Oxidative Stress and Regional Airway Remodeling and Fibrosis in Obese Asthma
Phase: PHASE2
Conditions: Asthma, Obesity
Sponsor: Bastiaan Driehuys

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Oxidative Stress and Regional Airway Remodeling and Fibrosis in Obese Asthma 
 
 
 
Research Protocol 
 
 
 
Version:  21MAY2024 
 
NCT04512547 
 
 
 
Funding Agency: 
National Institutes of Health (NIH) 
National Heart Lung and Blood Institute (NHLBI) 
 
1 R01 1HL153641-01 
 
 
 

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Study Team 
 
Principal Investigator: 
Loretta G. Que, MD 
Professor of Medicine 
Division of Allergy, Pulmonary & Critical Care Medicine 
Duke University Medical Center 
MSRB 1, Rm 279 
DUMC Box 2629 
Durham, NC  27710 
Office:  (919) 681-8551 
Cell:  (919) 796-5822 
Email: loretta.que@duke.edu  
 
 
Co-Investigators: 
 
Scott Shofer, M.D., Ph.D., 
Associate Professor of Medicine 
112 Hanes Bldg, Box 102356,  
Durham, NC 27710 
Duke Box 102356, Durham, NC 27710 
  
scott.shofer@duke.edu  
 
Bastiaan Driehuys, Ph.D. 
Professor of Radiology 
161-B Bryan Research, 311 Research Dr 
Durham, NC 27710 
Bastiaan.driehuys@duke.edu 
 
Jennifer Ingram, Ph.D. 
Assistant Professor of Medicine 
201 Research Dr.,  
Duke Box 2641 
Durham, NC 27710 
 
Purushothama Rao Tata, Ph.D. 
Assistant Professor of Cell Biology 
308 Nanaline Duke Bldg., Box 3709 
Duke University School of Medicine 
Durham, NC 27710 
purushothamarao.tata@duke.edu 
 
John Nouls, Ph.D. 
Assistant Professor of Radiology 
Duke University Bryan Research Building, Room 
141D 
311 Research Drive 
DUMC Box 3302 
Durham, NC 27710 
John.Nouls@duke.edu 
 
 
 
Joseph Mammarappallil, M.D., Ph.D. 
Assistant Professor of Radiology 
DUMC Box 3808 
Durham, NC 27710 
Joseph.Mammarappallil@duke.edu 
 
Cynthia Green, Ph.D. 
Assistant Professor of Biostatistics and 
Bioinformatics 
200 Morris Street,  
6112 200 Morris, Durham, NC 27701 
Duke Box 3850, Durham, NC 27710 
green054@mc.duke.edu  
 
Lead Study Coordinator: 
Shelby Martindale 
Division of Allergy, Pulmonary & Critical Care 
Medicine 
Duke University Medical Center 
1821 Hillandale Rd Suite 25A 
Durham, NC 27705 
Office: (919) 613-7699 
Email:shelby.martindale@duke.edu 
 
Other CRC’s:  
• 
Vlayka Liotcheva, Ph.D., Research Program 
Leader; (919) 681-6819; 
vlayka.liotcheva@duke.edu 
• 
Jennifer L Korzekwinski, Imaging Coordinator; 
(919) 681-7362; 
Jennifer.korzekwinski@duke.edu 
• 
Heather M Kuehn, Regulatory Assistant;  
(919) 479-0719; heather.kuehn@duke.edu 
 
 
 
 
 

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Other Study Personnel  
• 
Elianna A Bier, Graduate Student, Ph.D. 
candidate in Biomedical Engineering at Duke 
University 
• 
David G. Mummy, Ph.D., Research Associate 
• 
Monica Fernandez Desoto Arroyo, Associate in 
Research    
 
Laboratory Personnel: 
Zhonghui Yang, Ph.D. 
Division of Allergy, Pulmonary & Critical Care 
Medicine 
Duke University Medical Center 
DUMC Box 2629 
MSRB 1, Rm 267 
Durham, NC  27710 
Lab: (919) 668-0936 
Email: zhonghui.yang@duke.edu 
 
 
Other Scientific Contributors: 
Jeffrey Everitt, D.V.M. 
Professor, Department of Pathology 
Duke University Medical Center 
jeffrey.everitt@duke.edu 
 
 
 
 
 
 
 

 
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Study Summary: 
 
Title 
Oxidative Stress and Regional Airway Remodeling and Fibrosis in Obese 
Asthma 
 
Short Title 
Oxidative Stress in Asthma 
IRB Protocol Number 
Pro00104900 
Study Duration 
52 months 
Study Center(s) 
Duke  
Study Aims 
The human component of the study has two aims: 
Aim 1. Identify the pathology, structural cell profile (airway fibroblast and 
epithelial cell) and redox status corresponding to regional areas of fixed and 
reversible post-bronchodilator defects (BD) in obese asthmatics.  
Aim 2. Define the cellular requirement for redox-mediated TGF-β signaling 
between airway epithelial cells and fibroblasts driving regional remodeling in 
obese asthma.   
Number of Subjects 
Targeted enrollment number - 50 
Enrollment will consist of approximately: 
•  15-20 “Early Onset T2 high obese” asthma patients (diagnosed at age < 12 
years, and FeNO>25 ppb) 
•  15-20 “Late onset obese, non T2” asthma (diagnosed at age ≥12 years, 
FeNO < 25ppb, obese (BMI>30kg/m2) patients will be enrolled 
•  10 non-asthma obese controls who demonstrate no evidence of atopy or 
bronchodilator responsiveness will be also be enrolled 
Recruitment will include adults (18-65 years old)  with physician-diagnosed 
asthma 

 
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Asthma Cohort 
Inclusion/Exclusion 
Criteria 
INCLUSION 
1. Adequate completion of informed consent process with written 
documentation 
2. Male and female patients, 18 - 65 years old, inclusive 
3. Physician diagnosis of asthma for > 1 year 
4. Able to perform reproducible spirometry according to ATS criteria 
5. All racial/ethnic backgrounds may participate 
6. BMI ≥ 30 kg/m2 
7. Regular treatment with ICS or ICS/LABA and/or LAMA combination 
medication for at least 3 months; on a stable dose for the 4 weeks prior to 
Visit 0 
8. Smoking history <10 pack years and no smoking in the last 3 months 
9. Late onset asthma: Age of asthma onset (diagnosis) ≥12 years; 
10. FeNO < 25 ppb at Visit 0 
11. Early onset asthma: Age of asthma onset (diagnosis) <12 years 
• FeNO ≥ 25 ppb at Visit 0 
12. Positive Methacholine  challenge- Confirmation of asthma: Either (1) 12% or 
greater bronchodilator response (BDR) in FEV1 to albuterol at Visit 0 OR (2) 
PC20 methacholine ≤ 16 mg/ml at Visit 0 (historical results from previous studies 
or documented in clinical records within preceding year are acceptable 
alternatives)  
AND/OR Positive PD40 to X5 and R5 on IOS. 
 
EXCLUSION: 
13. Respiratory tract infection within the 4 weeks prior to Visit 0 
14. Oral or systemic corticosteroid burst (for any indication) within the 4 
weeks prior to Visit 0 
15. One-time doses, such as intra-articular injections into a shoulder or knee 
joint, require a 4-week washout prior to Visit 0 
16. Asthma-related ER visit within the previous 4 weeks of Visit 0 
17. History of ICU admission/intubation due to asthma in the past 1 year 
18. Three or more asthma exacerbations requiring treatment with systemic   
corticosteroids in the past year consistent with severe asthma 
19. Asthma exacerbation requiring systemic corticosteroids within the 4 
weeks prior to Visit 0 
20. Significant concomitant medical illness, including (but not limited to) 
heart disease, cancer, uncontrolled diabetes, other chronic lung diseases 
21. Chronic renal failure (creatinine > 2.0) at Visit 0 
22. Positive urine pregnancy test at Visit 0 or at any time during the study 
23. Untreated sleep apnea  
24. Participation in an intervention study (including, bronchoscopy) or use of 
investigative drugs within   the past 30 days or plans to enroll in such a 
trial during the study 
25. Unable or unlikely to complete study assessments in the opinion of the    
Investigator 
26. Study intervention poses undue risk to patient in the opinion of the 
Investigator 
27. Negative Methacholine  challenge (historical results from previous studies or 
documented in clinical records within preceding year are acceptable alternatives)  
      and negative PD40 to X5 and R5 on IOS 

 
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Inclusion/Exclusion 
Criteria for Non-
asthmatic controls 
INCLUSION 
1. No history of asthma or other chronic lung diseases 
2. Male and female patients, 18 - 65 years old, inclusive 
3. Not currently smoking or using other forms of tobacco-related products 
(including vaping) 
4. Smoking history <10 pack years and no smoking in the past 3 months 
5. FEV1 > 80% of predicted and FEV1/FVC > lower limit of normal. 
6. Ability to sign consent 
7. BMI ≥ 30 kg/m2 
8. Negative Methacholine  challenge (historical results from previous studies or 
documented in clinical records within preceding year are acceptable alternatives)  
and negative PD40 to X5 and R5 on IOS 
 
EXCLUSION 
9. Respiratory tract infection within the 4 weeks prior to Visit 0 
10. Oral or systemic corticosteroid burst (for any indication) within the 4 
weeks prior to Visit 0 
11. One-time doses, such as intra-articular injections into a shoulder or knee 
joint, require a 4-week washout prior to Visit 0 
12. Significant concomitant medical illness, including (but not limited to) 
heart disease, cancer, uncontrolled diabetes, other chronic lung diseases 
13. Chronic renal failure (creatinine > 2.0) at Visit 0 
14. Positive urine pregnancy test at Visit 0 or at any time during the study 
15. Untreated sleep apnea 
16. Participation in an intervention study (including bronchoscopy) or use of 
investigative drugs within the past 30 days or plans to enroll in such a 
trial during the study 
17. Unable or unlikely to complete study assessments in the opinion of the 
Investigator 
18. Study intervention poses undue risk to patient in the opinion of the    
Investigator 
19. Positive Methacholine  challenge- Confirmation of asthma: Either (1) 12% or 
greater bronchodilator response (BDR) in FEV1 to albuterol at Visit 0 OR (2) 
PC20 methacholine ≤ 16 mg/ml at Visit 0 (historical results from previous studies 
or documented in clinical records within preceding year are acceptable 
alternatives)  
AND/OR Positive PD40 to X5 and R5 on IOS. 
 

 
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Additional 
Inclusion/Exclusion 
Criteria for MRI 
All participants 
INCLUSION 
1. Outpatients of either gender, age > 18 
2. Willing and able to give informed consent and adhere to visit/protocol 
schedules. (Consent must be given before any study procedures are 
performed.) 
3. Women of childbearing potential must have a negative urine pregnancy 
test prior to MRI. 
EXCLUSION 
4. Medical or psychological conditions which, in the opinion of the 
investigator, might create undue risk to the subject or interfere with the 
subject’s ability to comply with the protocol requirements 
5. Conditions that will prohibit MRI scanning (metal in eye, claustrophobia, 
inability to lie supine, shoulder circumference >140 cm*.) *This 
measurement is not an absolute as it can vary based on weight 
distribution. 
 

 
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Study Schedule 
Following prescreening for eligibility, participants will provide consent, and 
undergo a screening visit (V 0) which includes:  
 
Screening visit (V 0) 
• 
Urine pregnancy for women of child-bearing potential  
• 
Anthropometrics and vital signs 
• 
Medical history including current and past medication review 
• 
Asthma control questionnaire (ACQ)  
• 
Skin allergy testing,  
• 
Blood draw for labs: 
o CBC with diff (LAB1748) 4.0 mL 
o fasting blood glucose (LAB81) 4.0 mL  
o total cholesterol (LAB60) 4.5 mL  
o triglycerides (LAB134) 4.5 mL  
o chemistries (LAB15) 4.5 mL, and  
o serum IGE (LAB74) 3.5 mL  
o CPT 8.0 mL  
o Total blood volume =  33 mL 
• 
Impulse oscillometry (IOS) measurements 
• 
Pulmonary function testing will be performed including:  
o exhaled nitric oxide (FeNO),  
 
May be repeated once if results are high 
o spirometry 
o Lung volumes via body plethysmograph  
o Methacholine challenge 
 
  Visit 1 (1-2 weeks after visit 0) 
• 
Urine pregnancy for women of child-bearing potential   
• 
Interim medical history and medications will be reviewed, and any 
AE’s evaluated. 
• 
Vital signs 
• 
ACQ 
• 
The participants will have a standard dose chest CT  
• 
Hyperpolarized Xe-MRI performed pre and post bronchodilator. 
• 
Approximately 24-hours after MRI, a follow up phone call. 
 
 
Visit 2 (1-2 weeks after visit 1) 
• 
Urine pregnancy for women of child-bearing potential  
• 
Standard of Care (SOC) Bronchoscopy consent 
• 
Vital signs and brief physical exam 
• 
Interim medical history and medications will be reviewed, and any 
AE’s evaluated. 
• 
The participants will have a bronchoscopy with broncho-alveolar 
lavage (BAL), brushes and biopsy.  
 
Visit 3  
• 
A follow up phone call. 
 

 
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Statistical 
Methodology 
For the in vivo assessments of airway remodeling and fibrosis (bronchodilator 
reversibility on 129XeMRI), we will compare biomarkers of fibrosis (e.g., 
trichrome or percent elastic fiber staining) between BD types (fixed and 
reversible airway segments) both within and between obese asthma groups 
(early onset, late onset and non-asthma control) using a repeated measures 
two-way ANOVA with appropriate distributional assumption. Main effects of 
BD type, obese asthma group and their interaction will be evaluated with 
repeating BD type within subject (random effect) with compound symmetry 
variance structure. If significant main effect differences are found, pairwise 
comparisons between BD types and obese asthma groups will be done using 
Tukey’s post-hoc test.  Assuming a 50% decrease in fibrosis between BD 
fixed and reversible types, a sample size of 10 per group provides our study 
with ≥80% power to detect an effect size of 0.53 within and 0.60 between 
groups, where effect size (f) is the size of the mean differences relative to the 
standard deviation and assumed correlation (p) within group of 0.30. A p-
value <0.05 will be considered statistically significant and all analyses will be 
performed using SAS version 9.4 or higher (SAS Institute, Inc., Cary, NC). 
Blinding to conditions and tissue sources will occur across the experimental 
design. In particular, tissues samples (post-BD fixed and BD reversible tissue 
based on region) will be blinded to the individual performing the experiments 
and the individual assays. The key for the tissue samples will be maintained 
by the program statistician and un-blinding will occur during data analysis by 
the statistician. The statistical models and analyses that are planned for the 
primary and secondary outcomes assume that the data are missing-at-random 
(MAR). 
Because likelihood-based methods will be applied, MAR data still yield valid 
estimates. Although not expected, if it appears that the MAR assumption is 
not reasonable, then non-ignorable statistical analyses, such as shared 
parameter modeling (Albert '19, Gottfredson '14), will be applied. 
 
Data Collection 
 
Data will be captured in REDCap, except for patient consent forms and 
AE/SAE forms, which will be on case report forms (CRFs) collected by the 
coordinators. REDCap is a software tool that does not require client local 
software and can be accessed from anywhere on the Internet and is secured 
on a Duke Health Technology Services (DHTS) server. Established systems 
for data entry, verification, and error-checking will be followed. 
 
Schedule of Events: 
 
Week 
-1 
0 
1 
1+ 
Visit number 
V0 
V1 
V2 
V3 
Study ICF 
x 
 
 
 
Bronchoscopy SOC consent 
 
  
x 
  
Vital signs 
x 
x 
x 
 
Baseline medical history, including 
current and past medication review 
x 
 
 
  
Interim medical and medications review 
and any AE’s evaluated 
 
x 
x 
 
Brief physical exam 
 
 
x 
 

 
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Week 
-1 
0 
1 
1+ 
Visit number 
V0 
V1 
V2 
V3 
ACQ 
x 
x 
  
  
Spirometry  
x 
  
  
  
Impulse oscillometry (IOS) 
x 
 
  
  
FeNo 
x 
  
  
Lung volumes via body plethysmograph  
x 
 
 
 
Methacholine challenge 
x 
 
 
 
Blood draw for clinical labs* 
x 
 
 
 
Skin Allergen panel 
x 
 
  
  
Anthropometrics  
x 
  
  
  
Pregnancy test** 
x 
x 
x 
  
CT Scan 
  
x 
  
  
129XeMRI pre and post bronchodilator 
  
x 
  
  
Bronchoscopy 
  
  
x 
  
Follow-up phone call 
  
 X (24 hours 
after MRI) 
  
X (24 hours 
after 
bronchoscopy) 
*Clinical labs: CBC with diff (LAB1748) 4.0 mL, fasting blood glucose (LAB81) 4.0 mL, total cholesterol (LAB60) 4.5 mL, 
triglycerides (LAB134) 4.5 mL, chemistries (LAB15) 4.5 mL, and serum IGE (LAB74) 3.5 mL, CPT 8.0 mL   
**for women of child-bearing potential 
 
The central hypothesis is that sites of abnormal ventilation on 129XeMRI represent areas of airway remodeling and 
fibrosis and are enriched with fibroblasts that are invasive, proliferative and fibrogenic. We further hypothesize that 
regional alterations in oxidant stress driving the production of transforming growth factor-beta (TGF-β) direct pro-
remodeling fibroblast functions. Lastly, we hypothesize that 129XeMRI will be a sensitive and specific biomarker 
of airway remodeling and fibrosis in obese asthmatics and rat models of obese asthma. By leveraging our excellence 
in clinical asthma, bronchoscopy, and translational expertise in cell function/signaling and 3D MR imaging in both 
patients and animal models, we will conduct both ex vivo cell-specific mechanistic studies and in vivo animal model 
studies to uncover the mechanisms of molecular and cellular function through the following Specific Aims: 
Aim 1. Identify the pathology, structural cell profile (airway fibroblast and epithelial cell) and redox status 
corresponding to regional areas of fixed and reversible post-bronchodilator defects (BD) in obese asthmatics. 
We hypothesize that post-BD fixed defects identified on 129XeMRI correspond to sites of increased oxidant stress 
and airway remodeling and fibrosis. We will use 129XeMR imaging to guide bronchoscopy sampling of airways in 
both BD fixed and BD reversible ventilation defects. We will relate the contribution of the oxidant milieu, structural 
cell phenotypes and histopathologic composition of airways to changes in ventilation defects observed on 
129XeMRI.   
Aim 2. Define the cellular requirement for redox-mediated TGF-β signaling between airway epithelial cells 
and fibroblasts driving regional remodeling in obese asthma. We hypothesize that regions of fixed defects on 
129XeMRI are composed of airway fibroblasts that are more proliferative, invasive and fibrogenic than fibroblasts 
in BD reversible defects and respond to the increased oxidative milieu and TGF-β signaling. We will assess 
epithelial cell signaling, airway fibroblast migration/invasiveness, proliferation and extracellular matrix (ECM) 
protein secretion with ex vivo cell culture models. These assays will establish significant associations between 
airway epithelial signaling and fibroblast function, changes in airway ventilation, and asthma endotypes. 
Aim 3  Define regional differences of spatial interactions of mesenchymal cells with epithelial cells in the 
airway submucosa. 
3A - Determine how transcriptional profiles change between lean vs obese asthma and controls;  

 
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3B - Determine how transcriptional profiles change in BD fixed vs responsive regions in obese asthma. 
We hypothesize that local gene expression changes and cell signaling among both structural cells and immune cells 
in airway tissue contributes to airway remodeling in asthma patients with obesity. We plan to utilize spatial 
transcriptomics techniques to analyze endobronchial tissue samples isolated from asthma and non-asthma 
participants with and without obesity to compare specific airway tissue gene expression patterns in asthma vs non-
asthma participants, participants with obesity vs non-obesity, and combined conditions. These gene expression 
patterns will be localized to pathologic features of asthma and obesity, including accumulation of submucosal 
eosinophils, fibrosis, smooth muscle and basement membrane thickening, etc in the tissue sections. 
  
We are collaborating with Dr. Alexander Misharin at Northwestern and Dr’s. Victor Roggli and Jeffrey Everitt 
(Duke pathologists) for this aim. Cryopreserved airway tissues from deidentified participants will be used for Aim 
3A and include tables containing the demographics, physiology, FeNO, ACQ and atopy status. Endobronchial 
biopsies obtained from participants in this study will be deidentified and included in Aim 3B. 
 
Purpose of the Study:   
The proposed study will determine if sites of abnormal ventilation on 129XeMRI represent areas of airway 
remodeling and fibrosis enriched with fibroblasts that are invasive, proliferative and fibrogenic. We hypothesize 
that regional alterations in oxidant stress drive the production of transforming growth factor-beta (TGF-β) which 
directs pro-remodeling fibroblast functions. We predict that 129XeMRI will be a sensitive and specific biomarker of 
airway remodeling and fibrosis in obese subjects with asthma.  
Primary outcomes: 
• 
We will identify the pathology, structural cell profile (airway fibroblast and epithelial cell) and redox 
status corresponding to regional areas of fixed and reversible post-bronchodilator defects (BD) in obese 
asthmatics;  
• 
We will define the cellular requirement for redox-mediated TGF-β signaling between airway epithelial 
cells and fibroblasts driving regional remodeling in obese asthma;  
• 
We will develop non-invasive 3D imaging techniques to assess airway regional remodeling in obese 
asthma.  
Secondary outcomes: 
• 
We predict that 129XeMRI will be a sensitive biomarker of asthma control and severity of disease.  
 
BACKGROUND & SIGNIFICANCE:  
Obese asthma. Obese asthma is a major public health concern. The prevalence of obesity continues to rise at an 
alarming rate and has reached epidemic proportions over the past several decades. Obesity is a risk factor for the 
development of asthma and is associated with approximately 250,000 cases per year in the U.S. 1. In patients with 
established asthma, obesity is associated with increased risk for exacerbations, worse respiratory symptoms and 
poor control 2,3. Obesity and weight gain are associated with increased asthma severity and a nearly 5-fold risk of 
hospitalization 4. This is a major public health concern, given that the Centers for Disease Control has estimated 
that 38.8% of asthmatics are obese.  
Obese asthma is broadly categorized into two distinct endotypes, early vs. late onset disease.  Early onset obese 
asthma is defined as being diagnosed at <12 years of age, associated with type 2 (T2) high disease, an allergic 
phenotype, and the development of obesity as a consequence of asthma. Late-onset obese asthma occurs after 12 
years of age or in adulthood 5 and is associated with the development of asthma subsequent to obesity 6. In early 
onset asthma, obesity contributes to disease severity and is associated with an increased incidence of fixed airway 
obstruction 7; this fixed obstruction is also referred to as airway remodeling and/or fibrosis (structural changes of 
the airway wall), and manifests as bronchodilator non-responsiveness. In contrast, the late onset obese asthmatic is 
a common and unique clinical phenotype that is not defined by T2-related airway inflammation. Despite having 
relatively preserved lung function and lower fractional exhaled nitric oxide (FeNO) levels, cluster analyses show 
that those asthmatics characterized by body mass index (BMI) >30 kg/m2 with late onset disease have a high 
symptom burden and high utilization of healthcare resources 8-10.  New biologics targeting T2 high asthma are less 
effective in obese asthmatics compared to lean patients and are not efficacious in non-T2 disease 11,12.  Therefore, 
we need to better understand how asthma differs in the obese population. Only with such understanding will it 
become possible to develop effective therapies that benefit these patients. 

 
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Airway Remodeling and Fibrosis. Airway remodeling and fibrosis in asthma represents a major cause of 
morbidity and mortality. It is notoriously difficult to diagnose with precision, to treat effectively, and to monitor 
effectively as the disease progresses. Clinical assessment of airway remodeling in the obese asthmatic patient is 
especially challenging as asthma in the obese is often attributed to mechanical effects caused by chronic lung 
compression and/or airway reactivity induced by breathing at low lung volumes. Airway remodeling can result from 
either accelerated, uncontrolled inflammation, as a response to injury or as a result of abnormal growth and 
development 13. Despite intensive research efforts, treatments for remodeling remain limited, reflecting a poor 
understanding of the common mechanisms underlying these processes. A central issue is that airway remodeling is 
heterogeneous.  Not only can remodeled tissue exhibit unique mechanisms based on the maturity of the remodeling, 
but also the extent of remodeling can differ across the landscape of the lung. This regional heterogeneity limits 
detection of airway remodeling and fibrosis and hampers the ability to test the efficacy of early interventions as 
global measures of lung function do not detect regional changes in air flow or functional compensation from 
surrounding tissues. Both these aspects of heterogeneity are addressed in this application.  
Obese asthma is associated with more severe airway remodeling and fibrosis than lean asthma 14,15. The epithelium 
is hypothesized to orchestrate the inflammatory and remodeling responses of the airway, as it serves as a rich source 
of TGF-β 16,17, a profibrotic growth factor. The epithelium also potentially serves as a source of myofibroblasts via 
epithelial-to-mesenchymal transition (EMT) 18. Fibroblasts and myofibroblasts are the main structural cells of the 
mesenchyme that mediate the majority of the events contributing to the subepithelial fibrosis in remodeled airways 
19-21. Following injury to the airway, epithelial cells secrete growth factors (e.g., TGF-β and epidermal growth factor) 
and become migratory by undergoing EMT. In parallel, resident fibroblasts in the airway migrate to sites of airway 
injury, proliferate, differentiate to myofibroblasts and deposit matrix 22-24. Indeed, fibroblasts isolated from the 
airways of asthmatic subjects make greater amounts of ECM proteins, including collagen 25 and hyaluronan 26, with 
lesser amounts of elastin 27, compared with fibroblasts isolated from non-asthmatics 28. Work by Hackett et al. 
suggests that asthmatic epithelium responds inappropriately to challenge resulting in dysregulated repair and airway 
remodeling and fibrosis 29. In T2 high disease, interleukin-13 (IL-13) has been shown to potentiate airway fibroblast 
invasion in the asthmatic airway through a mechanism involving TGF-β 1 and metalloproteinases 30.  Mechanisms 
regulating remodeling and fibrosis in nonT2 disease are not known, but this knowledge would greatly aid in the 
development of novel strategies to treat this predominant asthma endotype. 
Oxidative stress in Obese Asthma. Oxidative stress is increased in the obese patient because adipose tissue serves 
as a source of pro-inflammatory cytokines such as leptin, tumor necrosis factor-alpha (TNF-β, interleukin-1beta 
(IL-1β and interleukin-6 (IL-6) 31 which promote increased generation of reactive oxygen species (ROS) and 
reactive nitrogen species (RNS).  Oxidative stress is believed to play a critical role in the pathogenesis of asthma 
and can result from an imbalance between the production of ROS and RNS and/or a loss of antioxidant defense 
mechanisms. However, to date it remains unclear whether oxidative stress in obese asthma is secondary to increased 
inflammation or if it is causative to the development of asthma.  
Clinical Assessment of Airway Remodeling and Fibrosis in Obese Asthma. Conventional lung function testing 
lacks sensitivity to evaluate airway remodeling and fibrosis. Spirometry, though considered to be the gold standard, 
is limited in its use as it does not accurately measure airway resistance in peripheral small airways, the major site 
of airflow obstruction in obese asthma patients 32. Clinically the presence of fixed airflow obstruction as measured 
by lack of bronchodilator responsiveness (<200 cc or <12% improvement in forced expiratory volume (FEV1) or 
forced vital capacity (FVC) post-bronchodilator) is considered to be a late and irreversible manifestation of airway 
remodeling. To overcome this, forced oscillation technique (FOT) has been used to improve the sensitivity to detect 
small airways dysfunction, but FOT cannot quantify regional changes in small airway disease, which limits our 
ability to effectively monitor progression of disease and response. 
These 
limitations 
in 
assessing regional 
structure-function 
abnormalities have created a critical barrier to the advancement of 
our understanding of mechanisms driving airway remodeling in the 
obese asthmatic patient. 

 
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Hyperpolarized 129XeMRI to Assess Lung Structure Function. Hyperpolarized helium (3He) and xenon (129Xe) 
MR imaging of the lungs have emerged as novel mechanisms to probe changes in pulmonary ventilation, 
microstructure and gas exchange. 129Xe  has a high solubility in blood and lipid rich tissue. Inhaled 129Xe is absorbed 
quickly into the blood stream and distributed rapidly throughout the body by circulation 33. A unique quality of 
129Xe is that, once absorbed, it demonstrates distinct chemical shifts in the gas-phase, pulmonary barrier tissue, and 
red blood cells (RBCs) 34, so combining visualization of gas phase and dissolved phase allows us to simultaneously 
assess ventilation, interstitial abnormalities, and lung microstructure (Figure 1).  129XeMRI has been used to identify 
ventilation defects induced by exercise, methacholine challenge and those reversed by bronchodilators 35; these 
defects increase with asthma severity 36. Moreover, studies using methacholine to induce bronchoconstriction or 
albuterol as a bronchodilator demonstrate that some regional changes 
of airflow obstruction are relatively fixed in the lung while others are 
much more dynamic (months-years) 37,38. 129XeMRI has also been 
used to assess small airway dysfunction in asthma 33,39 and can 
identify and localize regional ventilation defects, even in asthmatics 
with normal spirometry, suggesting a potential role for 129XeMRI in 
preclinical detection of disease and as a biomarker of disease 
activity. We and others have found that 129XeMRI has a strong safety and tolerability profile in subjects with 
obstructive airway diseases; indeed, only transient hypoxia, dizziness, paresthesia/hypoesthesia and euphoria have 
been reported 40. Moreover, the test-retest variability of 129Xe MRI is exceptionally good (±1.5%) relative to that of 
the spirometric measure, FEV1 (±7.2%) 41.These studies support safety, tolerability and reproducibility of 
129XeMRI. 
Biomarkers in obese asthma. New precision medicine approaches to diagnose and monitor disease activity in the 
obese asthmatic patient are needed. Despite significant advances in our understanding of the pathobiology of 
asthma, most of what we have learned about asthma has been obtained studying lean asthmatics. In patients with 
early onset disease, T2 inflammatory biomarkers are being used to identify eosinophilic asthma phenotypes and to 
predict response to therapy 42-45. However, these biomarkers are poorly predictive of disease activity in obese 
asthma. This problem is amplified in the late onset obese asthma patient for whom there are no other biomarkers 
and very limited treatment options available. Indeed, biomarkers used to identify T2 driven disease in lean 
asthmatics have been found to be poorly predictive of eosinophilic airway inflammation in the T2 high obese 
asthmatic 46. As a result, a major limitation in studying obese asthma is lack of the ability to monitor and identify 
sites of disease activity. This project will advance our understanding of the different asthma endotypes and provide 
a noninvasive modality to regionally assess lung structure/function and response to therapy. 
Bronchoscopy and Clinical Phenotyping. 
By using 129Xe MRI to identify BD-fixed and reversible ventilation defects, this study will be the first to objectively 
evaluate the contribution of structural cell phenotypes, epithelial cell RNA transcriptomes, immune cells profiles 
and oxidant burden to obese asthma endotypes. Our use of co-registered chest CT/129XeMRI to identify BD fixed 
and reversible regional changes in ventilation and to guide airway tissue sampling for the study of underlying 
histopathology, cell composition and oxidative milieu is an innovative approach to studying the biological 
underpinnings regulating airway remodeling and fibrosis in obese asthma.  
 
All subjects enrolled will undergo bronchoscopy with endobronchial biopsy, brushings and bronchoalveolar lavage 
(BAL) and blood serum collection.  Six endobronchial biopsies and brushings per airway segment will be performed 
under direct visualization. Biopsy locations will be 129XeMRI-guided to airway regions of BD fixed and BD 
reversible defects. Two endobronchial biopsy specimens per airway segment will be fixed in 4% paraformaldehyde 
and embedded in paraffin. Two or three specimens per airway segment will be used for airway fibroblast culture. 
Epithelial cells harvested from airway brushes will be grown on air liquid interface for cell culture assays and airway 
epithelial cell RNA 
 
We will determine airway fibroblast phenotype (i.e. myofibroblast differentiation) in persistent MRI defects post-
BD using several methods: 
1) Airway fibroblasts will be cultured from fresh airway biopsy pieces following Ingram et al (34). 
Immuno-cytochemistry will be performed on cells at passage 0 cultured on collagen-coated chamber 
slides to determine reactivity with antibodies specific for myofibroblast markers (alpha smooth muscle 
Figure 1. 129Xe Gas Exchange MRI in a 
severe asthmatic. While the most prominent 
features are substantial ventilation defects 
(red), it is also noteworthy that numerous 
areas of the lung exhibit poor RBC transfer, 
while others exhibit exceptionally high transfer 

 
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actin (αSMA) and amine oxidase, copper containing 3 (AOC3) (77) as well as the fibroblast markers, 
Fibroblast specific protein-1 (FSP-1) and vimentin, and an epithelial cell marker, E-cadherin.  
2) Total RNA and protein lysates will be isolated from cells grown at air liquid at Passage 0. Western 
blotting and quantitative RT-PCR will be used to confirm protein and mRNA expression of αSMA (29) 
and AOC3 in myofibroblasts, and to rule out expression of epithelial cell protein, E-cadherin.  
3) To control for cell culture artifacts, fresh airway biopsy tissue will be digested with 
ethylenediaminetetraacetic acid disodium (EDTA) and we will perform fluorescence-activated cell 
sorting (FACS) for AOC3+ myofibroblasts and AOC3- fibroblasts. Cells will be fixed and stained with 
anti-vimentin and αSMA to confirm fibroblast and myofibroblast phenotype.  
1.3 We will perform single cell ribonucleic acid (RNA) sequencing (scRNAseq) analysis on brushings from airway 
segments corresponding to post-BD fixed and reversible defects on 129XeMRI. This will allow us to assess 
cellular heterogeneity and to determine it contribution to ventilation heterogeneity. We have performed 
scRNAseq of bronchial brushings previously using the Drop Seq method (78, 79). Drop-seq permits highly 
parallel genome-wide transcriptome profiling using unique barcodes that tags cellular mRNAs from individual 
cells. These unique barcodes will then be used to in silico reconstruct the single cell RNAs from tens of 
thousands of cells. For scRNA seq analysis, the FASTQ files will be processed using dropSeqPipe v0.3 and 
mapped on to the human genome. Unique molecular identifier (UMI) counts will then be further analyzed using 
an R package Seurat v3.0.6. UMI counts will be normalized using SCTransform v0.2. Principle components 
that are significant based on Jackstraw plots will be used for generating t-SNE and UMAP plots. Cell barcodes 
of clusters of interests will be extracted and utilized for velocyto run command in velocyto.py v0.17.15 as well 
as generating RNA velocity plots using velocyto.R v0.6 in combination with an R package SeuratWrappers 
v0.1.0. 
1.4 Determine the association between ventilation heterogeneity and airway physiology (pulmonary function 
testing (PFT) and impulse oscillometry) (80, 81). 
 
DESIGN & PROCEDURES:  
Subject Recruitment and Screening:  We anticipate 40 early and late onset obese asthma patients and 10 obese 
non-asthma volunteers will undergo Hyperpolarized 129XeMRI  and bronchoscopy after screening over the course 
of this study. Patients will undergo, twice in succession, an approximately 30-min MRI protocol, consisting of 
several breath-hold scans after hyperpolarized 129Xe or room air administration. The imaging will be repeated pre- 
and post-bronchodilator (BD), thus requiring 60-min to complete. The results of a co-registered CT of the chest and 
the pre-post bronchodilator Hyperpolarized 129XeMRI  will be reviewed by the Duke Imaging Team to identify 
segmental persistent post-BD ventilatory defects in obese asthmatics. The CT will provide precise structural 
information for segmental delineation, while MRI informs regional ventilatory function, interstitial uptake and lung 
microstructure and function. For these purposes, we need a single high-res (standard dose) CT scan at full 
inspiration. The Duke Imaging Team  will dictate to the bronchoscopist in a blinded fashion which segments contain 
BD fixed and BD reversible ventilatory defects. Bronchoalveolar lavage (BAL), endobronchial biopsies and 
brushings will be obtained from these segments.  
 
Study participants will be enrolled at the Duke University Health System from clinics, hospitals and communities 
via various IRB-approved recruitment methods, including Protocol Pro00102890; Pulmonary Medicine Healthy 
Volunteer Data Repository (HVDR) and Pro00103507; Pulmary Medicine  Lung Disease Volunteer Data 
Repository (LDVDR). 
 
Preliminary eligibility asthma diagnosis, medical history will be determined by pre-screening procedures that 
include a review of medical records (site specific electronic medical record (EMR) reports and search tools such as 
DEDUCE, slicer dicer and Mychart invitations, and manual review) and clinic schedules to identify potentially 
eligible participants. Each participant who is pre-screened (e.g. medical chart review is performed to assess potential 
eligibility prior to approaching the participant), approached for recruitment and screened for study participation will 
have the following information entered in a screening log: name, MRN, date of birth, race, ethnicity, contact 
information, date of pre-screen review, and enrollment status, along with the reason(s) for not enrolling in the study, 
if applicable. Participants will be enrolled into one of the three groups (late onset asthma, early onset asthma, and 
non-asthma controls) based on their profiles.  
 

 
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Informed Consent Process:  Participants will receive an overview of the study during an in person or phone pre-
screening process. A copy of the ICF can be sent to the potential participant via e-mail or mail prior to the screening 
visit.  Prior to the initiation of any clinical research procedures at study visits, designated research staff will describe 
the study’s purpose, all procedures, and risks/benefits and review the informed consent document with the potential 
participant. Subjects will be informed that their decision to participate in the study will have no impact on their 
medical care. Participants will be provided ample time to read the consent, ask questions, and deliberate their 
decision.  
 
Patients who are deemed eligible after pre-screening will be invited and scheduled for enrollment at Duke. A verbal 
consent will be performed to allow for placing orders in the EMR prior to written consent.   Willing participants 
will undergo informed written consent before any study-related procedures take place. Following consent, they will 
be assigned a study ID in consecutive order and record the assignment for ID on the log.  
 
The coordinator or PI will conduct the consent process with prospective participants. Dr. Driehuys will not 
personally obtain consent due to his conflict of interest. 
 
Enrollment, Study Visits & Procedures: 
 
Screening Visit 0 – 3-4 hours (week -1) 
Following Informed Consent, participants will be enrolled and will be assigned a study ID in consecutive order 
from the assignment log provided record the assignment on the study log. All enrolled participants will then 
perform: 
• 
Urine pregnancy for women of child-bearing potential  
• 
Anthropometrics and vital signs 
• 
Medical history review (including the following: age of asthma onset, prior hospitalizations, ER visits, 
steroid use, frequency of asthma exacerbations, antibiotic use, nocturnal awakenings, frequency of SABA 
use, immune desensitization, current and past medication use, self-described smoking history) 
o Demographics (race, ethnicity, gender, age) 
• 
ACQ 
• 
Skin allergy testing,  
• 
Blood draw for labs: 
o CBC with diff (LAB1748) 4.0 mL 
o fasting blood glucose (LAB81) 4.0 mL  
o total cholesterol (LAB60) 4.5 mL  
o triglycerides (LAB134) 4.5 mL  
o chemistries (LAB15) 4.5 mL 
o serum IGE (LAB74) 3.5 mL 
o CPT 8.0 mL   
o Total blood volume = 33 mL 
• 
IOS   
• 
Pulmonary function testing will be performed including:  
o exhaled nitric oxide (FeNO),  
o May be repeated once if results are high 
o spirometry 
o Lung volumes via body plethysmography 
o Methacholine challenge 
 
 
Visit 1 – 4 hours (week 0) 
• 
Urine pregnancy for women of child-bearing potential  
• 
Interim medical history and medications will be reviewed, and any AE’s evaluated  
• 
Vital signs  

 
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• 
ACQ 
• 
Standard dose chest CT (SDCT)  
• 
Hyperpolarized 129XeMRI pre and post bronchodilator.    
• 
Participants will receive a follow up phone call approximately 24 hours after the MRI with the 
coordinator, including a review of interim medical history and medications and any AE’s evaluated  
 
Visit 2 – 2 hours (week 1) 
• 
Standard of Care (SOC) Bronchoscopy consent 
• 
 Urine pregnancy for women of child-bearing potential  
• 
Vital signs and brief physical exam 
• 
Interim medical history and medications will be reviewed, and any AE’s evaluated. 
• 
Bronchoscopy with BAL 90 ml per airway in two airways for a total of 180 ml, and up to 6 brushes per 
airway (12 total) and endobronchial biopsies per airway segment (up to 12 total).   
• 
Post-procedure observation in the recovery area of 25 minutes to 1 hour.  
 
The participant will not be allowed to proceed with the bronchoscopy visit which includes moderate conscious 
sedation without an adult that comes with them during the check in process and is available to escort the 
participant home.    
 
Visit 3 – follow up (1 week after visit 2) 
• 
Participants will receive a follow up phone call with the coordinator, including a review of interim  
medical history and medications and any AE’s evaluated  
 
Asthma Cohort Inclusion / Exclusion Criteria are as follows: 
INCLUSION 
1. Adequate completion of informed consent process with written documentation 
2. Male and female patients, 18 - 65 years old, inclusive 
3. Physician diagnosis of asthma for > 1 year 
4. Able to perform reproducible spirometry according to ATS criteria 
5. All racial/ethnic backgrounds may participate 
6. BMI ≥ 30 kg/m2 
7. Regular treatment with ICS or ICS/LABA and/or LAMA combination medication for at least 3 months; 
on a stable dose for the 4 weeks prior to Visit 0 
8. Smoking history <10 pack years and no smoking in the last 3 months 
9. Late onset asthma: Age of asthma onset (diagnosis) ≥12 years; 
• 
FeNO < 25 ppb at Visit 0 
• 
Negative allergen skin test 
10. Early onset asthma: Age of asthma onset (diagnosis) <12 years 
• 
FeNO ≥ 25 ppb at Visit 0 
• 
Positive allergen skin test  
11.  Positive Methacholine  challenge- Confirmation of asthma: Either (1) 12% or greater bronchodilator response 
(BDR) in FEV1 to albuterol at Visit 0 OR (2) PC20 methacholine ≤ 16 mg/ml at Visit 0 (historical results from 
previous studies or documented in clinical records within preceding year are acceptable alternatives)  
       AND/OR Positive PD40 to X5 and R5 on IOS. 
 
 
EXCLUSION: 
1. Respiratory tract infection within the 4 weeks prior to Visit 0 
2. Oral or systemic corticosteroid burst (for any indication) within the 4 weeks prior to Visit 0 
• 
One-time doses, such as intra-articular injections into a shoulder or knee joint, require a 4-week washout 
prior to Visit 0 
3. Asthma-related ER visit within the previous 4 weeks of Visit 0 

 
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4. History of ICU admission/intubation due to asthma in the past 1 year 
5. Three or more asthma exacerbations requiring treatment with systemic corticosteroids in the past year consistent 
with severe asthma 
6. Asthma exacerbation requiring systemic corticosteroids within the 4 weeks prior to Visit 0 
7. Significant concomitant medical illness, including (but not limited to) heart disease, cancer, uncontrolled 
diabetes, other chronic lung diseases 
8. Chronic renal failure (creatinine > 2.0) at Visit 0 
9. Positive urine pregnancy test at Visit 0 or at any time during the study 
10. Untreated sleep apnea  
11. Participation in an intervention study (including, bronchoscopy) or use of investigative drugs within   the past 
30 days or plans to enroll in such a trial during the study 
12. Unable or unlikely to complete study assessments in the opinion of the Investigator 
13. Study intervention poses undue risk to patient in the opinion of the Investigator 
14. Negative Methacholine  challenge (historical results from previous studies or documented in clinical records within 
preceding year are acceptable alternatives)  
      and negative PD40 to X5 and R5 on IOS. 
 
 
Inclusion Exclusion Criteria for Non-asthmatic controls 
INCLUSION 
1. No history of asthma or other chronic lung diseases 
2. Male and female patients, 18 - 65 years old, inclusive 
3. Not currently smoking or using other forms of tobacco-related products (including vaping) 
4. Smoking history <10 pack years and no smoking in the past 3 months 
5. FEV1 > 80% of predicted and FEV1/FVC > lower limit of normal. 
6. Ability to sign consent 
7. BMI ≥ 30 kg/m2 
8. Negative allergen skin test 
9.  Negative Methacholine  challenge (historical results from previous studies or documented in clinical records 
within preceding year are acceptable alternatives) and negative PD40 to X5 and R5 on IOS. 
 
EXCLUSION 
1. Respiratory tract infection within the 4 weeks prior to Visit 0 
2. Oral or systemic corticosteroid burst (for any indication) within the 4 weeks prior to Visit 0 
• 
One-time doses, such as intra-articular injections into a shoulder or knee joint, require a 4-week washout 
prior to Visit 0 
3. Significant concomitant medical illness, including (but not limited to) heart disease, cancer, uncontrolled 
diabetes, other chronic lung diseases 
4. Chronic renal failure (creatinine > 2.0) at Visit 0 
5. Positive urine pregnancy test at Visit 0 or at any time during the study 
6. Untreated sleep apnea 
7. Participation in an intervention study (including bronchoscopy) or use of investigative drugs within the past 30 
days or plans to enroll in such a trial during the study 
8. Unable or unlikely to complete study assessments in the opinion of the Investigator 
9.    Study intervention poses undue risk to patient in the opinion of the Investigator 
10. Positive Methacholine  challenge (historical results from previous studies or documented in clinical         
records within preceding year are acceptable alternatives)  
      AND/OR positive PD40 to X5 and R5 on IOS. 
 
Additional Inclusion/Exclusion Criteria for MRI 
INCLUSION 
1. Outpatients of either gender, age > 18 

 
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2. Willing and able to give informed consent and adhere to visit/protocol schedules. (Consent must be given 
before any study procedures are performed.) 
3. Women of childbearing potential must have a negative urine pregnancy test prior to MRI. 
 
EXCLUSION 
1. Medical or psychological conditions which, in the opinion of the investigator, might create undue risk to 
the subject or interfere with the subject’s ability to comply with the protocol requirements 
2. Conditions that will prohibit MRI scanning (metal in eye, claustrophobia, inability to lie supine, shoulder 
circumference >140 cm*.) *This measurement is not an absolute as it can vary based on weight distribution. 
 
Descriptions of Evaluations and Measures: 
• 
Anthropometrics, height, weight and vital signs – Blood pressure is a determinant in classifying and 
understanding relative risk of cardiovascular disease. Blood pressure will be recorded using an automated 
blood pressure cuff. If, due to medical reasons, the participant cannot be measured for blood pressure on 
either arm, a brachial blood pressure reading with an appropriately sized cuff may be taken by experienced 
staff. To assess abdominal obesity, waist measurement should be made at the midpoint between the lower 
margin of the last palpable rib, and top of the iliac crest (the hip bone). Hip measurement will be measured 
at the widest point of the buttocks, with the tape measure parallel to the floor.  Height will be recorded to 
the nearest 1 mm. Weight will be recorded to the nearest 100 g or 0.1 kg and repeated 2 times. If the two 
readings are within 100g of each other, then these readings will be averaged and recorded. 
 
• 
Venipuncture - Blood samples will be obtained by venipuncture of an antecubital vein to determine the 
levels of study-related biomarkers and clinical labs.  
 
• 
Spirometry- a measure of pulmonary lung function.   
• 
Exhaled Nitric Oxide-This procedure involves exhaling gently into a small, handheld device that measures 
FeNO. Participants will also undergo measurement of exhaled nitric oxide according to standardized 
techniques. The risks of this test are minimal. Collection of FeNO involves inhalation to total lung capacity 
while in a seated position followed by slow exhalation against resistance for 10 seconds. Both children and 
adults can perform this without difficulty. A trained technician will be present to supervise and support the 
participant in order to minimize discomfort.  
 
• 
Impulse oscillometry: Using this procedure, the patient breathes through a pneumotachograph which 
generates a sound wave by a loudspeaker that is superimposed over the subject’s normal, quiet, tidal 
breathing. The patient’s airflow and sound wave response is transmitted to the apparatus and used to 
calculate the various components of resistance to breathing. The risks of this procedure are minimal. Both 
children and adults can perform the procedure without difficulty. 
 
• 
Lung volumes via body plethysmograph pre-bronchodilator - Body plethysmography Lung volumes 
will be used to assess the following lung volume parameters: Functional Residual Capacity (FRC), Vital 
Capacity (VC), and then Inspiratory Capacity (IC) 
 
• 
Methacholine challenge-Participants will be asked to come in for a methacholine challenge after 
consenting.  This test is often used to support the diagnosis of asthma.  Methacholine is a chemical that may 
cause the airways of asthmatics to narrow, but does not affect the airways of people without asthma.  The 
test consists of inhaling increasing doeses of methacholine through a nebulizer to assess how quickly your 
airways narrow.  Your airways may tighten slightly as the test proceeds.  We are performing the test to see 
how quickly your airways narrow.  We will start with very small doses, gradually increasing the dose of 
methacholine.  Spirometry will be performed after each dose to look for changes in lung function.  The 
procedure will be terminated if you demonstrate a significant change in your pulmonary function test (20% 
decrease in FEV1) or you reach the highest concentration of methacholine.  You will receive albuterol (an 
inhaler medicine that relaxes the airways) after the test is done to reverse any asthma symptoms.  The 
methacholine challenge test will be done by trained personnel and can take up to two hours to complete.  

 
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Before the test, you will be asked to withhold all short-acting bronchodilators for 4 hours, long-acting 
bronchodilators for 12 hours, and Tiotropium for 24 hours.  You will also be asked to avoid caffeine and 
alcohol for 8 hours. 
 
For challenge testing, methacholine will be administered via AeroEclipse BAN II nebulizer 
following guidelines recommended by the American Thoracic Society (ATS), with 1 additional dose, 
will be used: 0.03125 mg/mL, 0.0625 mg/ml, 0.1250 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 
mg/mL and 4 mg/mL.  
Solutions are prepared using Provocholine 100 mg in 20 ml, 1 vial will be required. 
 
 
The vials will be used in reverse order, starting from the lowest concentration.  
• Discard vials A and B.  
• Vials are prepared in the order A, B, C, D, E, F, G, H, I, and J but administered to the participant in the 
order J, I, H, G, F, E, D, and C.  
 
 
• 
Allergy testing - A skin prick test, also called an allergy skin test, checks for immediate allergic reactions 
to different substances at once. This test is usually done to identify allergies to trees, grasses and weeds. 
 
• 
Chest SDCT  
SDCT (Standard dose computed tomography) is a standard medical procedure in lung imaging, widely 
used clinically and in research. Risks to participants are minimal. The study will involve a small amount of 
radiation exposure to the subjects. There will be one SDCT scan performed during the study. The scan 
protocol will be adjusted based on the participant weight. The average weight adult radiation dose is 4 mSv 
per scan, (8mSv) for the study. This compares to the US limit on radiation workers which is 50 mSv. 
 
• 
129XeMRI  
Hyperpolarized 129Xe is treated as a drug by the FDA and is covered by our existing Investigational New 
Drug Filing (IND# 109,490), which has been active since 2011 and has reported no SAEs that required 
medical intervention. The proposed studies will use hyperpolarized 129Xe prepared in accordance with the 
Drug Master File that is part of the IND held by Polarean, Inc. We have been granted the rights by Polarean 
to cross-reference this IND for our own institutional IND filings. We will continue to follow a 129Xe 
administration protocol that is well established in our hands. 
 

 
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• 
Bronchoscopy with Bronchial alveolar lavage and Biopsy 
Bronchoscopy is an established clinical procedure that has been used as standard for diagnosis of lung 
lesions. Overall, research bronchoscopy in adult asthmatics is considered to be a safe and well-tolerated 
procedure 47. Furthermore, bronchoscopy with bronchoalveolar lavage, endobronchial biopsies and and 
airway brushings have been extensively used as clinical research tools in the last decade with complication 
rates not more than 2-3%.  
 
The Imaging Team will dictate in a blinded fashion to the bronchoscopist where the samples will be 
obtained based on the results of the co-registered CT and pre-post bronchodilator 129XeMRI ventilation 
images. Samples will be obtained from lung segments containing bronchodilator fixed and bronchodilator 
reversible defects in the following order to ensure standardization: 1) bronchial brushings, 2) 
bronchoalveolar lavage (BAL), 3) endobronchial biopsies.  
 
• 
Bronchoalveolar Lavage: The purpose of the BAL is to evaluate the inflammatory cell profile. 129XeMRI 
images will be used to guide where the bronchoscope will be placed and wedged for BAL collection.  Three 
40 ml aliquots of 37°C sterile 0.9% saline solution are instilled after the bronchoscope is wedged into each 
subsegment. The bath water temperature is continuously measured. The BAL fluid is harvested by 
immediate gently hand suction applied to each instilling syringe. Syringes are placed immediately in ice. 
The volume of the effluent is measured after the procedure and samples are combined with the exception 
of the first aliquot return which represents the “bronchial” portion of the lavage48. Reduction in oxygen 
saturation following bronchoalveolar lavage is common for several hours.  Supplemental O2 will be 
administered as required.  Our standard method will be used to count and differentiate cell types in the 
airway and alveolar fraction of the BAL 49 50 51. 
o 120 mL instilled in BD reversible and 120 mL instilled in BD fixed airways 
 
• 
Bronchial brushings: The purpose of this procedure is to obtain airway epithelia for ex vivo culture. A 
sheathed biopsy brush (ConMed Disposable Cytology Brushes, Cat #149) will be introduced through the 
bronchoscope into the appropriate subsegmental bronchus and the bronchial wall will be brushed. Site of 
brushings will be dictated by the Imaging Core in consultation with Dr. Mammarappalil, our radiologist. 
and the bronchial wall will be brushed.  The brush will be re-sheathed, withdrawn, and cut off into a sterile 
tube containing 10 ml RPMI medium for further processing.  Our previous experience indicates that we are 
able to retrieve approximately 1.5 million cells/brush (approximately 95% epithelial) using this technique.  
Cytospin preps will be prepared to evaluate the cell content.  A small aliquot of cells will be stained for 
cytokeratin to determine the percentage of epithelial cells with this technique.  
o up to 6/BD reversible airway and up to 6/BD fixed airway 
 
• 
Biopsy:  Endobronchial Biopsy: The purpose of this procedure is to obtain tissue for immunohistochemical 
staining and for isolation of fibroblasts for ex vivo cell culture experiments. Endobronchial biopsies are 
planned for Projects 1 and 4 and will be performed under direct visualization from the second to fourth 
generation airways. Site of biopsy will be dictated by Dr. Driehuys’ team.  Up to twelve biopsies will be 
taken from the subcarinae with an “alligator” type forceps. 
o 6 endobronchial biopsies in BD reversible and 6 endobronchial biopsies in BD fixed airway 
segments 
 
Descriptions of Questionnaires  
• 
Asthma Control Questionnaire (ACQ): This questionnaire asks about asthma symptoms during the past 
2 weeks. 
 
Subject’s Capacity to Give Legally Effective Consent:  Participants incapable of providing informed consent will 
not be enrolled. 
 
Costs to the Subject: There will be no costs to subjects. 

 
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Subject Compensation:  
• 
Screening Visit (V0): $75 
• 
Visit 1: $100 
• 
Visit 2: $450 
• 
Unscheduled visit:  $25 
• 
Total Potential Compensation: $650 
 
Compensation will be in the form of a ClinCard where compensation will be submitted at the completion of each 
visit.  In addition, parking passes will be provided for visit performed at the main Duke campus for the radiologic 
and bronchoscopy procedures.   
 
Study Interventions:  Study procedures include pulmonary function testing to include methacholine challenge,  
allergy testing, urine pregnancy tests, clinical interviews, questionnaires, a standard dose chest CT scan, 
Hyperpolarized 129XeMRI, and a venipuncture for clinical labs. 
 
RISK / BENEFIT ASSESSMENT: 
The risk/benefit ratio for this study is relatively low. All data collection procedures are minimally  
 
Description of and Steps to Minimize Study Risks: 
The potential risks associated with this study are as follows:  
 
1. Confidentiality   
Risks: Confidentiality is of critical importance, and we will take many precautions to protect against the 
possibility of a breach of confidentiality.  
Managing risk: In order to protect participants, we have obtained a Certificate of Confidentiality from 
DHHS. Thus, while we acknowledge that a breach of confidentiality is possible, the likelihood is very low 
with the above policies and procedures in place. 
 
2. Pulmonary function testing 
• 
Spirometry- 
Risks: The risks of spirometry are minimal. The possible risks include fatigue and precipitation of 
bronchospasm and light-headedness from repeated blowing attempts. 
Managing risk: Medical and nursing personnel and medications will be available at the study sites to treat 
and manage bronchospasm. Inhalation of a short-acting beta-2 adrenergic agonist (such as albuterol) will 
be used to assess reversibility at Visit 0 (if applicable) and to qualify participants for bronchoscopy at the 
relevant visits. The possible risks of inhaled beta-2 adrenergic agonists include tachycardia and hand 
tremors. These side effects are non-life threatening and are short-lived. The patient may stop the spirometry 
procedure at any time if he/she should feel shortness of breath or fatigue.  
 
• 
FeNO 
Risks: The risks of this procedure are minimal. Both children and adults can perform the procedure without 
difficulty. Possible risks include fatigue and precipitation of bronchospasm and light-headedness from 
repeated blowing attempts.  
Managing risk: Medical and nursing personnel and medications will be available at the study sites to treat 
and manage bronchospasm. Inhalation of a short-acting beta-2 adrenergic agonist (such as albuterol) will 
be used for subjects who experience bronchospasm. The possible risks of inhaled beta-2 adrenergic agonists 
include tachycardia and hand tremors. These side effects are non-life threatening and are short-lived. The 
patient may stop the procedure at any time if he/she should feel shortness of breath or fatigue. 
 
• 
Forced Oscillation 
Risks: There are no risks associated with this procedure. Both children and adults can perform the procedure 
without difficulty. 

 
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Managing risk:  None. 
 
• 
Methacholine Challenge 
Risks:  The major risk of methacholine challenge is the induction of severe bronchoconstriction.  As a 
precaution, participants will not undergo a methachline challenge if their FEV1 is less than 60% of 
predicted, or 1.5 liter. 
Managing Risk: Medical and nursing personnel and medications will be available at the study sites to treat 
and manage bronchospasm. Inhalation of a short-acting beta-2 adrenergic agonist (such as albuterol) will 
be used for subjects who experience bronchospasm. The possible risks of inhaled beta-2 adrenergic agonists 
include tachycardia and hand tremors. These side effects are non-life threatening and are short-lived. The 
patient may stop the procedure at any time if he/she should feel shortness of breath or fatigue 
 
3. Venous phlebotomy   
Risks: Risks associated with drawing blood include momentary discomfort, bruising, and/or dizziness. While 
infection, excess bleeding, clotting, and fainting are possible, these are unlikely to occur. To minimize these 
risks, staff will follow all laboratory safety procedures. 
Managing risk: Pressure will be applied to the venipuncture site to prevent bruising. Aseptic technique will be 
used to prevent infection. Blood will be obtained while the enrolled subjects are in a seated position and medical 
and nursing personnel will be available at the study sites to treat and manage vasovagal episodes.  
 
4.   Allergy Testing 
Risks: Risks from skin testing include allergy symptoms might occur during the test. The most common 
symptoms are itching and swelling of the skin where the tests are. In rare cases, shortness of breath can occur. 
Managing risk: To minimize the risk of bronchoconstriction, subjects are pretreated with albuterol and the 
procedure halted. 
 
5.  Hyperpolarized 129Xe MRI safety.   
a) Claustrophobia and anxiety  
Risk: Due to the size of the bore of the MRI scanner, there is an infrequent occurrence of claustrophobia (fear 
of being enclosed in tight spaces). These occurrences may be more likely in larger subjects. Claustrophobia is 
the most likely discomfort.  
Managing risk: Individuals with a self-reported history of claustrophobia will be assessed and a mock scan will 
be performed in cases where symptoms or history are of a concern. Also, technologists will do their best to help 
comfort any subject who is claustrophobic but chooses to continue, by using a cloth over the subject's eyes or 
a fan to provide cool air to the subject. Technologists will provide subjects with a squeeze ball alarm and instruct 
them to use it in case of any discomfort. During the actual MRI scan, staff will speak with subjects between 
scan sequences. Participants will know that study staff are continually present, and help can be immediately 
summoned. The technologist will also inform the subject that he or she is free to stop at any time, for any reason. 
Given the loud noises associated with scanning, hearing protection is provided. Participants will be told to alert 
the MRI technician immediately if the scanner is ever uncomfortably or painfully loud. 
 
b) Risks related to ferromagnetic objects  
Risk: Subjects will be comprehensively screened for objects within their bodies or on their persons that are 
ferromagnetic. Everyone must remove all metal objects before entering the scanner.  
Managing risk: To reduce risk, participants must remove any potentially dangerous objects prior to scanning.  
 
c) Risks related to inhaling hyperpolarized 129Xe 
Risk: Hyperpolarized 129Xe is treated as a drug by the FDA and is covered by our existing Investigational New 
Drug Filing (IND# 109,490), which has been active since 2011 and has reported no SAEs that required medical 
intervention. 129XeMRI is a non-invasive imaging modality that involves no ionizing radiation. Our center has 
to date conducted over 500 129Xe MRI exams in patients and volunteers, all of whom received at least 3-4 doses 
of 129Xe gas during the exam. The proposed studies will use hyperpolarized 129Xe prepared in accordance with 
the Drug Master File that is part of the IND held by Polarean, Inc.  

 
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Managing risk: The risks of 129XeMRI are minimal. Xenon is a general anesthetic when breathed continuously 
at concentrations greater than 70% for extended periods of time. In the proposed study, xenon will be delivered 
in a single breath, with alveolar concentrations below 25%. At these concentrations, subjects may experience 
transient effects including dizziness, slight tingling or numbness of the extremities, nausea, smelling of flowers, 
or a feeling of well-being and euphoria. These effects will wane within 1-2 minutes of exhaling the xenon and 
are documented in the consent forms. 
 
d) Incidental findings on 129XeMRI 
Risk: Since the MRI methods being tested are experimental, the MRI images will not be formally reviewed for 
incidental findings, abnormalities that are not expected to be seen that are identified by a reviewer.  
Managing risk: However, if there is a finding of concern noted by the study team, then the PI will approach the 
IRB for guidance on how to proceed on a case-by-case basis.  
The consent will clearly state that the MRI images will not be evaluated for incidental imaging findings. 
 
6.  Standard dose CT (SDCT) Safety 
SDCT is a standard medical procedure in lung imaging, widely used clinically and in research. Risks to 
participants are minimal. The study will involve a small amount of radiation exposure to the subjects. There 
will be one SDCT scan performed during the study. The scan protocol will be adjusted based on the participant 
weight. The average weight adult radiation dose is 4 mSv per scan, (8mSv) for the study. This compares to the 
US limit on radiation workers which is 50 mSv. 
Risk: The SDCT (lung scan) will be reviewed by a qualified person. There is a possibility that while reviewing 
the SDCT an incidental finding will be identified.  
Managing risk: The patient will be informed about the incidental finding. With permission from the patient, the 
information about the incidental finding will be forwarded to the primary doctor or the patient can be referred 
to an appropriate doctor for further evaluation.  
The patient will be provided the following information: 
• 
An incidental finding may cause you to feel anxious. 
• 
Since an incidental finding will be part of your medical record, it may affect your current or future 
life or health insurance coverage. This risk will vary depending on the type of insurance plan 
involved. 
• 
The costs for any care that will be needed to diagnose or treat an incidental finding would not be 
paid for by this research study. Any additional tests or treatments will be your choice; you or your 
insurer will be responsible for additional costs. 
 
7. Bronchoscopy:  
Risks: There is the small risk of bleeding, bronchospasm, irregular heartbeat, shortness of breath, infection or 
pneumonia in approximately 1-5% of individuals with asthma undergoing bronchoscopy47,52. There is a 
possibility that bronchospasm can worsen by this procedure. If this occurs, appropriate treatment including 
albuterol nebulization will be given. If the subject’s asthma symptoms continue, the procedure will be 
terminated, and additional medications will be given if clinically indicated. During the procedure, there may be 
a temporary drop in oxygen levels. Subjects will be given supplemental oxygen to keep levels normal. If a 
subject experiences difficulty necessitating continued observation, he/she will be hospitalized. Unstable patients 
will be transferred to the Medical Intensive Care Unit. Other risks of bronchoscopy include hemoptysis (occurs 
10-25% of the time and does not exceed more than 2 tablespoons of coughed-up blood in twenty-four hours), 
epistaxis and fever (occurring in less than 10-25% of subjects). One death has been reported in the literature, 
which was attributed to excessive lidocaine administration.  
Managing risk: Prior to the bronchoscopy, participant may be given an aerosolized lidocaine treatment by 
nebulizer or the nose, throat, and vocal cords will be sprayed with lidocaine to minimize coughing during the 
procedure.  During the bronchoscopy, patients are placed on supplemental oxygen and monitored continuously 
with an oxygen saturation monitor as well as electrocardiogram. The topical anesthesia and sedation dosages 
are monitored, and limits set and adhered to closely. Research bronchoscopies will be done with subjects with 
a pre-bronchodilator FEV1 > 50% of predicted. Patients will be instructed to remain on their long-term asthma 
medication controllers prior to undergoing bronchoscopy. Pre- and post-bronchoscopy care will be provided in 

 
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each participating institution and will include adequate nursing staff and standard post-sedation recovery care 
with direct PI oversight. Duke and University of Colorado have extensive training and experience in 
performance of such procedures both in the clinical and research setting. The expected duration of a 
bronchoscopy with brushing and biopsies is not more than 30 minutes. Based on our experience and published 
recommendations, we will limit the dose of administered lidocaine to a maximum of 9 mg/kg53. 
 
8. Risks of Principal Investigator Conflict of Interest:  
Dr. Driehuys is a founder of Polarean, Inc, a company that is commercializing hyperpolarized gas MRI 
technology. He and the company therefore potentially stand to benefit if the present work shows hyperpolarized 
129Xe MRI to be useful in detection or monitoring of gas exchange impairment. This potential conflict of interest 
is being actively managed by the Duke Research Integrity Office. Any risk specific to the proposed study is 
being managed in the following manner. Image reads will be done independently by the radiologists 
participating in the study, not Dr. Driehuys. Moreover, those readers will be blinded to clinical outcomes when 
they do their image assessments. Therefore, Dr Que will serve as principal investigators on all institutional 
review board applications at Duke. Hence, Dr. Driehuys will serve only in technical and study oversight roles, 
but not in image analysis or statistical analysis. Data generated under this protocol, will be published regardless 
of outcomes, and those efforts will be led by investigators other than Dr. Driehuys. 
 
DATA ANALYSIS & STASTICAL CONSIDERATIONS: For the ex vivo studies, conditions will be performed 
in triplicate and experiments will be independently replicated to ensure reproducibility. For the in vivo assessments 
of airway remodeling and fibrosis (bronchodilator reversibility on 129XeMRI), we will compare biomarkers of 
fibrosis (e.g., trichrome or percent elastic fiber staining) between BD types (fixed and reversible airway segments) 
both within and between obese asthma groups (early onset, late onset and non-asthma control) using a repeated 
measures two-way ANOVA with appropriate distributional assumption. Main effects of BD type, obese asthma 
group and their interaction will be evaluated with repeating BD type within subject (random effect) with compound 
symmetry variance structure. If significant main effect differences are found, pairwise comparisons between BD 
types and obese asthma groups will be done using Tukey’s post-hoc test.  Assuming a 50% decrease in fibrosis 
between BD fixed and reversible types, a sample size of 10 per group provides our study with ≥80% power to detect 
an effect size of 0.53 within and 0.60 between groups, where effect size (f) is the size of the mean differences 
relative to the standard deviation and assumed correlation (p) within group of 0.30. A p-value <0.05 will be 
considered statistically significant and all analyses will be performed using SAS version 9.4 or higher (SAS 
Institute, Inc., Cary, NC). Blinding to conditions and tissue sources will occur across the experimental design. In 
particular, tissues samples (post-BD fixed and BD reversible tissue based on region) will be blinded to the individual 
performing the experiments and the individual assays. The key for the tissue samples will be maintained by the 
program statistician and un-blinding will occur during data analysis by the statistician.  
 
Because of Dr. Bastiaan Driehuys's conflict of interest related to 129XeMRI, and as required by his Polarean 
management plan, data analysis and image analysis will be conducted by un-conflicted individuals. 
 
We hypothesize that the scRNA sequencing performed on epithelial brushings in Aim 1 will identify differentially 
expressed proteins in the airway epithelial cell secretomes (85) of early and late onset obese asthma and obese non-
asthma subjects, which drive differential fibroblast function. To perform this analysis, we will derive candidate 
proteins from pathway analysis in the epithelial brushings, correlate these to the secreted epithelial proteins and 
then confirm that pathways regulated by these proteins are active in either stimulating or suppressing fibroblast 
migration and invasion using transwell assays. Candidate genes in fibroblasts harvested from early and late onset 
obese asthmatics and non-asthma obese controls will be validated using RT-PCR. These studies have the potential 
to yield novel mechanistic insights into the pathogenesis of airway remodeling and fibrosis in obese asthma.  
 
We hypothesize that post-BD fixed defects identified on 129XeMRI in obese asthmatics correspond to regions of 
airway remodeling and fibrosis.  129XeMRI imaging-guided bronchoscopic brushings and biopsies will be used to 
obtain primary airway epithelial cells and fibroblasts to model early onset obese asthma (T2 high disease) ex vivo 
in cell culture. Epithelial cells will be grown in triplicate at air liquid interface (ALI) in 6-well transwells (86) at 
37
C in 5% CO2 until fully differentiated with a mucociliary phenotype then challenged in vitro with 50 ng/mL 
IL-13 ± 20 ng/mL leptin for 24-48 hours in serum free media (34) to mimic physiologic conditions of the T2 high 
obese asthmatic in vivo.   To model nonT2 disease in the late onset non-allergic obese asthmatic primary airway 

 
Pr o 0 0 1 0 1 4 9 0 0  
Pa ge 2 5  of 2 9  
Versi o n: 2 1 M A Y 2 0 2 4  
e pit helial cells har veste d fr o m B D fi xe d a n d B D re versi ble ve ntilat or y defects o n 1 2 9Xe M RI will be gr o w n i n 
tri plicate i n tra ns wells at A LI a n d c halle n ge d wit h 2 0 0 p p m oz o ne ( O3) f or 1 h o ur ± 2 0 n g/ m L le pti n ( O3 d ose a n d 
ti mi n g are base d o n pri or w or k).  I n  T 2 hi g h disease, T G Fβ 1 is u pre g ulate d a n d ass ociate d wit h e osi n o p hil b ur de n 
a n d ast h ma se verit y(8 9- 9 1 ). T G Fβ 1 m R N A e x pressi o n is als o u pre g ulate d b y O 3 i n h u ma n fi br o blasts (9 2 ).  T hese 
e x peri me nts will all o w us t o deter mi ne t he c o ntri b uti o n of air wa y e pit helial cells fr o m p ost - B D fi xe d a n d p ost-B D 
re versi ble re gi o ns o n T G Fβ1/ 2 e x pressi o n a n d a pical reacti ve o x y ge n s pecies ( R O S) pr o d ucti o n ( D C F H -D A, Cell 
Bi o La bs, I nc.).  Bas olateral cell s u per nata nts will be assesse d f or secrete d pr otei ns (8 5 , 9 3 ) t o gai n i nsi g ht i nt o 
w hic h pr otei ns are p ote ntiall y i n v ol ve d i n E C M de gra dati o n, matri x or ga nizati o n, a n d cell – cell i nteracti o ns a n d 
h o w t hese are altere d i n t he differe nt ast h ma e n d ot y pes.  
 
Pri mar y air wa y fi br o blasts o btai ne d b y e n d o br o nc hial bi o ps y fr o m p ost -B D fi xe d  a n d p ost -B D re versi ble re gi o ns 
will be c halle n ge d wit h 5 0 n g/ m L I L -1 3 ± 2 0 n g/ m L le pti n ( T 2 hi g h disease) vs 2 0 0 p p m oz o ne ± 2 0 n g/ m L le pti n 
( n o n-T 2 disease), res pecti vel y. Cell c ult ure me dia will be assesse d f or R O S pr o d ucti o n ( D C F H - D A, Cell Bi o La bs, 
I nc.) a n d s pecific secreti o n of T G Fβ 1/ 2, I L -1 3, elasti n, a n d T y pe I c olla ge n ( C ol 1 α ) ( E LI S A, R & D S yste ms) a n d 
fi br o blast secret o me t o discer n relati ve e x pressi o n le vels of E C M pr otei ns as well as matri x metall o pr otei nases 
( M M Ps) a n d tiss ue i n hi bit ors of metall o pr otei nases ( TI M Ps), w hic h are i m p orta nt f or pr o-fi br otic air wa y fi br o blast 
f u ncti o ns (2 9 , 3 1 , 3 4 ). Fi br o blast i n vasi o n, pr oliferati o n a n d mi grati o n will be assesse d usi n g pre vi o usl y descri be d 
met h o ds ( 3 4 , 7 4 ). T hese st u dies will all o w us t o deter mi ne t he fi br o blast s pecific res p o nses c o ntri b uti n g t o V D P 
heter o ge neit y. 
 
T Pri mar y air wa y e pit helial cells a n d fi br o blasts will be gr o w n i n c o -cult ure a n d c halle n ge d wit h eit her 5 0 n g/ m L 
I L-1 3 ± 2 0 n g/ m L le pti n f or 2 4 -4 8 h o urs or 2 0 0 p p m O 3 ± 2 0 n g/ m L le pti n f or 1 h o ur t o mi mic earl y ( T 2 hi g h) vs 
late ( n o n- T 2) o nset o bese ast h ma. R O S pr o d ucti o n a n d ge ne e x pressi o n i n air wa y e pit helial cells will be meas ure d 
a n d fi br o blast i n vasi o n, pr oliferati o n a n d mi grati o n assesse d as descri be d a b o ve.  T o t he n deter mi ne if air wa y 
fi br o blast f u ncti o ns (i n vasi o n vs. pr oliferati o n vs. mi grati o n) are de pe n de nt o n o xi dati ve stress, we will i nc u bate 
cells f or 4 h o urs wit h differe nt c o nce ntrati o ns ( 1, 3, 1 0 m M) of N -acet ylc ystei ne ( N A C) t o alter t he o xi dati ve milie u. 
Cells will t he n be was he d a n d i nc u bate d wit h 1 0 µ M D C F H -D A ( 2′, 7′ -dic hl or o di h y dr ofl u orescei n diacetate d ye; 
M olec ular Pr o bes) i n c ult ure me dia f or 3 0 mi n. Cells are t he n a nal yze d f or i ntracell ular R O S pr o d ucti o n b y fl o w 
c yt o metr y. St u dies will be re peate d usi n g differe nt T G F  ( S B 4 3 1 5 4 2; Ste m Cell Tec h n ol o gies a n d L Y- 3 6 4 9 4 7; 
Si g ma Al dric h) a n d N o x 4 ( G K T 1 3 7 8 3 1; Me d k o o Bi oscie nces, I nc) i n hi bit ors t o deter mi ne if fi br o blast f u ncti o n 
a n d o xi dati ve milie u are de pe n de nt o n T G F  si g nali n g a n d/ or N o x 4 e x pressi o n/acti vit y. 
 
B ot h e pit helial cells a n d fi br o blasts pr o d uce T G F β 1 a n d R O S u n der pr ofi br otic c o n diti o ns (7 6 ). We h y p ot hesize 
t hat air wa y p ost- B D fi xe d re gi o ns o n 1 2 9Xe M RI are c o m prise d of a s u bset of fi br o blasts t hat are a g gressi vel y m ore 
i n vasi ve, hi g hl y pr oliferati ve a n d m ore pat h o ge nic t ha n fi br o blasts fr o m p ost-B D re versi ble re gi o ns. T o deter mi ne 
if t he c ha n ges i n re gi o nal re m o deli n g a n d fi br osis are cell s pecific, B D re versi ble e pit helial cells will be gr o w n i n 
c o-c ult ure wit h B D fi xe d fi br o blasts a n d vice versa, e pit helial cells fr o m B D fi xe d re gi o ns will be gr o w n i n c o -
c ult ure wit h fi br o blasts fr o m B D re versi ble re gi o ns.  Cells will be c halle n ge d wit h 5 0 n g/ m L I L -1 3 + 2 0 n g/ m L 
le pti n  or 2 0 0 p p m O3 ± 2 0 n g/ m L le pti n  as descri be d i n Ai m 2. 1. E n d p oi nts t o be meas ure d i ncl u de fi br o blast 
f u ncti o nal assa ys (i n vasi o n, pr oliferati o n a n d mi grati o n) a n d ge ne a n d pr otei n e x pressi o n ( N o x 4, T G Fβ 1/ 2, I L - 1 3, 
elasti n a n d t y pe 1 c olla ge n) a n d R O S pr o d ucti o n. 
 
D at a a n al y si s will b e p erf or m e d u si n g a s uit e of pyt h o n -b a s e d t o ol s s uit a bl e f or h a n dli n g l ar g e -s c al e 
d at a s et s ( > 5 M c ell s), i n cl u di n g s c VI -t o ol s, S q ui d P y a n d C ell C h art er. Bri efly, aft er d at a Q C a n d filt eri n g o ut 
c ell s wit h l o w n u m b er of tr a n s cri pt s, w e will i nt e gr at e d at a fr o m all s a m pl e s t o g et h er, a n d p erf or m 
cl u st eri n g, si mil ar t o si n gl e -c ell R N A -s e q a n al y si s. T h e r e s ulti n g c ell cl u st er s will b e a n n ot at e d u si n g 
m a n u al r evi e w of m ar k er g e n e s or l a b el tr a n sf er fr o m si n gl e -c ell R N A -s e q atl a s e s u si n g tr a n sf er  l e ar ni n g 
t o ol s c Ar c h e s ( p art of s c VI-t o ol s a n d C ell C h art er), a n d t h e s e cl u st er s will b e pr oj e ct e d b a c k i nt o s p ati al 
c o nt ext. W e will c o m p ar e t h e r el ati v e a b u n d a n c e a n d a b s ol ut e d e n sity of s p e cifi c c ell ty p e s b et w e e n 
gr o u p s. W e will al s o extr a ct a d diti o n al  f e at ur e s, s u c h a s c ell siz e or m or p h ol o gi c al c h ar a ct eri sti c s. W e will 
u s e C ell C h art er t o p erf or m u n bi a s e d i d e ntifi c ati o n of s p ati al n ei g h b or h o o d s a n d t e st t h eir a s s o ci ati o n 
b et w e e n gr o u p s wit h a dj u st m e nt f or d e m o gr a p hi c c o v ari at e s.  
 
 

 
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DATA & SAFETY MONITORING: 
Patient safety and the integrity of the study will be monitored by an appointed university DSMB via review of 
adverse events and other safety parameters at their regularly scheduled meetings.  
 
Data Storage and Handling and Record Keeping:   
Any CRFs will be entered directly into REDCap by the study coordinator, with the support of Duke’s office of 
clinical research. 
 
Data will be captured in REDCap, except for patient consent forms, and AE/SAE forms, which will be on case 
report forms (CRFs) collected by the coordinators. REDCap is a software tool that does not require client local 
software and can be accessed from anywhere on the Internet and is secured on a Duke Health Technology Services 
(DHTS) server. All collected data are backed up daily, both on the local server and by the DHTS enterprise backup 
system. 
 
Source Documents and Records Retention.  Original source documents (consents) for study  participants will be 
maintained at the study site at the Duke Asthma Allergy Airway Center research offices in a badge accessed office 
in locked cabinets and will be accessible only to the study staff.  
 
Privacy, Data Storage, & Confidentiality:  The following precautions will protect the privacy of participants and 
maintain confidentiality of research data: (1) All staff will be well trained in confidentiality and data security 
procedures. (2) Privacy will be maintained by conducting all study procedures in closed rooms. (3) Each participant 
will be assigned a unique study ID number, and all data will be de-identified and coded with ID numbers only. The 
key linking participant names and ID numbers will be stored in a separate password protected document on Duke 
OIT approved servers and electronic storage locations, and only essential study staff will have access to it. (4) 
Documents such as informed consents, copies of laboratory results, and outside medical records obtained as part of 
the study will be securely stored in locked file cabinets in locked offices and in password protected documents on 
password protected computers and secure servers. Access to data storage areas and computers will be restricted to 
designated key personnel. (5) Analysis will occur on de-identified data only. (6) Data will only be stored for as long 
as necessary to complete the study, and for adherence to university, hospital, and federal regulations.  
 
The risk of loss of confidentiality is inherent in all research studies, and a statement to this effect will be included 
in the informed consent. Confidentiality will be assured by a number of procedures. First, all staff receive careful 
training in the importance of participant confidentiality, the separation of data from participant’s tracking 
information, and the use of locked offices, file cabinets, and computer accounts to prevent unauthorized access to 
the data or identifying information. Second, all study data will be treated confidentially. All questionnaires, and 
forms will be identified by participant ID (PID/SID) only. No identifying information will be included in the 
analytical database, nor in any presentations nor publication. All study materials and surveys will be maintained in 
locked filing cabinets in a secured office at all times. All records with personally-identifying information will be 
kept in a locked, limited access area (such as a locked file cabinet) available only to site principal investigators and 
designated study personnel.  
 
 

 
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