September - December 2004: 
Volume 17, Issue 3

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Inflammatory markers in monitoring response to treatment for asthma and chronic obstructive pulmonary disease
Monitoring of asthma and COPD patients is usually based on symptom scores, use of rescue medication and measurement of lung function. However, both asthma and COPD are chronic inflammatory diseases of the airways and there is a need to assess and monitor inflammation using markers directly implicated in the inflammatory process. Several invasive (bronchoscopy, nasal and skin biopsies) and non-invasive (bronchial hyperresponsiveness, blood, urine, induced sputum, exhaled air) biomarkers have been examined as potential markers of airway inflammation for the diagnosis and follow-up of asthma and COPD patients. The advantages and disadvantages of each one of them are discussed in this review. Presently, available data support the use of bronchial hyperresponsiveness (BHR) and sputum induction as the best available biomarkers to guide treatment in the majority of asthmatic patients, whereas in COPD patients none of the available inflammatory markers has gained wide acceptance among investigators till now. Larger and longer-term population-based studies focused on the relationship between markers of inflammation and treatment regimens are needed to determine the optimum biomarker for any given treatment. Pneumon 2004, 17(3):232-241.in
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In routine clinical practice, monitoring of patients with asthma or chronic obstructive pulmonary disease (COPD) relies primarily on symptom scores, use of reliever medication and measurement of lung function parameters (FEV1 and PEFR variation in asthma; FEV1 and IC in COPD). However, since both asthma and COPD are chronic inflammatory airway diseases,1,2 there is a strong need to assess and monitor inflammation using markers, i.e. cells and mediators, implicated in the inflammatory process. Preferably, these markers have to be non-invasive, reproducible, easy to measure sequentially, and inexpensive. Furthermore, they should accurately reflect the degree of the underlying inflammation and be sensitive to the effect of therapeutic intervention, exposure to irritants and exacerbations of the disease.

A method of direct evaluation and monitoring of the inflammatory process in the bronchi and lung parenchyma in asthma and COPD involves cell count and mediators' determinations in lung parenchyma biopsies or/and bronchoalveolar lavage (BAL) fluid. Therefore, bronchoscopy is an invaluable tool for obtaining biopsy samples and BAL fluid, that will then be investigated and thus contribute to the better understanding of the mechanisms involved in the abnormal inflammatory response characteristic of both conditions. Biopsy and BAL studies have shown that asthma is characterized by CD4-lymphocytic and eosinophilic inflammation, destruction of mucosal layer, submucosal thickening and remodeling changes,3 whereas the predominant finding in COPD is parenchymal infiltration by CD8-lymphocytes and polymorphonuclear cells with destruction of the normal architecture of alveolar spaces, and thickening of submucosal glands with intact epithelium,4,5 although eosinophilic inflammation has an important role in exacerbations of the disease.6 Corticosteroid treatment appears to suppress this inflammatory activity, more markedly in asthma,3,7 but there are also reports of partial response in COPD.8,9 Minimally invasive techniques such as nasal and skin biopsies have been used for the evaluation of systematic treatment, i.e. oral steroids and immunotherapy, in particular for asthma. Inflammatory changes in nasal and skin biopsies are considered to reflect lower airway inflammation,10 and have been used to demonstrate the effects of steroids11 and immunotherapy12 in patients with asthma. For all valuable information they provide, these invasive procedures cannot be incorporated in routine practice. The use of suitable markers in more easily obtained samples, such as blood, urine, sputum, and exhaled air as non-invasive alternatives for the evaluation of inflammation is considered appropriate in clinical practice. Bronchial hyperresponsiveness (BHR) is an additional non-invasive marker of underlying inflammation, especially in asthma.

Bronchial hyperresponsiveness (BHR) to a wide variety of bronchoconstrictors is one of the main features of asthma. It has been shown to be a good marker of inflammation that relates well to asthma severity.13 Metacholine, a direct bronchoconstrictor that acts on airway smooth muscle, is currently considered the standard for BHR challenge,13,14 since it is more sensitive than exercise or cold air challenge and is associated with less adverse events compared to histamine. In addition, a recent study showed that, compared to monitoring of symptom scores, FEV1, and PEFR variation, BHR to metacholine is more sensitive in predicting an imminent asthma attack and more useful in adjusting anti-inflammatory treatment.15 It has also been demonstrated that inhaled steroids14,15 and immunotherapy16,17 reduce BHR in patients with asthma and allergic rhinitis. Recent data suggest the use of AMP, a non-specific bronchoconstrictive agent acting indirectly through the release of inflammatory mediators from mast cells, as a more specific marker for atopic asthma18,19 that relates better to underlying inflammation20 and responds earlier to inhaled steroid therapy.21

However, BHR is characteristic not only for asthma; it also represents a significant finding in a high proportion of COPD patients, as demonstrated in the large Lung Health Study.22 This study included 5,877 COPD patients; 12-13% of these patients had a positive metacholine challenge test (PC20 <1 mg/mL), and an additional 34-37% gave uncertain test results. The presence of bronchial hyperresponsiveness in COPD patients is thought to relate to a more rapid fall of FEV123 and higher mortality rates.24 Treatment with inhaled steroids improves BHR;25 nevertheless, the effect of inhaled steroids on the course of the disease have not yet been determined.

Measurement of inflammatory markers in blood provides an alternative in the evaluation of the type and extent of airway inflammation, as well as treatment outcomes. Asthma patients show increased eosinophil counts in blood, which decrease following treatment with inhaled steroids26 or immunotherapy.27 In addition, eosinophilic cationic protein (ECP), a marker of eosinophil activation, seems to be associated with clinical symptoms and lung function parameters in asthma patients, whereas it declines significantly following anti-inflammatory treatment.28 The sole marker in blood that has been shown to have a fairly strong association with COPD (in particular, emphysema) in a sub-group of patients is α1-antitrypsin (α1-AT) deficiency.29 Replacement therapy restores normal α1-AT levels, and reduces inflammation.30 High levels of myeloperoxidase (MPO), a polymorphonuclear activation marker, in serum have been found in COPD patients, but they are not responsive to anti-inflammatory treatment.31 Several further inflammatory markers in serum have been evaluated in patients with asthma (sIL-2r, sICAM1, sVCAM1, cytokines IL-4 and IL-5 etc ) and COPD (TNFα, sVCAM1, cytokines IL-1β, IL-8 etc), but none has gained wide acceptance nor has been established as a useful marker in the evaluation of treatment.

Measurement of inflammatory markers in urine for the evaluation of inflammation has also been used in both clinical practice and research. In patients with asthma, eosinophilic peroxidase (EPX) in urine provides a less invasive means to evaluate eosinophilic inflammation, compared to serum ECP. High EPX levels have been found in symptomatic atopic patients, and shown to decline following anti-inflammatory treatment.32 Sequential EPX measurements may be particularly useful in monitoring children with asthma.33 Other mediators that can be determined in urine of patients with asthma include eicosanoid metabolites, such as leukotriene LTE4 and prostaglandin PGF2. Measurements of LTE4 in urine allow evaluation of the 5-lipoxygenase pathway activity, as well as the therapeutic effect of 5-lipoxygenase inhibitors.34 However, no significant effect of inhaled steroids on LTE4 levels in urine has been demonstrated.35 Both LTE4 and PGF2 levels in urine have been found to increase significantly in the late phase of allergen-induced asthmatic response; on the other hand, these levels fall significantly if allergen exposure is avoided.36 In COPD, the only adequately assessed marker in urine is desmosine, an elastin breakdown product. High desmosine levels have been reported in the urine of patients with COPD,37 which tend to rise further during an exacerbation of the disease.38 The effect of treatment on high desmosine levels has not been adequately addressed.

Induced sputum provides an additional non-invasive method for the evaluation of inflammation in asthma and COPD. It reflects inflammation in the lower airways more accurately than any other body fluid and is considered a good alternative to bronchoscopy. Cell counts, e.g. eosinophil count, are directly determined in sputum, and are closely related to findings from bronchial biopsies and BAL studies.39 High eosinophil counts, and ECP and IL-5 levels in induced sputum from asthma patients have been found in allergen-induced response and in seasons with high levels of pollen in the atmosphere,39,40 reflecting the late phase of allergic response. Both inhaled and oral steroids suppress eosinophilic inflammation in sputum and also reduce the release of pre-formed mediators from eosinophils,41 an effect that appears to be dose-dependent. Combination therapy with an inhaled steroid and a long-acting β2-agonist leads to a greater decline in sputum eosinophils compared to inhaled steroids alone.42 A decline in sputum eosinophil count has also been observed with the use of other anti-inflammatory agents, such as theophylline and leukotriene antagonists, but this effect is by far more prominent with steroids.42

Induced sputum is also a valuable tool in assessing inflammation in COPD. However, sputum findings seem to originate from different sources than relevant bronchoscopic findings.43 Most studies show a predominance of neutrophils in induced sputum and an inverse relationship between neutrophil count and FEV1 value and annual fall rate.44 Eosinophils are also present in a significant percentage of COPD patients; however, eosinophil counts that exceed 5% of total cell count are coupled with post-bronchodilatory reversibility in spirometry and response to steroid treatment.45 Induced sputum has further facilitated the understanding of the underlying pathophysiologic changes at times of COPD exacerbations. It appears that during episodes of COPD exacerbation neutrophil counts and mediators (MPO, IL-8) increase significantly and then return to lower levels during remission periods, irrespective of whether exacerbation was triggered by viral or bacterial infection.46 Another study suggests that cytokine (IL-8, IL-6) levels in the supernatant of sputum from patients with clinically stable disease may be predictive of the frequency and severity of exacerbations.47 Despite their ability to reduce the incidence of disease exacerbations, inhaled steroids have been shown to have no effect on a significant number of mediators implicated in a large part of the inflammatory process. A double blind placebo-controlled study showed no change in neutrophil percentage, nor in levels of neutrophilic elastase and other inflammatory mediators.48 On the other hand, theophylline seems to have a weak anti-inflammatory effect on neutrophilic cytokines, such as IL-8 and MPO, and may reduce neutrophil counts.49 Treatment outcome and induced sputum have been also used for the differential diagnosis of asthma and COPD. Inhaled steroids appear to reduce inflammation associated with asthma, as demonstrated by levels of mediators such as ECP and eosinophil peroxidase (EPO) in sputum supernatant. In COPD, however, steroids do not change levels of MPO and human neutrophil lipocalin (HNL), which are neutrophil inflammatory mediators in COPD.31 Nevertheless, the presence of eosinophilia in induced sputum of patients with COPD predicts response to steroids.50

Recent reports indicate that markers in exhaled air can be used for the evaluation and monitoring of inflammation in asthma and COPD. In asthma, the more extensively studied marker is nitric oxide (NO), which is elevated in patients with asthma;51 however, it is only of value in patients not treated with inhaled steroids. Exhaled NO levels increase significantly in the late phase of allergen-induced asthmatic response only in atopic patients; furthermore, this increase appears to be associated with sensitization to seasonal allergens.52,53 Exhaled NO levels may also be useful as an early non-invasive indicator of potential inflammation in the airways of atopic asymptomatic patients.54 Short-acting β2-agonists have no effect on exhaled NO,55 as contrasted with inhaled or oral steroids, which cause a decline in high NO levels.51 Low doses of inhaled steroids have been shown to effectively control exhaled NO levels in patients with mild to moderate asthma, as opposed to patients with severe asthma who do not benefit in terms of exhaled NO levels even if high-dose inhaled or oral steroids are used.56 Treatment with leukotriene antagonists has also been shown to reduce exhaled NO levels, although not as effectively as steroids do, but it additionally prevents NO increase during tapering steroid dosage.57

NO levels are less useful in the evaluation of COPD than in asthma. Exhaled NO levels in clinically stable COPD -irrespective of smoking status- are lower compared to those of non-asthmatic smokers.51 Low NO levels in these cases is probably due to endothelial NO synthase (eNOS) inhibition by smoke, or increased oxidative stress leading to NO consumption for peroxynitrate production. Increased NO levels in unstable disease or at times of disease exacerbation essentially reflect underlying infection, the potential presence of eosinophilic inflammation, and acidosis that occurs occasionally and causes increased NO release.58 A key observation relative to the value of NO levels in COPD is that COPD patients with partial post-bronchodilatory reversibility proportional to that characteristic of asthma have high NO levels. These patients demonstrate induced sputum eosinophilia and respond to treatment with inhaled steroids. Thus, it has been suggested that stable COPD patients with high NO levels respond to inhaled steroids.59

Other exhaled air markers that have been used in the evaluation of inflammation in asthma and COPD include carbon monoxide (CO), pentane and ethane, i.e. volatile molecules indicative of underlying lipid peroxidation. High CO, pentane and ethane levels in exhaled air have been found in asthmatic patients not receiving steroids.60 In both early and late phase of asthmatic response, exhaled air CO levels are increased, with a respective increase in nasal CO levels.60 Other studies, however, have failed to confirm these findings and report that there is no difference between asthmatics and controls in exhaled CO levels.61 Exhaled CO cannot be evaluated in COPD due to the concomitant smoking habit that affects it.62 Still, ex-smokers with COPD show higher CO levels compared to healthy controls, thus confirming the presence of inflammation and oxidative stress in developed disease.60 CO levels increase further at times of disease exacerbation. Similarly, pentane and ethane levels are increased in COPD, with the levels of the latter being associated with the degree of airway obstruction and tobacco use.63 Exhaled air temperature is an additional non-invasive marker for the evaluation of COPD. Low temperatures restore to normal with the use of vasodilating agents; this finding has led to the suggestion that exhaled air temperature reflects changes in bronchial blood flow.64

A recently proposed non-invasive alternative for the evaluation of airway inflammation is exhaled breath condensate (EBC), in which several volatile and non-volatile agents are quantitated. In asthma patients, EBC shows high levels of oxidative inflammatory markers, leukotrienes, NO metabolism products and cytokines.60 Atopy has not been found to affect the levels of these markers in EBC, indicating that atopy is not a significant contributor to the oxidative inflammatory process characteristic of asthma.65 On the contrary, anti-inflammatory treatment, in particular inhaled steroids, have been shown to favorably affect all inflammatory markers in EBC from patients with asthma.60 A recent study suggests that, since measurement of the above-described markers is quite difficult and time-consuming, pH determinations in EBC could be used as an easy and quick to obtain indicator of underlying inflammation in asthma, which has been shown to relate well to findings in induced sputum.66

The study of EBC in COPD has not been as comprehensive as in asthma. EBC studies have been primarily focused on oxidative inflammation, which however comprises one of the main pathophysiologic mechanisms of the disease. Hydrogen peroxide (H2O2), a key indicator of oxidative inflammation, is increased in stable COPD, and has been shown to increase further at times of disease exacerbation.67 Long-term administration of N-acetylocysteine reduces H2O2 levels in stable disease; it has therefore been suggested that this agent controls oxidative inflammation,68 an effect that has not been shown with the use of inhaled steroids.69 High levels of 8-isoprostane, another reliable marker of lipid peroxidation, are found in EBC from patients with COPD irrespective of smoking.70 This observation led to the suggestion that underlying inflammation rather than smoking is the main determinant of oxidative inflammation in COPD. A recent study, currently in press, shows that the evaluation of H2O2 in EBC from patients with COPD is more useful than 8-isoprostane, because H2O2 measurements are more consistent, better associated with disease staging according to GOLD, and strongly associated with underlying neutrophilia in induced sputum.71 Levels of leukotriene LTB4, a strong neutrophil chemoattractant, have been recently studied and were found to increase at disease exacerbation. The same study showed high 8-isoprostane levels. Both markers declined in the remission period; still, their levels did not restore to normal.72 From all EBC markers assessed in COPD till now, it appears that pH measurements are, as in the case of asthma, the most reliable and rapid marker of endogenous airway oxidation and are closely related to underlying neutrophilic inflammation in induced sputum and, consequently, to oxidative stress, as reflected in H2O2 and 8-isoprostane levels.71

Conclusively, many invasive and non-invasive markers have been used for the evaluation of airway inflammation in asthma and COPD. However, it is not yet clear which outcome evaluation method (inflammatory markers, lung function parameters, symptoms, etc) is more appropriate for monitoring response to anti-inflammatory treatment. Evaluation of disease using various outcome variables, with different patterns of response to therapeutic intervention, has led to inconsistent results. It is therefore necessary to conduct large, long-term clinical studies investigating the association between various inflammatory markers and treatment regimens. Such studies will indicate the most appropriate method to evaluate a given treatment. In addition, rapid and inexpensive quantitative assays of inflammatory markers should be developed. Presently, available data support the use of BHR and induced sputum as the best markers for evaluating treatment outcomes in the majority of asthma patients. The situation is less clear in COPD; no marker has as yet gained wide acceptance, although induced sputum appears to be gaining ground. Still, both measurement of bronchial hyperresponsiveness and induced sputum require time and expertise from medical staff, as well as compliant cooperation from the patient; for these reasons their use, although quite widespread in research, looms rather unfeasible in routine practice.



  1.  NHLBI/WHO Workshop report: Global Strategy for Asthma Management and Prevention. 2002; NIH Publication 02-3659.

  2.  GOLD: Global strategy for the diagnosis, management and prevention of chronic obstructive pulmonary disease. 2001; NIH Publication 01-2701.

  3.  Djukanovic R. Bronchoscopy as a research tool for the study of asthma pathogenesis and effects of antiasthma drugs. J Allergy Clin Immunol 1996; 98:S41-5; discussion S64-6.

  4.  Saetta M, Di Stefano A, Turato G, Facchini FM, Corbino L, Mapp CE, Maestrelli P, Ciaccia A, Fabbri LM. CD8+ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998; 157:822-6.

  5.  O'Shaughnessy TC, Ansari TW, Barnes NC, Jeffery PK. Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV1. Am J Respir Crit Care Med 1997; 155(3):852-7.

  6.  Saetta M, Di Stefano A, Maestrelli P, Turato G, Ruggieri MP, Roggeri A, Calcagni P, Mapp CE, Ciaccia A, Fabbri LM. Airway eosinophilia in chronic bronchitis during exacerbations. Am J Respir Crit Care Med 1994; 150:1646-52.

  7.  Lim S, Jatakanon A, John M, Gilbey T, O'Connor BJ, Chung KF, Barnes PJ. Effect of inhaled budesonide on lung function and airway inflammation. Assessment by various inflammatory markers in mild asthma. Am J Respir Crit Care Med 1999; 159:22-30.

  8.  Hattotuwa KL, Gizycki MJ, Ansari TW, Jeffery PK, Barnes NC. The effects of inhaled fluticasone on airway inflammation in chronic obstructive pulmonary disease: a double-blind, placebo-controlled biopsy study. Am J Respir Crit Care Med 2002; 165(12):1592-6.

  9.  Gamble E, Grootendorst DC, Brightling CE, Troy S, Qui Y, Zhu J, Parker D, Vignola AM, Kroegel C, Morell F, Hansel TT, Rennard SI, Compton C, Ohad A, Tri T, Edelson J, Pavord ID, Rabe KF, Barnes NC, Jeffery PK. Anti-inflammatory effects of the phosphodiesterase 4 inhibitor cilomilast (Ariflo) in COPD. Am J Respir Crit Care Med 2003 [Epub ahead of print].

10.  Gaga M, Lambrou P, Papageorgiou N, Koulouris N, Kosmas N, Fragakis N, Sofios C, Jordanoglou J. Eosinophils are a feature of upper and lower airway pathology in non-atopic asthma, irrespective of the presence of rhinitis. Clin Exp Allergy 2000; 30, 663-9.

11.  Varney V, Gaga M, Frew AJ, De Vos C, Kay AB. The effect of a single oral dose of prednisolone or cetirizine on inflammatory cells infiltrating allergen-induced cutaneous late-phase reactions in atopic subjects. Clin Exp Allergy 1992; 22:43-9.

12.  Varney VA, Hamid QA, Gaga M, Ying S, Jacobson M, Frew AJ, Kay AB, Durham SR. Influence of grass pollen immunotherapy on cellular infiltration and cytokine mRNA expression during allergen-induced late-phase cutaneous responses. J Clin Invest 1993; 92:644-51.

13.  Sterk PJ, Fabbri LM, Quanjer PH, Cockcroft DW, O'Byrne PM, Anderson SD, Juniper EF, Malo JL. Airway responsiveness. Standardized challenge testing with pharmacological, physical and sensitizing stimuli in adults. Report Working Party Standardization of Lung Function Tests, European Community for Steel and Coal. Official Statement of the European Respiratory Society. Eur Respir J 1993; 16(Suppl):53-83.

14.  Crapo RO, Casaburi R, Coates AL, Enright PL, Hankinson JL, Irvin CG, MacIntyre NR, McKay RT, Wanger JS, Anderson SD, Cockcroft DW, Fish JE, Sterk PJ. Guidelines for methacholine and exercise challenge testing-1999. This official statement of the American Thoracic Society was adopted by the ATS Board of Directors, July 1999. Am J Respir Crit Care Med 2000; 161:309-29.

15.  Sont JK, Willems LN, Bel EH, van Krieken JH, Vandenbroucke JP, Sterk PJ. Clinical control and histopathologic outcome of asthma when using airway hyperresponsiveness as an additional guide to long-term treatment. The AMPUL Study Group. Am J Respir Crit Care Med 1999; 159:1043-51.

16.  Grembiale RD, Camporota L, Naty S, Tranfa CM, Djukanovic R, Marsico SA. Effects of specific immunothe­rapy in allergic rhinitic individuals with bronchial hyperresponsiveness. Am J Respir Crit Care Med 2000; 162:2048-52.

17.  Walker SM, Pajno GB, Lima MT, Wilson DR, Durham SR. Grass pollen immunotherapy for seasonal rhinitis and asthma: a randomized, controlled trial. J Allergy Clin Immunol 2001; 107:87-93.

18.  Van Schoor J, Joos GF, Pauwels RA. Indirect bronchial hyperresponsiveness in asthma: mechanisms, pharmacology and implications for clinical research. Eur Respir J 2000; 16:514-33.

19.  Ludviksdottir D, Janson C, Bjornsson E, Stalenheim G, Boman G, Hedenstrom H, Venge P, Gudbjornsson B, Valtysdottir S. Different airway responsiveness profiles in atopic asthma, nonatopic asthma, and Sjogren's syndrome. BHR Study Group. Bronchial hyperresponsiveness. Allergy 2000; 55:259-65.

20.  Van den Berg, Meijer R, Kerstjens H, DeReus D, Koλter G, Kaufman H, Postma D. Pc(20) adenosine 5'-monophosphate is more closely associated with airway inflammation in asthma than pc(20) methacholine. Am J Respir Crit Care Med 2001; 163:1545-1550.

21.  Prosperini G, Rajakulasingam K, Cacciola RR, Spicuzza L, Rorke S, Holgate ST, Di Maria GU, Polosa R. J Changes in sputum counts and airway hyperresponsiveness after budesonide: monitoring anti-inflammatory response on the basis of surrogate markers of airway inflammation. Allergy Clin Immunol 2002; 110:855-61.

22.  Tashkin DP, Altose MD, Bleecker ER, Connett JE, Kanner RE, Lee WW, Wise R. The lung health study: airway responsiveness to inhaled methacholine in smokers with mild to moderate airflow limitation. The Lung Health Study Research Group. Am Rev Respir Dis 1992; 145:301-10.

23.  Rijcken B, Schouten JP, Xu X, Rosner B, Weiss ST. Airway hyperresponsiveness to histamine associated with accelerated decline in FEV1. Am J Respir Crit Care Med 1995; 151(5):1377-82.

24.  Hospers JJ, Postma DS, Rijcken B, Weiss ST, Schouten JP. Histamine airway hyper-responsiveness and mortality from chronic obstructive pulmonary disease: a cohort study. Lancet 2000; 356(9238):1313-7.

25.  Lung Health Study Research Group. Effect of inhaled triamcinolone on the decline in pulmonary function in chronic obstructive pulmonary disease. N Engl J Med 2000; 343(26):1902-9.

26.  Meijer RJ, Postma DS, Kauffman HF, Arends LR, Koeter GH, Kerstjens HA. Accuracy of eosinophils and eosinophil cationic protein to predict steroid improvement in asthma. Clin Exp Allergy 2002; 32:1096-103.

27.  Rak S, Heinrich C, Jacobsen L, Scheynius A, Venge P. A double-blinded, comparative study of the effects of short preseason specific immunotherapy and topical steroids in patients with allergic rhinoconjunctivitis and asthma. J Allergy Clin Immunol 2001; 108:921-8.

28.  Aziz I, Wilson AM, Lipworth BJ. Effects of once-daily formoterol and budesonide given alone or in combination on surrogate inflammatory markers in asthmatic adults. Chest 2000; 118:1049-58.

29.  Janus ED, Phillips NT, Carrell RW. Smoking, lung function, and alpha 1-antitrypsin deficiency. Lancet 1985; 1(8421):152-4.

30.  Stockley RA, Bayley DL, Unsal I, Dowson LJ. The effect of augmentation therapy on bronchial inflammation in alpha1-antitrypsin deficiency. Am J Respir Crit Care Med 2002; 165(11):1494-8.

31.  Keatings VM, Jatakanon A, Worsdell YM, Barnes PJ. Effects of inhaled and oral glucocorticoids on inflammatory indices in asthma and COPD. Am J Respir Crit Care Med 1997; 155(2):542-8.

32.  Kristjansson S, Strannegard IL, Strannegard O, Peterson C, Enander I, Wennergren G. Urinary eosinophil protein X in children with atopic asthma: a useful marker of antiinflammatory treatment. J Allergy Clin Immunol 1996; 97:1179-87.

33.  Labbe A, Aublet-Cuvelier B, Jouaville L, Beaugeon G, Fiani L, Petit I, Ouchchane L, Doly M. Prospective longitudinal study of urinary eosinophil protein X in children with asthma and chronic coug. Pediatr Pulmonol 2001; 31:354-62.

34.  B Dahlen, M Kumlin, E Ihre, O Zetterstrom, and SE Dahlen. Inhibition of allergen-induced airway obstruction and leukotriene generation in atopic asthmatic subjects by the leukotriene biosynthesis inhibitor BAYx 1005. Thorax 1997; 52: 342-347.

35.  O'Shaughnessy KM, Wellings R, Gillies B, Fuller RW. Differential effects of fluticasone propionate on allergen-evoked bronchoconstriction and increased urinary leukotriene E4 excretion. Am Rev Respir Dis 1993; 147:1472-6.

36.  O'Sullivan S, Roquet A, Dahlen B, Dahlen S, Kumlin M. Urinary excretion of inflammatory mediators during allergen-induced early and late phase asthmatic reactions. Clin Exp Allergy 1998; 28:1332-9.

37.  Gottlieb DJ, Stone PJ, Sparrow D, Gale ME, Weiss ST, Snider GL, O'Connor GT. Urinary desmosine excretion in smokers with and without rapid decline of lung function: the Normative Aging Study. Am J Respir Crit Care Med 1996; 154(5):1290-5.

38.  Fiorenza D, Viglio S, Lupi A, Baccheschi J, Tinelli C, Trisolini R, Iadarola R, Luisetti M, Snider GL. Urinary desmosine excretion in acute exacerbations of COPD: a preliminary report. Respir Med 2002; 96(2):110-4.

39.  Kips JC, Kharitonov SA, Barnes PJ. Non invasive assessment of airway inflammation in asthma. Eur Respir Mon 2003; 23:164-79.

40.  Keatings VM, O' Connor BJ, Wright LG, Huston D, Corrigan C, Barnes PJ. Late response to allergen is associated with increased concentrations of TNF-a and interleukin 5 in induced sputum. J Allergy Clin Immunol 1996; 99:693-98.

41.  Kips JC, Inman MD, Jayaram L, Bel EH, Parameswaran K, Pizzichini MM, Pavord ID, Djukanovic R, Hargreave FE, Sterk PJ. The use of induced sputum in clinical trials. Eur Respir J 2002; 37(Suppl):47s-50s.

42.  Djukanovic R. Airway inflammation in asthma and its consequences: implications for treatment in children and adults. J Allergy Clin Immunol 2002; 109:S539-48.

43.  Rutgers SR, Timens W, Kaufmann HF, van der Mark TW, Koeter GH, Postma DS. Comparison of induced sputum with bronchial wash, bronchoalveolar lavage and bronchial biopsies in COPD. Eur Respir J 2000; 15:109-15.

44.  Stanescu D, Sanna A, Veriter C Kostianev S, Calcagni PG, Fabbri LM, Maestrelli P. Airways obstruction, chronic expectoration and rapid decline of FEV1 in smokers are associated with increased levels of sputum neutrophils. Thorax 1996; 51:267-71.

45.  Pizzichini E, Pizzichini MM, Gibson P, Parameswaran K, Gleich GJ, Berman L, Dolovich J, Hargreave FE. Sputum eosinophilia predicts benefit from prednisone in smokers with chronic obstructive bronchitis. Am J Respir Crit Care Med 1998; 158:1511-7.

46.  Aaron SD, Angel JB, Lunau M, Wright K, Fex C, Le Saux N, Dales RE. Granulocyte inflammatory markers and airway infection during acute exacerbation of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001; 163:349-55.

47.  Bhowmik A, Seemungal TA, Sapsford RJ, Wedzicha JA. Relation of sputum inflammatory markers to symptoms and lung function changes in COPD exacerbations. Thorax 2000; 55:114-20.

48.  Culpitt SV, Maziak W, Loukides S, Nightingale JA, Matthews JL, Barnes PJ. Effect of high dose inhaled steroid on cells, cytokines, and proteases in induced sputum in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 160:1635-39.

49.  Culpitt SV, de Matos C, Russell RE, Donnelly LE, Rogers DF, Barnes PJ. Effect of theophylline on induced sputum inflammatory indices and neutrophil chemotaxis in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2002; 165:1371-6.

50.  Brightling CE, Monteiro W, Ward R, Parker D, Morgan MD, Wardlaw AJ, Pavord ID. Sputum eosinophilia and short-term response to prednisolone in chronic obstructive pulmonary disease: a randomised controlled trial. Lancet 2000; 356:1480-5.

51.  Kharitonov SA, Barnes PJ. Clinical aspects of exhaled nitric oxide. Eur Respir J 2000; 16:781-92.

52.  Kharitonov SA, O'Connor BJ, Evans DJ, Barnes PJ. Allergen-induced late asthmatic reactions are associated with elevation of exhaled nitric oxide. Am J Respir Crit Care Med 1995; 151:1894-99.

53.  Gratziou C, Lignos M, Dassiou M, Roussos C. Influence of atopy on exhaled nitric oxide in patients with stable asthma and rhinitis. Eur Respir J 1999; 14:897-901.

54.  Horvath I, Barnes PJ. Exhaled monoxides in asymptomatic atopic subjects. Clin Exp Allergy 1999; 29:1276-80.

55.  Yates DH, Kharitonov SA, Barnes PJ. Effect of short and long acting beta2-agonists on exhaled nitric oxide in asthmatic patients. Eur Respir J 1997; 10:1483-1488.

56.  Ricciardolo FLM. Multiple roles of nitric oxide in the airways. Thorax 2003; 58:175-82.

57.  Bisgaard H, Loland L, Oj JA. NO in exhaled air of asthmatic children is reduced by the leukotriene receptor antagonist montelukast. Am J Respir Crit Care Med 1999; 160:1227-31.

58.  Maziak W, Loukides S, Culpitt SV, Sullivan P, Kharitonov SA, Barnes PJ. Exhaled nitric oxide in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998; 157:998-1002.

59.  Silkoff PE, Martin D, Pak J, Westcott JY, Martin RJ. Exhaled nitric oxide correlated with induced sputum findings in COPD. Chest 2001; 119:1049-55.

60.  Barnes PJ, Kharitonov SA. Exhaled Markers of Pulmonary Disease. Am J Respir Crit Care Med 2001; 163: 1693-22.

61.  Khatri SB, Ozkan M, McCarthy K, Laskowski D, Hammel J, Dweik RA, Erzurum SC. Alterations in exhaled gas profile during allergen-induced asthmatic response. Am J Respir Crit Care Med 2001; 164:1844-8.

62.  Montuschi P, Kharitonov SA, Barnes PJ. Exhaled carbon monoxide and nitric oxide in COPD. Chest 2001; 120:496-501.

63.  Paredi P, Kharitonov SA, Leak D, Ward S, Cramer D, Barnes PJ. Exhaled ethane, a marker of lipid peroxidation, is elevated in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2000; 162:369-73.

64.  Paredi P, Caramori G, Cramer D, Ward S, Ciaccia A, Papi A, Kharitonov SA, Barnes PJ. Slower rise of exhaled breath temperature in chronic obstructive pulmonary disease. Eur Respir J 2003; 21:439-43.

65.  Loukides S, Bouros D, Patheodorou G, Panagou P, Siafakas NM. The Relationships Among Hydrogen Peroxide in Expired Breath Condensate, Airway Inflammation, and Asthma Severity. Chest 2002; 121:338-46.

66.  Kostikas K, Papatheodorou G, Ganas K, Psathakis K, Panagou P, Loukides S. pH in Expired Breath Condensate of Patients with Inflammatory Airway diseases. Am J Respir Crit Care Med 2002; 165:1364-70.

67.  Dekhuijzen PN, Aben KK, Dekker I, Aarts LP, Wielders PL, van Herwaarden CL, Bast A. Increased exhalation of hydrogen peroxide in patients with stable and unstable chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1996; 154:813-6.

68.  Kasielski M, Nowak D. Long-term administration of N-acetylcysteine decreases hydrogen peroxide exhalation in subjects with chronic obstructive pulmonary disease. Respir Med 2001; 95:448-56.

69.  Ferreira IM, Hazari MS, Gutierrez C, Zamel N, Chapman KR. Exhaled nitric oxide and hydrogen peroxide in patients with chronic obstructive pulmonary disease: effects of inhaled beclomethasone. Am J Respir Crit Care Med 2001; 164:1012-5.

70.  Montuschi P, Collins JV, Ciabattoni G, Lazzeri N, Corradi M, Kharitonov SA, Barnes PJ. Exhaled 8-isoprostane as an in vivo biomarker of lung oxidative stress in patients with COPD and healthy smokers. Am J Respir Crit Care Med 2000; 162:1175-77.

71.  Kostikas K, Papatheodorou G, Psathakis K, Panagou P, Loukides S. Oxidative stress in expired breath condensate of patients with COPD: Relationships with airway inflammation, and disease severity. Chest 2003; 124: 1373-80.

72.  Nowak D, Kasielski M, Antczak A, Pietras T, Bia­lasiewicz P. Increased content of thiobarbituric acid-reactive substances and hydrogen peroxide in the expired breath condensate of patients with stable chronic obstructive pulmonary disease: no significant effect of cigarette smoking. Respir Med 1999; 93:389-96.