Activated Macrophages and Ozone Toxicity
Phase: NA
Conditions: Air Pollution
Sponsor: Rutgers, The State University of New Jersey

Protocol Version 20– 12/9/21 
 
 
 
Title:  
 
 
 
 
Activated Macrophages and Ozone Toxicity 
 
Principal Investigator:  
Howard Kipen, MD, MPH 
 
Funding Source:  
 
National Institutes of Health 
 
1. Purpose/Specific Aims 
The purpose of the study is to better understand the mechanisms of lung injury from ozone 
exposure. Ozone is a ubiquitous urban air pollutant that causes adverse health effects in both 
healthy and susceptible individuals. The study will focus on the inflammatory mechanisms 
which follow ozone exposure. Specifically, the study will determine the role of different types 
of macrophages, proinflammatory M1 macrophages and anti-inflammatory reparative M2 
macrophages, in lung injury.  
 
The study will incorporate a pilot study to refine protocols and measure novel soluble 
inflammatory markers in sputum supernatant including cytokine, chemokines, eicosanoids and 
prostaglandins following ozone exposure. Samples from the pilot will be used to identify which 
markers will be measured in the main study. 
 
 
1.1 Objectives  
1. The phenotype of macrophages responding to ozone exposure will be assessed (main study). 
2. The objective of the pilot study is to pilot the study protocols. Additionally, the response of 
novel inflammatory markers to ozone exposure will be assessed.   
3. The response of the respiratory microbiome to ozone exposure will be assessed. 
 
 
1.2 Hypotheses  
The hypothesis for the main study is that ozone toxicity is a result of cytotoxic proinflammatory 
macrophages overwhelming the activity of anti-inflammatory reparative macrophages. 
 
2. Background and Significance 
Ozone is a ubiquitous air pollutant and the main component of photochemical smog.  It remains 
one of the most problematic air pollutants to control because it is formed from intermediates 
that arise from multiple sources [1-3].  Inhaled ozone has been shown to irritate and damage 
the lung in both healthy and susceptible individuals, including children and the elderly [3, 4]. 
Ozone causes inflammation and constriction of the airways, reducing pulmonary 
function. Ozone also exacerbates asthma and suppresses nonspecific immunity increasing the 
susceptibility of exposed individuals to respiratory infections [5, 6].  Epidemiologic studies in 
the U.S. have demonstrated that for every 10 ppb increase in peak ozone levels, the total death 
rate for the same day increases by 0.41% [4], while a more recent US/European study estimated 
0.56% [7]. Adverse effects of ozone are even greater in the developing world where ozone 
levels can be significantly higher than in the U.S. with a concomitantly greater health burden.  
In this regard, despite control attempts and much lower standards, daily maximum ozone 
concentrations have recently reached 128 ppb in Shanghai and 225 ppb in Mexico City [8, 9]. 

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 2 - 
 
Thus, new strategies to reduce lung injury from ozone and other air pollutants by elucidating 
specific pro- and anti-inflammatory mechanisms that contribute to ozone toxicity are key to 
public health.     
 
Accumulating evidence suggests that tissue injury induced by ozone is due, not only to direct 
effects of ozone on the lung, but also indirectly to the actions of resident and infiltrating 
macrophages [10-12]. These cells are activated to release a myriad of inflammatory mediators 
that fight infection, protect against xenobiotics, activate adaptive immunity, and promote 
wound healing. These diverse activities of macrophages are thought to be mediated by distinct 
subpopulations that develop in response to inflammatory signals within their 
microenvironment. Two major subpopulations have been identified: classically activated M1 
macrophages which release cytotoxic/proinflammatory mediators and alternatively activated 
M2 macrophages, which release mediators that suppress inflammation and initiate wound 
repair [13, 14]. 
 
Our overall goal is to analyze the distinct contributions of M1 and M2 macrophage 
subpopulations to ozone-induced lung injury.  Preliminary data demonstrate that both 
macrophage populations play a role in ozone toxicity. Whereas initially, classically activated 
M1 macrophages contribute to lung injury by releasing cytotoxic/proinflammatory mediators, 
subsequent release of anti-inflammatory and mitogenic mediators by M2 macrophages 
promotes tissue repair.  We hypothesize that ozone-induced injury and altered lung mechanics 
ensue when M1 macrophage proinflammatory/cytotoxic activity predominates over the 
antiinflammatory/reparative functions of M2 macrophages. To test this hypothesis, the 
phenotype of macrophages responding to ozone-induced injury will be analyzed in animals 
and humans. Additionally, mice with specific defects in M1 or M2 macrophages, or monocyte 
precursors, will be used to assess their contribution to toxicity. Lineage tracking strategies will 
be used to analyze the origin of macrophage subpopulations in the lung and mechanisms 
mediating their accumulation.  
 
The hypothesis that inhaled ozone causes a sequential accumulation of M1 and M2 
macrophages in humans and animals has not been previously tested. Findings that results in 
humans are comparable to mice would provide strong support for the relevance of our mouse 
models to human toxicity resulting from ozone exposure. For translational human studies, we 
will use induced sputum as a source of macrophages [15-17]. The advantage of induced sputum 
is that it is a safe and non-invasive technique; additionally, induced sputum selectively samples 
the surfaces of the large conducting airways, a primary target for ozone effects in humans [15, 
18].  
 
The supernatant from the induced sputum samples will be analyzed for markers of 
inflammation, including chemokines, cytokines, prostaglandins and eicosanoids. Eicosanoids, 
including 20-HETE and 15-HETE, have been shown to induce airway hyper-responsiveness 
and to be increased by oxidant exposure in animals [19]. In addition to directly inducing 
inflammation, oxidants, such as ozone, can generate free rad icals altering the 
oxidant/antioxidant balance within the respiratory tract.  The release of ROS leads to 
transcription of pro-inflammatory cytokine and chemokine genes, increased release of pro-
inflammatory mediators and up-regulation of adhesion molecules [20, 21].  Mononuclear cells 

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 3 - 
 
isolated from induced sputum in subjects with asthma express IL-4 and IL-10 levels to a greater 
degree than cells from healthy controls, suggesting an enhanced immunologically mediated 
inflammatory response [22].  Exhaled breath condensate samples, blood samples and urine 
samples will also be analyzed for markers of inflammation. 
 
The characterization of the lung microbiome is currently the focus of an ongoing study at 
Rutgers (Pro20140000953).  The study compares microbial communities in numerous samples 
from the respiratory tract (including lung cells collected by bronchoalveaolar lavage, induced 
sputum, oral wash, and oropharyngeal samples). Environmental exposures may alter the lung 
microbiome, affecting disease progression [23-25]. Samples will be collected by all of the 
above methods except bronchoalveolar wash to determine the response of the microbiome to 
ozone exposure. 
 
The current USEPA standard for ozone is 0.070 ppm averaged over an 8 hour day.  This is 
meant to protect not only healthy individuals, but those more sensitive to ozone’s effects such 
as infants, children, those with asthma, and those with other chronic lung conditions such as 
COPD.  The proposed exposure concentration of ozone (0.2 ppm, 3 hours) is almost twice 
maximal levels reported in the world’s most polluted cities (i.e., Mexico City); however, this 
concentration and higher concentrations (up to 0.4 ppm, 2 hours) with exercise have been used 
extensively for mechanistic studies at United States Environmental Protection Agency 
(USEPA), the University of Southern California (USC), the University of California, San 
Francisco (UCSF), the University of California, Davis (UC Davis), the University of 
Rochester, and other institutions for over 40 years with no adverse effects in hundreds of 
subjects, including asthmatics, beyond transient and expected physiologic declines in 
spirometry, reported [26-34]. For instance, one recent study at University of Rochester 
exposed healthy exercising subjects to 0.2 ppm of ozone for 3 hour, identical to our proposed 
protocol [27]. 7-10%declines in FEV1 were observed 0.5 hours after exposure had largely 
resolved by 4 hours following exposure.  Another study using the roughly equivalent 0.3 ppm 
ozone for 2 hours with exercise showed transient FEV1 declines of 7-15% [26]. Our exposures 
will be limited to 3 hours and will only occur once. Based on published studies, subjects are 
not expected to experience more than transitory effects including symptoms of mucosal 
irritation, cough or headache, but these are expected to be mild and dissipate within an hour 
after the exposure is terminated. 
 
3. Research Design and Methods 
The study is a single blind randomized cross-over design. Subjects (up to 68 healthy men and 
women) will participate in the study (48 non-smokers and 10 smokers in the main study and 
5-10 in the pilot study). Eligible subjects will have 5 clinic visits: one screening visit, 2 
exposure visits, and 2 post-exposure sample collection visits. Each subject will participate in 
2 exposure sessions, one to clean air (ozone<5ppb) and one to 0.2 ppm ozone in the Controlled 
Exposure Facility (CEF) at the Environmental and Occupational Health Sciences Institute 
(EOHSI). The order of the exposure sessions will be randomized. Up to 2 subjects may 
participate in each exposure session. Induced sputum samples will be collected at the baseline 
(during screening) then at one of three pre-selected time periods (24, 48 or 72 hours) following 
each exposure session. Breath, blood, oral washes, oropharyngeal swabs, and urine samples 
will be collected at each visit. 

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 4 - 
 
 
Subjects will undergo telephone screening. If eligible they will be scheduled for the screening 
visit. Subjects will be asked to refrain from antioxidants, over the counter anti-inflammatory 
medications, strenuous exercise, alcohol, home air purifiers, unvented household combustion 
sources, gases and fumes for 48 h prior to the screening visit and before each exposure visit 
through the post-sample collection period. Non-smokers will also be asked to avoid tobacco 
smoke. 
 
Subjects will be consented then scheduled for a screening visit to determine eligibility. 
Subjects will be asked to refrain from eating for 2 hours prior to the screening visit. On the day 
prior to the screening visit, subjects will be called/emailed/texted to confirm their appointment. 
If the subject reports a respiratory or other acute illness, including active allergies, during the 
four prior weeks or if they have taken any medication for allergies or respiratory illness, their 
visit will be rescheduled to a later date. The screening visit will include a complete medical 
history and physical examination (including height, weight, and three blood pressure 
measurements), lab tests (CBC with differential and platelet count, urinalysis, and standard 
metabolic screen), spirometry, impulse oscillometry (IOS), and ECG. A pregnancy test (urine 
hCG) will be administered to females at enrollment and before each exposure session. Exhaled 
breath condensate (EBC) samples will also be collected using an RTube™ (the condensate 
collection system manufactured 
by Respiratory Research, Inc.) or 
using the Jaeger EcoScreen. With 
the RTubeTM, each subject uses a 
new and separately packaged 
device to eliminate any risk of 
infection 
transmission. 
The 
samples are collected by having 
patients breathe through a one-
way valve into a cooled condenser 
for 5 to 15 minutes.  As much as 
1 ml of condensed liquid can be collected in a 5-minute period.  There is virtually no risk and 
no spuriously induced inflammation in the lung from this procedure, and it may be serially 
repeated [35]. Subjects will be asked for permission to store excess EBC sample for future use 
by the study investigators.  
 
Impulse oscillometry (IOS) is an additional noninvasive and effort-independent technique to 
measure airway resistance and capacitance.  The airway resistance and capacitance, reflecting 
airway compressibility, is measured at normal tidal breathing.  This physiologic technique also 
allows measurement of peripheral and central airway resistance.  IOS waves are measured for 
30 seconds of tidal breathing and the maneuver is repeated three times.  This technique has no 
known side effects and is a safe test providing a great deal of information regarding peripheral 
airway resistance. 
 
Oral washes and oropharyngeal swabs will be collected for microbiome analyses. For the oral 
wash (OW), participants will gargle with 10 ml sterile 0.9% saline and expel the sample into a 
specimen container. Oropharyngeal swabs (OS) will be collected using a new, sterile cotton 
Figure 1. R-tube (left); Ecoscreen (right) 

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 5 - 
 
swab.  While wearing gloves and depressing the tongue with a sterile tongue depressor, the 
swab will be inserted into the back of the oral cavity.  The participant will be asked to say 
"aahh” to elevate the uvula and with a sweeping motion the posterior pharyngeal wall and 
tonsilar pillars will be swabbed.  By applying a little force a large quantities of mucosa will be 
collected.  Care will be exercised to avoid swabbing the soft palate or touching the tongue with 
the swab tip.  The swab will immediately placed in a sterile tube and stored with the other 
samples for transport.  
 
In addition to the EBC, oral washes, and oropharyngeal swab samples, urine and blood 
(approximately 10 ml) samples will be collected at the screening visit. The urine samples will 
be analyzed for eicosanoids and other markers of inflammation. The urine samples of the 
smokers will also be analyzed for cotinine to confirm their smoking status. The blood samples 
will be processed by Dr. Laumbach’s lab at EOHSI. Plasma from the blood samples will be 
stored in the principal investigator’s private freezer for future analysis of markers of ozone 
response; no genetic analyses will be performed on these stored samples. Only study 
investigators will have access to these samples. 
 
At screening, subjects will be asked to provide an induced sputum specimen (baseline sample), 
which will be immediately analyzed for the presence of plugs. Using the method described by 
Pin et al. [36] and Alexis et al., [37] subjects inhale increasing concentrations of saline (3%, 
4% and 5%) for 3 consecutive 7-minute periods through a mouthpiece without a valve. To 
reduce the risk of bronchospasm, following the recommendations of the European Respiratory 
Commission [38], albuterol (180 mcg; 2 puffs of a standard metered inhaler) will be given to 
subjects 15 minutes prior to the start of the induction. Post-dose spirometry is performed to 
obtain a baseline FEV-1. The subject will be asked to breathe through his/her mouth during 
the induction. If the subject is observed to breathe through his/her nose, he/she will be asked 
to monitor his/her breathing and will be offered a nose clip to use during the induction. After 
each period of inhalation, an FEV-1 is measured for safety. If the FEV-1 decreases by 20% or 
more from the post-dose FEV-1, the procedure is stopped. After rinsing the mouth, swallowing 
water and blowing the nose to minimize contamination by saliva or post nasal drip, subjects 
will cough sputum into a sterile container.  Subjects unable to provide an adequate specimen 
(70-75 mg of plugs) will be excused from the study. In the main study, subjects will be assigned 
to a post-exposure sample collection time (24, 48, or 72 hours) to be used for both exposure 
sessions. In the pilot study and for all smokers, all subjects will be assigned to the 24 hour post-
exposure sample collection. The exposure sessions will be scheduled. 
 
Subjects may be screened (telephone and screening visits) year-round. For the main study, the 
exposure visits will be scheduled between October and June, consistent with low ambient 
ozone months in New Jersey. For the pilot study, since only acute responses are examined (24 
hours of the exposure), the screening and exposure visits will take place year-round. If over 
six months have passed between the screening visit and scheduling the exposures, those 
subjects will be re-consented and the screening visit will be repeated. 
 
Each exposure session will be 3 hours in duration. Subjects will be called the day prior to the 
visit to confirm the appointment. If unable to be reached by phone, reminder texts or emails 
may be sent from a Rutgers-authenticated computer. Subjects will be asked to report any 

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 6 - 
 
current or recent respiratory symptoms. If subjects report any symptoms within the previous 4 
weeks, the visit will be rescheduled. Subjects will be asked to refrain from eating for 2 hours 
prior to the exposure visit. At the start of the visit, the subject’s blood pressure, pulse, and 
temperature will be measured. IOS and spirometry will be performed before and after the 
exposure session. Two questionnaires will also be administered before the exposure session: a 
stress questionnaire and a symptom questionnaire. If the subject reports the presence of any 
respiratory symptoms, the exposure session will be canceled. During the exposure, heart rate 
will be measured using a wireless monitor (H7; Polar USA). Subjects will be asked to alternate 
(every 15 minutes) rest and mild/moderate exercise periods on a cycle ergometer to maintain 
an average heart rate of approximately 60% maximum heart rate (220-age). The exercise is 
necessary as healthy human subjects are relatively insensitive to ozone [39, 40]. The exercise 
schedule may be modified to maintain the ventilation rate or for the subject’s comfort. After 
the exposure session, the stress and symptom questionnaires will be administered. An exposure 
type questionnaire will also be administered immediately after the exposure. If the subject 
reports any physical symptoms or has a 20% decrease in his/her FEV1 after the exposure 
session, the study physician will be consulted. Subjects will be given verbal and written 
instructions to contact the Robert Wood Johnson Medical School Pulmonary Division if they 
experience any breathing problems after leaving EOHSI. Subjects will inform the responding 
pulmonologist that he/she is a study subject. As an additional precaution, subjects will be given 
a rescue inhaler to use only if directed to do so by the responding pulmonologist. Subjects will 
be shown how to use the inhaler prior to leaving the EOHSI clinic. The pulmonologist will 
inform Dr. Kipen of any calls made to the pulmonologist and any treatment. The symptom 
questionnaire will be administered to each subject the day after exposure. For subjects 
returning at 24 hours for the post-exposure sputum sample, the questionnaire will be 
administered in person. For all other subjects the questionnaire will be administered by phone. 
As a secondary check, the questionnaire will ask the study subject if he/she contacted the 
Pulmonary Division. If the subject reports the presence of any symptoms or called the 
Pulmonary Division, the study physician will be consulted, prior to the collection of any 
samples.  
 
Immediately before and after each exposure session EBC samples will be collected using the 
same methods as in the screening visits. In addition to the EBC samples, urine, oral washes, 
oropharyngeal swabs, and blood samples will be collected before and after each exposure 
session. During the second exposure visits, an additional blood sample will be collected for 
genotyping of GST polymorphisms. The urine samples will be analyzed for eicosanoids and 
other markers of inflammation. The blood samples will be processed by either Dr. Laumbach’s 
lab (research blood for future use) or the Rutgers University Cell and DNA Repository facility 
core at EOHSI (sample for genetic analysis).  
 
Subjects will be asked to return for a follow-up visit either 1, 2, or 3 days after each exposure 
visit. Subjects will be asked to refrain from eating for 2 hours prior to the visit. The blood 
pressure, pulse rate, and oral temperature will be checked. IOS and spirometry will be 
performed and an induced sputum sample will be collected. The symptom and stress 
questionnaire will be administered. The blood, urine, oral washes, oropharyngeal swabs, and 
EBC samples will also be collected.  
 

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 7 - 
 
Sputum samples obtained after the ozone exposure may be compared with samples collected 
either at the screening visit or after the clean air exposure. If the subject is randomized to ozone 
exposure first and is unable to produce a sputum sample after the ozone exposure, the subject 
will be withdrawn from the study. 
 
 
3.1. Duration of Study 
The study duration is expected to be four years. Each person will have 5 visits (one screening 
visit, two exposure visits, and two post-exposure visits) to the EOHSI clinic, for a total time of 
approximately15 hours.  
 
3.2 Study Sites 
The consent, screening, exposure sessions and sputum collection will take place at EOHSI. All 
consent forms and identifiable data will also be stored at EOHSI. Analysis of de-identified data 
will take place at the Ernest Mario School of Pharmacy and at the School of Public Health.  
 
3.3 Sample Size Justification  
Calculations are based on published human exposure studies [41, 42] demonstrating 2.3-fold 
and 1.6-fold increases (SE=8%) in mean fluorescence intensity of sputum leukocyte expression 
of markers of M1 (CD14) and M2 (CD16) macrophages following ozone exposure at 24 h 
compared to clean air. The SDs for changes [the square root of 2(1-rho)SD2] were calculated 
where rho is the intra-person correlation (0.5) between measurements. Based on this, 16 
subjects/exposure group are needed to achieve 90% power using 0.05 level two-group t-tests. 
 
3.4 Subject Selection  
 
Two subject populations will be studied: smokers (n=10) and non-smokers (n=70). The pilot 
study will recruit up to 10 people (non-smokers) and the main study will recruit 70 people (10 
smokers & 60 nonsmokers). 
 
3.4.1 Inclusion Criteria – both smokers and non-smokers. 
Healthy men and women (n= up to 80) between the ages of 18 and 40 will be recruited for the 
study. Subjects who participate in the pilot study will also be eligible to participate in the main 
study.  
 
Subjects must have been fully vaccinated for COVID-19 – people are considered fully 
vaccinated for COVID-19 ≥2 weeks after they have received the second dose in a 2-dose series 
(e.g., Pfizer-BioNTech or Moderna), or ≥2 weeks after they have received a single-dose 
vaccine (e.g., Johnson and Johnson). 
 
Additional Inclusion Criteria for Smokers: currently smoking at least one cigarette each 
day. 
 
3.4.2 Exclusion Criteria 
• 
Cardiovascular disease 

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 8 - 
 
• 
Respiratory disease 
• 
Recent (within 4 weeks) respiratory or COVID-19 symptoms 
• 
Diabetes 
• 
Pregnancy 
• 
HIV Infection 
• 
Orthopedic or rheumatologic conditions which would interfere with cycle use 
• 
Inability to produce a sputum plug 
• 
Daily use of antioxidant supplements, excluding those in a multivitamin. These 
supplements include Vitamin C or E, selenium, beta-carotene, lycopene, lutein, 
zeaxanthin and ginkgo biloba. Supplements taken less frequently but at least once a week 
will be reviewed by the principal investigator for eligibility determination. 
 
Additional Exclusion Criteria for Non-smokers: history of smoking within the past 5 years. 
 
4. Study Variables 
 
 
4.1 Independent Variables or Interventions  
The independent variable is the ozone exposure. The exposure will take place in the Controlled 
Exposure Facility at EOHSI. The CEF consists of three major components: 1) the exposure 
chamber; 2) the air pollutant generation system; and 3) the control systems. The CEF is a large 
stainless steel room (7.3 ft. high x 13.5 ft. wide x 9 ft. deep; volume= 887 cubic feet) in which 
the air flow, temperature and humidity can be varied and well controlled. A divider separates 
the chamber into two compartments, so that two subjects can be exposed simultaneously. There 
is a lavatory in the chamber for extended exposure studies. For the clean air exposure, ambient 
air that supplies the exposure chamber will be treated using a series of processes, which include 
cooling/heating, humidification/dehumidification, and filtration through a carbon filter, a 
KMnO4 purifier, and HEPA filters. The clean air enters the CEF through two diffusers located 
in the ceiling and exits through the perforated stainless steel floor without recirculation. 
 
For ozone exposure sessions, ozone will be generated in situ by an ozone arc-generator (Ozone 
Research & Equipment Corporation, Phoenix, AZ), mixed with the filtered clean air and will 
enter the CEF through the two air diffusers. The ozone concentration will be achieved by 
adjusting the ozone output and the air mixing flow through the chamber. Based on previous 
studies [43], ozone can be precisely controlled during an exposure session. The ozone 
concentration will be monitored in real time using an API Model 450 ozone analyzer (API, 
Inc., San Diego, CA). The sensitivity of the instrument is 1 ppb, and response time is less than 
20 sec.  Noise is less than 0.7 ppb.  
 
Additionally, during each exposure session, additional air pollutants will be monitored. CO 
concentration will be measured using a real-time Langan T15 CO gas monitor. NO/NO2 
concentration will be monitored by a TEI Model 42C TL Trace Level NO-NO2-NOx real-time 
Analyzer (Thermo Electron Corporation, Franklin, MA). PM2.5 mass concentration will be 
measured using a real-time personal PM monitor (SidePak, TSI Inc., MN).  
 
4.2 Dependent Variables or Outcome Measures 

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 9 - 
 
Induced sputum samples will be collected at the Environmental and Occupational Health 
Sciences clinic. The relative concentrations of M1 and M2 macrophages in the induced sputum 
sample will be assessed in the Rutgers University Flow Cytometry/Cell Sorting and Confocal 
Microscopy Core facility (http://www.flowcyt.rutgers.edu/). Cells will be separated from 
mucus plugs, stained with fluorescent antibodies and then analyzed by flow cytometry. 
Leukocytes will initially be separated from epithelial cells by light scatter and by CD45 
expression, and macrophages identified by HLA-DR expression [44]. Flow cytometry will also 
be used to assess macrophage phagocytosis and oxidative burst. To confirm their phenotype, 
sputum macrophage subpopulations will be sorted and expression of inflammatory proteins 
and additional M1/M2 macrophage markers not detectable by flow cytometry will be analyzed 
by RT-PCR and immunocytochemistry.  
 
Macrophage response may vary by genotype, specifically GST polymorphisms. The 
genotyping will be done by the Rutgers University Cell and DNA Repository (RUCDR) on a 
fee for service basis. De-identified samples will be sent to RUCDR. Any sample remaining 
after the genetic analyses will be destroyed. 
 
Supernatant from the induced sputum sample, EBC, and urine will be analyzed for cytokines, 
chemokines, prostaglandins and novel eicosanoids. The analysis will first be done in samples 
from the pilot study to identify markers of interest for the main study. All analyses will be 
done at the Rutgers Flow Cytometry Lab and in Dr. Laumbach’s lab in EOHSI. Subjects will 
be asked for permission to store excess samples for future use by the study investigators. 
 
Oral washes and oropharyngeal swabs as well as aliquots of induced sputum and EBC samples 
will be given to Dr. Lee Kerkhof for microbiome analysis.  
 
Specimens (induced sputum, exhaled breath condensate, and blood samples) will be analyzed 
for oxidized and nitrosylated variants of surfactant protein D. All analyses will take place in 
the Rutgers Flow Cytometry Lab. 
 
Data from the stress questionnaires, collected before and after the exposure and before the 
follow-up sputum induction, will be examined as possible modifiers of the response. Subject 
fitness, calculated by their heart rate recovery during the exposure visit, will also be examined 
as a possible modifier. 
 
 
4.4 Risks of Harm  
Ozone exposure: The proposed exposure concentration of ozone (0.2 ppm, 3 hours) is almost 
twice maximal levels reported in the world’s most polluted cities (i.e., Mexico City); however, 
this concentration and higher concentrations (up to 0.4 ppm, 2 hours) with exercise have been 
used extensively for mechanistic studies at EPA, USC, and other institutions for over 40 years 
with no adverse effects, beyond transient physiologic declines in spirometry, reported. Our 
exposures will be limited to 3 hours and will only occur once. Based on published studies, 
subjects are not expected to experience more than transitory effects including symptoms of 
mucosal irritation, cough or headache, but these are expected to be mild and dissipate within 
an hour after the exposure is terminated. Based on prior experiments looking at monitored 

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 10 - 
 
chamber exposures of smokers to ozone, it appears there is no increased risk of acute 
deterioration in pulmonary function, respiratory pattern, heart rate or symptomatology, even at 
ozone doses >3 times the concentration proposed in this protocol [45]. Thus, there does not 
appear to be an increased risk of harm by exposing smokers to ozone as described in this 
protocol. Prior to the exposure, subjects will complete a symptom questionnaire. If subjects 
report the presence of any symptoms, the exposure session will be canceled. During the 
exposure sessions, the participants are continually observed through a window to assure that 
they are not exhibiting any visual signs of physical problems and wear a sensor for continuous 
monitoring of heart rate.  A physician with a code cart is on-call during the entire procedure to 
respond to any subject complaints or situations. If a subject develops any symptoms of chest 
pain, dizziness, or other symptoms/signs of concern to the subject or the study technician, the 
physician will speak with and examine the subject and decide if the exposure session may 
continue. The covering physician will examine the subject, including EKG if indicated, and 
make an appropriate triage decision, including referral via ambulance to the emergency 
department. Regardless of the physician’s recommendation, the study subject may choose to end 
the session at any time. At the end of each exposure session, the subject will complete a 
symptom questionnaire. This questionnaire will be reviewed by the study technician and a 
physician to ensure that the subject is ready to resume daily activities. The subject will also be 
asked to complete the symptom questionnaire the next day (by phone or in person). If the subject 
reports any symptoms, he/she will be referred to the study physician. All subjects will be asked 
to return for their scheduled follow-up visit. 
 
As a precaution against delayed bronchospasm after an exposure session, after-hours response 
has been arranged with the pulmonary division Robert Wood Johnson Medical School. After 
each exposure session, subjects will be given written and verbal instructions to call the 
pulmonary division for any delayed respiratory problems. A physician will be available 24 
hours a day/7 days per week. Subjects will also be provided a rescue inhaler to be used only if 
advised by the responding pulmonologist. Prior to leaving EOHSI, subjects will be instructed 
in the use of the inhaler and asked to return the inhaler at their follow-up visit. At each follow-
up visit, the meter on the inhaler will be checked for use. Any use will be reported to the study 
physician to determine if the subject can continue with the study. The inhaler will be discarded 
once the subject has completed the study. 
 
The study has a single ozone exposure and a single clean air exposure. If the subject 
experiences bronchospasm either during or after the ozone exposure, the subject will be asked 
to return for his/her scheduled follow up visit for the sputum collection. Since no further ozone 
exposure will occur, the subject will be permitted to continue with the study and participate in 
the clean air exposure, if not done previously. In the unlikely event that the subject experiences 
bronchospasm either during or after the clean air exposure and has not yet had the ozone 
exposure, the subject will be considered ineligible and his/her participation will stop. If the 
ozone exposure session has been completed, the subject will be asked to continue in the study 
and return for a follow-up visit. 
 
Sputum induction: Sputum induction with hypertonic saline may cause reversible 
bronchoconstriction in some individuals.  Bronchoconstriction has been observed primarily in 
asthmatic subjects who have pre- existing bronchial hyperreactivity.  In order to reduce the risk 

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 11 - 
 
of bronchoconstriction, potential subjects who have a history of asthma, a history of symptoms 
consistent with bronchial hyperreactivity, or abnormal spirograms will be excluded from 
participation in the study. The sputum induction will be done only during clinic hours in 
EOHSI, when a physician is available. As a further precaution against bronchospasm, all 
subjects will be pretreated with albuterol 180 mcg or 2 puffs of a standard metered inhaler) 
prior to the start of induction. Post-albuterol spirometry will be done to determine a baseline 
FEV-1 during induction. Our induction protocol consists of 3 segments of inhalation of 
nebulized saline at increasing concentrations.  After each of the 3 segments spirometry is done. 
If the FEV-1 decreases from the baseline by more than 20% after a segment, , the procedure 
will be stopped. An additional 180mcg of albuterol will be administered and spirometry will 
be repeated 15 minutes afterward. If the FEV-1 has recovered (>=80% baseline FEV-1) the 
subject will be permitted to leave EOHSI. If the FEV-1 is still depressed (<80% baseline FEV-
1), the subject  will be referred to care at the EOHSI clinic and the referral will be documented 
as an adverse event. At any point during the induction, if the subject complains of any breathing 
difficulties, the induction will be stopped and the subject will be referred to care at the EOHSI 
clinic. The episode will be reported as an adverse event. 
 
Albuterol: Albuterol, a short-acting bronchodialator, will be administered before induction via 
a rescue inhaler to prevent bronchoconstriction and possibly afterward to treat difficulty 
breathing, wheezing or shortness of breath due to bronchoconstriction. Temporary side effects 
from albuterol include tremor, palpitations, and headache. These effects may last four to six 
hours. 
 
Genotyping: The selected assays currently have no immediate application to the diagnosis or 
treatment of any disease, but may relate to the inflammatory response. The primary risk from 
the testing is the loss of confidentiality. Every effort will be made to keep the results 
confidential.  
 
Venipuncture: Slight pain, some bleeding, or bruising may occur when blood is drawn for the 
routine laboratory tests. 
 
Oropharyngeal Swabs: The swabbing will be performed by a clinic nurse or medical assistant 
to reduce the possibility of a gag reflex. 
 
Oral wash: No physical risk but subjects may experience a salty taste. 
 
IOS: Subjects may experience brief, temporary lightheadedness during the IOS measurement.  
 
4.5 Potential for Benefit 
There are no direct benefits to individual subjects participating in this study, except to receive 
the results of the physical examination and routine lab tests to share with their personal 
physician.  Ozone is a ubiquitous air pollutant and the main component of photochemical smog.  
It remains one of the most problematic air pollutants to control because it is formed from 
intermediates that arise from multiple sources.  Inhaled ozone has been shown to irritate and 
damage the lung in both healthy and susceptible individuals, including children and the elderly. 

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 12 - 
 
This mechanistic public health research study has great potential to help us understand and 
ultimately prevent disease associated with common exposures such as ambient ozone. 
 
5.  Subject Recruitment and Enrollment Considerations 
 
5.1 Subject Recruitment 
Subjects will be recruited through posting flyers at approved locations on the Rutgers University 
Piscataway/New Brunswick campuses. Flyers will also be posted in nearby public and 
commercial buildings, including retail stores and community centers and made available at 
community events, with permission from the appropriate authority.  Additionally the study will 
be posted on the EOHSI website (http://eohsi.rutgers.edu/content/research-volunteers) and the 
EOHSI Facebook page. Announcements about the study will be posted periodically on the 
EOHSI Facebook page. The announcements will link to the website. EOHSI maintains a 
volunteer mailing list, for persons who wish to be notified when a new study is posted on the 
website. An email announcement will be sent to the mailing list subscribers. Through the 
website and the flyers, interested candidates will be instructed to call the study coordinator. The 
study coordinator or a research assistant will call the subject to explain the study and answer 
any questions. If the candidate wishes to participate, a brief telephone screening questionnaire 
will be administered. The screening questionnaire will ascertain the presence of major exposure-
study exclusions such as HIV infection, cardiovascular disease, stroke, diabetes, or asthma. The 
recruiter will ask about the COVID-19 vaccination status of the candidate, only fully vaccinated 
volunteers are eligible. The recruiter will inquire about a personal history of cigarette or other 
smoking within 5 years, regular use of vitamin E or C, selenium, or other supplements beyond 
a multivitamin, ingested herbal preparations, or any medication prescribed by a physician. The 
candidate will be asked to provide a list of his/her vitamins, supplements, and herbal 
preparations and their frequency of use. The supplements will be checked for antioxidant 
content and the candidate will be informed if he/she is eligible. If the candidate is not eligible, 
no identifying information will be recorded. If the candidate is eligible, contact information will 
be recorded and an appointment will be made for the screening visit. The candidate will be 
given, mailed or emailed (based on his/her preference) a copy of the consent form to review 
prior to the screening visit. A copy of the medical history questionnaire will also be given to the 
subject. The subject will be asked to bring the completed questionnaire to the screening visit. 
The subject will also be asked to bring proof of vaccination to the screening visit – this can be 
their physical vaccine card or a photo of it. If the subject is a Rutgers student or employee, they 
may show their myRutgers Dashboard vaccine status.  
 
5.2 Consent Procedures 
When subjects come to the EOHSI Clinical Center for screening visit, they will be provided the 
written informed consent to read and sign.  The study coordinator or a research assistant will 
review the consent form with the subject and answer any questions. If the subject wishes to 
participate, he/she will be asked to sign the consent form. The coordinator or research assistant 
will also sign the consent form. A copy of the signed consent form will be given to the subject. 
After the consent form is signed, the medical history questionnaire will be collected and the 
screening visit will begin. 
 

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 13 - 
 
After the screening visits, if the subject does not meet the research criteria, the recruiter will 
thank the individual and explain that for safety reasons, he/she cannot participate. The specific 
reason will be explained. For individuals who do not become subjects, personal identification 
information will be removed from the screening questionnaires, although demographic 
information and reasons for exclusion will be maintained for research purposes. If the subject 
does meet the research criteria, the first exposure and follow-up visit will be scheduled. 
 
5.3 Subject Costs and Compensation 
No costs are expected to be incurred by the subject. 
 
The subject will be compensated, in cash, on a pro-rated basis: 
 Screening visit - $50 
 Exposure visit - $125 
 Follow-up visit (for sputum collection) - $75 
 
If the subject completes all part of the study protocol, the total compensation will be $450. 
Subjects will receive compensation after the completion of their screening visit, and if 
applicable, at the conclusion of the study, according to the pro-rated schedule above. 
 
 
6.   Data Handling  
Confidentiality of participants will be protected by using identification numbers for all 
participants.  All hard copies of forms with personal data will be kept in a locked file in a 
private office (Room 127) at EOHSI. All data and specimens will be coded only by the study 
identification number. The link to identification of all participants will be kept in a password 
protected file on a secure shared drive at EOHSI and will be accessible to project staff directly 
involved in contacting participants and the Rutgers-RWJMS Institutional Review Board. The 
link will be destroyed when data entry is completed. All data (consent forms, etc.) will be kept 
for 6 years. De-identified data will be kept indefinitely. 
 
De-identified data will be entered into Excel worksheets. The data will be stored on the EOHSI 
secure shared drive. All data will be double-entered by two different investigators. All 
discrepancies in the data will be resolved by a third investigator. Only de-identified data will 
be transmitted between study investigators.. 
 
7. Statistical Analysis 
Descriptive statistics, including means, standard deviations, and histograms will summarize 
the distributions of markers stratified by time point and exposure. Analysis of covariance 
(ANCOVA) will be used to compare the means of markers of inflammation. Specifically, with 
baseline level as a covariate in the model, F-tests will test whether the means are significantly 
different following exposure to ozone or clean air at each follow-up time-point. The mean fold- 
changes (or log-fold-changes if necessary to meet normality assumptions) in M1 and M2 
macrophages will also be assessed at each time-point (24, 48 or 72 h) following ozone and 
compared to clean air in humans.             
 
8. Data and Safety Monitoring  

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 14 - 
 
All study procedures are conducted during regular business hours when a physician is available 
at the EOHSI clinic. All study staff are trained in study procedures by the principal investigator. 
During inductions, lung function will be assessed using spirometry. A significant decrease in 
lung function (FEV-1<80% baseline) will be treated with albuterol. If lung function does not 
recover, the subject will be referred to clinical care and the adverse event will be reported to 
the IRB. The pulmonary fellow on call at RWJBUH is available for backup if needed.  During 
the exposure sessions, the participants are continually observed through a window to assure 
that they are not exhibiting any visual signs of physical problems and wear a heart rate sensor 
(Polar.com) for continuous monitoring of rate. Regular voice communication between the 
study staff and the subject is maintained through an intercom system. A physician with a code 
cart is on-call during the entire procedure to respond to any subject complaints or situations. 
At the end of each exposure session, the subject will complete a symptom questionnaire and a 
spirometry. This questionnaire will be reviewed by the clinical technician and a physician to 
ensure that the subject is ready to resume daily activities.  If a subject develops symptoms of 
chest pain, dizziness, or other symptoms of concern to the subject or the physician, the study 
will be terminated and the covering physician will examine the subject, perform an EKG, and 
make an appropriate triage decision, including referral to the emergency department. In the 
case of a delayed reaction, subjects will be instructed to call the pulmonary division of RWJMS 
and be given a rescue inhaler. Any respiratory reaction requiring referral to the emergency 
department will be considered an adverse event and will be reported to the IRB within 1 week 
of occurrence. Although interim analyses are not appropriate for the study, a single occurrence 
of an adverse event will result in a suspension of exposure visits and sputum collection while 
procedures are reviewed. The visits will resume only after IRB review of the event and 
approval of any modified procedures. 
 
9. Reporting Results 
 
9.1 Individual Results 
All subjects will be given the results of their screening blood tests. For subjects who do not pass 
the physical examination or have significant findings on the blood tests, the physician will send 
a letter to them describing the reason for exclusion and to make diagnostic or treatment 
recommendations if needed. The physician will also be available to discuss findings if needed.  
 
9.2 Aggregate Results 
Aggregate results will be posted on the Center for Environmental Exposures and Disease 
website. The webpage is under development. 
 
9.3 Professional Reporting 
The results will be published in peer-reviewed journals and presented at professional 
conferences.  
 
10. Bibliography 
1. 
Flohr, C., et al., How well do questionnaires perform compared with physical 
examination in detecting flexural eczema? Findings from the International Study of 
Asthma and Allergies in Childhood (ISAAC) Phase Two. Br J Dermatol, 2009. 161(4): p. 
846-53. 

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 15 - 
 
2. 
Partridge, M.R., Has ISAAC told us as much as it can? Where now? Thorax, 2009. 64(6): 
p. 462-3. 
3. 
USEPA, U.S. EPA Air quality criteria for ozone and related photochemical oxidants, in 
Washington, DC. 2012. 
4. 
Fok, A.O. and G.W. Wong, What have we learnt from ISAAC phase III in the Asia-
Pacific rim? Curr Opin Allergy Clin Immunol, 2009. 9(2): p. 116-22. 
5. 
Lai, C.K., et al., Global variation in the prevalence and severity of asthma symptoms: 
phase three of the International Study of Asthma and Allergies in Childhood (ISAAC). 
Thorax, 2009. 64(6): p. 476-83. 
6. 
Annesi-Maesano, I., et al., [Exposure to fine air particles and occurrence of allergic 
diseases: results of ISAAC-France phase 2]. Arch Pediatr, 2009. 16(3): p. 299-305. 
7. 
Peng, C.X., et al., Influence of different fermentation raw materials on pyrolyzates of Pu-
erh tea theabrownin by Curie-point pyrolysis-gas chromatography-mass spectroscopy. 
Int J Biol Macromol, 2013. 54: p. 197-203. 
8. 
Montefort, S., et al., Increasing prevalence of asthma, allergic rhinitis but not eczema in 
5- to 8-yr-old Maltese children (ISAAC). Pediatr Allergy Immunol, 2009. 20(1): p. 67-71. 
9. 
Morales Suarez-Varela, M., et al., Parents' smoking habit and prevalence of atopic 
eczema in 6-7 and 13-14 year-old schoolchildren in Spain. ISAAC phase III. Allergol 
Immunopathol (Madr), 2008. 36(6): p. 336-42. 
10. 
Kudzyte, J., E. Griska, and J. Bojarskas, Time trends in the prevalence of asthma and 
allergy among 6-7-year-old children. Results from ISAAC phase I and III studies in 
Kaunas, Lithuania. Medicina (Kaunas), 2008. 44(12): p. 944-52. 
11. 
Ait-Khaled, N., et al., Global map of the prevalence of symptoms of rhinoconjunctivitis in 
children: The International Study of Asthma and Allergies in Childhood (ISAAC) Phase 
Three. Allergy, 2009. 64(1): p. 123-48. 
12. 
Al-Hegelan, M., et al., Ambient ozone and pulmonary innate immunity. Immunol Res, 
2011. 49(1-3): p. 173-91. 
13. 
Martinez, F.O., et al., Macrophage activation and polarization. Front Biosci, 2008. 13: p. 
453-61. 
14. 
Mantovani, A., et al., Macrophage plasticity and polarization in tissue repair and 
remodelling. J Pathol, 2013. 229(2): p. 176-85. 
15. 
Alexis, N.E., et al., Induced sputum derives from the central airways: confirmation using 
a radiolabeled aerosol bolus delivery technique. Am J Respir Crit Care Med, 2001. 
164(10 Pt 1): p. 1964-70. 
16. 
Masjedi, M.R., et al., Prevalence and Severity of Asthma Symptoms in Children of 
Tehran- International Study of Asthma and Allergies in Childhood (ISAAC). Iran J 
Allergy Asthma Immunol, 2004. 3(1): p. 25-30. 
17. 
Foliaki, S., et al., Prevalence of symptoms of childhood asthma, allergic 
rhinoconjunctivitis and eczema in the Pacific: the International Study of Asthma and 
Allergies in Childhood (ISAAC). Allergy, 2007. 62(3): p. 259-64. 
18. 
Alexis, N.E., et al., Low-level ozone exposure induces airways inflammation and modifies 
cell surface phenotypes in healthy humans. Inhal Toxicol, 2010. 22(7): p. 593-600. 
19. 
Cooper, P.R., et al., 20-HETE mediates ozone-induced, neutrophil-independent airway 
hyper-responsiveness in mice. PLoS One, 2010. 5(4): p. e10235. 

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 16 - 
 
20. 
MacNee, W., Airway infection does not accelerate decline in lung function in chronic 
obstructive pulmonary disease. American Journal of Respiratory and Critical Care 
Medicine, 2001. 164(10 Pt 1): p. 1758-1760. 
21. 
MacNee, W., Oxidative stress and lung inflammation in airways disease. European 
Journal of Pharmacology, 2001. 429(1-3): p. 195-207. 
22. 
Hamzaoui, A., et al., Inflammatory response in induced sputum mononuclear cells from 
patients with acute exacerbation of asthma. Mediators of Inflammation, 2000. 9(3-4): p. 
147-153. 
23. 
Shaw, J.G., et al., Biomarkers of progression of chronic obstructive pulmonary disease 
(COPD). Journal of Thoracic Disease, 2014. 6(11): p. 1532-1547. 
24. 
Ni, L., C.-C. Chuang, and L. Zuo, Fine particulate matter in acute exacerbation of 
COPD. Frontiers in Physiology, 2015. 6: p. 294. 
25. 
Johnson, L.W. and M.L. Wolbarsht, Mercury poisoning: a probable cause of Isaac 
Newton's physical and mental ills. Notes Rec R Soc Lond, 1979. 34(1): p. 1-9. 
26. 
Pearce, N., et al., Worldwide trends in the prevalence of asthma symptoms: phase III of 
the International Study of Asthma and Allergies in Childhood (ISAAC). Thorax, 2007. 
62(9): p. 758-66. 
27. 
Frampton, M.W., et al., Cardiovascular effects of ozone in healthy subjects with and 
without deletion of glutathione-S-transferase M1. Inhalation Toxicology, 2015. 27(2): p. 
113-119. 
28. 
Alfaro-Moreno, E.a.b., et al., Particulate matter in the environment: pulmonary and 
cardiovascular effects. Current Opinion in Pulmonary Medicine, 2007. 13(2): p. 98-106. 
29. 
Balmes, J.R., et al., Ozone induced decrements in FEV1 and FVC do not correlate with 
measures of inflammation. Am J Respir and Crit Care Med, 1996. 153(3): p. 904-909. 
30. 
McDonnell, W.F., et al., Prediction of lung function response for populations exposed to 
a wide range of ozone conditions. Inhalation Toxicology, 2012. 24(10): p. 619-633. 
31. 
Alexis, N.E., et al., Low level ozone exposure induces airway inflammation and modifies 
cell surface phenotypes in healthy humans. Inhalation Toxixology, 2010. 22(7): p. 593-
600. 
32. 
Holz, O., et al., Ozone-induced airway inflammatory changes differ between individuals 
and are reproducible. American Journal of Respiratory and Critical Care Medicine, 1999. 
159(3): p. 776-784. 
33. 
Alexis, N.E., et al., The glutathione-S-transferase Mu 1 null genotype modulates ozone-
induced airway inflammation in human subjects. Journal of Allergy & Clinical 
Immunology, 2009. 124(6): p. 1222-1228.e5. 
34. 
Christian, D., Chen LL, Scannell CH, Ferrando RE, Welch BS, Balmes JR, Ozone-
induced Inflammation is Attenuated with Multiday Exposure. American Journal of 
Respiratory & Critical Care Medicine, 1998. 158: p. 532-537. 
35. 
Kharitonov, S.A. and P.J. Barnes, Biomarkers of some pulmonary diseases in exhaled 
breath. Biomarkers, 2002. 7(1): p. 1-32. 
36. 
Pin, I., et al., Changes in the cellular profile of induced sputum after allergen-induced 
asthmatic responses. Am Rev Respir Dis, 1992. 145(6): p. 1265-9. 
37. 
Van Sickle, D., Perceptions of asthma among physicians: an exploratory study with the 
ISAAC video. Eur Respir J, 2005. 26(5): p. 829-34. 
38. 
Paggiaro, P.L., et al., Sputum induction. Eur Respir J Suppl, 2002. 37: p. 3s-8s. 

Activated Macrophages and Ozone Toxicity  
Howard Kipen, MD, MPH 
 
 
Protocol Version 19– 12/9/21 
 
- 17 - 
 
39. 
Burr, M.L., et al., Pollen counts in relation to the prevalence of allergic 
rhinoconjunctivitis, asthma and atopic eczema in the International Study of Asthma and 
Allergies in Childhood (ISAAC). Clin Exp Allergy, 2003. 33(12): p. 1675-80. 
40. 
Gharagozlou, M., et al., Gender similarity in low agreement between written and video 
ISAAC asthma questionnaires. Monaldi Arch Chest Dis, 2006. 65(4): p. 184-8. 
41. 
Sole, D., et al., Changes in the prevalence of asthma and allergic diseases among 
Brazilian schoolchildren (13-14 years old): comparison between ISAAC Phases One and 
Three. J Trop Pediatr, 2007. 53(1): p. 13-21. 
42. 
Lay, J.C., et al., Ozone enhances markers of innate immunity and antigen presentation on 
airway monocytes in healthy individuals. J Allergy Clin Immunol, 2007. 120(3): p. 719-
22. 
43. 
Fan, Z., et al., Ozone-initiated reactions with mixtures of volatile organic compounds 
under simulated indoor conditions. Environ Sci Technol, 2003. 37(9): p. 1811-21. 
44. 
Lipscomb, M.F., et al., Human alveolar macrophages: HLA-DR-positive macrophages 
that are poor stimulators of a primary mixed leukocyte reaction. J Immunol, 1986. 
136(2): p. 497-504. 
45. 
Shephard, R.J., et al., Interaction of ozone and cigarette smoke exposure. Environ Res, 
1983. 31(1): p. 125-37.