RICERCA ITALIANA     

Research program

BRONCHIECTASIS IN COPD PATIENTS : PREVALENCE AND BACTERIAL COLONIZATION.

University Co-ordinator

Università degli Studi di PADOVA - SCIENZE CARDIOLOGICHE, TORACICHE E VASCOLARI - PADOVA(PD)

Research Unit Leader

Graziella TURATO

Description

The research project aims to clarify the mechanisms responsible for the inflammatory and remodeling processes in the airway of subjects with COPD in different clinical conditions: stable COPD, COPD during an exacerbation and COPD associate to bronchiectasis.

This research unit will recruit 100 patients with COPD according to the protocol described in the part A of the project. Briefly, the subjects will undergo physical examination, lung function testing at baseline and post bronchodilator, and CT scan. In each patient exhaled breath condensate, venopuncture and sputum induction will be performed. All the collected material (X ray and biological samples) will be sent to the research unit number 1 for analysis. The patients included in the study will be divided in two groups: stable COPD and COPD with bronchiectasis.

One subgroup of patients with stable COPD (n=15) and an subgroup of patients with COPD and bronchiectasis (n=15) will undergo broncoscopy with biopsy collection. Moreover, bronchial biopsies obtained from a group of COPD subjects during an exacerbation (n=15), recruited from this research unit, will be examined. Exacerbations will be defined as increased dyspnea associated with increased cough and sputum production that cause the subjects to seek medical attention.The study will conform to the declaration of Helsinki and informed written consent will be obtained from each patient.
Subjects will be premedicated with atropine and anesthetized topically with lidocaine. Bronchoscopy will be performed with a flexible fiberoptic broncoscope. Bronchial biopsies will be taken through the bronchoscope with standard forceps from the carena of the basal segment bronchus of the right lower lobe. From this area two specimens will be obtained in each patients.

Biopsy analysis
Bronchial biopsies obtained from the 3 groups of patients will be prepared for electronic and light microscopy analysis. For light microscopy, biopsies will be fixed in 4% formaldehyde and, after dehydration, embedded in paraffin: serial sections will be cut and stained with histochemical and immunohistochemical techniques. With histochemical methods we will perform morphometric analysis to evaluate airway remodelling ( epithelial destruction and thickness of the basement membrane) while with immuhistochemical methods we will examine the expression of IL-6, IL-8, endothelin-1, bFGF and its receptor FGFR-1 in the epithelium and subepithelium. To avoid observer bias, the cases will be coded and measurements will be made without knowledge of clinical data.
Differences between groups will be analysed using the analysis of variance for clinical data and non parametric analysis for histologic data. Probability values of 0.05 or less will accepted as significant. Morphometric results will be correlated to clinical characteristics.
To evaluate the pathogenetic mechanisms responsible for epithelial metaplasia, bronchial biopsies from a subset of patients will be examined with electronic microscopy. Based on ultrastructural analysis of nuclear morphology, quantification of epithelial basal cells undergoing mitosis will be performed.

To better characterized the mechanisms responsible of airway remodeling in stable COPD, we will examine lobar bronchi obtained from subjects undergoing surgery for localized pulmonary lesions. Indeed, as opposed to bronchial biopsies which can sample only a small portion of the bronchial wall, specimens obtained at surgery allow analysis of the whole bronchial wall and therefore a better examination of the airway wall thickness, bronchial gland hypertrophy and angiogenesis.
We will examined lobar bronchi obtained from ten smokers with stable COPD (FEV1/FVC less than 80% after bronchodilator) and from ten smokers with normal lung function (FEV1 more than 80% predicted).
One ring from each subject will be fixed in 4% formaldehyde, embedded in paraffin and processed for immunohistochemical analysis. The expression of endothelin-1, bFGF and its receptor FGFR-1 will be quantified in the total airway wall as well as in the different airway compartments e.g. bronchial gland, bronchial smooth muscle submucosal vessels.
To evaluate the role of bFGF and endothelin-1 in angiogenesis, we will assess the number of vessels in the submucosa. Moreover, to evaluate bronchial gland enlargment, Reid's index will be calculated as ratio between the maximum thickness of each bronchial gland and the bronchial-wall thickness as measured from basement membrane to inner perichondrium along a single axis. Finally, to evaluate smooth muscle enlargment, smooth muscle proportion will be calculated as ratio between thickness of smooth muscle and bronchial-wall thickness.
To avoid observer bias, the cases will be coded and measurements will be made without knowledge of clinical data.
Differences between groups will be analysed using the analysis of variance for clinical data and non parametric analysis for histologic data. Probability values of 0.05 or less will accepted as significant. Morphometric results will be correlated to clinical characteristics.