RICERCA ITALIANA     

Research program

BRONCHIECTASIS IN COPD PATIENTS : PREVALENCE AND BACTERIAL COLONIZATION.

University Co-ordinator

Università degli Studi di MILANO - Tisiologia e malattie dell'apparato respiratorio - MILANO(MI)

Research Unit Leader

Francesco BLASI

Description

UO 1 will enrol around 100 COPD patients and perform the analysis of inflammatory markers on blood and Exhaled bronchial condensate of the whole population of the study. The same UO will perform, on the whole enrolled population, molecular biology techniques for the detection of bacterial DNA, PCR and RT-PCR, on sputum and bronchial samples. Standard microibiologic culture will be performed on sputum or brochoaspirate/BAL samples of the patients enrolled in the UO 1.
In collaboration with the radiologic unit of IRCCS Fondazione Policlinico-Mangiagalli-Regina Elena Milano Multislice CT scans will be performed, as described in the protocol, and recorded on CD rom and sent for further analysis to the UO 4(Zompatori). During the second year of the study the Uowill follow the bronchiectasic patient according to the randomization list of the study protocol.

The following methods are in use in the UO 1 and will be performed in the study:
Exhaled breath condensate (EBC)
The breath condensate samples are collected using a specially designed condensing chamber (Ecoscreen; Jaeger, Hoechberg, Germany); exhaled air enters and leaves the chamber though one-way inlet and outlet valves, thus keeping the chamber closed. The subjects wear noseclips and breath tidally through a mouthpiece connected to the condenser for ten minutes.
One millilitre aliquots of the sampled material were transferred to 2-ml plastic tube and stored at -70°C.

Citokines assays for serum and EBC
For all samples the two-dimensional electrophoresis will be performed to test the presence of salivary contamination of the condensate .
IL-6 and TNF-a concentrations in the serum and EBC samples will be measured using a specific enzyme immunoassay kit (EIA) (Cayman Chemical, Ann Arbor, USA). The assay will be directly validated by means of gas chromatography/mass spectrometry.
The detection limit of the IL-6 assay and TNF-a assay is 1.5 pg/ml after a two-hour development period. The reproducibility of repeat IL-6 and TNF-a measurements will be assessed by the Bland and Altman method and the coefficient of variation.

Proteinomic analysis
Proteomic analysis will be performed on the subset of samples obtained by the patients enrolled by the UO 1. The methods will be analysed in collaboration with Dipartimento di Farmacologia dell'Università degli Studi di Milano. Briefly, one-dimensional and two-dimensional electrophoresis will be performed and for qualitative evaluation and mass spectrometry analysis, the gels will be stained with silver nitrate, with or without glutaraldehyde in the sensitization step and formaldehyde in the impregnation solution. Spot volumes will be calculated using Image J 1.29x (W. Rasband, National Institutes of Health, Bethesda, Maryland) for one-dimensional gels, and with PDQUEST (Biorad, Hercules, California) for two-dimensional gels.

-Molecular biology on sputum/brochoaspirate/BAL
All the molecular biology tests will be applied on samples obtained from the whole population of enrolled patients. Bacterial DNA of Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Pseudomonas aeruginosa, Chlamydia pneumoniae and Mycoplasma pneumoniae will be detected. Qualitative PCR and quantitative Real Time-PCR will be performed according to standard protocols.

Peripheral Blood mononuclear cells will be collected and stored for further analysis (Elispot) to be performed by UO 2.
In collaboration with UO 3 samples of bronchial biopsies, from COPD and COPD with bronchiectasis patients, will be analysed by electron microscopy