Clinical trial NCT02464059
Conditions: Pulmonary Disease, Chronic Obstructive
Keywords: Cathelicidin Vitamin D
Sponsor: University of North Carolina, Chapel Hill
Start date: 2015-11-10; Completion date: 2020-04-15
Enrollment: 23 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This proposal will evaluate a potential mechanism of increased risk of lung function impairment, cathelicidin levels, as well as determine if vitamin D replacement can alter this pathway. This study will improve the understanding of factors which can lead to chronic lung disease. If effective, this application would also provide the justification to study vitamin D as a therapy to improve lung health.

Detailed description:
Understanding mechanisms leading to decrements in lung function, the physiologic hallmark of obstructive lung diseases including chronic obstructive pulmonary disease (COPD), are necessary to inform interventions to improve lung health. The antimicrobial peptide cathelicidin, and its primary regulator vitamin D, has been implicated in development and progression of chronic lung disease. In this study, the investigators will evaluate the effect of oral vitamin D supplementation on lung cathelicidin levels in humans. Cathelicidin has bactericidal and inflammatory activities in the lung and is regulated by vitamin D levels. The investigators hypothesize that oral vitamin D supplementation will raise cathelicidin levels in the pulmonary compartment, thereby restoring lung cathelicidin deficiency. To test this hypothesis, the investigators will recruit from two ongoing cohort studies: 1) At Johns Hopkins, the Subpopulations and Intermediate Outcome Measures in COPD Study study and 2)at University of North Carolina at Chapel Hill pulmonary and general medicine clinic. The investigators will measure blood and lung lavage cathelicidin levels in 40 vitamin D insufficient individuals (20 from each cohort) before and after eight weeks of oral vitamin D supplementation to determine the effect of vitamin D supplementation on cathelicidin levels.

Arms: arm 1: Oral supplementation with 50,000 IU vitamin D3 weekly for eight weeks
Interventions: intervention 1: Vitamin D3
Intervention descriptions: intervention 1: Oral supplementation with 50,000 IU vitamin D3 weekly for eight weeks in individuals with reduced vitamin D levels.

Protocol documents:
Prot_000.pdf: 
Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
PROTOCOL 
Cathelicidin and Vitamin D: Impact on Populations At-Risk and with 
COPD  
ClinicalTrials.Gov Registration NCT02464059 
UNC PROTOCOL 
Version 1.2 
Updated September 6 2017 
SECTION 1. Study Overview 
SECTION 2. Recruitment 
SECTION 3. Detailed study visit procedures 
SECTION 4. Reporting events 
SECTION 5. Study visit checklists 
SECTION 6. Study forms 
SECTION 7. Laboratory procedures 
SECTION 8. Bronchoscopy procedures 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
SECTION 1: Vitamin D Study Overview 
1.1: Introduction and Background: Understanding mechanisms leading to decrements in lung 
function, the physiologic hallmark of obstructive lung diseases including chronic obstructive 
pulmonary disease (COPD), are necessary to inform interventions to improve lung health.  The 
antimicrobial peptide cathelicidin, and its primary regulator vitamin D, has been implicated in 
the development and progression of chronic lung disease. Cathelicidin has bactericidal and 
inflammatory activities in the lung and is regulated by vitamin D levels.  
1.2: Purpose of the Study: The purpose of the study is to determine the effect of vitamin D 
replacement on blood and lung cathelicidin levels in populations at-risk or with established 
COPD. The antimicrobial peptide cathelicidin, and its primary regulator vitamin D, has been 
implicated in the development and progression of chronic lung disease.  We hypothesize that oral 
vitamin D supplementation will raise cathelicidin levels in the pulmonary compartment, thereby 
potentially restoring relative lung cathelicidin deficiency. To test this hypothesis, we will recruit 
from two sites: 1) Johns Hopkins (via the Subpopulations and Intermediate Outcome Measures 
in COPD Study (SPIROMICS) study of individuals with established COPD (NA_00035701: PI 
Hansel) and 2) UNC COPD Clinic for individuals at-risk for COPD (smokers with preserved 
lung function).  This MOP reflects procedures for the UNC site. 
At each site, we will measure blood and lung lavage cathelicidin levels in 20 vitamin D 
insufficient individuals (10 participants with serum 25-OH Vitamin D<10 ng/ml and 10 
participants with serum 25-OH vitamin D between 10-20) before and after eight weeks of oral 
vitamin D supplementation to determine the effect of vitamin D supplementation on cathelicidin 
levels. Completion of this project directly or indirectly works towards ultimately understanding 
if a readily measurable blood marker, cathelicidin, can inform who may benefit from vitamin D 
supplementation to prevent chronic lung disease. The results from this application would lead to 
a therapeutic intervention study that will provide further opportunities to improve our 
understanding of the relationship between vitamin D, cathelicidin and lung function. 
1.3 Objectives: 
To evaluate the effect of oral vitamin D supplementation on plasma and lung cathelicidin 
measures in individuals with vitamin D insufficiency. Our primary analysis will include 
comparison between pre- and post-supplementation cathelicidin levels using paired t-tests for 
repeated measures 
The study procedures outlined in this proposal specifically do not include any assessment of 
outcomes related to disease treatment, mitigation, cure, or prevention. We do not assess change 
in lung function over time, symptom scores, exacerbation risk, sputum production, pneumonia 
outcomes or any other assessment related to treatment/mitigation of COPD.  The only outcome 
collected if BAL and blood samples to measure cathelicidin pre- and post-vitamin D 
supplementation (along with standard safety outcomes that are not related to any disease 
process). The lack of any assessment of outcomes related to diagnosis, cure, mitigation, 
treatment or prevention of disease demonstrates that such outcomes are not the scope of the 
proposed study. 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
1.4 Overall Project Design: 
The design of the study to complete this aim will be in the format of a baseline and repeat 
bronchoscopy after oral supplementation with 50,000 IU vitamin D3 weekly for eight weeks in 
individuals with reduced vitamin D levels.  The specific study data collection is outlined in the 
table below.  
•
Participants will undergo a screening assessment (visit S0) to determine the presence of
vitamin D insufficiency (serum 25OH-VitD <20 ng/ml) as well as normal renal function
and calcium levels. Pre- and post-bronchodilator spirometry will be performed to
document normal lung function.
•
If eligible based on S0 data, participants will undergo baseline bronchoscopy with
bronchoalveolar lavage (BAL) for collection of lung cathelicidin levels (visit B1). Blood
for cathelicidin, vitamin D binding protein, vitamin D and PBMC will be collected at this
visit.
•
They will then return to the Meadowmont research clinic weekly for eight weeks of
directly observed vitamin D supplementation as well as assessment of adverse events
(visits V0-V7).
o
After the initial four weeks of treatment (V4), all patients will be evaluated and
have blood work obtained for calcium level to monitor for hypercalcemia.
o
Patients will be screened at each weekly visit for symptoms of hypercalcemia.
•
After 8 weeks of vitamin D treatment, serum 25OH-VitD levels will be measured (visit
V8) to ensure increased level prior to undergoing bronchoscopy. Individuals not
increasing vitamin D> 20 after 8 weeks will not be eligible for B2 bronchoscopy and will
be deemed to have completed study. Blood for BMP/ calcium level will also be collected.
•
A second BAL collection will occur after the eight week of vitamin D treatment to
measure cathelicidin in the plasma and the BAL (visit B2). Blood for cathelicidin,
vitamin D binding protein, vitamin D and PBMC will also be collected at this visit.
•
A 24 hour post-bronchoscopy follow-up phone call will occur after B1 and B2
•
All procedures will be research related. A visit window of ±1 day is acceptable for all
study visits except B1 and B2, where a 28 day window is permissible given potential
delay in laboratory results and bronchoscopy scheduling.
Table. Study Data Collection Schedule 
Visit 
S0 
B1 
V0 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
B2 
Time (weeks) 
-2
-1
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
Window (+/- days) 0 
28 
1 
1 
1 
1 
1 
1 
1 
1 
1 
28 
Visit Duration 
(hrs) 
2 
5 
1 
0.5 
0.5 
0.5 
1 
0.5 
0.5 
0.5 
1 
5 
Informed Consent 
• 
Spirometry 
• 
Vitamin D 
• 
• 
• 
Vit D binding 
prot/cathelicidin 
•
• 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
PBMC 
• 
• 
BMP/ calcium 
• 
• 
• 
BAL 
• 
• 
Drug 
administration 
• 
• 
• 
• 
• 
• 
• 
• 
Adverse events & 
interval history 
• 
• 
• 
• 
• 
• 
• 
• 
Key:Time= nominal visit time; S=screening visit, Bn= Bronchoscopy visit n, Vn=Study visit 
SECTION 2: RECRUITMENT 
2.1 Definition of Recruitment & Consent: Recruitment is establishing eligibility of the potential 
candidate and informing the candidate of their eligibility and signing of the informed consent 
form by the potential candidate.  
2.2 Sources of Recruitment: Potential participants will be identified from three sources: 1) 
patients referred by providers in the pulmonary subspecialty clinic, 2) patients referred by 
providers in the general medicine outpatient clinic, 3) self-referral from recruitment flyers. 
2.3 Process of Recruitment: Study team members will approach providers during clinic sessions 
for identification of potential participants. If the clinician is agreeable and obtains the approval 
for the patient, the recruitment team will approach the patient. For recruitment via face-to-face 
encounter, these conversations will occur in a private office with a trained study team member. 
Study information will be reviewed with the patient and a copy of the IRB approved consent 
form will be provided to them to review at their leisure (taking it home, if desired). After having 
the chance to consider the trial, a time/date will be scheduled for the consent process and 
screening visit. For participants self-referring from recruitment flyers, the study team will make 
contact via phone call using a telephone screening script.   
2.4 Study Inclusion and Exclusion Criteria: 
i.
Inclusion
i. Post-BD FEV1/FVC>=0.70
ii. Post-BD FEV1> 70% predicted
iii. Age 18-80
iv. Current smoker (current history of smoking at least 5 cigarettes daily)
v. Serum calcium<10.5mg/dl
vi. 25-OH VitD<20 ng/ml at screening
1. Note that target recruitment is at least 10 participants with 25-OH
VitD <10 ng/ml
vii. CrCl ≥60 mL/min as estimated by the Cockcroft-Gault equation
viii. Women of reproductive potential with negative serum or urine pregnancy
test and be willing to refrain from participating in a conception process
and subject/partner must use at least 2 reliable forms of contraceptives for
the duration of the study


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
ii.
Exclusion
i. Current use of vitamin D supplements
ii. Oxygen use >2L/min at rest
iii. Known allergy/sensitivity or any hypersensitivity to components of study
drugs or their formulations
iv. Pregnancy or currently breast-feeding
v. History of nephrolithiasis (kidney stones)
vi. Self-reported HIV positive serostatus
vii. Any condition that, in the opinion of the site investigator, would
compromise the subject’s ability to participate in the study.


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
SECTION 3: DETAILED STUDY VISIT PROCEDURES 
3.1 Chart/Phone Pre-Screening Process  
i.
The telephone screening script will be used to determine potential eligibility for all
participants contacted by phone.
ii.
If participant is eligible and interested, they will be scheduled for an in-person visit
for informed consent review/signing as well as screening laboratory data. If screening
is occurring in person, informed consent and blood work can be completed same day
provided the participant has had adequate time to review the consent form.
3.2 Screening Visit (S0) 
i.
Review/sign consent form
ii.
Assignment of study ID
a.
Using “VitDXXX” where
XXX= sequential numbers from 501-999
iii.
Completion of demographics form
iv.
Urine pregnancy test (for women of childbearing age)
v.
Performance of pre- and post-bronchodilator spirometry (4 puffs albuterol)
vi.
Completion of screening form to determine eligibility
vii.
If eligibility confirmed, obtain blood testing for screening visit (see laboratory 
processing section)
a.
One-3.5 ml SST to UNC McLendon Lab for eligibility screening
i. Serum 25OH-Vitamin D
ii. Serum Basic metabolic panel
b.
One 8.5 ml SST for serum storage
i. Serum for 25OH-Vitamin D and vitamin D binding protein
c.
Two 4 ml EDTA Lavender for plasma storage
i. Plasma for cathelicidin
viii.
Confirm contact number to reach participant after 25OH-VitD and BMP resulted.
ix.
Payment to participant
x.
After labs resulted, PI to review vitamin D/BMP results and determine eligibility
xi.
MD to contact person to arrange bronchoscopy 
3.3 Bronchoscopy (B1) 
i.
Before scheduling the first of the two bronchoscopy procedures, it should be
established that
i. Patient has signed IRB-approved consent for the research protocol
ii. Has met the inclusion criteria for the study.
iii. The benefits and risks of the procedure have been explained to the
participant and patient has given consent for the procedure.
ii.
The target windows for scheduling the bronchoscopy is 28 days after S0 visit for
the first procedure and 1 week after V8 for the second procedure.


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UNC PROTOCOL Version 1.2- Updated September 6 2017 
iii.
Day of bronchoscopy, the following bloods will be collected (processed at Alexis
lab):
a.
Two 10 ml Green Top Lithium tubes for PBMC storage
See “BRONCHOSCOPY PROCEDURES” for bronchoscopy procedure details 
3.4 Initial medication administration visit (V0) 
i.
Participant will present for first drug administration. At the time of visit, a bottle of
25-OH vitD will be removed for secured storage and label completed with:
a.
Participants study number (VitDXXX)
b.
Initials of the participant
c.
Date of V0 visit
ii.
Participant will be given one pill of Vitamin D 50,000 IU and asked to take pill with 
water at that time
iii.
Complete Vitamin D Subject Specific Accountability Record
iv.
Provide participant with Vitamin D informational handout
v.
Schedule V1 in 7 days
vi.
Payment to participant:  
3.5 Follow-up visits 
i.
Visits V1-V3
a.
Adverse events form is completed
b.
Participant will be given one pill of Vitamin D 50,000 IU and asked to take 
pill with water at that time
c.
Complete vitamin D administration log
d.
Schedule next visit in 7 days
e.
Payment to participant: 
ii.
Visit V4
a.
Adverse events form is completed
b. Participant will be given one pill of Vitamin D 50,000 IU and asked to take 
pill with water at that time
c.
Complete Vitamin D Subject Specific Accountability Record
d. Schedule next visit in 7 days
e.
Obtain blood (see laboratory processing section)
i. One-3.5 ml SST to UNC McLendon Lab for eligibility screening
1. Serum 25OH-Vitamin D
2. Serum Basic metabolic panel
f.
Confirm contact number to reach participant after 25OH-VitD and BMP 
resulted.
g. Payment to participant: 
h. Notify study MD when lab results completed 


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UNC PROTOCOL Version 1.2- Updated September 6 2017 
iii.
Visit V5-V7
a.
Adverse events form is completed
b.
Participant will be given one pill of Vitamin D 50,000 IU and asked to take 
pill with water at that time
c.
Complete Vitamin D Subject Specific Accountability Record
d.
Schedule next visit in 7 days
e.
Payment to participant: 
iv.
Visit V8
a.
Adverse events form is completed
b.
Schedule next visit in 7 days
c.
Obtain blood (see laboratory processing section)
i. One-3.5 ml SST to UNC McLendon Lab for eligibility screening
1. Serum 25OH-Vitamin D
2. Serum Basic metabolic panel
ii. One 8.5 ml SST for serum storage
1. Serum for 25 OH-Vitamin D and vitamin D binding protein
iii. Two 4 ml EDTA Lavender for plasma storage
1. Plasma for cathelicidin
d.
Confirm contact number to reach participant after 25OH-VitD and BMP 
resulted.
e.
Payment to participant: 
f.
Notify study MD when lab results completed
a.
If vitamin D not >20 ng/ml, participant not eligible for bronchoscopy 
and will have completed study
b.
If vitamin D>=20 ng/ml, study staff contact person to arrange 
bronchoscopy 
3.6 Bronchoscopy (B2) 
i.
The target windows for scheduling the bronchoscopy is 1 week after V8 for the
second procedure.
ii.
On day of bronchoscopy, the following blood will be obtained (processed at
Alexis lab):
a.
Two 10 ml Green Top Lithium tubes for PBMC storage
See “BRONCHOSCOPY PROCEDURES” for procedure details 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
SECTION 4: REPORTING EVENTS 
The proposed intervention study involves treatment of participants with vitamin D with the goal 
of determining if this intervention is efficacious in altering lung cathelicidin levels, and is 
therefore classified as a clinical trial.  Because this is a clinical trial, the PI will designate an 
independent monitor to perform independent review of ongoing study progress and safety. 
Adverse events (AE) will be reported both to our IRB and the Independent Monitor through 
annual progress reports or more frequently as described below. AE reports and annual summaries 
will not include subject- or group-identifiable material. Each report will only include the 
identification code. 
The Independent Monitor for this study is Dr. Sonali Bose at Johns Hopkins (sbose7@jhmi.edu) 
For this application, an adverse event is defined as any untoward medical occurrence in a subject 
during participation in the clinical study or with use of the intervention (vitamin D) being 
studied. An adverse finding can include a sign, symptom, abnormal assessment (laboratory test 
value, vital signs, etc.), or any combination of these. A serious adverse event (SAE) is any 
adverse event that results in one or more of the following outcomes: 
•
Death
•
A life-threatening event
•
Inpatient hospitalization or prolongation of existing hospitalization
•
A persistent or significant disability/incapacity
•
A congenital anomaly or birth defect
•
An important medical event based upon appropriate medical judgment
AEs will be labeled according to severity, which is based on their impact on the patient. An AE 
will be termed “mild” if it does not have a major impact on the patient, “moderate” if it causes 
the patient some minor inconvenience, and “severe” if it causes a substantial disruption to the 
patient’s well-being. AEs will be categorized according to the likelihood that they are related to 
the study intervention. Specifically, they will be labeled definitely unrelated, definitely related, 
probably related, or possibly related to the study intervention.  
The PI will review AE rates at least monthly. 
When an AE occurs, the AE will be reported to the Institutional Review Board at UNC, to the 
funding agency and to the Independent Monitor. Our procedure is for the study coordinator to 
notify the Principal Investigators and for the program staff to make necessary arrangements for 
the participant to see a medical provider. Most assessments will occur in research space adjacent 
to the Meadowmont clinic, and referrals would be made to the clinic acute provider. The 
Emergency Room is the first choice for emergent care, and we would arrange for transport or 
accompany the participant as needed.  SAEs that are unanticipated, serious, and possibly related 
to the study intervention will be reported to the Independent Monitor and IRB in accordance with 
requirements.  
Unexpected fatal or life-threatening AEs related to the intervention will be reported to the IRB 
and Independent Monitor within 3 days. Other serious and unexpected AEs related to the 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
intervention will be reported to the IRB and Independent Monitor within 14 days.  Anticipated or 
unrelated SAEs will be handled in a less urgent manner but will be reported to the Independent 
Monitor and IRB. In the annual AE summary, the Independent Monitor Report will be required 
to state that they have reviewed all AE reports.   
Study progress and safety will be reviewed monthly by the PI. Progress reports, including patient 
recruitment, retention/attrition, and AEs, will be provided to the Independent Monitor annually. 
An Annual Report will be compiled and will include a list and summary of AEs. In addition, the 
Annual Report will address (1) whether AE rates are consistent with pre-study assumptions; (2) 
reason for dropouts from the study; (3) whether all participants met entry criteria; (4) whether 
continuation of the study is justified on the basis that additional data are needed to accomplish 
the stated aims of the study; and (5) conditions whereby the study might be terminated 
prematurely. The Annual Report will be sent to the Independent and included in the Progress 
Report to the NIH. The IRB and other applicable recipients will review progress of this study on 
an annual basis.  
During the funding of this study, any action by the IRB, the Independent Monitor, or one of the 
study investigators that results in a temporary or permanent suspension of the study will be 
reported to the NHLBI Program Official within 1 business day of notification. 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
SECTION 5: STUDY VISIT CHECKLISTS 
The following pages include a separate page for each study visit with a checklist of items that 
must be completed at each visit. These pages should be printed and used as a step-by-step guide 
of each visit. 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
S0- Screening Visit Checklist 
□Review and sign consent form
□Assign study ID
□Complete demographics form
□Urine pregnancy test (for women of childbearing age)
□Pre- and post-bronchodilator spirometry
□Complete screening form
□If eligibility confirmed, obtain blood testing
□One-3.5 ml SST to UNC McLendon Lab for eligibility screening
(BMP, 25-OH vitamin D) 
□One 8.5 ml SST for serum storage (25-OH vitamin D, vitamin D binding protein)
□Two 4 ml EDTA lavender tube for plasma storage (cathelicidin)
□Confirm contact phone number
□Provide participant with “Bronchoscopy Handout”
□Payment to participant: 
□Notify MD of blood results for review and scheduling of bronchoscopy 
Review total recruitment: 
Eligibility: 25OH VitD <20ng/ml (with targeted recruitment of at least 10 participants 
with level <10 ng/ml) 
Bronchoscopy scheduling: Target 1 week ± 28 days 
□Once bronchoscopy is scheduled, V0 should be scheduled within 1 week ±1 day of
bronchoscopy) 
□Notify pharmacy of medication pickup
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
B1- Bronchoscopy #1 Visit Checklist 
□Review consent form for completion
□Obtain blood testing
□Two 10 ml Green Top Lithium tubes for PBMC storage
□Review labs to confirm vitamin D <20 ng/ml
□Escort patient to bronchoscopy
□Payment to participant: 
□MD to complete bronchoscopy form
□Staff to complete bronchoscopy sample processing form
□Confirm V0 is scheduled (within 1 week ±1 day of bronchoscopy) 
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
V0- Initial medication administration checklist 
□Go to pharmacy
□Provide participant one pill of 50,000 IU vitamin D to take with water at 
visit
□Provide participant informational flyer regarding vitamin D side effects
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V1 in 7±1 days
□Payment to participant:  
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
V1- Follow-up visit checklist 
□Go to pharmacy
□Complete adverse events form
□Provide participant one pill of 50,000 IU vitamin D to take with water at 
visit
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V2 in 7±1 days
□Payment to participant:  
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
V2- Follow-up visit checklist 
□Go to pharmacy
□Complete adverse events form
□Provide participant one pill of 50,000 IU vitamin D to take with water at 
visit
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V3 in 7±1 days
□Payment to participant:  
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
V3- Follow-up visit checklist 
□Go to pharmacy
□Complete adverse events form
□Provide participant one pill of 50,000 IU vitamin D to take with water at 
visit
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V4 in 7±1 days
□Payment to participant:  
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


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UNC PROTOCOL Version 1.2- Updated September 6 2017 
V4- Follow-up visit with lab draw checklist 
□Go to pharmacy
□Complete adverse events form
□Obtain blood testing
□One-3.5 ml SST to UNC McLendon Lab for eligibility screening
(BMP, 25-OH vitamin D) 
□Provide participant one pill of 50,000 IU vitamin D to take with water at visit
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V5 in 7±1 days
□Payment to participant:  
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
V5- Follow-up visit checklist 
□Go to pharmacy
□Complete adverse events form
□Provide participant one pill of 50,000 IU vitamin D to take with water at 
visit
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V6 in 7±1 days
□Payment to participant:  
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
V6- Follow-up visit checklist 
□Go to pharmacy
□Complete adverse events form
□Provide participant one pill of 50,000 IU vitamin D to take with water at 
visit
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V7 in 7±1 days
□Payment to participant:  
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___  
   Date: ______________ 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
V7- Follow-up visit checklist 
□Go to pharmacy
□Complete adverse events form
□Provide participant one pill of 50,000 IU vitamin D to take with water at 
visit
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V8 in 7±1 days
□Payment to participant:  
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
V8- Follow-up visit with lab draw checklist 
□Complete adverse events form
□Obtain blood testing
□One-3.5 ml SST to UNC McLendon Lab for eligibility screening
(BMP, 25-OH vitamin D) 
□One 8.5 ml SST for serum storage (25-OH vitamin D, vitamin D binding protein)
□Two 4 ml EDTA lavender tube for plasma storage (cathelicidin)
□Provide participant with “Bronchoscopy Handout”
□Confirm contact phone number
□Review labs to determine vitamin D level
If 25-OH-VitD≥20 mg/ml: Notify MD to schedule bronchoscopy 
If 25-OH-VitD<20 mg/ml: Participant not eligible for bronchoscopy; study is completed 
Bronchoscopy scheduling: Target 1 week ± 5 days 
□Payment to participant:  
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL Version 1.2- Updated September 6 2017 
B2- Bronchoscopy #2 Visit Checklist 
□Review consent form for completion
□Obtain blood testing
□Two 10 ml Green Top Lithium tubes for PBMC storage
□Review labs to confirm vitamin D ≥20 ng/ml
□Escort patient to bronchoscopy
□Payment to participant: 
□MD to complete bronchoscopy form
□Staff to complete bronchoscopy sample processing form 
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL 
SECTION 7: LABORATORY PROCEDURES 
1. Plasma processing for cathelicidin
1. Collect two 4 ml EDTA lavender top for plasma, invert 6-12 times after collection
2. Place sample on ice after collection
3. Centrifuge lavender top at 4°C at 1500rpm for 15 minutes.
4. Remove vacutainer tube from the centrifuge, carefully, protecting the layer and not
mixing.
5. Remove stopper from the vacutainer tube.
6. Remove plasma and transfer to fresh polypropylene tube. Be careful to not disturb white
cells in the buffy coat.
7. Recentrifuge the transferred plasma in order to avoid every contamination with white
blood cells: 1500xg at 4°C for 15 minutes
8. Aliquot into 250 microliter aliquots in polypropylene tubes labeled “plasma” (see table
below) with study ID
a.
Individual labels should also include study ID (VitDXXX) and date
9. Storage store at -80°C in polypropylene tubes.
2. Serum processing for 25-OH vitamin D and vitamin D binding protein
1. Collect one 8.5 ml SST for serum.
2. Allow samples to clot for 30 minutes at room temperature
3. Centrifuge for 15 minutes at 1000 x g.
4. Remove serum and aliquot into 250 microliter aliquots in polypropylene tubes labeled
“serum” (see table below) with study ID
a.
Individual labels should also include study ID (VitDXXX) and date
5. Store at -80°C in polypropylene tubes
3. PBMC Processing for storage
PBMC Processing 
1. Obtain the two 10 ml green-top tubes of heparinized whole blood. Samples should be
processed within two hours, and stored at room temperature prior to processing.


Cathelicidin and Vitamin D Study 
UNC PROTOCOL 
2. Pour each tube into 2 empty 50mL conicals such that each conical contains
approximately 10mL of whole blood.
3. Place the emptied green top tubes back on test tube rack. DO NOT discard vacutainer
tubes yet
4. Wash each vacutainer tube with about 3mL of Dulbecco’s Phosphate Buffered Saline
(DPBS) consecutively to ensure the collection of any residual cells along the tube walls.
Dispense the DPBS containing the residual cells into one of the 50mL conicals.
5. Repeat step 4 for all tubes.
a.
If the volume of each conical is less than 20mL, then proceed to step 9.
b. If the volume of each conical is 20mL or more, then proceed to step 6.
6. Dilute the blood to 50mL with DPBS and mix 2 times with a pipette.
7. Take conicals that have been pre-filled with 18mL of DPBS and place them behind the
sample conicals.
8. From the 50mL sample volume, separate out approximately 18mL into pre-filled
conicals.
9. Bring the volume of each conical to approximately 35mL of whole blood and DPBS.
10. Underlay Ficoll-Hypaque (F/H) by performing the following method:
a.
Insert a 10 ml pipette into a F/H bottle and draw up F/H to orange line of pipette.
b. Release the pipette from pipette gun to allow F/H to dispense slowly into each
conical.
c.
It is essential that the F/H be dispensed slowly so that there is no mixing of the
whole blood with the F/H layer.
11. Make sure the centrifuge break is OFF and centrifuge all tubes for 20 minutes at 2400
RPM at 22OC.
12. Harvest the interface which contains the mononuclear cells, with a 10 ml serological
pipette.
13. Pool the harvested cells into a clean, sterile 50 ml conical. Cells may be dispensed in two
clean 50mL conical if blood volume was received from more than 6 green top tubes.
Bring volume of each conical to 50 ml with DPBS.
14. Centrifuge 10 minutes at 500 G (1500 RPM)


Cathelicidin and Vitamin D Study 
UNC PROTOCOL 
15. Aspirate the DPBS with a 2 ml serological pipette into waste system. Leave
approximately 120µl of supernatant and vortex pellet gently.
16. If there are 2 conicals of harvested cells, combine into one conical.
17. Follow protocol for counting cells.
18. For the final wash, bring volume up to 35mL with sterile DPBS and centrifuge 5 minutes
at 1500 RPM.
PBMC Freezing 
1. After the final wash, aspirate and discard the supernatant from the PBMC pellet.
2. Re-suspend in Recovery Cell Culture media (freezing media) according to the PBMC
count (please refer to the Processing Table):
a.
Freeze in 10 million cells per mL of Recovery Cell Culture media using the
appropriate freezing racks
b. Aliquot 1mL of 10 million cells plus freezing media into the appropriately labeled
cryovial
c.
Place vials immediately into Mr. Frosty Cell Freezing Cans and into -80oC
overnight
d. On the following day, transfer PBMCs from cans in -80 oC to the -140 oC freezer
3. Bronchoalveolar lavage processing
A. BAL Supernatant
1. Spin the conical(s) filled with a volume of up to 80mL of BAL supernatant and cells at
1600 rpm for 6 minutes (4 oC)
2. After centrifugation, remove and save all the supernatant into the following (labeled
“Sup”):
a.
Aliquot 1mL into 6 vials. Store at -80 oC
b. Aliquot 10mL into 5-8 conicals depending upon remaining volume.Store at -80 oC
c.
Individual labels should include “Sup”, study ID (VitDXXX) and date
3. Re-suspend BAL Cells in 4mL HBSS media.
4. Add 35mL HBSS media to the BAL Cell conical, spin at 1500rpm for 5 minutes (22 oC)
5. After centrifugation, aspirate and discard the HBSS supernatant from the BAL Cells.


 
 
 
Cathelicidin and Vitamin D Study 
UNC PROTOCOL  
6. Re-suspend the cells in Recovery Cell Culture freezing media: 
a. Freeze in 10 million cells per mL of freezing media using freezing racks. 
b. Aliquot 1mL (10 million cells and freezing media) into appropriately labeled 
cryovial 
i. Individual labels should include “cells”, study ID (VitDXXX) and date 
c. Place vials immediately into Mr. Frosty Cell Freezing Cans and into -80oC for 
overnight incubation. 
d. On the following day, transfer cells from cans in -80 oC to the -140 oC freezer  
 
 
 
 
Processing Table 
Biospecimen 
(Parent 
tube) 
Volu
me 
Aliquot 
sample type 
Label 
Type 
Aliquot # 
Aliquot 
conc. & 
Volume 
Freezing 
Temp. 
(Celsius) 
BAL conical  80 
mL  
Supernatant 
(1mL aliquots) 
Sup 
6 
1.0mL 
minus 80 º 
Supernatant 
(10mL 
aliquots) 
Sup 
Depends on 
Supernatant 
volume 
10.0mL 
minus 80 º 
Bal Cells 
cells 
Depends on 
Bal Cell 
count 
10x106 
Bal 
Cells/ 
1.0mL 
minus 80º 
overnight 
to minus 
140º 
Green top 
tube 
20 
ml 
PBMC 
PBMC 
Depends on 
PBMC count 
10x106 
PBMC/ 
1.0mL 
minus 80º 
overnight 
to minus 
140º 
Lavender top 
EDTA tube 
for plasma 
10 
ml 
Plasma (250 
microliter 
aliquots) 
Plasma As many as 
possible 
250 
microlit 
minus 80 º 
SST top for 
serum 
8 ml 
Serum (250 
microliter 
aliquots) 
Serum 
As many as 
possible 
250 
microlit 
minus 80 º 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL 
SECTION 8: BRONCHOSCOPY PROCEDURES 
i.
Patient preparation
a.
The participant should be interviewed prior to the procedure to ensure that 
there are no contraindications to performing the bronchoscopy such as
i. recent acute or serious illness
ii. anticoagulation or bleeding disorder
iii. fever
iv. oxygen desaturation on room air
v. allergy to local anesthetics or sedation
vi. food intake within 6 hours.
b.
Patients should be instructed to take their usual morning oral medications 
with a small sip of water.
c.
Patients should also have a companion or reliable escorted means of 
transportation home after the procedure so that they can safely return 
home after the procedure.
d.
The patient should be informed that the procedure is being performed for 
research purposes and is not for clinical care.
e.
The details of the procedure should be explained to the participant and 
consent obtained in accordance with local hospital or clinic practices and 
procedures.
f.
Risks related to the procedure:
i. allergic or adverse reaction to sedation or local anesthesia
ii. nose-bleed
iii. sore-throat
iv. coughing
v. chest soreness from coughing
vi. fever
vii. infection
g.
Participant will be reimbursed in the form of a check, given to participant 
immediately prior to procedure
ii.
Conduct of the procedure
a.
Sedation
i. Before sedation, a “time-out” should be performed to verify that 
the patient is correctly identified and is undergoing a 
bronchoscopy.
ii. The bronchoscopy insertion orifice should be ascertained (right or 
left nostril or mouth) and local anesthetic applied in accordance 
with local practice.
iii. The patient is moderately sedated during monitoring of vital signs 
and oxygenation according to local procedures.
iv. Typically a benzodiazepine (e.g midazolam) and a narcotic (e.g. 
fentanyl) are used. 


Cathelicidin and Vitamin D Study 
UNC PROTOCOL 
1. Upper limits of sedation
a.
Fentanyl 400 mcg IV
b. Versed 6 mg IV
b. Method
i. The bronchoscope is inserted and the upper airway is anesthetized
with 1-2 mL aliquots of lidocaine 1% or 2% solution.
ii. The lower airways are anesthetized with aliquots of lidocaine to
diminish or prevent coughing.
iii. The amount of lidocaine instilled should be monitored. Typically
no more than 500 mg should be administered in a single procedure.
iv. In order to minimize the use of lidocaine, the inspection of the left
lung airways should be brief or limited.
c.
Collection of specimens
i. The bronchoscope is advanced into the right mainstem bronchus
and the BAL is collected by instillation of 50 mL x 2 in the right
middle lobe (either segments). If necessary to obtain at least 50 cc
BAL return, additional 50 cc aliquots can be instilled in alternative
segments of the RML or anterior RUL, to a maximum of 300 cc
BAL instillation.
ii. Return of BAL fluid can sometimes be facilitated by ensuring that
the return suction is not too high (< 100 cm H2O), by turning the
patient with the right lung superior, or by asking the patient to take
a deep breath and cough.
d. Completion of procedure
i. The BAL form is completed which records the results of the BAL.
ii. The research coordinator should be notified that the procedure is
completed.
iii. If the patient has a post-bronchoscopy fever, the use of anti-
pyretics are advised.
e.
Post bronchoscopy care
i. The patient should be monitored in supervised environment until
they have recovered from sedation, and vital signs and oxygenation
are stable.
ii. The patient should be instructed not to take any food or liquid by
mouth for at least two hours after the procedure or until the gag-
reflex has returned.
iii. The patient should be notified to contact the clinic or study
physician if any symptoms of concern are noted after the
procedure.
i.
Sample Handling


Cathelicidin and Vitamin D Study 
UNC PROTOCOL 
a.
BAL samples should be transported within 30 minutes at room temperature. If
transport time will be 30-60 min, samples should be placed on ice. Transport
time should not exceed 60 minutes to maximize cell viability.


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
PROTOCOL 
Cathelicidin and Vitamin D: Impact on Populations At-Risk and with 
COPD 
ClinicalTrials.Gov Registration NCT02464059 
SPIROMICS/BAYVIEW PROTOCOL 
Version 1.7 
Updated April 20 2016 
SECTION 1. Study Overview 
SECTION 2. Recruitment 
SECTION 3. Detailed study visit procedures 
SECTION 4. Reporting events 
SECTION 5. Study visit checklists 
SECTION 6. Study forms 
SECTION 7. Laboratory procedures 
SECTION 8. Bronchoscopy procedures 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
SECTION 1: Vitamin D Study Overview 
1.1: Introduction and Background: Understanding mechanisms leading to decrements in lung 
function, the physiologic hallmark of obstructive lung diseases including chronic obstructive 
pulmonary disease (COPD), are necessary to inform interventions to improve lung health.  The 
antimicrobial peptide cathelicidin, and its primary regulator vitamin D, has been implicated in 
the development and progression of chronic lung disease. Cathelicidin has bactericidal and 
inflammatory activities in the lung and is regulated by vitamin D levels.  
1.2: Purpose of the Study: The purpose of the study is to determine the effect of vitamin D 
replacement on blood and lung cathelicidin levels in populations at-risk or with established 
COPD. The antimicrobial peptide cathelicidin, and its primary regulator vitamin D, has been 
implicated in the development and progression of chronic lung disease.  We hypothesize that oral 
vitamin D supplementation will raise cathelicidin levels in the pulmonary compartment, thereby 
potentially restoring relative lung cathelicidin deficiency. To test this hypothesis, we will recruit 
from two ongoing cohort studies at Johns Hopkins, the SHIELD study of African-American 
individuals at high risk for development of obstructive lung diseases (OLDs) (NA_00019960: PI 
Kirk), and the Subpopulations and Intermediate Outcome Measures in COPD Study 
(SPIROMICS) study of individuals with established COPD (NA_00035701: PI Hansel). We will 
also recruit participants from prior COPD studies NA_00009880, NA_00035381, 
NA_00085617, NA_00068035, and IRB00047215. This MOP reflects procedures for the 
SPIROMICS site. 
We will measure blood and lung lavage cathelicidin levels in 20 vitamin D insufficient 
individuals (10 participants with serum 25-OH Vitamin D<10 ng/ml and 10 participants with 
serum 25-OH vitamin D between 10-20) before and after eight weeks of oral vitamin D 
supplementation to determine the effect of vitamin D supplementation on cathelicidin levels 
Completion of this project directly or indirectly works towards ultimately understanding if a 
readily measurable blood marker, cathelicidin, can inform who may benefit from vitamin D 
supplementation to prevent chronic lung disease. The results from this application would lead to 
a therapeutic intervention study that will provide further opportunities to improve our 
understanding of the relationship between vitamin D, cathelicidin and lung function. 
1.3 Objectives: 
To evaluate the effect of oral vitamin D supplementation on plasma and lung cathelicidin 
measures in individuals with vitamin D insufficiency. Our primary analysis will include 
comparison between pre- and post-supplementation cathelicidin levels using paired t-tests for 
repeated measures 
1.4 Overall Project Design: 
The design of the study to complete this aim will be in the format of a baseline and repeat 
bronchoscopy after oral supplementation with 50,000 IU vitamin D3 weekly for eight weeks in 
individuals with reduced vitamin D levels.  The specific study data collection is outlined in the 
table below.  


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
•
Participants will undergo a screening assessment (visit S0) to determine the presence of
vitamin D insufficiency (serum 25OH-VitD <20 ng/ml) as well as normal renal function
and calcium levels. Pre- and post-bronchodilator spirometry will be performed.
•
If eligible based on S0 data, participants will undergo baseline bronchoscopy with
bronchoalveolar lavage (BAL) for collection of lung cathelicidin levels (visit B1). Blood
for cathelicidin, vitamin D binding protein, vitamin D and PBMC will be collected at this
visit.
•
They will then return to the SPIROMICS research clinic weekly for eight weeks of
directly observed vitamin D supplementation as well as assessment of adverse events
(visits V0-V7).
o
After the initial four weeks of treatment (V4), all patients will be evaluated and
have blood work obtained for calcium level to monitor for hypercalcemia.
o
Patients will be screened at each weekly visit for symptoms of hypercalcemia.
•
After 8 weeks of vitamin D treatment, serum 25OH-VitD levels will be measured (visit
V8) to ensure increased level prior to undergoing bronchoscopy. Individuals not
increasing vitamin D> 20 after 8 weeks will not be eligible for B2 bronchoscopy and will
be deemed to have completed study. Blood for BMP/ calcium level will also be collected.
•
A second BAL collection will occur after the eight week of vitamin D treatment to
measure cathelicidin in the plasma and the BAL (visit B2). Blood for cathelicidin,
vitamin D binding protein, vitamin D and PBMC will also be collected at this visit.
•
All procedures will be research related. A visit window of ±1 day is acceptable for all
study visits except B1 and B2, where a 28 day window is permissible given potential
delay in laboratory results and bronchoscopy scheduling.
Table. Study Data Collection Schedule 
Visit 
S0 
B1 
V0 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
B2 
Time (weeks) 
-2
-1
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
Window (+/- days) 0 
28 
1 
1 
1 
1 
1 
1 
1 
1 
1 
28 
Visit Duration 
(hrs) 
2 
5 
1 
0.5 
0.5 
0.5 
1 
0.5 
0.5 
0.5 
1 
5 
Informed Consent 
• 
Spirometry 
• 
Vitamin D 
• 
• 
• 
Vit D binding 
prot/cathelicidin/ 
PBMC 
• 
• 
BMP/ calcium 
• 
• 
• 
BAL 
• 
• 
Drug 
administration 
• 
• 
• 
• 
• 
• 
• 
• 
Adverse events & 
interval history 
• 
• 
• 
• 
• 
• 
• 
• 
Key:Time= nominal visit time; S=screening visit, Bn= Bronchoscopy visit n, Vn=Study visit 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
SECTION 2: RECRUITMENT 
2.1 Definition of Recruitment & Consent: Recruitment is establishing eligibility of the potential 
candidate and informing the candidate of their eligibility and signing of the informed consent 
form by the potential candidate.  
2.2 Sources of Recruitment: Participants of the SPIROMICS study (Protocol NA_00035701- PI 
Hansel) additional approved studies including prior COPD studies NA_00009880, 
NA_00035381, NA_00085617, NA_00068035, and IRB00047215. Total goal N=20. 
Recruitment happens at the Bayview Lung Health Study (SPIROMICS)  
2.3 Process of Recruitment: Study team members from this protocol will receive a list of 
research participants from the above listed protocols who have previously indicated willingness 
to be contacted for future studies. This study team will contact research participants. These 
contacts will be made via phone call or in face-to-face encounters during scheduled study visits 
for the ongoing approved protocols. For recruitment via face-to-face encounter, these 
conversations will occur in a private office with a trained study team member.   
2.4 Study Inclusion and Exclusion Criteria: 
i.
Inclusion
i. Post-BD FEV1/FVC<0.70
ii. Post-BD FEV1> 50% predicted
iii. Age 18-80
iv. Current or former smokers
v. Serum calcium<10.5mg/dl
vi. 25-OH VitD<20 ng/ml at screening
1. Note that target recruitment is at least 10 participants with 25-OH
VitD <10 ng/ml
vii. CrCl ≥60 mL/min as estimated by the Cockcroft-Gault equation
viii. Women of reproductive potential with negative serum or urine pregnancy
test and be willing to refrain from participating in a conception process
and subject/partner must use at least 2 reliable forms of contraceptives for
the duration of the study
ii.
Exclusion
i. Current use of vitamin D supplements
ii. Oxygen use >2L/min at rest
iii. Known allergy/sensitivity or any hypersensitivity to components of study
drugs or their formulations
iv. Pregnancy or currently breast-feeding
v. History of nephrolithiasis (kidney stones)
vi. Self-reported HIV positive serostatus
vii. Any condition that, in the opinion of the site investigator, would
compromise the subject’s ability to participate in the study.


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
SECTION 3: DETAILED STUDY VISIT PROCEDURES 
3.1 Chart/Phone Pre-Screening Process 
i.
Potential individuals will be identified through screening database for following
eligibility
a.
Post-BD FEV1/FVC<0.70
b. Post-BD FEV1> 50% predicted
c.
Age 18-80
d. Current or former smokers
ii.
Individuals will be contacted via phone or in person during in-person visit.
iii.
The telephone screening script will be used to determine further eligibility for all
participants.
iv.
If participant is eligible and interested, they will be scheduled for an in-person visit
for informed consent review/signing as well as screening laboratory data. If screening
is occurring in person, informed consent and blood work can be completed same day.
3.2 Screening Visit (S0) 
i.
Review/sign consent form
ii.
Assignment of study ID
a.
Using “VitDXXX” where
XXX= sequential numbers from 001-999
iii.
Completion of demographics form
iv.
Urine pregnancy test (for women of childbearing age)
v.
Performance of pre- and post-bronchodilator spirometry (4 puffs albuterol)
vi.
Completion of screening form to determine eligibility
vii.
If eligibility confirmed, obtain blood testing for screening visit (see laboratory 
processing section)
a.
Two 3.5 ml SST to JHH for eligibility screening
i. Serum 25OH-Vitamin D
ii. Serum Basic metabolic panel
viii.
Confirm contact number to reach participant after 25OH-VitD and BMP resulted.
ix.
Payment to participant
x.
After labs resulted, PI to review vitamin D/BMP results and determine eligibility
xi.
MD to contact person to arrange bronchoscopy 
3.3 Bronchoscopy (B1) 
i.
Before scheduling the first of the two bronchoscopy procedures, it should be
established that
i. Patient has signed IRB-approved consent for the research protocol
ii. Has met the inclusion criteria for the study.
iii. The benefits and risks of the procedure have been explained to the
participant and patient has given consent for the procedure.


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
ii.
The target windows for scheduling the bronchoscopy is 1 week after S0 visit for
the first procedure and 1 week after V8 for the second procedure.
iii.
Day of bronchoscopy, the following bloods will be collected:
a.
Two 3.5 ml SST for serum storage
i. Serum for 25OH-Vitamin D and vitamin D binding protein
b. Two 4 ml EDTA Lavender for plasma storage
i. Plasma for cathelicidin
c.
Two 10 ml Green Top Lithium tubes for PBMC storage
i. PBMC to be processed at Mark Liu’s lab
See “BRONCHOSCOPY PROCEDURES” for bronchoscopy procedure details 
3.4 Initial medication administration visit (V0) 
i.
Participant will present for first drug administration. At the time of visit, a bottle of
25-OH vitD will be removed for secured storage and label completed with:
a.
Participants study number (VitDXXX)
b.
Initials of the participant
c.
Date of V0 visit
ii.
Participant will be given one pill of Vitamin D 50,000 IU and asked to take pill with 
water at that time
iii.
Complete Vitamin D Subject Specific Accountability Record
iv.
Provide participant with Vitamin D informational handout
v.
Schedule V1 in 7 days
vi.
Payment to participant: 
3.5 Follow-up visits 
i.
Visits V1-V3
a.
Adverse events form is completed
b.
Participant will be given one pill of Vitamin D 50,000 IU and asked to take 
pill with water at that time
c.
Complete vitamin D administration log
d.
Schedule next visit in 7 days
e.
Payment to participant
ii.
Visit V4
a.
Adverse events form is completed
b.
Participant will be given one pill of Vitamin D 50,000 IU and asked to take 
pill with water at that time
c.
Complete Vitamin D Subject Specific Accountability Record
d.
Schedule next visit in 7 days
e.
Obtain blood (see laboratory processing section)
i. Two 3.5 ml SST to JHH for safety screening
1. Serum 25OH-Vitamin D 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
2. Serum Basic metabolic panel
f.
Confirm contact number to reach participant after 25OH-VitD and BMP 
resulted.
g. Payment to participant
h. Notify study MD when lab results completed 
iii.
Visit V5-V7
a.
Adverse events form is completed
b.
Participant will be given one pill of Vitamin D 50,000 IU and asked to take 
pill with water at that time
c.
Complete Vitamin D Subject Specific Accountability Record
d.
Schedule next visit in 7 days
e.
Payment to participant:
iv.
Visit V8
a.
Adverse events form is completed
b.
Schedule next visit in 7 days
c.
Obtain blood (see laboratory processing section)
i. Two 3.5 ml SST to JHH for safety screening
1. Serum 25OH-Vitamin D
2. Serum Basic metabolic panel
d.
Confirm contact number to reach participant after 25OH-VitD and BMP 
resulted.
e.
Payment to participant
f.
Notify study MD when lab results completed
a.
If vitamin D not >20 ng/ml, participant not eligible for bronchoscopy 
and will have completed study
b.
If vitamin D>=20 ng/ml, study staff contact person to arrange 
bronchoscopy 
3.6 Bronchoscopy (B2) 
i.
The target windows for scheduling the bronchoscopy is 1 week after V8 for the
second procedure.
ii.
On day of bronchoscopy, the following blood will be obtained:
a.
Two 3.5 ml SST for serum storage
i. Serum for 25OH-Vitamin D and vitamin D binding protein
b. Two 4 ml EDTA Lavender for plasma storage
i. Plasma for cathelicidin
c.
Two 10 ml Green Top Lithium tubes for PBMC storage
i. PBMC to be processed at Mark Liu’s lab
See “BRONCHOSCOPY PROCEDURES” for procedure details 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
SECTION 4: REPORTING EVENTS 
The proposed intervention study involves treatment of participants with vitamin D with the goal 
of determining if this intervention is efficacious in altering lung cathelicidin levels, and is 
therefore classified as a clinical trial.  Because this is a clinical trial, the PI will designate an 
independent monitor to perform independent review of ongoing study progress and safety. 
Adverse events (AE) will be reported both to our IRB and the Independent Monitor through 
annual progress reports or more frequently as described below. AE reports and annual summaries 
will not include subject- or group-identifiable material. Each report will only include the 
identification code. 
The Independent Monitor for this study is Dr. Sonali Bose at Johns Hopkins (sbose7@jhmi.edu) 
For this application, an adverse event is defined as any untoward medical occurrence in a subject 
during participation in the clinical study or with use of the intervention (vitamin D) being 
studied. An adverse finding can include a sign, symptom, abnormal assessment (laboratory test 
value, vital signs, etc.), or any combination of these. A serious adverse event (SAE) is any 
adverse event that results in one or more of the following outcomes: 
•
Death
•
A life-threatening event
•
Inpatient hospitalization or prolongation of existing hospitalization
•
A persistent or significant disability/incapacity
•
A congenital anomaly or birth defect
•
An important medical event based upon appropriate medical judgment
AEs will be labeled according to severity, which is based on their impact on the patient. An AE 
will be termed “mild” if it does not have a major impact on the patient, “moderate” if it causes 
the patient some minor inconvenience, and “severe” if it causes a substantial disruption to the 
patient’s well-being. AEs will be categorized according to the likelihood that they are related to 
the study intervention. Specifically, they will be labeled definitely unrelated, definitely related, 
probably related, or possibly related to the study intervention.  
The PI and Independent monitor will review AE rates at least monthly. 
When an AE occurs, the AE will be reported to the Institutional Review Board at Johns Hopkins 
Hospital and Bloomberg School of Hygiene, to the funding agency and to the Independent 
Monitor. Our procedure is for the study coordinator to notify the Principal Investigators and for 
the program staff to make necessary arrangements for the participant to see a medical provider. 
Most assessments will occur in research space adjacent to the Hopkins clinics, and referrals 
would be made to the clinic acute provider. The Johns Hopkins Emergency Room is the first 
choice for emergent care, and we would arrange for transport or accompany the participant as 
needed.  SAEs that are unanticipated, serious, and possibly related to the study intervention will 
be reported to the Independent Monitor and IRB in accordance with requirements.  
Unexpected fatal or life-threatening AEs related to the intervention will be reported to the IRB 
and Independent Monitor within 3 days. Other serious and unexpected AEs related to the 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
intervention will be reported to the IRB and Independent Monitor within 14 days.  Anticipated or 
unrelated SAEs will be handled in a less urgent manner but will be reported to the Independent 
Monitor and IRB. In the annual AE summary, the Independent Monitor Report will be required 
to state that they have reviewed all AE reports.   
Study progress and safety will be reviewed monthly by the PI. Progress reports, including patient 
recruitment, retention/attrition, and AEs, will be provided to the Independent Monitor following 
each of the quarterly reviews. An Annual Report will be compiled and will include a list and 
summary of AEs. In addition, the Annual Report will address (1) whether AE rates are consistent 
with pre-study assumptions; (2) reason for dropouts from the study; (3) whether all participants 
met entry criteria; (4) whether continuation of the study is justified on the basis that additional 
data are needed to accomplish the stated aims of the study; and (5) conditions whereby the study 
might be terminated prematurely. The Annual Report will be sent to the Independent Monitor 
and will be forwarded to the IRB and included in the Progress Report to the NIH. The IRB and 
other applicable recipients will review progress of this study on an annual basis.  
During the funding of this study, any action by the IRB, the Independent Monitor, or one of the 
study investigators that results in a temporary or permanent suspension of the study will be 
reported to the NHLBI Program Official within 1 business day of notification. 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
SECTION 5: STUDY VISIT CHECKLISTS 
The following pages include a separate page for each study visit with a checklist of items that 
must be completed at each visit. These pages should be printed and used as a step-by-step guide 
of each visit. 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
S0- Screening Visit Checklist 
□Review and sign consent form
□Assign study ID
□Complete demographics form
□Urine pregnancy test (for women of childbearing age)
□Pre- and post-bronchodilator spirometry
□Complete screening form
□If eligibility confirmed, obtain blood testing
□Two 3.5 ml SST to JHH (BMP, 25-OH vitamin D)
□Confirm contact phone number
□Provide participant with “Bronchoscopy Handout”
□Payment to participant
□Notify MD of blood results for review and scheduling of bronchoscopy 
Review total recruitment: 
Eligibility: 25OH VitD <20ng/ml (with targeted recruitment of at least 10 participants 
with level <10 ng/ml) 
If 10 participants have been recruited with level ≥ 10 and <20, then a participant with 
level ≥10 and <20 is no longer eligible. At that point, only individuals with level <10 are 
eligible. 
Bronchoscopy scheduling: Target 1 week ± 5 days 
□Once bronchoscopy is scheduled, V0 should be scheduled within 1 week ±1 day of
bronchoscopy) 
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
B1- Bronchoscopy #1 Visit Checklist 
□Review consent form for completion
□Obtain blood testing
□Two 3.5 ml SST for serum storage (25-OH vitamin D, vitamin D binding protein)
□Two 4 ml EDTA lavender tube for plasma storage (cathelicidin)
□Two 10 ml Green Top Lithium tubes for PBMC storage (Mark Liu’s lab)
□Review labs to confirm vitamin D <20 ng/ml
□Escort patient to bronchoscopy
□Payment to participant
□MD to complete bronchoscopy form
□Staff to complete bronchoscopy sample processing form
□Confirm V0 is scheduled (within 1 week ±1 day of bronchoscopy) 
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
V0- Initial medication administration checklist 
□Label new bottle of vitamin D with participant study ID, initials, date
□Provide participant one pill of 50,000 IU vitamin D to take with water at visit
□Provide participant informational flyer regarding vitamin D side effects
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V1 in 7±1 days
□Payment to participant 
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
V1- Follow-up visit checklist 
□Complete adverse events form
□Provide participant one pill of 50,000 IU vitamin D to take with water at visit
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V2 in 7±1 days
□Payment to participant 
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
V2- Follow-up visit checklist 
□Complete adverse events form
□Provide participant one pill of 50,000 IU vitamin D to take with water at visit
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V3 in 7±1 days
□Payment to participant 
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
V3- Follow-up visit checklist 
□Complete adverse events form
□Provide participant one pill of 50,000 IU vitamin D to take with water at visit
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V4 in 7±1 days
□Payment to participant 
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
V4- Follow-up visit with lab draw checklist 
□Complete adverse events form
□Obtain blood testing
□Two 3.5 ml SST to JHH (BMP, 25-OH vitamin D)
□Provide participant one pill of 50,000 IU vitamin D to take with water at visit
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V5 in 7±1 days
□Payment to participant 
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
V5- Follow-up visit checklist 
□Complete adverse events form
□Provide participant one pill of 50,000 IU vitamin D to take with water at visit
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V6 in 7±1 days
□Payment to participant 
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
V6- Follow-up visit checklist 
□Complete adverse events form
□Provide participant one pill of 50,000 IU vitamin D to take with water at visit
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V7 in 7±1 days
□Payment to participant 
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
V7- Follow-up visit checklist 
□Complete adverse events form
□Provide participant one pill of 50,000 IU vitamin D to take with water at visit
□Complete Vitamin D Subject Specific Accountability Record
□Schedule V8 in 7±1 days
□Payment to participant 
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
V8- Follow-up visit with lab draw checklist 
□Complete adverse events form
□Obtain blood testing
□Two 3.5 ml SST to JHH (BMP, 25-OH vitamin D)
□Provide participant with “Bronchoscopy Handout”
□Confirm contact phone number
□Review labs to determine vitamin D level
If 25-OH-VitD≥20 mg/ml: Notify MD to schedule bronchoscopy 
If 25-OH-VitD<20 mg/ml: Participant not eligible for bronchoscopy; study is completed 
Bronchoscopy scheduling: Target 1 week ± 5 days 
□Payment to participant 
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
B2- Bronchoscopy #2 Visit Checklist 
□Review consent form for completion
□Obtain blood testing
□Two 3.5 ml SST for serum storage (25-OH vitamin D, vitamin D binding protein)
□Two 4 ml EDTA lavender tube for plasma storage (cathelicidin)
□Two 10 ml Green Top Lithium tubes for PBMC storage (Mark Liu’s lab)
□Review labs to confirm vitamin D ≥20 ng/ml
□Escort patient to bronchoscopy
□Payment to participant
□MD to complete bronchoscopy form
□Staff to complete bronchoscopy sample processing form 
Assigned ID:  VitD ___ ___ ___     Participant Initials:  ___ ___ ___ 
   Date: ______________ 


 
 
 
Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
SECTION 7: LABORATORY PROCEDURES 
 
1. Plasma processing for cathelicidin 
1. Collect two 4 ml EDTA lavender top for plasma, invert 6-12 times after collection 
2. Place sample on ice after collection 
3. Centrifuge lavender top at 4°C at 1500rpm for 15 minutes.   
4. Remove vacutainer tube from the centrifuge, carefully, protecting the layer and not 
mixing. 
5. Remove stopper from the vacutainer tube. 
6. Remove plasma and transfer to fresh polypropylene tube. Be careful to not disturb white 
cells in the buffy coat.  
7. Recentrifuge the transferred plasma in order to avoid every contamination with white 
blood cells: 1500xg at 4°C for 15 minutes 
8. Aliquot into 250 microliter aliquots in polypropylene tubes labeled “plasma”  (see table 
below) with study ID 
a. Individual labels should also include study ID (VitDXXX) and date 
9. Storage store at -80°C in polypropylene tubes. 
2. Serum processing for 25-OH vitamin D and vitamin D binding protein 
1. Collect two 3.5 ml SST for serum. 
2. Allow samples to clot for 30 minutes at room temperature 
3. Centrifuge for 15 minutes at 1000 x g.  
4. Remove serum and aliquot into 250 microliter aliquots in polypropylene tubes labeled 
“serum” (see table below) with study ID 
a. Individual labels should also include study ID (VitDXXX) and date 
5. Store at -80°C in polypropylene tubes 
3. PBMC Processing for storage (Hui Qing in Mark Liu’s lab) 
PBMC Processing 
1. Obtain the two 10 ml green-top tubes of heparinized whole blood. Samples should be 
processed within two hours, and stored at room temperature prior to processing. 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
2. Pour each tube into 2 empty 50mL conicals such that each conical contains
approximately 10mL of whole blood.
3. Place the emptied green top tubes back on test tube rack. DO NOT discard vacutainer
tubes yet
4. Wash each vacutainer tube with about 3mL of Dulbecco’s Phosphate Buffered Saline
(DPBS) consecutively to ensure the collection of any residual cells along the tube walls.
Dispense the DPBS containing the residual cells into one of the 50mL conicals.
5. Repeat step 4 for all tubes.
a.
If the volume of each conical is less than 20mL, then proceed to step 9.
b. If the volume of each conical is 20mL or more, then proceed to step 6.
6. Dilute the blood to 50mL with DPBS and mix 2 times with a pipette.
7. Take conicals that have been pre-filled with 18mL of DPBS and place them behind the
sample conicals.
8. From the 50mL sample volume, separate out approximately 18mL into pre-filled
conicals.
9. Bring the volume of each conical to approximately 35mL of whole blood and DPBS.
10. Underlay Ficoll-Hypaque (F/H) by performing the following method:
a.
Insert a 10 ml pipette into a F/H bottle and draw up F/H to orange line of pipette.
b. Release the pipette from pipette gun to allow F/H to dispense slowly into each
conical.
c.
It is essential that the F/H be dispensed slowly so that there is no mixing of the
whole blood with the F/H layer.
11. Make sure the centrifuge break is OFF and centrifuge all tubes for 20 minutes at 2400
RPM at 22OC.
12. Harvest the interface which contains the mononuclear cells, with a 10 ml serological
pipette.
13. Pool the harvested cells into a clean, sterile 50 ml conical. Cells may be dispensed in two
clean 50mL conical if blood volume was received from more than 6 green top tubes.
Bring volume of each conical to 50 ml with DPBS.
14. Centrifuge 10 minutes at 500 G (1500 RPM)


 
 
 
Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
15. Aspirate the DPBS with a 2 ml serological pipette into waste system. Leave 
approximately 120µl of supernatant and vortex pellet gently. 
16. If there are 2 conicals of harvested cells, combine into one conical.  
17. Follow protocol for counting cells. 
18. For the final wash, bring volume up to 35mL with sterile DPBS and centrifuge 5 minutes 
at 1500 RPM.  
PBMC Freezing  
1. After the final wash, aspirate and discard the supernatant from the PBMC pellet. 
2. Re-suspend in Recovery Cell Culture media (freezing media) according to the PBMC 
count (please refer to the Processing Table): 
a. Freeze in 10 million cells per mL of Recovery Cell Culture media using the 
appropriate freezing racks 
b. Aliquot 1mL of 10 million cells plus freezing media into the appropriately labeled 
cryovial 
c. Place vials immediately into Mr. Frosty Cell Freezing Cans and into -80oC 
overnight 
d. On the following day, transfer PBMCs from cans in -80 oC to the -140 oC freezer 
(SHIELDLUNG BAL PBMC) 
 
3. Bronchoalveolar lavage processing (Hui Qing in Mark Liu’s lab) 
A. BAL Supernatant  
1. Spin the conical(s) filled with a volume of up to 80mL of BAL supernatant and cells at 
1600 rpm for 6 minutes (4 oC) 
2. After centrifugation, remove and save all the supernatant into the following (labeled 
“Sup”): 
a. Aliquot 1mL into 6 vials. Store at -80 oC 
b. Aliquot 10mL into 5-8 conicals depending upon remaining volume.Store at -80 oC 
c. Individual labels should include “Sup”, study ID (VitDXXX) and date 
3. Re-suspend BAL Cells in 4mL HBSS media. 


 
 
 
Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
4. Add 35mL HBSS media to the BAL Cell conical, spin at 1500rpm for 5 minutes (22 oC) 
5. After centrifugation, aspirate and discard the HBSS supernatant from the BAL Cells. 
6. Re-suspend the cells in Recovery Cell Culture freezing media: 
a. Freeze in 10 million cells per mL of freezing media using freezing racks. 
b. Aliquot 1mL (10 million cells and freezing media) into appropriately labeled 
cryovial 
i. Individual labels should include “cells”, study ID (VitDXXX) and date 
c. Place vials immediately into Mr. Frosty Cell Freezing Cans and into -80oC for 
overnight incubation. 
d. On the following day, transfer cells from cans in -80 oC to the -140 oC freezer  
 
 
Processing Table 
Biospecime
n (Parent 
tube) 
Volu
me 
Aliquot 
sample type 
Label 
Type 
Aliquot # 
Aliquot 
conc. & 
Volume 
Freezing 
Temp. 
(Celsius) 
BAL conical  80m
L  
Supernatant 
(1mL aliquots) 
Sup 
6 
1.0mL 
minus 80 
º 
Supernatant 
(10mL 
aliquots) 
Sup 
Depends on 
Supernatant 
volume 
10.0mL 
minus 80 
º 
Bal Cells 
cells 
Depends on 
Bal Cell 
count 
10x106 
Bal 
Cells/ 
1.0mL 
minus 80º 
overnight 
to minus 
140º 
Green top 
tube 
20 ml PBMC 
PBMC 
Depends on 
PBMC 
count 
10x106 
PBMC/ 
1.0mL 
minus 80º 
overnight 
to minus 
140º 
Lavender top 
EDTA tube 
for plasma 
10 ml 
Plasma (250 
microliter 
aliquots) 
Plasma 
As many as 
possible 
250 
microlit 
minus 80 
º 
SST top for 
serum 
8 ml 
Serum (250 
microliter 
aliquots) 
Serum 
As many as 
possible 
250 
microlit 
minus 80 
º 


Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
SECTION 8: BRONCHOSCOPY PROCEDURES 
i.
Patient preparation
a.
The participant should be interviewed prior to the procedure to ensure that 
there are no contraindications to performing the bronchoscopy such as
i. recent acute or serious illness
ii. anticoagulation or bleeding disorder
iii. fever
iv. oxygen desaturation on room air
v. allergy to local anesthetics or sedation
vi. food intake within 6 hours.
b.
Patients should be instructed to take their usual morning oral medications 
with a small sip of water.
c.
Patients should also have a companion or reliable escorted means of 
transportation home after the procedure so that they can safely return 
home after the procedure.
d.
The patient should be informed that the procedure is being performed for 
research purposes and is not for clinical care.
e.
The details of the procedure should be explained to the participant and 
consent obtained in accordance with local hospital or clinic practices and 
procedures.
f.
Risks related to the procedure:
i. allergic or adverse reaction to sedation or local anesthesia
ii. nose-bleed
iii. sore-throat
iv. coughing
v. chest soreness from coughing
vi. fever
vii. infection
g.
Participant will be reimbursed in the form of a check, given to participant 
immediately prior to procedure
ii.
Conduct of the procedure
a.
Sedation
i. Before sedation, a “time-out” should be performed to verify that 
the patient is correctly identified and is undergoing a 
bronchoscopy.
ii. The bronchoscopy insertion orifice should be ascertained (right or 
left nostril or mouth) and local anesthetic applied in accordance 
with local practice.
iii. The patient is moderately sedated during monitoring of vital signs 
and oxygenation according to local procedures. 


 
 
 
Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
iv. Typically a benzodiazepine (e.g midazolam) and a narcotic (e.g. 
fentanyl) are used.  
1. Upper limits of sedation 
a. Fentanyl 400 mcg IV 
b. Versed 6 mg IV 
b. Method 
i. The bronchoscope is inserted and the upper airway is anesthetized 
with 1-2 mL aliquots of lidocaine 1% or 2% solution.  
ii. The lower airways are anesthetized with aliquots of lidocaine to 
diminish or prevent coughing. 
iii. The amount of lidocaine instilled should be monitored. Typically 
no more than 500 mg should be administered in a single procedure. 
iv. In order to minimize the use of lidocaine, the inspection of the left 
lung airways should be brief or limited.   
c. Collection of specimens 
i. The bronchoscope is advanced into the right mainstem bronchus 
and the BAL is collected by instillation of 50 mL x 2 in the right 
middle lobe (either segments). If necessary to obtain at least 50 cc 
BAL return, additional 50 cc aliquots can be instilled in alternative 
segments of the RML or anterior RUL, to a maximum of 300 cc 
BAL instillation.  
ii. Return of BAL fluid can sometimes be facilitated by ensuring that 
the return suction is not too high (< 100 cm H2O), by turning the 
patient with the right lung superior, or by asking the patient to take 
a deep breath and cough. 
d. Completion of procedure 
i. The BAL form is completed which records the results of the BAL. 
ii. The research coordinator should be notified that the procedure is 
completed. 
iii. If the patient has a post-bronchoscopy fever, the use of anti-
pyretics are advised.  
e. Post bronchoscopy care 
i. The patient should be monitored in supervised environment until 
they have recovered from sedation, and vital signs and oxygenation 
are stable.  
ii. The patient should be instructed not to take any food or liquid by 
mouth for at least two hours after the procedure or until the gag-
reflex has returned.  
iii. The patient should be notified to contact the clinic or study 
physician if any symptoms of concern are noted after the 
procedure.  
 
i. 
Sample Handling 


 
 
 
Cathelicidin and Vitamin D Study 
SPIROMICS/BAYVIEW PROTOCOL Version 1.7- Updated April 20 2016 
a. BAL samples should be transported within 30 minutes at room temperature. If 
transport time will be 30-60 min, samples should be placed on ice. Transport 
time should not exceed 60 minutes to maximize cell viability.  

SAP_001.pdf: 
Statistical Analysis Plan for Protocol  
“Cathelicidin and Vitamin D: Impact on Populations At-Risk and with COPD” (NCT02464059) 
Version 27 April 2020 
This document serves as an addendum to the approved protocols related to NCT02464059 [“Cathelicidin 
and Vitamin D: Impact on Populations At-Risk with COPD” (Protocol version 1.2; 9/6/17; IRB 16-2200) 
and “Cathelicidin and Vitamin D: Impact on Populations with COPD (Protocol 8/23/17; IRB 17-2170)” to 
outline the detailed statistical analysis plan for the prespecified outcomes of this study. In the event that 
information in this document is discrepant from the original protocols (as it relates to endpoints or 
analyses), this detailed statistical analysis plan supersedes the original protocols.  
Analysis populations 
Full analysis set (FAS): All screened patients with informed consent and at least baseline 
bronchoscopy. 
 
Descriptive Summary of Study Cohort 
Using the FAS, the following variables will be summarized using descriptive statistics (mean and 
standard deviation, min, max, median) and graphical displays (boxplots, histograms) as appropriate.   
 
Demographics and clinical variables 
 
Baseline pre- and post-bronchodilator spirometry measurements 
 
Specific pre-defined outcomes are defined below along with the planned analytical approach. 
1.1. Primary Outcomes 
 
1.1.1. Change from Baseline in Lung Cathelicidin Level at 8 Weeks [ Time Frame: Baseline and 8 weeks   
• 
Change from baseline to 8 weeks in bronchoscopic lavage lung cathelicidin levels after vitamin D 
supplementation (comparison between pre- and post-supplementation cathelicidin levels using 
paired t-tests for repeated measures) 
2.1. Secondary Outcomes 
2.1.1.  Change from Baseline in Blood Cathelicidin at 8 Weeks [ Time Frame: Baseline and 8 weeks] 
• 
Change from baseline to 8 weeks in blood cathelicidin levels after vitamin D 
supplementation (comparison between pre- and post-supplementation cathelicidin levels 
using paired t-tests for repeated measures) 
Analysis plan 
Paired sample (dependent) t-test to evaluate for differences in BAL cathelicidin (primary outcome) and 
plasma cathelicidin (secondary outcome) between pre- to post-vitamin D supplementation. Specifically, 
we will compare mean cathelicidin levels (log transformed as needed for normality) from pre- and post-
supplementation BAL samples, and separately from plasma samples. P-values and 95% confidence 
intervals will be calculated for all estimates.   
All tests with p value < 0.05 will be interpreted nominally as statistically significant.  There will be no 
imputation for missing data.