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1.  A Large Scale, Ion Current based Proteomics Investigation of Bronchoalveolar Lavage Fluid in Chronic Obstructive Pulmonary Disease Patients 
Journal of proteome research  2013;13(2):627-639.
Proteomic analysis of bronchoalveolarBlavageBfluid (BALF) in chronic obstructive pulmonary disease (COPD) patients may provide new biomarkers and deeper understanding of the disease mechanisms but remains challenging. Here we describe an ionBcurrentBbased strategy for comparative analysis of BALF proteomes from patients with moderate and stable COPD vs. healthy controls. The strategy includes an efficient preparation procedure providing quantitative recovery and a nanoBLC/MS analysis with a long, heated column. Under optimized conditions, high efficiency and reproducibility were achieved for each step, enabling a “20Bplex” comparison of clinical subjects (n=10/group). Without depletion/fractionation, a total of 423 unique protein groups were quantified under stringent criteria with at least two quantifiable peptides. SeventyBsix proteins were determined as significantlyBaltered in COPD, which represent a diversity of biological processes such as alcohol metabolic process, gluconeogenesis/glycolysis, inflammatory response, proteolysis, and oxidation reduction. Interestingly, altered alcohol metabolism responding to oxidant stress is a novel observation in COPD. The prominently elevated key enzymes involved in alcohol metabolism (e.g. ADH1B, ALDH2&ALDH3A1) may provide a reasonable explanation for a bewildering observation in COPD patients known for decades: the underestimation of the blood alcohol concentrations through breath tests. These discoveries could provide new insights for identifying novel biomarkers and pathological mediators in clinical studies.
PMCID: PMC4073647  PMID: 24188068
Biomarker Discovery; Bronchoalveolar Lavage Fluid; Chronic Obstructive Pulmonary Disease; Peptide Extracted Ion Current
2.  Airway Microbiome Dynamics in Exacerbations of Chronic Obstructive Pulmonary Disease 
Journal of Clinical Microbiology  2014;52(8):2813-2823.
Specific bacterial species are implicated in the pathogenesis of exacerbations of chronic obstructive pulmonary disease (COPD). However, recent studies of clinically stable COPD patients have demonstrated a greater diversity of airway microbiota, whose role in acute exacerbations is unclear. In this study, temporal changes in the airway microbiome before, at the onset of, and after an acute exacerbation were examined in 60 sputum samples collected from subjects enrolled in a longitudinal study of bacterial infection in COPD. Microbiome composition and predicted functions were examined using 16S rRNA-based culture-independent profiling methods. Shifts in the abundance (≥2-fold, P < 0.05) of many taxa at exacerbation and after treatment were observed. Microbiota members that were increased at exacerbation were primarily of the Proteobacteria phylum, including nontypical COPD pathogens. Changes in the bacterial composition after treatment for an exacerbation differed significantly among the therapy regimens clinically prescribed (antibiotics only, oral corticosteroids only, or both). Treatment with antibiotics alone primarily decreased the abundance of Proteobacteria, with the prolonged suppression of some microbiota members being observed. In contrast, treatment with corticosteroids alone led to enrichment for Proteobacteria and members of other phyla. Predicted metagenomes of particular microbiota members involved in these compositional shifts indicated exacerbation-associated loss of functions involved in the synthesis of antimicrobial and anti-inflammatory products, alongside enrichment in functions related to pathogen-elicited inflammation. These trends reversed upon clinical recovery. Further larger studies will be necessary to determine whether specific compositional or functional changes detected in the airway microbiome could be useful indicators of exacerbation development or outcome.
PMCID: PMC4136157  PMID: 24850358
3.  Phagocytic Dysfunction of Human Alveolar Macrophages and Severity of Chronic Obstructive Pulmonary Disease 
The Journal of Infectious Diseases  2013;208(12):2036-2045.
Background. Alveolar macrophages in chronic obstructive pulmonary disease (COPD) have fundamental impairment of phagocytosis for nontypeable Haemophilus influenzae (NTHI). However, relative selectivity of dysfunctional phagocytosis among diverse respiratory pathogens: NTHI, Moraxella catarrhalis (MC), Streptococcus pneumoniae (SP), and nonbacterial particles, as well as the contribution of impaired phagocytosis to severity of COPD, has not been explored.
Methods. Alveolar macrophages, obtained from nonsmokers (n = 20), COPD ex-smokers (n = 32), and COPD active smokers (n = 64), were incubated with labeled NTHI, MC, SP, and fluorescent microspheres. Phagocytosis was measured as intracellular percentages of each.
Results. Alveolar macrophages of COPD ex-smokers and active smokers had impaired complement-independent phagocytosis of NTHI (P = .003) and MC (P = .0007) but not SP or microspheres. Nonetheless, complement-mediated phagocytosis was enhanced within each group only for SP. Defective phagocytosis was significantly greater for NTHI than for MC among COPD active smokers (P < .0001) and ex-smokers (P = .028). Moreover, severity of COPD (FEV1%predicted) correlated with impaired AM phagocytosis for NTHI (P = .0016) and MC (P = .01).
Conclusions. These studies delineate pathogen- and host-specific differences in defective alveolar macrophages phagocytosis of respiratory bacteria in COPD, further elucidating the immunologic basis for bacterial persistence in COPD and provide the first demonstration of association of impaired phagocytosis to severity of disease.
PMCID: PMC3836465  PMID: 23908477
nontypeable Haemophilus influenzae; Moraxella catarrhalis; Streptococcus pneumoniae; alveolar macrophage; phagocytosis; COPD
4.  Measuring respiratory symptoms of COPD: performance of the EXACT- Respiratory Symptoms Tool (E-RS) in three clinical trials 
Respiratory Research  2014;15(1):124.
Symptomatic relief is an important treatment goal for patients with COPD. To date, no diary for evaluating respiratory symptoms in clinical trials has been developed and scientifically-validated according to FDA and EMA guidelines. The EXACT – Respiratory Symptoms (E-RS) scale is a patient-reported outcome (PRO) measure designed to address this need. The E-RS utilizes 11 respiratory symptom items from the existing and validated 14-item EXACT, which measures symptoms of exacerbation. The E-RS total score quantifies respiratory symptom severity, and 3 domains assess breathlessness, cough and sputum, and chest symptoms.
This study examined the performance of the E-RS in each of 3 controlled trials with common and unique validation variables: one 6-month (N = 235, US) and two 3-month (N = 749; N = 597; international). Subjects completed the E-RS as part of a daily eDiary. Tests of reliability, validity, and responsiveness were conducted in each dataset.
In each study, RS-Total score was internally consistent (Cronbach α) (0.88, 0.92, 0.92) and reproducible (intra-class correlation) in stable patients (2 days apart: 0.91; 7 days apart: 0.71, 0.74). RS-Total scores correlated significantly with the following criterion variables (Spearman’s rho; p < 0.01, all comparisons listed here): FEV1% predicted (−0.19, −0.14, −0.15); St. George’s Respiratory Questionnaire (SGRQ) (0.65, 0.52, 0.51); Breathlessness, Cough, and Sputum Scale (BCSS) (0.89, 0.89); modified Medical Research Council dyspnoea scale (mMRC) (0.40); rescue medication use (0.43, 0.42); Functional Performance Inventory Short-Form (FPI-SF) (0.43); 6-minute walk distance (6-MWT) (−0.30, −0.14) and incremental shuttle walk (ISWT) (−0.18) tests. Correlations between these variables and RS-Breathlessness, RS-Cough and Sputum, RS-Chest Symptoms scores supported subscale validity. RS-Total, RS-Breathlessness, and RS-Chest Symptoms differentiated mMRC levels of breathlessness severity (p < 0.0001). RS-Total and domain scores differentiated subjects with no rescue medication use and 3 or more puffs (p < 0.0001). Sensitivity to changes in health status (SGRQ), symptoms (BCSS), and exercise capacity (6MWT, ISWT) were also shown and responder definitions using criterion- and distribution-based methods are proposed.
Results suggest the E-RS is a reliable, valid, and responsive measure of respiratory symptoms of COPD suitable for use in natural history studies and clinical trials.
Trial registration
MPEX: NCT00739648; AZ1: NCT00949975; AZ 2: NCT01023516
Electronic supplementary material
The online version of this article (doi:10.1186/s12931-014-0124-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4203869  PMID: 25287629
COPD; Respiratory symptoms; Clinical trials; Dyspnoea; Cough; Sputum; Chest symptoms
5.  Lung flute improves symptoms and health status in COPD with chronic bronchitis: A 26 week randomized controlled trial 
Chronic obstructive pulmonary disease (COPD) is characterized by mucus hypersecretion that contributes to disease related morbidity and is associated with increased mortality. The Lung Flute® is a new respiratory device that produces a low frequency acoustic wave with moderately vigorous exhalation to increase mucus clearance. We hypothesized that the Lung Flute, used on a twice daily basis will provide clinical benefit to patients with COPD with chronic bronchitis.
We performed a 26 week randomized, non-intervention controlled, single center, open label trial in 69 patients with COPD and Chronic Bronchitis. The primary endpoint was change in respiratory symptoms measured with the Chronic COPD Questionnaire (CCQ). Secondary endpoints included health status, assessed by the St. George Respiratory questionnaire (SGRQ), BODE (Body-Mass Index, Airflow Obstruction, Dyspnea, and Exercise Capacity)
index score and exacerbation frequency.
While the control patients did not demonstrate any significant changes in the primary endpoint (CCQ change at 26 weeks of +0.01, p = 0.8), a trend (p = 0.08) to decrease (improvement) in the CCQ (-0.23 at 26 weeks) was seen with the Lung Flute. Furthermore, a significant improvement in the symptom domain of the CCQ was seen only with the lung flute (-0.42, p = 0.004). Health status (SGRQ) improvement, was also only seen with the Lung Flute (-3.23, p = 0.03). The BODE score increased in the control group (3.31 at baseline, 4.14 at 26 weeks), however it remained stable in the Lung Flute arm (3.16 at baseline and 26 weeks), with the changes from baseline being significantly different between the 2 arms (p = 0.01). There was a trend for less exacerbations in the Lung Flute group (p = 0.07). Adverse effects were minor, with only 1 patient discontinuing treatment because of lack of efficacy. Serious adverse effects seen were all determined to be unrelated to the device use.
The Lung Flute is a safe and effective treatment in COPD with chronic bronchitis, providing a wide array of benefits. Identifier
PMCID: PMC4299801  PMID: 25625006
Mucus clearance; COPD; Oscillatory device; Chronic bronchitis
6.  Design and validation of a supragenome array for determination of the genomic content of Haemophilus influenzae isolates 
BMC Genomics  2013;14:484.
Haemophilus influenzae colonizes the human nasopharynx as a commensal, and is etiologically associated with numerous opportunistic infections of the airway; it is also less commonly associated with invasive disease. Clinical isolates of H. influenzae display extensive genomic diversity and plasticity. The development of strategies to successfully prevent, diagnose and treat H. influenzae infections depends on tools to ascertain the gene content of individual isolates.
We describe and validate a Haemophilus influenzae supragenome hybridization (SGH) array that can be used to characterize the full genic complement of any strain within the species, as well as strains from several highly related species. The array contains 31,307 probes that collectively cover essentially all alleles of the 2890 gene clusters identified from the whole genome sequencing of 24 clinical H. influenzae strains. The finite supragenome model predicts that these data include greater than 85% of all non-rare genes (where rare genes are defined as those present in less than 10% of sequenced strains). The veracity of the array was tested by comparing the whole genome sequences of eight strains with their hybridization data obtained using the supragenome array. The array predictions were correct and reproducible for ~ 98% of the gene content of all of the sequenced strains. This technology was then applied to an investigation of the gene content of 193 geographically and clinically diverse H. influenzae clinical strains. These strains came from multiple locations from five different continents and Papua New Guinea and include isolates from: the middle ears of persons with otitis media and otorrhea; lung aspirates and sputum samples from pneumonia and COPD patients, blood specimens from patients with sepsis; cerebrospinal fluid from patients with meningitis, as well as from pharyngeal specimens from healthy persons.
These analyses provided the most comprehensive and detailed genomic/phylogenetic look at this species to date, and identified a subset of highly divergent strains that form a separate lineage within the species. This array provides a cost-effective and high-throughput tool to determine the gene content of any H. influenzae isolate or lineage. Furthermore, the method for probe selection can be applied to any species, given a group of available whole genome sequences.
PMCID: PMC3723446  PMID: 23865594
7.  Significance of the microbiome in obstructive lung disease 
Thorax  2012;67(5):456-463.
The composition of the lung microbiome contributes to both health and disease, including obstructive lung disease. Because it has been estimated that over 70% of the bacterial species on body surfaces cannot be cultured by currently available techniques, traditional culture techniques are no longer the gold standard for microbial investigation. Advanced techniques that identify bacterial sequences, including the 16S ribosomal RNA gene, have provided new insights into the depth and breadth of microbiota present both in the diseased and normal lung. In asthma, the composition of the microbiome of the lung and gut during early childhood development may play a key role in the development of asthma, while specific airway microbiota are associated with chronic asthma in adults. Early bacterial stimulation appears to reduce asthma susceptibility by helping the immune system develop lifelong tolerance to innocuous antigens. By contrast, perturbations in the microbiome from antibiotic use may increase the risk for asthma development. In chronic obstructive pulmonary disease, bacterial colonisation has been associated with a chronic bronchitic phenotype, increased risk of exacerbations, and accelerated loss of lung function. In cystic fibrosis, studies utilising culture-independent methods have identified associations between decreased bacterial community diversity and reduced lung function; colonisation with Pseudomonas aeruginosa has been associated with the presence of certain CFTR mutations. Genomic analysis of the lung microbiome is a young field, but has the potential to define the relationship between lung microbiome composition and disease course. Whether we can manipulate bacterial communities to improve clinical outcomes remains to be seen.
PMCID: PMC3578398  PMID: 22318161
9.  A Clonal Group of Nontypeable Haemophilus influenzae with Two IgA Proteases Is Adapted to Infection in Chronic Obstructive Pulmonary Disease 
PLoS ONE  2011;6(10):e25923.
Strains of nontypeable Haemophilus influenzae show enormous genetic heterogeneity and display differential virulence potential in different clinical settings. The igaB gene, which encodes a newly identified IgA protease, is more likely to be present in the genome of COPD strains of H. influenzae than in otitis media strains. Analysis of igaB and surrounding sequences in the present study showed that H. influenzae likely acquired igaB from Neisseria meningitidis and that the acquisition was accompanied by a ∼20 kb genomic inversion that is present only in strains that have igaB. As part of a long running prospective study of COPD, molecular typing of H. influenzae strains identified a clonally related group of strains, a surprising observation given the genetic heterogeneity that characterizes strains of nontypeable H. influenzae. Analysis of strains by 5 independent methods (polyacrylamide gel electrophoresis, multilocus sequence typing, igaB gene sequences, P2 gene sequences, pulsed field gel electrophoresis) established the clonal relationship among the strains. Analysis of 134 independent strains collected prospectively from a cohort of adults with COPD demonstrated that ∼10% belonged to the clonal group. We conclude that a clonally related group of strains of nontypeable H. influenzae that has two IgA1 protease genes (iga and igaB) is adapted for colonization and infection in COPD. This observation has important implications in understanding population dynamics of H. influenzae in human infection and in understanding virulence mechanisms specifically in the setting of COPD.
PMCID: PMC3187821  PMID: 21998721
10.  A novel study design for antibiotic trials in acute exacerbations of COPD: MAESTRAL methodology 
Antibiotics, along with oral corticosteroids, are standard treatments for acute exacerbations of chronic obstructive pulmonary disease (AECOPD). The ultimate aims of treatment are to minimize the impact of the current exacerbation, and by ensuring complete resolution, reduce the risk of relapse. In the absence of superiority studies of antibiotics in AECOPD, evidence of the relative efficacy of different drugs is lacking, and so it is difficult for physicians to select the most effective antibiotic. This paper describes the protocol and rationale for MAESTRAL (moxifloxacin in AECBs [acute exacerbation of chronic bronchitis] trial; NCT00656747), one of the first antibiotic comparator trials designed to show superiority of one antibiotic over another in AECOPD. It is a prospective, multinational, multicenter, randomized, double-blind controlled study of moxifloxacin (400 mg PO [ per os] once daily for 5 days) vs amoxicillin/clavulanic acid (875/125 mg PO twice daily for 7 days) in outpatients with COPD and chronic bronchitis suffering from an exacerbation. MAESTRAL uses an innovative primary endpoint of clinical failure: the requirement for additional or alternate treatment for the exacerbation at 8 weeks after the end of antibiotic therapy, powered for superiority. Patients enrolled are those at high-risk of treatment failure, and all are experiencing an Anthonisen type I exacerbation. Patients are stratified according to oral corticosteroid use to control their effect across antibiotic treatment arms. Secondary endpoints include quality of life, symptom assessments and health care resource use.
PMCID: PMC3133509  PMID: 21760724
AECOPD; moxifloxacin; amoxicillin/clavulanic acid; clinical trial design; exacerbation; antibiotic
11.  Lung endothelial monocyte-activating protein 2 is a mediator of cigarette smoke–induced emphysema in mice 
The Journal of Clinical Investigation  2011;121(6):2470-2479.
Pulmonary emphysema is a disease characterized by alveolar cellular loss and inflammation. Recently, excessive apoptosis of structural alveolar cells has emerged as a major mechanism in the development of emphysema. Here, we investigated the proapoptotic and monocyte chemoattractant cytokine endothelial monocyte-activating protein 2 (EMAPII). Lung-specific overexpression of EMAPII in mice caused simplification of alveolar structures, apoptosis, and macrophage accumulation, compared with that in control transgenic mice. Additionally, in a mouse model of cigarette smoke–induced (CS-induced) emphysema, EMAPII levels were significantly increased in murine lungs. This upregulation was necessary for emphysema development, as neutralizing antibodies to EMAPII resulted in reduced alveolar cell apoptosis, inflammation, and emphysema-associated structural changes in alveoli and small airways and improved lung function. The mechanism of EMAPII upregulation involved an apoptosis-dependent feed-forward loop, since caspase-3 instillation in the lung markedly increased EMAPII expression, while caspase inhibition decreased its production, even in transgenic EMAPII mice. These findings may have clinical significance, as both current smokers and ex-smoker chronic obstructive pulmonary disease (COPD) patients had increased levels of secreted EMAPII in the bronchoalveolar lavage fluid compared with that of nonsmokers. In conclusion, we suggest that EMAPII perpetuates the mechanism of CS-induced lung emphysema in mice and, given its secretory nature, is a suitable target for neutralization antibody therapy.
PMCID: PMC3104742  PMID: 21576822
12.  Nontypeable Haemophilus influenzae in chronic obstructive pulmonary disease and lung cancer 
Chronic obstructive pulmonary disease (COPD) is predicted to become the third leading cause of death in the world by 2020. It is characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lungs to noxious particles and gases, most commonly cigarette smoke. Among smokers with COPD, even following withdrawal of cigarette smoke, inflammation persists and lung function continues to deteriorate. One possible explanation is that bacterial colonization of smoke-damaged airways, most commonly with nontypeable Haemophilus influenzae (NTHi), perpetuates airway injury and inflammation. Furthermore, COPD has also been identified as an independent risk factor for lung cancer irrespective of concomitant cigarette smoke exposure. In this article, we review the role of NTHi in airway inflammation that may lead to COPD progression and lung cancer promotion.
PMCID: PMC3048087  PMID: 21407824
COPD; NTHi; inflammation
13.  Molecular Basis of Increased Serum Resistance among Pulmonary Isolates of Non-typeable Haemophilus influenzae 
PLoS Pathogens  2011;7(1):e1001247.
Non-typeable Haemophilus influenzae (NTHi), a common commensal of the human pharynx, is also an opportunistic pathogen if it becomes established in the lower respiratory tract (LRT). In comparison to colonizing isolates from the upper airway, LRT isolates, especially those associated with exacerbations of chronic obstructive pulmonary disease, have increased resistance to the complement- and antibody-dependent, bactericidal effect of serum. To define the molecular basis of this resistance, mutants constructed in a serum resistant strain using the mariner transposon were screened for loss of survival in normal human serum. The loci required for serum resistance contribute to the structure of the exposed surface of the bacterial outer membrane. These included loci involved in biosynthesis of the oligosaccharide component of lipooligosaccharide (LOS), and vacJ, which functions with an ABC transporter encoded by yrb genes in retrograde trafficking of phospholipids from the outer to inner leaflet of the cell envelope. Mutations in vacJ and yrb genes reduced the stability of the outer membrane and were associated with increased cell surface hyrophobicity and phospholipid content. Loss of serum resistance in vacJ and yrb mutants correlated with increased binding of natural immunoglobulin M in serum as well as anti-oligosaccharide mAbs. Expression of vacJ and the yrb genes was positively correlated with serum resistance among clinical isolates. Our findings suggest that NTHi adapts to inflammation encountered during infection of the LRT by modulation of its outer leaflet through increased expression of vacJ and yrb genes to minimize recognition by bactericidal anti-oligosaccharide antibodies.
Author Summary
Haemophilus influenzae generally colonizes the human upper respiratory tract. When isolated from the lower respiratory tract, this opportunistic pathogen is associated with inflammatory conditions such as pneumonia and exacerbations of chronic obstructive pulmonary disease (COPD). Here we show that one of the adaptations made by H. influenzae isolated from the lower respiratory tract is increased resistance to the bactericidal effect of antibody and complement. To define the mechanism for increased resistance, mutants were screened to identify the complete set of genes required to inhibit killing by antibody and complement. These included multiple genes that all contribute to biosynthesis of the organism's surface oligosaccharide (lipooligosaccharide), which is targeted by bactericidal antibody. Our results also revealed a novel function for additional genes that maintain the lipid asymmetry of the surface membrane and thereby limit recognition of the pathogen by anti-oligosaccharide antibodies.
PMCID: PMC3017122  PMID: 21253576
14.  Epitope mapping immunodominant regions of the PilA protein of nontypeable Haemophilus influenzae (NTHI) to facilitate the design of two novel chimeric vaccine candidates 
Vaccine  2009;28(1):279-289.
We designed and tested three PilA-derived vaccine candidates in a chinchilla model of ascending nontypeable Haemophilus influenzae (NTHI)-induced otitis media (OM). Delivery of antiserum directed against each immunogen conferred varying degrees of protection. Presentation of a B-cell epitope derived from the OMP P5 adhesin at the N-terminus of recombinant soluble PilA protein (as opposed to the C-terminus), resulted in a protective chimeric immunogen that combined epitopes from two distinct NTHI adhesins (type IV pili and OMP P5). Incorporating protective epitopes derived from two NTHI adhesins/virulence determinants into a single pediatric vaccine candidate to prevent OM has multiple potential inherent advantages.
PMCID: PMC2787809  PMID: 19699813
chinchilla; otitis media; COPD
16.  Use of Moraxella catarrhalis Lipooligosaccharide Mutants To Identify Specific Oligosaccharide Epitopes Recognized by Human Serum Antibodies▿  
Infection and Immunity  2009;77(10):4548-4558.
Moraxella catarrhalis is a causative agent of otitis media in children and lower respiratory tract infections in adults suffering from chronic obstructive pulmonary disease (COPD). This strict human pathogen continues to be a significant cause of disease in this broad spectrum of patients because there is no available vaccine. Although numerous putative vaccine antigens have been described, little is known about the human immune response to M. catarrhalis infection in vivo. Human serum antibodies are directed at a number of surface proteins, and lipooligosaccharides (LOS) and detoxified LOS may be an effective immunogen in mice. In this study, we used a specific LOS-based enzyme-linked immunosorbent assay (ELISA), containing the three major M. catarrhalis serotypes together with a complete series of truncated LOS mutants, to detect the development of new antibodies to specific regions of the oligosaccharide molecule. We compared serum samples from COPD patients who had recently cleared an M. catarrhalis infection to serum samples collected prior to their infection. Variability in the antibody response to LOS was observed, as some patients developed serotype-specific antibodies, others developed antibodies to the LOS of each serotype, others developed broadly cross-reactive antibodies, and some did not develop new antibodies. These newly developed human antibodies are directed at both side chains and core structures in the LOS molecule. This LOS-based ELISA can be used to dissect the human antibody response to both internal and external carbohydrate epitopes, thus providing a better understanding of the humoral immune response to M. catarrhalis LOS epitopes developed during natural infection.
PMCID: PMC2747917  PMID: 19651870
17.  Pulsed moxifloxacin for the prevention of exacerbations of chronic obstructive pulmonary disease: a randomized controlled trial 
Respiratory Research  2010;11(1):10.
Acute exacerbations contribute to the morbidity and mortality associated with chronic obstructive pulmonary disease (COPD). This proof-of-concept study evaluates whether intermittent pulsed moxifloxacin treatment could reduce the frequency of these exacerbations.
Stable patients with COPD were randomized in a double-blind, placebo-controlled trial to receive moxifloxacin 400 mg PO once daily (N = 573) or placebo (N = 584) once a day for 5 days. Treatment was repeated every 8 weeks for a total of six courses. Patients were repeatedly assessed clinically and microbiologically during the 48-week treatment period, and for a further 24 weeks' follow-up.
At 48 weeks the odds ratio (OR) for suffering an exacerbation favoured moxifloxacin: per-protocol (PP) population (N = 738, OR 0.75, 95% confidence interval (CI) 0.565-0.994, p = 0.046), intent-to-treat (ITT) population (N = 1149, OR 0.81, 95% CI 0.645-1.008, p = 0.059), and a post-hoc analysis of per-protocol (PP) patients with purulent/mucopurulent sputum production at baseline (N = 323, OR 0.55, 95% CI 0.36-0.84, p = 0.006).
There were no significant differences between moxifloxacin and placebo in any pre-specified efficacy subgroup analyses or in hospitalization rates, mortality rates, lung function or changes in St George's Respiratory Questionnaire (SGRQ) total scores. There was, however, a significant difference in favour of moxifloxacin in the SGRQ symptom domain (ITT: -8.2 vs -3.8, p = 0.009; PP: -8.8 vs -4.4, p = 0.006). Moxifloxacin treatment was not associated with consistent changes in moxifloxacin susceptibility. There were more treatment-emergent, drug related adverse events with moxifloxacin vs placebo (p < 0.001) largely due to gastrointestinal events (4.7% vs 0.7%).
Intermittent pulsed therapy with moxifloxacin reduced the odds of exacerbation by 20% in the ITT population, by 25% among the PP population and by 45% in PP patients with purulent/mucopurulent sputum at baseline. There were no unexpected adverse events and there was no evidence of resistance development.
Trial registration number, NCT00473460 (
PMCID: PMC2834642  PMID: 20109213
18.  Moraxella catarrhalis acquisition, airway inflammation and protease-antiprotease balance in chronic obstructive pulmonary disease 
Moraxella catarrhalis causes approximately 10% of exacerbations in chronic obstructive pulmonary disease (COPD) and also colonizes the lower airway in stable patients. Little is known about the effects of colonization by M. catarrhalis on airway inflammation and protease-antiprotease balance, and how these changes compare to those seen during exacerbations. Since COPD is a progressive inflammatory disease, elucidating the effects of bacterial colonization and exacerbation on airway inflammation is relevant to understanding disease progression in COPD. Our aims were (1) Analyze changes in airway inflammation in colonization and exacerbation of COPD due to M. catarrhalis; (2) Explore protease-antiprotease balance in colonization and exacerbation due to M. catarrhalis. Our hypothesis were (1) Acquisition of a new strain of M. catarrhalis in COPD increases airway inflammation from baseline and alters the protease-antiprotease balance towards a more proteolytic environment; (2) These changes are greater during exacerbations associated with M. catarrhalis as compared to colonization.
Thirty-nine consecutive COPD patients with 76 acquisitions of a new strain of M. catarrhalis over a 6-year period were identified in a prospective study. Seventy-six pre-acquisition sputum supernatant samples, obtained just before acquisition of M catarrhalis, and 76 acquisition samples (34 were associated with exacerbation, 42 with colonization) were analyzed for IL-8, TNF-α, Neutrophil Elastase (NE) and Secretory leukocyte protease inhibitor (SLPI). Changes were compared in paired samples from each patient.
IL-8, TNF-α and NE were significantly elevated after acquisition of M. catarrhalis, compared to pre-acquisition samples (p =< 0.001 for all three). These changes were present in colonization (p = 0.015 for IL-8; p =< 0.001 for TNF-α and NE) as well as in exacerbation (p =< 0.001 for all three), compared to pre-acquisition levels. SLPI was significantly lower after acquisition (p =< 0.001), in colonization (p =< 0.001) as well as in exacerbation (p = 0.004), compared to pre-acquisition levels. SLPI levels correlated negatively with NE levels (R2 = 0.07; p = 0.001).
Acquisition of M. catarrhalis in COPD causes increased airway inflammation and worsening protease-antiprotease imbalance during exacerbations and also in colonization, even in the absence of increased symptoms. These effects could contribute to progression of airway disease in COPD.
PMCID: PMC2780445  PMID: 19912665
19.  Identification of Domains of the Hag/MID Surface Protein Recognized by Systemic and Mucosal Antibodies in Adults with Chronic Obstructive Pulmonary Disease following Clearance of Moraxella catarrhalis▿  
Moraxella catarrhalis is a common cause of respiratory tract infection in the setting of chronic obstructive pulmonary disease (COPD). Adults with COPD acquire and clear strains of M. catarrhalis from the respiratory tract continuously and develop strain-specific protection following clearance of a strain. In previous work, we identified Hag/MID (Moraxella immunoglobulin D-binding protein), a large multifunctional surface protein that acts as an adhesin and hemagglutinin, as a target of antibody responses in adults with COPD after clearance of M. catarrhalis. The goal of the present study was to characterize the domains of Hag/MID to which humans make antibodies, including both systemic and mucosal antibody responses. Analysis of recombinant peptide constructs, which spanned the M. catarrhalis strain O35E Hag/MID protein, with well-characterized serum and sputum samples revealed that most adults with COPD made antibodies directed toward a region of the molecule bounded by amino acids 706 to 863. Serum immunoglobulin G (IgG) and IgA purified from sputum both recognized the same domain. Some flanking sequence of this fragment was necessary for the epitope(s) in this region to maintain its conformation to bind human antibodies. These results reveal that humans consistently generate both systemic and mucosal antibody responses to an immunodominant region of the Hag/MID molecule, which was previously shown to overlap with several biologically relevant domains, including epithelial cell adherence, IgD binding, collagen binding, and hemagglutination.
PMCID: PMC2681595  PMID: 19321697
20.  Serial Isolates of Persistent Haemophilus influenzae in Patients with Chronic Obstructive Pulmonary Disease Express Diminishing Quantities of the HMW1 and HMW2 Adhesins▿  
Infection and Immunity  2008;76(10):4463-4468.
In patients with chronic obstructive pulmonary disease (COPD), the lower respiratory tract is commonly colonized by bacterial pathogens, including nontypeable Haemophilus influenzae. The H. influenzae HMW1 and HMW2 adhesins are homologous proteins that promote bacterial adherence to respiratory epithelium and are the predominant targets of the host immune response. These adhesins undergo graded phase variation, controlled by the numbers of 7-bp repeats upstream of the HMW1 and HMW2 structural genes (hmw1A and hmw2A, respectively). In this study, we examined the levels of HMW1 and HMW2 expressed by H. influenzae isolates collected serially from patients with COPD. We found that expression of HMW1 and HMW2 in a given strain decreased over time in a majority of patients, reflecting progressive increases in the numbers of 7-bp repeats and associated with high serum titers of HMW1/HMW2-specific antibodies. We speculate that the presence of high titers of antibodies against the HMW1 and HMW2 adhesins and other immune factors in the lower respiratory tracts of patients with COPD may result in gradual selection for bacteria with reduced levels of HMW1 and HMW2.
PMCID: PMC2546813  PMID: 18678658
21.  The problems of meta-analysis for antibiotic treatment of chronic obstructive pulmonary disease, a heterogeneous disease: a commentary on Puhan et al 
BMC Medicine  2008;6:29.
Exacerbations are a major cause of morbidity and mortality in chronic obstructive pulmonary disease. Exacerbations can be of bacterial, viral or mixed etiology, with bacteria involved in 50% of exacerbations. Consequently, current management of exacerbations frequently involves the use of antibiotics. The paper by Puhan et al published this month in BMC Medicine examines the benefit of antibiotics in placebo-controlled trials in mild to moderate outpatient exacerbations. The authors use a meta-analytic approach and rightly conclude that more trials are needed in this area. However, the heterogeneity of chronic obstructive pulmonary disease patients and exacerbations and the limited end-points in past trials do not allow firm conclusions to be drawn about antibiotic use in outpatient exacerbations based on this meta-analysis. Future trials need to take into account this heterogeneity as well as incorporate novel end-points to address this important issue.
PMCID: PMC2569059  PMID: 18847482
22.  Optimizing antibiotic selection in treating COPD exacerbations 
Our understanding of the etiology, pathogenesis and consequences of acute exacerbations of chronic obstructive pulmonary disease (COPD) has increased substantially in the last decade. Several new lines of evidence demonstrate that bacterial isolation from sputum during acute exacerbation in many instances reflects a cause-effect relationship. Placebo-controlled antibiotic trials in exacerbations of COPD demonstrate significant clinical benefits of antibiotic treatment in moderate and severe episodes. However, in the multitude of antibiotic comparison trials, the choice of antibiotics does not appear to affect the clinical outcome, which can be explained by several methodological limitations of these trials. Recently, comparison trials with nontraditional end-points have shown differences among antibiotics in the treatment of exacerbations of COPD. Observational studies that have examined clinical outcome of exacerbations have repeatedly demonstrated certain clinical characteristics to be associated with treatment failure or early relapse. Optimal antibiotic selection for exacerbations has therefore incorporated quantifying the risk for a poor outcome of the exacerbation and choosing antibiotics differently for low risk and high risk patients, reserving the broader spectrum drugs for the high risk patients. Though improved outcomes in exacerbations with antibiotic choice based on such risk stratification has not yet been demonstrated in prospective controlled trials, this approach takes into account concerns of disease heterogeneity, antibiotic resistance and judicious antibiotic use in exacerbations.
PMCID: PMC2528209  PMID: 18488427
COPD; exacerbation; bronchitis; antibiotics
23.  Modulation of Airway Inflammation by Haemophilus influenzae Isolates Associated with Chronic Obstructive Pulmonary Disease Exacerbation 
PMCID: PMC2647636  PMID: 16921118
airway epithelial cells; bacterial adherence; interleukin-8; nuclear factor-κB; p38 mitogen-activated protein kinase
24.  Impaired Alveolar Macrophage Response to Haemophilus Antigens in Chronic Obstructive Lung Disease 
Rationale: Interactions of nontypeable Haemophilus influenzae (NTHI) with macrophages are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the immunologic mechanisms that mediate NTHI-macrophage inflammation are poorly understood. Outer membrane protein (OMP) P6 and lipooligosaccharide (LOS) of NTHI are potent immunomodulators. We theorized that alveolar macrophages in COPD possess fundamental immune defects that permit NTHI to evade host responses.
Objective: To test this hypothesis, we obtained human alveolar and blood macrophages from exsmokers with COPD, exsmokers without COPD, and nonsmokers.
Methods: Alveolar and blood macrophages from each donor were incubated with purified LOS and OMP P6 and with OMP P2 and the total outer membrane preparation (0.1–1 μg/ml).
Measurements: Supernatants (24 h) were assayed for IL-1β, TNF-α, IL-10, IL-12, and IL-8 by multianalyte multiplexed flow cytometry.
Results: Comparative induction of COPD and non-COPD alveolar macrophages by LOS and OMP P6 revealed diminished IL-8, TNF-α, and IL-1β responses of COPD alveolar macrophages (p ⩽ 0.03 for each). COPD alveolar macrophages also had diminished responses to total outer membrane (p ⩽ 0.03 for each). In contrast, COPD blood macrophages had no significant differences among donor groups in IL-8, TNF-α, or IL-1β responsiveness to NTHI antigens. Diminished IL-12 responses of COPD blood macrophages to NTHI antigens, compared with nonsmokers, could not be independently dissociated from group differences in age and pack-years.
Conclusions: These findings support a paradigm of defective immune responsiveness of alveolar macrophages, but not blood macrophages, in COPD.
PMCID: PMC2662920  PMID: 16574934
chronic obstructive pulmonary disease; macrophage, alveolar; nontypeable Haemophilus influenzae; phagocytosis
25.  Airway Inflammation and Bronchial Bacterial Colonization in Chronic Obstructive Pulmonary Disease 
Rationale: Inflammation is now recognized as an integral part of the pathogenesis of chronic obstructive pulmonary disease (COPD). In contrast to the sterile airways of normal lungs, bacterial pathogens are often isolated from the airways in stable COPD. This “colonization” of the tracheobronchial tree, currently believed to be innocuous, could serve as an inflammatory stimulus, independent of current tobacco smoke exposure.
Objective: To test the hypothesis that bacterial colonization is associated with airway inflammation in stable COPD.
Methods: Bronchoscopy with bronchoalveolar lavage (BAL) was performed in three groups of subjects: 26 ex-smokers with stable COPD (COPD), 20 ex-smokers without COPD (ex-smokers), and 15 healthy nonsmokers (nonsmokers). Quantitative bacterial cultures, cell counts, chemokine, cytokine, proteinase/antiproteinase, and endotoxin levels in the BAL fluid were compared.
Results: Potentially pathogenic bacteria were recovered at ⩾ 100 cfu/ml in 34.6% of COPD, 0% of ex-smokers, and in 6.7% of nonsmokers (p = 0.003). All values are expressed as median (interquartile range). Subjects with colonized COPD had significantly greater relative (12.0 [28.4] vs. 3.0 [7.8]%, p = 0.03) and absolute (4.98 [5.26] × 104/ml vs. 3.04 [2.82] × 104/ml, p = 0.02) neutrophil counts, interleukin 8 (33.8 [189.8] vs. 16.9 [20.1] pg/ml, p = 0.005), active matrix metalloproteinase-9 (2.16 [4.30] vs. 0.84 [0.99] U/ml, p = 0.03), and endotoxin (36.0 [72.6] vs. 3.55 [7.17] mEU/ml, p = 0.004) levels in the BAL than the subjects with noncolonized COPD. These inflammatory constituents of BAL were also significantly elevated in subjects with colonized COPD when compared with ex-smokers and nonsmokers.
Conclusions: Bacterial colonization is associated with neutrophilic airway lumen inflammation in ex-smokers with COPD and could contribute to progression of airway disease in COPD.
PMCID: PMC2662918  PMID: 16474030
bacterial colonization; chronic obstructive pulmonary disease; neutrophilic inflammation

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