RICERCA ITALIANA     

Research program

BRONCHIECTASIS IN COPD PATIENTS : PREVALENCE AND BACTERIAL COLONIZATION.

University Co-ordinator

Università degli Studi di MODENA e REGGIO EMILIA - DIAGNOSTICA PER IMMAGINI - MODENA(MO)

Research Unit Leader

Pietro TORRICELLI

Description

In the first part of the research project, 100 patients with chronic obstructive pulmonary disease (COPD) will be recruited. Patients enrolled will be included has to present the following inclusion criteria: 1. smokers or former smokers of at least 10 pack-years; 2. COPD diagnosed by forced spirometry with FEV1 > 0,7 L, FEV1 post-broncho dilation <60% and FEV1/FVC < 70%; 3. bronchodilator test performed at inclusion or no more than 6 months before inclusion should have been negative (increase in FEV1 < 200 ml and 12%, 10 minutes after administration of 2 puffs of salbutamol); 4. stable phase defined by clinical criteria of the attending investigator, but at least 6 weeks from the last exacerbation; 5. informed consent. The following patients will be excluded: 1. patients receiving oral corticosteroids at any dose or another immunosuppression; 2. contraindication for sputum collection or impossibility to obtain a sample of sputum valid for analysis.
During the first visit, medical history will be recorded and subjects will undergo a physical examination and lung function testing at baseline and after bronchodilator administration (salbutamol 200 mcg). Lung function testing will be performed using body plethysmograph (Jager); DLCO determination will be also performed. Computed Tomography (CT) scan will be performed to evaluate the presence of bronchiectasis, using the following protocol: 1. MSCT in inspiration at TLC with slice of 1 mm every 10 + 3 espiratory slices at RV using three predetermined levels (aortic arch, tracheal carena, right basal common vein); 2. technical parameters: 120 kV, 220 mA, filtro bone, FOV includine both lung, lung parenchima window (-600/1600 HU); 3. all CT will be saved on CD rom.
Our Unit will perform a double-blind bronchiectasis evaluation of all the CT scans together with Unit 04. Exhaled breath condensate will be collected to assess inflammation markers (IL-6, IL-8, TNF- and cysteinyl-leukotrienes). Sputum samples will be obtained for bacterial colonization assessment; two different aliquots will be collected: one for bacterial examination in the local Microbiology Laboratory and the other will be frozen for reverse-transcription polymerase chain reaction (RT-PCR) analysis.
A 25 ml peripheral venous blood sample is collected at admission for isolation of peripheral blood mononuclear cells (PBMCs). The tube is immediately inverted 4 times to mix anticoagulant additive with blood and kept at room temperature. Following centrifugation, red blood cells deposit below a gel band in tube, whereas PBMCs form a pellet above the gel. Serum aliquots will be arranged in duplicate and stored at -20°C until the time of analysis.
The ELISPOT assay, based on the principle of a sandwich capture ELISA, detects IFN- molecules in the immediate vicinity of the T cell from which they are secreted, while they are still at a high concentration. After development, each resulting spot thus represents the "footprint" of an individual antigen-specific IFN- secreting T cell, or spot-forming cell (SFC). The ex vivo ELISPOT assay for IFN- is sufficiently sensitive to detect antigen-specific T cells directly from peripheral blood, without the need for a prior in vitro stimulation step. Moreover, since the ex vivo ELISPOT assay enumerates antigen-specific T cells with rapid effector function, only short incubation periods are required. In the present study, PBMCs will be stimulated with specific antigens of Haemophilus influenzae, Streptococcus pneumoniae, Staphilococcus aureus, Moraxella catharralis and Pseudomonas aeruginosa. Our Unit will receive PBMCs isolated from all the other Units and will perform all the ELISPOT tests.
In the second part of the research project, all the procedures described above will be repeated only in COPD patients with bronchiectasis after randomization in 3 groups to receive: 1. standard of care; 2. azithromycin 500 mg OD 3 days a week (Monday, Wednesday, Friday) for 6 months and then inhaled steroids (fluticasone 500 ug bid) for 6 months; 2. inhaled steroids (fluticasone 500 ug bid) for 6 months and then azithromycin 500 mg OD 3 days a week (Monday, Wednesday, Friday) for 6 months.