Modulation Of Airway Reactivity With Chronic Mechanical Strain
Phase: NA
Conditions: Asthma
Sponsor: Indiana University

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MODULATION OF AIRWAY REACTIVITY WITH CHRONIC MECHANICAL STRAIN 
 
 
 
Principal Investigator: 
 
Robert S. Tepper, MD, PhD 
 
705 Riley Hospital Drive, ROC 4270 
 
Indianapolis, IN 46202 
 
 
 
 
 
 
Co-Investigators: 
 
Susan Gunst, Ph.D. 
Hasna Jalou, M.D. 
Nadia Krupp, M.D. 
 
 
Dated: March 17, 2016 
 
 
 
 
Indiana University School of Medicine 
 
Department of Pediatrics 
 
Pulmonology, Allergy, and Sleep Medicine 
 
Riley Hospital for Children at Indiana University Health 
 

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Table of Contents: 
 
Study Schema 
1.0 
Background, Rationale, and Specific Aims 
2.0 
Inclusion/Exclusion Criteria 
3.0 
Enrollment/Randomization 
4.0 
Study Procedures 
5.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to Participants 
or Others 
6.0 
Study Withdrawal/Discontinuation  
7.0 
Statistical Considerations 
8.0 
Privacy and Confidentiality Issues 
9.0 
Follow up and Record Retention 
10.0 Administrative, Ethical, Regulatory Considerations 
 
 
 

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1.0 
Background 
 
Asthma is characterized by chronic airway inflammation, repeated episodes of reversible airway 
obstruction and airway hyper-reactivity. Airway inflammation and clinical symptoms of asthma are most 
often initiated by IgE-mediated responses following exposure to environmental allergens. Inflammatory 
cell recruitment into the lungs and airway hyper responsiveness are key components of the allergen-induced 
inflammatory response, which results from the interaction of resident airway cells and inflammatory cells that 
release local mediators. Patients with severe, recurrent asthma also have remodeling of the airway wall with 
increased airway smooth muscle, increased inflammatory cells and collagen deposition. 
For many decades, corticosteroids have been used to control airway inflammation, while β-adrenergic 
bronchodilators have been the primary treatment for bronchoconstriction.  Although these therapies 
are effective for the vast majority of asthmatics, they are limited by high cost, poor adherence, and 
increasing concern about long-term adverse effects. The FDA has issued a black-box warning linking long-
acting β- adrenergic bronchodilators to asthma deaths, and other studies have linked corticosteriods to 
fracture risk and growth retardation10-12. Recently introduced therapies that focus on inhibiting a single 
component of the allergic inflammatory response, such as anti-lL-5 or anti-IL-13 antibodies, have had limited 
efficacy13. Thus, there is a compelling need for new, safe and effective approaches to asthma treatment, 
particularly in children with severe asthma, where there is generally a lifelong burden of disease, use of 
medications, and accounts for the majority of health care expenditures among all children with asthma. The 
development of a therapeutic approach that could reduce both airway responsiveness and airway 
inflammation would be an important advance in the treatment of asthma. 
During the previous funding period of this project, our laboratory demonstrated that chronic mechanical 
strain imposed on the airways in vivo using continuous positive airway pressure (CPAP) results in a dramatic 
reduction in airway reactivity in vivo in mice, ferrets and rabbits1-3. Lungs, airways and airway smooth muscle 
(ASM) tissues isolated from CPAP-treated animals studied in vitro exhibited lower responsiveness to 
bronchoconstrictors1-3. We also observed this suppression of airway responsiveness by chronic 
mechanical strain in a rabbit model of allergic asthma5. These animal studies led to a small clinical trial in 
which adults with asthma were treated with nocturnal CPAP for 1 week.  CPAP caused a significant 
reduction in airway reactivity in these patients6. This novel approach for treating airway hyper-reactivity is 
currently being evaluated in a NIH multi-center Phase II clinical trial of adults with mild to moderate asthma 
(U01 HL108730). 
We now propose to determine the efficacy of chronic mechanical strain as an inhibitor of airway 
inflammation. Our preliminary studies have demonstrated that chronic mechanical strain can suppress the 
responses of airway tissues to allergic inflammatory stimuli and suppress the activation of signaling 
molecules involved in these responses3,14,15. Based on these studies, we hypothesize that chronic 
mechanical strain will inhibit signaling processes in airway tissues that lead to airway inflammation, 
hyper-reactivity and remodeling. We propose a novel molecular mechanism for the inhibitory effects of 
chronic mechanical strain on the responses of ASM to inflammatory stimuli. Anti-inflammatory agents such 
as corticosteroids remain the primary treatment for severe chronic asthma. The consequences of long term 
steroid therapy can be extremely deleterious, particularly in children, for whom steroids can have a 
significant adverse impact on growth and development. Our proposed study will provide the basis for a new 
non-pharmacologic therapy for the control of asthma that may be particularly important for the treatment 
of children with severe steroid-dependent disease. We will determine whether mechanical strain, as 
delivered using CPAP, is an effective therapy for suppressing airway hyper-reactivity and inflammation in 
pediatric subjects with severe asthma. The concept that a non-pharmacologic therapy may play such a 
pivotal role in suppressing ASM contraction, inflammation and tissue remodeling is novel and 
exciting. Furthermore, elucidation of the molecular mechanisms by which strain suppresses inflammatory 
signaling pathways could also provide a basis for the development of new pharmacologic targets for asthma 
therapy. 
  
Pathophysiology of airway inflammation in asthma. Th2 cytokines derived from CD4+ T lymphocytes 
play a pivotal role in the airway pathology associated with asthma16-19. The structurally related Th2 cell 
derived cytokines interleukin (IL)-4 and IL-13 stimulate allergic and eosinophilic inflammation as well as 

4 
 
 
epithelial and smooth muscle changes, and they are widely recognized as mediators of allergen-induced 
airway hyperresponsiveness and airway remodeling8,18-21. Th2 cytokines can induce airway hyper-
reactivity without inflammatory cell recruitment which indicates that many of the pathophysiologic effects of 
allergic inflammation in asthma result from the direct actions of IL-4 or IL-13 on airway cells9,19,20,22-24. The 
incubation of ASM with IL- 
13 or IL-4 in vitro causes ASM hypercontractility, inhibits relaxation to isoproterenol and stimulates release of 
the eosinophil chemo-attractant eotaxin and IL-57,9,25-28. The cytokines secreted by ASM and other airway 
cells have paracrine effects on inflammatory cells and thus contribute to airway inflammation29-31. 
Phenotypic alterations in airway cells induced by the direct actions of cytokines may be a primary 
mechanism for airway remodeling in asthma. Changes in gene expression induced by IL-13 or IL-4 have 
been demonstrated in airway cells including ASM, fibroblasts and airway epithelial cells15,32,33. Both IL-4 and 
IL-13 activate signaling pathways that regulate cell growth and proliferation as well as the expression of 
responsive genes34. The direct stimulation of resident airway cells with inflammatory cytokines in vitro 
stimulates cell growth and proliferation and the secretion of matrix proteins13,35,36. In our previous animal 
studies, , we  evaluated the effects of chronic mechanical strain on the physiologic responses of ASM tissues 
to inflammatory mediators in vitro, and on airway inflammation and remodeling in vivo using murine models 
of asthma. 
 
Molecular mechanisms for the direct effects of inflammatory cytokines on ASM cells. 
IL-4 and IL-13 both signal through a heterodimeric receptor complex that 
consists of an IL-4Rα chain and an IL-13Rα chain (Fig.1). In response to 
stimulation, the IL-4/IL-13 receptor subunits associate with Janus kinases 
(Jak) that phosphorylate the IL4Rα and IL13 receptor side chains, 
activating two distinct signaling proteins that bind to the phosphorylated 
IL-4Rα receptor side chain: insulin receptor-2 (IRS-2) and “signal 
transducer and activator of transcription 6”(STAT6)34. IRS2 activates the 
PI3 kinase-dependent protein kinase Akt, a regulator of cell growth and 
hypertrophy, as well as ERK1/2 MAP kinase and other MAP kinases.  
After 
phosphorylation by Jak, activated STAT6 dimerizes and migrates to the 
nucleus where it initiates the transcription of IL-4/IL-13 responsive genes. 
Stimulation of ASM cells or tissues by IL-4/IL-13 in vitro results in the 
phosphorylation of Akt, ERK1/2 and  STAT6,  the  synthesis  and  
secretion  of  eotaxin  and  IL-5,  and suppresses the expression of smooth muscle phenotype-specific 
proteins7,8,15,26,37-40 (Figs.1,4-6). Our previous studies and preliminary data have shown that chronic 
mechanical strain suppresses the activation of Akt and ERK, and that strain inhibits eotaxin secretion and IL-
13-mediated gene transcription (Figs. 5,6,9).  
 
Chronic mechanical strain suppresses airway responsiveness and ASM contractility. 
Mechanical strain  of  the  lungs  during  breathing  has  an  important  role  in  reducing  airway  
responsiveness41.  Deep inspirations and tidal breathing decrease airway responsiveness acutely in 
healthy adults and animals and their absence increases airway responsiveness42-47. Similar effects of 
mechanical strain have been observed in isolated airway segments and trachealis smooth muscle strips 
studied in vitro, demonstrating that the effects of mechanical strain on airway reactivity reflect its direct effects 
on ASM45,48-52. Although the effects of deep inspiration on airway reactivity are short-lived, these observations 
suggest 
that 
mechanical 
strain 
could 
be 
a 
useful 
approach 
for 
suppressing 
airway 
hyperresponsiveness46,47,53,54. 
We initially explored the effects of imposing mechanical strain on the airways for prolonged time periods 
to determine whether it could cause a longer term reduction of airway responsiveness. Chronic mechanical 
strain for 1-2 days applied to bronchial segments or tracheal muscle strips in vitro resulted in a decrease 
in their contractile responsiveness to ACh as assessed subsequently under unstrained conditions4. These 
results suggest that chronic strain can induce persistent alterations in the functional properties of the ASM 
tissues. 
We subsequently tested the effects of chronic strain in vivo by applying CPAP to rabbits and ferrets for 
periods of 4 days to 2 weeks and found that CPAP suppressed airway reactivity to ACh in vivo1,2. Lobes, 

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airway segments and tracheal smooth muscle tissues 
excised from the lungs of these CPAP treated animals 
also exhibited reduced contractility in vitro. The effects 
of chronic mechanical strain on airway reactivity were 
persistent and could be detected for at least 24 hours 
after CPAP5. In addition, the continuous use of CPAP 
was not necessary for it to be effective: limiting CPAP to 
nocturnal periods for 4 days in rabbits was similarly 
effective in reducing airway responsiveness (Fig. 2A). 
We also tested the efficacy of chronic mechanical strain 
at reducing airway hyperresponsiveness in the presence of allergic airway inflammation. In Ova-sensitized 
and challenged rabbits continuous CPAP administered for 4 days was effective at suppressing airway 
reactivity5 (Fig 2B). We recently extended this study to human asthmatics:  Adults with stable asthma and 
normal spirometry used nocturnal CPAP for 7 days6.  CPAP resulted in a significant decrease in airway 
reactivity while a Sham-treated group of asthmatics had no significant change in airway reactivity.  
 
Effects of chronic mechanical strain on ASM phenotype and its responses to inflammatory mediators. 
ASM cells in culture modulate their properties between a synthetic and differentiated phenotype84-86.  
Integrin proteins are widely recognized to be important regulators of cell phenotype, and can mediate 
transitions in the phenotypic status of cells in response to changes in the composition or stiffness of the 
extracellular matrix. Changes in ECM have been shown to modulate the secretion of eotaxin by ASM cells in 
culture in response to stimulation with IL-1387. 
Our data indicate that mechanical strain imposed on ASM 
tissues is a potent stimulus for regulating the phenotype and 
synthetic status ASM tissues14,15. When we imposed chronic 
mechanical strain on ASM tissues in vitro, the activation of 
pathways mediated by PI3-kinase dependent Akt were 
suppressed while the expression of smooth muscle phenotype- 
specific proteins was potentiated15  (Fig. 4). We confirmed 
this effect on the airways of mice subjected to chronic 
mechanical strain in vivo: the activation of Akt was 
significantly depressed in animals subjected to prolonged high 
mechanical strain using PEEP as compared with control-
treated animals (Fig. 13C)3. Because Akt is a critical mediator 
of pathways activated by inflammatory stimuli such as IL-13, 
we hypothesized that mechanical loads would suppress 
the inflammatory responses of ASM to local mediators and 
might thereby mitigate airway inflammation (Fig.4). 
We performed  studies  on  ASM tissues  in  vitro  to  evaluate  the  effects  of  mechanical  strain on  the 
responses of ASM tissues to stimulation with the inflammatory cytokine IL-1314,15. The administration of IL-
13 to ASM tissues in vitro stimulated the synthesis and expression of the chemokine, eotaxin, and suppressed 
the expression of smooth muscle phenotype-specific proteins (Figs. 5,6). Subjecting ASM tissues to 
mechanical strain suppressed IL-13 stimulated eotaxin synthesis and secretion and potentiated the 
expression of smooth muscle specific proteins, promoting the differentiated phenotype. Thus, while IL-13 
promotes chemokine synthesis and the synthetic phenotype of ASM, mechanical strain opposes this 
effect, suppressing IL-13 stimulated eotaxin synthesis and promoting the differentiated phenotype. 
(Fig. 4) 
Our studies have also established the role of integrin signaling in the regulation of ASM differentiation and 
mechano-sensitive signaling to the nucleus. We found that integrin-linked kinase (ILK), a multi-domain 
β integrin-binding protein kinase that regulates Akt, is a critical upstream regulator of signaling pathways 
that regulate the synthetic and differentiation functions of ASM tissues88. ILK forms a stable heterotrimeric 
complex with PINCH, an adaptor protein, and α-parvin, which binds to actin filaments89. ILK also binds 
directly to β integrin proteins and to the mechano-sensitive adhesome protein paxillin, and paxillin binding is 
necessary for the localization of ILK to integrin adhesomes90. ILK regulates the PI3 kinase-dependent 

6 
 
 
activation of Akt91. We found that Akt activation inhibits the transcriptional regulator, serum response factor 
(srf) in ASM, thereby suppressing the expression of smooth muscle phenotypic proteins88. Mechanical loads 
inhibit the ILK- dependent activation of Akt, thus promoting the differentiated phenotype15(Fig. 4). Our 
preliminary data also show that the ILK complex also plays a critical role in regulating the mechanosensitivity 
of eotaxin synthesis in response to the stimulation of ASM with IL-1314 (Fig. 10). 
 
Summary. 
We have shown that chronic mechanical strain suppresses allergen-induced hyper-responsiveness in 
vivo in animal models of asthma and the effectiveness of nocturnal CPAP at reducing airway hyper-reactivity 
in adults with mild-moderate asthma. Our studies of ASM tissues indicate that mechanical strain is sensed 
and transduced by integrin adhesomes to signaling pathways that regulate the synthetic and differentiation 
status of ASM. Our proposed studies will use in vitro models to assess the effects of strain on the 
inflammatory responses of ASM tissues (SA #1), will evaluate the suppression of airway inflammation by 
chronic strain in murine models of asthma (SA #2), and will test the efficacy of mechanical strain as 
therapy to suppress airway inflammation and hyper-reactivity in children with severe asthma (SA #3). 
 
SPECIFIC AIM #3: 
DETERMINE WHETHER CPAP SUPPRESSES AIRWAY HYPER-REACTIVITY AND 
AIRWAY INFLAMMATION IN CHILDREN WITH SEVERE ASTHMA. 
 
RATIONALE.  Whereas some children with asthma have intermittent symptoms that are improved with 
short-acting bronchodilators, many have persistent symptoms requiring daily treatment with inhaled 
corticosteroids. Children with severe asthma often have ongoing symptoms, airway inflammation and hyper-
reactivity despite treatment with high doses of inhaled corticosteroids. They are at high risk for asthma 
exacerbation requiring systemic corticosteroids, emergency room visits and hospitalization. Children with 
severe asthma are often highly atopic with increased peripheral blood eosinophilia, aeroallergen sensitivity, 
elevated serum IgE concentrations, elevated eNO, and eosinophilia in induced sputum and BALF120-125. A 
novel non-pharmacologic treatment that suppresses airway hyper-reactivity and inflammation in children 
with severe asthma could provide a significant advance in the therapy for these difficult to treat patients. We 
hypothesize that chronic mechanical strain will suppress airway reactivity and inflammation in 
children with severe asthma. In SA #3 we will determine whether the use of nocturnal CPAP for 1 month in 
children with severe asthma suppresses airway reactivity as assessed by bronchial challenge, and airway 
inflammation by induced sputum. 
 
PRELIMINARY DATA. Our recent study of adults with mild-moderate clinically stable asthma demonstrated that 
treatment with nocturnal CPAP for 1 week decreased airway reactivity (increase in PC20) compared to a 
Sham-treated group6.These results provided the pilot data for our current NIH phase II multi-center 
clinical trial designed to assess the effectiveness of using nocturnal CPAP to suppress airway reactivity in 
subjects with mild-moderate asthma. However, this trial is restricted primarily to adult patients who may 
not be steroid- dependent and are less likely to have persistent airway inflammation or exhibit airway 
remodeling. Therefore we plan to evaluate children with severe asthma, as CPAP therapy may be particularly 
beneficial to this group. Our study will also assess airway inflammation which is not included in previous or 
ongoing clinical trials.  
 
Fig.16. Nocturnal CPAP suppressed airway hyper-reactivity in an 8 yr old girl.  
We assessed the effect of 1 month treatment with nocturnal CPAP (8 cmH2O) or Sham (0 cm H2O) in two 8 
year old girls who had PC20 <1 mg/ml, which is indicative of airway hyper-reactivity. The child treated with 
CPAP showed a marked decrease in airway reactivity (increased PC20), while the ham-treated child maintained 
airway. This demonstrates our ability to recruit and evaluate young children, and is consistent with our 
hypothesis. 
 
 
 
 
 

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2.0 
Inclusion/Exclusion Criteria 
 
SUBJECT RECRUITMENT 
 
Inclusion Criteria:   
1. Children 8–17 yrs olds with severe asthma (N=120) will be recruited from the Pediatric High Risk 
Asthma Clinic and Pulmonary Clinics at Riley Hospital for Children at Indiana University Health.  
2. Severe asthma will be defined by the need for medication therapies following steps 4-6 according 
to the National Institutes of Health’s Asthma Care Quick Reference, September 2012 or high dose 
of inhaled corticosteroids.   
3. Children on a stable regimen of asthma medications for at least 8 wks prior to enrollment without 
systemic corticosteroids for >4 wks will be eligible.  
Exclusion Criteria:  
1. Obese (>95% predicted BMI).  
2. Congenital heart disease or chronic lung disease.  
3. History of pneumothorax.  
4. Inability to perform pulmonary function testing.  
5. Oxygen saturation <93%.  
6. FEV1 <70% predicted.  
7. PC20 >16 mg/ml of methacholine.  
 
3.0 
Enrollment/Randomization 
 
TREATMENT:  
1. Nocturnal CPAP (8-12 cmH2O) or Sham (0 cmH2O).  
2. Subjects will be randomized to use CPAP or Sham CPAP at night for a total of 28 days (+/- 3 
days) with a minimum of 4 hrs/night for at least 5 days/wk. If patient is having asthma symptoms at 
time of final visit, treatment may be extended up to 14 additional days. If patient receives oral 
steroids at any time during treatment with nocturnal CPAP, the final visit will be extended to at 
least 21 days (3 weeks) after the last dose of oral steroid was given. Adherence to the use of 
CPAP will be checked by having the patient mail the SD data card from their machine to the 
Investigator on day 7 and day 14 of CPAP usage and by downloading the data at the time of the final 
visit. Adherence and tolerance of CPAP will be evaluated by periodic phone calls and documented 
daily calendar diary with hours of use. 
 
4.0 Study Procedures: 
 
1. Spirometry: Spirometry will be performed after withholding bronchodilators. Forced vital capacity 
(FVC) and forced expiratory volume in 1 second (FEV1) will be measured using ATS guidelines.  
2. Airway Reactivity assessed by MCh Bronchial Challenge: Testing will use the 5 breath dosimeter 
protocol recommended by ATS126. Following inhalation of saline, MCh will be inhaled in quadrupling 
concentrations starting with 0.0625 mg/ml and continuing until the MCh concentration required for FEV1 
to decrease by 20% from baseline (PC20) is achieved or a maximum MCh concentration of 16 mg/ml is 
inhaled.  
3. Airway Inflammation assessed by Exhaled Nitric Oxide (eNO): eNO will be measured at a constant 
expiratory flow of 50 ml/s with a chemiluminescence analyzer (NIOX; Aerocrine) using ATS 
guidelines127.  
4. Airway Inflammation assessed by Induced Sputum. Sputum induction will be performed after MCh 
challenge and treatment with albuterol to return FEV1 to > 70% predicted. Sputum will be induced by 
inhaling increasing concentrations (3 to 7%) of saline at 5 min intervals. Oscillating positive expiratory 
pressure (PEP) therapy may be used if needed to assist with sputum expectoration in conjunction with 

8 
 
 
saline treatments for up to 20 min or until an adequate sample is produced. Spirometry is performed 
after each expectoration to check that the FEV1 has not decreased by >20%, in which case the child 
would be given bronchodilator and no more hypertonic saline. Sputum will be processed for differential 
cell counts and supernatants stored at -80ºC for subsequent analysis of eotaxin and eosinophil cationic 
protein128. 
5. Asthma Symptom control: Asthma control will be assessed by using the Asthma Control Test129. 
6. Berlin score: Questionnaire will be used to determine high or low risk status for obstructive sleep 
apnea130. 
 
5.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to Participants or Others 
 
Adverse Events 
An adverse event (AE) is any untoward medical occurrence in a clinical investigation of a patient that does not 
necessarily have a causal relationship with the treatment.  An AE is therefore any unfavorable and unintended 
sign (including an abnormal laboratory finding), symptom or disease temporally associated with the 
administration of an investigational product, whether or not related to that investigational product.   
 
The Investigator will probe, via discussion with the subject, for the occurrence of AEs during each subject visit 
and record the information in the site’s source documents.  Adverse events will be recorded in the subject 
internal data collection forms.  Adverse events will be described by duration (start and stop dates and times), 
severity, outcome, treatment and relation to study medication, or if unrelated, the cause. 
 
Serious Adverse Experiences (SAE) 
An SAE is defined as any untoward medical occurrence that: 
 
 
Results in death 
 
Is considered life threatening (i.e., in the view of the investigator the adverse experience places the 
subject at immediate risk of death from the reaction, as it occurred; it does not include a reaction that, 
had it occurred in a more severe form, might have caused death) 
 
Requires hospital admission or prolongation of an existing hospitalization 
 
Results in persistent or significant disability/incapacity (i.e., a substantial disruption of a person’s ability 
to conduct normal life functions) 
 
Is a congenital anomaly/birth defect 
 
Is an important medical event (i.e., when based upon appropriate medical judgment, the adverse 
experience may jeopardize the subject and may require medical or surgical intervention to prevent one 
of the above listed outcomes) 
 
Data Safety Monitoring Plan 
 
Data Safety Monitor Gregory Montgomery, MD will monitor this study on an annual basis.  Data to be 
monitored includes:  data quality, subject recruitment, accrual and retention and adverse events.  The results 
of related studies that impact subject safety and privacy will also be assessed.   
 
Data monitoring will also include the interpretation of the data and confirming that the data is kept in a secure 
database.  The co-investigators of the study will be doing an interim analysis and interpretation of the data on 
an annual basis. 
 
Adverse events will be dealt with on a case by case basis and all institutions (IRB/ NIH) involved in the study 
will be notified by either a formal letter or memo. 
 
The following information will be reported to the IRB on an annual basis with the continuing review: 
1. Dates of monitoring 
2. Summary of cumulative adverse events 

9 
 
 
3. Assessment of external factors that impact the safety of subjects 
4. Summary of subject privacy and research data confidentiality 
5. Any changes in risk-benefit ratio 
 
6.0 
Study Withdrawal/Discontinuation 
 
If the parent/guardian chooses to withdraw the subject from the study, then the parent/guardian will inform a 
member of the research team.  At that time, no further testing will be obtained.  Samples already obtained from 
the subject prior to withdrawal will still be included in analysis.  
 
Management of Increased Asthma Symptoms: If a subject’s asthma symptoms worsen during the course of 
the treatment period, they will be directed to their pediatric pulmonologist or the High Risk Asthma clinic for 
management.  Any changes in therapy, including controller medications or courses of systemic steroids will be 
recorded. As the enrolled subjects have severe asthma, one course of systemic steroids in 4 weeks would not 
be considered unusual and would not trigger withdrawal from the study. However, an inpatient hospitalization 
or two courses of systemic steroids in the 4 week treatment period will trigger withdrawal from the study 
 
7.0 
Statistical Considerations 
 
ANALYSIS:  
Primary Outcome: The change in airway reactivity (logPC20) measured prior to and after 4 wks of either 
CPAP or SHAM treatment. We will compare the change in PC20 between two groups using an analysis of 
covariance (ANCOVA) model, adjusting for baseline PC20. We anticipate that PC20 will increase for the 
CPAP treated group, but not change for the SHAM treated subjects. 
Secondary Outcomes: Airway Inflammation assessed as the change in the percentage eosinophils in the 
induced sputum measured prior to and after 4 wks CPAP or SHAM treatment. We will compare the change 
between two groups using an ANCOVA model adjusting for baseline percentage eosinophils. We 
anticipate that eosinophilia will decrease for the CPAP treated group, but not in the SHAM treated subjects. 
 
Justification of Sample Size:  Change in Airway Responsiveness: We calculated the sample size based on 
the difference of logPC20 changes between groups. Our previous study showed a difference of change in 
logPC20 between CPAP and SHAM groups as 0.41 with a standard deviation of 0.46. With a 1 month 
intervention period in our study (compared to 1 week in previous study), we expect a larger effect size. 
Therefore, we conservatively assume a 0.41 difference and a 0.46 standard deviation. With 30 patients in 
each group, we expect 27 of them will complete the study. We will have more than 85% power to detect 
such a difference with a 5% Type I error rate using a two-sample t-test. ANCOVA model will be more 
powerful than the t-test. Change in Airway Inflammation: We calculated the smallest changes in airway 
inflammation that can be detected with the sample size generated above. With 27 patients completing the 
study, we have 80% power to detect an effect size as small as 78% (difference of changes is 78% of the 
standard deviation) using a t-test.  
 
 
ANTICIPATED RESULTS.  
Based upon our previous experience that CPAP suppresses airway reactivity in adults with  mild-
moderate  asthma,  our  preliminary data  in  children  and  our  animal  models,  we  anticipate  that 
nocturnal CPAP will suppress airway reactivity in children with severe asthma. These findings would have 
important clinical implications and justify a multi-center trial for much longer periods of CPAP treatment to 
evaluate its potential in decreasing asthma symptoms and medication usage in children with severe asthma. 
We also expect CPAP to suppress airway inflammation. Although our primary measurement of airway 
inflammation is induced sputum eosinophilia, we will evaluate additional inflammatory markers. It is 
possible that our sample size will be too small to obtain statistical significance for these markers. However, 
if we find that CPAP shows a tendency to decrease airway inflammation, our results could be used to power a 
larger clinical trial. It is possible that CPAP will increase airway inflammation, b u t  this seems unlikely as 

10 
 
 
CPAP in adults with obstructive sleep apnea has been found to decrease systemic inflammatory 
markers131,132. 
 
8.0 
Privacy/Confidentiality Issues 
 
Each study subject will be assigned a subject number.  All samples will be identified only by subject number 
when submitted to the research laboratory for analysis.  The enrollment list containing both the subject number 
and identifiable information will be kept on a password protected server.  
 
Security and confidentiality 
To comply with HIPAA guidelines, processes and procedures have been documented and implemented to 
ensure the security and protection of the study data within the computer operations center, the server, and the 
database.  IU uses a three level security model to secure and protect data collected and stored within 
REDCap.  This model is as follows:  1) an individual must register an account within the Indiana CTSI Hub and 
either be assigned a username/password if external to IU or use their IU login credentials and authenticate via 
IU’s Central Authentication Service (CAS); 2) once a Hub account has been established, an individual must 
submit a request for a REDCap account; the request must be approved and then a REDCap account is 
established; and, 3) an individual must have their REDCap account be granted appropriate access and 
privileges to the specific project database by the project PI or delegate.  In addition, since REDCap is web-
based, IU utilizes SSL (https) encryption for secure connectivity. 
 
9.0 
Follow-up and Record Retention 
 
Enrollment: 
Enrolling sufficient subjects is always challenging; however we were successful at this in our previous 
CPAP study6. Riley Hospital for Children is the primary referral center for all of Indiana and our pediatric 
pulmonary group manages most of the severe pediatric asthma, so we do not expect problems recruiting 
subjects for this study. In multiple previous studies we recruited more than 100 subjects133-137. 
 
CPAP Measures: 
In our preliminary studies, the use of CPAP for an average of 4 hrs/night was effective. Our CPAP devices can 
monitor the number of hours that CPAP is actually being used by measuring respiratory pressure fluctuations. 
Daily usage statistics can be stored on an SD card that is downloaded after 1 week, 2 weeks and 4 weeks of 
treatment. To reinforce adherence, we insure mask comfort at the initial visit, use humidification to 
prevent nasal drying and reinforce adherence with regular telephone calls and documented daily calendar 
diary with of hours of use. 
 
Data Capture System 
The Research Electronic Data CAPture (REDCap) database system will be used to collect data for this study.  
The REDCap software toolset provides a secure, web-based environment that is flexible enough to be used 
for a variety of types of research, provides an intuitive interface for users to enter data and have real time 
validation rules (with automated data type and range checks) at the time of entry.  The system offers easy 
data manipulation with logged auditing, functionality for reporting, monitoring and querying patient records, 
and an automated export mechanism to common statistical packages such as:  SPSS, SAS, Stata, R/S-Plus.  
REDCap has been used within Indiana University and affiliates since April 2009 and it currently supports 177 
projects and over 380 users within IU, Purdue, Notre Dame, IU Health, VA, Rehabilitation Hospital of Indiana, 
and Wishard Health.  The input data will be stored in secured server for storage and analysis. 
 
Backup 
At all times, appropriate backup copies of the database and related software files will be maintained and the 
information will be appropriately protected from illegitimate access. At critical junctures of the protocol (e.g., 

11 
 
 
production of interim reports and final reports), a permanent archive of the database will be made. Archived 
versions of the database will be saved for at least three years after the end of the study. 
 
Availability and Retention of Investigational Records: 
The Investigator must make study data accessible to authorized representatives of the NIH and IRB/IEC upon 
request.  A file for each subject must be maintained that includes the signed Informed Consent, HIPAA 
Authorization and copies of all source documentation related to that subject.  The Investigator must ensure the 
reliability and availability of source documents from which the information on the internal data collection forms 
was derived.  All study documents (subject files, signed informed consent forms, copies of internal data 
collection forms, Study File Notebook, etc.) must be kept secured for a period of three years.   
 
10.0 
ADMINISTRATIVE, ETHICAL, REGULATORY CONSIDERATIONS 
 
The study will be conducted according to the Declaration of Helsinki, Protection of Human Volunteers (21 CFR 
50), Institutional Review Boards (21 CFR 56), and Obligations of Clinical Investigators (21 CFR 312). 
To maintain confidentiality, all lung function data, evaluation forms, reports and other records will be identified 
by a coded number and initials only.  All study records will be kept in a locked file cabinet and code sheets 
linking a subject’s name to a subject identification number will be stored separately in another locked file 
cabinet.  Clinical information will not be released without written permission of the subject, except as necessary 
for monitoring by the FDA.  The Investigator must also comply with all applicable privacy regulations (e.g., 
Health Insurance Portability and Accountability Act of 1996, EU Data Protection Directive 95/46/EC). 
 
Protocol Amendments 
Any amendment to the protocol will be written by the Study Principal Investigators and submitted to the IRB for 
their approval. 
 
Publications  
The preparation and submittal for publication of manuscripts containing the study results shall be in 
accordance with a process determined by mutual written agreement among the study Sponsor and 
participating institutions.  The publication or presentation of any study results shall comply with all applicable 
privacy laws, including, but not limited to, the Health Insurance Portability and Accountability Act of 1996.  
 

12 
 
 
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