Asthma and Obesity Diet Versus Exercise

Our primary hypothesis is that inflammatory SNPs may regulate the degree of the inflammatory
response, with obesity modifying the severity of the disease. In this instance, asthma that
develops in the context of obesity demonstrates the potential deleterious relationship
between a specific proinflammatory state (obesity) and the genetic regulators of inflammation
(SNPs).

Our secondary hypothesis proposes that short-term (12-weeks) weight loss by diet alone, but
not exercise alone, will reduce lung specific inflammation and diminish the pro-inflammatory
responses in female African American obese adolescents with asthma compared to a waiting list
control group who after their initial 12 weeks then receive a combined 12-week diet plus
exercise program (waiting list control/combined). A third exploratory hypothesis proposes
that the frequency of identified SNPs will be significantly related to the amount of fat loss
through diet, exercise or combined program and will further be mediated by specific airway
and, pro-and-anti-inflammatory markers. Specific aims include:

1. To determine the frequency of single nucleotide polymorphisms and SNP haplotypes in pro-
and anti-inflammatory genes in female African American obese and non-obese asthmatic and
non-asthmatic adolescents, 13-19 years or age.

2. To examine the effects of diet or exercise on lung specific inflammation (exhaled nitric
oxide, [eNO]) and pro-and-anti-inflammatory responses in female African-American obese
asthmatic and non-asthmatic adolescents compared to a waiting list control/ combined
group.

3. To determine the effects of the inflammatory SNPs in the modulation of several
inflammatory markers and lung specific inflammation (eNO) in female African-American
obese asthmatic and non-asthmatic adolescents before and after weight loss through diet,
exercise or both.

We will conduct a cross-sectional analysis of 4 groups of African American adolescent (13-19
years) females as follows: Group 1A: Obese with asthma Group 1B: Non-obese with asthma Group
1C: Obese non-asthmatics Group 1D: Non-obese, non-asthmatics

Patients included in this part of the study will be female African American obese and
non-obese asthmatic adolescents, 13-19 years, as defined by United States Centers for Disease
control (USCDC 2000 sex specific Body Mass Index (BMI)-for-age growth charts. As controls we
will include female African American obese, non asthmatic adolescents and healthy age matched
controls (non-obese, non-asthmatics).

Assuming a dominant model, for a haplotype of 5 SNPs, a sample of 100 individuals per group
(matching 1:1) would achieve at least 80% power to detect an OR of 1.5 for the association of
the haplotype with group membership (such as obese with asthma vs. non-obese with asthma), at
0.05 significance level. All other group comparisons would assume the same conditions, thus
samples of 100 for each of the groups are required.

After parent/guardian has signed the informed consent, a general medical exam and history is
taken, and spirometry testing is performed to assess the lung function of each participant,
obesity status will be assessed. For girls found to be non-obese during visit 1,
approximately 30 ml of peripheral blood will be obtained from 100 asthmatics and 100
non-asthmatic healthy age matched controls. For obese participants (100 asthmatic and 100
non-asthmatics), peripheral blood draws will be obtained during visit 2 (baseline).
Peripheral blood will be tested for a Hemoglobin A1-C (HA1C), lipid profile and to determine
the presence of inflammation and genetic markers. An additional blood sample from all African
American adolescent girls participating in the study will be collected into heparin tubes (2
10ml tubes). The mononuclear layer will be separated using density gradients and will
characterize the circulating MDS. Flow cytometry will be used to determine the percentage of
monocytic or granulocytic MDSC using several fluorescence-labeled antibodies, including
anti-CD11b, CD14, CD15, CD33, CD66 and HLA-DR. The DNA will be extracted by DNAzol
(Invitrogen Corp; Grand Island, NY). For the SNP analysis we plan to use the humanCNV370-Quad
bead chip from lllumina (lllumina Inc, San Diego, CA). This chip allows the analysis of more
than 370,000 SNPs simultaneously and uses only 200 ng of genomic DNA. We will initially focus
our analysis in the pro- and anti-inflammatory cytokine gene SNPs (/LS, TNF, TGFB, ILIO, IL8,
IL6, PTGS2, ARG1) and SNPs in leptin, leptin receptor and adiponectin (Table 11) and the
results will be confirmed by TaqMan as follows: genomic DNA (5 ng) will be denatured at 95°C
for 10 min and amplified for 40 cycles of 15 sec at 92°C and 1 min at 58°C, in the presence
of 2X TaqMan Universal Master Mix (Applied Biosystems), water, and the respective primer and
reporter probe mix (labeled with either FAM or VIC). The reaction will be analyzed using a
7900 HT instrument (Applied Biosystems), for the presence of VIC or FAM fluorescence marker,
or both, using the Sequence Detection System (Applied Biosystems) to determine the genotype.
Controls will include individuals of known genotype and blanks without DNA. In addition, 15%
of the samples will be run twice in separate assays and the allele classification compared.
The individuals of known genotype and blanks without DNA will be included for each SNP in
every batch.

Chi-square, Fisher's exact, and student-t tests will be used to assess the statistical
significance of the differences in the frequencies of the different SNPs between obese
asthmatics, non-obese asthmatics, obese non-asthmatics and healthy controls. Haplotype
frequencies will be inferred using log-linear modeling embedded within an
expectation-maximization algorithm. Quality control procedures (including evaluation of
genotyping efficiency and Hardy-Weinberg equilibrium), assessment of linkage disequilibrium
between markers as well as haplotype association analysis will be performed separately in
obese versus nonobese and asthmatic versus non-asthmatics.

After enrollment, eligible participants will undergo a baseline visit to include
approximately 30ml peripheral blood draw, Dual Energy X-RAY Absorptiometry (DEXA) scan,
exhaled nitric oxide (eNO), and study eligibility per the study physician. Following this
baseline visit, obese participants with and without asthma will be randomized to:

1) diet only, 2) exercise only or 3) waiting list/combined intervention (no intervention for
12 weeks followed by combined diet and exercise).

Approximately 110 subjects will be recruited per treatment and control group. Assuming an
attrition rate of 20%, we will maintain 360 subjects, 90 per group which will provide
adequate statistical power to:

1. detect significant differences in eNO between diet only, exercise only, combined
program, or a waiting list control group of female African American obese asthmatics and
non-asthmatics as per the sample size power calculations below and;

2. as part of the exploratory aim 3, determine the effects of the inflammatory SNPs in the
modulation of inflammatory markers and eNO in female African-American obese asthmatic
and non-asthmatic adolescents before and after weight loss through diet, exercise or
both as per the sample size power calculations).

Intervention Methods:

Treatment groups will receive the Trim Kids behavior modification program but the
intervention materials will be specific to diet only or exercise only treatments. Non-obese
asthmatics and non-obese, non-asthmatics will serve as controls for the exploratory aim and
participate in a general medical exam, spirometry and blood testing only. Parents and
children attend a weekly two-hour comprehensive session for a recommended time period.
Medical supervision and guidance are provided by a physician or nurse at each session in
order to increase compliance and monitor side effects.

Management of asthma: We will exclude persons with severe or uncontrolled asthma. In the case
of acute asthma attacks participants will be managed according to a written and reviewed
asthma action plan, with the use of asthma medications or S-agonists like Albuterol, and if
necessary with a short course of other medications. Asthmatic participants involved in the
intervention group will also be required to use a pre-exercise dose of asthma medication
before participation in each exercise session or will be excluded from that session. Any
exacerbation will be recorded and participants will be able to contact a health provider, and
if needed will be referred to emergency services. In case of a more severe exacerbation or
progressive worsening, the participant will be treated according to standard of care, and
taken off the study if chronic or prolonged anti-inflammatory treatment is considered a
priority.

Nutrition education includes a series of learning activities, which are specific to needs of
each group as they progress through the different stages of the treatment program. The
children are provided with easy-to-use food frequency checklists to fill in each week.

The exercise protocols to be utilized in this study will be based on the Trim Kids exercise
protocol, which tailors the recommendations to the child's medical condition (co-morbidities
such as asthma), weight status and fitness level. All exercise recommendations will follow
the guidelines of the American Thoracic Society (ATS), American Academy of Pediatrics (AAP)
and American Academy of Sports Medicine (ACSM). Exercise sessions at the LSUHSC Wellness
center will be supervised by ACSM certified instructors and personal trainers. During each
12-week session of the program, the exercise activities correspond with the group's physical
condition of obesity and ability to comprehend, synthesize and apply health and fitness
Information to daily life situations. Children receive an exercise video containing an
exercise routine to perform at home. They are also given cards to record the number of
minutes of physical activity, they perform each week. Compliance is monitored by observing
these physical activity record cards and monitoring of heart and breathing rate. Behavior
modification and psychosocial education are integrated into educational sessions by a
psychologist.

Behavior modification skills are taught stressing how discussion, modeling, role playing and
guided problem solving are used. Topics such as self-monitoring, commitment, limit setting,
habit formation, goal setting and action plans, decision-making skills and assertiveness
training are discussed.

The intervention will be conducted at the Louisiana State University (LSU) Health Sciences
Center Wellness Facility. Both diet and exercise groups will meet for medical monitoring and
weigh in a clinical area adjacent to a private room with a scale during the first 30 minutes
of each weekly class.

The diet only group will meet in a classroom after the 30-minute medical monitoring and
weigh-in. Behavior modification class will follow for 30 minutes and Nutrition education for
approximately 30-60 minutes. Study participants enrolled in the diet only intervention will
be placed on either a protein modified fast diet plus supplementation of vitamins and
minerals, followed by a balanced, hypo-caloric diet and/or a moderate intensity, progressive
exercise program, and given instruction on behavior modification.

The exercise only group will meet in the large multi-purpose room after the 30 minute medical
monitoring and weigh in. Behavior modification class will follow for 30 minutes. Participants
will utilize the aerobic equipment, attend culturally specific dance classes, participate in
strength training, and outdoor family field sports in an adjacent community park area for
approximately 30-60 minutes. The participants exercise in a playful, sporadic, intermittent
manner while the instructor discusses key concepts using symbols, music and various props.

Following a 12 week waiting period, participants in the combined program will meet in a
separate multi-purpose room and will participate in diet, exercise and behavior modification
sessions once per week.

Inclusion Criteria:

- African American Females with an age of 13-19 years will be recruited. Our aim is to
target high school age youth who are most likely to exhibit autonomy and authority to
control both their nutrition and exercise regimens. In addition this age range is
representative of the age group most likely to access school based health centers for
recruitment purposes. Lastly, the study includes the collection of blood samples for
the purpose of examining varied physiologic parameters that are affected by
maturation. Participants above the age of 13 are most likely pubertal and thus, less
likely to be affected by maturation issues.

Exclusion Criteria:

- Presence of other chronic pulmonary or systemic disease.

- Presence of moderate or severe atopic dermatitis or allergic rhinitis.

- Severe asthmatic based upon American Thoracic Society (ATS) standards and/or NIH
guidelines.

- Acute lower respiratory infection in the last 2 weeks before baseline eNO evaluation.

- Pregnant are nursing mothers Study participants and parents (s) will be advised of the
known risks and consequences associated with the testing and also of the reasonably
known risks and consequences of not undergoing testing.

Severe or uncontrolled asthmatics will not be allowed to participate.

Participants with a recent (in the last 2 weeks) lower respiratory infection will not be
allowed to participate.