RICERCA ITALIANA     

Non-invasive methods to assess biological and mechanistic bases of respiratory diseases: clinical and epidemiological applications.

Università degli Studi di Parma

Abstract

Respiratory disorders are a leading cause of death worldwide, and further increase in mortality is expected in the near future. In Europe, respiratory diseases rank second (after cardiovascular diseases) in terms of mortality, incidence, prevalence and costs. Whereas there has been a substantial reduction of infective lung diseases, such as tuberculosis and pneumonia, over the last decades there has been a progressive increase of inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Chronic respiratory disorders, particularly asthma and COPD are becoming a leading medical problem worldwide.
The concept that inflammation leads to bronchial hyper-responsiveness, airflow limitation and mucus hypersecretion in chronic pulmonary diseases, especially asthma and chronic obstructive pulmonary disease (COPD) has led to a widening search for the types of inflammatory cells and mediators that are responsible for the cascade of events linking the initial stimulus to the final abnormality in airway function.
In the last few years, such non invasive techniques as induced sputum analysis and exhaled nitric oxide (NO) measurement have been successfully introduced in the evaluation of asthma and COPD. Recently, analysis of exhaled breath condensate (EBC) has been introduced among the techniques to evaluate airways inflammation. EBC is very easy to perform, non-invasive, can be repeated within short period of time also in ventilated subjects. Exhaled breath consists of a gaseous phase, which contains gases (e.g., NO and CO) volatile compounds such as , and water vapour containing aerosol particles, that can be condensed by breathing through a cooling system. The fluid known as EBC contains many non-volatile substances including proteins, lipids e oxidation products that seem to reflect the composition of bronchoalveolar extracellular lining fluid.
In this project, we will apply the most sensitive, selective and specific reference analytical techniques (LC-MS/MS, GC-MS, ICP-MS) for the analysis of non invasive lung biological matrices.
Relying on the collaboration of researchers with different background (Occupational Medicine, Respiratory Medicine, Toxicology, Industrial Hygiene), the present research project is aimed at: i) validation of noninvasive methods (exhaled air and EBC collection) applicable to the study of lung response in subjects with asthma and COPD, either at steady state and after inhaled stimuli with allergens and pollutants;; ii)validation of inflammatory and/or oxidative stress biomarkers present in EBC, both by intra assay evaluation (reproducibility, sensitivity and accuracy) and by their correlation with clinical and functional parameters. Both diagnostic and prognostic validity of EBC analysis in lung diseases will be assessed, particularly for those mediators known to be specific for asthma and COPD and sensitive to inflammatory changes; iii) development of noninvasive methods to assess target tissue dose and early lung events in healthy subjects exposed to pneumotoxic substances. <<<

Principal Investigator

Antonio MUTTI Università degli Studi di PARMA

Research Objectives

Relying on the collaboration of researchers with different background (Occupational Medicine, Respiratory Medicine, Toxicology, Industrial Hygiene), the present research project is aimed at:

a) validation of inflammatory and/or oxidative stress biomarkers present in EBC, both by intra assay evaluation (reproducibility, sensitivity and accuracy) and by their correlation with clinical and functional parameters. Both diagnostic and prognostic validity of EBC analysis in lung diseases will be assessed, particularly for those mediators known to be specific for asthma and COPD and sensitive to inflammatory changes;

b) validation of noninvasive methods (exhaled air and EBC collection) applicable to the study of lung response in subjects with asthma and COPD, either at steady state and after inhaled stimula with allergens and pollutants;

c) development of noninvasive methods to assess target tissue dose and early lung events in healthy subjects exposed to pneumotoxic substances;

d) comparison between biological results obtained by EBC analysis and those obtained by standard techniques (induced sputum analysis and exhaled NO measurement) as well as by clinical-functional parameters;

e) application of the most sensitive, selective and specific reference analytical techniques (LC-MS/MS, GC-MS, ICP-MS) for a better understanding of molecular mechanisms leading to lung damage. <<<

Timescale

24 months

National and international background

Respiratory disorders are a leading cause of death worldwide, and further increase in mortality is expected in the near future. In Europe, respiratory diseases rank second (after cardiovascular diseases) in terms of mortality, incidence, prevalence and costs (1-3). Whereas there has been a substantial reduction of infective lung diseases, such as tuberculosis and pneumonia, over the last decades there has been a progressive increase of inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Chronic respiratory disorders, particularly asthma and COPD are becoming a leading medical problem worldwide (4).

COPD is the 4th cause of death in the USA, its incidence being increasing despite the decreasing prevalence of smokers. In Italy, an estimated 2.6 million men and women have COPD, and the disease causes around 18 000 deaths each year. In addition to mortality, morbidity from COPD results in substantial use of secondary healthcare resources. The burden of COPD in Italy may be due to the underdiagnosis of the disease by healthcare professionals, particularly in the early stages of the disease, and a lack of awareness among physicians of recommended treatment practices (4).

Undoubtedly, the most important risk factor in the development of COPD is cigarette smoking. The effects of cigarette smoke on the lung are manifold. Cigarette smoke has been found to attract inflammatory cells into the lungs and stimulates the release of of the proteolytic enzyme elastase from these cells. Any occupation in which the local environment is polluted with the aforementioned gases and particulates increases the risk of developing of COPD. In addition, there is evidence that cadmium and silica also increase the risk of COPD. This is especially true if the subject smokes. Occupations at risk include coal miners, construction workers who handle cement, metal workers, grain handlers, cotton workers and workers in paper mills. However, the effect of smoking far outweighs any influences from the work environment (2,4-8).

Bronchial asthma is a leading medical problem worldwide. Asthma is a chronic inflammatory disorder of the airways. It is a serious disease, sometimes lethal, which involves people of each age and all nations. The incidence of asthma is increasing worldwide, mainly among children and the its mortality is increased of 42% in the last 10 years (9). Asthma has dramatically risen worldwide over the past decades, particularly in developed countries, and experts are puzzled over the cause of this increase. The mechanisms that cause asthma are complex and vary among population groups and even from individual to individual. Many asthma sufferers have allergies, and some researchers are targeting common factors in both these conditions. Not all people with allergies have asthma, however, and not all cases of asthma can be explained by allergic response. Other contributing causes need to be studied (9).

Asthma is most likely to be caused by a convergence of factors that can include genes (probably several) and various environmental and biologic triggers (e.g., infections, dietary patterns, hormonal changes in women, and allergens). The medical and socio-economic impact that COPD and asthma have on the community is enormous. Consequently, measures aimed at primary and secondary prevention of COPD and asthma, including smoking cessation, abating air pollution and identification of smokers at risk for developing COPD and asthma, are highly desirable. Unfortunately, these goals remain elusive and COPD and asthma represent a challenging unmet medical need (10).

Several tools have been developed for diagnosing, monitoring and evaluating COPD and asthma. Lung function tests are essential tools to provide insights into the physiological derangements in static and dynamic lung volumes and gas exchange (11). Radiologic techniques, especially high resolution computed tomographic scanning, provide insight into the loss of lung tissue (12). Both these methods are indirect measurements of adverse effects occurring in a biological system as a long-term consequence of exposure to toxic or noxious agents.

The concept that inflammation leads to bronchial hyperresponsiveness, airflow limitation and mucus hypersecretion in chronic pulmonary diseases, especially asthma and chronic obstructive pulmonary disease (COPD) has led to a widening search for the types of inflammatory cells and mediators that are responsible for the cascade of events linking the initial stimulus to the final abnormality in airway function. However, there is significant evidence that COPD is an inflammatory process just as is bronchial asthma, however, it seems that there are different patterns of lung inflammation in these patients (13-16).

COPD is a debilitating inflammatory disease of the lungs characterized by an increased presence of neutrophils and macrophages in the airways of affected patients. Neutrophils produce proinflammatory mediators, cytokines, and proteases and recruitment of neutrophils to the airways involves chemotactic stimuli such as interleukin (IL)-8 and adhesion of neutrophils to pulmonary and bronchial epithelial cells with subsequent migration into the airways and alveoli by integrin-dependent mechanisms (14,16).

Inflammatory process in asthma is characterised by activation of airway resident cells and infiltration with other cell populations recruited by chemotactic factors for different cell populations (IL-5, eotaxin, RANTES, etc.) (9). In the airways, activated inflammatory cells release cytokines and inflammatory mediators contributing to the ongoing of the inflammatory process. Eosinophils seem to play a relevant role in asthma and are considered as the main hallmark of asthma. Eosinophils are potent pro-inflammatory cells, secreting many inflammatory mediators as leukotrienes, with strong bronchoconstrictor activity, and basic proteins as MBP (major basic protein), EPO (eosinophilic peroxidase), ECP (eosinophilic cationic protein) that induce direct damage to the airway epithelium, promote bronchial hyperresponsiveness and the release of more mediators from other inflammatory cells. Eosinophils are also able to produce proinflammatory cytokines (TGFbeta, TNFa, IL-4,-5,-6,-8, GM-CSF, RANTES, eotaxin, etc), and thereby amplify the inflammatory process (16). High eosinophil levels are commonly found also in blood of symptomatic asthmatic subjects.

Many attempts to directly evaluate inflammation in asthma have been tried using bronchial biopsy, bronchial lavage or bronchoalveolar lavage, giving important informations on physiopathological mechanisms of bronchial asthma. These methods are quite invasive and may be performed mainly as a research tool. In the last few years less invasive techniques as induced sputum analysis (17) and exhaled nitric oxide (NO) measurement (18) have been successfully introduced in the evaluation of asthma and COPD.

Induced sputum collection and analysis is generally well tolerated by patients. Induction and collection of induced sputum, by inhalation of hypertonic saline, can give direct information on the kind and degree of bronchial inflammation and brought to a better understanding of the role of inflammatory mediators found in the airways of asthmatic patients. A good correlation between inflammatory mediators levels and clinical and functional findings was found in asthmatic subjects (19, 20). The evaluation of these hallmarks of bronchial inflammation can help to better characterise asthmatic patients, in addition to clinical and functional findings.

Exhaled NO measurement is another technique widely adopted in monitoring asthma inflammation. NO is a common biological and neural trasmitter, very important in normal airways and blood vasal tone regulation; in the airways is produced by many cells, both resident or recruited during the inflammatory process. Its physiopathological meaning is still unclear (21), but it has been demonstrated that NO levels in exhaled air are higher in asthmatic in comparison with healthy subjects, that its levels raise during spontaneous or induced asthma exacerbation and decrease after anti-inflammatory treatment (21).

Recently, analysis of exhaled breath condensate (EBC) has been introduced among the techniques to evaluate airways inflammation. EBC is very easy to perform, non-invasive, can be repeated within short period of time also in ventilated subjects. Exhaled breath consists of a gaseous phase, which contains volatile compounds such as NO and carbon monoxide (CO), and water vapour containing aerosol particles, that can be condensed by breathing through a cooling system. The fluid known as EBC contains many non-volatile substances including proteins, lipids e oxidation products that seem to reflect the composition of bronchoalveolar extracellular lining fluid (22). Several inflammatory and/or oxidation markers have been measured in EBC of healthy subjects and patients with different diseases including asthma. High levels of cys-leukotriens (LTE4, LTC4 e LTD4) and LTB4 have been found in EBC of patients with moderate to severe asthma, with further increase after corticosteroid therapy withdrawal. Levels of 8-isoprostane seem to be correlated with the severity of asthma irrespectively from the presence of corticosteroid therapy, while malondialdehyde levels decrease with corticosteroid therapy. High levels of 3-nytrotirosine and nitrosothiols are also reported in patients with asthma, and an increase in H2O2 concentrations is reported in severe and unstable asthma (22, 23).

The use of EBC is a very promising technique in the assessment of airways inflammation. Nevertheless there are still many open problems; among those, the lack of a standard method of collection remains the most important: humidity of inspired air, ventilation volume per unit time, condenser temperature, time of collection, use of a nose clip, all these aspects may have influence on the volume of EBC collected, and maybe on EBC markers levels too. More should be known on patient intra individual variability and on the clinical utility of EBC in pulmonary disease. Moreover there are still few reports, also if rapidly increasing, on EBC markers measurement and significance.

In this project, we will apply the most sensitive, selective and specific reference analytical techniques (LC-MS/MS, GC-MS, ICP-MS) for the analysis of non invasive lung biological matrices. <<<