Urban Environment and Childhood Asthma (URECA)
Conditions: Asthma, Allergy
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

NIAID Natural History Protocol Template Version 1.0 
January 31, 2008 
 
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URECA 
 
Urban Environment and Childhood Asthma 
 
 
NIAID Protocol Number: ICAC-07 
 
 
Sponsored by: 
National Institute of Allergy and Infectious Diseases (NIAID) 
 
 
Principal Investigator: James E. Gern 
 
 
Version Number: 30.0 
Protocol Addendum 
Continuation of URECA Study to Age 17 
 
 
21 January 2021 
 
 

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Statement of Compliance 
This clinical study will be conducted using good clinical practice (GCP), as delineated in 
Guidance for Industry: E6 Good Clinical Practice: Consolidated Guidance E6(R2), and 
according to the criteria specified in this study protocol.  Before study initiation, the protocol and 
the informed consent documents will be reviewed and approved by the central IRB.  Any 
amendments to the protocol or to the consent materials will also be approved by the central IRB 
before they are implemented. 
 
 

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Table of Contents 
 
Statement of Compliance ............................................................................................................ 3 
Table of Contents ........................................................................................................................ 4 
List of Abbreviations .................................................................................................................... 7 
Protocol Summary ...................................................................................................................... 8 
Schematic of Study Design ....................................................................................................... 10 
Key Roles ................................................................................................................................. 11 
1 
Background Information and Scientific Rationale ............................................................ 12 
 
Background Information .............................................................................................. 12 
 
Rationale .................................................................................................................... 12 
 
Potential Risks and Benefits ....................................................................................... 14 
1.3.1 
Risks of Study Procedures ................................................................................... 14 
1.3.2 
Potential Benefits ................................................................................................. 15 
2 
Study Objectives ............................................................................................................ 16 
 
Primary Objective........................................................................................................ 16 
 
Secondary Objectives ................................................................................................. 16 
 
Exploratory Objectives ................................................................................................ 16 
3 
Study Design .................................................................................................................. 16 
 
Description of the Study Design .................................................................................. 16 
 
Study Endpoint ........................................................................................................... 17 
3.2.1 
Primary Endpoint ................................................................................................. 17 
3.2.2 
Secondary Endpoints ........................................................................................... 17 
4 
Study Population ............................................................................................................ 17 
 
Population Description ................................................................................................ 17 
 
Participant Inclusion Criteria ....................................................................................... 18 
5 
Study Procedures/Evaluations ........................................................................................ 18 
 
Clinical Evaluations ..................................................................................................... 18 
5.1.1 
Dust Analysis for Allergen and Microbial Products ............................................... 18 
5.1.2 
Allergen Skin Testing ........................................................................................... 19 
5.1.3 
Pre/Post Bronchodilator Pulmonary Function Testing .......................................... 19 
5.1.4 
Methacholine Challenge ...................................................................................... 19 
5.1.5 
Exhaled Nitric Oxide Measurement ...................................................................... 19 
5.1.6 
Induced Sputum ................................................................................................... 19 
5.1.7 
Nasal Epithelial Cell Collection ............................................................................ 20 
5.1.8 
Collection of Nasal Lining Fluid ............................................................................ 20 
5.1.9 
Fitness Testing .................................................................................................... 20 
 
Laboratory Evaluations ............................................................................................... 20 
5.2.1 
Clinical and Research Laboratory Evaluations and Specimen Collection ............. 20 
5.2.2 
Specimen Preparation, Handling and Shipping .................................................... 21 
 
Substudies .................................................................................................................. 22 
6 
Research Use of Stored Human Samples, Specimens or Data ...................................... 22 
 
Use of Stored Samples/Data ....................................................................................... 22 
 
Disposition of Stored Samples/Data ............................................................................ 22 
7 
Study Schedule .............................................................................................................. 22 
 
Informed Consent ....................................................................................................... 23 

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14/15-Year Clinic Visit A ............................................................................................. 23 
 
14/15-Year Clinic Visit B ............................................................................................. 23 
 
16/17-Year Clinic Visit A ............................................................................................. 23 
 
16/17-Year Clinic Visit B ............................................................................................. 23 
 
Quarterly Phone Calls ................................................................................................. 24 
 
Methacholine Challenge Visit ...................................................................................... 24 
 
Home Environmental Exposure Evaluation ................................................................. 24 
 
Additional Visits or Contacts ....................................................................................... 24 
 Re-contact of Subjects after Study Termination .......................................................... 25 
8 
Assessment of Safety ..................................................................................................... 25 
 
Definition of an Adverse Event (AE) ............................................................................ 25 
 
Definition of a Serious Adverse Event (SAE) ............................................................... 25 
 
Death Not Related to Study Procedures ..................................................................... 26 
 
Methods and Timing for Assessing, Recording, Analyzing and Adverse Events ......... 26 
8.4.1 
Methods and Timing for Assessment ................................................................... 26 
8.4.2 
Recording/Documentation.................................................................................... 27 
8.4.3 
Analysis/Management .......................................................................................... 27 
 
Reporting Procedures ................................................................................................. 27 
8.5.1 
Serious Adverse Event Reporting ........................................................................ 27 
8.5.2 
Adverse Event Reporting ..................................................................................... 27 
8.5.3 
Reporting to the IRB ............................................................................................ 28 
8.5.4 
Reporting Pregnancy ........................................................................................... 28 
 
Type and Duration of the Follow-up of Participants after Adverse Events ................... 28 
 
Participant Discontinuation .......................................................................................... 28 
 
Replacement of a Participant Who Discontinues Study Participation .......................... 28 
9 
Clinical Monitoring Structure ........................................................................................... 29 
 
Site Monitoring Plan .................................................................................................... 29 
 
Safety Monitoring ........................................................................................................ 29 
9.2.1 
Medical Monitor Review ....................................................................................... 29 
9.2.2 
DSMB Review ...................................................................................................... 29 
10 
Statistical Considerations ............................................................................................... 29 
 Overview ..................................................................................................................... 29 
 Endpoints .................................................................................................................... 30 
10.2.1 
Primary Endpoints ............................................................................................... 30 
10.2.2 
Secondary Endpoints ........................................................................................... 30 
 Measures to Minimize Bias ......................................................................................... 30 
 Analysis Plan .............................................................................................................. 31 
10.4.1 
Primary Analysis of Primary Objective ................................................................. 31 
10.4.2 
Analysis of Secondary Objectives ........................................................................ 31 
10.4.3 
Analysis of Exploratory Endpoints ........................................................................ 32 
 Sample Size Considerations ....................................................................................... 33 
11 
Quality Control and Quality Assurance ........................................................................... 33 
12 
Ethics/Protection of Human Subjects .............................................................................. 34 
 The Belmont Report .................................................................................................... 34 
 Institutional Review Board ........................................................................................... 34 
 Informed Consent Process .......................................................................................... 34 
 Assent Process ........................................................................................................... 34 

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 Participant Confidentiality............................................................................................ 34 
 Study Discontinuation ................................................................................................. 35 
13 
Data Handling and Record Keeping ............................................................................... 35 
 Data Management Responsibilities ............................................................................. 35 
 Data Capture Methods ................................................................................................ 35 
 Types of Data ............................................................................................................. 36 
 Source Documents and Access to Source Data/Documents ....................................... 36 
 Timing/Reports ........................................................................................................... 36 
 Study Records Retention ............................................................................................ 36 
14 
Publication Policy ........................................................................................................... 36 
Appendix A: Scientific References ............................................................................................ 37 
Appendix B: Schedule of Procedures/Evaluations ..................................................................... 39 
 
 
 

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List of Abbreviations 
 
ACRN 
Asthma Clinical Research Network 
AE 
Adverse Event/Adverse Experience 
ANOVA 
Analysis of Variance 
APIC 
Asthma Phenotypes in the Inner City (trial) 
ATS 
American Thoracic Society 
BIA 
Bioelectrical Impedance Analysis 
BMI 
Body Mass Index 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
CMP 
Clinical Monitoring Plan 
DAIT 
Division of Allergy, Immunology, and Transplantation 
DMP 
Data Management Plan 
DNA 
Deoxyribonucleic Acid 
DSMB 
Data and Safety Monitoring Board 
eCRF 
Electronic Case Report Form 
EDC 
Electronic Data Capture 
eNO 
Exhaled Nitric Oxide 
FDA 
Food and Drug Administration 
FEV1 
Forced Expiratory Volume in one second 
FWA 
Federal Wide Assurance 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
Human Immunodeficiency Virus 
ICAC 
Inner City Asthma Consortium 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
ICU 
Intensive Care Unit 
IgE 
Immunoglobulin E 
IgG 
Immunoglobulin G 
IOS 
Impulse Oscillometry 
IRB 
Institutional Review Board 
LCA 
Latent Class Analyses 
MDI 
Metered-Dose Inhaler 
MOP 
Manual of Operations 
mRNA 
Messenger RNA (ribonucleic acid) 
N 
Number (typically refers to participants) 
NHLBI 
National Heart, Lung, and Blood Institute 
NIAID 
National Institute of Allergy and Infectious Diseases 
NIH 
National Institutes of Health 
PBMC 
Peripheral Blood Mononuclear Cells 
PD20 
Provocative Dose that causes a 20% fall in FEV1 
PI 
[Site] Principal Investigator 
RNA 
Ribonucleic Acid 
SACCC 
Statistical and Clinical Coordinating Center 
SAE 
Serious Adverse Event/Serious Adverse Experience 
 

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Protocol Summary 
 
Full Title: 
Urban Environment and Childhood Asthma 
Short Title: 
URECA 
Conducted by: 
National Institute of Allergy and Infectious Diseases 
Principal Investigator: 
James Gern, MD 
Sample Size: 
N=609 originally enrolled 
Study Population: 
Inner-city children at high risk for development of asthma 
due to family history, plus a small sample of inner-city 
children without family history. 
Accrual Period: 
N/A – Study participants were accrued from 2005-2007 
Study Design: 
URECA is a longitudinal birth cohort study which is being 
extended to follow study children to age 17 years. 
Study Duration: 
Start Date: 1 April 2021  
End Date: 31 December 2024 
Primary Objective: 
To determine the wheezing, asthma and atopy 
phenotypes in minority children growing up in poor urban 
neighborhoods as they develop from birth through 
adolescence. 
Secondary Objectives: 
1. To longitudinally analyze the development of lung 
function and its relationship to asthma. 
2. To identify which urban exposures (allergens, 
pollutants, microbes, viral infections, stress) from early 
life onward affect the development and natural history 
through adolescence of asthma, allergic rhinitis and 
allergic sensitization.  
3. To determine the association of pre-pubertal obesity 
and the effect of pubertal changes in adiposity and sex 
hormones on lung growth and asthma onset, 
prevalence and morbidity. 

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Exploratory Objectives: 
1. To provide samples for analysis and outcomes data 
for use in mechanistic studies to understand 
relationships between environmental exposures, 
changes in immune development, alterations in DNA 
methylation patterns of airway cells, and the 
development of allergic sensitization, allergic rhinitis 
and asthma. 
2. To work collaboratively with investigators outside of 
the NIAID asthma network on mechanistic or clinical 
studies to test novel hypotheses and to validate, 
compare and contrast findings from URECA with those 
in other cohorts or clinical studies. 
Endpoints: 
Incidence and prevalence of asthma and occurrence of 
specific phenotypes of asthma 
 
 
 

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Key Roles 
 
PROTOCOL CHAIR – JAMES GERN, MD 
 
 
 
 
 
 
 
 
NIAID MEDICAL MONITOR – 
 
 
 
 
 
 
 
 
NIAID PROJECT MANAGER – 
 
 
 
 
 
 
 
 
STATISTICIAN – 
 
 
 
 
 
 
 
 
 
 

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The results of these studies will link the pivotal early life exposures to airway epithelial cell 
DNAm and gene expression patterns, and ultimately to clinical expression of allergic diseases 
and asthma. These integrated data can be used to detect at-risk children at an early age, and 
from these pathways, develop and test interventions in early life to prevent allergy and asthma 
in urban children, and thus relieve a lifetime burden of asthma in this high-risk urban children.   
 
Potential Risks and Benefits 
1.3.1 
Risks of Study Procedures 
The URECA study has been designated as an observational birth cohort study by the CAUSE 
consortium and DAIT. As such, this study involves minimal active intervention outside of the 
normal standard of care for the participant. No study drugs are used in the URECA study. All 
risks of the URECA study are limited to study procedures, i.e. blood draws, nasal sample 
collection, allergen skin testing, sputum collection, or pulmonary function tests not included as 
part of standard of care for this group of patients. 
1.3.1.1 Allergy Skin Testing 
Redness, swelling, and itching of the skin are likely to occur during and 1-2 hours after an 
allergen skin test. These symptoms could occur up to one or two days after the skin test. The 
study doctor may provide oral or topical antihistamines to treat these symptoms. There is also a 
very rare chance that the participant may have asthma symptoms or faint during the test. A 
medical provider trained in treating anaphylaxis will be available to provide immediate treatment 
in the event that a participant experiences an allergic reaction. Stopping antihistamines before 
skin testing may make allergy (but not asthma) symptoms worse. Participants will be told they 
can take their medications if they need them, but the test will need to be rescheduled.  
1.3.1.2 Blood Collection 
The risks associated with taking blood include possible pain from the stick, as well as bleeding, 
bruising, and infection of the skin. Lightheadedness and fainting rarely occur during non-fasting 
blood collections, but are more likely during fasting blood collections. To minimize these risks, a 
staff member who is trained to draw blood from children will collect the samples. Additionally, 
investigative sites may apply an analgesic medication such as EMLA® to the skin before the 
blood draw to reduce the pain of the stick. Side effects from this medication include erythema, 
burning, paleness at the skin site, edema, and alterations in temperature. Reactions are mild 
and transient. There is a potential for allergic reactions.  
1.3.1.3 Spirometry 
Spirometry can cause coughing or lightheadedness, which will go away shortly after the test is 
finished. The albuterol that is given during reversibility testing can cause increased heart rate 
and blood pressure, nausea, headache, and a jittery or nervous feeling. These symptoms 
usually resolve in less than an hour.  
Participants will be asked to withhold their asthma medications for a period of time before the 
procedure depending on the medication. Withholding of asthma medications before testing may 
cause a worsening of asthma symptoms.  Participants will be informed that they can take their 
asthma medications if needed, and the procedure will be rescheduled. Exact medications to 

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withhold, length of abstention and procedures are described in the URECA V Manual of 
Operations (MOP) for ICAC-07. 
1.3.1.4 Methacholine Challenge 
The methacholine challenge can cause coughing, chest tightness, shortness of breath, and 
wheezing.  A medication to open the airways (albuterol) will be promptly given to help reverse 
these effects. There is a rare risk that a severe asthma episode may occur.  A study clinician 
experienced in asthma care will be available should such an episode occur. 
Participants will be asked to withhold their asthma medications for a period of time before the 
procedure, depending on the medication. Withholding of asthma medications before testing may 
cause a worsening of asthma symptoms.  Participants will be informed that they can take their 
asthma medications if needed, and the procedure will be rescheduled. Exact medications to 
withhold, length of abstention and procedures are described in the URECA V MOP for ICAC-07. 
1.3.1.5 Sputum Induction 
Sputum induction may result in wheezing, coughing, or chest tightness. Participants will be pre-
medicated with albuterol in order to minimize this risk. This procedure may cause throat 
irritation. Participants may rinse their mouths or gargle with water after the procedure to reduce 
these symptoms. Less commonly, nausea, vomiting, and headache can occur. 
1.3.1.6 Exhaled Nitric Oxide Measurement 
This test may cause dizziness which will go away soon after the test is finished. 
1.3.1.7 Nasal Epithelial Cell Collection 
The risks associated with the nasal cell collection procedure include discomfort or pain, 
transient nosebleed, sneezing, tearing of the eyes, runny nose, and postnasal drip. 
1.3.1.8 Collection of Nasal Lining Fluid 
The collection of nasal lining fluid may produce mild, temporary discomfort such as a tickle nose 
and/or tearing of the eyes.  
1.3.1.9 Questionnaires 
There is a possibility that participants may find the questions too personal.  Participants may 
refuse to answer any questions that make them feel uncomfortable. 
1.3.2 
Potential Benefits 
Those participants with asthma may benefit by receiving frequent asthma assessments and 
care from a study clinician, as well as asthma education including select environmental control 
measures. The participant’s asthma may or may not improve while in this study. 

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2 Study Objectives 
 
Primary Objective 
The primary objective of the continuation of URECA to age 17 is to determine the wheezing, 
asthma and atopy phenotypes in minority children growing up in poor urban neighborhoods as 
they develop from birth through adolescence. 
 
Secondary Objectives 
Secondary objectives are: 
1. To determine whether early life exposure to allergens in the home in the absence of a 
diverse microbiome is associated with sensitization to those allergens and respiratory 
symptoms (perennial rhinitis and allergic asthma phenotypes). 
2. To determine the association of pre-pubertal obesity and the effect of pubertal changes 
in adiposity and sex hormones on lung growth and asthma onset, prevalence and 
morbidity. 
 
Exploratory Objectives 
URECA has several exploratory objectives to use data and samples collected from the study 
participants: 
1. To determine the relationships between DNAm and gene expression in AECs collected 
from URECA children at ages 11 and 15 years, genotypes, and respiratory and allergy 
phenotypes to age 17 years. 
2. To identify which urban exposures (allergens, pollutants, microbes, viral infections, 
stress) from early life onward are associated with AEC DNAm and gene expression 
(ages 11 and 15 years) and respiratory and allergy phenotypes to age 17 years.  
3. To work collaboratively with investigators in CREW, ECHO and other networks on 
mechanistic or clinical studies to test novel hypotheses and to validate, compare and 
contrast findings from URECA with those in other cohorts or clinical studies. 
3 Study Design 
 
Description of the Study Design 
The continuation protocol retains many of the successful features of the ongoing protocol; 
children will continue to be monitored through quarterly phone calls and yearly clinic visits (see 
Appendix B). Interim goals are to maintain a high level of participation in study procedures, and 
retain the current subjects to age 17 years. The quarterly calls serve a dual purpose in providing 
frequent assessments of respiratory symptoms, while also enabling staff to maintain close 
contact with the families to promote retention and adherence with study procedures. We will 
continue to conduct environmental sampling, immunologic profiling, and studies of body 
composition and changes in sex hormones and adipokines during puberty that will enable a 
longitudinal analytic approach.  

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We anticipate that the eventual expression of asthma will continue to evolve between ages 14 
and 17 years, and thus, this stage of URECA will collect data on not only the development and 
resolution of asthma but also asthma morbidity and changes in asthma severity. Finally, in 
school-aged children, we will be able to identify risk factors for the onset of allergic rhinitis and 
the transition in some children from allergic rhinitis to allergic asthma.    
 
Study Endpoint 
3.2.1 
Primary Endpoint 
The primary outcome of URECA through age 7 was the development of asthma, which was 
determined using an algorithm that evaluated reported physician diagnosis, lung function, 
symptoms, healthcare utilization and medication use. At age 7, 29% of the URECA study 
participants met this definition for asthma.4 We also identified five specific phenotypes based on 
longitudinal trajectories of symptoms, allergen sensitization, and lung function using data 
through age 7 years.5 These trajectories were refined at age 10 years, where an additional 
phenotype with high symptoms, high sensitization, and significant lung function deficits was 
found (Altman M et al, under review).  
Data regarding asthma diagnosis, the frequency and severity of cough and wheezing illnesses, 
healthcare utilization, and medication use will continue to be collected during each of the 
quarterly telephone calls. Examinations will also be conducted as part of the scheduled study 
visits to age 17 years, and questions about wheezing and asthma will be asked at these visits 
as well. All of this information will be recorded into a centralized database for analysis.  
3.2.2 
Secondary Endpoints 
A number of secondary outcome variables will also be assessed as follows: 
1. Methacholine responsiveness – this will be ascertained at the 14/15-year time point.  
2. Pulmonary function – pre- and post-bronchodilator spirometry and impulse oscillometry 
will be measured at age 14/15 and again at age 16/17.   
3. Allergic sensitization – defined as a dichotomous variable (aeroallergen-specific IgE by 
skin test or serum test) or as a continuum (number of positive skin or sIgE tests, sum of 
sIgE values). 
4. Allergic rhinitis – chronic seasonal or perennial rhinitis and corresponding allergen-
specific IgE or skin test.  
4 Study Population 
 
Population Description 
To address the primary objectives of the URECA V protocol, all the participants active in the 
URECA IV protocol in early 2021 will be invited to continue under the new protocol into age 17 
years.  
The URECA study is being conducted at the Johns Hopkins University School of Medicine, 
Baltimore, MD; Boston University School of Medicine, Boston, MA; Children’s Hospital of New 
York-Presbyterian, New York, NY; and Washington University School of Medicine, St. Louis, 

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MO. At the time of enrollment, study participants had to live in the inner city, defined for this 
study as specific contiguous neighborhoods where at least 20% of the population is below the 
federal poverty level. The resulting study populations in Baltimore and St. Louis are 
predominantly African-American. The populations in Boston and New York are more evenly 
divided between African-American and Hispanic (Table 2). 
Table 2.  URECA Study Population by Ethnicity and Race of Child and Site 
 
Hispanic 
African-American 
Mixed Race 
White, Asian, 
Other 
Baltimore 
2% 
90% 
4% 
4% 
Boston 
31% 
54% 
11% 
4% 
New York 
61% 
36% 
3% 
0% 
St. Louis 
2% 
88% 
8% 
1% 
Total 
21% 
70% 
7% 
3% 
 
The URECA study enrolled 560 inner-city children at high risk for developing allergic diseases 
and asthma on the basis of a parental history of asthma, allergic rhinitis or atopic dermatitis. A 
small comparison sample of 49 babies without allergic family history was also enrolled. Babies 
born at 34 weeks gestation or later were allowed into the study, provided that there was no 
significant respiratory distress as defined by the exclusion criteria, and they were otherwise 
healthy. Infants with other lung diseases such as respiratory distress syndrome, 
bronchopulmonary dysplasia and pneumonias were not enrolled due to confounding effects on 
respiratory symptoms and lung function. Babies of HIV-infected mothers were excluded due to 
immunomodulatory effects of HIV infection, and of anti-retroviral medications such as 
zidovudine (AZT). 
 
Participant Inclusion Criteria 
Participants to be included in URECA V are those who are active in the URECA IV protocol at 
approximately age 14-16. 
5 Study Procedures/Evaluations 
 
Clinical Evaluations 
A number of procedures are currently on hold due to the COVID pandemic, and we will maintain 
flexibility in scheduling based on when research restrictions related to the pandemic are lifted. 
For example, if any of the scheduled assessments do not occur at a visit, that assessment can 
be completed at the next visit after restrictions are lifted. The study assessments and 
techniques employed in the original URECA protocol will continue to be followed as described 
below. 
5.1.1 
Dust Analysis for Allergen and Microbial Products 
Dust specimens will be collected at the age 16/17-year time point following procedures 
described in the URECA V MOP for ICAC-07. One combined dust sample will be collected from 
the participant’s bed (defined as the location where the participant sleeps the most) and 

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bedroom floor. Handling of specimens and laboratory assays will remain the same as in the 
original protocol. 
5.1.2 
Allergen Skin Testing 
Skin testing to a panel of aeroallergens will be performed at the 14/15-year time point.  This will 
include a panel of well-characterized antigens that have been associated with respiratory 
allergies and asthma in this age group. Only personnel that are trained in the procedures 
established by the URECA V MOP for ICAC-07 will perform this test. Skin testing will be done 
by the prick technique using the GreerPick system (Greer; Lenoir, NC) in accordance with 
generally accepted guidelines.6 Tests will be read after 15 minutes by measuring the wheal for 
each antigen and for the controls. Participants will be asked to stop taking antihistamines for a 
period of time specified in the URECA V MOP prior to the test to limit interference with the 
results of the skin test. 
5.1.3 
Pre/Post Bronchodilator Pulmonary Function Testing 
The 14/15- and 16/17-year time points will each include one pre/post bronchodilator testing 
session. After completion of spirometry as described in the main protocol, albuterol via MDI with 
spacer will be administered. Fifteen minutes later the spirometry and IOS will be repeated. 
5.1.4 
Methacholine Challenge 
Participants will undergo methacholine challenge testing one time. Airway responsiveness will 
be measured by assessing the dose of methacholine required to produce a drop in FEV1 of 20% 
(PD20) after the administration of increasing concentrations of methacholine using the small 
volume nebulizer-tidal breathing technique. A trained and certified pulmonary function 
technician will perform the test based on the procedures outlined in the URECA V MOP for 
ICAC-07. These procedures will be used due to the documented safety of the approach in large 
pediatric asthma populations. Provocholine® will be used as the commercial source of 
methacholine, since it is an FDA approved product for children as young as 5 years of age. 
Testing criteria are described in the MOP for ICAC-07 that ensure the quality of the data 
collected and the safety of the study participants.  
5.1.5 
Exhaled Nitric Oxide Measurement 
Measurement of eNO will be obtained at two visits, once in the 14/15-year time point and once 
in the 16/17-year time point. The procedure will be conducted prior to the measurement of 
spirometry. Exhaled NO will be measured employing a technique modified after Silkoff et al.7 
and following American Thoracic Society (ATS) guidelines for eNO assessment.8 Nitric oxide 
concentrations will be measured using a commercially-available analyzer and a procedure 
described in detail in the URECA V MOP for ICAC-07. 
5.1.6 
Induced Sputum 
Sputum will be induced at the age 16/17-year time point by inhalation of hypertonic saline 
solution using the method that was used in the Asthma Clinical Research Network (ACRN).9 
Safety monitoring per spirometry and symptom report will be performed during and after sputum 
induction. Processing will be performed according to the URECA V MOP for ICAC-07. Slides 
will be read at a central site for cellular determinations. Residual sputum cells will be processed 

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to analyze cell markers and RNA and DNA isolation for expression and epigenetic studies. 
Sputum supernatants will be collected, aliquoted, and stored pending further analysis.  
5.1.7 
Nasal Epithelial Cell Collection 
Nasal epithelial cell samples will be obtained by brushing the nasal mucosa at the age 14/15-
year time point. A trained clinician will conduct the procedure. The inferior turbinate of one nasal 
passage will be sampled to obtain an adequate number of epithelial cells for mRNA and DNA 
isolation. The sampled area will be observed for hemostasis. Procedural details and instructions 
for processing the samples are included in the URECA V MOP for ICAC-07. 
5.1.8 
Collection of Nasal Lining Fluid 
Nasal lining fluid will be collected at the age 14/15-year time point, by placing absorbent filter 
paper just inside the nasal cavity, allowing for passive collection of secretions. This sample will 
be obtained for future measurements of metabolites, proteins, and possibly microbiome. 
5.1.9 
Fitness Testing 
The Six-Minute Walk Test will be used at the 14/15-year time point to assess the participant’s 
level of fitness.10 For this test the participant is asked to walk around two cones placed in a 
hallway 60 meters apart at a fast pace for six minutes. The number of meters walked is 
measured, as well as the participant’s pulse and respiratory rate. The participant is asked to rate 
his or her level of fatigue on a visual analogue scale. 
 
Laboratory Evaluations 
5.2.1 
Clinical and Research Laboratory Evaluations and Specimen 
Collection 
5.2.1.1 Urine Pregnancy Testing 
All females who have reached menarche will be required to have a urine pregnancy test prior to 
any procedures that entail any risk, but not prior to a blood draw. Results of all pregnancy tests 
will be given to the participant and/or caretaker following state laws.   
Pregnancy will not result in withdrawal from the study, but will be reported on the appropriate 
CRFs and followed to outcome. Pregnant participants may not participate in some procedures; 
refer to the URECA V MOP for ICAC-07. If the pregnancy results in anything other than a 
normal birth or elective abortion of a healthy fetus, it will be reported as a SAE. 
5.2.1.2 Urine Collection 
Urine will be collected at the 14/15, and 16/17-year time points for measurement of urinary 
cotinine, a marker for tobacco smoke exposure. A urine sample will be collected following 
procedures described in the URECA V MOP for ICAC-07. 
5.2.1.3 Blood Collection 
Blood will be collected while participants are active in URECA V to measure serum markers of 
adiposity and systemic inflammation (e.g., leptin, adiponectin, hsCRP, IL-6, TNF-α), levels of 

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sex hormones (e.g. estrogen, testosterone), and insulin resistance (e.g. fasting insulin and 
glucose), as well as total and allergen-specific IgE.  Blood is also used for analysis of 
immunoregulatory mRNA and protein responses as a measurement of individual and 
developmental immunologic patterns. Plasma and serum proteins will also be analyzed, and 
these assays will be performed in a multiplex format whenever possible. In addition, systems 
approaches (e.g. microarray, proteomics, epigenetics) will also be used, as these technologies 
are developed to assess global patterns in immunoregulatory factors in the blood or in blood 
cells. Additional analysis may be done for potential biomarkers related to asthma and allergies. 
The amount of blood drawn will follow the NIH Guidelines for Limits of Blood Drawn for 
Research Purposes in the Clinical Center (5/2012). For pediatric patients, no more than 5 mL/kg 
may be drawn for research purposes in a single day, and no more than 9.5 mL/kg may be 
drawn over any eight-week period. For adults the amount shall not exceed 10.5 mL/kg or 550 
mL, whichever is smaller, over any eight-week period. 
5.2.1.4 Nasal Fluid 
Nasal fluids will be analyzed for proteins (e.g. cytokines) or metabolites using methods as listed 
above. 
5.2.1.5 Immunologic Studies 
The overall goal of the immunologic studies is to define effects of environmental stimuli on 
immune development, and then identify patterns of immune development that are associated 
with the development of asthma. These studies will be conducted using cells and fluids from 
blood, the upper airway, and possibly urine. 
5.2.2 
Specimen Preparation, Handling and Shipping 
5.2.2.1 Instructions for Specimen Storage 
Most samples that are collected at the clinical sites will be shipped directly to processing 
laboratories. However, samples that will need to be stored for at least 3 months (specimens of 
DNA, plasma, etc.), as well as specimens that are generated by laboratories that are awaiting 
further analysis, will be sent to a central storage facility pending analysis. This facility will 
maintain a computerized inventory of all of the specimens to be analyzed, and will ship the 
specimens to designated laboratories for analysis upon request.  In order to protect subjects, all 
samples are stored and shipped under code so that neither the participant nor their caregivers 
can be identified individually. 
5.2.2.2 Specimen Shipment 
Instructions for sample preparation, handling, storage, and shipping are included in the URECA 
V MOP for ICAC-07.  Principal Investigators will be responsible for knowing about and 
observing all the regulations for classification, packaging and labeling, permits or authorizations, 
and personnel training for shipment of biological and hazardous materials required for the 
conduct of this study. 

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Substudies 
URECA also provides an outstanding opportunity to conduct studies of mechanisms related to 
the onset, progression and prevention of allergic rhinitis and asthma, and we have designed 
extensive collaborative studies with research groups identified by the CAUSE Leadership 
Center, including: 
1. 
Studies to define mechanisms of microbial effects on the risk of allergic diseases and 
asthma (collaboration with the University of California San Francisco),  
2. 
A systems-based analysis to integrate environmental exposures, epigenetic changes 
via methylation, gene transcription, immune development and asthma/allergy clinical 
outcomes and biomarkers (collaboration with the Benaroya Research Institute, 
Seattle, WA and the University of Chicago), and 
3. 
Analysis of epitope recognition and functional characteristics of allergen-specific T 
cells, and relationship to allergen exposure, sensitization, and expression of clinical 
disease (collaboration with the La Jolla Institute of Allergy and Immunology). 
Each substudy or ancillary study involving URECA participants or samples from participants will 
be accompanied by a proposal specifying the objectives of the substudy, the sample 
requirements, and the analytic plan. If additional samples or procedures not included in the main 
study consent are required for the substudy, an informed consent addendum will need to be 
signed by the participant’s caretaker/guardian, and the participant will sign an assent form. 
6 Research Use of Stored Human Samples, Specimens or 
Data 
 
Use of Stored Samples/Data 
Extra research samples remaining once the specified analyses are conducted will be stored 
long-term for future research. Participants will be asked to give permission for long-term storage 
and future use during the consent process. 
 
Disposition of Stored Samples/Data 
Stored samples will be maintained in long-term storage until they are used or until permission is 
obtained from the CAUSE Steering Committee and DAIT/NIH to destroy them. 
7 Study Schedule 
The COVID pandemic has caused unpredictable delays in the visit schedule. To maximize 
participation and facilitate the collection of complete data sets in URECA V, we have designed 
the protocol with increased flexibility in scheduling. This will include two visits in the 14/15-year 
time frame, two visits in the 16/17-year time frame, and one standalone visit for a subset of 
participants. Many procedures that cannot be completed – due to COVID-related restrictions, for 
example – at their intended visits may be completed at the subsequent visit (see Appendix B).  
 

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Informed Consent 
Once the URECA V protocol is approved, informed consent to continue participation will be 
obtained by the URECA staff at the earliest time that the participant’s parent/guardian is seen in 
person. If the family has not been seen in person and the participant reaches 16 years of age, a 
telephone consenting procedure will be used to continue with quarterly phone calls, according to 
IRB requirements. In these cases, written consent will be obtained at the next clinic visit, before 
any study activity. We will also obtain written assent from participating children according to 
local IRB guidelines before enrollment into URECA V.  
 
14/15-Year Clinic Visit A 
At the 14/15-year Visit A, questionnaires about the participant’s physical health, habits, and 
environment will be administered. Questionnaires pertaining to stress and mental health will 
also be administered. A brief physical examination will be conducted, to focus on ears, nose, 
throat, chest and skin, and including Tanner staging. Urine and blood samples will be collected, 
supplies and instructions for home dust collection will be distributed, and exhaled nitric oxide will 
be measured. IOS and spirometry will be performed. Allergen skin testing to a panel of common 
indoor and outdoor allergens will be performed. Methacholine challenge is planned, with post-
bronchodilator spirometry and IOS reversibility being done only when participants do not meet 
the criteria for methacholine challenge. 
 
14/15-Year Clinic Visit B 
At the 14/15-year Visit B, questionnaires about the participant’s physical health, habits, and 
environment will be administered. A brief physical examination will be conducted, to focus on 
ears, nose, throat, chest and skin. Urine samples will be collected for post-menarcheal female 
participants. BIA will be performed to assess adiposity. In addition, the Six-Minute Walk Test will 
be performed to assess fitness. Nasal lining fluid will be collected in addition to nasal epithelial 
cells. IOS and spirometry will be performed. Participants who were unable to conduct the 
methacholine challenge at Visit A will attempt to conduct the procedure at this visit, again with 
post-bronchodilator spirometry and IOS reversibility done if the participant does not meet the 
criteria for the challenge. If the methacholine challenge is not conducted at either Visit A or Visit 
B during this time point, it will be conducted at a separate visit to occur any time after the 14/15-
year Visit B.  
 
16/17-Year Clinic Visit A 
At the 16/17-year Visit A, questionnaires about the participant’s physical health, habits, and 
environment will be administered. Questionnaires pertaining to stress and mental health will 
also be administered. A brief physical examination will be conducted, to focus on ears, nose, 
throat, chest and skin. Urine and blood samples will be collected. Spirometry and sputum 
induction will be performed. Supplies and instructions for the home dust collection will be 
provided to the families.  
 
16/17-Year Clinic Visit B 
At the 16/17-year Visit B, questionnaires about the participant’s physical health, habits, and 
environment will be administered. A brief physical examination will be conducted, to focus on 

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ears, nose, throat, chest and skin. Urine samples will be collected for post-menarcheal female 
participants. BIA will be performed to assess adiposity. Spirometry and impulse oscillation 
spirometry with reversibility will be performed. Exhaled nitric oxide will be measured.  
 
Quarterly Phone Calls 
Phone calls will be placed to the family every 3 months between clinic visits to collect data 
related to study outcomes, and also to continue to engage the family in the study and provide 
informational updates. If the final 16/17-year visit (Visit B) has occurred, but the child has not yet 
reached the age of 17 years, quarterly calls may continue until the 17th year. Questionnaires to 
be administered will collect data on respiratory symptoms, environmental exposures and diet, 
health care utilization, and medication use. These phone calls will continue many questionnaires 
from the original URECA study, in addition to new questions appropriate for the older age group. 
 
Methacholine Challenge Visit 
Participants who are unable to complete the methacholine challenge at their 14/15-year Visit A 
or Visit B will be asked to come to clinic for a separate visit. During this visit, questionnaires will 
be administered to assess for changes to the participant’s health and medications. The full set 
of questionnaires for annual clinic visits is not expected, although some may be administered if 
previously missed. IOS and Spirometry will be performed, followed by methacholine challenge. 
If the participant does not meet criteria to perform the challenge, post-bronchodilator spirometry 
and IOS with reversibility may be completed, if not previously conducted at the 14/15-year time 
point. 
 
Home Environmental Exposure Evaluation 
At the 16/17-year Visit A, the family will be given a dust collection kit, which includes instructions 
on how to collect a dust sample (refer to the URECA V Dust Collection Instructions Handout). A 
combined dust sample from the participant's bedroom floor and the participant's bed will be 
collected. The room where the participant sleeps most nights will be considered the bedroom. 
Measuring templates will be used to delineate the areas to be vacuumed. Dust will be collected 
using a vacuum cleaner with a special dust collection filter attached. The dust collector will be 
placed into a sealable plastic bag and mailed back to the study center for temporary storage 
(frozen). Crude samples will be batched and shipped to a central laboratory by express mail for 
sieving, extraction, and analysis. The dust specimens will be assayed to measure the 
concentration allergens such as: Der p 1, Der f 1, Bla g 1, Bla g 2, Fel d 1, Can f 1, Alt a 1, and 
Mus m 1. Additional allergens of interest and markers of fungal and microbial exposure may be 
measured.  In addition, the caretaker or participant will complete a dust collection questionnaire, 
which will be mailed back with the dust collector.  
When problems arise with the family doing the environmental sample collections, staff may go 
to the home to collect the dust samples. 
 
Additional Visits or Contacts 
Additional visits or telephone contacts may be made for any of the following reasons: 
1. 
A visit was conducted but 1 or more planned procedures were not completed, or a 
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2. 
Contact information needs to be updated (no more often than every 6 months). 
Any of the assessments listed above may be conducted at additional visits or by telephone (as 
appropriate)—either for the first time or for reassessment. 
During the consent process, the participants will be asked to consent or assent to the possibility 
of additional visits or contacts as described in this section.  No participant will be subjected to an 
assessment for which he or she has not provided written consent or assent, and no informed 
consent/assent form will contain any information that has not been approved by the IRB of 
record. 
 Re-contact of Subjects after Study Termination 
The caretaker/guardian of the participant is asked during the informed consent process to 
indicate that contact for future research studies is permissible. 
8 Assessment of Safety 
 
Definition of an Adverse Event (AE) 
The URECA study has been designated as an observational birth cohort study by the ICAC and 
DAIT. As such, this study involves minimal active intervention outside of the normal standard of 
care for the participant. No study drugs are used in the URECA study.  Adverse events are 
limited to any occurrence or worsening of an undesirable or unintended sign (including an 
abnormal laboratory finding), symptom, or disease in a study subject that is specifically 
associated (probably, possibly, or definitely – defined in section 8.4.1.2) with a study procedure 
that is not part of the normal standard of care for the participant. In the URECA study, adverse 
events are those related to the blood draws, nasal sample collection, allergen skin testing, 
sputum collection, or pulmonary function tests not included as part of standard of care for this 
group of patients. 
 
Definition of a Serious Adverse Event (SAE) 
A Serious Adverse Event (SAE) is defined as an AE resulting in one of the following outcomes: 
 
Death during the period of protocol-defined surveillance 
 
Life Threatening Event (defined as an event that places a participant at immediate risk of 
death) 
 
Inpatient hospitalization or prolongation of existing hospitalization during the period of 
protocol-defined surveillance  
 
Congenital anomaly or birth defect 
 
Persistent or significant disability/incapacity 
Any other condition which, in the judgment of the investigator, represents a significant hazard, 
such as an important medical event that does not result in one of the above outcomes, may be 
considered a serious adverse event when the event jeopardizes the participant or requires 
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Death Not Related to Study Procedures 
A death occurring in a study participant not associated with blood draws, nasal sample 
collection, allergen skin testing, sputum collection, or pulmonary function tests will be reported 
as a serious adverse event not related to study procedures. The reporting process will follow the 
SAE reporting process. 
 
Methods and Timing for Assessing, Recording, Analyzing 
and Adverse Events 
8.4.1 
Methods and Timing for Assessment 
8.4.1.1 Grading of Adverse Events/Serious Adverse Events 
Each adverse event will be assessed for severity and classified into one the categories below: 
 
Grade 1 (Mild): Event requires minimal or no treatment and do not interfere with the 
participant’s daily activities. 
 
Grade 2 (Moderate): Event results in a low level of inconvenience or concern with the 
therapeutic measures.  Moderate events may cause some interference with functioning. 
 
Grade 3 (Severe): Event interrupts a subject’s usual daily activity or functioning and 
may require systemic drug therapy or other treatment.  Severe events are usually 
incapacitating. 
 
Grade 4 (Life threatening):  Any adverse drug experience that places the participant, in 
the view of the investigator, at immediate risk of death from the event as it occurred. 
 
Grade 5 (Death) 
8.4.1.2 Attribution of Adverse Event to Study Procedure 
For the purpose of this study, only AEs related to a study procedure (Section 8.1) will be 
reportable and by definition will always be assessed as related. The degree of certainty about 
relatedness will be graded using the two categories below. 
 
Definitely Related: There is clear evidence to suggest a causal relationship, and other 
possible contributing factors can be ruled out. The clinical event, including an abnormal 
laboratory test result, occurs within a reasonable timeframe after study procedure(s) and 
cannot be explained by concurrent disease or other drugs or chemicals.  
 
Possibly Related: There is some evidence to suggest a causal relationship (e.g., the 
event occurred within a reasonable time after study procedure(s)).  However, the 
influence of other factors may have contributed to the event (e.g., the subject’s clinical 
condition, other concomitant events). Although an adverse event may be judged only as 
“possibly related” soon after discovery, it can be flagged as requiring more information 
and later be upgraded to “definitely related”. 

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8.4.2 
Recording/Documentation 
Adverse events and serious adverse events related to the study procedures will be recorded on 
an appropriate eCRF. The event, the procedure to which it is related, start date, stop date, and 
severity of each reportable event will be recorded on the eCRF. 
8.4.3 
Analysis/Management 
The rate of AEs has historically been low in this study; therefore, the primary analysis of AEs will 
occur by the medical monitor as described in Section 8.5.  Additional management is not 
expected to be necessary. 
 
Reporting Procedures 
All adverse events related to the blood draws, nasal sample collection, allergen skin testing, 
sputum collection, or pulmonary function tests occurring during the study will be reported to the 
SACCC. The SACCC reports to the NIAID Medical Monitor who is responsible for passing the 
information on to the NIAID Data Safety and Monitoring Board (DSMB). Adverse events will be 
followed until resolved or considered stable. 
8.5.1 
Serious Adverse Event Reporting 
Adverse event reporting requirements to the NIAID DSMB for this protocol are as follows: 
 
Investigators will submit a completed serious adverse event report to the NIAID DSMB 
within 7 days after becoming aware of a subject death, a potentially life-threatening 
(Grade 4) serious adverse event that is possibly or definitely related to study 
procedure(s), an urgent inpatient hospitalization or transfer to the ICU. 
 
Investigators will submit a completed serious adverse event report to the NIAID DSMB 
within 15 days after becoming aware of any Grade 3 (severe) adverse event that is 
possibly or definitely related to study procedure(s), or an inpatient hospitalization (other 
than elective), a persistent or significant disability/incapacity, or a congenital 
anomaly/birth defect. 
 
Investigators will report within 15 days on any other event or condition regardless of 
grade, which in their judgment represents an event reportable to the NIAID DSMB. 
 
Investigators will forward all safety reports and related communications to the NIAID 
DSMB within 15 days of receipt. 
 
A summary of all adverse events will be reported to the NIAID DSMB with a continuing 
review submission. 
8.5.2 
Adverse Event Reporting 
Adverse events which are not SAEs or Grade 2 or higher unexpected AEs, will be recorded on 
the appropriate case report form and sent to the SACCC for incorporation into annual reporting 
to the NIAID and DSMB.  

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8.5.3 
Reporting to the IRB 
The Principal Investigator (or delegate) must report adverse events and serious adverse events 
to the central IRB promptly in accordance with local regulations or policies, in addition to 
providing the information to the SACCC. 
8.5.4 
Reporting Pregnancy 
A pregnancy will not be reported as an adverse event for follow-up purposes.  All pregnancies 
that are identified during the study will be followed to conclusion and the outcome of each will be 
reported. If the pregnancy results in anything other than a normal birth or elective abortion of a 
healthy fetus, it will be reported as a serious adverse event. 
 
Type and Duration of the Follow-up of Participants after 
Adverse Events 
The site investigator must apply his or her clinical judgment as to whether an AE is of sufficient 
severity to require that the participant immediately be removed from the study. The investigator 
must institute any necessary medical therapy to protect a participant from any immediate 
dangers. 
An AE will be followed until any of the following takes place:  a) it is resolved, b) participant is 
stable, or c) a minimum of 30 days after participant is discontinued from the study, whichever 
comes first. 
 
Participant Discontinuation 
Participants may choose to withdraw from the study at any time, during a study visit, or 
afterwards in person, by telephone, or in writing. 
Participants may be prematurely terminated from the study for the following reasons: 
1. 
The participant elects to withdraw consent from all future study activities. 
2. 
The participant is “lost to follow-up” (i.e., no further follow-up is possible because 
attempts to re-establish contact with the participant have failed). 
3. 
The investigator no longer believes participation is in the best interest of the 
participant. 
Participants will be notified, if possible, if they are discontinued from the study.  No additional 
follow-up will be required. 
 
Replacement of a Participant Who Discontinues Study 
Participation 
URECA study participants will not be replaced after discontinuation. However, “lost” study 
participants who are subsequently relocated and who provide informed consent, may be re-
activated. 

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9 Clinical Monitoring Structure 
 
Site Monitoring Plan 
Clinical site monitoring will be conducted according to the URECA Clinical Monitoring Plan 
(CMP) for ICAC-07 to ensure that human subject protection, study procedures, and laboratory 
and data collection processes are of high quality and meet sponsor, International Conference on 
Harmonisation, Good Clinical Practice, and regulatory guidelines.  Representatives from the 
National Institute of Allergy and Infectious Diseases (NIAID) and/or the SACCC will visit each 
clinical site or meet with each clinical site via telephone during a specified timeframe according 
to the URECA CMP for ICAC-07. Key study personnel must be available to assist the visitors 
during these visits or attend the call if completed via telephone.  Additional details regarding 
clinical site monitoring, including remote monitoring, are outlined in the URECA CMP and MOP 
for ICAC-07. 
 
Safety Monitoring 
Safety monitoring will be performed following the URECA Safety Management Plan (SMP). 
9.2.1 
Medical Monitor Review 
The DAIT Medical Monitor will review all serious adverse events and Grade 2 or higher 
unexpected adverse events immediately upon notification by the SACCC. The Medical Monitor 
will review reports prepared by the SACCC of all adverse events at least quarterly. 
9.2.2 
DSMB Review 
The DSMB will review any events as requested by the Investigators, SACCC, or DAIT Medical 
Monitor. They will review a listing of all adverse events once per year. Further, the DSMB will be 
informed of expedited SAEs at the same time as the central IRB. 
10 Statistical Considerations 
 Overview 
The URECA continuation into age 17 years has the following specific aims that will be 
accomplished under this protocol. Mechanistic and substudy aims will be addressed in separate 
analytic plans.  
1. To determine the wheezing, asthma and atopy phenotypes in minority children growing 
up in poor urban neighborhoods as they develop from birth through adolescence.  
2. To longitudinally analyze the development of lung function and its relationship to asthma. 
3. To identify which urban exposures (allergens, pollutants, microbes, viral infections, 
stress) from early life onward affect the development and natural history through 
adolescence of asthma, allergic rhinitis and allergic sensitization.  
4. To determine the association of pre-pubertal obesity and the effect of pubertal changes 
in adiposity and sex hormones on lung growth and asthma onset, prevalence and 
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 Endpoints 
10.2.1 Primary Endpoints 
The primary endpoints for this phase of the URECA study are wheezing, asthma and atopy 
phenotypes in minority children. 
10.2.2 Secondary Endpoints 
Secondary endpoints include: 
1. 
Methacholine responsiveness – this will be ascertained at age 14/15.  
2. 
Pulmonary function – pre- and post- bronchodilator spirometry and impulse 
oscillometry will be measured once at age 14/15, and once at age 16/17.   
3. 
Allergic sensitization – defined as a dichotomous variable (aeroallergen-specific IgE 
by skin test or serum test) or as a continuum (number of positive skin or sIgE tests, 
sum of sIgE values). 
4. 
Allergic rhinitis – chronic seasonal or perennial rhinitis and corresponding allergen-
specific IgE or skin test.  
 Measures to Minimize Bias 
The primary threat to validity in a cohort study such as URECA is missing data due to loss-to-
follow-up or missed visits and procedures. The URECA study to date has done a remarkable job 
of retaining the participants, with 73% of the original cohort still active at age 14/15. In recent 
phases of URECA, >85% of expected visits have been completed.  
Missing data create problems because fewer observations will reduce the power of the study. If 
the data are nonrandomly missing, i.e. associated with either the predictor or the outcome, 
estimates generated in the analyses may be biased, possibly leading to invalid inferences. For 
example, children with asthma symptoms might miss their spirometry measurement. This can 
lead to nonrandomly missing data and can complicate analyses.  
The pattern of missing data will be explored for every analysis. Missing data can generally be 
characterized as being in 1 of 3 classes--missing completely at random, missing at random, and 
not missing at random (also called nonignorably missing or informatively missing).11-13 If data 
are missing completely at random or missing at random, we will select the most appropriate 
available statistical method, typically a linear or generalized linear model or survival model that 
addresses the specific question. If the data are informatively missing, selection of the 
appropriate technique will require further consideration. Currently the statistical community has 
not reached a consensus on how to best address the issue of informatively missing data, but 
many approaches have been proposed.12-18 Analyses conducted by Rho statisticians for multiple 
projects suggest in many cases the best approach is to use a linear mixed model even in the 
face of informatively missing data.19 URECA analyses to date have shown most missing data to 
be the result of missed visits or sample analysis problems, and not informatively missing. 
 

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 Analysis Plan 
10.4.1 Primary Analysis of Primary Objective 
The purpose of the primary analysis in the fourth phase of URECA will be to identify wheezing 
and asthma phenotypes in the children through the age of 17 years. Furthermore, we will 
identify risk factors for the asthma phenotypes related to environmental exposures, immune 
responses and biomarkers, weight and fitness status, and airway and environmental microbiota.  
Using the extensively and longitudinally characterized URECA cohort, we will: 
1. 
Categorize URECA subjects into already described early life patterns of wheezing 
(Tucson Children’s Respiratory Study, Avon Longitudinal Study of Parents and 
Children, Prevention and Incidence of Asthma and Mite Allergy) and development of 
atopic disorders (Manchester) to determine if these patterns are indeed observed 
among inner-city American children. 
2. 
Using longitudinal data obtained from the URECA cohort through the age of 17 
years, we will perform a cluster analysis to identify patterns of wheezing, allergic 
sensitization and lung function over time.  In URECA the comprehensive longitudinal 
measurements within a child allow the application of techniques such as latent class 
analysis where periods have the greatest impact on the development and 
maintenance of asthma through the teen years. In particular, symptoms, 
exacerbations, allergy, immunologic measures, BMI, fitness, psychosocial factors 
and objective measurements of lung function, spirometry, eNO and IOS will define 
clusters of asthma phenotypes based on a combination of these variables.  We will 
consider latent class analysis and other clustering techniques to identify phenotypes 
in URECA V. 
10.4.2 Analysis of Secondary Objectives 
10.4.2.1 Early life exposure to household allergens 
To determine whether early life exposure to allergens in the home in the absence of a diverse 
microbiome is associated with sensitization to those allergens and respiratory symptoms 
(perennial rhinitis and allergic asthma phenotypes), we will examine the association between 
early-life exposures to allergens and diversity in the microbiome towards allergic sensitization. 
The assumption is that children with high levels of exposure and low microbiome diversity will 
have a higher likelihood of early sensitizations. Early sensitizations will be obtained by latent 
class mixed model analysis combining both skin tests and specific IgEs to aeroallergens. Then 
a multinomial logit model, with possible adjustments for race and gender, will test the 
(multiplicative & additive) interaction between these two predictors and the likelihood of early 
sensitization. Furthermore, these prolonged exposures can have a compound effect in 
sensitization, and summary measures such as cumulative exposures may not fully capture 
these relationships. We proposed to extend the model suggested above by using a distributed 
lag linear (and non-linear) model with flexible exposure-response and lag-response functions to 
examine the association between sensitization and exposures in early life. 
Pathway analysis will be used to determine how a host of environmental factors contribute to 
the development of rhinitis with specific classifications. Based on medical evidence in the 

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published literature we will develop and test a conceptual model to describe how different risk 
factors domains (i.e. allergic inflammation, pulmonary physiology, environmental exposures, 
etc.) are linked to rhinitis. A casual network analysis will be implemented using structural 
equations models to simultaneously perform (1) a confirmatory factor analysis in each domain to 
estimate the relationship between the domain of interest and each observed variable used to 
measure the domain and (2) a series of regression models to estimate the standardized direct 
effect of each independent domain onto rhinitis. 
10.4.2.2 Effect of pubertal changes in adiposity and sex hormones 
on lung growth and asthma onset, prevalence and 
morbidity 
Levels of steroid hormones, steroid binding proteins, leptin, adiponectin and serum inflammatory 
indicators (IL-6, TNF-α, CRP) at 8, 10, 12, 14/15, and 16/17 years of age will be compared to 
asthma status by ANOVA. These analyses will also be stratified by sex, and we will test whether 
sex hormones and sex hormone binding proteins modify the relationship between leptin and 
asthma prevalence in post-pubertal girls and boys. Body habitus and measurements of fitness 
(6-minute walk in clinic) will also be compared among children with vs. without asthma. We 
expect that children at greatest risk for asthma will be those with low fitness and activity, high 
BMI, increased serum inflammatory markers, and atopy. 
We will use trajectory analysis to examine whether changes in weight or BMI are related to 
asthma prevalence in children from age 10 to 17 years. We will use semiparametric mixture 
modeling—which combines latent growth curve and mixture modeling—to identify growth 
trajectories over time separately for boys and girls. Trajectory parameters will be estimated 
using the maximum likelihood approach built upon a binary logit model, using the BIC (Bayesian 
information criterion) to identify the best model. The objective of model selection is to 
summarize the distinctive features as parsimoniously as possible. The resulting growth 
trajectories will be used as predictor variables in a logistic regression model predicting 
development of asthma. The model will be tested for effect modification by sex. 
10.4.3 Analysis of Exploratory Endpoints 
The overall approach to identify phenotypes will be similar to that used with the 7-year and 10-
year data sets, using latent class mixed models to identify homogenous subpopulations within 
sets of longitudinal variable trajectories. Variables characterizing the trajectory clusters (e.g. 
symptoms, allergic sensitization, lung function and BMI) and other relevant predictors (including 
demographics) will be used to represent subjects in a Gower distance matrix, which can handle 
mixed data types. The Akaike information criterion will be used to select the number of clusters 
(i.e. number of subpopulations) for each variable.  These variables will be evaluated for 
collinearity and variable sets that are highly collinear will be represented by a constituent 
variable that is most representative of each subset. Children will be clustered to identify distinct 
phenotypes selected using four criteria: 1) a generalization of the within-clusters sum of squares 
measure, 2) average silhouette width, 3) Dunn index, and 4) the ratio of average-within versus 
average-between clusters. In this aim, we will identify asthma endotypes by linking respiratory 
allergy and asthma phenotypes to mechanistic data, including the patterns of airway epithelial 
cell DNA methylation and gene expression at 11 and 15 years that will be calculated in Aim 1.  
Our approach will first assign each subject to one of the phenotypes defined by the approach for 
Subaim 3.1.  We will then conduct an analysis to identify gene expression patterns and 

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methylation patterns (from ages 11 and 15), and environmental exposures that are 
characteristic of each phenotype and distinguish among them.   
To identify endotypes, we will use a sparse multinomial logistic regression model Variables for 
these models include the DNA methylation and expression modules (Aim 1), and environmental 
exposures (e.g. viral infections, allergens, microbes, tobacco smoke). Thus, the model will 
represent endotypes by mapping from biological states and environmental exposures to clinical 
phenotypes. In a sparse model, the learning algorithm is biased towards selecting a relatively 
small number of variables. Thus, although we will include a moderately large number of 
candidate variables in the models, only a sufficient number of variables to distinguish the 
phenotypes are selected. We will use well established sparse learning methods, such as 
LASSO and elastic net, which are well suited to high-dimensional data sets that include many 
potentially irrelevant variables.  
We will next identify biomarkers for predicting respiratory allergy and asthma phenotypes that 
we observe at ages 11 and 15.  This analysis will start with subjects clustered according to the 
phenotypes that we defined in Aim 3.1 and will proceed by inferring functions composed from 
individual early-life biomarkers that are predictive of the phenotype an individual subject will 
exhibit. Sparse multinomial logistic regression models will be used to evaluate early life 
biomarkers. The candidate predictor variables for these models will include (i) demographics, (ii) 
clinical variables such as wheeze frequency, (iii) gene expression measurements from nasal 
epithelial cells, and (iv) DNAm measurements from nasal epithelial cells.  Aside from 
demographics, these variables will come from measurements taken during the first three years 
of life. 
 Sample Size Considerations 
The sample size for the URECA cohort is determined by the original recruitment, and retention 
to this point (442 subjects at age 14 years, which represents 73% of the original 609 recruited 
subjects) has been excellent. This sample size enabled identification of phenotypes at 7 and 10 
years of age. 
11 Quality Control and Quality Assurance 
Training of study staff will be conducted prior to beginning any new procedures.  All staff 
members will be required to complete certification and quality control in all applicable study 
procedures as outlined in the URECA V MOP for ICAC-07.  The site principal investigator and 
study coordinator(s) will be responsible for ensuring that all procedures are performed according 
to the protocol. Periodic reviews of procedures will be conducted by the study coordinator or 
other trained personnel according to an individual schedule for each staff member that is based 
on the activities they are responsible for conducting.  Details of the quality control plan, including 
certifications and quality control of study procedures, are provided in the URECA V MOP for 
ICAC-07. 

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12 Ethics/Protection of Human Subjects 
 The Belmont Report 
In accordance with the FWA00005897: “This institution assures that all of its activities related to 
human subject research, regardless of funding source, will be guided by the ethical principles of 
The Belmont Report.”  Additionally, the investigator assures that all activities of this protocol will 
be guided by the ethical principles of The Belmont Report, 45 CFR 46 and all of its subparts (A, 
B, C and D). 
 Institutional Review Board 
A copy of the protocol, informed consent forms, other information to be completed by 
participants, such as survey instruments or questionnaires, and any proposed advertising/ 
recruitment materials will be submitted to the IRB for written approval. 
All subsequent amendments to the protocol, informed consent documents, and other study 
documentation referenced above must be reviewed and approved by the IRB before 
implementation.  The annual Continuing Review must also be reviewed and approved by the 
IRB throughout the duration of the study. 
The IRB will be notified of SAEs and protocol violations. 
 Informed Consent Process 
The informed consent and assent forms are a means of providing information about the study to 
a prospective participant/guardian to allow for an informed decision about participation in the 
study. The parent or legal guardian of the participating child (or their legally acceptable 
representative) must read, sign, and date the informed consent form before continuing the study 
or undergoing any study-specific assessments. Consent materials for participants who do not 
speak or read English will be translated into Spanish for clinical sites with Spanish-speaking 
staff. 
The informed consent and assent forms will be revised whenever important new safety 
information is available, whenever the protocol is amended with changes that require re-
consent/re-assent by participants, and/or whenever any new information becomes available that 
may affect participation in the study. 
A copy of the informed consent form (and assent form, if applicable) will be given to a 
prospective participant/caretaker for review. The prospective participant/caretaker will be told 
that being in the study is voluntary and that he or she may withdraw from the study at any time 
for any reason. 
 Assent Process 
All URECA V participants will sign a written assent form as described above. 
 Participant Confidentiality 
Following Health Insurance Portability and Accountability Act guidelines, a participant’s privacy 
and confidentiality will be respected throughout the study.  Each participant will be assigned a 

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sequential identification number and this number, rather than a name, will be used to collect, 
store, and report participant information. Data reported in medical journals or scientific meetings 
will be presented in aggregate for participants as a whole. No individual participant will be 
identified in any way. 
Participant confidentiality will be strictly held in trust by the participating investigators, their staff, 
and the sponsor(s) and their agents. This confidentiality is extended to cover testing of biologic 
samples in addition to the clinical information relating to participating subjects. 
The study protocol, documentation, data, and all other information generated will be held in 
strict confidence. No information concerning the study or the data will be released to any 
unauthorized third party without prior written approval of the sponsor. 
The study monitor or other authorized representatives of the sponsor may inspect all documents 
and records required to be maintained by the investigator, including but not limited to medical 
records (office, clinic, or hospital) for the participants in this study. The clinical site will permit 
access to such records. 
 Study Discontinuation 
There are no study halting rules. However, the study may be discontinued at any time at the 
discretion of the NIH. 
13 Data Handling and Record Keeping 
Study data will be entered in a login-secured, web-based electronic data capture (EDC) system. 
The system operates on all Internet browser platforms and is designed to handle numerous 
simultaneous studies within a program. Physical, logical, and operational security related to the 
data is implemented by a network operation center. Additional details regarding the EDC system 
are provided in the Data Management Plan (DMP) for ICAC-07. 
 Data Management Responsibilities 
The data management tasks required for this study are a joint responsibility of the SACCC and 
clinical site staff. The clinical site staff are responsible for collecting and entering study data into 
the EDC system per the protocol and specific guidelines in the URECA V MOP for ICAC-07, for 
ensuring the accuracy of these data, and for maintaining and organizing all original source 
documents and any additional sources of data. The EDC system has been validated and is 
compliant with Part 11 Title 21 of the Code of Federal Regulations. The site principal 
investigator is responsible for supervising the data collection and data management processes 
at the clinical site to ensure the overall quality of the data generated by all clinical site staff. The 
SACCC is responsible for ensuring the quality of the data at all the clinical sites. The specific 
responsibilities of the clinical site staff, site principal investigators, and the SACCC are included 
in the DMP for ICAC-07. All data management activities at the SACCC and clinical sites will be 
conducted in accordance with the DMP. 
 Data Capture Methods 
Data will be captured onto paper CRFs for later entry into eCRFs, or directly into eCRFs. The 
first recording of any information captured for the study will be considered the source document, 

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which may be, but is not limited to, a medical record, a laboratory or clinical report, a paper 
CRF, or an eCRF.  
The details regarding the electronic verification of all data fields, including univariate and 
multivariate validation (i.e., range checks and cross-field and cross-form checks), validation of 
data omission, and query management are listed in the DMP. 
 Types of Data 
Clinical, demographic, laboratory, and AE data will be collected for this study. 
 Source Documents and Access to Source Data/Documents 
The clinical sites participating in this study will maintain the highest degree of confidentiality 
permitted for the clinical and research information obtained from the participants. Medical and 
research records will be maintained at each clinical site in the strictest confidence. However, as 
a part of the quality assurance and legal responsibilities of an investigation, the clinical site must 
permit authorized representatives of the sponsor to examine (and when required by applicable 
law, to copy) clinical records for the purpose of quality assurance reviews, audits, and 
evaluations of the study safety and progress. Unless required by the laws that permit copying of 
records, only the coded identity associated with documents or with other participant data may 
be copied (and all personally identifying information must be obscured). Authorized 
representatives as noted above are bound to maintain the strict confidentiality of medical and 
research information that is linked to individuals. The clinical site will normally be notified before 
auditing visits occur. 
 Timing/Reports 
Data will be monitored by staff at the SACCC. Status reports on the progress of the study and 
data collection will be generated regularly. Reports will be sent to the NIAID project manager 
and medical officer on a regular basis. 
 Study Records Retention 
Study documents must be maintained at the clinical site or a local storage facility for at least 5 
years following the completion of the study. Study documents that must be retained include all 
hard copies of CRFs, IRB approval documentation and related correspondence, and signed 
informed consent forms. 
14 Publication Policy 
Presentations and publication of the results of this study will be governed by the CAUSE 
Publication Policy. 
 
 

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Appendix A: Scientific References 
1. 
Gern JE. The Urban Environment and Childhood Asthma study. J Allergy Clin Immunol 
2010;125:545-9. 
2. 
Gern JE, Visness CM, Gergen PJ, Wood RA, Bloomberg GR, O'Connor GT, et al. The 
Urban Environment and Childhood Asthma (URECA) birth cohort study: design, 
methods, and study population. BMC Pulm Med 2009;9:17. 
3. 
Zook PM, Jordan C, Adams B, Visness CM, Walter M, Pollenz K, et al. Retention 
strategies and predictors of attrition in an urban pediatric asthma study. Clin Trials 
2010;7:400-10. 
4. 
O'Connor GT, Lynch SV, Bloomberg GR, Kattan M, Wood RA, Gergen PJ, et al. Early-
life home environment and risk of asthma among inner-city children. J Allergy Clin 
Immunol 2018;141:1468-75. 
5. 
Bacharier LB, Beigelman A, Calatroni A, Jackson DJ, Gergen PJ, O'Connor GT, et al. 
Longitudinal Phenotypes of Respiratory Health in a High-Risk Urban Birth Cohort. Am J 
Respir Crit Care Med 2019;199:71-82. 
6. 
Demoly P, Piette V, Bousquet J. In vivo methods for the study of allergy. In: Adkinson 
NFJ, Yunginger JW, Busse WW, Bochner BS, Holgate ST, Simons FER, eds. 
Middleton's Allergy: Principles and Practice. Philadephia: Mosby; 2003:631-55. 
7. 
Silkoff PE, McClean PA, Slutsky AS, Furlott HG, Hoffstein E, Wakita S, et al. Marked 
flow-dependence of exhaled nitric oxide using a new technique to exclude nasal nitric 
oxide. Am J Respir Crit Care Med 1997;155:260-7. 
8. 
American Thoracic Society. Recommendations for standardized procedures for the on-
line and off-line measurement of exhaled lower respiratory nitric oxide and nasal nitric 
oxide in adults and children-1999. This official statement of the American Thoracic 
Society was adopted by the ATS Board of Directors, July 1999. Am J Respir Crit Care 
Med 1999;160:2104-17. 
9. 
Fahy JV, Boushey HA, Lazarus SC, Mauger EA, Cherniack RM, Chinchilli VM, et al. 
Safety and reproducibility of sputum induction in asthmatic subjects in a multicenter 
study. Am J Respir Crit Care Med 2001;163:1470-5. 
10. 
American Thoracic Society. ATS statement: guidelines for the six-minute walk test. Am J 
Respir Crit Care Med 2002;166:111-7. 
11. 
Laird NM. Missing data in longitudinal studies. Statistics in medicine 1988;7:305-15. 
12. 
Little RJA. Modeling the drop-out mechanism in repeated-measures studies. Journal of 
the American Statistical Association 1995;90:1112-21. 
13. 
Little RJA, Rubin DB. Statistical Analysis with Missing Data. New York: Wiley; 1987. 
14. 
Bowman FD, Stewart PW, Sen PK, Helms RW. Making inferences about projected 
completors in longitudinal studies. Journal of biopharmaceutical statistics 2004;14:947-
67. 
15. 
Diggle P, Kenward MG. Informative Drop-Out in Longitudinal Data Analysis. Applied 
Statistics 1994;43:49-93. 

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16. 
Glynn RJ, Laird NM, Rubin DB. Multiple Imputation in Mixture Models for Nonignorable 
Nonresponse With Follow-ups. Journal of the American Statistical Association 
1993;88:984-93. 
17. 
Schluchter MD. Methods for the analysis of informatively censored longitudinal data. 
Statistics in medicine 1992;11:1861-70. 
18. 
Wu MC, Bailey KR. Estimation and comparison of changes in the presence of 
informative right censoring: conditional linear model. Biometrics 1989;45:939-55. 
19. 
Wildasin HA, Wallace D, Kesler K. Assessing Dropout Bias with Mixed-Effects and 
pattern Mixture Models.  28th Annual Meeting of the Society for Clinical Trials; 2007 May 
2007; Montreal, Canada. 
 
 

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instead, bronchodilator reversibility is missed at Year 14 in favor of the methacholine challenge, reversibility will be 
done at the Year 15 visit. 
6 At the standalone Methacholine Challenge Visit, staff will conduct Post-Bronchodilator IOS/Spirometry only when 
the participant both does not meet ATS guidelines for the Methacholine Challenge and did not perform Post-
Bronchodilator IOS/Spirometry at the last formally scheduled instance.