Chest ACCP Education Calendar
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Guest Access | Sign In via User Name/Password
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Article Archive
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (34)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Garey, K. W.
Right arrow Articles by Rubinstein, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Garey, K. W.
Right arrow Articles by Rubinstein, I.
(Chest. 2004;125:22-26.)
© 2004 American College of Chest Physicians

Markers of Inflammation in Exhaled Breath Condensate of Young Healthy Smokers*

Kevin W. Garey, PharmD; Melinda M. Neuhauser, PharmD; Richard A. Robbins, MD; Larry H. Danziger, PharmD and Israel Rubinstein, MD, FCCP

* From the University of Houston College of Pharmacy (Drs. Garey and Neuhauser), Houston, TX; University of Illinois at Chicago Colleges of Pharmacy and Medicine (Drs. Rubinstein and Danziger), Chicago, IL; VA Chicago Health Care System (Dr. Rubinstein), Chicago, IL; and Southern Arizona VA Healthcare Center and the University of Arizona (Dr. Robbins), Tucson, AZ.

Correspondence to: Israel Rubinstein, MD, FCCP, Department of Medicine (M/C 719), University of Illinois at Chicago, 840 South Wood St, Chicago, IL 60612-7323; e-mail: IRubinst{at}uic.edu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Introduction: Although a strong correlation exists between long-term cigarette smoking, pulmonary inflammation, and COPD, efforts to identify populations at risk of acquiring COPD have so far been unsuccessful. To this end, noninvasive detection and monitoring of biomarkers of pulmonary inflammation in young healthy smokers may assist in this task.

Study objectives: The purpose of this study was to determine the concentrations of total protein, nitrites, interleukin (IL)-1ß, and tumor necrosis factor (TNF)-{alpha}, and neutrophil chemotactic activity in exhaled breath condensate (EBC) collected from healthy college student smokers and nonsmokers.

Design: EBC was collected from 20 volunteers (9 nonsmokers and 11 smokers) during tidal breathing for 20 min. EBC was also collected from smokers 30 min after smoking one filtered cigarette. The concentrations of total protein, nitrite, IL-1ß, and TNF-{alpha} in EBC was determined by enzyme-linked immunosorbent assay. Neutrophil chemotactic activity in EBC was determined in vitro using the blind-well technique.

Results: The concentrations of total protein and nitrite, and neutrophil chemotactic activity were significantly higher in EBC of smokers in comparison to nonsmokers (p < 0.05). The concentrations of total protein and nitrite in the condensate of smokers did not change significantly after smoking one cigarette. The concentrations of IL-1ß and TNF-{alpha} in EBC were similar in nonsmokers and smokers.

Conclusions: Concentrations of certain inflammatory mediators and neutrophil chemotactic activity are increased in EBC of young healthy smokers. Collection and analysis of EBC may assist in early detection of cigarette smoke-induced pulmonary inflammation and identifying populations at risk for acquiring COPD.

Key Words: airway • biomarkers • cigarette smoking • cytokines • epithelial lining fluid • lung • mediators • neutrophil chemotaxis • noninvasive testing


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It is well established that long-term cigarette smoking is associated with activation of a cascade of inflammatory response in the lung that leads to tissue injury and dysfunction and manifested clinically as COPD.1 Previous studies2 3 4 5 using bronchoscopy and BAL have documented altered cytokine regulation and increased protein concentration in the lung of long-term smokers. Whether an inflammatory response is also present in the lung of young healthy individuals after a relatively short-term exposure to cigarette smoke is uncertain.

This notion is important because a substantial number of young adults pursue regular cigarette smoking. Consequently, there is an urgent need to develop smoking tobacco-related biomarkers of early pulmonary inflammation and dysfunction using simple, noninvasive methods so populations at risk could be identified and appropriate interventions instituted.6

To this end, exhaled breath condensate (EBC) is an emerging, simple, and noninvasive method to sample the lower respiratory tract for various compounds in humans7 8 9 Several mediators implicated in the inflammatory cascade have been detected in EBC, including proteins, nitrite, and cytokines.10 11 12 This approach provides real-time longitudinal sampling of the extracellular lining fluid of the lung for biomarkers of inflammation in the same individual, thereby facilitating monitoring of this detrimental process.

The purpose of this study was to begin to assess the utility of EBC in monitoring the extent of pulmonary inflammation in young healthy smokers by determining the concentrations of total protein, nitrite, interleukin (IL)-1ß, and tumor necrosis factor (TNF)-{alpha}, two ubiquitous and seminal pro-inflammatory cytokines thought to play a seminal role in cigarette smoke-induced lung injury, and neutrophil chemotactic activity in EBC collected from healthy college student smokers and nonsmokers.2 3


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
Nine life-long nonsmoking healthy volunteers (5 men and 4 women; mean age, 22.0 ± 1.9 years [± SD]) and 11 health volunteers (5 men and 6 women; mean age, 23.4 ± 0.9 years) with a 2.0 ± 1.2–pack-year cigarette smoking history were recruited from students attending the University of Illinois at Chicago. All participants had no history of chronic respiratory tract disorders, including asthma and COPD, and denied symptoms of acute respiratory illness within 4 weeks preceding the study. No biological tests were conducted to ascertain the life-long nonsmoking status of nonsmokers recruited to this study. The Institutional Review Board of the University of Illinois at Chicago approved the study, and all individuals gave written informed consent to participate.

Collection of EBC
EBC was collected from all 20 participants for 20 min at the same time of the day (9 to 11 AM). Smokers were then asked to smoke one filtered cigarette of their choice, and 30 min thereafter EBC was collected once again.

The apparatus and methods used to collect EBC have been previously described in detail.10 Briefly, EBC was collected by suspending a 1.5-m Teflon perfluoroalkoxy tube with a 0.5-cm internal diameter installed in a polystyrene foam container filled with ice and connected to a 10-mL polypropylene test tube. While sitting comfortably, subjects breathed tidally for 20 min with inspiration through the nose and expiration through the mouth into the tube. At least 3 mL of condensate were collected for all cases within the 20-min time period. Subjects were instructed to form a complete seal around the tube with their mouth, maintain a dry mouth during collection by periodically swallowing excess saliva, and report any adverse events during or after collection. The EBC was collected into a labeled polypropylene test tube with no preservatives added and stored immediately (within 5 min) at -70°C until analyzed.

Analysis of EBC
All EBC samples were analyzed within 2 months of collection. Amylase concentrations were determined in all 31 samples of EBC collected from the 9 nonsmokers and 11 smokers (before and after smoking one cigarette) as a measure of salivary contamination of the condensate. After thawing, the concentration of amylase in a 200-µL sample of condensate was determined in duplicate using a commercially available kit (Sigma; St. Louis, MO) by spectrophotometry. The lower detection limit of the assay was 7 IU/mL. Total amount of protein in a 100-µL thawed sample of EBC was determined in duplicate according to the method of Bradford13 using a commercially available kit (Pierce; Rockford, IL). The concentration of nitrite in a 100-µL thawed sample of EBC was determined by a modified Griess reaction as previously described in our laboratory.14 Briefly, the sample was incubated with Escherichia coli nitrate reductase (0.5 U/mL) at room temperature for 10 min to convert nitrate to nitrite in the sample being tested. Thereafter, an equal volume of the Griess reagent was added and incubated at room temperature for 10 min. Absorbency was determined at 540 nm using a spectrophotometer. The concentration of nitrite in each sample was determined in duplicate using an automated thermoregulated enzyme-linked immunosorbent assay (ELISA) microplate reader.

The concentration of IL-1ß and TNF-{alpha} in a 100-µL thawed sample of EBC was determined in duplicate by ELISA using commercially available kits according to the instructions of the manufacturer (R&D Systems; Minneapolis, MN) and an automated thermoregulated ELISA microplate reader. The lower detection limit of IL-1ß and TNF-{alpha} was 2 pg/mL and the interassay and intra-assay variations were < 5%. In preliminary studies, we determined that recovery of exogenous IL-1ß and TNF-{alpha} added to EBC and stored for 2 months at - 70°C was within the reproducibility of the measurements (data not shown).

On a separate day, EBC was collected from four nonsmokers and four smokers, and neutrophil chemotactic activity was determined using a 48-well microchemotaxis chambers (Neuroprobe; Cabin John, MD) as previously described.15 16 The bottom of the cells were filled with 25 µL of the thawed condensate sample in triplicate. A 10-µm thick polyvinylpyrrolidone-free polycarbonate filter with a pore size of 3 µm was placed over the samples. The silicon gasket and the upper pieces of the chamber were applied, and 50 µL of the cell suspension was placed in the upper wells. The chambers were incubated in humidified air in 5% CO2 37°C for 30 min. Nonmigrated cells were wiped away from the filter. The filter was immersed in methanol for 5 min, stained with a modified Wright stain, and mounted on a glass slide. Cells that had completely migrated through the filter were counted by using light microscopy. Chemotaxis was expressed as the mean number of cells per high-power field (HPF) from duplicate wells.

Statistical Analysis
Data are expressed as means ± SD where appropriate. Comparisons between smokers (before and after smoking) and nonsmokers were performed by the Kruskal-Wallis analysis of variance and paired comparisons by the Mann-Whitney test because the data were not normally distributed. Pearson correlation was utilized to determine the relationship between measured variables. A statistical software package (SYSTAT Version 7.0; SYSTAT Software; Richmond, CA) was used. Statistical significance was defined as p < 0.05.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
No adverse effects were reported during the course of the study. The volume of EBC collected from the 20 participants in this study over the 20-min period was 3 to 5 mL. Amylase was not detected in any of the 31 EBC samples collected from smokers either before and after smoking one cigarette and nonsmokers. Total protein concentration in EBC was significantly lower in nonsmokers (5.7 ± 2.5 µg/mL) than smokers either before (28.8 ± 35.8 µg/mL) or after smoking one cigarette (25.9 ± 33.9 µg/mL; Fig 1 ; p < 0.05). Total protein concentration was similar before and after smoking one cigarette. Nitrite concentration in EBC was significantly lower in nonsmokers (16,156 ± 7,029 nmol/L) than smokers either before (24,672 ± 7,534 nmol/L) or after (23,043 ± 4,051 nmol/L) acute cigarette smoking (p < 0.05). There was no significant difference in nitrite concentration before and after smoking one cigarette.



View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Total protein and nitrite concentration in smokers and nonsmokers. *p < 0.05 when compared to nonsmokers.

 
The concentration of IL-1ß in EBC was similar in nonsmokers and smokers before smoking one cigarette (1.5 ± 1.0 pg/mL and 1.6 ± 1.8 pg/mL, respectively; p > 0.5). However, IL-1ß concentration in EBC significantly decreased after smoking one cigarette in comparison to nonsmokers (Fig 2 ; p < 0.05). The concentration of TNF-{alpha} in EBC was higher in smokers before smoking in comparison to nonsmokers (7.4 ± 17.5 pg/mL vs 3.9 ± 8.5 pg/mL, respectively; p < 0.05). The concentration of TNF-{alpha} in EBC increased after smoking one cigarette although the difference was not statistically significant. Neutrophil chemotactic activity was increased in EBC of smokers relative to nonsmokers (Fig 3 ; n = 4; p < 0.05). Smokers averaged 98 ± 38 cells per HPF while nonsmokers averaged 43 ± 6 cells per HPF.



View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. IL-1ß and TNF-{alpha} concentrations in smokers and nonsmokers. *p < 0.05 when compared to nonsmokers.

 


View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Chemotactic activity of smokers and nonsmokers. *p < 0.05 when compared to nonsmokers.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There are three new findings of this study. Firstly, we found that the concentrations of total protein and nitrite were significantly increased in EBC of young healthy cigarette smokers as compared to nonsmokers. Mean protein concentration increased by over fivefold (5.7 to 28.8 µg/mL) and nitrite concentration by > 50% (16,156 to 24,672 nmol/L) in smokers relative to nonsmokers of a similar age. Secondly, smoking one cigarette was associated with a significant decrease in IL-1ß concentration in EBC of these smokers. Although the relevance of this finding to the proposed pathophysiology of cigarette smoke-induced lung disease is uncertain, it may predispose smokers to recurrent lung infections given the pivotal role of IL-1ß in combating invading pathogens.17 18 19

Lastly, neutrophil chemotactic activity was increased in EBC of young healthy smokers relative to nonsmokers. This is an important observation because it is well established that long-term cigarette smoking and COPD are associated with airway inflammation characterized by intense neutrophilic infiltration in the airway mucosa.4 5 20 This aberrant response observed in EBC may represent a functional method to detect early and potentially reversible pulmonary injury in young healthy smokers. Clearly, additional studies are warranted to characterize neutrophil chemotactic activity detected in EBC of young healthy smokers, including nicotine, and the effects smoking cessation has on this response.

The inflammatory mediators detected in EBC in this study were derived from subglottic region(s) based on lack of salivary contamination.7 21 Whether they are derived from the airway and/or the lung parenchyma is uncertain. Conceivably, the increased nitrite concentration in EBC of smokers could have emanated from tobacco smoke. However, this possibility seems unlikely as nitrite concentration in EBC of smokers did not increase after smoking one cigarette.

The effects of cigarette smoking on the concentrations of IL-1ß and TNF-{alpha} in BAL have been previously investigated.2 3 21 For instance, Brown et al3 obtained alveolar macrophages from healthy volunteers who were either nonsmokers, light smokers (< 10 pack-year), or heavy smokers (> 10 pack-year). Similar to our results, IL-1ß release from alveolar macrophages stimulated with lipopolysaccharide was significantly decreased in individuals with a smoking history in comparison to nonsmokers. In the study by Brown et al,3 macrophages of light smokers displayed intermediate values for IL-1ß release upon stimulation compared to heavy smokers, suggesting a cumulative effect of cigarette smoking.

Contrary to our results, McCrea et al2 showed less release of TNF-{alpha} from isolated, lipopolysaccharide-stimulated alveolar macrophages of healthy smokers in comparison to nonsmokers; however, these authors did not investigate the effects of cigarette smoking in young individuals as we did. Further studies to elucidate the effects of relatively short-term cigarette smoking on the concentrations of IL-1ß and TNF-{alpha} in EBC are warranted.

It is well established that metabolites of nitric oxide, a potent proinflammatory mediator, are present in cigarette smoke. However, we found that smoking one cigarette did not increase nitrite and nitrate concentrations in EBC of young healthy smokers. By contrast, Balint et al22 showed that acute smoking is associated with a transient increase in the concentrations of these metabolites in EBC of healthy smokers. Moreover, they found no significant difference in nitrite and nitrate concentrations in EBC of nonsmokers and healthy smokers. In addition, Corradi et al23 showed that nitrate levels are increased more than sixfold in EBC of healthy smokers, but not in patients with COPD, relative to nonsmoking healthy control subjects. Although the reasons underlying these discrepant results are uncertain, we studied younger individuals with a shorter smoking history than did Balint et al.22 Conceivably, the effects of cigarette smoking on nitric oxide metabolites in healthy smokers could be manifested early in the course of the exposure while other oxidative products could be expressed later on. Clearly, additional studies, including individuals exposed to passive smoking, are warranted to support or refute this hypothesis.

In summary, we found that concentrations of certain inflammatory mediators and neutrophil chemotactic activity are increased EBC of young healthy smokers. Collection and analysis of EBC may assist in early detection of cigarette smoke-induced pulmonary inflammation and identifying populations at risk for acquiring COPD.


   Footnotes
 
Abbreviations: EBC = exhaled breath concentrate; ELISA = enzyme-linked immunosorbent assay; HPF = high-power field; IL = interleukin; TNF = tumor necrosis factor

This study was supported in part by VA Merit Review and National Institutes of Health grant HL072323.

Received for publication August 13, 2002. Accepted for publication July 8, 2003.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Roth, MD, Arora, A, Barsky, SH, et al (1998) Airway inflammation in young marijuana and tobacco smokers. Am J Respir Crit Care Med 157,928-937[Abstract/Free Full Text]
  2. McCrea, KA, Ensor, JE, Nall, K, et al Altered cytokine regulation in the lungs of cigarette smokers. Am J Respir Crit Care Med 1994;150,696-703[Abstract]
  3. Brown, GP, Iwamoto, GK, Monick, MM, et al Cigarette smoking decreases interleukin 1 release by human alveolar macrophages. Am J Physiol 1989;256,C260-C264
  4. Gotoh, T, Ueda, S, Nakayama, T, et al Protein components of bronchoalveolar lavage fluids from non-smokers and smokers. Eur J Respir Dis 1983;64,369-377[Medline]
  5. Morrison, D, Strieter, RM, Donnelly, SC, et al Neutrophil chemokines in bronchoalveolar lavage fluid and leukocyte-conditioned medium from nonsmokers and smokers. Eur Respir J 1998;12,1067-1072[Abstract]
  6. Wechsler, H, Rigotti, NA, Gledhill-Hoyt, J, et al Increased levels of cigarette use among college students: a cause for national concern. JAMA 1998;280,1673-1678[Abstract/Free Full Text]
  7. Mutlu, GM, Garey, KW, Robbins, RA, et al Collection and analysis of exhaled breath condensate in humans. Am J Respir Crit Care Med 2001;164,731-737[Free Full Text]
  8. Ho, LP, Faccenda, J, Innes, JA, et al Expired hydrogen peroxide in breath condensate of cystic fibrosis patients. Eur Respir J 1999;13,103-106[Abstract]
  9. Montuschi, P, Kharitonov, SA, Ciabattoni, G, et al Exhaled 8-isoprostane as a new non-invasive biomarker of oxidative stress in cystic fibrosis. Thorax 2000;55,205-209[Abstract/Free Full Text]
  10. Ho, LP, Innes, JA, Greening, AP Nitrite levels in breath condensate of patients with cystic fibrosis is elevated in contrast to exhaled nitric oxide. Thorax 1998;53,680-684[Abstract/Free Full Text]
  11. Dekhuijzen, PN, Aben, KK, Dekker, I, et al Increased exhalation of hydrogen peroxide in patients with stable and unstable chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1996;154,813-816[Abstract]
  12. Nowak, D, Kasielski, M, Pietras, T, et al Cigarette smoking does not increase hydrogen peroxide levels in expired breath condensate of patients with stable COPD. Monaldi Arch Chest Dis 1998;53,268-273[Medline]
  13. Bradford, M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1975;72,248-254[CrossRef]
  14. Onyuksel, H, Ikezaki, H, Patel, M, et al A novel formulation of VIP in sterically stabilized micelles amplifies vasodilation in vivo. Pharm Res 1999;16,155-160[CrossRef][ISI][Medline]
  15. Hatipoglu, U, Gao, X, Verral, S, et al Sterically stabilized phospholipids attenuate human neutrophils chemotaxis in vitro. Life Sci 1998;63,693-699[Medline]
  16. Sato, E, Simpson, KL, Grisham, MB, et al Inhibition of MIP-1{alpha}-induced human neutrophil and monocyte chemotactic activity by reactive oxygen and nitrogen metabolites. J Lab Clin Med 2000;135,161-169[CrossRef][ISI][Medline]
  17. Wewers, MD, Diaz, PT, Wewers, ME, et al Cigarette smoking in HIV infection induces a suppressive inflammatory environment in the lung. Am J Respir Crit Care Med 1998;158,1543-1549[Abstract/Free Full Text]
  18. Sher, ME, Bank, S, Greenberg, R, et al The influence of cigarette smoking on cytokine levels in patients with inflammatory bowel disease. Inflamm Bowel Dis 1999;5,73-78[ISI][Medline]
  19. Ouyang, Y, Virasch, N, Hao, P, et al Suppression of human IL-1ß, IL-2, IFN-{gamma}, and TNF-{alpha} production by cigarette smoke extracts. J Allergy Clin Immunol 2000;106,280-287[CrossRef][ISI][Medline]
  20. Bridges, RB, Wyatt, RJ, Rehm, SR Effects of smoking on inflammatory mediators and their relationship to pulmonary dysfunction. Eur J Respir Dis Suppl 1986;146,145-152[Medline]
  21. Dandrea, T, Tu, B, Blomberg, A, et al Differential inhibition of inflammatory cytokine release from cultured alveolar macrophages from smokers and non-smokers by NO2. Hum Exp Toxicol 1997;16,577-588[Medline]
  22. Balint, B, Donnelly, LE, Hanazawa, T, et al Increased nitric oxide metabolites in exhaled breath condensate after exposure to tobacco smoke. Thorax 2001;56,456-461[Abstract/Free Full Text]
  23. Corradi, M, Pesci, A, Casana, R, et al Nitrate in exhaled breath condensate of patients with different airway diseases. Nitric Oxide 2003;8,26-30[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 ChestHome page
J.-L. Corhay, L. Hemelaers, M. Henket, J. Sele, and R. Louis
Granulocyte Chemotactic Activity in Exhaled Breath Condensate of Healthy Subjects and Patients With COPD
Chest, June 1, 2007; 131(6): 1672 - 1677.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
I. Horvath, J. Hunt, P. J. Barnes, and On behalf of the ATS/ERS Task Force on Exhaled Bre
Exhaled breath condensate: methodological recommendations and unresolved questions
Eur. Respir. J., September 1, 2005; 26(3): 523 - 548.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
J. L. Kreindler, A. D. Jackson, P. A. Kemp, R. J. Bridges, and H. Danahay
Inhibition of chloride secretion in human bronchial epithelial cells by cigarette smoke extract
Am J Physiol Lung Cell Mol Physiol, May 1, 2005; 288(5): L894 - L902.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Article Archive
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (34)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Garey, K. W.
Right arrow Articles by Rubinstein, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Garey, K. W.
Right arrow Articles by Rubinstein, I.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS