Project Yeah

Research Questions: Slide 1• Does 3 months of yoga improve fitness, depression, cardiovascular

indicators, weight, etc. in previously sedentary individuals?• Do experienced yoga practitioners have better fitness, depression,

weight, bp than non-exercisers?• Does 3 months of yoga increase parasympathetic tone in novices?• Do experienced yoga users have more parasympathetic tone than

non-exercisers?

Research Questions: Slide 2• Does 3 months of yoga reduce inflammation in systemic, molecular,

and cellular measures in novices?• Do experienced yoga practitioners have less inflammation than non-

exercisers?• Is PNS tone associated with inflammation in systemic, molecular

and cellular measures?

Design• 1. Prospective 3 month intervention for non-exercisers randomized

to yoga or aerobic walking. Comparison of pre-post, and between groups.

• 2. Cross sectional study comparing experienced yoga practitioners with non-exercisers

Inclusion criteria: Non-exercisers

• Female• 25-45 y/o• Not currently engaged in regular physical

activity

Inclusion Criteria: Yoga experts

• Female• 20-45 y/o• Have practiced yoga for 3+ years• Currently practicing at least twice a week

Exclusion criteria

• Alcohol abuse• Drug use/smoking• Prescription drugs considered on case by case• Heart disease• Postmenopausal• Pregnant• Most chronic diseases• Major mental health diseases

Recruitment strategies

• Flyers• Outreach• Listserve• Outreach to yoga studios• Incentives• Total number of ‘touches’ to recruit the

participants: 152 for intervention and 21 for experienced (control) group

RecruitmentPHASE 1: 152 people contacted to recruit 34

– 23% success rate– 21 outside age range (9 old, 12 young)– 50 had schedule conflicts– 11 had exclusionary diseases– 33 “other” (included drug user, male, unwilling to donate blood, pregnant)

Of the 36 recruited, in cohort 1 (n=19)3 no-shows, 1 refused to give blood, 8 randomized to yoga—1 dropped after cited scheduling conflicts7 randomized to walking—4 dropped after (1 moved away, 3 said they thought they would get to do yoga)

In cohort 2 (n=15) 4 no shows All put in yoga group—4 later drops all cited schedule

PHASE 2: Experienced yogi’s 21 people contacted to recruit 10 (details of why not eligible not kept)

IRB Process

• First submission• Consents approved and stamped• Surveys approved• Flyers approved• Revised submission• Process repeated (4 times—including

extension)

Variables

• Demographic and Social History• Behavior• Complementary and Alternative Medicine use • Fitness measures• Biological measures• Cardiac measures• Biological blood work• Inflammatory markers

Basic Biometrics

• Weight• Height• BMI

Demographic & Social History

• Age• Gender• Education• Income• Social status• Medications• Reproductive history• Chronic diseases

Fitness and Cardiovascular Measures

• 3 minute step test • Blood Pressure and Heart Rate at rest, peak, and

during recovery measured by EKG

• Heart Rate Variability gathered via Zephyr Bioharness IV

Behavior & Lifestyle

• Exercise• Smoking history• Drug use• Physical Activity

• Questionnaire• Accelerometer (pre and post)

• Sedentariness

Use of Complementary & Alternative Medicine

• Nutritional modalities• Mind body modalities• Movement modalities• Supplements

Blood based markers of Inflammation

• Complete Blood Count (CBC) Complete Metabolic Panel (CMP) HgA1C: Measure of long-term blood

glucose control. C-Reactive Protein (CRP) Cortisol serum

Cellular and Molecular Markers of Inflammation

Stat3 – cell growth and apoptosis

1:http://www.cellsignal.com/productListing?Ntt=stat3

• required for murine fetal development.

• possesses oncogenic potential

• anti-apoptotic activities.1

Nuclear membrane

Surface receptor

Cell Surface

Participants

• Pre-Post novice yoga completers: 13• 14 completers, but one participant removed from

analysis due to development of diabetes during intervention period.

• Pre-Post novice walkers completers: 3• Pre-novices non-completers: 9

• 5 yoga and 4 walking• Experienced yoga practitioners: 10

YOGA INTERVENTION

• Participants practiced of a blend of Ashtanga and Hatha forms of yoga with a strong focus on breathing throughout the practice.

• Participants were asked to attend 2 classes and practice at least once a week on their own using the provided video. –The video was a 20 minute practice. –Classes lasted for 60 minutes.

RESULTS

Characteristics of Participants at Baseline All (n=26) Yoga C (n=13) Yoga NC (n=5) Aerobic C (n=3) Aerobic NC (n=4)

Age 31.75 5.72 33.75 4.47 28.67 3.09 25.33 0.58 25.00 n/aHt (cm) 164.60 7.41 167.96 7.28 162.52 8.18 162.63 5.94 160.15 5.80

Wt (lbs) 149.54 33.33 150.19 40.38 156.12 21.52 127.67 29.30 142.50 68.43

BMI 25.24 6.37 25.35 7.28 27.25 5.94 21.77 3.42 25.11 3.84

HR Rest (bpm) 77.57 11.32 75.31 10.36 73.00 12.30 89.00 10.58 69.00 n/aHR Peak (bpm) 164.87 11.31 161.54 12.55 167.00 5.29 162.00 2.00 180.00 n/a

%MVPA 3.58 2.19 3.85 2.31 3.92 1.90 3.37 2.11 n/a n/a

%Sed 67.32 10.14 66.07 8.92 69.18 19.35 73.85 1.83 n/a n/a

pNN50 11.82 9.95 10.88 8.82 15.58 9.95 5.09 2.83 n/a n/a

* Values are reported as means and standard deviations

Univariate: yoga novices (pre/post)Variable Pre-

(13-14)Post- (13-14)

Mean SD Mean SD p-value

SBP rest 118 5.8 116 7.9 0.912

Weight 150 40.3 150 42.5 0.00

SBP_pk 117.7 5.9 115.5 7.5 0.515

HR rest 75 10.3 71 10.0 0.102

HR peak 161.5 12.5 155.5 16.3 0.002

HR rec1 123 18.4 117 17.1 0.034

HR rec2 103 16.3 99.3 17.2 0.001

pNN50 10.88 8.82 9.72 6.43 0.340

A1C 5.2 0.25 5.4 0.24 0.043

*Calculated using T-testClinical Significance: • Rest HR down 4 bpm• Peak HR dwon 6 bpm (statistically

significant too)

May also be meaningful

• Sedentary time (self report) down 8%

OTHER MARKERS OF INFLAMMATIONPre-yoga (14) Post-yogaMean SD Mean SD p-value

Cortisol (12) 14.25 5.40 16.44 6.50 0.198

CRP (6) 0.640 0.625 0.403 0.471 0.443

WBC (13) 6.3 1.87 5.86 1.234 0.297

55

60

65

70

75

80

85

90

95

100HR REST (PRE_POST)

TIME POINT (LEFT PRE, RIGHT POST)

HR (B

PM)

120

130

140

150

160

170

180

190

HR PEAK

TIME POINT (LEFT =PRE RIGHT=POST)

HR (B

PM)

Characteristics of Yogis

Advanced Yogis (n=10)Mean SD

Age 37.50 6.84Ht (cm) 165.90 10.31Wt (lbs) 140.39 16.75BMI 23.26 2.68

Bivariate: novice vs experiencedVariable Cases (non exercise)

19-20Controls (yogi) 9 p-value

Mean SD Mean SD

SBP (rest) 117.7 5.879 110.2 6.36 0.010

SBP (rec-2 min)

121.8 3.4 118.2 8.45 0.194

HR (rest) 75.31 10.35 66.22 6.3 0.030

HR (peak) 161.5 12.09 152.11 10.73 0.082

HR (rec-2 min) 103.4 16.32 91.11 15.77 0.094

pNN50 11.53 8.77 16.93 12.91 0.132

Clinical Significance: • HR Recovery less by 17 bpm at min 1 & 2• Less time doing sedentary activity (accelerometer 4%, self-

report 14.5%)• Pnn50 50% greater in experienced (11.5% vs 16.9%)

*Calculated using non-paired T-test

System Level ConclusionsModest changes in key variables in 12 weeks, but encouraging trends

Large differences in key variables between experienced and non

CELLULAR & MOLECULAR TESTING

Goal: Develop a molecular workflow to analyze the immune response in human blood to Yoga versus other exercise.

For this project• Feasibility• Generate preliminary data on which later grant applications

can be based• Generate Bio-repository for future gene expression studies

Analysis

Cell Signaling

Cell responsiveness to Nicotine and IL-6

• Nicotine- classic agonist for a7 nACHR

• IL-6- classic agonist for STAT3 pathway

Analysis

Cell Signaling

Biorepository

Cell responsiveness to Nicotine and IL-6

Magnetic bead column separation Quality Control

Monocyte and T Cell count

• Nicotine- classic agonist for a7 nACHR

• IL-6- classic agonist for STAT3 pathway

RNA analysis through RT-PCR

Flow Cytometry

Baseline

Post-Exercise

Draw Antibody Staining

Cell Activation Analysis

2

How:1. Cell Signaling

Flow Cytometry

BaselinePost-Exercise

Blood draw Antibody Staining

Magnetic Bead Separation

Cell Type and Activation Analysis

RNA Analysis

10 2

How:

2. Biorepository

Cell Signaling• Stat3 – cell growth and apoptosis

1:http://www.cellsignal.com/productListing?Ntt=stat3

• required for murine fetal development.

• possesses oncogenic potential

• anti-apoptotic activities.1

Nuclear membrane

Surface receptor

Cell Surface

Cell Signaling• Stat3 – cell growth and apoptosis

SignalingStat3

Low Signal

High Signal

Low Cell Count

High Cell Count

Median Fluorescent Intensity (MFI)• “middle” of curve• Very standard statistic in literature (>10,000 events)• Less affected by outliers

Fluorescent Intensity

Quantification

*n=6 P=0.0122, Paired t-test

*

Nicotine P-STAT3 response ratio reduced post-YOGA (n=6)

Change in response to nicotine after exercise• Each point on scatterplot expressed as ratio of MFI post Nicotine to

MFI sham• Statistically significant Decrease in STAT3 activation after exercise

Raw data…

Patient ID Baseline Pre Nicotine Pre Ratio Baseline Post Nicotine Post Ratio201-7 3250 4998 1.537846154 993 1130 1.13796576

201-9 1472 2020 1.372282609 551 612 1.110707804

201-11 585 1379 2.357264957 276 549 1.989130435

201-19 1013 1878 1.853899309 729 375 0.514403292

201-21 953 2209 2.317943337 632 747 1.181962025

201-23 1454 3008 2.068775791 2740 2062 0.752554745

Baseline Raw data…

*n=6 P=0.315, Paired t-test

*

*n=6 P=0.0084, Paired t-test

*

IL-6 P-STAT3 response ratio increased post-YOGA (n=6)

Raw data…

Patient ID Baseline Pre IL-6 Pre Ratio Baseline Post IL-6 Post Ratio201-27 2054 5600 2.726387537 1508 6833 4.531167109

201-29 4096 7440 1.81640625 1649 4266 2.587022438

201-33 4063 9689 2.384691115 1704 6429 3.772887324

201-35 1972 4703 2.384888438 1352 6173 4.565828402

201-39 7625 5738 0.75252459 5976 15085 2.524263722

201-41 6508 9969 1.531807007 720 4683 6.504166667

Change in response to IL-6 after exercise• Each point on scatterplot expressed as raw MFI post IL-6• Statistically significant decrease in response ratio and Baseline

Baseline Raw data…

*n=6 P=0.0313, Paired t-test

*

Conclusion 1 of 2

• We have a way to assess the responsiveness of a cell to an agonist that is relevant to the human inflammation signaling before and after exercise

• There is preliminary data to suggest that Yoga might lower STAT3 activation

Analysis

Cell Signaling

Cell responsiveness to Nicotine and IL-6

• Nicotine- classic agonist for a7 nACHR

• IL-6- classic agonist for STAT3 pathway

Biorepository

Magnetic bead column separation Quality Control

Monocyte and T Cell count

RNA analysis through RT-PCR

Flow Cytometry

Baseline

Post-

Exercise

Blood Draw Antibody Staining

Magnetic Bead Separation

Cell Type and Activation Analysis

RNA Analysis

10 2

How:

Gene Expression• Stained for CD14+ monocytes• Average 3.3% of blood• Purification of CD14+ monocytes through magnetic bead separation

for RNA analysis

Biorepository

Conclusion 2 of 2• We can detect gene expression in fractions of peripheral blood

• 10mL is likely a rate limiting amount for magnetic bead separation

Next Steps?• Continue with BioRepository and RT-PCR

• Correlations with Blood draw data and demographics

• Finish analysis of Experienced practitioners

• What else needed for publication?

Other outcomes

Acknowledgements• David Wing – blood collection• Ann-Marie Hageny – cell staining and flow cytometry• Emelie Amburn – RNA prep• Glory BUI – cDNA, PCR and gel• Matthew Eliceiri – Design, Analysis and Summary• Dr. Linda Hill-Study PI• CalIT2-Funding Support

TEAMLinda HillJeanne NicholsAndrew BairdBrian EliceiriAnne Marie HagenyDenise GallagherDavid WingElizabeth FriedmanEmelie AmburnGlory BUIMatthew Eliceiri Walking leaders/Yoga teachersTravis NguyenGraeme BairdJill WaalenLaura Schmalzl

Additional input• Deborah Forster• Ramesh Rao• Paul Mills• Giorgio• Etc, need to list

Analysis challenges

• HRV–zephyr–bliss buzzer

• Multiple variables• Two labs: Hillcrest/Quest• Yoga studies: synergy, variation in type**

Other challenges and limitationsBudgetRecruitmentStaffingBuilding limitations

Next steps• Publications

• Changes in fitness, depression, activity• Changes in inflammation• Challenges with HRV

• Grant proposals