Phase I/II Randomized Cross-over Study of Hypertonic Saline on Airway Inflammatory Response to Inhaled Wood Smoke
Phase: NA
Conditions: Airway Inflammation
Sponsor: University of North Carolina, Chapel Hill

 
 
 
Complete Title:  
PHASE I/II RANDOMIZED CROSS-OVER STUDY OF HYPERTONIC 
SALINE ON AIRWAY INFLAMMATORY RESPONSE TO INHALED 
WOOD SMOKE 
Short Title:   
Hypertonic saline for wood smoke-induced airway inflammation 
 
NCT Number:  
NCT03851406 
Drug Name(s): 
Sodium Chloride 5% solution for inhalation (Hypertonic Saline) 
Funding Agency:  
Department of Defense (DoD) 
Protocol Date: 
March 6, 2025 
 
 
 
 
 
 
 
 
 
Study Principal Investigator: Terry Noah, MD 
UNC Center for Environmental Medicine, Asthma and Lung Biology 
104 Mason Farm Road, CB 7310 
Chapel Hill NC 27599-7310 
Phone (919) 966-1055 
email: terry_noah@med.unc.edu 
 
 
 
 
 
 

2 
 
TABLE OF CONTENTS 
TABLE OF CONTENTS 
Table of Contents ................................................................................................................. 2 
Abbreviations and Definitions of Terms ............................................................................ 3 
Protocol Synopsis ............................................................................................................... 4 
1 BACKGROUND  AND RATIONALE ................................................................................. 7 
2 STUDY OBJECTIVES ...................................................................................................... 8 
3 INVESTIGATIONAL PLAN ............................................................................................... 8 
4 STUDY PROCEDURES ................................................................................................. 11 
5 STUDY EVALUATIONS AND MEASUREMENTS ......................................................... 15 
6 STATISTICAL CONSIDERATIONS ............................................................................... 19 
7 STUDY INTERVENTION (DEVICE OR OTHER INTERVENTION) ............................... 22 
8 STUDY INTERVENTION ADMINISTRATION ................................................................ 22 
9 SAFETY MANAGEMENT ............................................................................................... 22 
10  DATA COLLECTION AND MANAGEMENT .................................................................. 26 
11 RECRUITMENT STRATEGY ......................................................................................... 27 
12 CONSENT PROCESS ................................................................................................... 27 
13 REFERENCES ............................................................................................................... 27 
Appendix ............................................................................................................................. 30 
 
 
 
 
 
 
 
 
 
 
 
 
 

3 
 
ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation 
Definition 
AE 
Adverse event 
AED  
Automated external defibrillator 
ADL 
Activities of daily living 
ATS 
American Thoracic Society 
BMI 
Body mass index 
BP 
Blood pressure 
BSA 
Body Surface Area 
CBC 
Complete blood count 
CEMALB 
Center for Environmental Medicine, Asthma, and Lung Biology 
Co57 
Cobalt 57 
DSMB 
Data Safety Monitoring Board 
EBC 
Exhaled breath condensate 
CRP 
 C- Reactive Protein 
EKG 
Electrocardiogram 
FA 
Filtered air 
FEF25-75 
Maximal mid-expiratory flow rate 
FEV1 
Forced vital capacity in one second 
FVC 
Forced vital capacity 
GSH 
Reduced glutathione 
GSSG 
Oxidized glutathione 
GSTM1 
Glutathione-s-transferase mu 1 
HR 
Heart rate 
HS 
Hypertonic saline 
HSF 
Human Studies Facilities 
IUD 
Intrauterine device 
LPS 
Lipopolysaccharide 
MCC 
Mucociliary clearance 
mCi 
Microcurie 
NCI-CTCAE 
National Cancer Institute’s Common Terminology Criteria for Adverse 
Events 
NHANES 
National Health and Nutritional Examination Survey 
NSAIDs 
Non-steroidal anti-inflammatory drugs 
O3 
Ozone 
PM 
Particulate matter 
PMN 
Peripheral mononuclear cells (i.e. neutrophils) 
RR 
Respiratory rate 
SAE 
Serious adverse event 
Tc99m-SC 
Sulfur coloid 
TLR 
Toll-like receptor 
WSP 
Wood smoke particles 
 
 
 

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PROTOCOL SYNOPSIS 
Study Title 
Phase I/II Randomized Cross-Over Study of Hypertonic Saline on  
Airway Inflammatory Response to Inhaled Wood Smoke 
Funder 
Department of Defense 
Clinical Phase 
Phase I/II 
Study Rationale 
Deployment of military personnel has been associated with increased 
respiratory morbidity likely due, in part, to inhalation of novel particulate 
matter (PM), such as from burn pits. Inflammation is a key initial 
response to inhaled particulates. Our center has developed a protocol 
using inhaled wood smoke particles (WSP) as a model agent to study 
PM-induced airway inflammation. One approach to mitigating this PM-
induced airway inflammation is to rapidly clear inhaled particles from 
airway surfaces. We have previously shown that a single treatment of 
inhaled hypertonic saline (HS) followed by a cough maneuver performed 
during sputum inductions accelerated mucociliary clearance (MCC) after 
exposure to lipopolysaccharide (LPS), another inhaled pro-inflammatory 
agent. Therefore, we hypothesize that increased MCC induced by HS 
will reduce airway inflammation following WSP exposure.  
Study Objective(s) 
Primary  
• 
To determine the efficacy of nebulized 5% HS (v. no treatment) in 
mitigating WSP-induced neutrophilic airway inflammation, assessed 
via sputum %PMNs, in healthy adults 
Secondary 
• 
To assess the safety of HS in healthy adults by measuring symptom 
questionnaires and spirometry 
• 
To determine the effect of HS on mucus based-parameters following 
WSP exposure 
• 
To determine the effect of HS on MCC using gamma scintigraphy  
• 
To determine the effect of HS on airway inflammatory mediators and 
markers of oxidative stress in exhaled breath condensates (EBC) 
and induced sputum following WSP exposure 
• 
To determine the effect of HS on systemic inflammatory markers and 
serum cytokines  
Study Drug/Device 
(If Applicable) 
Sodium Chloride 5% solution for inhalation (hereafter referred to as 
Hypertonic Saline or HS). 
Study Design 
 
Healthy adults demonstrating a >10% increase in sputum %PMNs 
following WSP exposure in a separate screening protocol will be invited 
back to participate in this randomized open-label cross-over study. 
Those eligible for participation will undergo a baseline MCC, EBC, 
sputum induction, spirometry, and venipuncture. Participants will then 
undergo exposure to WSP after which they will be randomized to receive 
either a single treatment of inhaled HS or no treatment. MCC, EBC, 
sputum induction, and venipuncture are repeated 6 hours after WSP 
exposure to assess for primary and secondary endpoints. Spirometry 

5 
 
and symptom questionnaires are also performed before and after WSP 
exposure and HS treatment to assess for safety endpoints. After at least 
a 1-month washout period, participants will receive the alternative 
treatment during the cross-over period.  
Subject Population 
KEY criteria for 
Inclusion and 
Exclusion: 
Inclusion Criteria 
• 
Subjects age 18-45 years, inclusive, of both sexes 
• 
No physician-diagnosed asthma or any symptoms consistent 
with asthma; FEV1 of at least 80% predicted and FEV1/FVC ratio 
of >70 
• 
Ability to provide an induced sputum sample 
• 
Demonstrate a >10% increase from baseline in sputum %PMNs 
following inhaled WSP exposure in a separate screening 
protocol 
Exclusion Criteria 
• 
Any chronic illness, which may impact safety or study results 
• 
Any infections within 4 weeks of WSP exposure 
• 
Inability to discontinue medications, which may impact study 
results 
• 
Pregnant/lactating women, and children <18 years of age 
Number Of Subjects  14  
Study Duration 
Each subject’s participation will last up to 6 months. The entire study is 
expected to last 4 years.  
Study Phases 
Screening 
Study Treatment 
Follow-Up   
(1) Screening: Potential participants will undergo a general screening 
protocol (IRB #98-0799) to identify healthy adults suitable for WSP 
exposure and a second screening protocol (IRB #15-1775) to identify 
subjects with a >10% increase in sputum %PMNs after WSP exposure. 
Following enrollment, a baseline and training visit will include: an 
informed consent; medical screening for eligibility including vital signs; 
spirometry; and baseline sputum induction, and MCC.  
(2) Intervention: Participants will undergo baseline venipuncture and 
EBC followed by WSP exposure after which MCC assessment will be 
initiated. At the beginning of the MCC scan, subjects will receive 5% HS 
via nebulization (vs. no treatment). Measurement of MCC will continue 
for a total of 120 minutes. EBC, venipuncture, and sputum induction will 
be obtained 4 hours post-WSP exposure. Pre- and post-exposure 
spirometry and symptom questionnaires will also be conducted.  
(3) Follow-up: Medical screening for safety endpoints, spirometry, 
sputum, venipuncture, and lung retention imaging will be performed the 
day following WSP exposure. Participants will also be contacted 3-7 
days after exposure to assess for any delayed adverse events.  
(4) Cross-over: After a one-month wash out period, participants will be 
crossed over to the alternative treatment. A discontinuation visit will be 
performed 5 to 10 days after the last study visit.  

6 
 
Efficacy Evaluations The effect of HS on measures of WSP-induced airway and systemic 
inflammation compared to baseline measures will be assessed as 
follows:  
a) sputum induction: sputum % PMNs (primary endpoint) and mucus-
based parameters 
b) gamma scintigraphy scan of radiolabeled Tc99 sulfur colloid: MCC  
c) sputum induction and EBC: airway inflammatory cytokines and 
markers of oxidative stress 
d) venipuncture: systemic inflammatory markers and cytokines 
All measurements will be performed at baseline and again 4 hours post-
WPS exposure, except for MCC which will be performed immediately 
following treatment with HS. Sputum induction will also be performed 24 
hours post WSP challenge. The change in these measurements 
(postWSP – preWSP) will be compared for each interventional arm (HS 
v. no treatment).  
Safety Evaluations 
Vital signs, symptom questionnaire, spirometry 
Statistical And 
Analytic Plan 
Our primary endpoint is the change in sputum %PMNs from baseline to 
4 hours post-WSP exposure, comparing inhaled HS treatment to no 
treatment. For this analysis, after checking that there is no carry-over 
effects, we will use the one sample t-test to test for the treatment effect. 
Confidence intervals for the potential treatment effects will also be 
provided. We will also explore other analytic approaches such as the 
random effects model as outlined in further detail below. Analysis of 
secondary endpoints (change in %PMNs 24 hours post WSP challenge, 
total mucin concentration, mucus composition, MCC, sputum and EBC 
inflammatory and oxidative stress markers, and systemic inflammatory 
markers, comparing inhaled HS treatment to no treatment) will be 
performed similarly. In all analyses, criterion for significance will be 
p≤0.05. 
DATA AND SAFETY 
MONITORING PLAN 
A study coordinator will enter data into REDCap, and then a second 
person will verify the data. Subjects will be monitored in real time for 
safety by study staff. A study physician will be immediately available 
during inhalation and treatment procedures and will determine the 
severity and relatedness of any adverse events (AEs). The PI will 
conduct ongoing assessment of safety with strict adherence to pre-
specified study-suspension rules. All serious adverse events (SAEs) 
will be reported to UNC IRB and funding agency within 24 hours, and 
all AEs will be reported to UNC IRB and funding agency annually. A 
formal DSMB charter meeting DOD approval will be established prior 
to commencing study. The DSMB will address issues regarding safety 
concerns, efficacy concerns, termination of the trial due to pre-
specified stopping criteria, and ethical concerns. The DSMB will be 
furnished with relevant information by the Principal Investigator to 
make these decisions. 
 
 

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1 
BACKGROUND AND RATIONALE 
1.1 
Introduction 
Military deployment is associated with exposure to novel particulate matter (PM), such as 
from burn pits, aeroallergens, and increased cigarette consumption (1-5).  Warfighters 
exposed to these inhalational exposures exhibit immediate and chronic respiratory 
morbidity (5). For example, military service personnel surveyed in both the Republic of 
Korea (ROK) and Kabul, Afghanistan reported a general increase in respiratory morbidity, 
including asthma and chronic bronchitis, associated with their deployment (1, 6, 7).  Air 
contaminants in the ROK were characterized by elevated levels of both PM0.5-2.5 and PM2.5-
10. Similarly, exposures in Kabul were characterized by multiple airborne PM exposures, 
including those from burn pits. Burn pit PM includes metals, bioaerosols, organic by-
products, and biomass combustion particles. These findings indicate that inhaled PM is a 
likely cause of respiratory morbidity in the field.  
 
Inflammation is a key initial response to inhaled particulates. Wood smoke particles (WSP) 
serve as a model agent to study PM-induced bronchitis. WSP inhalation generates 
reactive oxidant (and nitrosative) species which cause local injury of airway epithelial cells 
and release of damage-associated molecular patterns (DAMPs) that activate toll-like 
receptors (TLR) and IL-1-mediated innate immune responses by resident airway 
macrophages. Contamination of PM with bioaerosols, which contain lipopolysaccharide 
(LPS), also activates innate immune responses through TLR4 activation of resident airway 
macrophages. These complementary processes result in recruitment of neutrophils 
(PMN), which mediate luminal airway inflammation with release of toxic mediators such 
as neutrophil elastase and myeloperoxidase that promote acute and chronic bronchitis 
(8).  
 
Therefore, mitigation of PM-induced airway neutrophilic inflammation should be a key 
focus in order to reduce the respiratory morbidity of military personnel. We have studied 
a number of pro-inflammatory inhaled agents, such as nebulized LPS (9-11), ozone (O3) 
(12), and WSP, as models of acute neutrophilic bronchitis against which to test a number 
of therapeutic agents. To this effect, we have reported that inhaled fluticasone inhibits O3-
induced (12) and LPS-induced (13) neutrophilic inflammation, and that parenteral 
anakinra (14) and oral γ-tocopherol (15, 16)  inhibit neutrophilic responses to inhaled LPS. 
In addition to agents with inherent anti-inflammatory and anti-oxidant properties, rapid 
clearance of inhaled particles from airway surfaces is a complementary approach to 
reduce PM-induced airway inflammation. In this study, we propose the use of 5% 
hypertonic saline (HS) to enhance mucociliary clearance (MCC) following WSP exposure 
to mitigate airway inflammation to WSP.  
 
1.2 
Name and Description of Investigational Product or Intervention  
Sodium Chloride 5% solution (HS) for inhalation (prepared from 7% Hypersal®, PARI 
Respiratory Equipment, Inc, Midlothian, VA). 
 
1.3 
Non-Clinical and Clinical Study Findings 
MCC is dependent on airway secretory cells and submucosal glands that produce a 
mucin-rich fluid layer on the airway surface and ciliated cells that hydrate and propel 

8 
 
mucus out of the lung and into the upper airway. Rates of MCC are dependent on ciliary 
beat frequency, hydration, and the rheologic properties of mucus. In vitro studies have 
demonstrated that HS, through an osmotic effect on airway surfaces, improved hydration 
and mucus rheologic properties, and accelerated mucus transport rates. In addition, our 
data over the last 30 years has shown that inhaled hypertonic saline (HS) plus cough (17, 
18) is the most effective method for acutely clearing the bronchial airways of inhaled, 
deposited particles. The combined effect is greater than either HS (19) or cough (20) 
alone. In our studies of asthmatics, we examined the ability of a single HS treatment with 
a coached cough maneuver to acutely clear radiolabeled Tc99m sulfur colloid particles 
from airways following LPS exposure (21). Following HS inhalation and cough clearance 
maneuvers developed to recover sputum samples for analysis, we observed a rapid 
clearance of >50% of the inhaled radiolabelled particles. We hypothesize that if other pro-
inflammatory particles (PMs, burn pit particles) were cleared similarly via HS-induced 
acceleration of MCC shortly after exposure, there would be reductions in acute PM-
induced inflammation. Thus, in this study, we will assess the effectiveness of inhaled 5% 
HS, a dose well tolerated by asthmatics at baseline and after inhaled LPS/allergen 
challenges (21), in mitigating WSP-induced airway neutrophilic inflammation in healthy 
volunteers. Normal saline 0.9% (NS) is not going to be used as a placebo treatment in this 
study, as inhalation of NS itself impacts the rheologic properties of mucus and MCC and 
thus would not be a suitable placebo.   
 
1.4 
Relevant Literature and Data 
Much of the published data relevant to this proposal has been generated by investigators 
in the UNC Center for Environmental Medicine, Asthma and Lung Biology (CEMALB) and 
has been referenced in above discussions. This body of literature also highlights our 
extensive experience in performing the evaluation tools used in this study, including, but 
not limited to, WSP exposure, sputum induction and processing, MCC assessment, and 
spirometry. Complete references for these publications have been provided in Section 14. 
 
2 
STUDY OBJECTIVE 
  
2.1 
Primary Objective 
The primary objective of this study is to examine the effectiveness of a single treatment of 
inhaled 5% HS on mitigating WSP-induced airway inflammation, measured by sputum 
%PMNs, in healthy adults 6 hours post-WSP exposure.  
 
2.2 
Secondary Objective 
Secondary objectives of this study include examining the effect of inhaled 5% HS on 
additional features of airway and systemic inflammation such as mucus-based 
parameters, mucociliary clearance (MCC), inflammatory and oxidative stress markers 
from sputum and exhaled breath condensate (EBC), and systemic inflammatory markers. 
Additionally, effects of 5% HS on WSP-induced neutrophilic airway inflammation 24 hours 
post-WSP exposure will also be assessed.  
 
3 
INVESTIGATIONAL PLAN 
 
3.1 
Study Design 

9 
 
• 
Type of design: Randomized, open-label, cross-over study 
 
• 
Study phases:  
a) Screening: Prior to enrollment in this study, participants will take part in two 
separate screening protocols: a general screening protocol (IRB #98-0779) and a 
screening WSP exposure protocol (IRB #15-1775). The general screening protocol 
will identify healthy volunteers suitable for WSP exposure. These healthy adults 
will undergo the screening WSP exposure protocol to identify those individuals with 
a >10% increase in sputum %PMNs following WSP exposure, who will then be 
invited to participate in this study.  
 
b) Visits 0, 1, and 2 – Baseline (Period 1) and Training Day: Along with an informed 
consent, a medical screening, including vital signs and spirometry, will be 
performed to confirm eligibility. Volunteers will be trained on the cycle ergometer 
to reach an exercise level producing a minute ventilation of 20 L/min/m2 body 
surface area (BSA) while being monitored with telemetry (EKG) per our 
established WSP exposure procedure. Finally, a baseline MCC scan, sputum 
induction, and venipuncture will be obtained, which will provide the “pre-WSP” 
measurements for primary and secondary data analyses. A 30 minute retention 
scan will be performed 24 hours after baseline MCC to determine amount of 
Tc99m-SC retained in airways. 
 
c) Visits 3 (Period 1) and 7 (Period 2) - WSP exposure: Participants will return within 
4 weeks of Visit 0 to undergo WSP exposure. Spirometry, venipuncture, and EBC 
will be obtained prior to exposure. After WSP exposure, subjects will inhale 
radiolabeled Tc99m sulfur colloid (Tc99m-SC) to start MCC assessment. PM 
deposition imaging will be obtained, followed by treatment with 5% HS (15 ml) x15 
min delivered by a PeriLC star nebulizer with a coached cough maneuver (vs. no 
treatment x15 min). Participants will have MCC measured for a total of 120 
minutes. EBC, venipuncture, and sputum induction will be obtained 6 hours post-
WSP exposure, which will provide the “post-WSP” measurements for primary and 
secondary data analyses. Spirometry and symptom questionnaires will be 
obtained pre- and post-exposure to assess for safety endpoints.  
 
d) Visits 4 (Period 1) and 8 (Period 2) - 24 hours post-exposure follow-up: Any 
symptoms post-WSP exposure will be reviewed and spirometry repeated along 
with EBC, venipuncture and sputum induction. Participants will undergo a 30-
minute scan to determine amount of Tc99m-SC retained in airways. 
 
e) Visits 5 and 6 – Cross-over and Baseline (Period 2): Participants will return after 
at least a 2 week wash-out period. Interval medical history will be reviewed and 
repeat MCC, spirometry, venipuncture and sputum induction obtained as pre-WSP 
measurements for period 2. A retention scan will be performed 24 hours after 
baseline MCC for period 2. Participants will then be crossed over to the alternative 
treatment group and undergo visits 7 and 8 as above. 
 

10 
 
f) Visit 9 – Study Completion: Five to 10 days after the second exposure, participants 
will return for a study completion visit in which medical history and symptom 
questionnaires will be reviewed. If the participant has any health symptoms or 
concerns, they will receive a physical examination and evaluation by a study 
physician. 
 
g) Unscheduled Visits: We do not anticipate any unscheduled visits. Subjects will be 
rescheduled if they have a change in health status that requires a delay in study 
visits. If this causes a visit to be out of window, the subject can have an 
unscheduled visit at the discretion of the PI. 
 
3.2 
Allocation to Treatment Groups and Blinding 
This is an open-label study. Dr. Haibo Zhou, the study biostatistician, will have a colleague 
prepare the randomization schedule using permuted block randomization with a block size 
of 4 (2 HS, 2 no treatment for the first treatment period of the protocol).  The colleague will 
send this schedule directly to the UNC investigational drug service. 
 
3.3 
Study Duration, Enrollment and Number of Subjects 
Each subject will have 10 study visits (excluding the two separate screening protocols – 
the general screening protocol and the screening WSP exposure protocol – as detailed 
above) and remain in the study for approximately three months. The expected duration of 
the entire study is 4 years. A total of 14 subjects will be enrolled into this study.  
 
3.4 
Study Population 
Inclusion Criteria: 
1. Age 18-45 years, inclusive, of both genders 
2. Negative pregnancy test for females who are not s/p hysterectomy with 
oopherectomy 
3. No history of episodic wheezing, chest tightness, or shortness of breath consistent 
with asthma, or physician-diagnosed asthma. 
4. FEV1 of at least 80% of predicted and FEV1/FVC ratio of ≥0.70.  
5. Oxygen saturation of ≥93% 
6. Ability to provide an induced sputum sample, determined by screening protocol 
IRB#98-0799 
7. Subject must demonstrate a ≥ 10% increase in sputum %PMNs 6 hours following 
inhaled WSP exposure, when compared to baseline sputum (to be completed in a 
separate protocol IRB# 15-1775). 
8. Documentation of Covid vaccination based on current public health guidelines.  
 
Exclusion Criteria: 
Subjects who meet any of these criteria are not eligible for enrollment as study 
participants: 
1. Clinical contraindications: 
a. Any chronic medical condition considered by the PI as a contraindication 
to the exposure study including significant cardiovascular disease, 
diabetes, chronic renal disease, chronic thyroid disease, history of chronic 
infections/immunodeficiency. 

11 
 
b. Viral upper respiratory tract infection within 4 weeks of challenge. 
c. Any acute infection requiring antibiotics within 4 weeks of exposure or fever 
of unknown origin within 4 weeks of challenge. 
d. Abnormal physical findings at the baseline visit, including but not limited to 
abnormalities on auscultation, temperature of 37.8° C, Systolic BP > 
150mm Hg or < 85 mm Hg; or Diastolic BP > 90 mm Hg or < 50 mm Hg, or 
pulse oximetry saturation reading less than 93%. 
e. Physician diagnosis of asthma 
f. 
If there is a history of allergic rhinitis, subjects must be asymptomatic of 
allergic rhinitis at the time of study enrollment. 
g. Mental illness or history of drug or alcohol abuse that, in the opinion of the 
investigator, would interfere with the participant’s ability to comply with 
study requirements. 
h. Medications which may impact the results of the WSP exposure, interfere 
with any other medications potentially used in the study (to include steroids, 
beta antagonists, non-steroidal anti-inflammatory agents) 
i. 
Cigarette smoking > 1 pack per month 
j. 
Unwillingness to use reliable contraception if sexually active (IUD, birth 
control pills/patch, condoms). 
k. Use of immunosuppressive or anticoagulant medications including routine 
use 
of 
NSAIDS. 
Oral 
contraceptives 
are 
acceptable, 
as 
are 
antidepressants and other medications may be permitted if, in the opinion 
of the investigator, the medication will not interfere with the study 
procedures or compromise safety and if the dosage has been stable for 1 
month. 
l. 
Orthopedic injuries or impediments that would preclude bicycle or treadmill 
exercise. 
m. Inability to avoid NSAIDS, Multivitamins, Vitamin C or E or herbal 
supplements for 4 days prior to study visits. 
n. Allergy/sensitivity to study drugs or their formulations 
o. Positive Covid test within 90 days of the study visit.  
2. Pregnant/lactating women and children (< 18 years as this is age of majority in 
North Carolina) will also be excluded since the risks associated with WSP 
exposure to the fetus or child, respectively, are unknown and cannot be justified 
for this non-therapeutic protocol. Individuals over 45 years of age will not be 
included due to the increased possibility of co-morbidities and need for prohibited 
medications. 
3. Inability or unwillingness of a participant to give written informed consent 
 
4 
STUDY PROCEDURES  
 
4.1 
Screening Visits:  
General Screening: To identify healthy volunteers suitable for WSP exposure, subjects will 
complete a general screening protocol (UNC IRB approved study #98-0799, Database and 
Screening Protocol for Research Studies of the Center for Environmental Medicine & Lung 
Biology). 
 

12 
 
Screening WSP exposure: This protocol will identify subjects who experience a ≥ 10% increase 
in sputum %PMNs after WSP exposure, compared to baseline sputum (UNC IRB approved study 
#15-1775, To identify persons who are susceptible to WSP-induced inflammation and examine 
the role of GSTM1 and other factors in this susceptibility).  
4.2 
Visit 0: Baseline Visit (at least 2 weeks after screening WSP exposure): 
1. Informed consent 
2. Medical history 
3. Vital signs, including oxygen saturation 
4. Urine pregnancy test 
5. Physical exam 
6. 12-lead electrocardigram (EKG) 
7. Spirometry 
 
4.3 
Visit 1: Training Day 
1. Urine pregnancy test (if not within 7 days of visit 0) 
2. Vital signs, including oxygen saturation 
3. Volunteers will be trained on the cycle ergometer to reach an exercise level producing 
a minute ventilation of 20 L/min/m2 body surface area (BSA). Subjects will be 
monitored with a standard 3-lead EKG during exercise (15 minutes). 
4. MCC 
5. Sputum induction 
 
4.4 
Visit 2: Retention Scan (24 hours after Visit 1) 
1. MCC retention scan (30 minutes) 
 
4.5 
Visits 3 (Period 1) and 7 (Period 2): WSP exposure (within 6 weeks of Visit 0 for Period 
1 and Visit 5 for Period 2) 
1. Vital signs, oxygen saturation 
2. Symptom questionnaire 
3. Urine Pregnancy Test (if not previously performed within 7 days) 
4. Venipuncture [CBC with differential, serum cytokines, serum CRP, GSTM1 genotype 
(Period 1 only)] 
5. EBC 
6. Spirometry 
7. 500 μg/m3 WSP chamber exposure for 2 hours, with intermittent exercise and rest. 
Subjects will alternate 15 minutes of exercise on a cycle ergometer (intensity needed 
to achieve 20 L/min/m2 BSA determined at baseline visit) with 15 minutes of rest. 
8. Symptom questionnaire (immediately prior to exit from the chamber) 
9. Immediately following exit from chamber, subjects will inhale radiolabeled Tc99m-SC 
after which PM deposition imaging will be obtained and MCC will be started.  
10. At the start of the MCC scan, subjects will inhale 15 ml of 5% HS x 15 minutes 
delivered by a PeriLC star nebulizer with a coached cough maneuver (vs no treatment, 
including no cough maneuver) 
11. Symptom questionnaire following completion of HS treatment (if received) 
12. Complete MCC for a total of 120 minutes.  
13. Symptom questionnaire (4 hours post-WSP exposure) 
14. EBC (4 hours post-WSP exposure) 
15. Spirometry (4 hours post-WSP exposure) 
16. Sputum Induction (4 hours post-WSP exposure) 

13 
 
17. Venipuncture for CBC with differential, serum cytokines, CRP (4 hours post-WSP 
exposure) 
 
4.6 
Visits 4 (Period 1) and 8 (Period 2) (24 hours post-WSP exposure) 
1. Review any adverse events 
2. Vital signs, oxygen saturation 
3. Symptom questionnaire 
4. EBC 
5. Retention scan 
6. Spirometry 
7. Venipuncture (CBC with differential, serum cytokines, serum CRP) 
8. Sputum Induction 
Participants will be contacted by phone 3-7 days following Visit 4 to assess for any delayed 
adverse events. No phone call will be made after Visit 8 as participants will be returning for a 
study completion visit as below.  
A 2 week washout is required between Period 1 and Period 2. 
4.7 
Visit 5: Baseline (Period 2) (following a minimum 2 week washout) 
1. Review medical history and adverse events 
2. Urine pregnancy test 
3. Vital signs, oxygen saturation 
4. MCC 
5. Spirometry 
6. Venipuncture (CBC with differential only) 
7. Sputum Induction 
 
4.8 
Visit 6: Retention Scan (Period 2) (24 hours after visit 5) 
1. MCC retention Scan (30 minutes) 
 
4.9 
Visit 9: Study Completion [5-10 days following WSP exposure during Period 2 (Visit 7)]  
1. Review any adverse events 
2. Vital signs, oxygen saturation 
3. If the participant has any health symptoms or concerns, they will receive a physical 
examination and evaluation by a study physician. 
 
4.10 
Unscheduled visits 
Any subject who has an unexpected adverse event (AE) as a result of the study treatment 
will have access to a study physician. Subjects will be invited back to the lab for vital signs 
collection, physical exam, and spirometry. If the subject has any AE not associated with 
the study, he/she will not complete the study until the AE resolves such that it does not 
interfere with the study or with safety. If this causes a visit to be out of window, the subject 
can have an unscheduled visit at the discretion of the PI. 
 
4.11 
Concomitant Medication documentation 
Concomitant medications are collected at the screening visit and throughout the study 
visits.   
 
4.12 
Rescue medication administration 

14 
 
As the study population consists of healthy volunteers, we do not anticipate subjects 
requiring any rescue medication administration. However, in the event that subjects 
experience bronchospasm with symptoms of cough, chest tightness, or wheezing 
associated with WSP exposure or HS treatment, the subject will be evaluated by a study 
physician. If necessary, rescue medication in the form of albuterol, a short-acting 
bronchodilator, will be readily available and administered per study physician 
recommendations. There is also a code cart immediately available with epinephrine 
autoinjectors. Oxygen is available for supplementation if required. 
 
4.13 
Subject Withdrawal and Study Suspension Procedures 
Subjects may withdraw at any point in the study and will be asked to return to the lab for 
a final visit if they withdraw from the study prior to completion of all study procedures to 
monitor safety.   Subjects may be withdrawn at any time for safety concerns.   
Criteria for safety prior to initiation of wood smoke inhalation challenge: (Subjects not 
meeting these criteria the morning of challenge will not proceed with wood smoke 
inhalation challenge): 
1. FEV1 of at least 80% of predicted and FEV1/FVC ratio of ≥ 0.70.  
2. Baseline oxygen saturation of at least 93%. 
3. No history of physician-diagnosis asthma or frequent cough, wheezing, or shortness 
of breath consistent with a history of asthma. 
4. No history of viral respiratory tract symptoms within 4 weeks of challenge. 
5. No current symptoms of rhinorrhea, sneezing, nasal or ocular pruritus consistent with 
allergic rhinoconjunctivitis. 
Criteria for safety of a given individual following WSP exposure and treatment which would 
suspend the individual from further participation in the study will include: (Subjects will be 
withdrawn for the following reasons): 
1. Failure of return of FEV1 without treatment to within 90 % of baseline within 6 hours. 
2. A symptom score greater than 45 (representing a ranking of “moderate” on each of 15 
criteria, with a maximum score of 60, see appendix) without improvement to a score 
of 15 or less within 6 hours without therapy. 
3. Specifically, a symptom score greater than “moderate” (3 on a scale of 0-4) for 
shortness of breath or cough at 24 hours post-challenge. 
4. A reduction in oxygen saturation to <93% during the 6 hour observation period post-
WSP exposure 
5. Need for rescue albuterol therapy during the 6 hours observation period post-WSP 
exposure 
6. If a subject contracts a Covid infection during the time they are participating in the 
study, they will be paused for 90 days, and they will be re-evaluated for eligibility after 
that time.  
 
Criteria for safety within the entire protocol (failure of which would result in suspension of 
further study until consultation with study sponsor, UNC IRB, and DSMB) will include the 
following: (Study will be suspended for the following reasons):  
 
1. If 3 of the first 10 participants fail the individual safety criteria outlined above 

15 
 
2. Once 10 subjects have been enrolled, study suspension will occur if more than 25% 
of all participants fail the individual safety criteria outlined above 
3. Occurrence of any study-related Serious Adverse Event (SAE) 
4.14 
Screen failure procedures 
For subjects who do not meet enrollment criteria, all study related activity will stop once 
it is determined that the subject does not meet criteria. 
 
5 
STUDY EVALUATIONS AND MEASUREMENTS 
 
• 
List variables that will be abstracted from medical charts: none 
 
• 
Describe baseline evaluation 
As listed above for baseline visit and training visit of period 1 (Section 4.2), vital signs, 
including HR, RR, temperature and BP will be collected after a thorough review of the 
medical history. Oxygen saturation levels will also be noted followed by a brief physical 
examination, including but not limited to the cervical lymph nodes, eyes, ears, nose, 
throat, cardiovascular and respiratory systems. Laboratory evaluations will include 
urine pregnancy test and venipuncture (see below for details). In addition, baseline 
spirometry, MCC, EBC, and sputum induction will be performed as detailed below. 
Subjects will also undergo training on the cycle ergometer to reach an exercise level 
producing a minute ventilation of 20 L/min/m2 BSA in preparation for the WSP 
exposure during Visits 3 (period 1) and 7 (period 2). Baseline visit for period 2 (Section 
4.5 above) will include all of the measurements obtained for baseline visit and training 
visit of period 1, but subjects will not require additional training on the cycle ergometer.  
 
• 
Describe how measurements will be taken. 
a) Woodsmoke particle chamber exposure: We will employ the exposure protocol 
as described by Ghio and colleagues (22). We will heat red oak wood on an 
electric heating element (Brinkmann, Dallas, Texas, USA) in a Quadrafire 3100 
woodstove (Colville, Washington, USA) at 800°F to generate wood smoke. The 
smoke will be extracted from the chimney and injected into the chamber air 
stream, and WSP concentration will be controlled using a tapered element 
oscillating microbalance (TEOM; Thermo Fisher Scientific, Franklin, 
Massachusetts, USA) to measure the chamber concentration. A Model DR-
4000 DataRAM (Thermo Fisher Scientific) will also be used to measure the 
concentration. The particle number concentration will be measured with a 
Model 3775(TSI, Shoreview, Minnesota, USA) and the number and size 
distribution will be measured with a Model 3936L75 SMPS (TSI). The particle 
concentration will be verified by weighing filters obtained using a versatile air 
pollution sampler (VAPS; URG, Chapel Hill, North Carolina, USA). The 
chamber temperature and humidity will be controlled to approximately 22°C 
and 40%, respectively. The concentrations of WSP we will use for this study 
will not exceed 500 μg/m3, with mild exercise to increase minute ventilation (20 
L/min/m2 BSA) for 15 minute periods, alternating with 15 minute rest periods 
across an overall 2 hour total exposure period. Subjects will be trained during 
Visit 0 on the cycle ergometer to reach this minute ventilation. A 12 lead-EKG 
will be performed prior to training for safety purposes, and subjects will be 
monitored with a standard 3 lead EKG during exercise.  
 

16 
 
b) Sputum Induction: Sputum samples will be collected using a specific sputum 
induction protocol in which FEV1 and FVC will be measured to determine the 
baseline FEV1 and FVC values. The FEV1 values that match a 10% and 20% 
fall from baseline will be calculated. An ultrasonic nebulizer will be filled with 
15 mL of 3% hypertonic saline (inhalation grade for respiratory use only) to 
begin the test. The nebulizer will be set to the maximum output setting and 
turned on. The subject will be instructed to breathe normally (i.e. tidal breaths) 
through the nebulizer mouthpiece for 7 minutes. The saline will be nebulized 
through the mouthpiece in a jet stream and inhaled. The nose will not be 
occluded for this procedure. Prior to expectoration, subjects will blow their 
nose, rinse their mouth with water, and clear their throat to avoid the inclusion 
of non-airway fluid samples. The subject will also be encouraged to come off 
the mouthpiece at any time to cough if a sputum sample from the lower airways 
(i.e. not from the back of the throat) is ready for expectoration. The sample will 
be coughed into a sterile specimen jar and capped. Following repeat 
measurement of FEV1 after the first inhalation period, the concentration of 
saline will be increased from 3% to 4%, provided the FEV1 falls by <10% from 
the baseline value. The same procedure will be followed for the final inhalation 
period using 5% hypertonic saline. The nebulization will be stopped at that 
point, or earlier, if a sputum sample of good quality is obtained (i.e. visible 
sputum plugs). The 4% and 5% saline solutions will be mixed for each induction 
by study staff just prior to induction and a volume of 15-20 mL will be used on 
each occasion. Participants will not be pre-treated with Albuterol as is our 
standard procedure with healthy volunteers. Albuterol will be available if 
needed. Sputum sample will be used to evaluate for inflammatory cell 
composition and mucus-based parameters, such as total mucin concentration, 
% solid content, and MUC5AC/MUC5B.  
 
c) Spirometry: Spirometry will be performed according to American Thoracic 
Society (ATS) guidelines. Subjects will inhale as deeply as possible then 
exhale as rapidly and completely as possible into the spirometer. 
Measurements obtained from each maneuver include the FVC, the FEV1, the 
maximal mid-expiratory flow rate (FEF25-75) and the peak flow. The largest FVC 
and FEV1, from at least 3 acceptable trials will be selected for analysis and 
subject must meet the appropriate inclusion/exclusion criteria based on 
comparison of the subject’s measurements to the predicted value for an 
individual of his/her gender, age, race and height with NHANES III as the 
predicted set. 
 
d) Exhaled breath condensate: EBC will be collected at individual study sites via 
established protocols (23) using the commercially available RTube 
(Respiratory Research, Inc., Charlottesville, VA)—a handheld, self-contained, 
single-use device. The device consists of a mouthpiece connected to a one-
way valve that directs exhaled breath through a condenser tube cooled by a 
cooling sleeve. Gaseous phase liquid and aerosols in exhaled breath 
condense into liquid form on the inside surface of the chilled condenser tube 
and can be extracted using a plunger device supplied by the manufacturer. 
The specific collection protocol would include chilling the aluminum cooling 
sleeve to -20º C, then placing this cooling sleeve with insulated cover over the 
RTube device immediately before collection. Once the cooling sleeve is in 

17 
 
place, the subject breathes tidally through the mouthpiece for 15 minutes. EBC 
samples are stored at -80º C pending analysis. 
e) Mucociliary clearance: Prior to each MCC study, a transmission Cobalt57 
(Co57) scan will be performed to define the lung boundaries, to assign regions 
of interest, and to normalize these regions for lung volume differences. A 
rectangular phantom containing the radioisotope Co57 (<25 microcurie (mCi)) 
will be placed in front (5cm) of the subject sitting with his/her back to the gamma 
camera for 30 seconds. Prior to the transmission scan before each MCC 
assessment, we will place 2 spot markers of Americium241 (0.9 mCi each, 
gamma 66 kilo electron volts) on the upper and lower back of each subject 
during scanning (both Tc99m-SC deposition/retention and Co57 transmission). 
With dual isotope imaging, these spot markers will allow alignment of images 
for more accurate determination of regional deposition/retention. These very 
low radiation sources have been obtained from commercially available home 
smoke alarms. The placement of these markers will be determined to be 
outside the lung field during the transmission scan. Their location will be 
marked in semi-permanent ink for later placement during Tc99m 
deposition/retention scans. The shielded side of this source will be 
placed/taped onto the subject’s skin. 
 
Radiolabeled Tc99m-SD will be delivered using a modified Pari-LC Star 
nebulizer (MMAD 9.5 um). This is a closed delivery system that produces 80 
ml/sec air flow, and therefore limits the inspiratory flow rate to this value.  While 
seated in front of a gamma camera subjects will perform single inhalations 
lasting ~10 seconds each from the delivery system, and will exhale at 500 
ml/sec (using feedback from a flow meter in the breathing circuit).  
Approximately 5 of these inhalation maneuvers will be required to deposit an 
adequate isotope dose to the lung.  Subjects will be allowed to breathe 
normally (off the nebulizer) in between each inspiratory maneuver. Each 
volunteer will practice these maneuvers prior to the actual radioaerosol 
inhalation to guarantee his/her proficiency. The activity of Tc99m-SC loaded in 
the nebulizer will be adjusted to provide an estimated 40 mCi deposited in the 
lung for each MCC scan. A single crystal detector will be placed at the subject’s 
back during inhalation to monitor dose to the lung. Total inhalation time should 
be less than 5 minutes in all cases. Immediately following isotope inhalation, 
the subject will gargle and drink water to clear activity that deposited in the 
mouth into the stomach. The subject will then (within a minute of final inhalation 
maneuver) be seated in front of a large-field-of-view gamma camera to begin 
acquiring particle retention images.  
 
For each MCC measurement, the gamma image capture will capture 
continuously for the first 34 minutes. In the WSP exposure visit in which 
subjects are randomized to receive HS treatment (either visit 3 or 7), 
participants will inhale HS as described in Section 7 at the onset of gamma 
image capture. Image capture will be continuous during HS inhalation (~15 
minutes) and coached cough maneuver and will continue for a total 34 minutes, 
after which 2 consecutive 2-minute images will be obtained at the start of every 
10-minute period until 2 hours have passed. The subject will also return the 
day following each MCC measurement to sit in front of the gamma camera for 
30 minutes to obtain images of the lungs to visualize retention of Tc99m-SC 

18 
 
particles. No additional Tc99m-SC will be administered at this visit. MCC will 
be obtained in the same manner (and for the same length of time) when 
subjects are randomized to receive no treatment (HS inhalation and cough 
maneuver are omitted).  
 
• 
Describe rating scales, tests, psychological tools, laboratory evaluations, etc. 
a) Urine pregnancy test (UPT): A UPT will be obtained on all females who are not 
s/p hysterectomy with oophorectomy at visits 0, 1, 3, 5, and 7. If positive, these 
women will be excluded from the study. UPTs are valid for 7 days. 
b) Venipuncture: Blood will be drawn to evaluate a CBC with differential, CRP, 
and inflammatory cytokine profile pre- and post-WSP exposure at Visits 3 and 
7. At the time of each venipuncture, up to 50 cc will be drawn, and the total for 
the study will be no more than 200 cc. 
c) Symptom questionnaire: To access for safety throughout the study, symptom 
questionnaires will be performed pre-WSP exposure (visits 3 and 7), 
immediately post-WSP exposure, post-HS treatment (if received), 6 hours 
post-WSP exposure, and 24 hours post-WSP exposure (visit 4 and 8).The 
questionnaire consists of 15 symptoms, requiring a rating from 0-4 with a 
minimum score of 0 and maximum score of 60. See Appendix for symptom 
questionnaire. 
 
5.1 
Efficacy Evaluation 
The effect of HS on WSP-induced airway and systemic inflammation will be assessed with 
the following measurements:  
a) sputum induction: sputum % PMNs (primary endpoint) and mucus production and 
composition 
b) gamma scintigraphy scan of radiolabeled Tc99 sulfur colloid: MCC  
c) sputum induction and EBC: airway inflammatory cytokines and markers of oxidative 
stress 
d) venipuncture: systemic inflammatory markers and cytokines 
All measurements will be performed at baseline and again 6 hours post-WSP exposure, 
except for MCC which will be performed immediately following treatment with HS. An 
additional sputum induction will also be performed 24 hours post-WSP exposure to 
evaluate airway inflammatory cell composition. The change in these measurements 
(postWSP – preWSP) will be compared for each interventional arm (HS v. no treatment). 
See Section 6 for detailed primary and secondary endpoints.  
5.2 
Pharmacokinetic Evaluation: n/a 
 
5.3 
Safety Evaluations 
Safety evaluations will be performed with the following measures with strict adherence to 
subject withdrawal and study suspension criteria (Section 4.10):  
a) Physical exam and vital signs: In addition to a limited physical exam of the eyes, 
ears, nose, throat, and respiratory and cardiovascular systems, we will also check 
vital signs (temperature, HR, BP, RR, and oxygen saturations) at each baseline 
visit (0 and 5), pre-WSP and 6 hours post-WSP exposure (visits 3 and 7), and 24-
hours post-WSP (visits 4 and 8). Additional exams and vital signs will be performed 
at physician discretion if needed based on the participant’s symptoms.  

19 
 
 
b) Spirometry: Spirometry will also be performed at each baseline visit (0 and 5), pre-
WSP and 6 hours post-WSP exposure (visits 3 and 7), and 24-hours post-WSP 
(visits 4 and 8). Healthy volunteers have been shown to tolerate WSP exposure 
well without any changes in lung function (22). Additionally, while HS may 
potentially induce bronchospasm, this is also generally well-tolerated in healthy 
volunteers without underlying airway hyper-responsiveness. Therefore, we will 
only obtain spirometry 6 hours following WSP exposure and HS treatment (if 
received). However, if participants assign a score greater than 3 (“moderate”) to 
symptoms attributable to bronchospasm (cough, wheezing, chest tightness, 
shortness of breath, or pain on deep inspiration), then they will be evaluated by the 
study physician to determine if additional spirometry and/or rescue treatment with 
albuterol is necessary. Additional spirometry will be performed at physician 
discretion.  
 
c) Symptom questionnaire (see Appendix): This will be performed pre-WSP, 
immediately before exiting WSP chamber, following HS treatment (if received), and 
6 hours post-WSP exposure (visits 3 and 7), and 24-hours post-WSP (visits 4 and 
8). Symptoms may be accessed at other time points during the study per physician 
discretion.  
 
d) Direct observation: Participants will remain under the direct observation of a study 
coordinator and study physician throughout each study visit and will be monitored 
for a minimum of 6 hours following each WSP exposure and HS treatment (if 
received).  
 
6 
STATISTICAL CONSIDERATION    
 
6.1 
Primary Endpoint 
Change in sputum %PMNs [Δ (postWSP – preWSP)[prednisone] vs Δ (postWSP – 
preWSP)[placebo]] 4 hours post-WSP exposure and at 24 hours post -WSP exposure 
 
6.2 
Secondary Endpoints 
1)  Induced Sputum: 
a) Granulocyte (e.g.eosinophil) numbers/mg and percentages at 4 and 24 hours post-
WSP exposure 
b) cytokine and chemokine concentrations (TH1 and TH2) via Mesoscale platform 
(pg/ml)   
6.3 Exploratory Outcome measures: 
1) Induced sputum: 
a) Oxidative stress markers [e.g. GSH/GSSG ratio, 8-isoprostane(pg/ml)] 
b) Mucus-based parameters including: total mucin concentration (µg/ml), % solid 
content,  MUC5AC and MUC5B concentrations (pmol/ml) and ratio, biophysics 
(cohesion; macro- and micro-bead rheology) 

20 
 
2)  EBC:   
a) Sialic acid/urea ratio  
b) purine mediators (µM) 
c) amino acids and dipeptides (µM) 
d) GSH/GSSG ratio 
3)  MCC measures: 
a) the central (C) vs. peripheral (P) deposition ratio (C/P), for regional airway 
deposition   
b) MCC, rate at which radiolabeled particles are cleared from the lung (reported as 
% particle retention vs time) 
c) skew, a measure of the particle deposition heterogeneity   
4)  Blood: 
a) Systemic inflammatory markers (granulocyte numbers/mL and percentages) 
b) Serum cytokine concentrations via Mesoscale platform (pg/mL) 
 
6.3 
Statistical Methods 
Our analytic plan was developed in collaboration with Dr. Zhou, the biostatistician for the 
CEMALB, who will oversee all statistical analyses. For analysis of our primary endpoint, 
change in sputum %PMNs at 6 hours post-WSP exposure, as well as other secondary 
endpoints within each treatment group, we will use (i) descriptive analysis, (ii) one-sample 
comparison test, and (iii) random effects regression analysis designed to take advantage 
of the crossover design (24). Specifically, we will use the tabular and graphic methods to 
describe/explore the basic relationship between the groups, including confidence intervals 
for all statistical estimates. We will then follow up by comparing the Δ sputum %PMNs 
(post WSP – pre WSP [HS]) vs (post WSP – pre WSP [no treatment]) using a one-sample 
t-test. Examination of the normality assumption will be incorporated into the sensitivity 
analyses, and nonparametric test will be conducted in the case of evidence of extreme 
departure from normality. Finally, we will explore a random effect regression modeling 
technique (Equation 1) used in previous studies that allows us to assess treatment effect 
while accounting for the period effect (14, 16). In all analyses, criterion for significance will 
be p≤0.05, and all results exceeding this threshold will be reported as inconclusive. In 
addition, all statistical estimates will be tabulated with corresponding confidence intervals. 
For secondary analyses, we will employ multiple variable regression modeling techniques 
to determine if specific individual risk factors (e.g.,BMI) impact either the effect of WSP 
exposure or the effect of HS treatment on the primary or secondary endpoints, similar to 
approaches previously described to assess genotype effects on pollutant responses (24). 
The secondary analyses will only be conducted after the primary hypothesis is tested and 
solely for exploratory purposes.  
 
Equation1 : Linear mixed model approach for global unified analysis of data for secondary and 
exploratory outcomes: 
 
 
 
 
 
Y_11k=μ+π_1+τ_1+βX_k+s_1k+ϵ_11k; Y_12k=μ+π_2+τ_2+λ_1+βX_k+s_1k+ϵ_12k 
 
Y_21k=μ+π_1+τ_2+βX_k+s_2k+ϵ_21k; Y_22k=μ+π_2+τ_1+λ_2+βX_k+s_2k+ϵ_22k     
μ is the grand mean, π1 and π2 are period effects, τ1 and τ2 are treatment effects, λ1 and λ2 
are carryover effects for different sequences, ς1k and ς2k are individual random effects within 
each sequence, and ε's are random errors. Subscript k is for subjects. 

21 
 
 
 
6.4 
Sample Size and Power 
Our primary endpoint is the change in sputum %PMNs pre- and 6 hours post-WSP 
exposure, comparing HS treatment to no treatment. In our ongoing WSP study, we found 
a mean increase from baseline in sputum %PMNs of 27.9% with SD of 14.3%. A previous 
study with 1 pre-treatment dose with high dose inhaled corticosteroids prior to 0.4 ppm 
O3-exposure produced a 35% reduction in mean increase of sputum %PMNs (12). 0.4 
ppm O3 yields a similar PMN response as 500 µg/m3 WSP. Assuming that rapid airway 
clearance with a single dose of HS has a similar effect (at least a 35% reduction in sputum 
%PMNs after WSP exposure), α=0.05, β=0.8, the SD is the same, and each pair of 
measurements on the same subject will have a correlation 0.5, a sample size of 20 would 
be required for a two-sided test. Based on our past experience with intervention studies, 
we have inflated this sample size to 24 participants for attrition related to cross-over 
studies and missing sputum samples. While we do not anticipate a significant problem 
with missing data, we will determine whether there is differential dropout, and use multiple 
imputation methods to address missing data as previously described (25).   
 
New information added March 25, 2022: The separate “Smokescreen” study (IRB 15-1775), 
from which these  subjects are drawn, was able to complete 50 screening exposures, 
determining that 28 met the predefined “responder” definition for eligibility for this project.  
These samples were collected pre-pandemic and were processed during the pandemic pause, 
yielding results that the responder group has a mean baseline of 24% PMN which increases to 
40% at 6 hrs and 52% 24 hrs post exposure [1; personal communication, D. Peden].   The 
availability of these new data have led to concern that the enrollment target should be amended, 
since the new dataset should allow a more accurate sample estimate.   
 
There are no published or preliminary data on effect size for hypertonic saline, but in 
collaboration with Dr. Haibo Zhou, using the “Smokescreen” sputum data, estimating the saline 
effect size to be a 50% inhibition of WSP-induced sputum neutrophilia based on a previously 
published study of prednisone effect on sputum neutrophilia after controlled ozone exposure [2], 
and assuming a more conservative standard deviation for the prednisone effect size than the 
one reported in that study, we estimate that the N for this project to detect a significant saline 
effect should be 12 rather than 20.  Anticipating 5-10% of subjects will produce inadequate 
samples, we propose a goal of completing 14 subjects to obtain 12 complete datasets.  
 
 
6.5 
Interim Analysis 
We do not anticipate HS will cause toxicity based on our data that 5% HS is well tolerated 
by asthmatics at baseline or after inhaled LPS/allergen challenges (21). However, as HS 
has not been tested post-WSP, we will undertake an interim analysis to assess safety and 
futility after 12 subjects have completed the study. 
 
7 
STUDY INTERVENTION  
 

22 
 
• 
Description 
Sodium chloride 5% solution, which is diluted from commercially available sodium 
chloride 7% solution (Hypersal®; PARI Respiratory Equipment, Inc, Midlothian, VA), 
followed by coached cough maneuver 
 
• 
Receipt/Storage 
HS will be delivered and stored within the CEMALB. 
 
• 
Packaging/Labeling 
This is an open label study. 
 
• 
Dosing 
Fifteen ml of 5% HS is nebulized and inhaled until the dose is complete, typically 15 
minutes. The subject will be instructed on the proper use of the PARI LC Star Nebulizer 
System, an approved device for aerosol inhalation. The subject will use the nebulizer 
until the HS dose is completed followed by a coached cough maneuver (similar to a 
sputum induction).  
 
• 
Treatment compliance and Adherence 
Subjects are observed during all dosing periods. 
 
• 
Drug Return/Destruction: n/a 
 
• 
Drug Accountability: n/a 
 
8 
STUDY INTERVENTION ADMINISTRATION 
 
• 
Randomization procedures 
The randomization schedule will be generated by one of Dr. Zhou’s colleagues using 
permuted block randomization with a block size of 4 (2 HS, 2 no treatment for the first 
treatment period of the protocol).   
 
• 
Blinding procedures 
This is an open-label study.  
 
• 
Unblinding procedures: n/a 
 
9 
SAFETY MANAGEMENT 
 
• 
Definition of Adverse Event (AE) and Serious Adverse Event (SAE) 
An AE for a given volunteer will be defined as failure of any of the safety criteria 
outlined above. Any symptoms that induce a volunteer to seek medical attention from 
any provider within 96 hours of a study visit will also be considered an AE. A SAE will 
be defined as any event that requires hospitalization or results in life threatening illness 
or injury, permanent (or likely to be permanent) illness or injury, or death if these events 
occur within 96 hours of a study visit (or if the clinical scenario leading up to 
hospitalization, illness, injury or death begins within 96 hours of a study treatment visit).   

23 
 
 
• 
Grading criteria 
• 
In addition to determining whether an adverse event fulfills criteria for a SAE or 
not, the severity of AEs experienced by study participants will be graded according 
to the criteria set forth in the National Cancer Institute’s Common Terminology 
Criteria for Adverse Events (NCI-CTCAE) Version 4.0. This document provides a 
common language to describe levels of severity, to analyze and interpret data, and 
to articulate the clinical significance of all adverse events.   
• 
All adverse events whether or not listed in the NCI-CTCAE will be graded on a 
scale from 1 to 5 according to the following standards in the NCI-CTCAE manual 
(A semi-colon indicates ‘or’ within the description of the grade.): 
o Grade 1 = Mild; asymptomatic or mild symptoms; clinical or diagnostic 
observations only; intervention not indicated. 
o Grade 2 = Moderate; minimal, local or noninvasive intervention indicated; 
limiting age-appropriate instrumental activities of daily living (ADL) 
(preparing meals, shopping for groceries or clothes, using the telephone, 
managing money, etc). 
o Grade 3 = Severe or medically significant but not immediately life-
threatening; hospitalization or prolongation of hospitalization indicated; 
disabling; limiting self-care ADL (bathing, dressing and undressing, feeding 
self, using he toilet, taking medications, and not bedridden).  
o Grade 4 = Life-threatening consequences; or urgent intervention indicated. 
o Grade 5 = Death related to AE. 
 
• 
Adverse Event/Serious Adverse Event monitoring procedures 
Evaluations for safety throughout study visits are detailed in Section 5.3. In addition to 
these, review and classification of AEs will occur at each study visit with ongoing 
monitoring of safety throughout the entirety of the study by the PI. A formal DSMB 
charter meeting DOD approval will be established prior to commencing the proposed 
studies. Additionally, we have also identified a research monitor (RM), UNC 
pulmonologist Dr. Scott Donaldson, who will be responsible for overseeing the safety 
of the research subjects and report observations/findings to the IRB. 
• 
Adverse Event/Serious Adverse Event reporting procedures 
All SAEs will be reported to UNC IRB, DSMB, and funding agency within 24-48 hours, 
and all AEs will be reported to UNC IRB and DSMB at regular, predetermined intervals 
per protocol. Per DoD guidelines, only the following events will be reported to the 
HRPO: (1) All unanticipated problems involving risk to subjects or others; (2) 
Suspensions, clinical holds (voluntary or involuntary), or terminations of this research 
by the IRB, the institution, the sponsor, or regulatory agencies; (3) Any instances of 
serious or continuing noncompliance with the federal regulations or IRB requirements; 
(4) The knowledge of any pending compliance inspection/visit by the Food and Drug 
Administration (FDA), Office for Human Research Protections, or other government 
agency concerning this clinical investigation or research; (5) The issuance of inspection 
reports, FDA Form 483, warning letters, or actions taken by any government regulatory 
agencies; (6) Change in subject status when a previously enrolled human subject 
becomes a prisoner must be promptly reported to the USAMRMC ORP HRPO with a 
report that includes actions taken by the institution and the IRB. The duties of the RM 
specifically will be to review all unanticipated problems involving risks to subjects or 

24 
 
others associated with the protocol and provide an independent report of the event to 
the IRB and DSMB. The Research Monitor may discuss the research protocol with the 
investigators; shall have authority to stop a research protocol in progress, remove 
individual human subjects from a research protocol, and take whatever steps are 
necessary to protect the safety and well-being of human subjects until the IRB can 
assess the monitor’s report; and shall have the responsibility to promptly report their 
observations and findings to the IRB, DSMB, and the HRPO. 
 
• 
Medical Emergency procedures 
There is a study physician available for study visits, including WSP exposure and HS 
inhalation procedures. A code cart and Automated External Defibrillator (AED) are 
available in the event of a cardiopulmonary event.  Albuterol, both inhalers and 
nebulizers, are available in the event of bronchospasm. If a subject has an event that 
is does not immediately respond to care in the research lab, he/she will be transported 
via EMS to UNC Healthcare for treatment. 
 
• 
Data Safety Monitoring Plan 
Data is initially recorded on paper documents, and the documents are maintained in a 
binder, which is kept in a locked office within the EPA Human Studies Facility (HSF). 
The EPA HSF is a secure facility with a guarded entrance and requires identification 
for entry. This data is then entered into REDCap by an initial data entry person, and 
confirmed by a second user. 
 
• 
Potential Risks: 
a) WSP exposure: Concentrations of wood smoke exhaust proposed for this study 
(500 μg/m3) are below those reported for individuals using biomass burning for 
heating and food preparation and those living in the area of a forest fire or 
agricultural burning. No major adverse health effects were observed in a 
recently completed study, which exposed healthy volunteers to WSP (22). The 
mean PM was 485 + 84 μg/m3 during the WSP exposure (vs. “below the 
detectable limit” for filtered air (FA)), and analyses of chamber concentrations 
of total hydrocarbon and carbon monoxide during WSP exposure were also 
increased compared to FA (4.8 ± 0.5 vs. 2.0 ± 0.1 ppm and 2.7 ± 0.7 vs. 0.0 ± 
0.0 ppm, respectively). However, these healthy subjects had no significant 
change in pulmonary function and no nasal irritation, rhinitis, inspiratory pain, 
shortness of breath, cough, wheezing or chest tightness after exposure to WSP 
or filtered air. There were complaints of fatigue, headache and eye irritation, but 
these were noted by volunteers as frequently before as after both the filtered air 
and particle exposures. Pulmonary function testing demonstrated no significant 
changes from pre-exposure values following exposures to either filtered air or 
WSP. The moderate exercise on the ergometer has a rare risk of causing leg 
cramps or soreness. The EKG patches cause a rare risk of skin irritation or 
blistering. 
b) Hypertonic Saline inhalation: Inhalation of HS carries a small risk of throat 
irritation and bronchospasm, which may lead to the onset of asthma-like 
symptoms. This is uncommon in healthy adults without a history of underlying 
airway hyper-reactivity, which is the target population of this study.  
c) Sputum induction: The saline solutions (3%, 4% and 5%) nebulized during 
sputum induction may cause throat irritation, but it is uncommon for the duration 
of discomfort to last more than a few minutes post procedure. The procedure 

25 
 
also has a minimal risk of inducing bronchospasm, which is less of a concern in 
healthy adults.  
d) Spirometry:  Potential risks include possible lightheadedness or wheezing. 
e) Mucociliary clearance scan: The radiation exposure from the MCC scan, 
including the Co57 transmission scan and the Americium 241 disks, which are 
used as fiducial markers, is approximately 177 mRems. This is less than the 
natural environmental radiation that adults receive every year, which in Chapel 
Hill is about 300 mRems. 
f) Exhaled breath condensate: There are no additional risks associated with the 
EBC procedure as study subjects are simply asked to exhale into a chilled tube.  
• 
Protections to Minimize Risk: 
a) WSP exposure: Strict adherence to inclusion and exclusion criteria will be 
followed prior to subject enrollment, and medical history updated throughout 
study visits to affirm continued study eligibility. If participants develop any 
infectious processes during the washout period, period 2 will be delayed for a 
minimum of 4 weeks (as per exclusion criteria) following resolution of illness 
and/or completion of antibiotics. During WSP exposure, subjects will be 
monitored by direct observation or via closed-circuit television. Subjects will 
have EKG telemetry leads attached to monitor cardiac rate and rhythm during 
exercise throughout the exposure. Subjects will be aware that they can 
terminate their exposure for any reason and still receive compensation for their 
participation up to that point. A physician familiar with the protocol will be 
available for all challenge procedures. The investigator or duty physician will 
end the exposure if the subject is found to be suffering from any major adverse 
effect. An emergency “crash cart” with standard emergency medications, IV 
fluids, and a defibrillator are is also readily available at the CEMALB in the 
unlikely event of a medical emergency during any challenge or study visit. 
Pulse oximetry will be performed during the exposures to wood smoke and 
filtered air, and the subject will be withdrawn from the chamber if the value is 
<90%. The risk of discomfort associated with exercise will be limited because 
ability and response to exercise will be assessed at the training visit, and 
subjects with orthopedic issues that limit ability to pedal a bicycle will be 
excluded. Subjects will be encouraged to remain well hydrated to minimize 
risks from exercise. Should skin irritation develop from EKG patches, a topical 
steroid for symptomatic relief will be recommended.  
b) Hypertonic saline inhalation: Only healthy adults will be recruited to participate 
in this study and have a minimal risk of bronchospasm to HS. A CEMALB 
physician is present in the building at all times during HS inhalation. Symptom 
questionnaire will be completed immediately following HS inhalation. If at any 
time during the study participants assign a score greater than 3 (“moderate”) 
to symptoms attributable to bronchospasm (cough, wheezing, chest tightness, 
shortness of breath, or pain on deep inspiration), then they will be evaluated 
by the study physician to determine if additional evaluation via spirometry 
and/or rescue treatment with albuterol is necessary. This procedure is similar 
to a sputum induction. 
c) Sputum Induction: Throat irritation will be minimized by providing the subject 
with a drink (water, juice etc) and a snack after induction. An albuterol 
multidose inhaler will be immediately available in the event the subject 
experiences symptoms of bronchoconstriction; however as only healthy 
subjects are being enrolled in this study, no albuterol pre-treatment will be 
given. A physician on duty in the facility will be available during sputum 

26 
 
inductions. If the covering physician feels that the subject’s respiratory status 
is such that providing an induced sputum sample would place them at 
increased risk for significant bronchospasm, sputum sampling will be deferred. 
Baseline FEV1 and FVC will be measured before the start of the induction and 
at the end of each level (concentration) of saline inhalation. The FEV1 values 
that match a 10% and 20% reduction from baseline will be calculated and 
recorded. Subjects who have a 10-20% decrease in FEV1 after the 3% or 4% 
level of saline will not be advanced to a higher concentration of saline and will 
continue at the same level. If the FEV1 drops by > 20%, the induction will be 
stopped and the subject will be treated with 2 puffs of albuterol. Additional 
albuterol may be given if needed. 
d) Spirometry: Subjects will be seated in a non-rolling chair when spirometry is 
performed and standard methodology conforming to the ATS guidelines for 
measurement of spirometry will be used.  Subjects will be instructed to notify 
the study staff if they feel lightheaded, and albuterol will be available in the 
event the subject experiences any unanticipated bronchoconstriction. 
e) Mucociliary Clearance: Radiation history is collected. Any subject who will 
exceed safe annual exposure limits will not be enrolled.  
f) EBC: Standard procedures are followed for EBC. No additional risk is present 
from an EBC. 
 
10 
DATA COLLECTION AND MANAGMENT  
 
• 
Monitoring Plan 
We will use RedCap for data management. The data will be entered by one person 
(typically the coordinator or, for lab analysis, a lab staff member) and then checked by 
a second person. The data is not marked "complete" until the 2nd person verifies the 
entry. REDCap creates a data dictionary when the database is established, and this is 
used as the codebook.  
 
• 
Database documentation 
All databases using equipment generated have a date and user attached to the 
electronic file. Data will be linked with a codebook (i.e. sample ID) using Excel. A 
second entry is done manually in a lab notebook, which includes date, samples 
assayed, assay used, any issues (like missing samples/data), reference to where data 
is stored, and a printout of the data (all as hardcopies in a lab notebook).  Adherence 
to the codebook is ensured by having a second individual to check the entries. The PI 
takes responsibility for data management computations. 
 
• 
Case report forms 
Case report forms will be developed by the study team, using templates from previous 
studies. These forms are maintained by study coordinators, and reviewed by 
investigators as needed.  
 
• 
Maintaining Confidentiality 
Subjects will be issued a subject number when they enroll into the study, and this 
number will only be used to label samples. All protected healthy information will be 
maintained by the study coordinator or the investigators and will be kept in locked areas 
when not in use. The identifiers that go into REDCap will only be accessible to those 
who need them for their job, specifically study physicians, investigators and 

27 
 
coordinators. Subjects sometimes undergo procedures – such as sitting in front of the 
gamma camera – at the same time, however other than basic introductions, nothing 
about one subject is disclosed to the other subject.  
 
11 
RECRUITMENT STRATEGY 
Informational emails at UNC will be used to recruit subjects.   
 
12 
CONSENT PROCESS 
 
• 
Describe the procedure that will be used to obtain informed consent/HIPAA 
authorization and assent 
The study will be described in detail to the subject, including why the study is being 
conducted, the intervention being studied, the risks and benefits, and what is expected 
of the subject.  The subject will be given adequate time to read the consent, and consent 
will be obtained prior to any study procedures.  Consent may take place on a separate 
day from the baseline procedures. 
 
• 
Who will obtain consent 
Consent will be obtained by a study coordinator or a study physician.  
 
• 
Where will consent process take place 
The consent will take place at the UNC CEMALB, located in the EPA HSF on Mason 
Farm Road in Chapel Hill. 
 
• 
How will investigator assure that subjects comprehend the nature of the study, 
procedures, the risks and benefits 
The subject will be encouraged to ask questions regarding the study and procedures 
involved. Open ended questions will be asked of the subject to solicit correct responses, 
to help ensure that the subject understands the study commitment, procedures and 
risks and benefits.  
 
 
13 
REFERNECES  
1. 
Pugh MJ, Jaramillo CA, Leung KW, Faverio P, Fleming N, Mortensen E, et al. Increasing 
Prevalence of Chronic Lung Disease in Veterans of the Wars in Iraq and Afghanistan. Military 
medicine. 2016;181(5):476-81. 
2. 
Korzeniewski K, Nitsch-Osuch A, Konior M, Lass A. Respiratory tract infections in the 
military environment. Respiratory physiology & neurobiology. 2015;209:76-80. 
3. 
Baird CP. Review of the Institute of Medicine report: long-term health consequences of 
exposure to burn pits in Iraq and Afghanistan. US Army Medical Department journal. 2012:43-7. 
4. 
Gomez JC, Yamada M, Martin JR, Dang H, Brickey WJ, Bergmeier W, et al. Mechanisms 
of interferon-gamma production by neutrophils and its function during Streptococcus pneumoniae 
pneumonia. American journal of respiratory cell and molecular biology. 2015;52(3):349-64. 
5. 
Barth SK, Dursa EK, Bossarte R, Schneiderman A. Lifetime Prevalence of Respiratory 
Diseases and Exposures Among Veterans of Operation Enduring Freedom and Operation Iraqi 
Freedom Veterans: Results From the National Health Study for a New Generation of U.S. 
Veterans. Journal of occupational and environmental medicine. 2016;58(12):1175-80. 
6. 
Szema AM. Occupational Lung Diseases among Soldiers Deployed to Iraq and 
Afghanistan. Occupational medicine & health affairs. 2013;1. 

28 
 
7. 
Morris MJ, Lucero PF, Zanders TB, Zacher LL. Diagnosis and management of chronic 
lung disease in deployed military personnel. Therapeutic advances in respiratory disease. 
2013;7(4):235-45. 
8. 
Auerbach A, Hernandez ML. The effect of environmental oxidative stress on airway 
inflammation. Curr Opin Allergy Clin Immunol. 2012;12(2):133-9. 
9. 
Alexis NE, Brickey WJ, Lay JC, Wang Y, Roubey RA, Ting JP, et al. Development of an 
inhaled endotoxin challenge protocol for characterizing evoked cell surface phenotype and 
genomic responses of airway cells in allergic individuals. Annals of allergy, asthma & immunology 
: official publication of the American College of Allergy, Asthma, & Immunology. 2008;100(3):206-
15. 
10. 
Hernandez ML, Harris B, Lay JC, Bromberg PA, Diaz-Sanchez D, Devlin RB, et al. 
Comparative airway inflammatory response of normal volunteers to ozone and lipopolysaccharide 
challenge. Inhal Toxicol. 2010;22(8):648-56. 
11. 
Hernandez M, Brickey WJ, Alexis NE, Fry RC, Rager JE, Zhou B, et al. Airway cells from 
atopic asthmatic patients exposed to ozone display an enhanced innate immune gene profile. 
The Journal of allergy and clinical immunology. 2012;129(1):259-61 e1-2. 
12. 
Alexis NE, Lay JC, Haczku A, Gong H, Linn W, Hazucha MJ, et al. Fluticasone propionate 
protects against ozone-induced airway inflammation and modified immune cell activation markers 
in healthy volunteers. Environmental health perspectives. 2008;116(6):799-805. 
13. 
Alexis NE, Peden DB. Blunting airway eosinophilic inflammation results in a decreased 
airway neutrophil response to inhaled LPS in patients with atopic asthma: a role for CD14. J 
Allergy Clin Immunol. 2001;108(4):577-80. 
14. 
Hernandez ML, Mills K, Almond M, Todoric K, Aleman MM, Zhang H, et al. IL-1 receptor 
antagonist reduces endotoxin-induced airway inflammation in healthy volunteers. J Allergy Clin 
Immunol. 2015;135(2):379-85. 
15. 
Hernandez ML, Wagner JG, Kala A, Mills K, Wells HB, Alexis NE, et al. Vitamin E, gamma-
tocopherol, reduces airway neutrophil recruitment after inhaled endotoxin challenge in rats and in 
healthy volunteers. Free Radic Biol Med. 2013;60:56-62. 
16. 
Burbank AJ, Duran CG, Pan Y, Burns P, Jones S, Jiang Q, et al. Gamma tocopherol-
enriched supplement reduces sputum eosinophilia and endotoxin-induced sputum neutrophilia in 
volunteers with asthma. J Allergy Clin Immunol. 2017. 
17. 
Alexis NE, Hu SC, Zeman K, Alter T, Bennett WD. 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):1964-70. 
18. 
Alexis NE, Lay JC, Zeman KL, Geiser M, Kapp N, Bennett WD. In vivo particle uptake by 
airway macrophages in healthy volunteers. American journal of respiratory cell and molecular 
biology. 2006;34(3):305-13. 
19. 
Sood N, Bennett WD, Zeman K, Brown J, Foy C, Boucher RC, et al. Increasing 
concentration of inhaled saline with or without amiloride: effect on mucociliary clearance in normal 
subjects. Am J Respir Crit Care Med. 2003;167(2):158-63. 
20. 
Bennett WD, Foster WM, Chapman WF. Cough-enhanced mucus clearance in the normal 
lung. J Appl Physiol (1985). 1990;69(5):1670-5. 
21. 
Alexis NE, Bennett W, Peden DB. Safety and benefits of inhaled hypertonic saline 
following airway challenges with endotoxin and allergen in asthmatics. J Asthma. 2017:1-4. 
22. 
Ghio AJ, Soukup JM, Case M, Dailey LA, Richards J, Berntsen J, et al. Exposure to wood 
smoke particles produces inflammation in healthy volunteers. Occup Environ Med. 
2012;69(3):170-5. 
23. 
Esther CR, Jr., Lazaar AL, Bordonali E, Qaqish B, Boucher RC. Elevated airway purines 
in COPD. Chest. 2011;140(4):954-60. 
24. 
Jones B, and Kenward, M.G. . Design and analysis of cross-over trials. Third ed: CRC 
Press; 2015. 

29 
 
25. 
Alexis NE, Zhou H, Lay JC, Harris B, Hernandez ML, Lu TS, et al. The glutathione-S-
transferase Mu 1 null genotype modulates ozone-induced airway inflammation in human subjects. 
The Journal of allergy and clinical immunology. 2009;124(6):1222-8 e5 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
APPENDIX 
a) Table of Study Procedures:  
 
V0 
(Baseline) 
V1 
(Training) 
V2 (post-
MC) 
Pre-WSP 
V3 (Prd 1) 
V7 (Prd 2) 
Post-WSP 
V3 (Prd 1) 
V7 (Prd 2) 
V4 (Prd 1) 
V8 (Prd 2) 
V5 
(Baseline- 
Prd 2) 
V6 (post-
MCC) 
V9 
(Completion 
visit) 

30 
 
Consent 
X 
 
 
 
 
 
 
 
 
Review 
history/AE’s 
X 
 
 
X 
X 
X 
X 
 
X 
Vital Signs 
X 
X 
 
X 
 
X 
X 
 
X 
Urine hCG 
(females only) 
X 
X 
 
X 
 
 
X 
 
 
Symptom 
Questionnaire 
 
 
 
X 
3X (post-
WSP, post-
HS, 6hr 
post) 
X 
 
 
 
Spirometry 
(FEV1) 
X 
 
 
X 
X 
X 
X 
 
 
EBC 
 
 
 
X 
X 
X 
 
 
 
Physical Exam 
X 
 
 
X 
 
X 
X 
 
 
Venipuncture 
 
 
 
X 
X 
 
 
 
 
Sputum 
Induction 
 
 
X 
 
 
X 
X 
X 
 
 
2 hour WSP 
chamber 
exposure 
 
 
 
X 
 
 
 
 
 
MCC 
 
X 
 
 
X 
 
X 
 
 
MCC 
Retention 
Scan 
 
 
X 
 
 
X 
 
X 
 
Ergometric 
Cycle Train 
 
X 
 
 
 
 
 
 
 
HS treatment 
vs no 
treatment 
 
 
 
 
X 
 
 
 
 
b) Symptom Questionnaire: 

31 
 
Please indicate if you are experiencing any of the symptoms or restrictions listed below, using the 
following scale to indicate the severity. Circle the number. 
 
0 = NONE 
 
 
(not present) 
1 = TRACE/NOTICED 
(barely detectable) 
2 = MILD/LIGHT 
 
(present, but not annoying) 
3 = MODERATE 
 
(present, but somewhat annoying) 
4 = SEVERE/HEAVY  
(present and very annoying and painful) 
Symptoms 
None 
Trace 
Mild 
Moderate 
Severe 
1. Headache 
0 
1 
2 
3 
4 
2. Irritation of the Nose 
0 
1 
2 
3 
4 
3. Stuffy Nose/Sinus Congestion 
0 
1 
2 
3 
4 
4. Runny Nose 
0 
1 
2 
3 
4 
5. Dry or Sore Throat 
0 
1 
2 
3 
4 
6. Pain on Deep Inspiration 
0 
1 
2 
3 
4 
7. Unusual Fatigue 
0 
1 
2 
3 
4 
8. Eye Irritation 
0 
1 
2 
3 
4 
9. Shortness of Breath 
0 
1 
2 
3 
4 
10. Sneezing 
0 
1 
2 
3 
4 
11. Coughing 
0 
1 
2 
3 
4 
12. Wheezing/Whistling of Chest 
0 
1 
2 
3 
4 
13. Chest Tightness 
0 
1 
2 
3 
4 
14. Sweating 
0 
1 
2 
3 
4 
15. Other: 
0 
1 
2 
3 
4