CORRESPONDENCE References 1. Dye C, Williams BG, Espinal MA, Raviglione MC. Erasing the world’s slow stain: strategies to beat multidrug-resistant tuberculosis. Science 2002;295:2042–2046. 2. Ottenhoff TH, Kaufmann SH. Vaccines against tuberculosis: where are we and where do we need to go? PLoS Pathog 2012;8: e1002607. 3. Garton NJ, Waddell SJ, Sherratt AL, Lee SM, Smith RJ, Senner C, Hinds J, Rajakumar K, Adegbola RA, Besra GS, et al. Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum. PLoS Med 2008;5:e75. 4. Deb C, Lee C-M, Dubey VS, Daniel J, Abomoelak B, Sirakova TD, Pawar S, Rogers L, Kolattukudy PE. A novel in vitro multiple-stress dormancy model for Mycobacterium tuberculosis generates a lipid-loaded, drug-tolerant, dormant pathogen. PLoS ONE 2009;4:e6077. 5. Daniel J, Deb C, Dubey VS, Sirakova TD, Abomoelak B, Morbidoni HR, Kolattukudy PE. Induction of a novel class of diacylglycerol acyltransferases and triacylglycerol accumulation in Mycobacterium tuberculosis as it goes into a dormancy-like state in culture. J Bacteriol 2004;186:5017–5030. 6. Downing KJ, Mischenko VV, Shleeva MO, Young DI, Young M, Kaprelyants AS, Apt AS, Mizrahi V. Mutants of Mycobacterium tuberculosis lacking three of the five rpf-like genes are defective for growth in vivo and for resuscitation in vitro. Infect Immun 2005;73: 3038–3043. 7. Kana BD, Gordhan BG, Downing KJ, Sung N, Vostroktunova G, Machowski EE, Tsenova L, Young M, Kaprelyants A, Kaplan G, et al. The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro. Mol Microbiol 2008;67: 672–684. 8. Biketov S, Mukamolova GV, Potapov V, Gilenkov E, Vostroknutova G, Kell DB, Young M, Kaprelyants AS. Culturability of Mycobacterium tuberculosis cells isolated from murine macrophages: a bacterial growth factor promotes recovery. FEMS Immunol Med Microbiol 2000;29:233–240. 9. Wu X, Yang Y, Han Y, Zhang J, Liang Y, Li H, Li B, Wang L. Effect of recombinant Rv1009 protein on promoting the growth of Mycobacterium tuberculosis. J Appl Microbiol 2008;105:1121–1127. 10. Telkov MV, Demina GR, Voloshin SA, Salina EG, Dudik TV, Stekhanova TN, Mukamolova GV, Kazaryan KA, Goncharenko AV, Young M, et al. Proteins of the Rpf (resuscitation promoting factor) family are peptidoglycan hydrolases. Biochemistry (Mosc) 2006;71:414–422. 11. Cohen-Gonsaud M, Barthe P, Bagneris ´ C, Henderson B, Ward J, Roumestand C, Keep NH. The structure of a resuscitation-promoting factor domain from Mycobacterium tuberculosis shows homology to lysozymes. 2005;12(3):270–3. 12. Kana BD, Mizrahi V. Resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling. FEMS Immunol Med Microbiol 2010;58:39–50. 13. Shleeva MO, Kudykina YK, Vostroknutova GN, Suzina NE, Mulyukin AL, Kaprelyants AS. Dormant ovoid cells of Mycobacterium tuberculosis are formed in response to gradual external acidification. Tuberculosis (Edinb) 2011;91:146–154.
Copyright © 2014 by the American Thoracic Society
Club Cell Protein and Chronic Obstructive Pulmonary Disease Progression: The Unrealized Potential of a Peripheral Lung Biomarker To the Editor: Park and coworkers (1) recently reported the results of a 9-year longitudinal study assessing the prognostic value of serum CC16 614
in patients with chronic obstructive pulmonary disease (COPD). These authors found that serum CC16 levels were inversely related to the decline of FEV1. As the association was rather weak, they concluded that serum CC16 might be of value for predicting COPD progression, but only as an adjunct to classical risk factors. Using CC16-deficient mice, Park and colleagues failed to demonstrate a role of CC16 in COPD progression, which they interpret as another limitation (1). I would like here to express some caution about these conclusions, and to recall the diagnostic potential of CC16 and the conditions required to realize it. Serum CC16 is a biomarker developed in the early 1990s (2) that largely inspired the pneumoproteinemia concept (3)—that is, the evaluation of airways epithelium integrity by measuring lung-specific proteins in serum. What really makes the CC16 pneumoprotein of diagnostic interest is its sensitivity to two types of critical changes in distal airways. First, the protein rapidly rises in serum as the airways permeability increases, which is frequent in lung disorders. Second, when the epithelial barrier is preserved, serum CC16 closely correlates with the number of Club cells, well-known targets of lung toxicants, including tobacco smoke. Thus, contrary to the belief underlying the experiment of Park and coworkers (1) on CC16-deficient mice, the significance of serum CC16 resides not in its biological function, but in the nature and extent of airways damage mirrored by changes in its serum levels. In other terms, the inverse association between serum CC16 and the FEV1 decline over time might simply mean that the progression of COPD is related to the severity of airways damage caused by chronic smoking. When using serum CC16, it is also important to keep in mind that this protein is a peripheral marker of events taking place in the deep lung. This means that circulating levels of CC16 are determined not only by the intrapulmonary pool of CC16, but also by the rate at which the protein leaks from the lungs and at which it is cleared from plasma. Because of its small size, CC16 is rapidly eliminated from plasma by glomerular filtration, and as a corollary its serum level rises in parallel with serum creatinine. In healthy subjects, variations in renal function already account for 25% of serum CC16 variations, which is almost equivalent to the variations caused by smoking (4). No doubt that the risk of confounding by renal function was much higher in the study by Park and colleagues, given the characteristics of the studied population (1). Another metabolic confounder of serum CC16 is the time of blood sampling, which can generate up to 20 to 25% variation depending on whether blood is taken in the morning or in the afternoon (5). Finally, patients with COPD frequently present an increased airway permeability that may abolish the decrease of serum CC16 induced by smoking, unless an adjustment is made with a more specific permeability biomarker (e.g., surfactantassociated proteins D or B). Because serum levels of CC16 were unadjusted for variations in renal function and sampling time, the study by Park and coworkers could not accurately estimate the diagnostic potential of this biomarker and even the impact of chronic smoking (1). This probably explains why this study failed to replicate the dose-dependent decrease of serum CC16 with the number of pack-years that was consistently reported by previous studies. Associations of serum CC16 with age and body mass index are most probably secondary associations
American Journal of Respiratory and Critical Care Medicine Volume 189 Number 5 | March 1 2014
CORRESPONDENCE driven by the decline of renal function accompanying ageing and obesity (6). n Author disclosures are available with the text of this letter at www.atsjournals.org. Alfred Bernard, Ph.D. Catholic University of Louvain Brussels, Belgium
References 1. Park HY, Churg A, Wright JL, Li Y, Tam S, Man SP, Tashkin D, Wise RA, Connett JE, Sin DD. Club cell protein 16 and disease progression in chronic obstructive pulmonary disease (COPD). Am J Respir Crit Care Med 2013;188:1413–1439. 2. Bernard A, Marchandise FX, Depelchin S, Lauwerys R, Sibille Y. Clara cell protein in serum and bronchoalveolar lavage. Eur Respir J 1992;5:1231–1238. 3. Hermans C, Bernard A. Lung epithelium-specific proteins: characteristics and potential applications as markers. Am J Respir Crit Care Med 1999;159:646–678. 4. Hermans C, Aly O, Nyberg B-I, Peterson C, Bernard A. Determinants of Clara cell protein (CC16) concentration in serum: a reassessment with two different immunoassays. Clin Chim Acta 1998;272: 101–110. 5. Helleday R, Segerstedt B, Forsberg B, Mudway I, Nordberg G, Bernard A, Blomberg A. Exploring the time dependence of serum Clara cell protein as a biomarker of pulmonary injury in humans. Chest 2006;130:672–675. 6. de Boer IH, Katz R, Fried LF, Ix JH, Luchsinger J, Sarnak MJ, Shlipak MG, Siscovick DS, Kestenbaum B. Obesity and change in estimated GFR among older adults. Am J Kidney Dis 2009;54:1043–1051.
Copyright © 2014 by the American Thoracic Society
Reply From the Authors: We thank Professor Bernard for his interest in our article and his insightful comments. Because our study did not take into account
Correspondence
renal function of the subjects or the timing of the blood draw, we may have underestimated the biomarker potential of club (Clara) cell protein 16 (CC-16) to predict disease progression in chronic obstructive pulmonary disease (COPD) (1). As suggested by Bernard, we measured surfactant protein D (SFTPD) as a potential surrogate of airway permeability in the Lung Health Study (2). In this cohort, serum SFTPD was not significantly associated with serum CC-16 (P = 0.240, adjusted for age and sex), which suggests that airway permeability was not a major confounder to our analysis. Finally, we agree with Bernard’s suggestion that CC-16 is unlikely to be a major molecular driver of COPD. In our study, we exposed CC-16–deficient mice to 6 months of cigarette smoke and found that these were not protected from emphysema or small airway remodeling (1). In sum, we share Bernard’s enthusiasm for CC-16 as a very promising biomarker but not as a therapeutic target in COPD. n Author disclosures are available with the text of this letter at www.atsjournals.org. Hye Yun Park, M.D. Samsung Medical Center Seoul, Korea Don D. Sin, M.D. St. Paul’s Hospital Vancouver, Canada
References 1. Park HY, Churg A, Wright JL, Li Y, Tam S, Man SF, Tashkin D, Wise RA, Connett JE, Sin DD. Club cell protein 16 and disease progression in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2013;188:1413–1419. 2. Hill J, Heslop C, Man SF, Frohlich J, Connett JE, Anthonisen NR, Wise RA, Tashkin DP, Sin DD. Circulating surfactant protein-D and the risk of cardiovascular morbidity and mortality. Eur Heart J 2011;32: 1918–1925.
Copyright © 2014 by the American Thoracic Society
615
Copyright © 2014 by the American Thoracic Society
Club Cell Protein and Chronic Obstructive Pulmonary Disease Progression: The Unrealized Potential of a Peripheral Lung Biomarker To the Editor: Park and coworkers (1) recently reported the results of a 9-year longitudinal study assessing the prognostic value of serum CC16 614
in patients with chronic obstructive pulmonary disease (COPD). These authors found that serum CC16 levels were inversely related to the decline of FEV1. As the association was rather weak, they concluded that serum CC16 might be of value for predicting COPD progression, but only as an adjunct to classical risk factors. Using CC16-deficient mice, Park and colleagues failed to demonstrate a role of CC16 in COPD progression, which they interpret as another limitation (1). I would like here to express some caution about these conclusions, and to recall the diagnostic potential of CC16 and the conditions required to realize it. Serum CC16 is a biomarker developed in the early 1990s (2) that largely inspired the pneumoproteinemia concept (3)—that is, the evaluation of airways epithelium integrity by measuring lung-specific proteins in serum. What really makes the CC16 pneumoprotein of diagnostic interest is its sensitivity to two types of critical changes in distal airways. First, the protein rapidly rises in serum as the airways permeability increases, which is frequent in lung disorders. Second, when the epithelial barrier is preserved, serum CC16 closely correlates with the number of Club cells, well-known targets of lung toxicants, including tobacco smoke. Thus, contrary to the belief underlying the experiment of Park and coworkers (1) on CC16-deficient mice, the significance of serum CC16 resides not in its biological function, but in the nature and extent of airways damage mirrored by changes in its serum levels. In other terms, the inverse association between serum CC16 and the FEV1 decline over time might simply mean that the progression of COPD is related to the severity of airways damage caused by chronic smoking. When using serum CC16, it is also important to keep in mind that this protein is a peripheral marker of events taking place in the deep lung. This means that circulating levels of CC16 are determined not only by the intrapulmonary pool of CC16, but also by the rate at which the protein leaks from the lungs and at which it is cleared from plasma. Because of its small size, CC16 is rapidly eliminated from plasma by glomerular filtration, and as a corollary its serum level rises in parallel with serum creatinine. In healthy subjects, variations in renal function already account for 25% of serum CC16 variations, which is almost equivalent to the variations caused by smoking (4). No doubt that the risk of confounding by renal function was much higher in the study by Park and colleagues, given the characteristics of the studied population (1). Another metabolic confounder of serum CC16 is the time of blood sampling, which can generate up to 20 to 25% variation depending on whether blood is taken in the morning or in the afternoon (5). Finally, patients with COPD frequently present an increased airway permeability that may abolish the decrease of serum CC16 induced by smoking, unless an adjustment is made with a more specific permeability biomarker (e.g., surfactantassociated proteins D or B). Because serum levels of CC16 were unadjusted for variations in renal function and sampling time, the study by Park and coworkers could not accurately estimate the diagnostic potential of this biomarker and even the impact of chronic smoking (1). This probably explains why this study failed to replicate the dose-dependent decrease of serum CC16 with the number of pack-years that was consistently reported by previous studies. Associations of serum CC16 with age and body mass index are most probably secondary associations
American Journal of Respiratory and Critical Care Medicine Volume 189 Number 5 | March 1 2014
CORRESPONDENCE driven by the decline of renal function accompanying ageing and obesity (6). n Author disclosures are available with the text of this letter at www.atsjournals.org. Alfred Bernard, Ph.D. Catholic University of Louvain Brussels, Belgium
References 1. Park HY, Churg A, Wright JL, Li Y, Tam S, Man SP, Tashkin D, Wise RA, Connett JE, Sin DD. Club cell protein 16 and disease progression in chronic obstructive pulmonary disease (COPD). Am J Respir Crit Care Med 2013;188:1413–1439. 2. Bernard A, Marchandise FX, Depelchin S, Lauwerys R, Sibille Y. Clara cell protein in serum and bronchoalveolar lavage. Eur Respir J 1992;5:1231–1238. 3. Hermans C, Bernard A. Lung epithelium-specific proteins: characteristics and potential applications as markers. Am J Respir Crit Care Med 1999;159:646–678. 4. Hermans C, Aly O, Nyberg B-I, Peterson C, Bernard A. Determinants of Clara cell protein (CC16) concentration in serum: a reassessment with two different immunoassays. Clin Chim Acta 1998;272: 101–110. 5. Helleday R, Segerstedt B, Forsberg B, Mudway I, Nordberg G, Bernard A, Blomberg A. Exploring the time dependence of serum Clara cell protein as a biomarker of pulmonary injury in humans. Chest 2006;130:672–675. 6. de Boer IH, Katz R, Fried LF, Ix JH, Luchsinger J, Sarnak MJ, Shlipak MG, Siscovick DS, Kestenbaum B. Obesity and change in estimated GFR among older adults. Am J Kidney Dis 2009;54:1043–1051.
Copyright © 2014 by the American Thoracic Society
Reply From the Authors: We thank Professor Bernard for his interest in our article and his insightful comments. Because our study did not take into account
Correspondence
renal function of the subjects or the timing of the blood draw, we may have underestimated the biomarker potential of club (Clara) cell protein 16 (CC-16) to predict disease progression in chronic obstructive pulmonary disease (COPD) (1). As suggested by Bernard, we measured surfactant protein D (SFTPD) as a potential surrogate of airway permeability in the Lung Health Study (2). In this cohort, serum SFTPD was not significantly associated with serum CC-16 (P = 0.240, adjusted for age and sex), which suggests that airway permeability was not a major confounder to our analysis. Finally, we agree with Bernard’s suggestion that CC-16 is unlikely to be a major molecular driver of COPD. In our study, we exposed CC-16–deficient mice to 6 months of cigarette smoke and found that these were not protected from emphysema or small airway remodeling (1). In sum, we share Bernard’s enthusiasm for CC-16 as a very promising biomarker but not as a therapeutic target in COPD. n Author disclosures are available with the text of this letter at www.atsjournals.org. Hye Yun Park, M.D. Samsung Medical Center Seoul, Korea Don D. Sin, M.D. St. Paul’s Hospital Vancouver, Canada
References 1. Park HY, Churg A, Wright JL, Li Y, Tam S, Man SF, Tashkin D, Wise RA, Connett JE, Sin DD. Club cell protein 16 and disease progression in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2013;188:1413–1419. 2. Hill J, Heslop C, Man SF, Frohlich J, Connett JE, Anthonisen NR, Wise RA, Tashkin DP, Sin DD. Circulating surfactant protein-D and the risk of cardiovascular morbidity and mortality. Eur Heart J 2011;32: 1918–1925.
Copyright © 2014 by the American Thoracic Society
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