthma participants had a significantly increased proportion and number of CD62Lbright/CD16dim neutrophils (median: 5.4% and 0.14 × 109/L, respectively), in comparison with healthy (3.54% and 0.12 × 109/L, respectively); p

Netsanet A Negewo,1,2 Peter G Gibson,1–3 Peter AB Wark,1–3 Jodie L Simpson,1,2 Vanessa M McDonald1–4 1Priority Research Centre for Healthy Lungs, 2Hunter Medical Research Institute, Faculty of Health and Medicine, The University of Newcastle, Callaghan, 3Department of Respiratory and Sleep Medicine, John Hunter Hospital, Newcastle, 4School of Nursing and Midwifery, Faculty of Health and Medicine, The University of Newcastle, Callaghan, NSW, Australia Background: COPD patients are often prescribed multiple medications for their respiratory disease and comorbidities. This can lead to complex medication regimens resulting in poor adherence, medication errors, and drug–drug interactions. The relationship between clinical outcomes and medication burden beyond medication count in COPD is largely unknown.Objectives: The aim of this study was to explore the relationships of medication burden in COPD with clinical outcomes, comorbidities, and multidimensional indices.Methods: In a cross-sectional study, COPD patients (n=222) were assessed for demographic information, comorbidities, medication use, and clinical outcomes. Complexity of medication regimens was quantified using the validated medication regimen complexity index (MRCI).Results: Participants (58.6% males) had a mean (SD) age of 69.1 (8.3) years with a postbronchodilator forced expiratory volume in 1 second % predicted of 56.5 (20.4) and a median of five comorbidities. The median (q1, q3) total MRCI score was 24 (18.5, 31). COPD-specific medication regimens were more complex than those of non-COPD medications (median MRCI: 14.5 versus 9, respectively; P

Netsanet A Negewo,1,2 Vanessa M McDonald,1–4 Katherine J Baines,1,2 Peter AB Wark,1–3 Jodie L Simpson,1,2 Paul W Jones,5 Peter G Gibson1–3 1Priority Research Centre for Healthy Lungs, Faculty of Health and Medicine, The University of Newcastle, Callaghan, NSW, Australia; 2Hunter Medical Research Institute, Faculty of Health and Medicine, The University of Newcastle, Callaghan, NSW, Australia; 3Department of Respiratory and Sleep Medicine, John Hunter Hospital, Newcastle, NSW, Australia; 4School of Nursing and Midwifery, Faculty of Health and Medicine, The University of Newcastle, Callaghan, NSW, Australia; 5Institute for Infection and Immunity, St George’s, University of London, London, UK Introduction: Sputum eosinophilia occurs in approximately one-third of stable chronic obstructive pulmonary disease (COPD) patients and can predict exacerbation risk and response to corticosteroid treatments. Sputum induction, however, requires expertise, may not always be successful, and does not provide point-of-care results. Easily applicable diagnostic markers that can predict sputum eosinophilia in stable COPD patients have the potential to progress COPD management. This study investigated the correlation and predictive relationship between peripheral blood and sputum eosinophils. It also examined the repeatability of blood eosinophil counts.Methods: Stable COPD patients (n=141) were classified as eosinophilic or noneosinophilic based on their sputum cell counts (≥3%), and a cross-sectional analysis was conducted comparing their demographics, clinical characteristics, and blood cell counts. Receiver operating characteristic curve analysis was used to assess the predictive ability of blood eosinophils for sputum eosinophilia. Intraclass correlation coefficient was used to examine the repeatability of blood eosinophil counts.Results: Blood eosinophil counts were significantly higher in patients with sputum eosinophilia (n=45) compared to those without (0.3×109/L vs 0.15×109/L; P

Background: Cell-free DNA reflects both normal and tumor-derived DNA released into the circulation through cellular necrosis and apoptosis. The authors sought to determine the role of preoperative total plasma cell-free DNA levels in predicting clinical outcome in patients with ovarian cancer.Methods: After institutional review board consent, DNA was extracted from plasma of 164 women with invasive epithelial ovarian carcinoma (EOC), 49 with benign ovarian neoplasms, and 75 age-matched controls. The samples were randomly divided into training (n = 144) and validation (n = 144) sets. Quantification of cell-free DNA was performed using real-time polymerase chain reaction for beta-globin, and the number of genome equivalents (GE) per milliliter of plasma was determined. Cell-free DNA was correlated with clinicopathologic parameters.Results: The training and validation sets were similar in terms of demographic features. In the training set, EOC patients had a median preoperative cell-free DNA level of 10,113 GE/mL, compared with patients with benign ovarian neoplasms (median, 2365 GE/mL; P < .0001) and controls (median, 1912 GE/mL, P < .0001). Cell-free DNA >22,000 GE/mL was significantly associated with decreased patient survival (P < .001). After adjusting for other clinical variables, preoperative cell-free DNA >22,000 GE/mL was an independent predictor (P = .02) for disease-specific survival. Analysis of the validation set confirmed significantly higher cell-free DNA levels in EOC (median, 13,672 GE/mL) and that cell-free DNA >22,000 GE/mL was associated with a 2.83-fold increased risk of death from disease (P < .001).Conclusions: Preoperative plasma total cell-free DNA levels are significantly elevated in patients with EOC. Elevated plasma cell-free DNA is an independent predictor for death from disease in ovarian cancer. [ABSTRACT FROM AUTHOR]

There are several well-documented indications for using this technology to diagnose a genetic disorder before implantation, says this distinguished expert. But when should obstetricians recommend PGD aneuploidy testing to women of advanced maternal age? [ABSTRACT FROM AUTHOR]

Array-based comparative genomic hybridization detects significantly more and smaller abnormalities than standard chromosome analysis. Already in limited use, the test may well replace karotyping for prenatal diagnosis of genetic disorders. [ABSTRACT FROM AUTHOR]

Rationale: Asthma is characterized by both chronic inflammation and remodeling of the airways. Proteases are important mediators of inflammation, cytokine activation, and tissue remodeling. Objectives: This study investigated matrix metalloproteinase-9 (MMP-9) and neutrophil elastase (NE) enzyme activity in the sputum of subjects with different inflammatory phenotypes of asthma (eosinophilic, neutrophilic, and paucigranulocytic asthma) and in healthy control subjects. Methods and Measurements: Nonsmoking adults with asthma and healthy control sujects underwent hypertonic saline challenge and sputum induction. Selected sputum portions were dispersed with dithiothreitol and assayed for MMP-9 and NE enzyme activity. Main Results: Subjects with eosinophilic asthma had significantly more active MMP-9 (39 ng/ml) compared with those with neutrophilic asthma (10 ng/ml) and control subjects (2.5 ng/ml, p < 0.01). Although there were high levels of total MMP-9 in neutrophilic asthma (5,273 ng/ml), most (> 99%) was inactivated (and bound to tissue inhibitor of metalloproteinase-1). In neutrophilic asthma, more subjects had NE activity (39%) compared with both healthy control subjects (0%), subjects with eosinophilic asthma (6%), or subjects with paucigranulocytic asthma (0%, p < 0.05). There were strong and consistent positive correlations between interleukin-8, neutrophils, and proteolytic enzymes. MMP-9 was inversely correlated with NE (r = -0.93). Conclusions: Proteolytic enzyme activity in asthma is dependent on the underlying inflammatory phenotype and is differentially regulated with MMP-9 activity a feature of eosinophilic inflammation, and active NE in neutrophilic inflammation. [ABSTRACT FROM AUTHOR]

Objective: Early pregnancy losses increase with marked hyperglycemia in diabetic pregnancy. However, mean loss rates do not differ from those of nondiabetic pregnancy. This observation might be explained by increased fetal losses at the extremes of glycemia in diabetic and nondiabetic pregnancy. To test this hypothesis, we examined relationships of proximate measures of prior glycemia, glycated protein and fructosamine, to pregnancy loss.Research Design and Methods: A total of 389 diabetic and 429 nondiabetic pregnant subjects participated in the Diabetes In Early Pregnancy study. Glycated protein and fructosamine measurements were standardized as multiples of control values for each center (Z score). The logarithm of odds of pregnancy loss were plotted against Z scores and tested by logistic models.Results: Mean pregnancy loss rates were 12% in diabetic and 13% in normal pregnancies. However, over six intervals of glycated protein in diabetic pregnancy, fetal loss rates at the upper and lower extremes (24 and 33%, respectively) were approximately threefold higher than the four intervening rates (8-14%). The odds ratio of pregnancy loss for these extreme intervals to the intervening intervals is 3.0 (P = 0.01). Nondiabetic losses showed a similar pattern. In confirmation, logit pregnancy losses were increased in a J-shaped curve at the glycemic extremes in normal (P < 0.019) and diabetic (P < 0.015) pregnancy. The upper glycemic extreme in diabetic pregnancy was two- to fivefold higher than in control pregnancy.Conclusions: Pregnancy losses are increased at the extremes of glycemia in both normal and diabetic pregnancy but at higher levels in diabetic pregnancy. The data suggest defensive adaptations against hyperglycemia in diabetic pregnancy. [ABSTRACT FROM AUTHOR]

Induced sputum 8-iso-prostaglandin F(2alpha) (PGF(2alpha)) concentrations may be a useful marker of oxidative stress in airways disease. This study examines oxidative stress (measured by 8-iso-PGF(2alpha)) in airway disease according to disease type (asthma and bronchiectasis), disease activity (stable and acute asthma), and disease pattern (intermittent, mild, moderate, and severe persistent asthma). We compared subjects with stable asthma (n = 71) and bronchiectasis (n = 23) with healthy control subjects (n = 29). Another group of patients with asthma (n = 39) were assessed during and after acute exacerbation. Induced sputum 8-iso-PGF(2alpha) concentrations were validated and found to be elevated in subjects with stable asthma and bronchiectasis versus control subjects (median [interquartile range] 216 [103-389] and 698 [264-1,613] ng/L vs. 123 [41-290] ng/L, p < 0.001) and increased as clinical asthma pattern worsened (intermittent 115 [42-153], mild persistent 116 [89-229] ng/L, moderate persistent 183 [110-317] ng/L, severe persistent 387 [102-587] ng/L; p = 0.010). Sputum 8-iso-PGF(2alpha) concentrations were elevated during acute asthma and decreased with recovery (458 [227-950] ng/L vs. 214 [148-304] ng/L, p = 0.0002). We conclude that 8-iso-PGF(2alpha) is involved in the pathophysiology of inflammatory airway diseases, being related to disease type, pattern, and activity. Analysis of 8-iso-PGF(2alpha) concentrations in induced sputum provides a useful tool for monitoring oxidative stress and investigating strategies aimed at reducing oxidative stress in airways disease. [ABSTRACT FROM AUTHOR]

The latest official guidelines on cystic fibrosis screening have some clinicians bewildered--and others looking for an easy way to put them to good use. A top expert in the field provides practical advice on how to individualize the recommendations. [ABSTRACT FROM AUTHOR]

Isolation and analysis of intact fetal cells in maternal blood is an attractive method of non-invasive prenatal diagnosis; however, detection levels are not optimal. The poor sensitivity and inconsistent recovery of fetal cells is compounded by small numbers of circulating fetal cells and loss of fetal cells during enrichment procedures. Optimizing selection criteria by utilizing less complicated methods for target cell enrichment is essential. We report here salutary results using a simple density-based depletion method that requires neither MACS (magnetic-activated cell sorting) nor flow cytometric separation for enrichment of progenitor cells. Maternal blood samples (n = 81) were obtained from women prior to invasive prenatal genetic diagnostic procedures and processed randomly within 24 h using one of two density-based enrichment methods. For progenitor cell enrichment, samples (n = 49) were labeled with a RosetteSep™ progenitor antibody cocktail to remove unwanted mature T-cells, B-cells, granulocytes, natural killer cells, neutrophils and myelomonocytic cells. For CD45-negative cell enrichment, samples (n = 14) were labeled with RosetteSep™ CD45 antibody to remove unwanted maternal white cells. The desired cellular fraction was collected and analyzed by either fluorescent in situ hybridization (FISH) or real-time PCR for the presence of intact fetal cells and to quantify Y-chromosome-specific DYS1 sequences, respectively. Overall, FISH and real-time PCR correct detection rates for the progenitor cell enrichment approach were 53% and 89% with 3% (1 out of 30 cases) and 0% false–positive detection, respectively. Fetal sequences were detected in the range from 0.067 to 1.167 genome equivalents per milliliter of blood. No fetal cells were detected using the CD45-negative enrichment method. Flow cytometric analysis of cord blood showed that a unique myeloid population of cells was recovered using... [ABSTRACT FROM AUTHOR]

Although successful pregnancies have been reported in women with this enzyme deficiency, patients are at increased risk for deteriorating respiratory function and IUGR. [ABSTRACT FROM AUTHOR]