RICERCA ITALIANA     

Research program

New insights of inflammation and remodeling in airway obstructive deseases

University Co-ordinator

Università degli Studi di PARMA - SCIENZE CLINICHE - PARMA(PR)

Research Unit Leader

Alfredo Antonio CHETTA

Description

METHODS


Subjects

Twenty-five non smoking patients with mild to moderate asthma and 25 COPD patients will be recruited from the oupatient clinic of the Department of Clinical Sciences, Respiratory Disease Section, of the Parma University.

Asthmatic and COPD patients must meet the conventional diagnostic criteria for asthma and COPD (14), respectively. The presence of a persistent cough is not necessary for inclusion in the study. All patients must be clinically stable and will not receive corticosteroids during the 3 months before the study and must be free from respiratory infections in the 4 weeks preceding the study.

The degree of asthma severity will be assessed according to an asthma severity score based on symptoms, bronchodilator use and daily peak expiratory flow variability, measured during the three weeks prior to the methacholine challenge test day. Possible scores range from 0 to 12 (15). Atopy will be assessed by skin prick tests with a battery of 10 common inhalant allergens.

Ten healthy non smoking volunteers will be enrolled, as a control group. All subjects will provide written informed consent to enter the study.


Lung Function Study and Methacholine Challenge

Baseline FEV1 will be measured in triplicate by a flow-sensing spirometer connected to a computer for data analysis (Vmax 22, Sensor Medics, Yorba Linda, US). Methacholine inhalation challenge will be done according to the European Respiratory Society guidelines (16). Doubling increasing concentrations of methacholine (Lofarma, Milan, Italy) from 0.03 to 32 mg/ml will be delivered by a dosimeter (output: 9 μl/puff; Mefar, Brescia, Italy) and inhaled. Inhalations will be interrupted when FEV1 decreases by 20% from its postsaline value. The concentration provoking a 20% decrease in FEV1 (PD20 M, in μmol) will be determined by linear interpolation of the last two experimental points.


Capsaicin Cough Challenge

Capsaicin cough challenge will be done according to the method previously described (17). Doubling increasing concentration of capsaicin (Sigma Chemical Co, St Louis, Missouri, USA) from 2 to 500 μM will be delivered by a dosimeter (output: 9 μl/puff; Mefar, Brescia, Italy) and inhaled. Capsaicin challenge will be interrupted when a given inhalation causes five cough (C5). This dose will be repeated to ensure that a reproducible C5 response is attained and, if so, that the value will be recorded as the subject's value (C5 capsaicin).


Fiberoptic Bronchoscopy

Fiberoptic bronchoscopy will be performed according to a previously described protocol (9). Premedication consists of atropine (0.5 mg) and diazepam (10 mg), both given by intramuscular injection. Local anaesthesia will be obtained with a tetracaine tablet (20 mg) given 15 min before bronchoscopy. An additional aliquot of 2% lignocaine will be aerosolized into the upper airways and applied topically to the pyriform sinuses and vocal cords to prevent coughing and as a local anaesthetic. Nebulized salbutamol (1.25 mg) and ipratropium bromide (0.25 mg) will be inhaled 5 min prior to bronchoscopy (Model 1T10;Olympus, Tokyo, Japan). Three to five mucosal biopsy specimens will be taken with alligator forceps at the subcarinae in the middle and lower lobes of the right lung. Administration of nebulized salbutamol and ipratropium bromide will be repeated after bronchoscopy when necessary. Patients will be closely monitored after bronchoscopy in the outpatient clinic, and will be discharged when the effects of sedation disappear and lung function returns to the baseline values.


Biopsy Processing

Mucosal biopsies will be immediately transferred into ice-cold acetone containing the protease inhibitor iodoacetamide (20 mM) and phenylmethylsulfonylfluoride (2 mM) for fixation; they will be then stored at –20°C for 24 hours and processed into the water-soluble resin, glycolmethacrylate (Polysciences, Northampton, UK), for embedding (18). Sections of 2 μm will be cut and stained using monoclonal antibodies against type IV collagen to outline the endothelial basement membrane (Novocastra Lab., Newcastle, UK), tryptase (DAKO, Glostrup, Denmark) and EG2 (Pharmacia and Upjohn Diagnostic AB, Uppsala, Sweden) to identify mast cells and eosinophils respectively, and VEGF (NeoMarkers, Fremont, California) to recognize the 121, 165 and 189 isoforms of VEGF. Light microscopy will be performed with a Leica microscope (Leica DMLB, Werzlar, Germany) at 1000x magnification.

The number of vessels, mast cells, eosinophils and VEGF+ cells will be counted in all nonoverlapping high power fields in the lamina propria. The latter is defined as a zone 100 μm below the epithelial basement membrane, and expressed as number of vessels and cells per square millimeter of lamina propria. In each section, all available nonoverlapping high power fields with intact tissue covered by intact basement membrane will be examined. Vascular area is expressed as percentage of the area of the assessed lamina propria. The mean size of vessels is estimated by dividing the total vascular area by the total number of vessels. Basement membrane thickness will be measured as described previously (19). Final values represent the mean of at least two sections from two different biopsies in each subject. A single observer who has no knowledge of patient characteristics will evaluate tissue sections using an image analysis system (Leica Q500C; Leica Cambridge Ltd., Cambridge, UK). An average of eight power fields will be examined for each subject included in the study.


Statistical Analysis

Values will be presented as mean±standard deviation (SD). Analysis of variance and post-hoc test will be used for comparison between patients and control subjects. Relationships will be estimated by the Spearman's rank correlation coefficient (rs). A p value less than 0.05 will be taken as significant.