Clinical Features and Airways Inflammation in Never Smokers and Smokers With COPD

NCT ID: NCT03274791

Last Updated: 2017-09-07

Basic Information

• Recruitment Status: COMPLETED
• Total Enrollment: 68 participants
• Study Classification: OBSERVATIONAL
• Study Start Date: 2013-09-30
• Study Completion Date: 2017-01-31

Brief Summary

The aim of this study was to investigate the airway inflammatory profile and the clinical presentation of chronic obstructive pulmonary disease (COPD) in never smokers compared to smokers with COPD.

Detailed Description

40 COPD patients (21 smokers and 19 never smokers) and 28 healthy never smokers were included in the present study. Information about respiratory symptoms and comorbidities were collected. Subjects underwent pulmonary function tests and COPD was defined according to Global Initiative for Chronic Obstructive lung Disease spirometric criteria. Induced sputum was collected to determine total and differential inflammatory cells counts as well as inflammatory mediators (Interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α)). The Mann-Whitney U and the χ2 tests were used for results comparisons. Correlations between symptoms, spirometric parameters, cytokines levels, inflammatory cells and risk factors of COPD were examined with Spearman's rank correlation test.

Conditions

COPD; Airway Obstruction; Inflammation

Study Design

• Observational Model Type: CASE_CONTROL
• Study Time Perspective: RETROSPECTIVE

Study Groups

Healthy subjects

Healthy non-smoking subjects were never smokers with normal spirometry and did not have a history of lung disease or chronic respiratory symptoms.

Information about respiratory symptoms and comorbidities were collected. Healthy subjects underwent pulmonary function tests (Spirometry). Induced sputum was collected to determine total and differential inflammatory cells counts as well as inflammatory mediators (Interleukin-8 and tumor necrosis factor-alpha).

Analysis of induced sputum

Intervention Type: DIAGNOSTIC_TEST

Induced sputum was collected and analysed in the three arms (Healthy subjects, Never smokers with COPD and smokers with COPD) The volume of the sputum sample, was treated with an equal volume of 0.1% dithiothreitol. The samples were then vortexed and placed in a shaking water bath at 37°C for 15 min to ensure complete homogeneisation. The suspensions were centrifuged at 400× g and the sputum supernatant stored at -80°C until cytokines analysis. The cell pellet was resuspended in 0.5% bovine serum albumin in phosphate buffered saline and cytospin were made by putting 100 µL of the cell suspension in the funnels.

Slides for differential cell counts were stained with May-Grünwald Giemsa.

Lung Function test

Intervention Type: DIAGNOSTIC_TEST

All the subjects underwent pulmonary function tests (Healthy subjects, Never smokers with COPD and smokers with COPD).

Pulmonary function parameters were measured using a portable spirometer (Easy One ndd. Medizintechnik; Zurich, Switzerland) according to the ATS criteria (American Thoracic Society). Reversibility test was performed 15 min after inhalation of 400 µg of Salbutamol (Ventolin, GlaxoSmithKline, Middlesex, UK). All spirometry data were graded for quality and only tests that met high quality scores were used for the final analysis.

Enzyme-linked Immunosorbent Assay

Intervention Type: DIAGNOSTIC_TEST

IL-8 and TNF-α were measured in the sputum supernatant of all the subjects (Healthy subjects, Never smokers with COPD and smokers with COPD).

Commercially available kits were used to detect IL-8 (Human IL-8 Immuno-Biological Laboratories ELISA Kit) and TNF-α (Human TNF-alpha Sigma-Aldrich ELISA Kit) concentrations in the sputum supernatants. The absorbance was measured at 450 nm. The lower limits detection were 2 pg/mL for IL-8 and 5 pg/mL for TNF-α. The intra assay coefficients of variability were 8.7% for IL-8 and 7.7% for TNF- α.

COPD Never smokers

Never smokers referred to subjects who had never smoked Airflow limitation in COPD was defined as a post-bronchodilator forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC) ratio <70% and FEV1 reversibility of <12% and 200 mL from baseline values after inhalation of 400 µg of Salbutamol.

Information about respiratory symptoms and comorbidities were collected. Never smoking subjects with COPD underwent pulmonary function tests (Spirometry). Induced sputum was collected to determine total and differential inflammatory cells counts as well as inflammatory mediators (Interleukin-8 and tumor necrosis factor-alpha).

Analysis of induced sputum

Intervention Type: DIAGNOSTIC_TEST

Induced sputum was collected and analysed in the three arms (Healthy subjects, Never smokers with COPD and smokers with COPD) The volume of the sputum sample, was treated with an equal volume of 0.1% dithiothreitol. The samples were then vortexed and placed in a shaking water bath at 37°C for 15 min to ensure complete homogeneisation. The suspensions were centrifuged at 400× g and the sputum supernatant stored at -80°C until cytokines analysis. The cell pellet was resuspended in 0.5% bovine serum albumin in phosphate buffered saline and cytospin were made by putting 100 µL of the cell suspension in the funnels.

Slides for differential cell counts were stained with May-Grünwald Giemsa.

Lung Function test

Intervention Type: DIAGNOSTIC_TEST

All the subjects underwent pulmonary function tests (Healthy subjects, Never smokers with COPD and smokers with COPD).

Pulmonary function parameters were measured using a portable spirometer (Easy One ndd. Medizintechnik; Zurich, Switzerland) according to the ATS criteria (American Thoracic Society). Reversibility test was performed 15 min after inhalation of 400 µg of Salbutamol (Ventolin, GlaxoSmithKline, Middlesex, UK). All spirometry data were graded for quality and only tests that met high quality scores were used for the final analysis.

Enzyme-linked Immunosorbent Assay

Intervention Type: DIAGNOSTIC_TEST

IL-8 and TNF-α were measured in the sputum supernatant of all the subjects (Healthy subjects, Never smokers with COPD and smokers with COPD).

Commercially available kits were used to detect IL-8 (Human IL-8 Immuno-Biological Laboratories ELISA Kit) and TNF-α (Human TNF-alpha Sigma-Aldrich ELISA Kit) concentrations in the sputum supernatants. The absorbance was measured at 450 nm. The lower limits detection were 2 pg/mL for IL-8 and 5 pg/mL for TNF-α. The intra assay coefficients of variability were 8.7% for IL-8 and 7.7% for TNF- α.

COPD Smokers

Smokers were defined as persons who had smoked > 20 packs of cigarettes in a lifetime and who continue smoking every day.

Airflow limitation in COPD was defined as a post-bronchodilator forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC) ratio <70% and FEV1 reversibility of <12% and 200 mL from baseline values after inhalation of 400 µg of Salbutamol.

Information about respiratory symptoms and comorbidities were collected. Smoking subjects with COPD underwent pulmonary function tests (Spirometry). Induced sputum was collected to determine total and differential inflammatory cells counts as well as inflammatory mediators (Interleukin-8 and tumor necrosis factor-alpha).

Analysis of induced sputum

Intervention Type: DIAGNOSTIC_TEST

Induced sputum was collected and analysed in the three arms (Healthy subjects, Never smokers with COPD and smokers with COPD) The volume of the sputum sample, was treated with an equal volume of 0.1% dithiothreitol. The samples were then vortexed and placed in a shaking water bath at 37°C for 15 min to ensure complete homogeneisation. The suspensions were centrifuged at 400× g and the sputum supernatant stored at -80°C until cytokines analysis. The cell pellet was resuspended in 0.5% bovine serum albumin in phosphate buffered saline and cytospin were made by putting 100 µL of the cell suspension in the funnels.

Slides for differential cell counts were stained with May-Grünwald Giemsa.

Lung Function test

Intervention Type: DIAGNOSTIC_TEST

All the subjects underwent pulmonary function tests (Healthy subjects, Never smokers with COPD and smokers with COPD).

Pulmonary function parameters were measured using a portable spirometer (Easy One ndd. Medizintechnik; Zurich, Switzerland) according to the ATS criteria (American Thoracic Society). Reversibility test was performed 15 min after inhalation of 400 µg of Salbutamol (Ventolin, GlaxoSmithKline, Middlesex, UK). All spirometry data were graded for quality and only tests that met high quality scores were used for the final analysis.

Enzyme-linked Immunosorbent Assay

Intervention Type: DIAGNOSTIC_TEST

IL-8 and TNF-α were measured in the sputum supernatant of all the subjects (Healthy subjects, Never smokers with COPD and smokers with COPD).

Commercially available kits were used to detect IL-8 (Human IL-8 Immuno-Biological Laboratories ELISA Kit) and TNF-α (Human TNF-alpha Sigma-Aldrich ELISA Kit) concentrations in the sputum supernatants. The absorbance was measured at 450 nm. The lower limits detection were 2 pg/mL for IL-8 and 5 pg/mL for TNF-α. The intra assay coefficients of variability were 8.7% for IL-8 and 7.7% for TNF- α.

Interventions

Analysis of induced sputum

Induced sputum was collected and analysed in the three arms (Healthy subjects, Never smokers with COPD and smokers with COPD) The volume of the sputum sample, was treated with an equal volume of 0.1% dithiothreitol. The samples were then vortexed and placed in a shaking water bath at 37°C for 15 min to ensure complete homogeneisation. The suspensions were centrifuged at 400× g and the sputum supernatant stored at -80°C until cytokines analysis. The cell pellet was resuspended in 0.5% bovine serum albumin in phosphate buffered saline and cytospin were made by putting 100 µL of the cell suspension in the funnels.

Slides for differential cell counts were stained with May-Grünwald Giemsa.

Intervention Type: DIAGNOSTIC_TEST

Lung Function test

All the subjects underwent pulmonary function tests (Healthy subjects, Never smokers with COPD and smokers with COPD).

Pulmonary function parameters were measured using a portable spirometer (Easy One ndd. Medizintechnik; Zurich, Switzerland) according to the ATS criteria (American Thoracic Society). Reversibility test was performed 15 min after inhalation of 400 µg of Salbutamol (Ventolin, GlaxoSmithKline, Middlesex, UK). All spirometry data were graded for quality and only tests that met high quality scores were used for the final analysis.

Intervention Type: DIAGNOSTIC_TEST

Enzyme-linked Immunosorbent Assay

IL-8 and TNF-α were measured in the sputum supernatant of all the subjects (Healthy subjects, Never smokers with COPD and smokers with COPD).

Commercially available kits were used to detect IL-8 (Human IL-8 Immuno-Biological Laboratories ELISA Kit) and TNF-α (Human TNF-alpha Sigma-Aldrich ELISA Kit) concentrations in the sputum supernatants. The absorbance was measured at 450 nm. The lower limits detection were 2 pg/mL for IL-8 and 5 pg/mL for TNF-α. The intra assay coefficients of variability were 8.7% for IL-8 and 7.7% for TNF- α.

Intervention Type: DIAGNOSTIC_TEST

Other Intervention Names

spirometry; ELISA

Eligibility Criteria

Inclusion Criteria

• Subjects with COPD, according to GOLD criteria (smokers and never smokers). Moreover, inhaled short-acting β2-agonists were stopped at least 8h before the test and inhaled long-acting β2-agonist were stopped at least 48h before the test.
• Healthy control subjects : never smokers with normal spirometry and did not have a history of lung disease or chronic respiratory symptoms.

Exclusion Criteria

• COPD patients were excluded from the study if they had an exacerbation, a respiratory tract infection or if they used a systemic form of corticosteroid preparation (oral or intravenous injection therapy) or antibiotics within the two months prior to the study entry. Patients with other respiratory disorder like pneumonia, pulmonary emboli, congestive heart failure, lung cancer or tuberculosis were also excluded.

• Minimum Eligible Age: 40 Years
• Eligible Sex: ALL
• Accepts Healthy Volunteers: Yes

Sponsors

Ministry of Scientific Research, Tunisia

OTHER_GOV

Sponsor Role: lead

Responsible Party

Myriam Denguezli

Assistant Professor

Responsibility Role: PRINCIPAL_INVESTIGATOR

Principal Investigators

Zouhair Tabka, MD PhD

Role: STUDY_DIRECTOR

Faculty of Medicine of Sousse

Amina Mrizak, MSc

Role: PRINCIPAL_INVESTIGATOR

Faculty of Medicine of Sousse

Other Identifiers

COPDNS

Identifier Type: -

Identifier Source: org_study_id