Conditions: Asthma
Keywords: World Trade Center WTC Induced Sputum Exhaled Breath Condensate
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 18
Healthy volunteers: yes

Brief summary:
Asthma is a heterogeneous disease, and although much is understood about mechanisms of inflammation in allergic asthma, less is known about mechanisms of irritant-induced asthma (IA). Understanding the underlying similarities and differences in mechanisms of these two types of asthma will help focus current treatments and lead to development of new therapies. There is a longstanding NYU/Bellevue Asthma registry (NYUBAR), with a large population (N = 900) of asthma cases and controls, a program that has been housed at the CTSI (formerly GCRC). The destruction of the World Trade Center (WTC) resulted in massive dust, gas and fume exposures to local residents, workers and cleanup workers and individuals involved in rescue and recovery and adverse respiratory health effects of this disaster are reported more than 7 years after 9/11. Many responders, as well as those exposed as residents or local workers, have developed IA, asthma that arises after a lag from an environmental exposure . The WTC Environmental Health Center (WTC EHC) is one of the three New York City (NYC) WTC Centers of Excellence and the only one that focuses on treatment and monitoring of local workers and residents. As such, it has a large population of individuals with irritant-induced asthma. It has been proposed to use participants from the NYUBAR and the WTC EHC to expand the knowledge of irritant and allergic asthma. Non-invasive studies allow for the assessment of airway inflammation, a non-specific response to environmental exposure and injury. Recent technologies also allow for assessment of microRNA (miRNA), small RNAs that regulate gene expression at the post-transcriptional level and thus serve as a pathway to regulation of inflammation. The hypothesis will be tested in that airway inflammation in irritant and allergic asthma may be similar, but result from divergent miRNA regulatory pathways expressed in sputum cells. These studies will provide preliminary data for future studies that will help identify biological pathways to categorize these asthma phenotypes and target future treatment interventions.

Detailed description:
This study will have two asthma phenotypes and one control population. Patients will be recruited from the WTC EHC = 30 with WTC dust cloud exposure, patients will be recruited from the NYUBAR (n=30) and have asthma as defined by NIH guidelines, and control patients will be recruited from the NYUBAR (n=30) and will have no respiratory sx, no asthma diagnosis, or no WTC dust exposure.

The study will entail two to three visits. On visit 1 (V1) all individuals will sign informed consent to participate in the study under an NYU IRB approved protocol. A questionnaire will be completed with standardized questions that include information on WTC exposures, demographics, presence and severity of respiratory symptoms, tobacco history and past medical history. Individuals will undergo spirometry with inhaled bronchodilator. Individuals will undergo methacholine challenge test (visit 1a) if they have normal spirometry or no bronchial hyperresponsiveness. On visit 2, individuals will return to undergo ENO, EBC and spirometry with pre and post bronchodilator maneuvers and induced sputum. Blood will be obtained for CBC with differential cell count, and assessment of total IgE and allergen-specific IgE. Blood will also be stored for future analysis of inflammatory markers. Based on experience, there have been individuals unable to produce enough sputum and thus yield too small a number of cells. These individuals are excluded from data analysis. In addition, there will also be subjects who are able to produce sputum and return for repeat sputum testing.

Arms:
arm 1: None arm 2: None

Interventions: intervention 1: Fluticasone propionate HFA 220
Intervention descriptions: intervention 1: Subjects will take two (2) puffs of Fluticasone propionate HFA 220 twice a day.

Protocol document:
Prot_SAP_000.pdf: 
 
 
 
 
Kazeros, PI 
March 17, 2016 
1
 
 
H#: 10-01191 
NCT02740543 
 
Title: Biomarkers of Irritant-Induced and Allergic Asthma – Phase I and Phase II 
 
Principal Investigator: 
 
Angeliki Kazeros, MD  
Co-Investigator: 
 
 
Joan Reibman, MD  
Professional Study Personnel: 
Bertram Bleck, PhD 
 
 
 
 
 
Mengling Liu, PhD 
 
 
 
 
 
Karen Carapetyan, MA 
 
 
 
 
 
Maria L Cotrina, PhD 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Specific Aims 
Asthma is a heterogeneous disease, and although much is understood about mechanisms of inflammation in allergic 
asthma, less is known about mechanisms of irritant-induced asthma (IA) (5, 7). Understanding the underlying similarities 
and differences in mechanisms of these two types of asthma will help focus current treatments and lead to development of 
new therapies. We have a longstanding NYU/Bellevue Asthma registry (NYUBAR), with a large population (N = 900) of 
asthma cases and controls, a program that has been housed at the CTSI (formerly GCRC). The destruction of the World 
Trade Center (WTC) resulted in massive dust, gas and fume exposures to local residents, workers and cleanup workers 
and individuals involved in rescue and recovery and adverse respiratory health effects of this disaster are reported more 
than 7 years after 9/11 (1-4). Many responders, as well as those exposed as residents or local workers, have developed IA, 
asthma that arises after a lag from an environmental exposure (1, 5, 6). The WTC Environmental Health Center (WTC 
EHC) is one of the three New York City (NYC) WTC Centers of Excellence and the only one that focuses on treatment 
and monitoring of local workers and residents. As such, it has a large population of individuals with irritant-induced 
asthma. We now propose to use participants from the NYUBAR and the WTC EHC to expand our knowledge of irritant 
and allergic asthma. Non-invasive studies allow for the assessment of airway inflammation, a non-specific response to 
environmental exposure and injury. Recent technologies also allow for assessment of microRNA (miRNA), small RNAs 
that regulate gene expression at the post-transcriptional level and thus serve as a pathway to regulation of inflammation. 
We will test the hypothesis that airway inflammation in irritant and allergic asthma may be similar, but result 
from divergent miRNA regulatory pathways expressed in sputum cells. These studies will provide preliminary data 
for future studies that will help identify biological pathways to categorize these asthma phenotypes and target future 
treatment interventions. We propose the following aims: 
 
Specific Aim 1: To test the hypothesis that irritant–induced asthma is characterized by elevated markers of inflammation 
in a manner similar to that described for allergic asthma.  
SA1.a We will identify and phenotype individuals with WTC dust cloud-exposed irritant-induced asthma (IA), individuals 
with allergic asthma (AA) and healthy unexposed individuals (Controls). 
SA1.b We will measure biomarkers of inflammation using noninvasive techniques for exhaled nitric oxide (ENO) and 8-
isoprostane from exhaled breath condensate (EBC) in WTC irritant-induced asthma (IA), individuals with allergic asthma 
(AA) and in healthy unexposed individuals (Controls). 
SA1.c We will measure inflammatory cells in induced sputum from individuals with IA, AA and Controls.  
 
Specific Aim 2: To test the hypothesis that the miRNA expression in airway epithelial cells will differ between IA, AA 
and Control individuals.  
SA2.a. We will isolate RNA from epithelial cells and inflammatory cells in induced sputum and assess the miRNA 
expression profile using microarray technology among individuals with irritant-induced asthma (IA), individuals with 
allergic asthma (AA) and healthy unexposed individuals (Controls). 
SA2.b. We will identify the mRNA targets of the differentially expressed miRNAs by using publicly available databases 
and correlate miRNA and mRNA expression.  


 
 
 
 
Kazeros, PI 
March 17, 2016 
2
 
Specific Aim 3: To test the hypothesis that levels of RNA of targets in human sputum cells will differ before and after the 
administration of inhaled corticosteroids in asthmatics (both IA, AA). The importance of this hypothesis in the 
characterization of irritant induced and allergic asthma is to further characterize the regulatory pathways expressed in 
sputum cells by identifying which inflammatory markers are steroid sensitive and steroid resistant.  
 
SA3.a. We will isolate RNA targets in discrete sputum cells from induced sputum in IA and AA individuals and compare 
levels before and after administration of inhaled corticosteroids. 
 
 
I. Background and Significance 
The destruction of the World Trade Center (WTC) resulted in massive dust, gas and fume exposures to local residents, 
workers and cleanup workers as well as to individuals involved in rescue and recovery. Local residents and workers were 
caught in the massive dust cloud as the buildings collapsed and a major proportion of individuals returned to work or their 
homes one week after the event when many office buildings and residences were only incompletely cleaned. Responders, 
local workers, and residents had potential for ongoing dust and fume exposure as indoor and outdoor WTC dust was 
recirculated and the fires burned for four months. Adverse respiratory health effects from this unprecedented disaster 
continue to be reported more than 7 years after 9/11 by the Centers of Excellence in New York (1, 3, 8). Data from 
firefighters who worked at the site demonstrate persistent respiratory symptoms and airway hyperresponsiveness one-year 
post 9/11 suggesting that they initially developed reactive airways disease (3). Data from subsequent studies of 
occupationally-exposed individuals, as well as those exposed as residents or local workers, suggest that many have also 
developed irritant-induced asthma, a form of asthma that can arise after a lag from an environmental exposure (1, 5, 6).  
Why study irritant-induced asthma (IA) 
Asthma, a complex syndrome with many clinical phenotypes, is characterized by a variable degree of airflow obstruction, 
airway hyperresponsiveness, and airway inflammation which results in symptoms of cough, shortness of breath, wheezing 
and chest tightness (9). Asthma is a heterogeneous disease with some patients having a clear allergic component, whereas 
others non-allergic asthma, some of which arises after irritant exposure (dust, gas, vapors or fumes). In the WTC 
population there is a high prevalence of asthma-like symptoms that developed after 9/11 which are treated with typical 
asthma medications despite the uncertainty of the underlying mechanisms and pathophysiology. We know from 
individuals under treatment in the WTC EHC that respiratory symptoms continue to persist more than 5 years after the 
event and although most individuals with respiratory symptoms have normal screening spirometry, there is a high rate of 
airway hyperresponsiveness consistent with irritant-induced asthma (1, 10). The underlying pathology and inflammatory 
response in irritant-induced is unclear and may differ from classic allergic asthma. Little is known about mechanisms of 
irritant-induced asthma. 
Why study non-invasive markers of inflammation 
There are ongoing efforts to define biomarkers to characterize airway inflammation in asthma. Presently the “gold 
standard” to measure airway inflammation and biomarkers of inflammation in humans involves direct airway sampling by 
bronchoscopy (11). The invasive nature of bronchoscopy makes it impractical and thus there is much interest in 
implementation of noninvasive methods. Approaches using exhaled nitric oxide (ENO), exhaled breath condensate (EBC) 
and induced sputum (IS) as surrogates of the lung have many advantages including sampling simplicity, subject comfort, 
analytical ease and safety for longitudinal studies. Few studies have been performed on non-invasive markers of irritant-
induced asthma. A recent report demonstrated a similar inflammatory cell profile of increased eosinophils and neutrophils 
in induced sputum in individuals with acute irritant-induced asthma from accidental inhalational injury compared to 
individuals with allergic occupational asthma (12).  
Exhaled NO increases in individuals with asthma, decreases with inhaled corticosteroid treatment and asthma control and 
correlates with eosinophil count in induced sputum and bronchoscopic sampling of airway inflammatory cells (13-19). 
ENO has been suggested as a more sensitive and reliable screening tool for the diagnosis of asthma than spirometry(11, 
20). Whether ENO levels in irritant-induced asthma are also elevated has not been studied therefore making this 
noninvasive biomarker an ideal start in characterizing the inflammatory response in this type of asthma.  
Exhaled breath condensate (EBC) is collected by the condensation of warm breath in a cold condensing tube. Levels of 
select markers in the condensed phase breakdown products reflect changes in the composition of the airway lining fluid. 
Biomarkers from EBC have been used to study asthma, chronic obstructive lung disease (COPD) and lung cancer (11, 21-
26). The application of EBC in human exposure studies is sparse. Markers of inflammation and oxidative stress that have 


 
 
 
 
Kazeros, PI 
March 17, 2016 
3
been identified in EBC include isoprostanes and pH (11)(27, 28). The measurement of EBC components (pH, and 8-
isoprostane) may help characterize injury in response to inhalational exposure from WTC fumes and dust.  
Induced sputum (IS) can be collected after inhalation of hypertonic saline. Measurements of IS, including cell counts, 
cytokines and chemokines have been used to assess lung inflammation extensively in individuals with asthma and COPD 
(11, 29, 30). Measurement of eosinophils in IS has been proposed as a diagnostic tool in occupational asthma (31, 32)(33-
36). IS contains exfoliated airway epithelial cells and is easily accessible and can serve as a direct measure of the lower 
respiratory tract. A simple inexpensive and effective method to limit the cellular heterogeneity of IS by enriching airway 
epithelial cells in a sample has been described by our group and others (37, 38). Only one study has examined the ability 
to both isolate and analyze microRNA from sputum in patients with lung cancer suggesting a need for further 
investigation of other lung diseases, particularly asthma (38).  
Why study microRNA and mRNA 
Studies suggest that the end result of airway injury, whether due to an allergic or irritant process is similar, and is 
characterized by airway eosinophilia and markers of “remodeling.” We and others have described that epithelial cells 
participate in the development and persistence of airway inflammation due to allergic and environmental exposures (39-
41).The biologic pathways that produce airway inflammation, although mediated by airway epithelial cells, may differ 
between the irritant and allergic asthma phenotypes. MicroRNAs (miRNAs) are a class of small non-protein-coding 
RNAs that posttranscriptionally regulate the expression of hundreds of target genes, thereby controlling a wide range of 
biological functions including cellular proliferation, differentiation and apoptosis (42, 43). MiRNAsdownregulate gene 
expression by suppression of translation or mRNA degradation. Identification of aberrant expression of miRNA may 
serve as valuable diagnostic or prognostic disease markers (42). Most studies to date have linked miRNAs to cancer 
because of their potential to function as tumor suppressors or oncogenes (44). Airway biopsies in individuals who smoke 
demonstrate miRNAs that are differentially expressed and inversely correlated with their predicted mRNA targets when 
compared to never smokers suggesting that miRNAs may be contributing to smoking-induced mRNA expression changes 
in airway epithelial cells (45). A recent study failed demonstrate a difference in miRNA expression from bronchial 
biopsies in mild asthmatics compared to those of healthy individuals but the possibility exists that differences may have 
been masked by the degree of cellular heterogeneity in bronchial biopsies (46). We propose to examine miRNA 
expression in epithelial cells derived from induced sputum to directly evaluate regulation of epithelial cell-directed lung 
inflammation.  
Our studies of irritant-induced asthma and allergic asthma using non-invasive methods (ENO, EBC and IS) and miRNA 
expression will characterize similarities and differences in these asthma phenotypes that can lead to improved biologic 
understanding of disease and potential targets for treatment interventions.  
Preliminary data 
WTC and asthma cohorts 
The WTC EHC is a program developed for the evaluation, treatment and monitoring of individuals with presumed WTC 
illness. Initiated as an unfunded community collaboration, it eventually received funding from the American Red Cross, 
and subsequently from the City of New York and most recently, federal funding from the National Institute of 
Occupational Safety and Health (NIOSH). The program targets local workers, residents and clean-up workers and as such, 
is the only one in NYC with this population. We have recently described the respiratory symptoms and findings in this 
population of individuals with diverse exposures Fig. 1 (1). Although most of the WTC EHC population had normal lung 
function, clear evidence of airway obstruction was noted in others (6 %), and some had reduced lung volume (20%). 
These findings suggest that exposures to WTC dust, fumes and gasses resulted in heterogeneous patterns of injury. To 
date, there are over 3,000 patients enrolled in the Bellevue component of the program, who have signed consent to be 
recontacted and have information available to us on our WTC EHC database. The NYUBAR has over 900 asthma cases 
and controls enrolled and phenotyped with consent to be recontacted. Thus the populations described are available to us.  
Irritant asthma in the WTC-exposed population 
Initial and persistent airway hyperresponsiveness has been well-described in the firefighter population involved in WTC 
rescue and response (3, 4, 47) To date, our WTC EHC data suggest that respiratory symptoms have persisted for more 
than 5 years after the event (1, 2). Our preliminary data show that among individuals in our WTC clinic with persistent 
lower respiratory symptoms that began after 9/11 and had normal spirometry (n=103), 50% demonstrate a positive 
methacholine challenge test. Individuals who were caught in the WTC dust clouds created as the towers collapsed on 9/11 
were at highest risk for a positive methacholine test (odds ratio = 4.1; p<0.006). These data suggest persistent irritant-
induced asthma in a large population of symptomatic WTC EHC patients.  
Non-invasive biomarkers in the WTC-exposed population 


 
 
 
 
Kazeros, PI 
March 17, 2016 
4
An early case-report of a firefighter with severe lung disease showed a remarkable eosinophilic pneumonia (48). An 
analysis of IS performed on firefighters 10 months after their WTC exposure showed an increase in inflammatory cells 
consisting of neutrophils and eosinophils(33). Few other studies of biomarkers have been performed in the WTC 
population. Our initial studies suggest that there is an increase in peripheral blood eosinophils in our patients who have 
persistent symptoms of wheeze (submitted as an abstract to American Thoracic Society). These data suggest that our 
patients with irritant-induced asthma may have an inflammatory response characterized by an increase in eosinophils.  
Isolated epithelial cells 
We will use isolated epithelial cells from IS. We have ample experience isolating bronchial epithelial cells from in vitro 
cultures of epithelial cells and dendritic cells. Fluorescence-activated cell sorting (FACS) analysis of these cells confirms 
our ability to separate human bronchial epithelial cells and to confirm their purity. We have experience with induced 
sputum and subsequent cell isolation, as described in our recent publication (Bleck et al., 2015) 
Microarray  
We propose microarray analysis of miRNA. We have previously published microarray analysis of alveolar macrophages 
obtained from smokers and nonsmokers (49) as well as those obtained from small airways (50). We have previously 
published microarray analysis demonstrating upregulation of MER tyrosine kinase (MERTK) apoptotic cell removal 
receptor in smokers and confirmed upregulation with RT-PCR (49).  
 
Research Design and Methods 
Specific Aim 1: To test the hypothesis that irritant–induced asthma is characterized by elevated markers of inflammation 
in a manner similar to that described for allergic asthma.  
SA1.a We will identify and phenotype individuals with WTC dust cloud-exposed irritant-induced asthma (IA), individuals 
with allergic asthma (AA) and healthy unexposed individuals (Controls) (N=90). 
We will study two asthma phenotypes and a control population. Our asthma phenotypes and control population will be 
defined in the following manner: 
IA: Patients will be recruited from the WTC EHC (N = 30) and will have WTC dust cloud exposure, New symptoms after 
9/11, and symptoms of wheeze and shortness of breath > 2x / week) in the 4 weeks before inclusion (persistent 
symptoms). 
AA: These patients will be recruited from the NYUBAR (N = 30) and advertising and will have asthma as defined by NIH 
guidelines (51), persistent symptoms, and absence of WTC dust exposure. 
Control: These patients will be recruited from the NYUBAR (N = 30) and advertising and will have no respiratory sx, no 
asthma diagnosis, no WTC dust exposure. Additional appropriate inclusion and exclusion criteria will be developed for 
each group including tobacco use, other lung disease, timing of inhaled corticosteroids etc.  
 
The study will entail two to three visits. On visit 1 (V1) all individuals will sign informed consent to participate in the 
study under an NYU IRB approved protocol. A questionnaire will be completed with standardized questions that include 
information on WTC exposures, demographics, presence and severity of respiratory symptoms, tobacco history and past 
medical history. Individuals will undergo spirometry with inhaled bronchodilator. Individuals will undergo methacholine 
challenge test (visit 1a) if they have normal spirometry or no bronchial hyperresponsiveness. On visit 2, individuals will 
return to undergo ENO, EBC and spirometry with pre and post bronchodilator maneuvers and induced sputum. Blood will 
be obtained for CBC with differential cell count, and assessment of total IgE and allergen-specific IgE. Blood will also be 
stored for future analysis of inflammatory markers. Based on our experience, we have had individuals unable to produce 
enough sputum and thus yield too small a number of cells. These individuals are excluded from data analysis. In addition, 
we also have subjects who are able to produce sputum and return for repeat sputum testing. 
  
SA1.b We will measure biomarkers of inflammation using noninvasive techniques for exhaled nitric oxide (ENO) and 8-
isoprostane from exhaled breath condensate (EBC) in WTC IA, AA and controls.  
We will use the handheld NIOX MINO® to measure ENO (Aerocrine, Sweden) (17, 18, 20, 52). The NIOX MINO® 
fulfills the ATS recommendations and provides reliable readings and is portable and we have experience with it (53). 
Most healthy individuals have ENO levels in the range of 5-35 ppb (some variation with age, gender, race/ethnicity) (54). 
Individuals with asthma have ENO levels that can range from 25-80 ppb and even higher (18, 61, 62). 
EBC will be collected using a commercially manufactured condenser R tube® (Respiratory Research, Inc., 
Charlottesville, VA) for this study because of its transportability and price. Again, we have experience with this 
technique. Measurements will be made according to published methods (28). Samples will be transported immediately 


 
 
 
 
Kazeros, PI 
March 17, 2016 
5
and frozen (-70˚ C) to improve stability. Our initial studies will focus on measurements of 8-isoprostane, a reliable 
biomarker of oxidative stress (24, 64). 8-isoprostane will be measured by commercially available ELISA. We will also 
measure pH of EBC (28, 67). EBC pH is reduced in individuals with asthma and COPD. Changes in pH may be used as 
diagnostic and monitoring modalities in inflammatory airway diseases (68). We will measure pH in degassed samples as 
described (69). Samples will also be stored for future studies of inflammatory mediators including cytokines/chemokine 
studies.  
SA1.c We will measure inflammatory cells in induced sputum from individuals with IA, AA and Controls.  
Increased numbers of eosinophils and neutrophils have been identified in IS in allergic asthma and occupational asthma 
and have been used to monitor asthma disease activity (31, 33, 34, 36, 70).  We propose to perform sputum induction, 
collection and processing according to established protocols on each individual (71, 72). IS will be collected after 
inhalation of hypertonic saline (3%, every 2minute intervals for up to 20minutes) with repeated monitoring of FEV1 as a 
safety measure. Sputum will be processed immediately to obtain total and cell differential cell counts (73). Our initial 
evaluationincluded analysis of cell counts, with measurements of eosinophils and neutrophils (Bleck et al., 2015). We 
consider significant sputum eosinophilia when sputum eosinophils are > 2% and significant sputum neutrophilia when 
sputum neutrophils are > 60%, cut off values, that have been described in the literature (12). We will also examine cell 
counts as continuous variables.  
Specific Aim 2: To test the hypothesis that the miRNA expression in airway epithelial cells will differ between IA, AA 
and Control individuals.  
SA2.a. We will isolate RNA from epithelial cells and inflammatory cells in induced sputum and nasal brushing and assess 
the miRNA and mRNA target genes and  expression profile using microarray technology among individuals with IA, AA 
and Controls. 
The use of sputum for molecular genetic analyses is limited by its cellular heterogeneity which includes 1% of bronchial 
epithelial cells and greater than 95% macrophages and neutrophils (74).  Enrichment of bronchial epithelial cells has been 
achieved by magnetic cell sorting (MACS) or fluorescent cell sorting techniques. We will perform positive selection of 
epithelial cells and inflammatory cells using magnetic bead preparations or fluorescent cell sorting techniques. Human 
epithelial cells will be purified from sputum according to the expression of CD326 (human epithelial antigen, HEA, or 
epithelial cell adhesion molecule (EpCAM) using the magnetic bead CD326 (EpCAM) Tumor Cell Enrichment and 
Detection Kit (Miltenyi Biotech). To confirm purity of our isolation for selected samples, epithelial cell identification will 
be demonstrated by intracellular pan-cytokeratin staining. Cells will immediately be placed in RNAprotect Cell Reagent 
(QIAGEN) and RNA isolated and fractionated into low molecular weight (LMW) and high molecular weight (HMW) 
using the miRNeasy mini kit (QIAGEN). RNA will be quantified and quality assessed. We will collaborate with the NYU 
Cancer Institute (CI) Genomics Services. We will have access to high-throughput genomic and computational 
methodologies specifically for microRNA. We will use the GeneChip® miRNA Array. Briefly, LMW RNA will be 
labeled with a FlashTag labeling kit (Genisphere, Inc.) according to the manufacturer’s protocol. Samples will be 
incorporated into a hybridization mix and applied to an affymetrix chip and scanned. Computational analysis with 
techniques such as principal component analysis will be performed with analytic services provided through the NYU CI 
Genomics core facility. Data will be log transformed and only miRNA replicates that are detected at levels of 2 standard 
deviations above the average background will be considered for further analysis. These studies will provide us with 
distinct miRNAs that may differ between IA, AA and controls, or IA and AA. If specific miRNAs are identified that are 
different between IA, AA and controls, or IA and AA, we will further validate expression of the miRNA using 
quantitative real time PCR.  
SA2.b. We will identify the mRNA targets of the differentially expressed miRNAs by using publicly available databases 
and correlate miRNA and mRNA expression.  
Most miRNA bind to target-3’UTR and function as translational repressors most often by promoting the degradation of 
mRNA. Each miRNA may regulate up to 200 target genes (42). We therefore propose to correlate patterns of miRNA 
changes in our defined populations, with mRNA expression arrays. HMW RNA from isolated epithelial cells will be used 
for hybridization to Affymetrix HG-U133 Plus 2.0 microarrays. Images will be analyzed in the core facility and data will 
be normalized as follows: (1) per array, by dividing the raw data by the 50th percentile of all measurements; and (2) per 
gene, by dividing the raw data by the median expression level for all the genes across all arrays in a data set. We will 
determine whether up or down-regulated mRNAs are potential targets for previously identified miRNAs.  
 
Specific Aim 3: To test the hypothesis that levels of RNA of targets in human sputum cells will differ before and after the 
administration of inhaled corticosteroids in asthmatics (both IA, AA). The rationale for this aim of the study is to 


 
 
 
 
Kazeros, PI 
March 17, 2016 
6
investigate which markers are steroid sensitive and which are steroid resistant which would provide information for 
therapeutic targeting in asthma. 
 
 
SA3.a. We will isolate RNA targets in discrete sputum cells from induced sputum in IA and AA individuals and compare 
levels before and after administration of inhaled corticosteroids. 
 
In this second phase of the study, (Phase II) we will enroll AA or IA individuals who have fit enrollment criteria for the 
phase I part of the study and participated in phase I of the study and were able to provide us with an acceptable sputum 
sample. These participants will be recontacted for participation in Phase II if they signed permission to be re-contacted. 
We will obtain a repeat sputum sample as baseline for the Phase II study. An inhaled corticosteroid (Fluticasone HFA 220 
mcg, 1 puff twice daily) and large volume spacer will be provided. Inhalation technique and a daily diary will be 
reviewed. Subjects will be instructed to use Fluticasone propionate 220 mcg q puff twice daily and to record their use in a 
written diary A repeat sputum sample will be obtained after 2 weeks of Fluticasone propionate use.  
 
Sputum samples will be processed for discrete cells and RNA levels (mRNA, miRNA) will be analyzed to understand the 
effect of ICS use on RNA expression in discrete sputum cell populations.  
 
Statistical Analysis: 
We were able to develop basic techniques and measurement from 10 patients in each group as initially proposed in the 
previous version of this protocol. We now propose to increase our enrollment from 30 to 50 subjects in each group based 
on the recruitment and enrollment data of about 30% of screen failures (33 subjects screen failed out of 108 total). In 
addition, there have been subjects who have not been able to produce sputum or provide adequate number of sputum cells 
for analysis (38 subjects out of 65 accrued gave adequate sputum samples). This represents about 60% success rate for 
sample quality. Therefore, we will need to increase our enrollment numbers to achieve an n=30 per group.  
Power Analysis: Based on a similar rate of screen failures and inadequate samples, the analysis is now based on 
50patients in each group. Our group sample sizes of 50 and 50 achieve 90% power to detect a difference of 0.73 given the 
null hypothesis that the two-group difference is 0 and the estimated group standard deviations are 1.0 and 1.0, with a 
significance level (alpha) of 0.05 using a two-sided two sample t-test. We will work with Dr. Mengling Liu, our 
biostatistician for our statistical analysis. 
 
For ENO and EBC analyses, we will express data as means + SD for normally distributed variables. For variables that are 
not normally distributed we will use medians and the interquartile range. We will use statistical analysis including 
unpaired t tests, Kruskal-Wallis test, and regression coefficients as appropriate. P values of < 0.05 will be considered 
significant. Differential expression of miRNA and hierarchical clustering will be performed with the genomics core. 
Statistics using t tests or algorithms designed specifically for data mining and biochip interpretation will be used. Pathway 
analyses will also be performed by the genomics computational core.  
Anticipated results and alternative approaches 
We will continue to recruit patients with WTC exposure or with asthma based on our experience in our asthma registry or 
our WTC clinic, in which most people are willing and anxious to participate in any study that might help characterize their 
symptoms.  
We plan to also submit an advertisement to the IRB for approval and to use research match (researchmatch.org) a national 
registry which is a secure, web-based approach as a recruitment method, and will post the advertisement. 
We have chosen to use individuals with WTC dust cloud exposure as our model of initial high dose exposure to enhance 
our ability to identify patients with irritant asthma. We suspect that other more prolonged exposures also induce irritant 
asthma, but our previous studies and those of other investigators suggest that this exposure provides the highest risk.   
We have proposed a positive selection strategy for isolation of bronchial epithelial cells from IS. The possibility exists 
that this technique may provide too low a yield of cells. Should that be the case, we can use a negative selection strategy 
using anti-CD14 and anti-CD16 antibody beads to specifically deplete macrophages and neutrophils respectively (37). We 
may also need to obtain duplicate sputum samples from patients, i.e. have patients return for a second sputum induction.  
Our initial analyses will be between our healthy controls and asthma in general (AI and AA) and subsequently between 
each type of asthma (AI and AA) to optimize our ability to detect differences in sputum markers and miRNA. The 


 
 
 
 
Kazeros, PI 
March 17, 2016 
7
possibility exists that we will find no difference between measurements of markers of inflammation in exhaled breath 
between AI and AA. Should that be the case, we will have to examine whether our pilot study was powered to identify a 
small difference.  
We are basing our miRNA and mRNA studies on those that have been performed in epithelial cells derived from cigarette 
smokers (45). These studies have shown a correlation between mRNA and miRNa expression and thus there is precedent 
for our studies. Because of the high price of each of these chips, and the cost of core analysis time, we initially propose to 
use 5 subjects for each condition to obtain a global pattern scale. These studies will subsequently be extended to get a 
reasonable statistical power.  
From our pilot studies and recent publication we will continue to investigate mechanisms of asthma and irritant-induced 
asthma. The studies are high risk but have potential for high yield, and will provide data for subsequent NIH applications 
for replication and further evaluation for a junior faculty member. In addition, they have potential to provide information 
that could help target specific interventions for asthma of different phenotypes as we proposed in phase II of the study. As 
such, they fit the mandate of the CTSI grants.  
 
II. Characteristics of the Research Population 
 
1. Number of Subjects: 90 (this includes the total number of subjects enrolled in phase I and II of the study that are able 
to produce sputum)  
[Number of subjects in Phase II: 40] 
     The estimated number of subjects to be enrolled (signed consent, including screen failures) is 300. 
 
2.  Gender of Subjects: Men and Women (50 % each) 
 
3.  Age of Subjects: 18-80 years of age 
 
4.  Racial and Ethnic Origin: The sample population of approximately 80 participants is expected to include a distribution 
of subjects by race/ethnicity in relation to our local population which includes a high percentage of minority subjects 
(36% White, 35% Black, 50% Hispanic) 
 
5.  Inclusion Criteria: 
Inclusion for the WTC population with Irritant-Induced Asthma (IA):  
Patients will be recruited from the WTC EHC and will have WTC dust cloud exposure, new symptoms after 9/11, 
and symptoms of wheeze and shortness of breath (> 2x / week) in the 4 weeks before inclusion (persistent 
symptoms).We will be recruiting new subjects for phase II of the study but participants who will have completed  
Phase I of the study and were able to produce adequate sputum samples may also be contacted for Phase II. 
 
Inclusion for Allergic Asthma population(AA): Patients will be recruited from the NYUBAR or advertisement 
and will have asthma as defined by NIH guidelines, persistent symptoms, absence of WTC dust exposure. 
Participants who will have completed the Phase I of the study and were able to produce adequate sputum samples 
may also be contacted for Phase II. 
  
Inclusion of Control Population: Patients will be recruited from the NYUBAR and will have no respiratory 
symptoms, no asthma diagnosis, no WTC dust exposure, no current tobacco use, < 5 p-y history of tobacco use, 
and normal spirometry with no bronchodilator response and negative methacholine challenge in past 6 months. 
 
Additional inclusion criteria for individuals with IA and AA:> 18 years of age, current nonsmoker, < 5 pack year 
(p-y) history of tobacco use, spirometry in the past 6 months or on day of evaluation with a bronchodilator 
response of ≥ 12% and 200 ml improvement in FEV1 or a positive methacholine challenge test (decrease in FEV1 
≥ 20% (PC20) after inhalation of < 16 mg/ml of methacholine), inhaled corticosteroid use in previous 1 month or 
more will be allowed. 
 
Inclusion criteria for Phase II 
Successfully completed Phase I 
Has asthma according to Phase I diagnostic criteria 


 
 
 
 
Kazeros, PI 
March 17, 2016 
8
Signed consent to be re-contacted 
 
6.  Exclusion Criteria: 
Exclusion criteria: current smoker, pulmonary diseases such as Chronic Obstructive Pulmonary Disease (COPD) 
or Interstitial Lung Disease , cardiac disease, inability to perform lung function or other maneuvers, upper 
respiratory tract infection within the last 4 weeks, FEV1 <60% predicted normal pre-bronchodilator, oral 
corticosteroid treatment within the last 4 weeks 
 
7. Vulnerable Subjects: No vulnerable subjects will be part of this study. 
 
III. Methods and Procedures 
 
1. Methods and Procedures: 
We will use the handheld NIOX MINO® to measure ENO (Aerocrine, Sweden) (17, 18, 20, 52). The NIOX MINO® 
fulfills the ATS recommendations and provides reliable readings and is portable and we have experience with it (53). 
Most healthy individuals have ENO levels in the range of 5-35 ppb (some variation with age, gender, race/ethnicity) (54). 
Individuals with asthma have ENO levels that can range from 25-80 ppb and even higher (18, 61, 62). 
 
We propose to perform sputum induction, collection and processing according to established protocols on each individual 
(71, 72). IS will be collected after inhalation of hypertonic saline (3%, every 4 minute intervals for up to 12 minutes) with 
repeated monitoring of FEV1 as a safety measure. Sputum will be processed immediately to obtain total and cell 
differential cell counts (73). Our initial evaluations will include analysis of cell counts, with measurements of eosinophils 
and neutrophils. We consider significant sputum eosinophilia when sputum eosinophils are > 2% and significant sputum 
neutrophilia when sputum neutrophils are > 60%, cut off values, that have been described in the literature (12). We will 
also examine cell counts as continuous variables.  
 
The use of sputum for molecular genetic analyses is limited by its cellular heterogeneity which includes 1% of bronchial 
epithelial cells and greater than 95% macrophages and neutrophils (74).  Enrichment of bronchial epithelial cells has been 
achieved by magnetic cell sorting (MACS). We will perform positive selection of discrete sputum cell populations 
including epithelial cells and dendritic cells using magnetic bead preparations or via flow cytometry. Human epithelial 
cells will be purified from sputum according to the expression of CD326 (human epithelial antigen, HEA, or epithelial 
cell adhesion molecule (EpCAM) using the magnetic bead CD326 (EpCAM) Tumor Cell Enrichment and Detection Kit 
(Miltenyi Biotech). To confirm purity of our isolation for selected samples, epithelial cell identification will be 
demonstrated by intracellular pan-cytokeratin staining. Cells will immediately be placed in RNAprotect Cell Reagent 
(QIAGEN) and RNA isolated and fractionated into low molecular weight (LMW) and high molecular weight (HMW) 
using the miRNeasy mini kit (QIAGEN). RNA will be quantified and quality assessed. We will collaborate with the NYU 
Cancer Institute (CI) Genomics Services. We will have access to high-throughput genomic and computational 
methodologies specifically for microRNA. We will use the GeneChip® miRNA Array. Briefly, LMW RNA will be 
labeled with a FlashTag labeling kit (Genisphere, Inc.) according to the manufacturer’s protocol. Samples will be 
incorporated into a hybridization mix and applied to an affymetrix chip and scanned. Computational analysis with 
techniques such as principal component analysis will be performed with analytic services provided through the NYU CI 
Genomics core facility. Data will be log transformed and only miRNA replicates that are detected at levels of 2 standard 
deviations above the average background will be considered for further analysis. These studies will provide us with 
distinct miRNAs that may differ between IA, AA and controls, or IA and AA. If specific miRNAs are identified that are 
different between IA, AA and controls, or IA and AA, we will further validate expression of the miRNA using 
quantitative real time PCR.  
 
We will obtain epithelial cells from the lining of the nose using a technique called nasal brushing. This is a 
simple, nonsurgical procedure and is well tolerated by most people. We will gently brush the inside of the 
lateral wall of each nostril with a small, delicate, sterile brush. This procedure takes about 5 seconds for each 
nostril. 
 


 
 
 
 
Kazeros, PI 
March 17, 2016 
9
We also propose to correlate patterns of miRNA changes in our defined populations, with mRNA expression arrays. 
HMW RNA from isolated epithelial cells will be used for hybridization to Affymetrix HG-U133 Plus 2.0 microarrays. 
Images will be analyzed in the core facility and data will be normalized as follows: (1) per array, by dividing the raw data 
by the 50th percentile of all measurements; and (2) per gene, by dividing the raw data by the median expression level for 
all the genes across all arrays in a data set. We will determine whether up or down-regulated mRNAs are potential targets 
for previously identified miRNAs.  
 
Phase II 
Only subjects with Irritant asthma or Allergic asthma who have successfully completed the first phase of the study and 
have signed consent to be re-contacted will be recruited for the second phase of the study (Phase II). Phase II will involve 
two visits over a 2 week time period. For Phase II, we will first obtain sputum samples for a baseline analysis of cells. 
Study subjects will be provided with an inhaled corticosteroid, Fluticasone propionate HFA 220 mcg, 2 puffs twice a day 
(Treatment), or no treatment (No treatment). Fluticasone HFA will be provided by the study physician through the CTSI 
and the Bellevue Pharmacy. Treatment cases will be instructed to self administer the medication with a large volume 
spacer that will be provided to them. Inhaler teaching will be performed by study physician or coordinator at the time that 
the subject is provided the inhaler. Treatment Cases and No treatment controls will be provided a diary to record 
fluticasone inhaler. Treatment cases and No treatment controls will be allowed to use their usual rescue bronchodilator 
(albuterol) on an as needed basis. Treatment Cases and No treatment controls will return after 2 weeks for a repeat sputum 
induction.  
 
2. Data Analysis and Data Monitoring: 
We will start with 10 patients in each group to develop the basic techniques and measurements. Our group sample sizes of 
10 and 10 achieve 80% power to detect a difference of 1.3 given the null hypothesis that the two-group difference is 0 and 
the estimated group standard deviations are 1.0 and 1.0, with a significance level (alpha) of 0.05 using a two- sided two-
sample t-test. Based on results from these studies we will work with Dr. Mengling Liu, our biostatistician and the NYU CI 
Genomics core for our statistical analysis. We put in a small number of patients as this is a pilot study to develop 
information that will allow for a subsequent appropriate power analysis.  
For ENO and EBC analyses, we will express data as means + SD for normally distributed variables. For variables that are 
not normally distributed we will use medians and the interquartile range. We will use statistical analysis including 
unpaired t tests, Kruskal-Wallis test, and regression coefficients as appropriate. P values of < 0.05 will be considered 
significant. Differential expression of miRNA and hierarchical clustering will be performed with the genomics core. 
Statistics using t tests or algorithms designed specifically for data mining and biochip interpretation will be used. Pathway 
analyses will also be performed by the genomics computational core.  
For Phase II, the primary outcome is the change in TSLP gene expression in epithelial cells after 2 weeks of treatment of 
inhaled corticosteroid compared to no treatment. We hypothesize that a significant reduction in the gene expression will 
be observed in the treatment group of inhaled corticosteroid compared with the untreated group. The difference between 
two randomized groups will be compared using the two-group t-test if data are normally distributed, and the 
nonparametric Wilcoxon test will be used for skewed data. The effect of inhaled corticosteroids on newly identified 
epithelial targets (gene expression array) will also be examined. This is an exploratory goal and will be performed after 
cell stabilization and short-term culture cells to insure appropriate RNA quality.  
Sample size and power:  Group sample sizes of 15 and 15 achieve 80% power to detect a difference of 1.3SD against the 
null hypothesis that both group means are equal with a significance level (alpha) of 0.01 using a two-sided two-sample t-
test assuming equal variance. 
 
 
3: Data Storage and Confidentiality: 
Care will be taken to ensure confidentiality. Research records will be held in locked cabinets or secure storage rooms. All 
transmission of data to the coordinating center for analysis will be by study ID code only. 
Sample specimens will be stored for up to 10 years for future use or until the end of the study.  Participants will be able to 
decide if they want any of their specimens stored for research done at a later time.  
 
IV. Risk/Benefit Assessment 
 


 
 
 
 
Kazeros, PI 
March 17, 2016 
10
1. Risk: Minimal Risk 
Spirometry, ENO, IOS and EBC. 
Participation in these studies will pose minimal risk of physical or psychological harm.  Spirometry, impedance 
oscillometry, ENO and exhaled breath condensate collection are minimally invasive.   
 
Methacholine testing: MCT has been performed with no serious side effects. Participants may develop transient side 
effects such as cough, mild dyspnea, and chest tightness although many experience no symptoms. Inhaled methacholine 
can cause bronchoconstriction and safety of each subject is considered prior to testing. A physician and the medical 
director of the pulmonary lab are trained to treat acute bronchospasm, including appropriate use of resuscitation 
equipment, and a physician is present during the procedure. Our pulmonary function lab in Bellevue Hospital is well 
trained in performing MCT in the clinical and research setting. If bronchospasm occurs appropriate medications will be 
administered as per the lab protocol (oxygen, nebulizers, subcutaneous epinephrine). If a participant in the study develops 
an acute asthma episode during any part of this procedure, the procedure will be stopped and albuterol will be 
administered to improve breathing. The bronchodilator will be provided by a physician involved in the study and the 
participant will be monitored until their breathing has returned to its baseline.  
 
For all women of childbearing ability, a pregnancy test will be performed before the methacholine challenge 
study. Pregnant subjects will be excluded from the study. 
 
Induced sputum: Sputum induction involves inhalation of hypertonic saline aerosol, a stimulus known to cause 
bronchoconstriction in asthmatic subjects. To ensure participant safety during sputum induction we will follow the 
following protocol: procedure will be performed in a hospital setting by highly trained and experienced staff, 
administration of a bronchodilator can be given to inhibit hypertonic saline-induced bronchoconstriction, we will limit the 
sputum induction to 20 minutes and monitor the participant’s symptoms closely with repeat spirometry 
every 2 minutes after induction. If bronchoconstriction occurs we will follow the protocol discussed under MCT.  
 
Nasal brushing: Nasal brushing. We do not anticipate any untoward risk of performing this procedure in normal 
individuals of in those with asthma. This procedure is not usually painful but some people experience a moment of 
discomfort for about 5 seconds during the brushing. There may be minor irritation such as reflex tearing from your eyes, 
or sneezing. There may be a rare chance that there is a small amount of bleeding as if you had a scrape. These possible 
discomforts resolve on their own. 
 
Administration of inhaled corticosteroid –We are providing high dose inhaled corticosteroid (Fluticasone propionate HFA 
220 mcg) for a short (2 week) period of time. This medication poses no more than minimal risk to research subjects and 
the risk is minimized by the short treatment period and the use of a large volume spacer. This is an approved treatment for 
asthma. The most common symptoms experienced by patients who have taken fluticasone are: upper respiratory 
infections (25-29%), hoarseness or dysphonia (1-4%), inflammation of throat, nose or sinuses (1-12%),  oral candidiasis 
(2%) , nausea and vomiting (1%), other gastrointestinal symptoms (1-2%), and muscle or bone pain (3%). Subjects will be 
instructed to on avoidance of oral candidiasis by rinsing their mouth after administration of the medication.  
 
 
2. Protection against Risks: 
All participants will be provided with a contact physician capable of providing sources of appropriate medical or 
professional intervention in the event of adverse effects to the subject. This person will be available on a 24 hour contact 
basis through the New York University/Bellevue Hospital Asthma Clinic hot line.  
 
3: Potential Benefit to the Subjects: 
 
The subject will be informed that the proposed research will be of no immediate benefit to him/her but may help advance 
our knowledge of irritant and allergic asthma and help others in the future.  
 
V. Investigator Qualifications and Experience 
 


 
 
 
 
Kazeros, PI 
March 17, 2016 
11
Joan Reibman, MD. Dr. Reibman is a Professor of Medicine and Environmental Medicine and will oversee in procedure, 
analysis and will be responsible for overall analysis and presentation of data. 
 
Angeliki Kazeros, MD, Dr. Kazeros is a junior faculty member in the Division of Pulmonary, Critical Care and Sleep 
Medicine. She will supervise patient recruitment, obtain consent, and oversee the noninvasive procedures. 
 
Bertram Bleck, PhD. Dr. Bleck will oversee cell studies including cell analysis, epithelial cell isolation, FACS analysis 
and will perform ELISAs.  
 
Karen Carapetyan, M.A. Ms. Carapetyan is a clinical coordinator for the asthma and WTC studies. She will help with 
recruitment of patients, maintenance of IRB forms and in the performance of non-invasive procedures.  
 
Maria L Cotrina, Ph.D. is a clinical coordinator for the asthma and WTC studies. She will help with recruitment of patient, 
maintenance of IRB forms and in the performance of non-invasive procedures. 
 
Mengling Liu, PhD. Dr. Liu is a biostatistician in the Department of Environmental Medicine. She currently works with 
the WTC EHC and will now help in analysis of biologic data.  
 
 
VI. Subject Identification, Recruitment and Consent/Assent 
 
1. Method of Subject Identification and Recruitment 
The WTC-EHC clinic and the asthma clinic at Bellevue will be the recruitment source. The physicians will screen the 
patients and refer them to the study. 
 
Investigators will also reach out to potential participants via ResearchMatch.Org, which is a national registry developed 
by institutions affiliated with the Clinical and Translational Science Awards (CTSA). This provides a secure, web-based 
approach to address a key barrier to advancing research: participant recruitment. We also have an IRB-approved 
advertisement and will post this at various sites.  
 
2. Process of Consent 
The consent form will be written and oral. When it is needed the services of a translator will be provided. Attached is a 
copy of the informed consent that will be presented to subjects.A second consent form will be provided to asthmatic 
patients who have completed Phase I of the study and wish to continue on to Phase II of the study with administration of 
inhaled corticosteroid.  Attached is the copy of the consent form for phase II of the study that will be presented to 
subjects. The consent process will take place in the CTSI rooms. Confidentiality and privacy of the patients will be 
maintained. 
The 
patient 
will 
have 
the 
opportunity 
to 
ask 
questions 
to 
the 
physician. 
The Clinical Coordinator and the physician will obtain consent from the subjects. Time will be allotted for a period of 
Question and Answer in which the patient will have the opportunity to have all their concerns addressed by the physician 
regarding the study. Primary recruitment will be with patients who understand English. The questionnaires are only 
available 
in 
English, 
and 
if 
required 
translations 
services 
will 
be 
requested.  
 
3. Subject Capacity: 90 subjects (accrued and able to be included in the data analysis) 
 
4. Subject /Representative Comprehension: Time will be allotted for a period of Question and Answer in which the patient 
will have the opportunity to have all their concerns addressed by the physician regarding the study. 
 
5. Debriefing Procedures: no information will be withheld from the subject since no psychological studies will be 
performed. 
 
6. Consent Forms: 
All participants will have consent obtained according to the NYU-IRB consent form. Consent will be sought by a clinical 
coordinator and signed consent forms will be maintained for all subjects. Complete information on the procedure and 
potential risks of the procedure will be provided to the patient. The participant will be informed that the procedure will be 
of no immediate benefit to him/her but may help advance our knowledge of mechanisms of airway inflammation 
 
7. Documentation of Consent: All consents will be signed. 


 
 
 
 
Kazeros, PI 
March 17, 2016 
12
 
8. Costs to the Subject: There will be no cost to the subject.  
 
9. Payment for Participation: The subject will receive $50.00 per visit. 
 
 
 
References 
 
1. 
Reibman J, Liu M, Cheng Q, Liautaud S, Rogers L, Lau S, Berger KI, Goldring RM, Marmor M, Fernandez-Beros 
ME, et al. Characteristics of a residential and working community with diverse exposure to world trade center dust, 
gas, and fumes. J Occup Environ Med 2009;51:534-541. 
2. 
Reibman J, Lin S, Hwang SA, Gulati M, Bowers JA, Rogers L, Berger KI, Hoerning A, Gomez M, Fitzgerald EF. 
The world trade center residents' respiratory health study: New-onset respiratory symptoms and pulmonary function. 
Environ Health Perspect 2005;113:406-411. 
3. 
Prezant DJ, Weiden M, Banauch GI, McGuinness G, Rom WN, Aldrich TK, Kelly KJ. Cough and bronchial 
responsiveness in firefighters at the world trade center site. N Engl J Med 2002;347:806-815. 
4. 
Banauch GI, Dhala A, Alleyne D, Alva R, Santhyadka G, Krasko A, Weiden M, Kelly KJ, Prezant DJ. Bronchial 
hyperreactivity and other inhalation lung injuries in rescue/recovery workers after the world trade center collapse. 
Crit Care Med 2005;33:S102-106. 
5. 
Mapp CE, Boschetto P, Maestrelli P, Fabbri LM. Occupational asthma. Am J Respir Crit Care Med 2005;172:280-
305. 
6. 
de la Hoz RE, Shohet MR, Chasan R, Bienenfeld LA, Afilaka AA, Levin SM, Herbert R. Occupational toxicant 
inhalation injury: The world trade center (wtc) experience. Int Arch Occup Environ Health 2008;81:479-485. 
7. 
Maestrelli P, Boschetto P, Fabbri LM, Mapp CE. Mechanisms of occupational asthma. J Allergy Clin Immunol 
2009;123:531-542; quiz 543-534. 
8. 
Herbert R, Moline J, Skloot G, Metzger K, Baron S, Luft B, Markowitz S, Udasin I, Harrison D, Stein D, et al. The 
world trade center disaster and the health of workers: Five-year assessment of a unique medical screening program. 
Environ Health Perspect 2006;114:1853-1858. 
9. 
Busse WW, Lemanske RF, Jr. Asthma. N Engl J Med 2001;344:350-362. 
10. Tonorezos E, Caplan-Shaw C., Cheng Q, Liu M, Fernandez-Beros M, Kazeros A, Segal L, Berger K, Goldring R, 
Reibman J. Methacholine challenge testing in a population with world trade center dust and fume exposure and 
persistent respiratory symptoms. American Thoracic Society 2009. 
11. Murugan A, Prys-Picard C, Calhoun WJ. Biomarkers in asthma. Curr Opin Pulm Med 2009;15:12-18. 
12. Malo JL, L'Archeveque J, Castellanos L, Lavoie K, Ghezzo H, Maghni K. Long-term outcomes of acute irritant-
induced asthma. Am J Respir Crit Care Med 2009;179:923-928. 
13. Kharitonov SA, Yates D, Robbins RA, Logan-Sinclair R, Shinebourne EA, Barnes PJ. Increased nitric oxide in 
exhaled air of asthmatic patients. Lancet 1994;343:133-135. 
14. Kharitonov SA, Yates D, Springall DR, Buttery L, Polak J, Robbins RA, Barnes PJ. Exhaled nitric oxide is increased 
in asthma. Chest 1995;107:156S-157S. 
15. Silkoff P. Exhaled nitric oxide as a diagnostic tool. Am J Respir Crit Care Med 2003;167:665-666; author reply 665-
666. 
16. Silkoff PE, Trudeau JB, Gibbs R, Wenzel S. The relationship of induced-sputum inflammatory cells to bal and 
biopsy. Chest 2003;123:371S-372S. 
17. Smith AD, Taylor DR. Is exhaled nitric oxide measurement a useful clinical test in asthma? Curr Opin Allergy Clin 
Immunol 2005;5:49-56. 
18. Smith AD, Cowan JO, Brassett KP, Herbison GP, Taylor DR. Use of exhaled nitric oxide measurements to guide 
treatment in chronic asthma. N Engl J Med 2005;352:2163-2173. 
19. Carraro S, Rusalen F, Stefani S, Zanconato S, Baraldi E. [measurement of exhaled nitric oxide]. Minerva Pediatr 
2009;61:99-102. 
20. Smith AD, Cowan JO, Filsell S, McLachlan C, Monti-Sheehan G, Jackson P, Taylor DR. Diagnosing asthma: 
Comparisons between exhaled nitric oxide measurements and conventional tests. Am J Respir Crit Care Med 
2004;169:473-478. 
21. Corradi M, Pesci A, Casana R, Alinovi R, Goldoni M, Vettori MV, Cuomo A. Nitrate in exhaled breath condensate 
of patients with different airway diseases. Nitric Oxide 2003;8:26-30. 


 
 
 
 
Kazeros, PI 
March 17, 2016 
13
22. Corradi M, Rubinstein I, Andreoli R, Manini P, Caglieri A, Poli D, Alinovi R, Mutti A. Aldehydes in exhaled breath 
condensate of patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2003;167:1380-1386. 
23. Montuschi P, Corradi M, Ciabattoni G, Nightingale J, Kharitonov SA, Barnes PJ. Increased 8-isoprostane, a marker 
of oxidative stress, in exhaled condensate of asthma patients. American journal of respiratory and critical care 
medicine 1999;160:216-220. 
24. Montuschi P. Analysis of exhaled breath condensate in respiratory medicine: Methodological aspects and potential 
clinical applications. Therapeutic advances in respiratory disease 2007;1:5-23. 
25. Montuschi P, Barnes P, Roberts LJ, 2nd. Insights into oxidative stress: The isoprostanes. Curr Med Chem 
2007;14:703-717. 
26. Carraro S, Corradi M, Zanconato S, Alinovi R, Pasquale MF, Zacchello F, Baraldi E. Exhaled breath condensate 
cysteinyl leukotrienes are increased in children with exercise-induced bronchoconstriction. The Journal of allergy 
and clinical immunology 2005;115:764-770. 
27. Accordino R, Visentin A, Bordin A, Ferrazzoni S, Marian E, Rizzato F, Canova C, Venturini R, Maestrelli P. Long-
term repeatability of exhaled breath condensate ph in asthma. Respir Med 2008;102:377-381. 
28. Hunt J. Exhaled breath condensate: An evolving tool for noninvasive evaluation of lung disease. J Allergy Clin 
Immunol 2002;110:28-34. 
29. Malerba M, Ragnoli B, Radaeli A, Tantucci C. Usefulness of exhaled nitric oxide and sputum eosinophils in the 
long-term control of eosinophilic asthma. Chest 2008;134:733-739. 
30. Kim HB, Kim CK, Iijima K, Kobayashi T, Kita H. Protein microarray analysis in patients with asthma: Elevation of 
the chemokine parc/ccl18 in sputum. Chest 2009;135:295-302. 
31. Lemiere C, Pizzichini MM, Balkissoon R, Clelland L, Efthimiadis A, O'Shaughnessy D, Dolovich J, Hargreave FE. 
Diagnosing occupational asthma: Use of induced sputum. Eur Respir J 1999;13:482-488. 
32. Lemiere C, Walker C, O'Shaughnessy D, Efthimiadis A, Hargreave FE, Sears MR. Differential cell counts in sputum 
in respiratory epidemiology: A pilot study. Chest 2001;120:1107-1113. 
33. Fireman EM, Lerman Y, Ganor E, Greif J, Fireman-Shoresh S, Lioy PJ, Banauch GI, Weiden M, Kelly KJ, Prezant 
DJ. Induced sputum assessment in new york city firefighters exposed to world trade center dust. Environ Health 
Perspect 2004;112:1564-1569. 
34. Fireman E, Lerman Y, Stark M, Schwartz Y, Ganor E, Grinberg N, Frimer R, Landau DA, Zilberberg M, Barenboim 
E, et al. Detection of occult lung impairment in welders by induced sputum particles and breath oxidation. Am J Ind 
Med 2008;51:503-511. 
35. Dragonieri S, Musti M, Izzo C, Esposito LM, Foschino Barbaro MP, Resta O, Spanevello A. Sputum induced 
cellularity in a group of traffic policemen. Sci Total Environ 2006;367:433-436. 
36. Deykin A, Lazarus SC, Fahy JV, Wechsler ME, Boushey HA, Chinchilli VM, Craig TJ, Dimango E, Kraft M, Leone 
F, et al. Sputum eosinophil counts predict asthma control after discontinuation of inhaled corticosteroids. J Allergy 
Clin Immunol 2005;115:720-727. 
37. Qiu Q, Todd NW, Li R, Peng H, Liu Z, Yfantis HG, Katz RL, Stass SA, Jiang F. Magnetic enrichment of bronchial 
epithelial cells from sputum for lung cancer diagnosis. Cancer 2008;114:275-283. 
38. Xie Y, Todd NW, Liu Z, Zhan M, Fang H, Peng H, Alattar M, Deepak J, Stass SA, Jiang F. Altered mirna 
expression in sputum for diagnosis of non-small cell lung cancer. Lung Cancer 2009. 
39. Bleck B, Tse DB, Curotto de Lafaille MA, Zhang F, Reibman J. Diesel exhaust particle-exposed human bronchial 
epithelial cells induce dendritic cell maturation and polarization via thymic stromal lymphopoietin. J Clin Immunol 
2008;28:147-156. 
40. Bleck B, Tse DB, Jaspers I, Curotto de Lafaille MA, Reibman J. Diesel exhaust particle-exposed human bronchial 
epithelial cells induce dendritic cell maturation. J Immunol 2006;176:7431-7437. 
41. Holgate ST, Davies DE. Rethinking the pathogenesis of asthma. Immunity 2009;31:362-367. 
42. Miller RL, Ho SM. Environmental epigenetics and asthma: Current concepts and call for studies. Am J Respir Crit 
Care Med 2008;177:567-573. 
43. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed rnas. 
Science 2001;294:853-858. 
44. Calin GA, Croce CM. Microrna signatures in human cancers. Nat Rev Cancer 2006;6:857-866. 
45. Schembri F, Sridhar S, Perdomo C, Gustafson AM, Zhang X, Ergun A, Lu J, Liu G, Bowers J, Vaziri C, et al. 
Micrornas as modulators of smoking-induced gene expression changes in human airway epithelium. Proc Natl Acad 
Sci U S A 2009;106:2319-2324. 


 
 
 
 
Kazeros, PI 
March 17, 2016 
14
46. Williams AE, Larner-Svensson H, Perry MM, Campbell GA, Herrick SE, Adcock IM, Erjefalt JS, Chung KF, 
Lindsay MA. Microrna expression profiling in mild asthmatic human airways and effect of corticosteroid therapy. 
PLoS One 2009;4:e5889. 
47. Banauch GI, Alleyne D, Sanchez R, Olender K, Cohen HW, Weiden M, Kelly KJ, Prezant DJ. Persistent 
hyperreactivity and reactive airway dysfunction in firefighters at the world trade center. Am J Respir Crit Care Med 
2003;168:54-62. 
48. Rom WN, Weiden M, Garcia R, Yie TA, Vathesatogkit P, Tse DB, McGuinness G, Roggli V, Prezant D. Acute 
eosinophilic pneumonia in a new york city firefighter exposed to world trade center dust. Am J Respir Crit Care Med 
2002;166:797-800. 
49. Kazeros A, Harvey BG, Carolan BJ, Vanni H, Krause A, Crystal RG. Overexpression of apoptotic cell removal 
receptor mertk in alveolar macrophages of cigarette smokers. Am J Respir Cell Mol Biol 2008;39:747-757. 
50. Vanni H, Kazeros A, Wang R, Harvey BG, Ferris B, De BP, Carolan BJ, Hubner RH, O'Connor TP, Crystal RG. 
Cigarette smoking induces overexpression of a fat-depleting gene azgp1 in the human. Chest 2009;135:1197-1208. 
51. Expert panel report 3 (epr-3): Guidelines for the diagnosis and management of asthma-summary report 2007. J 
Allergy Clin Immunol 2007;120:S94-138. 
52. Pijnenburg MW, Bakker EM, Hop WC, De Jongste JC. Titrating steroids on exhaled nitric oxide in children with 
asthma: A randomized controlled trial. Am J Respir Crit Care Med 2005;172:831-836. 
53. Hemmingsson T, Linnarsson D, Gambert R. Novel hand-held device for exhaled nitric oxide-analysis in research and 
clinical applications. J Clin Monit Comput 2004;18:379-387. 
54. Olin AC, Bake B, Toren K. Fraction of exhaled nitric oxide at 50 ml/s: Reference values for adult lifelong never-
smokers. Chest 2007;131:1852-1856. 
55. ATS/ERS recommendations for standardized procedures for the online and offline measurement of exhaled lower 
respiratory nitric oxide and nasal nitric oxide, 2005. Am J Respir Crit Care Med 2005;171:912-930. 
56. Kharitonov S, Alving K, Barnes PJ. Exhaled and nasal nitric oxide measurements: Recommendations. The european 
respiratory society task force. Eur Respir J 1997;10:1683-1693. 
57. Terada A, Fujisawa T, Togashi K, Miyazaki T, Katsumata H, Atsuta J, Iguchi K, Kamiya H, Togari H. Exhaled nitric 
oxide decreases during exercise-induced bronchoconstriction in children with asthma. Am J Respir Crit Care Med 
2001;164:1879-1884. 
58. Silkoff PE, Wakita S, Chatkin J, Ansarin K, Gutierrez C, Caramori M, McClean P, Slutsky AS, Zamel N, Chapman 
KR. Exhaled nitric oxide after beta2-agonist inhalation and spirometry in asthma. Am J Respir Crit Care Med 
1999;159:940-944. 
59. Kharitonov SA, Yates D, Barnes PJ. Increased nitric oxide in exhaled air of normal human subjects with upper 
respiratory tract infections. Eur Respir J 1995;8:295-297. 
60. Olin AC, Aldenbratt A, Ekman A, Ljungkvist G, Jungersten L, Alving K, Toren K. Increased nitric oxide in exhaled 
air after intake of a nitrate-rich meal. Respir Med 2001;95:153-158. 
61. Sorkness CA, Lemanske RF, Jr., Mauger DT, Boehmer SJ, Chinchilli VM, Martinez FD, Strunk RC, Szefler SJ, 
Zeiger RS, Bacharier LB, et al. Long-term comparison of 3 controller regimens for mild-moderate persistent 
childhood asthma: The pediatric asthma controller trial. J Allergy Clin Immunol 2007;119:64-72. 
62. Silkoff PE, Carlson M, Bourke T, Katial R, Ogren E, Szefler SJ. The aerocrine exhaled nitric oxide monitoring 
system niox is cleared by the us food and drug administration for monitoring therapy in asthma. J Allergy Clin 
Immunol 2004;114:1241-1256. 
63. Horvath I, Hunt J, Barnes PJ, Alving K, Antczak A, Baraldi E, Becher G, van Beurden WJ, Corradi M, Dekhuijzen 
R, et al. Exhaled breath condensate: Methodological recommendations and unresolved questions. Eur Respir J 
2005;26:523-548. 
64. Montuschi P, Nightingale JA, Kharitonov SA, Barnes PJ. Ozone-induced increase in exhaled 8-isoprostane in 
healthy subjects is resistant to inhaled budesonide. Free Radic Biol Med 2002;33:1403-1408. 
65. Montuschi P. Exhaled breath condensate analysis in patients with copd. Clin Chim Acta 2005;356:22-34. 
66. Barreto M, Villa MP, Olita C, Martella S, Ciabattoni G, Montuschi P. 8-isoprostane in exhaled breath condensate 
and exercise-induced bronchoconstriction in asthmatic children and adolescents. Chest 2009;135:66-73. 
67. Vaughan J, Ngamtrakulpanit L, Pajewski TN, Turner R, Nguyen TA, Smith A, Urban P, Hom S, Gaston B, Hunt J. 
Exhaled breath condensate ph is a robust and reproducible assay of airway acidity. Eur Respir J 2003;22:889-894. 
68. Hunt JF, Fang K, Malik R, Snyder A, Malhotra N, Platts-Mills TA, Gaston B. Endogenous airway acidification. 
Implications for asthma pathophysiology. Am J Respir Crit Care Med 2000;161:694-699. 
69. Hunt J. Exhaled breath condensate ph assays. Immunol Allergy Clin North Am 2007;27:597-606; vi. 


 
 
 
 
Kazeros, PI 
March 17, 2016 
15
70. Green RH, Brightling CE, McKenna S, Hargadon B, Parker D, Bradding P, Wardlaw AJ, Pavord ID. Asthma 
exacerbations and sputum eosinophil counts: A randomised controlled trial. Lancet 2002;360:1715-1721. 
71. Pizzichini E, Pizzichini MM, Efthimiadis A, Evans S, Morris MM, Squillace D, Gleich GJ, Dolovich J, Hargreave 
FE. Indices of airway inflammation in induced sputum: Reproducibility and validity of cell and fluid-phase 
measurements. Am J Respir Crit Care Med 1996;154:308-317. 
72. Pin I, Gibson PG, Kolendowicz R, Girgis-Gabardo A, Denburg JA, Hargreave FE, Dolovich J. Use of induced 
sputum cell counts to investigate airway inflammation in asthma. Thorax 1992;47:25-29. 
73. Spanevello A, Beghe B, Bianchi A, Migliori GB, Ambrosetti M, Neri M, Ind PW. Comparison of two methods of 
processing induced sputum: Selected versus entire sputum. Am J Respir Crit Care Med 1998;157:665-668. 
74. Belda J, Leigh R, Parameswaran K, O'Byrne PM, Sears MR, Hargreave FE. Induced sputum cell counts in healthy 
adults. Am J Respir Crit Care Med 2000;161:475-478. 
75.  Bleck B, Kazeros A, Bakal K, Garcia-Medina L, Adams A, Liu M, Lee RA, Tse DB, Chiu A, Grunig G, Egan III JP, 
Reibman J. Coexpression of type 2 immune targets in sputum-derived epithelial and dendritic cells from asthmatic 
subjects. J Allergy Clin Immunology 2015; 136:619-627.