Hedgehog signaling in the airway epithelium of patients with chronic obstructive pulmonary disease.

Genome-wide association studies have linked gene variants of the receptor patched homolog 1 (PTCH1) with chronic obstructive pulmonary disease (COPD). However, its biological role in the disease is unclear. Our objective was to determine the expression pattern and biological role of PTCH1 in the lungs of patients with COPD. Airway epithelial-specific PTCH1 protein expression and epithelial morphology were assessed in lung tissues of control and COPD patients. PTCH1 mRNA expression was measured in bronchial epithelial cells obtained from individuals with and without COPD. The effects of PTCH1 siRNA knockdown on epithelial repair and mucous expression were evaluated using human epithelial cell lines. Ptch1+/- mice were used to assess the effect of decreased PTCH1 on mucous expression and airway epithelial phenotypes. Airway epithelial-specific PTCH1 protein expression was significantly increased in subjects with COPD compared to controls, and its expression was associated with total airway epithelial cell count and thickness. PTCH1 knockdown attenuated wound closure and mucous expression in airway epithelial cell lines. Ptch1+/- mice had reduced mucous expression compared to wildtype mice following mucous induction. PTCH1 protein is up-regulated in COPD airway epithelium and may upregulate mucous expression. PTCH1 provides a novel target to reduce chronic bronchitis in COPD patients.

Characteristics of COPD in never-smokers and ever-smokers in the general population: results from the CanCOLD study.

BACKGROUND: There is limited data on the risk factors and phenotypical characteristics associated with spirometrically confirmed COPD in never-smokers in the general population. AIMS: To compare the characteristics associated with COPD by gender and by severity of airway obstruction in never-smokers and in ever-smokers. METHOD: We analysed the data from 5176 adults aged 40 years and older who participated in the initial cross-sectional phase of the population-based, prospective, multisite Canadian Cohort of Obstructive Lung Disease study. Never-smokers were defined as those with a lifetime exposure of <1/20 pack year. Logistic regressions were constructed to evaluate associations for 'mild' and 'moderate-severe' COPD defined by FEV1/FVC <5th centile (lower limits of normal). Analyses were performed using SAS V.9.1 (SAS Institute, Cary, North Carolina, USA). RESULTS: The prevalence of COPD (FEV1/FVC

Prediction of the rate of decline in FEV(1) in smokers using quantitative Computed Tomography.

BACKGROUND: A study was undertaken to determine if quantitative CT estimates of lung parenchymal overinflation and airway dimensions in smokers with a normal forced expiratory volume in 1 s (FEV(1)) can predict the rapid decline in FEV(1) that leads to chronic obstructive pulmonary disease (COPD). METHODS: Study participants (n = 143; age 45-72 years; 54% male) were part of a lung cancer screening trial, had a smoking history of >30 pack years and a normal FEV(1) and FEV(1)/forced vital capacity (FVC) at baseline (mean (SD) FEV(1) 99.4 (12.8)%, range 80.2-140.7%; mean (SD) FEV(1)/FVC 77.9 (4.4), range 70.0-88.0%). An inspiratory multislice CT scan was acquired for each subject at baseline. Custom software was used to measure airway lumen and wall dimensions; the percentage of the lung inflated beyond a predicted maximal lung inflation, the low attenuation lung area with an x ray attenuation lower than -950 HU and the size distribution of the overinflated lung areas and the low attenuation area were described using a cluster analysis. Multiple regression analysis was used to test the hypothesis that these CT measurements combined with other baseline characteristics might identify those who would develop an excessive annual decline in FEV(1). RESULTS: The mean (SD) annual change in FEV(1) was -2.3 (4.7)% predicted (range -23.0% to +8.3%). Multiple regression analysis revealed that the annual change in FEV(1)%predicted was significantly associated with baseline percentage overinflated lung area measured on quantitative CT, FEV(1)% predicted, FEV(1)/FVC and gender. CONCLUSION: Quantitative CT scan evidence of overinflation of the lung predicts a rapid annual decline in FEV(1) in smokers with normal FEV(1).

Elastin expression in very severe human COPD.

Alveolar elastic fibres are key targets of proteases during the pathogenesis of chronic obstructive pulmonary disease (COPD). In the current study, we hypothesised that a response to injury leads to enhanced alveolar elastin gene expression in very severe COPD. Lung samples obtained from 43 patients, including 11 with very severe COPD (stage 4), 10 donors, 10 with moderate/severe COPD (stage 2-3) and 12 non-COPD subjects, were analysed for elastin mRNA expression by real-time RT-PCR and in situ hybridisation. Alveolar elastic fibres were visualised using Hart's staining of sections of frozen inflated lungs obtained from 11 COPD stage 4 patients and three donor lungs. Compared with donors, non-COPD and stage 2-3 COPD, elastin mRNA expression was significantly increased in very severe COPD lungs (12-fold change), and localised in situ hybridisation induced elastin expression to alveolar walls. Compared with donors, alveolar elastic fibres also comprised a greater volume fraction of total lung tissue in very severe COPD lungs (p<0.01), but elastic fibre content was not increased per lung volume, and desmosine content was not increased. The present study demonstrates enhanced alveolar elastin expression in very severe COPD. The efficiency of this potential repair mechanism and its regulation remain to be demonstrated.

Lung structure and function in COPD.

The pathogenesis of chronic obstructive pulmonary disease (COPD) is related to a chronic innate and adaptive inflammatory immune response to inhaled toxic particles and gases, primarily as a result of the tobacco smoking habit. This inflammatory immune process develops in the lungs of everyone that smokes, and there is an association between the extent and severity of this tissue response and the severity of airflow limitation present in the fraction of the smoking population that develops COPD. This infiltration of inflammatory immune cells into the lung tissue is inextricably linked to a tissue repair and remodeling process that enlarges the bronchial mucus glands, thickens the walls and narrows the lumen of conducting airways <2 mm in diameter. A multivariate analysis has shown that thickening of the walls of the small conducting airways and occlusion of their lumen by inflammatory exudates containing mucus explains more of the variance in the association between FEV1 decline and histology in COPD than the infiltration of the tissue by any inflammatory cell type. Emphysematous destruction of the gas exchanging tissue also contributes to the airflow limitation by reducing the elastic recoil pressure available to drive air out of the lung during forced expiration. This tissue destruction begins in the respiratory bronchioles in very close proximity to the small conducting airways that become the major site of obstruction in COPD. The mechanism(s) that allow small airways to thicken in such close proximity to lung tissue undergoing emphysematous destruction remain a puzzle that needs to be solved. As the accumulation of tissue responsible for thickening the small conducting airways is a very different pathological process from the emphysematous destruction of surrounding gas exchanging tissue, we need a better understanding of the pathogenesis of both processes and better methods of separating their relative contribution to airflow limitation in individuals to adequately prevent and treat COPD.

Induction of mesenchymal cell phenotypes in lung epithelial cells by adenovirus E1A.

Epithelial-mesenchymal transformation is now recognised as an important feature of tissue remodelling. The present report concerns the role of adenovirus infection in inducing this transformation in an animal model of chronic obstructive pulmonary disease. Guinea pig primary peripheral lung epithelial cells (PLECs) transfected with adenovirus E1A (E1A-PLECs) were compared to guinea pig normal lung fibroblasts (NLFs) transfected with E1A (E1A-NLFs). These cells were characterised by PCR, immunocytochemistry, electron microscopy, and Western and Northern blot analyses. Electrophoretic mobility shift assays were performed in order to examine nuclear factor (NF)-kappaB and activator protein (AP)-1 binding activities. E1A-PLECs and E1A-NLFs positive for E1A DNA, mRNA and protein expressed cytokeratin and vimentin but not smooth muscle alpha-actin. Both exhibited cuboidal morphology and junctional complexes, but did not contain lamellar bodies or express surfactant protein A, B or C mRNAs. These two cell types differed, however, in their NF-kappaB and AP-1 binding after lipopolysaccharide stimulation, possibly due to differences in the expression of the subunits that comprise these transcriptional complexes. E1A transfection results in the transformation of peripheral lung epithelial cells and normal lung fibroblasts to a phenotype intermediate between that of the two primary cells. It is postulated that this intermediate phenotype may play a major role in the remodelling of the airways in chronic obstructive pulmonary disease associated with persistence of adenovirus E1A DNA.

Computed tomographic estimation of lung dimensions throughout the growth period.

The aim of the current study was to use computed tomography (CT) to estimate airway wall and lumen, and arterial and parenchyma dimensions in children throughout the growth period, and to provide normative data to study alterations caused by pulmonary disease. Clinical CT scans reported as normal that were performed in children for nonpulmonary and noncardiac reasons were analysed for lung weight, gas volume, lung expansion, lung surface/volume ratio, airway wall area, airway lumen area, airway lumen perimeter, arterial area and airway surface length/area ratio. The age range of the 50 subjects was 0-17.2 yrs. The data showed only little increase in lung expansion throughout childhood (n = 32). There was substantial variability in lung expansion between subjects. Airway wall and lumen and arterial area were exponentially associated with subjects' height (n = 50). Airway surface length/area ratio was linearly associated to alveolar surface/volume ratio. The data from the current study provide normative computed tomography estimates of airway wall and lumen, and arterial and parenchyma dimensions throughout the growth period that may be useful for the study of alterations in disease.

Convergence of the epidemiology and pathology of COPD.

The epidemiology of chronic obstructive pulmonary disease (COPD) has been dominated by one hypothesis stating that cigarette smoking and chronic bronchitis were the key to pathogenesis and another that asthma, chronic bronchitis, and even emphysema are related to different expressions of a primary airway abnormality. The first hypothesis was rejected in the late 1960s based on a longitudinal study of working men where only a fraction of smokers developed COPD and where development of COPD was independent of the absence or presence of chronic bronchitis. Chronic bronchitis in more advanced COPD was subsequently associated with a more rapid decline in lung function and more frequent exacerbations. The second hypothesis is more difficult to test but longitudinal studies have shown that the presence of bronchial hyperresponsiveness may predict the subjects who go on to develop COPD. This brief review attempts to reconcile these findings with the pathology found in the lung.

Report of a workshop: quantitative computed tomography scanning in longitudinal studies of emphysema.

It has been reported that quantitative computed tomography (CT) scanning of the lungs showed decreased progression of emphysema in a randomised clinical trial in patients with severe alpha1-antitrypsin (alpha1-AT) deficiency receiving monthly intravenous augmentation therapy with human alpha1-AT. Comparable results were not obtained using rate of decline of forced expiratory volume in one second. Accordingly, the Alpha-1 Foundation convened a workshop to explore the feasibility of using quantitative CT data as a primary outcome variable in trials of drugs for treating alpha1-AT deficiency. This report reviews the following: the principles for the use of modern CT scanners for quantifying emphysema; the methods and data on validation by comparison with measurements of severity of emphysema in inflation-fixed specimens of lungs; and the possibility of decreasing radiation dosage from CT to make it safe and ethically possible to use CT in longitudinal studies. The workshop concluded that it is feasible, safe and ethically possible to use computed tomography in longitudinal studies of emphysema. It recommended that the primary end-point should be a significant shift in the 15th percentile of lung density.

Estimation of lung growth using computed tomography.

Anatomical studies suggest that normal lungs grow by rapid alveolar addition until about 2 yrs of age followed by a gradual increase in alveolar dimensions. The aim of this study was to examine the hypothesis that normal lung growth can be monitored by computed tomography (CT). Therefore, the gas volume per gram of lung tissue was estimated from measurements of lung density obtained from CT scans performed on children throughout the growth period. CT scans were performed on 17 males and 18 females, ranging in age from 15 days-17.6 yrs. CT-measured lung weight was correlated with predicted post mortem values and CT measured gas volume with predicted values of functional residual capacity. The median value for lung expansion was 1.86 mL x g(-1) at 15 days, decreased to 0.79 mL x g(-1) by 2 yrs and then increased steadily to 5.07 mL x g(-1) at 17 yrs. Computed tomography scans can be used to estimate lung weight, gas volume and expansion of normal lungs during the growth period. The increase in the lung expansion after the age of 2 yrs suggests progressive alveolar expansion with increasing lung volume.

Selection of patients for lung volume reduction surgery using a power law analysis of the computed tomographic scan.

BACKGROUND: A study was undertaken to test the hypothesis that patients respond better to lung volume reduction surgery (LVRS) if their emphysema is confluent and predominantly located in the upper lobes. METHODS: A density mask analysis was used to identify voxels inflated beyond 10.2 ml gas/g tissue (-910 HU) on preoperative and postoperative CT scans from patients receiving LVRS. These hyperinflated regions were considered to represent emphysematous lesions. A power law analysis was used to determine the relationship between the number (K) and size (A) of the emphysematous lesions in the whole lung and two anatomical regions using the power law equation Y=KA(-D). RESULTS: The analysis showed a positive correlation between the change in the power law exponent (D) and the change in exercise (Watts) after surgery (r=0.47, p=0.03). There was also a negative correlation between the power law exponent D in the upper region of the lung preoperatively and the change in exercise following surgery (r=-0.60, p<0.05). CONCLUSIONS: These results confirm that patients with large upper lobe lesions respond better to LVRS than patients with small uniformly distributed disease. Power law analysis of lung CT scans provides a quantitative method for determining the extent and location of emphysema within the lungs of patients with COPD.

Chronic obstructive pulmonary disease - part 2: pathology and biochemistry of emphysema.

The pathology and pathogenesis of emphysema are reviewed, with particular reference to the proteinase-antiproteinase hypothesis.

Infection of Gammarus duebeni populations by two vertically transmitted microsporidia; parasite detection and discrimination by PCR-RFLP.

We screened a population of the brackish water crustacean Gammarus duebeni from the Isle of Cumbrae for the presence of vertically transmitted microsporidia. We compared 2 screening techniques; light microscopy and PCR-based detection using generic 16S rDNA microsporidian primers. Fifty percent of females from this population tested positive for vertically transmitted microsporidia. The PCR screen was 100% efficient in comparison with existing LM based screening. In addition, the PCR screen produced bands of 2 sizes suggesting that more than 1 species of microsporidian was present. Sequencing revealed 2 distinct species of vertically transmitted microsporidia; 33% of females were infected with the feminizer Nosema granulosis and 17% were infected with a new species which we provisionally designate Microsporidium sp. On the basis of sequence information, we developed a discriminatory PCR-RFLP test based on MspI and HaeIII digests. This screen allows rapid detection and discrimination of vertically transmitted microsporidia in natural field populations. We applied the PCR-RFLP screen to a second G. duebeni population from the Isle of Man. This population also hosted these 2 parasite species. In total 45% of females harboured N. granulosis and 10% harboured Microsporidium sp. No dual-infected individuals were found in either population. The occurrence of 2 vertically transmitted parasites within a population has implications for our understanding of parasite-host relationships in the field and we discuss factors affecting the dynamics of parasite-parasite competition and coexistence.

Age-related changes in the calcium homeostasis of adherent neutrophils.

The elderly are susceptible to infections and show a decline in neutrophil (PMN) functions that are regulated by cytosolic calcium [Ca2+]i. This study measures [Ca2+]i in suspended and adherent PMN of young and elderly individuals by using flow cytometry, confocal microscopy, the bacterial peptide fMLP, and the fluorescent Ca2+ indicator fluor-3/acettoxymethyl ester. PMN from both age groups show a steep and transient fMLP-induced Ca2+ increase. This increase is independent of external divalent cations and is desensitized by a subsequent exposure to the same agonist. Adherent PMN of the elderly express elevated [Ca2+]i before (basal) and after fMLP activation but show reduced ability to mobilize Ca2+ into and from the cytosol. PMN of the elderly take longer (13.7 +/- 3 s) to attain the maximal response compared to those of young adults (5.7 +/- 0.8 s). PMN from both age groups show heterogeneity in the time and magnitude of this response. However, PMN of the elderly show a decrease in the proportion of cells with prompt and effective reaction and an increase in the representation of a cell subpopulation manifesting delayed response. We conclude that age-related delayed and reduced PMN response to a bacterial peptide could hamper functional activities that are essential in host protection against infections.

Core to rind distribution of severe emphysema predicts outcome of lung volume reduction surgery.

Computed tomography (CT) has shown that emphysema is more extensive in the inner (core) region than in the outer (rind) region of the lung. It has been suggested that the concentration of emphysematous lesions in the outer rind leads to a better outcome following lung volume reduction surgery (LVRS) because these regions tend to be more surgically accessible. The present study used a recently described, computer-based CT scan analysis to quantify severe emphysema (lung inflation > 10.2 ml gas/g tissue), mild/moderate emphysema (lung inflation = 10.2 to 6.0 ml gas/g tissue), and normal lung tissue (lung inflation < 6.0 ml gas/g tissue) present in the core and rind of the lung in 21 LVRS patients. The results show that the quantification of severe emphysema independently predicts change in maximal exercise response and FEV(1). We conclude that a greater extent of severe emphysema in the rind of the upper lung predicts greater benefit from LVRS because it identifies the lesions most accessible to removal by LVRS.

Role of latent viral infections in chronic obstructive pulmonary disease and asthma.

Acute viral respiratory tract infections are well known to precipitate asthma attacks and acute exacerbations of chronic obstructive pulmonary disease, but their role in the pathogenesis of chronic disease is poorly defined. Double-stranded DNA viruses have the ability to persist in airway epithelial cells long after the acute infection has cleared. During these latent infections, viral genes are expressed at the protein level without replication of a complete virus. The expression of the adenoviral trans-activating protein has been demonstrated in the airway epithelium of both human and animal lungs and is associated with an amplification of the cigarette smoke-induced inflammatory response. Studies of cultured human airway epithelial cells have also shown that transfection with this viral gene upregulates the expression of intercellular adhesion molecule 1 and interleukin 8 by these cells when they are challenged with endotoxin. In guinea pigs, cigarette smoke-induced emphysema is amplified by latent adenoviral infection. Furthermore, this infection independently increased the number of CD-8 cells, whereas the cigarette smoke independently increased the number of CD-4 cells in the inflammatory infiltrate. On the other hand, allergen-induced lung inflammation was uninfluenced by latent adenoviral infection in the guinea pig, but the latent infection caused the eosinophilic component of this response to become steroid resistant. These studies suggest that latent adenoviral infections may have a role in the pathogenesis of obstructive airway disease by amplifying the response to cigarette smoke and inducing steroid resistance.