Phase I/II Randomized, Double-blind, Placebo-controlled Cross-over Study of Prednisone on Airway Inflammatory Response to Inhaled Wood Smoke
Phase: PHASE1
Conditions: Airway Inflammation
Sponsor: University of North Carolina, Chapel Hill

 
 
PHASE I/II RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED 
CROSS-OVER STUDY OF PREDNISONE ON AIRWAY INFLAMMATORY 
RESPONSE TO INHALED WOOD SMOKE 
 
 
NCT number NCT03861390 
Document Date 04/23/23 
 

 
 
 
Complete Title:  
PHASE I/II RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED 
CROSS-OVER STUDY OF PREDNISONE ON AIRWAY 
INFLAMMATORY RESPONSE TO INHALED WOOD SMOKE 
Short Title:   
Prednisone for wood smoke-induced airway inflammation  
Drug Name(s): 
Prednisone 
Funding Agency:  
Department of Defense (DoD) 
Protocol Date: 
April 23, 2023 
 
 
 
 
 
 
 
 
 
Study Principal Investigator:  
Terry L. Noah, MD 
Professor and Vice Chair for Faculty Development, UNC Department of Pediatrics 
Division Chief, Pediatric Pulmonology 
Investigator, UNC Center for Environmental Medicine, Asthma and Lung Biology 
Campus Box 7220 
Email:terry_noah@med.unc.edu 
Telephone (919) 966-1505 
 
 
 
 
 

 
2 
 
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 ......................................................... 14 
6 STATISTICAL CONSIDERATIONS ............................................................................... 19 
7 STUDY INTERVENTION (DEVICE OR OTHER INTERVENTION) ............................... 21 
8 STUDY INTERVENTION ADMINISTRATION ................................................................ 21 
9 SAFETY MANAGEMENT ............................................................................................... 22 
10  DATA COLLECTION AND MANAGEMENT .................................................................. 24 
11 RECRUITMENT STRATEGY ......................................................................................... 25 
12 CONSENT PROCESS ................................................................................................... 25 
13 REFERENCES ............................................................................................................... 26 
Appendix ............................................................................................................................. 28 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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 
CRP 
C – reactive protein 
DSMB 
Data Safety Monitoring Board 
EBC 
Exhaled breath condensate 
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 
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 
 
 
 

 
4 
 
PROTOCOL SYNOPSIS 
Study Title 
Phase I/II Randomized, Double-blind, Placebo-controlled Cross-Over 
Study of Prednisone 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. Prednisone is a well-known anti-
inflammatory agent used in the treatment of airway inflammatory 
diseases, such as asthma. Efficacy of prednisone has not been studied 
in the context of PM-induced airway inflammation, such as that seen in 
military personnel. We hypothesize that a single dose of oral prednisone 
will reduce neutrophilic airway inflammation following WSP exposure.  
Study Objective(s) 
Primary  
• 
To determine the efficacy of single dose of oral prednisone in 
mitigating WSP-induced neutrophilic airway inflammation, assessed 
via sputum %PMNs, in healthy adults 
Secondary 
• 
To assess the safety of WSP exposure in healthy adults by 
measuring vitals, symptom questionnaires, and spirometry 
• 
To determine the effect of a single dose of oral prednisone on mucus 
based-parameters following WSP exposure 
• 
To determine the effect of prednisone on MCC using gamma 
scintigraphy  
• 
To determine the effect of prednisone 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 prednisone on systemic inflammatory 
markers and serum cytokines  
Study Drug/Device 
(If Applicable) 
Prednisone 60 mg 
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, double-blind, placebo-controlled 
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 oral dose of prednisone or placebo. 
Repeat MCC will be obtained starting 2 hours after WSP exposure, and 
EBC, sputum induction, and venipuncture are repeated 4 hours after 

 
5 
 
WSP exposure to assess for primary and secondary endpoints. 
Spirometry and symptom questionnaires are also performed before and 
after WSP exposure 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 4 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; spirometry; and 
baseline sputum induction, MCC, and venipuncture. 
(2) Intervention: Participants will undergo baseline spirometry, 
venipuncture, and EBC followed by WSP exposure for a total of two 
hours after which they will be randomized to receive either a single dose 
of prednisone or placebo. MCC assessment will be initiated 2 hours 
post-exposure with measurements taken 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, 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 a single dose of prednisone 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 starting 2 hours 
after WSP exposure with measurements taken for a total of 120 minutes. 
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 (prednisone vs placebo).  
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 a single dose of prednisone to 
placebo. This analysis will rely on a linear mixed-effects model that 
accounts for the treatment effect and the period effect. 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 systematic inflammatory 
markers, comparing a single dose of oral prednisone to matching 
placebo) will be performed similarly. The experiment-wide rate of Type I 
errors will be controlled at level α = 0.05. Non-significant tests will be 
reported as being inconclusive. 
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 NC TracS Data Safety Monitoring Board (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. 
 
1 
BACKGROUND AND RATIONALE 
1.1 
Introduction 

 
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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 this study, we will evaluate the efficacy of oral prednisone, a readily available anti-
inflammatory medication commonly used in airway inflammatory diseases, in mitigating 
WSP-induced airway inflammation.  
 
1.2 
Name and Description of Investigational Product or Intervention  
Prednisone 20 mg tablets USP (total dose of 60 mg proposed in this study) and 
Matching Placebo tablets 
 
1.3 
Non-Clinical and Clinical Study Findings 
Systemic corticosteroids are a cost-effective and readily available therapeutic option to 
treat a number of inflammatory conditions. Guidelines-based care for inflammatory 
respiratory diseases, such as asthma and COPD, support the use of oral systemic 
steroids for acute exacerbations of these illnesses (17, 18). Additionally, meta-analyses 
have also highlighted the benefit of oral steroids in certain infectious diseases of the 
upper and lower airways in which neutrophilic inflammation is a key finding (19, 20). Our 
own group has demonstrated the use of an inhaled corticosteroid, fluticasone, in 

 
8 
 
mitigating neutrophilic airway inflammation secondary to inhaled O3 (12) and LPS (13) 
challenges. As the respiratory morbidity of military personnel is driven by PM-induced 
neutrophilic airway inflammation as discussed previously, it is imperative to identify 
readily-available, cost-effective, and rapidly efficacious therapies that reduce airway 
inflammation for these individuals. Thus, in this study and using WSP exposure as a 
model of PM-induced bronchitis, we will assess the effectiveness of a single dose of 60 
mg of prednisone, a standard adult dose used in the treatment of other inflammatory 
airway diseases, in mitigating WSP-induced airway neutrophilic inflammation in healthy 
volunteers. As a secondary endpoint, we will also determine whether a single dose of 
prednisone alters mucociliary clearance patterns after WSP-exposure.  
 
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 oral dose of 
prednisone 60 mg (v. placebo) on mitigating WSP-induced airway inflammation, measured 
by sputum %PMNs, in healthy adults 4 hours post-WSP exposure. 
 
2.2 
Secondary Objective 
Secondary objectives of this study include examining the effect of oral prednisone (v. 
placebo) 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 
• 
Type of design: Randomized, double-blind, placebo-controlled 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.  

 
9 
 
 
b) Visits 0, 1, and 2 – Baseline (Period 1) and Training Day: Along with an informed 
consent, a medical screening, including 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 a 12 lead electrocardiogram (EKG) per our established WSP 
exposure procedure. Finally, a baseline MCC scan and sputum induction will be 
obtained, which will provide some of 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 receive either 
a single dose of prednisone 60 mg or matching placebo. Four hours after WSP 
exposure, participants will then inhale radiolabeled Tc99m sulfur colloid (Tc99m-
SC) to start MCC assessment. MCC measurements will be taken for a total of 120 
minutes. EBC, venipuncture, and sputum induction will be obtained 4 
 
d)  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.  
 
e) 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 and sputum induction. Participants will undergo a 30-minute scan to 
determine amount of Tc99m-SC retained in airways. 
 
f) Visits 5 and 6 – Cross-over and Baseline (Period 2): Participants will return after a 
minimum 4 week wash-out period, which is the amount of time allotted for 
resolution of airway inflammation post-WSP exposure. Interval medical history will 
be reviewed and repeat MCC, spirometry, 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. 
 
g) 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. 
 
h) Unscheduled Visits: We do not anticipate any unscheduled visits. However, 
subjects will be provided with contact information for a study physician in the event 
of any delayed adverse events outside of direct observation during study visits and 
re-evaluated as deemed necessary by the study physician. Subjects will be 
rescheduled if they have a change in health status that requires a delay in study 

 
10 
 
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 a randomized, double-blind, placebo-controlled 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 prednisone, 2 placebo for the first treatment 
period of the protocol). The colleague will send this schedule directly to the UNC 
investigational drug service, who will prepare study drug or placebo for administration. 
 
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 sexes 
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. 
7. Subject must demonstrate a ≥ 10% increase in sputum %PMNs 4 hours following 
inhaled WSP exposure, when compared to baseline sputum (to be completed in a 
separate protocol IRB# 15-1775). 
8. Proof of being fully vaccinated to Covid-19 
 
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. 
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 

 
11 
 
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. 
n. Allergy/sensitivity to study drugs or their formulations 
o. Positive Covid-19 test in the past 90 days 
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). 
 
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 1 month after screening WSP exposure): 
1. Informed consent 
2. Medical history 

 
12 
 
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. 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). 
3. MCC 
4. 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. Physical Exam  
4. Urine Pregnancy Test (if not previously performed within 7 days) 
5. Venipuncture (CBC with differential, serum cytokines, CRP) 
6. EBC 
7. Spirometry 
8. 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. 
9. Symptom questionnaire (immediately prior to exit from the chamber) 
10. Immediately following exit from chamber, subjects will receive either 60 mg of 
prednisone or placebo per randomization schema 
11. MCC (2 hours post-exposure) 
12. Symptom questionnaire (6 hours post-WSP exposure) 
13. EBC (4 hours post-WSP exposure) 
14. Spirometry (4 hours post-WSP exposure) 
15. Sputum Induction (4 hours post-WSP exposure) 
16. 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. Physical exam 
5. EBC 
6. Retention scan 
7. Spirometry 
8. Sputum Induction 
9. Venipuncture for CBC with differential, serum cytokines, CRP 

 
13 
 
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 4 week washout is required between Period 1 and Period 2 for resolution of airway inflammation 
elicited following WSP exposure in Period 1.  
4.7 
Visit 5: Baseline (Period 2) (following a minimum 4 week washout) 
1. Review medical history and adverse events 
2. Urine pregnancy test 
3. Vital signs, oxygen saturation 
4. Physical Exam  
5. MCC 
6. Spirometry 
7. Sputum Induction 
8. Venipuncture (safety labs only – CBC w/ diff) 
 
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 and contact information for a study physician. Subjects will be invited 
back to the lab for vital signs collection, physical exam, and spirometry as deemed 
necessary by the study physician. 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 
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, 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. 
 

 
14 
 
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 4 hour observation period post-
WSP exposure 
5. Need for rescue albuterol therapy during the 4 hours observation period post-WSP 
exposure 
 
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 
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 

 
15 
 
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 (21). 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.  
 
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 

 
16 
 
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 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 (22) 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 
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 

 
17 
 
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 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 particles. No additional Tc99m-SC will be 
administered at this visit.  
 
• 
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 at pre-WSP exposure (visits 3 and 7), 
immediately post-WSP exposure, 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 

 
18 
 
The effect of prednisone 60 mg (v. placebo) 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 4 hours post-WPS exposure, 
except for MCC which will be performed 2 hours post-WSP and will be completed by 4 
hours post-WSP exposure. The change in these measurements (postWSP – preWSP) will 
be compared for each interventional arm (prednisone vs placebo). 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 4 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.  
 
b) Spirometry: Spirometry will also be performed at each baseline visit (0 and 5), pre-
WSP and 4 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 (21). 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) following WSP exposure, 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, and 4 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 5 hours following each WSP exposure.  
 
e) Post-study visit safety plan: We do not anticipate any delayed adverse events. 
However, all participants will have access to and contact information for a study 

 
19 
 
physician during the duration of their participation. Per study physician 
recommendations, they will be re-evaluated with an unscheduled visit as detailed 
in Sections 3.1 and 4.10. Additionally, all subjects will be contacted via phone by 
a study coordinator 3-7 days after visit 4 to access for any delayed adverse events 
as noted in Section 4.6 above.  
 
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) 
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) 
 
 
 
 
 

 
20 
 
6.4      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 4 hours post-WSP exposure, we will employ a linear mixed-
effects model used in previous studies that allows us to assess treatment effect while 
accounting for any period effect (14, 16). The parameter estimates obtained by fitting this 
model (i.e., inter- and intra-subject variance, period effect, and treatment effect) will be 
used to compute estimates (e.g., effect sizes, means, standard errors, confidence 
intervals) and to compute a p-value for the primary null hypothesis that the difference in 
treatment effects is exactly zero in the target population. The same linear mixed-effects 
regression modeling technique will be used to analyze secondary endpoints as well. 
Additionally, we will use tabular and graphic methods to describe/explore the basic 
relationship between the treatment groups, including confidence intervals for all statistical 
estimates. Subsequently, as part of sensitivity analyses, we will compare the Δ sputum 
%PMNs (post WSP – pre WSP [prednisone]) vs (post WSP – pre WSP [placebo]) using a 
paired-sample T-Test. Examination of the normality assumption will be performed, and a 
nonparametric Wilcoxon signed-rank test will be conducted in the case of evidence of 
extreme departure from normality. For all of the aforementioned analyses, criterion for 
significance will be p≤0.05, and results exceeding this threshold will be reported as 
inconclusive. Lastly, 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 treatment on the primary or secondary endpoints, similar to 
approaches previously described to assess genotype effects on pollutant responses (23). 
These analyses will only be conducted after the primary hypothesis is tested and solely 
for exploratory purposes.  
 
6.5      Sample Size and Power 
Our primary endpoint is the change in sputum %PMNs pre- and 4 hours post-WSP 
exposure, comparing prednisone to placebo. This study originally targeted enrollment of 24 
subjects to yield complete 20 datasets, enrolled a total of 7 subjects and completed 6 prior to 
the study being put on hold for the pandemic from March 2020 till July 2021.  No samples had 
been analyzed from this study yet.   Meanwhile our ongoing WSP screen protocol was able to 
complete 50 screening exposures, determining that 28 met the predefined “responder” 
definition. 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. The availability of these new data 
have led to concern that the Smokisone enrollment target should be amended, since the new 
dataset should allow a more accurate sample estimate.  Using our ongoing WSP screen sputum 
data, estimating the prednisone 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 
study to detect a significant prednisone 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.  

 
21 
 
 
6.6      Interim Analysis 
We do not plan on conducting an interim analysis. Prednisone dosing is being used for its 
indication as an anti-inflammatory treatment. Because we do not anticipate safety 
concerns attributable to prednisone treatment for 1 dose, no interim analysis will be 
needed. Additionally, our study will be reviewed and surveyed by the Data and Safety 
Monitoring Board for external safety monitoring. 
 
7 
STUDY INTERVENTION  
 
• 
Description 
Prednisone 60 mg (supplied as three Prednisone 20 mg tablets USP) 
 
• 
Receipt/Storage 
IDS will be responsible for providing prednisone and matching placebo for each study 
subject and for each treatment period in a blinded fashion.  
 
• 
Packaging/Labeling 
This is a randomized, double blind study so packaging and labeling by IDS is such that 
both study participants and study personnel will be blinded to the treatment (either 
prednisone or placebo) given.  
 
• 
Dosing 
Study participants will receive 60 mg of prednisone (3 tablets of prednisone 20 mg) or 
matching placebo during period 1. They will then crossover to period 2 and receive the 
alternative treatment. Prednisone 60 mg is the standard clinical daily dose used to treat 
exacerbations of airway diseases, such as asthma and COPD.  
 
• 
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 prednisone, 2 placebo) for the 
first treatment period of the protocol).   
 
• 
Blinding procedures 

 
22 
 
• 
This is a randomized, double blind study so packaging and labeling by IDS is such that 
both study participants and study personnel (coordinators and physicians) will be 
blinded to the treatment (either prednisone or placebo) given.  
 
• 
Unblinding procedures: Unblinding will occur at the conclusion of the study or earlier 
if needed for safety purposes and as dictated by the UNC IRB, study sponsor, and 
DSMB.   
 
 
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).   
 
• 
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. The UNC DSMB will 
be employed to review AEs throughout the study.  The PI will report to the DSMA at 
least biannually.  Any SAE;s will be immediately reported to the DSMB.    

 
23 
 
 
• 
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 
others associated with the protocol and provide an independent report of the event to 
the IRB and DSMB. The PI will stop a research protocol in progress, remove individual 
human subjects from a research protocol, and will protect the safety and well-being of 
human subjects until the IRB and DSMB can assess the s report. All findings from these 
events will be promptly reported to the HRPO. 
 
• 
Medical Emergency procedures 
There is a study physician available for study visits, including WSP exposure. 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 (21). 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 

 
24 
 
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) Oral prednisone: Long-term use of prednisone is associated with a number of 
complications such as increased risk of infections, fluid and electrolyte 
disturbances, gastrointestinal complications, dermatoses, ocular complications 
(cataracts, glaucoma), adrenal suppression, increased risk of osteoporosis, 
myopathy, personality changes, insomnia, among other things. However, a 
single dose of prednisone, as proposed in this study, is associated with minimal 
side effects, and may only cause temporary euphoria or irritability, hunger, or 
insomnia. As the half-life of prednisone is 3-4 hours, these minor side effects 
will last no longer than 24 hours.  
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 
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 

 
25 
 
<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) Oral prednisone: Dosing will be limited to a single dose with minimal side 
effects as noted above. In addition, those with hypersensitivity to prednisone 
will be excluded from the study as noted in Section 3.4.   
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 
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 

 
26 
 
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 
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  

 
27 
 
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. 
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. 
Expert Panel Report 3 (EPR-3): Guidelines for the Diagnosis and Management of Asthma-
Summary Report 2007. The Journal of allergy and clinical immunology. 2007;120(5 Suppl):S94-138. 

 
28 
 
18. 
Walters JA, Tan DJ, White CJ, Wood-Baker R. Different durations of corticosteroid therapy for 
exacerbations of chronic obstructive pulmonary disease. The Cochrane database of systematic reviews. 
2018;3:Cd006897. 
19. 
Stern A, Skalsky K, Avni T, Carrara E, Leibovici L, Paul M. Corticosteroids for pneumonia. The 
Cochrane database of systematic reviews. 2017;12:Cd007720. 
20. 
Venekamp RP, Thompson MJ, Hayward G, Heneghan CJ, Del Mar CB, Perera R, et al. Systemic 
corticosteroids for acute sinusitis. The Cochrane database of systematic reviews. 2014(3):Cd008115. 
21. 
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. 
22. 
Esther CR, Jr., Lazaar AL, Bordonali E, Qaqish B, Boucher RC. Elevated airway purines in COPD. 
Chest. 2011;140(4):954-60. 
23. 
Jones B, and Kenward, M.G. . Design and analysis of cross-over trials. Third ed: CRC Press; 2015. 
24. 
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. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
29 
 
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) 
Consent 
X 
 
 
 
 
 
 
 
 
Review 
history/AE’s 
X 
 
 
X 
X 
X 
X 
 
X 
Vital Signs 
X 
 
 
X 
 
X 
X 
 
X 
Urine hCG 
(females only) 
X 
X 
 
X 
 
 
X 
 
 
Symptom 
Questionnaire 
 
 
 
X 
2X (post-
WSP, hr 
post-WSP) 
X 
 
 
 
Spirometry 
(FEV1) 
X 
 
 
X 
X 
X 
X 
 
 
EBC 
 
 
 
X 
X 
X 
 
 
 
Physical Exam 
X 
 
 
X 
 
X 
X 
 
 
Venipuncture 
 
 
 
X 
X 
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 
 
 
 
 
 
 
 
Prednisone vs 
placebo 
 
 
 
 
X 
 
 
 
 

 
30 
 
b) Symptom Questionnaire: 
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