AGENDA UF Pepper OAIC Safety Committee Conference Call ... · 3/22/2013 · compound Garcinia Cambogia (hydroxycitric acid) on food intake, satiety, body weight, and oxidative stress

Pepper‐Safety Committee‐AGENDA01‐2013‐03‐22

AGENDA UF Pepper OAIC Safety Committee Conference Call

Friday, March 22, 2013 3:00 p.m. – 4:00 p.m. (Eastern)

Dial-in #: 1-866-851-9754

Guest code: 155034 Host: 1550345 (UF OAIC)

1. Review and approval of minutes from 06/04/12 conference call

2. Summary Table of Closed Studies

3. Safety Review of Active Studies – ACTION REQUIRED

Protocol Study PI Primary DSMB Reviewer

1 *Continuing Study* Skeletal Muscle Apoptosis and Physical Performance, Oxidative DNA/DNA Damage and Repair in Aged Human Muscle (429‐2005)

Christiaan Leeuwenburgh, PhD

John Meuleman, MD

2 *New Study* Cortical control of walking: assessment, mechanisms and functional implications (Think‐2‐Walk Study) (212‐2012)

David Clark, PhD Jing Cheng, PhD

3 *New Study* Establishment of Age‐Specific Induced Pluripotent Stem (iPS) Cells from Dermal Fibroblasts of Young and Elderly Individuals: Potential Model of Aging (Aging iPS Cell Study) (381‐2012)

Anna‐Marie Joseph, PhD

Stephen Kritchevsky, PhD

4. Review of Independent DSMB Reports

a. The LIFE Study (Field Center PI: Todd Manini, PhD) b. The Testosterone Trial (T‐Trial) (Field Center PI: Marco Pahor, MD) c. Aspirin in Reducing Events in the Elderly (ASPREE) (Field Center PI: Stephen Anton, PhD)

5. Closed Session (Safety Committee and Marco Pahor, MD)

6. Next Conference Call – September 2013 (date and time TBD)

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Meeting Minutes

Project: Pepper Committee: UF Pepper DSMB Chair: Stephen Kritchevsky, PhD Date: 06/04/12 Start Time: 1:00 PM EST Adjourn Time: 2:00 PM EST Meeting: In person__ Call_x_ Video __ Location if in person: Present: Drs. Steve Anton, Thomas Buford, Jing Cheng, Stephen Kritchevsky, Christiaan Leeuwenburgh, Todd Manini, and John Meuleman, Minutes prepared by: Laura Pons File name: Pepper-Safety-MNTS2012-06-04 # Topic Discussion Action Due date Responsible

person (s) 1 Approval of Prior

Minutes The minutes from the February 4, 2011 Pepper Center Safety Committee meeting were approved as distributed.

Approved

2 Research of Botanicals Obesity Reduction and Nutrition – The ReBorn Study PI: Stephen Anton, PhD

Primary Reviewer: Jing Cheng, PhD The intention of this study is to research the effects of two doses of the botanical compound Garcinia Cambogia (hydroxycitric acid) on food intake, satiety, body weight, and oxidative stress levels. Dr. Cheng reported that 19 participants have enrolled with 60 participants as the original goal. Of the 19 participants enrolled, 5 withdrew and only 3 completed the study. Dr. Anton confirmed that this study has been suspended because of an issue with the research capsules. There were no adverse events reported.

This study has been suspended. It will be revisited on an ad-hoc basis if a new protocol is implemented.

3 Role of Skeletal Muscle Blood Flow and Regeneration in Sarcopenia PI: Thomas Buford, PhD

Primary Reviewer: Stephen Kritchevsky, PhD This study is to examine the role of skeletal muscle angiogenesis and perfusion on satellite cell basal numbers, evaluate the differences in age and how it affects satellite cell responsiveness to proliferative stimuli, and examine the role of muscle perfusion and satellite cell number in physical function. Dr. Kritchevsky reported that 68 participants have been enrolled with a target enrollment of 36. Of the 68 participants, 30 completed, and 22 withdrew. Dr. Buford reported the study has been completed, and the IRB has reviewed all the safety data from the 30 participants who completed the study.

This study has been completed. There were no adverse events or safety concerns related to the intervention.

4 Task Specific Exercise for the Pre-Clinically Disabled PI: Todd Manini, PhD

Primary Reviewer: Jing Cheng, PhD This study was also reviewed in February 2011 with no safety concerns noted. The study is to determine the short and long-term responses of task-specific exercise in the pre-clinically disabled, and to explore mechanisms of adaptation following task-specific exercise. Dr. Manini reported that 71 participants

Data analysis for this study is almost complete. There was no safety concerns noted.

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completed the study out of 287 enrolled. Although the target was 72 subjects, the study was completed with only 71. The recruitment and enrollment is closed, but data analysis is still being completed.

5 Chemotherapy-induced Muscle Weakness and Functional Limitation in Older Breast Cancer Survivors (PI: Todd Manini, PhD)

Primary Reviewer: Stephen Kritchevsky, PhD This study was also reviewed in February 2011 with no safety concerns noted. This study is to evaluate muscle weakness, physical function and quality of life in elderly breast cancer survivors, and to determine the role of DNA/RNA oxidation and inflammation for predicting muscle weakness and quality of life. Dr. Manini noted that 34 participants were completed through collaboration with the Moffitt Cancer Center and 23 subjects completed the study at Gainesville. This study has been completed as far as recruitment and participants. There were no adverse events reported, and no safety concerns noted. Any future multi-site studies should have their own DSMB. However, if no DSMB is available, this committee will review information on a case-by-case basis.

This study has been completed. There were no adverse events or safety concerns noted.

6 Locomotor Reserve: A Novel Approach for Detecting Mobility Deficits with Aging PI: David Clark, PhD

Primary Reviewer: Jing Cheng, PhD This is a new study that will examine the relationship between walking speed reserve, step length reserve, short physical performance battery, and 400 m walk time. The recruitment information was not available for review, and the committee specifically wanted to know how many participants have been enrolled. Laura Pons will follow up with Dr. Clark for the status of this study, and then she will report back to the committee.

Laura Pons will follow up with Dr. Clark for the status of this study, and then she will report back to the committee.

7 Respiratory Sarcopenia in Elderly Women and Chronic Lung Infections PI: Kevin Fennelly, PhD

Primary Reviewer: John Meuleman, MD This study will assess whether or not serum leptin concentrations are significantly different between elderly women with stable or resolved non-tuberculos mycobacterial lung infections and controls without a history of respiratory disease matched for age and body mass index. It will also compare body composition, nutritional indices, serum concentrations of C-reactive protein, and general muscle strength. Dr. Fennelly was not present on the call. The committee specifically questioned the protocol on page 6, in the first paragraph, where it explains how 5 different test solutions will be presented during 3 trials of each to the subjects. The total amount of

This study has not started, so there are no adverse events or safety concerns reported. The committee specifically questioned the protocol on page 6, in the first paragraph, where it explains how 5 different test solutions will be presented during 3 trials of each to the subjects. The total amount of presentations should be 15 instead of 9. Laura Pons will follow up with Dr. Fennelly for clarification, and then she will report back to the committee.

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presentations should be 15 instead of 9. Laura Pons will follow up with Dr. Fennelly for clarification, and then she will report back to the committee.

8 Skeletal Muscle Apoptosis and Physical Performance, Oxidative RNA/DNA Damage and Repair in Aged Human Muscle PI: Christiaan Leeuwenburgh, PhD

Primary Reviewer: Stephen Kritchevsky, PhD This study was also reviewed in February 2011 with no safety concerns noted. The purpose of this study is to determine the extent of muscle apoptosis during aging in young healthy people and in high-functioning and low-functional older subjects. Dr. Leeuwenburgh reported that 31 of the original 34 subjects enrolled are still participating in the study.

Discomfort from the biopsy was reported, but none posed safety concerns. The committee reported no concerns related to this study.

9 Resveratrol for Improved Performance in the Elderly: The RIPE Trial; Resveratrol Supplementation to Improve Memory Dysfunction in Older Adults; Resveratrol for Reduced Muscle Lipid Content in Older Adults (PI/Leader: Todd Manini, PhD; Steve Anton, PhD)

Primary Reviewer: John Meuleman, MD This study was reviewed in February 2011 with no safety concerns noted. Overall, there were 14 reports related to diarrhea symptoms, but no safety concerns. This study is now complete.

This study has been completed.

10 Molecular Mechanisms of Skeletal Muscle Loss in HIV-infected Older Persons (PI: Todd Manini, PhD)

Primary Reviewer: John Meuleman, MD This study was reviewed in February 2011 with no safety concerns noted. This study is now complete.


11 Mitochondrial Function and Fatigue (PI: Todd Manini, PhD)

Primary Reviewer: Stephen Kritchevsky, PhD This study was also reviewed in February 2011 with no safety concerns noted. This study is now complete.


12 Other Business Dr. Kritchevsky will forward a reporting form to be completed for each study being reviewed that will be helpful for future DSMB calls. The form will also facilitate the recruitment report for each study including the number of participants enrolled, completed, and withdrawn. The planned end date of each study would also be helpful to the committee. In addition, a summary table of adverse events detailing how many adverse events, SAEs, and how many were related to the study should also be provided. The committee also stressed that studies no longer recruiting or dealing with participants

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should not be reviewed by the DSMB.

13 Next Conference Call

The next conference call will be scheduled December 2012.

Version 2013-03-14 Page 1 of 4

Summary Table of Closed Studies

Protocol Title & PI Status Resulting Grants & Publications

Research on Botanicals Obesity Reduction and Nutrition (The ReBorn Study) (Anton)

Study was terminated early due to problems with the investigational product. An updated protocol will not be submitted for consideration.

Grants: • R21 submission planned

Publications: • Investigations of botanicals on food intake, satiety, weight loss and

oxidative stress: study protocol of a double-blind, placebo-controlled, crossover study. Anton SD, Shuster J, Leeuwenburgh C, Zhong X, Yi J, He XB. 2011 Nov;9(11):1190-8. PMID: 2208858

Resveratrol for Improved Performance in the Elderly: The RIPE Trial (Anton & Manini)

Data collection completed; In data analysis

Grants: • R01 AT007564, Anton PI—fundable score

Publications: • Naugle K, Higgins T, Manini TM. Energy Metabolism and Diet: Effects on

Healthspan in Bioactive Foods and Aging. Published by Elsevier. Maryland Heights, MO USA.

Resveratrol for Reduced Muscle Lipid Content in Older Adults (Manini). This is an ancillary study to the RIPE trial.


Grants: • R01 AT007564, Anton PI—fundable score

Publications: • Naugle K, Higgins T, Manini TM. Energy Metabolism and Diet: Effects on

Healthspan in Bioactive Foods and Aging. Published by Elsevier. Maryland Heights, MO USA.



Role of Skeletal Muscle Blood Flow and Regeneration (Buford)


Grants: • R21 AR062612. Manini- Co-I. Novel exercise interventions to improve trunk

muscle in low back pain. Scored and resubmitted Publications: • Blood flow restriction enhances post-resistance exercise angiogenic gene

expression. Larkin KA, Macneil RG, Dirain M, Sandesara B, Manini TM, Buford TW. Med Sci Sports Exerc. 2012 Nov; 44(11): 2077-83. PMID: 22677927

• Blood flow restricted exercise and skeletal muscle health. Manini TM, Clark BC. Exerc Sport Sci Rev. 2009 Apr;37(2):78-85. PMID:19305199

• Delayed-onset muscle soreness induced by low-load blood flow-restricted exercise. Umbel JD, Hoffman RL, Dearth DJ, Chleboun GS, Manini TM, Clark BC. Eur J Appl Physiol. 2009 Dec;107(6):687-95. PMID: 19727801

• Manini TM, Vincent KV, Leeuwenburgh CL, Borst SE, Clark BC. Myogenic and proteolytic mRNA expression following blood flow restricted exercise. Acta Physiologica Scandinavia. Vol 201(2): 25-63. 2011.

Locomotor adaptability: a novel approach for detecting mobility deficits with aging (Clark)


Publications : • Neuromuscular contributions to age-related weakness. Clark DJ, Fielding

RA. J Gerontol A Biol Sci Med Sci. 2012 Jan;67(1):41-7. PMID: 21415261

Chemotherapy-induced muscle weakness and function limitation in older breast cancer survivors (Extermann & Manini)


Publications: • Models of accelerated sarcopenia: critical pieces for solving the puzzle of

age-related muscle atrophy. Buford TW, Anton SD, Judge AR, Marzetti E, Wohlgemuth SE, Carter CS, Leeuwenburgh C, Pahor M, Manini TM. Ageing Res Rev. 2010 Oct;9(4):369-83. PMID: 20438881


Respiratory Sarcopenia and Chronic Lung Infections in Elderly Women: An Investigator-Initiated Outpatient Pilot Study (Fennelly)

Withdrawn

Mitochondrial Function and Fatigue (Manini)


Grants: • R01 AG042525, Manini PI—fundable score • Work on this project led to being a co-investigator on the Women’s Health

Initiative, Manini, Early-stage Investigator. Publications: • Skeletal Muscle Mitochondrial Energetics Are Associated With Maximal

Aerobic Capacity and Walking Speed in Older Adults. Coen PM, Jubrias SA, Distefano G, Amati F, Mackey DC, Glynn NW, Manini TM, Wohlgemuth SE, Leeuwenburgh C, Cummings SR, Newman AB, Ferrucci L, Toledo FG, Shankland E, Conley KE, Goodpaster BH. J Gerontol A Biol Sci Med Sci. 2012 Oct 31. PMID: 23051977

• The impact of aging on mitochondrial function and biogenesis pathways in skeletal muscle of sedentary high- and low-functioning elderly individuals. Joseph AM, Adhihetty PJ, Buford TW, Wohlgemuth SE, Lees HA, Nguyen LM, Aranda JM, Sandesara BD, Pahor M, Manini TM, Marzetti E, Leeuwenburgh C. Aging Cell. 2012 Oct;11(5):801-9. PMID: 22681576

• Mitochondrial DNA sequence variation is associated with free-living activity energy expenditure in the elderly. Tranah GJ, Lam ET, Katzman SM, Nalls MA, Zhao Y, Evans DS, Yokoyama JS, Pawlikowska L, Kwok PY, Mooney S, Kritchevsky S, Goodpaster BH, Newman AB, Harris TB, Manini TM, Cummings SR; Health, Aging and Body Composition Study. Biochim Biophys Acta. 2012 Sep;1817(9):1691-700. PMID: 22659402



Molecular Mechanisms of Skeletal Muscle in HIV-Infected Older Persons (Manini)


Publications: • Models of accelerated sarcopenia: critical pieces for solving the puzzle of

age-related muscle atrophy. Buford TW, Anton SD, Judge AR, Marzetti E, Wohlgemuth SE, Carter CS, Leeuwenburgh C, Pahor M, Manini TM. Ageing Res Rev. 2010 Oct;9(4):369-83. PMID: 20438881

Task Specific Exercise for the Pre-Clinical Disabled (Manini)


Publications: • Role of self-efficacy (SE) and anxiety among pre-clinically disabled older

adults when using compensatory strategies to complete daily tasks. Higgins TJ, Janelle CM, Naugle KM, Knaggs J, Hoover BM, Marsiske M, Manini TM. Arch Gerontol Geriatr. 2012 Nov-Dec;55(3):611-24. PMID: 22770713

• Efficacy of resistance and task-specific exercise in older adults who modify tasks of everyday life. Manini T, Marko M, VanArnam T, Cook S, Fernhall B, Burke J, Ploutz-Snyder L. J Gerontol A Biol Sci Med Sci. 2007 Jun;62(6):616-23. PMID: 17595417

• Mobility decline in old age: a time to intervene. Manini TM. Exerc Sport Sci Rev. 2013 Jan;41(1):2. PMID: 23262463

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PEPPER CENTER OAIC DATA SAFETY MONITORING REPORT

Please complete the following form and attach the most recent external DSMB report if not previously reported (if applicable).

New Study Continuing Study

Protocol Title Skeletal muscle apoptosis and physical performance; Oxidative RNA/DNA damage and repair in aged human muscle

PI Christiaan Leeuwenburgh, PhD IRB # 429-2005

Most recent IRB approval date 10/23/12 (Expires on 10/20/13)

Target Enrollment Total 65 (N=20 young adults ages 20-35; N=25 old high-functioning adults ages 70-99; N=20 old low-functioning adults ages 70-99)

Anticipated Study Completion Date October 2013

ABSTRACT OF STUDY (Hypothesis and Design): Abstract: The age-related loss of muscle mass and strength, also termed sarcopenia, is a commonly recognized consequence of aging and has been associated with frailty, functional loss, hospitalization, and increased mortality among older people. Sarcopenia and its consequences have a considerable economic impact, since it has been estimated that the healthcare cost attributable to sarcopenia in the US in 2000 was $ 18.5 billions. Furthermore, this expenditure is expected to dramatically rise due to the estimated increased number of elderly. Despite the clinical and socioeconomic impact of sarcopenia, the mechanisms underlying this syndrome are still poorly understood. Preclinical animal models strongly suggest that apoptosis, a programmed cell death, might play a prominent role in the age-related muscle wasting. Hence, within this development project we propose to investigate the extent of cell death from apoptosis in human skeletal muscle and its contribution to sarcopenia and physical impairment in older subjects. Furthermore, we propose to investigate the involvement of energy deficits and apoptosis arising from mitochondrial dysfunction in the pathophysiology of the age-related muscle loss. This development project will test if apoptosis of muscle cells is a key mechanism in the development of sarcopenia, physical function decline and disability in older adults and if this is linked to energy deficits. In specific aim one, we will assess the extent of muscle apoptosis in muscle biopsies obtained from the vastus lateralis muscle of young control subjects (ages 20-35) and high-performance and low-performance older subjects (age range 70-99 years). In specific aim 2, we will investigate the role of Poly(ADP-ribose) polymerase 1 (PARP-1) and apoptosis-inducing factor (AIF) in the induction of skeletal muscle apoptosis. In specific aim 3, we propose to investigate the contribution of the muscle energy deficit, due to the age-related mitochondrial dysfunction, in the development of muscle wasting. Finally, in specific aim 4, we propose to reassess after four years physical performance, muscle mass and the extent of muscle apoptosis, in the high-performing participants, in order to correlate eventual decline in physical function, muscle mass and functional status, with changes in muscle apoptosis and in biochemical parameters in this very old population. Physical performance will be established according to the summary performance score obtained in the Short Form Physical Performance Battery (SPPB). In addition to the SPPB we will also employ hand grip strength and knee extensor strength tests and we will quantify muscle contractile area using 3D magnetic

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resonance imaging. Disability will be assessed using a self-report questionnaire. These studies will enhance our understanding of the biology and pathophysiology underlying the geriatric syndrome of sarcopenia and provide significant and novel insights that will enable us to identify new potential targets for interventions aimed at preventing and treating sarcopenia and functional impairment in older adults. Research Plan: Using a cross-sectional design (age 20-35 and 70-99 years) we will quantify the level of apoptosis in the vastus lateralis muscle in a healthy (see inclusion/exclusion criteria) population. Apoptosis of irreplaceable post-mitotic cells may be important mechanism in the development of sarcopenia and has not been investigated in healthy humans with age. A variety of chronic diseases have documented cases of apoptosis in human skeletal muscle; i.e., chronic pulmonary diseases and patients with heart failure. However, it is essential to establish baseline levels of apoptosis in relatively healthy humans before proceeding to more complex conditions, such as disease conditions and frailty. In addition, we will determine knee extensors strength, the myocyte volume, extra cellular space and the myonuclear domain (cross-sectional muscle area/nucleus) by quantifying the number of nuclei within a cross-sectional area of muscle. This specific aim will establish baseline levels of apoptosis and assess standard measures to assess sarcopenia.

Study design. To accomplish our aims, we propose two separates, yet complementary studies

1. In order to gain insights into changes of the apoptosis potential with age and its contribution to muscle mass and strength loss, we will quantify the level of apoptosis in the vastus lateralis muscle in young (age 20-35 years; N =20), old high-functioning, (age 70-99 years; N = 25), and old low-functioning (age 70-99 years; N = 20) subjects. Muscle biopsy and MRI scan to quantify quadriceps contractile area will be performed in the study participants. Muscle strength will be assessed by hand grip strength test and the isokinetic knee extensor test.

2. Preliminary data on the contribution of skeletal muscle apoptosis to sarcopenia, physical

function loss in advanced age will be gathered by quantifying the extent of muscle apoptosis in the vastus lateralis muscle of old (age 70-99 years), high-functioning (N = 25) and low-functioning (N = 20) subjects, along with measures of muscle mass (MRI), muscle strength (hand grip and knee extension test), and overall physical performance (Short Physical Performance Battery, SPPB (67)). In addition, we will collect preliminary data regarding the role mitochondrial-driven apoptosis in muscle wasting and functional loss at old age. A brief physical exam will be administered to all study participants, and major biological signs (body weight, standing height, body temperature, blood pressure, and pulse) will be recorded. A basic questionnaire regarding participants’ health history will also be administered

After four years the old participants will be revaluated for physical performance, functional status muscle mass (MRI), muscle apoptosis, and biochemical parameters Disability will also be assessed using a self-report disability questionnaire. In order to gain information regarding changes in physical performance and functional status with time and to maximize retention, an annual visit employing SPPB, hand grip and knee extensor testing, will be scheduled for the old participants.

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INCLUSION / EXCLUSION CRITERIA (Please list in box below) Inclusion criteria. The following inclusion criteria will be adopted to select the study participants:

• males and females aged 20-35 and 70-99 years • sedentary lifestyle (i.e., the subject has spent less than 20 minutes per week in the past

2 month performing structured physical activity, such as exercising at a gym and/or weight training)

• willing and able to give informed consent. Exclusion criteria:

• history of smoking in the prior 12 months • active treatment for cancer or history of cancer in the past 3 years • congestive heart failure NYHA Class III or IV • previous stroke with upper and/or lower extremities involvement within the last 6 months • peripheral vascular disease Fontaine Class III/IV • History of life-threatening cardiac arrhythmias, stroke, severe Parkinson’s disease or

severe neurological disorders likely to interfere with physical function • cognitive impairment (i.e., MMSE ≤ 23) • renal disease requiring dialysis • lung disease requiring steroids • lower extremity amputation • severe osteoarthritis that interferes with physical function • Complicated diabetes • inflammatory diseases such as active rheumatoid arthritis, vasculitis, autoimmune

disorders, and inflammatory bowel disease • life-threatening illnesses with an estimated life expectancy less than 1 year • history of drug or alcohol abuse • taking GH and/or estrogen replacement therapy • Testosterone medication • Anticoagulant therapy • involved in active weight loss > 5 kg in prior 3 months • planning to relocate out of the study area in the next 4 years (ages 70-99 only) • pregnancy • SPPB score of 8-10 • Lidocaine allergy • MRI exclusions, such as pregnancy, claustrophobia, heart pacemaker / defibrillator,

heart valve prosthesis, aneurysm clip, metallic stent, neurostimulation system, cochlear implants or inner ear prosthesis, insulin pump or other infusion pump, metal slivers in the orbital area/eye socket

Temporary exclusion criteria:

• recent bacterial infection (< 2 weeks) • acute febrile illness in prior 2 months • high blood pressure (i.e., BP ≥ 180/110 mm Hg) at the screening visit (subject will be

referred to his/her physician and reevaluated after appropriated therapy being instituted) • major surgery or hip/knee replacement in the past 6 months

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Current Enrollment

Please place an “X” the box for each:

Ongoing Complete * N/A

Recruitment X^ Intervention X

Participant Contact X ^ Please see Protocol Revision section on page 9 of this report for pending IRB submission. *If you have marked complete for each row, this will be the last report for the DSMB.

Enrollment Summary (as of 3/5/13)

Active Completed Withdrawn or Lost to Follow-up Deceased Total Enrolled

14 42 21 3 80 Participants Who Have Withdrawn Consent or Lost to Follow Up (List in order of most recent to oldest since start of study.

Date:

Acrostic:

Reason for withdrawal or loss to follow up and state if it was related to the intervention or an adverse event. (attach additional information if needed):

02/12/13 lsooox Participant withdrew consent - moved to Maryland. 12/04/12 wvedhn Participant withdrew consent due to personal reasons. 12/04/12 ldwvlv Participant withdrew consent due to personal reasons. 04/23/12 fahwem Participant withdrew consent due to health reasons. 09/15/11 emiqzr Participant completed Visit Years 1-3, however could not be contacted for the

Year 4 visits. Participant was lost to follow-up. 03/29/10 btmkeo At visit 2, participant’s blood pressure was found to be high. Participant was

asked to follow-up with PCP for clearance to continue in the study. Participant never received clearance to return for additional visits. Participant was lost to follow-up.

02/08/10 irrbft Participant withdrew consent due to personal reasons. 10/07/09 fcyxzc Participant completed first study visit, and then could not be contacted for next

visit. Participant was lost to follow-up. 10/02/09 zburuh Participant withdrawn from study by PI (aversion to blood draws). 07/13/09 hqvayy Participant withdrew consent due to health reasons. 06/18/09 axhxem Participant withdrew consent due to health and personal reasons. 05/07/09 pryocf Participant withdrew consent due to personal reasons. 04/14/09 srvnch Participant withdrew consent due to personal reasons. 11/24/08 shqzsg Participant withdrew consent due to personal reasons. 11/14/08 hyrtqe Participant qualified for study, but was put on waitlist as high functioning group

was filling quickly. Participant withdrew from waitlist. 07/15/08 xmwudi Participant completed first two study visits, and then could not be contacted for

next visit. Participant was lost to follow-up. 07/03/08 hasnhi Participant completed first three study visits, and then could not be contacted for

next visit. Participant was lost to follow-up. 06/10/08 ofzrwc Participant withdrew consent- on advice of PCP due to nerve damage in back and

leg. 05/27/08 dbbfzf Participant withdrew consent- moved out of area. 05/06/08 epghas Participant completed first two study visits, and then could not be contacted for

next visit. Participant was lost to follow-up. 02/22/08 gfzynw Participant withdrew consent- too nervous to complete biopsy.

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Total Number of Protocol Deviations: _14___ (List in order of most recent to oldest since start of study)

Date:

Acrostic:

Explanation:

03/2013 N/A In review of all study records in preparation for this report, it was noted that we enrolled a larger number of participants than initially targeted (80 v. 65 participants).

07/2012 lsooox Missed year 3 visit. Participant will complete measurement at next visit. 02/2012 fahwem Missed year 3 visit. Participant will complete measurement at next visit. 12/2011 vlosvl Missed year 3 visit. Participant will complete measurement at next visit. 07/2011 lsooox Missed year 2 visit. Participant will complete measurement at next visit. 06/2011 evyppv Missed year 3 visit. Participant will complete measurement at next visit. 03/2011 ufalxv Missed year 2 visit. Participant will complete measurement at next visit. 02/18/11 xsaymp Year 3 gift card was given at Year 4 visit 1. Participant received the missed

compensation at the next appointment 12/2010 ivbjma Missed year 3 visit. Participant will complete measurement at next visit. 12/2010 vlosvl Missed year 2 visit. Participant will complete measurement at next visit. 09/2010 ofmaos Missed year 3 visit. Participant will complete measurement at next visit. 06/2010 evyppv Missed year 2 visit. Participant will complete measurement at next visit. 05/17/10 srvqef Year 3 hand grip measurement was not collected. Participant still enrolled and

will complete measurement at Year 4 visit. 12/11/09 scxxhq Physical Exam occurred after visit 2. Participant completed study with no

complications. AE INFORMATION: Please use table below to report any Adverse Events that are related to the study (Possibly, Probably, or Definitely). They do not have to be reportable to IRB to be included (i.e. they also do not need to be unexpected or related). If blinded, you do not need to complete intervention arm. If there is not more than one intervention it may be left blank. TOTAL Number of AEs__47______ (List in order of most recent to oldest since start of study) * shaded entries represent events that have previously been reviewed by this Committee

Date of Onset Date Ended Acrostic Event Relationship

with Study Explanation

If related 11/27/12 11/28/12 vzizsq Discomfort near the

biopsy site Yes The participant took

Tylenol on 11/27/12. Reported being better on 11/28/12. No further follow-up is required.

11/27/12 11/27/12 vzizsq Dizziness after biopsy procedure

Yes The participant did not have any breakfast that morning, was given water and felt better before leaving. No further follow-up is required.

07/18/2012 coqrlv Low Blood Pressure

No Participant experienced low blood pressure and did not complete

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the assessment visit. Was advised to f/u with PCP and report back to study staff.

4/12/12 Exact date is unknown

itwmhd A little tender near the biopsy site

Yes On 4/12/12 the participant reported feeling a little tender near the biopsy site, otherwise felt great. No further follow-up by the study team beyond phone call 4/13/12 and phone message asking to call back on 4/16/12.

June/July 2012 (exact date is unknown)

June/July 2012 (exact date is unknown)

jrsyyg Fell off tractor & injured left shoulder

No Participant seen by PCP and orthopedics. No treatments or hospitalization required.

April/May 2012 (exact date is unknown)

April/May 2012 (exact date is unknown)

jrsyyg Fell & hit head with slight concussion.

No ER visit, not hospitalized.

11/23/11 11/23/11 vzizsq Dehydration No 5 hour stay in hospital for dehydration. Released same day.


rxgoyz Little soreness near the biopsy site

Yes No further follow-up by the study team beyond the phone call on 8/12/2011

June /2011 (exact date is unknown)

June/2011 (exact date is unknown)

sqtvsf Skin cancer removed from back of left leg

No Outpatient procedure with no complications.

01/25/2011 01/25/2011 zvmraf Fainted due to blood pressure of 80/60

No Participant was seen at ER. Removed from blood pressure medication, given CT scan and IV fluids.

01/02/2011 Unknown ofmaos Fell at home & injured right ankle.

No Participant had an x-ray on 1/5/11. Pt is waiting for the results as to whether or not she needs a cast.

12/17/2010 12/17/2010 olftun Muscle soreness Yes 05/08/2010 07/08/2010 sqtvsf Fell out of golf cart No Participant received

12 stitches in the finger and leg.

04/01/2010 04/04/2010 fahwem Soreness from biopsy Yes

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03/04/2010 03/04/2010 xgnfzz Non-Hodgkin’s Lymphoma Cyst Removed

No Outpatient procedure to remove cyst from the throat.

03/17/2009 03/18/2009 orwrvn soreness when stretching leg-from biopsy

Yes Participant took 1 Tylenol

02/24/2009 03/03/2009 pxhftk Bruising from biopsy & lump under skin

Yes


wvedhn a tiny bruise around biopsy site

Yes No pain was reported. No further follow-up is required (phone call by the study team on 2/11/09.


ldwvlv a little sore Yes No further follow-up is required (phone call by the study team on 1/29/09).


emiqzr sensitive when going from sitting to standing position

Yes No further follow up by the study team beyond the phone call on 12/4/08.

11/04/2008 11/10/08 vzizsq Soreness, bruising and swelling at biopsy site

Yes Resolved (the participant called 11/10/08) No further follow-up is required.


ofmaos tenderness at biopsy site

Yes Participant took 2 aspirin. Resolved (the study team followed-up during MRI visit on 11/20/2008)

10/16/2008 10/16/2008 jqvgew little sore post biopsy Yes 10/14/2008 10/15/2008 isepja stiffness & bruising at

biopsy site Yes

10/14/2008 10/14/2008 isepja Light headed after biopsy

Yes

10/02/2008 10/03/2008 itwmhd Small knot after biopsy w/ bruising & sensitive to touch

Yes Participant took 3 Tylenol

09/17/2008 09/19/2008 qdxslj Soreness from biopsy Yes 08/20/2008 08/22/2008 wqbsaf Soreness from biopsy Yes 08/20/2008 08/20/2008 wqbsaf light headed before

biopsy Possible

08/14/2008 08/16/2008 syfszr Soreness, hematoma and bruising from biopsy

Yes

07/29/2008 07/29/2008 rdmnly Dizziness after biopsy Yes Participant was given Gatorade. Dizziness resolved within a few minutes.

07/21/2008 08/21/2008 srvqef Bruising from biopsy Yes 07/10/2008 07/11/2008 srvqef Soreness from biopsy Yes 07/10/2008 07/11/2008 orpdxb Soreness from biopsy Yes

Page 8 of 9

06/19/2008 06/20/2008 dmdazg Soreness from biopsy Yes 06/10/2008 06/12/2008 idpfoh Soreness from biopsy Yes 05/29/2008 Unknown bwvfxw numbness 1.5 inches

below biopsy site Yes Referred to PCP,

neurologist, and finally orthopedist. Prescribed Naproxin with no benefit, Next step MRI (1/29/09). No further follow-up by the study team. Outcome is unknown

05/29/2008 Unknown bwvfxw Soreness from biopsy Yes Resolved (no mentioning of soreness during 6/3/08 visit). No further follow-up is required.

05/29/2008 05/29/2008 zglvdg Felt faint during biopsy Yes Participant given food and Gatorade. Faintness resolved within a few minutes.

05/29/2008 05/30/2008 zglvdg Soreness from biopsy Yes 05/29/2008 06/13/2008 cifbau Extreme soreness from

biopsy Yes

05/27/2008 05/27/2008 nkjkxx tongue numb post biopsy

Possible

05/27/2008 05/28/2008 nkjkxx Soreness from biopsy Yes 05/15/2008 05/15/2008 dmdazg Car accident-no injuries No 04/28/2008 06/28/2008 olftun Reported hematoma

from biopsy Yes

03/25/2008 03/26/2008 rygoyx Soreness from biopsy Yes 01/22/2008 02/04/2008 olftun Fever and Bronchitis No SAE INFORMATION: Please use table below to report all events that are Serious since the start of the study. They do not have to be reportable to IRB to be included (i.e. they also do not need to be unexpected or related). If blinded, you do not need to complete intervention arm. If there is not more than one intervention it may be left blank. TOTAL Number of SAEs___10_____ (List in order of most recent to oldest since start of study) * shaded entries represent events that have previously been reviewed by this Committee

Date of Onset Date Ended Acrostic Event Relationship


If related 02/16/2013 02/16/2013 ofmaos Death No Participant

hospitalized for pneumonia on 2/13/13.

06/01/2012 Ongoing lymcxa Liver Cancer No Diagnosed with liver cancer in June 2012.

03/16/2011 03/16/2011 zlxvvr Death No Throat, lung and colon cancers

Page 9 of 9

12/09/2010 12/22/2010 ofmaos Fluid in the lungs No Fluid in the lungs was drained and participant given prescription for 1 week.

01/25/2010 02/01/2010 zrxnbb Infection in Right knee No Given IVs and antibiotics. Released 1 week later from hospital.

12/29/2009 12/31/2009 zrxnbb Right knee replacement No Discharged from hospital 12/31/09

04/04/2010 04/14/2010 jgvgew Colon Cancer No Surgery to remove cancer on 4/14/10; Recovered.

01/21/2010 01/25/2010 olftun Pneumonia No Participant given antibiotics and IV.

09/10/2009 Unknown evyppv Cyst removed from back No No longer able to walk. Exact end unknown, however participant seen in clinic on 12/6/12 and completed 400 m walk.

10/20/2008 10/20/2008 dmdazg Death No Cause of death liver and lung cancers.

PROTOCOL CHANGES: Include all changes, since last DSMB review. Explain if safety related.

Date of change Protocol Change Made

Explanation of Safety Relation

Pending Funding was not obtained to complete

microbiopsies in 20 participants (10 newly recruited young participants and 10 existing older participants). The protocol has been revised to remove this aspect of the study. Recruitment to enroll these participants will not be conducted.

No relation to participant safety.

10/17/12 New investigators and staff were added to the study protocol.

No relation to participant safety.

Protocol Page 1 of 6 IRB version 03.09.04 PI version 3/18/2013 10:59 AM

Protocol

1. Project Title Cortical control of walking: assessment, mechanisms and functional implications

2. Investigator(s): David J. Clark, ScD (Principal Investigator) Evangelos Christou, PhD (Co-Investigator) 3. Abstract: Walking is a complex sensorimotor task requiring coordination of numerous muscles, yet healthy adults easily control steady state walking with little direct cortical control of movement details. This “automatic” control is facilitated by sub-cortical networks that coordinate walking. Sub-cortical control is beneficial because the cortex remains relatively unencumbered, and thus available for other important information-processing such as ambulating safely in a community setting. We propose that any disruption to this sub-cortical control will necessitate a compensatory increase in the role of the cortex to successfully control walking. An abundance of evidence indicates that older adults have an increased reliance on the cortex for control of walking. Classically, this increased cortical demand has been shown by marked decrements in performance on dual-task paradigms, in which a secondary attention-demanding task is combined with walking. Furthermore, increased cortical activation has been observed during walking and during other coordinated tasks in older adults. The reasons for increased cortical demand are poorly understood, though increased cortical activity in regions associated with sensorimotor monitoring of task performance suggest aberrant processing of sensory information by sub-cortical centers. Sensory information is known to be a critical input to the sub-cortical networks that coordinate walking. Peripheral sensory impairments are common in older adults and have been implicated as a determinant of walking deficits. Based on this cumulative evidence, our overarching hypothesis is that peripheral sensory impairments in older adults disrupt sub-cortical control of walking, leading to increased cortical demand of walking and concomitant deficits in mobility function. 4. Background: Accumulating evidence indicates that cortical demand of walking is increased with aging, but the specific reason(s) for this increase remain unclear. We propose that a variety of age-related impairments can disrupt the normal operation of sub-cortical networks that control walking (e.g., brainstem centers contributing to balance and locomotion6, spinal cord circuitry for pattern generation3,15, peripheral sensory receptors3,12, etc). In order to maintain mobility function, cortical involvement in the control of walking must then increase to compensate for sub-cortical disruptions. In the present study, we focus on the potential role of peripheral sensory impairments.


Peripheral sensory information is a critical source of input to the spinal circuitry that facilitates sub-cortical control of walking3,9,10. It is therefore notable that peripheral sensory impairments are common in older adults and may disrupt sub-cortical control. A major study of almost 800 older adults reports that more than one-third of adults age 70 and above exhibit bilateral peripheral sensory impairments based on clinical evaluation11. In many cases the sensory impairment is not accompanied by a known disease/injury11,16, and the individual may not be overtly aware of the sensory impairment. Among the most compelling evidence that impaired peripheral sensation increases cortical demand of walking comes from a recent study which showed that dual-task performance was more compromised in diabetic patients with peripheral neuropathy relative to diabetic patients without peripheral neuropathy13. But even in non-diabetic older adults, sensory impairments have been implicated in walking deficits11.

Dual-task paradigms offer a clinically feasible approach for assessing cortical demand of walking, and involve walking while performing a secondary task requiring cortical involvement17. Dual task interference should be low if control of walking is predominantly sub-cortical, and higher if walking requires substantial cortical resources. Findings from dual-task paradigms are valuable, but this approach is nevertheless plagued by a variety of complicating factors. Most notably, 1) different secondary tasks yield considerably different amounts of dual-task interference and 2) difference outcome measure have considerably different sensitivity for detecting dual-task interference (i.e., walking speed vs. gait parameters vs. secondary task performance, etc2,18,19). Currently, there is no standard by which to choose the secondary task and outcome measure. The findings of the proposed study may yield a gold standard.

Dual-task performance depends largely on the “supply and demand” framework described by Seidler et al 20. “Supply” refers to the availability of cognitive resources (i.e., cognitive function). “Demand” refers to the cognitive (i.e., cortical) demands of walking. Either reduced cognitive supply or increased cognitive demand can impair walking ability, and if both occur then walking deficits are further exacerbated21. In this proposal, we roughly control for “supply” by recruiting only older adults with high cognitive function. We instead focus on “demand” by assessing whether cortical demand of walking increases in response to impaired peripheral sensation.

There are some alternatives to dual-task paradigms for probing cortical contributions during walking, including EEG and fNIRS. However, a weakness of these techniques is that it isn’t possible to know whether altered brain activity actually corresponds to an altered neuromuscular activation. In this project we will use two novel EMG-based measures to specifically assess altered neuromuscular activation. EMG spinal map analysis will be used in conjunction with dual-task paradigms and EMG cross-wavelet spectral analysis will be used as an independent measure of cortical demand. This project leverages from the expertise and infrastructure provided by the PI’s joint affiliation with the UF Pepper Center and VA Brain Rehabilitation Research Center. The resources available to the PI present a unique opportunity to launch this innovative line of research quickly and at low cost.


5. Specific Aims: Specific Aim 1a: To determine if peripheral sensory impairment increases cortical demand of walking. Specific Aim 1b: To determine if increased cortical demand of walking reduces mobility function. Specific Aim 2: Test the hypothesis that electromyographic (EMG) measures are more sensitive than gait parameters to determine cortical demand using a dual-tasking paradigm in older adults.

6. Research Plan: Non-VA Research: Participant recruitment and enrollment criteria. Participants will be recruited with assistance from the Pepper Center Recruitment, Adherence and Retention Core. Volunteers who respond to our recruitment ads/flyers will undergo a preliminary screening by telephone. Volunteers who meet the following criteria will be invited to our study site for further screening and assessment:

• age 65-85 • body mass index from 19-32 (based on self-reported weight and height) • no use of cane/walker (based on self-report) • report physical fatigue when walking a quarter mile, climbing two flights of stairs

or performing household chores • no substantial involuntary weight gain/loss in the past six months • blood pressure below 160/95 (based on self report) • no broken/fractured bones in past year • no central nervous system injury • no terminal illness • no pain when walking

At visit 1 we consent and enroll participants, who will then undergo further screening and assessment. All participants will undergo the following assessments: height and weight assessment for BMI verification, blood pressure peripheral sensory function, 10 meter preferred walking speed, 10 meter fast walking speed, 400 meter preferred walking speed, Short Physical Performance Battery, Berg Balance Scale, Modified Gait Efficacy Scale, Mini Mental State Examination. Twenty Individuals meeting the following criteria will be invited to also participate in a second assessment visit at the VA Brain Rehabilitation Research Center (detailed in the VA Research section):

• body mass index from approximately 19-32 • blood pressure below 160/95 • walking speed < 1.0 m/s, • half of the participants will have peripheral sensory impairments (see next

section) • Mini Mental State Exam score ≥ 21 • self-report of some difficulty/fatigue during daily tasks. • Berg Balance Scale ≥41


If all criteria are met, the participant will then complete the VA informed consent form for Visit 2. Both men and women will be recruited, with a goal of 50% enrollment for each sex. We expect that recruitment of minorities will be consistent with the population in the greater Gainesville area. Non-VA Research: Peripheral sensory testing and functional assessments. Peripheral sensory function will be assessed using clinically accepted techniques11. A person will be considered to have a sensory impairment if one or both lower limbs meet any of the following criteria: 1) impaired lower limb proprioception assessed by a standard clinical limb matching test; 2) inability to consistently detect a 10-g Semmes Weinstein monofilament measured at the following sites: first and fifth metatarsal heads, pulp of the hallux, heel; 3) inability to consistently detect vibratory sensation of the great toe using a tuning fork; or 4) absence of ankle tendon reflex. Half of our sample will demonstrate peripheral sensory impairment and half will not demonstrate sensory impairment. VA Research: Neuromechanical assessment of walking. In addition to the twenty older adults who will undergo the VA research procedures, we will also test up to 10 healthy young adults (<35 years old). Young adults will be required to have good functional capability (no difficulty performing daily tasks according to self report). Participants will be invited to the VA Brain Rehabilitation Center’s Human Motor Performance Laboratory for a neuromechanical assessment of walking. Participants will walk at a self-selected comfortable speed on our instrumented treadmill, which acquires ground reaction forces independently from each leg. Kinematic data (e.g., joint angles and velocities) will be acquired at 100 Hz by a 12 camera motion capture system (Vicon Motion Systems) from reflective markers placed on the body using a modified Helen Hayes marker set. Surface electromyography (EMG) will be recorded bilaterally at 2000 Hz by a commercially available acquisition system (Motion Lab Systems, Inc) using surface electrodes placed over the tibialis anterior, soleus, medial gastrocnemius, vastus medialis, rectus femoris, medial hamstrings and lateral hamstrings. Each walking trial will be videotaped. During treadmill walking, participants will wear a safety harness to prevent falling. Data for each walking condition will be recorded for 30-60 seconds. Participants will also walk overground on a 14 foot instrumented walkway at comfortable pace. This walkway provides spatial and temporal measures of gait (e.g., speed, cadence, step length, step width, duration of gait cycle phases, etc). Participants will perform multiple passes over the walkway such that a minimum of 15 gait cycles are acquired for each walking condition. During both treadmill and overground walking, participants will walk normally and under dual-task conditions: Walking + hand/arm motor task: The participant will hold a clipboard in one hand. On the clipboard will be a sheet of paper on which two squares are drawn (with dimensions of 2”x 2”), one near the top of the sheet and one near the bottom. A few pennies will be placed within the top box on the clipboard. The participant will be instructed to slide each penny, one by one, from the top box to the bottom box using the index finger. He/she will then slide each penny back to the top box, and continue this pattern for the duration of the walking trial.


Walking + cognitive task (auditory 2-back test): a list of letters will be read to the participant. One letter will be read at the instant of heel strike for every 2nd gait cycle of the dominant leg. If the participant hears the same letter read twice with only one other letter in between, he/she will say the repeated letter. Participants may be asked to walk barefoot for a small number of walking trials, the cumulative time of which will not exceed 5 minutes. Participants may also be asked to wear textured plastic insoles in their regular shoes for a small number of walking trials. Participants will also be asked to perform other tasks while walking, such as taking a long step, high step, short step, step over an obstacle, accelerating, decelerating, stooping to pick up an object, reaching overhead, walking over a soft mat, walking with dim lighting and/or wearing a weighted vest (not to exceed 15% of body weight or participant comfort, whichever is less). We will ask participants to wear fNIRS sensors on their scalp during the data collection procedures. fNIRS (functional near infrared spectroscopy) is a technology that uses near-infrared light to measure changes in the concentration of oxygenated and deoxygenated hemoglobin in tissue, such as the brain. fNIRS is a safe, non-invasive, portable, relatively inexpensive alternative to other neuroimaging technologies. We may ask participants to wear skin conductance sensors on their fingers during the data collection procedures. Skin conductance, also known as galvanic skin response, is a safe measure that is associated with psychological or physiological arousal/stress. We may ask participants to perform some tasks outside of the Human Motor Performance Lab, but still on VA property. These tasks may include walking on grass, walking up/down a wheelchair ramp, walking up/down stairs, walking in a busy corridor of the medical center and walking on a concrete pathway. Participants will be videotaped during the data collection session with their approval. If they approve, the camera will be positioned such that the head and face of the participant are cut out of the picture. In the event that faces are captured, we have software the can be used to obscure the face prior to using videos for approved purposes. List of VA Equipment: instrumented treadmill, overhead safety harness, GAITRite instrumented walkway, fNIRS, electromyography, motion analysis system, video camera, textured plastic shoe insole.

7. Possible Discomforts and Risks: The proposed locomotor assessments are routine and pose low risk to study participants. Treadmill walking studies has been conducted extensively for many years in various populations without significant occurrence of adverse advents. As with all physical activity, there is small risk of injury (e.g., strained muscle or tendon) or falling. However, participants will wear a safety harness during treadmill walking to prevent falls. There is a slight risk of skin irritation with the use of surface EMG and fNIRS


electrodes/sensors and tape. Fatigue and/or muscle soreness may occur due to physical exertion associated with testing. Sufficient rest will be provided to minimize discomfort for participants. Participation in these procedures is not required and there are no alternative procedures. Assessment of skin conductance has no known substantial risk.

8. Possible Benefits: Participants will receive no immediate or guaranteed benefits from participation in the study. However, as a result of their participation we will have improved understanding of neural factors that limit mobility function with aging. This knowledge will be used to develop appropriate interventions for avoiding mobility disability.

9. Conflict of Interest: There are no conflicts of interest.

1

Newly Supported Pepper Studies Data Safety Monitoring Plan

Protocol title: Cortical control of walking: assessment, mechanisms and functional implications PI: David Clark, ScD Please attach latest stamped informed consent. Describe the plan to monitor participant safety by addressing the following elements. If any of these do not apply, explain why. 1. What is the risk level for your study (check only 1 box)? No Greater than Minimal Risk

The probability and magnitude of harm or discomfort anticipated in the research are not greater than those ordinarily encountered in daily life or during the performance of routine physical and psychological examinations or tests. This category includes protocols that pose “no greater than minimal risk” according to federal regulations. Requires Minimal-Intensity Monitoring.

Minor Increase over Minimal Risk Research involves a minor increase over minimal risk. There is an increased probability of a low-severity event that is reversible (e.g., muscle/joint soreness or sprain from exercising) or the likelihood of serious harm occurring is extremely rare (e.g., fatal anaphylaxis from skin testing). Requires Low-Intensity Monitoring.

Moderate Risk Risks are greater than minimal, but are not considered high. There is an increased probability of a moderate-severity event that is reversible (e.g., hypoglycemic episode, bronchoconstriction or infection), but there is adequate surveillance and protections to identify adverse events (AEs) promptly and to keep their effects minimal. The likelihood of serious harm is extremely rare to very rare. Requires Moderate- Intensity Monitoring.

High Risk The study risk is greater than a moderate-risk study due to the increased probability for generating SAEs. There is an increased probability for the occurrence of a study related event that is serious and prolonged or permanent, or there is significant uncertainty about the nature or likelihood of adverse events. Requires High-Intensity Monitoring.

2. What level of safety monitoring will be used for your study (check only 1 box)?

Minimal-Intensity Monitoring -The PI will monitor the study (interpreting test results, determining levels of severity and association for adverse events), with prompt reporting of AEs and other study related information to the IRB, GCRC, sponsor, and other agencies as appropriate. Other information to be reported includes continuing annual renewal updates, any protocol deviations, changes to the protocol or consent form, etc.

Low-Intensity Monitoring -The PI will monitor the study as noted under “Minimal-Intensity Monitoring.” Team meetings by the PI and his/her staff will be conducted on a routine basis when appropriate. The IRB and the GCRC will be promptly notified if the risk/benefit ratio changes.

Moderate-Intensity Monitoring - The PI will monitor the study on a day-to-day basis. This includes all monitoring activities described above in “Low-Intensity Monitoring.” In addition, most drug trials will require well-described criteria for dose escalation, criteria defining maximum tolerated dose (MTD), and/or criteria for stopping the trial or discontinuing involvement of a study subject. A Safety Monitor or a Data Monitoring Committee must also be utilized to review adverse events.

High-Intensity Monitoring - The PI monitors the study on a day-to-day basis. This includes all monitoring activities described above in “Moderate-Intensity Monitoring.” Most high-risk protocols will also require a Data Monitoring Committee (DMC). High-risk, double-blinded trials must have an independent DMC. An independent DMC is also required by NIH Guidelines for all NIH-funded multicenter Phase III clinical trials involving greater than minimal risk of harm or gene transfer or gene therapy. If no other DMC is in place for a single-institution clinical trial, the PI may use the GCRC Human Subject Protection Committee as an independent board to fulfill this function.

2

3. Who in addition to the Pepper Center DSMB will be reviewing the accumulating study wide

safety data (across all study participants) for your study? PI only Safety monitor(s) – provide names / affiliations below Study affiliated DMC* Independent DMC* * If checked, please remember to forward these reports along with your study report for the

Pepper DSMB to review. 4. How frequently will the accumulating study wide safety data (across all study participants) be

reviewed by the above noted individual(s) or committee (e.g., quarterly, every 6 months)? Every six months. 5. Study Risks and Safety

a. List all expected risks (physical, psychological, social, risks to confidentiality, etc.) associated with the study intervention(s) (e.g., drug, procedures, device) and/or the study procedures/tests. Quantify risks whenever possible (e.g., tremors: 5% of subjects; tachycardia: common vs. rare). All risks must be included in the study consent form. As with all physical activity, there is small risk of injury (e.g., strained muscle or tendon) or falling. There is a slight risk of skin irritation with the use of surface EMG and fNIRS electrodes/sensors and tape. Muscle soreness may occur due to physical exertion associated with testing. Sufficient rest will be provided to minimize discomfort for participants. There is a slight risk that information about a participant could be revealed inappropriately or accidentally. Depending on the nature of the information, such a release could embarrass the participant or compromise information security.

b. How will the expected risks described in 5a above be minimized? Participants will wear a safety harness during treadmill walking to prevent falls. c. How will the expected risks described in 5a above be treated, should they occur? Emergency medical treatment is available at Shands Medical Center and/or the Malcom Randall

VA Medical Center, as appropriate. Non-emergency medical treatment, if necessary, will be handled on a case by case basis depending on the severity and nature of the injury.

d. What tests/measurements (and their frequencies) will be performed to monitor participant

safety (e.g., labs, x-rays/scans, PE, questionnaires, performance measures, ECG, pathology)? Blood pressure and heart rate will be assessed at the beginning of each study visit prior to

participating in study activities. Additional blood pressure and heart rate measurements may be made if the participant appears uncomfortable, fatigued, etc.

e. Once subjects are enrolled in the study, what pre-specified lab alert values or other clinical

criteria would lead to discontinuation or alteration of treatment (e.g., ALT>3x ULN: decrease dose by 50%)?

Systolic blood pressure above 160 or diastolic blood pressure above 95.

UF I Institutional Review Board UNIVERSITY of FLORIDA

INFORMED CONSENT FORM to Participate in Research, and

AUTHORIZATION to Collect, Use, and Disclose Protected

Health Information (PHI)

University of Florida Health Center

Institutional Review Board APPROVED FOR USE

From 1 /t iJ /-. 0 I ;l_ Through ?/9� 0 { a

�

INTRODUCTION

Name of person seeking your consent: _________________ _

Place of employment & position:--------------------

Please read this form which describes the study in some detail. A member of the research team will describe this study to you and answer all of your questions. Your participation is entirely voluntary. If you choose to participate you can change your mind at any time and withdraw from the study. You will not be penalized in any way or lose any benefits to which you would otherwise be entitled if you choose not to participate in this study or to withdraw. If you have questions about your rights as a research subject, please call the University of Florida Institutional Review Board (IRB) office at (352) 273-9600.

GENERAL INFORMATION ABOUT THIS STUDY

1. Name of Participant ("Study Subject")

2. What is the Title of this research study?

Cortical control of walking: assessment, mechanisms and functional implications (The Think-2-Walk Study).

IRB Project#: 212-2012 I RB Version: 1 0/19/2011 PI Version: 7/2/2012

Page 1 of 9

3. Who do you call if you have questions about this research study?

Principal Investigator: David J. Clark, SeD

352-376- 16 11 x5244 (office) 352-443-0655 (cell)

William Marena, Aging and Rehabilitation Research Center (ARRC) Manager

352-273-92 12 (office)

4. Who is paying for this research study?

The sponsor of this study is National Institutes of Health (NIH) through University of Florida Claude D. Pepper Older Americans Independence Center.

5. Why is this research study being done?

The purpose of this research study is to understand why some older people develop problems with walking. We will evaluate whether your walking performance is related to certain characteristics of the nervous system, the muscular system, and the way that these systems control movement.

You are being asked to be in this research study because you are an older (65- 85 years old) or younger ( < 35 years old) adult who meets the established study criteria.

WHAT CAN YOU EXPECT IF YOU PARTICIPATE IN THIS STUDY?

6. What will be done as part of your normal clinical care (even if you did not participate in this research study)?

Nothing in this research study is part of your normal clinical care.

7. What will be done only because you are in this research study?

Study Visit 1: Physical Testing and Screening (at the ARRC at the University of Florida) This visit will take approximately 2.5 hours. You will undergo a number of tests including:

• peripheral sensory function of your legs/feet • memory and mental problem solving • how fast or far you can walk • balance and other mobility-related tests • questionnaires

IRB Project#: 212-2012

IRB Version: 10/19/2011

PI Version: 7/2/2012

Page 2 of 9

These tests will all be performed during a single visit to our research site. Based on the results of these tests, some participants will qualify to move on to other parts of this research. You will be notified by telephone within two weeks of the date on this form whether you are eligible to continue in the study.

If you have any questions now or at any time during the study, please contact one of the research team members listed in question 3 of this form.

8. How long will you be in this research study?

Some participants will be in the research study for only one day. However, those who meet certain study requirements will be asked to make an additional study visit. In unusual cases resulting from equipment malfunction or other unexpected events, we may ask you to make an additional visit to complete the study procedures. The total duration of participation in the study will not exceed two months. All of the research will be performed at the University of Florida or across the street at the Malcom Randall VA Medical Center.

9. How many people are expected to take part in this research study?

Up to 145 people may be enrolled in this research study, but most will be excluded from participation after the first study visit. 30 people are expected to participate in the second visit.

WHAT ARE THE RISKS AND BENEFITS OF THIS STUDY AND

WHAT ARE YOUR OPTIONS?

10. What are the possible discomforts and risks from taking part in this research study?

There is a risk of falling while we test your walking and balance ability. It is possible that you may experience muscle soreness or fatigue due to the physical activity involved with this study. These are normal responses to exercise and generally disappear within 2-3 days. .

Researchers will take appropriate steps to protect any information they collect about you. However, there is a slight risk that information about you could be revealed inappropriately or accidentally. Depending on the nature of the information, such a release could upset or embarrass you, or possibly affect your insurability or employability. Questions 17-21 in this form discuss what information about you will be collected, used, protected, and shared.

This study may include risks that are unknown at this time.

IRB Project#: 212-2012 IRB Version: 10/19/2011 PI Version: 7/2/2012

Page 3 of 9

Participation in more than one research study or project may further increase the risks to you. If you are already enrolled in another research study, please inform one of the research team members listed in question 3 of this form or the person reviewing this consent with you before enrolling in this or any other research study or project.

Throughout the study, the researchers will notify you of new information that may become available and might affect your decision to remain in the study.

If you wish to discuss the information above or any discomforts you may experience, please ask questions now or call one of the research team members listed in question 3 in this form.

11a. What are the potential benefits to you for taking part in this research study?

There is no direct benefit for participating in this research study.

11 b. How could others possibly benefit from this study?

Others may benefit from this study if the findings contribute to improved understanding of why some older adults experience difficulty walking.

11c. How could the researchers benefit from this study?

In general, presenting research results helps the career of a scientist. Therefore, the Principal Investigator listed in question 3 of this form may benefit if the results of this study are presented at scientific meetings or in scientific journals.

12. What other choices do you have if you do not want to be in this study?

The option to taking part in this study is doing nothing. If you do not want to take part in this study, tell the Principal Investigator and do not sign this Informed Consent Form.

13a. Can you withdraw from this study?

You are free to withdraw your consent and to stop participating in this study at any time. If you do withdraw your consent, you will not be penalized in any way and you will not lose any benefits to which you are entitled.

If you decide to withdraw your consent to participate in this study for any reason, please contact one of the research team members listed in question 3 of this form. They will tell you how to stop your participation safely.

If you have any questions regarding your rights as a research subject, please call the Institutional Review Board (IRB) office at (352) 273-9600.




Page 4 of 9

13b.lf you withdraw, can information about you still be used and/or collected?

If you withdraw from this study, the Principal Investigator would still like to use any information about you that is helpful to the study. If you withdraw from the study before it is completed, then data collected might be used for analysis, but no further data will be collected.

13c. Can the Principal Investigator withdraw you from this study?

You may be withdrawn from the study without your consent for the following reasons:

• The Principal Investigator decides that continuation could be harmful to you. • You do not qualify to be in the study because you do not meet the study

requirements. Ask the Principal Investigator if you would like more information. • A change in your health and physical functioning making it difficult for you to

comply with the protocol • You need treatment not allowed in the study

• Other reasons affecting administration of the research project.

WHAT ARE THE FINANCIAL ISSUES IF YOU PARTICIPATE?

14. If you choose to take part in this research study, will it cost you anything?

The Sponsor will pay for all medical services and activities required as part of your participation in this study as described above in the question "What Will Be Done Only Because You Are In This Research Study".

If you receive a bill for these services, please contact David J. Clark, SeD at (352) 376-1611 x5244 (office) or William Marena, Aging and Rehabilitation Research Center (ARRC) Manager at (352) 273-9212 (office).

Any other medical services you receive would have been provided to you even if you were not in the study. These services will be billed to you or your insurance company. You will be responsible for paying any deductible, co-insurance, and/or co-payments for these services, and any non-covered or out-of-network services.

Some insurance companies may not cover costs associated with studies. Please contact your insurance company for additional information.

15. Will you be paid for taking part in this study?

Yes. You will be given a $25 gift card for each day that you visit our research site for testing. If you live 20 miles or more from the University of Florida Health Science




Page 5 of 9

Center you will also be given a $20 gas card for each day that your visit our research site for testing.

16. What if you are injured because of the study?

If you are injured as a direct result of your participation in this study, only the professional services that you receive from any University of Florida Health Science Center healthcare provider will be provided without charge. These healthcare providers include physicians, physician assistants, nurse practitioners, dentists or psychologists. Any other expenses, including Shands hospital expenses, will be billed to you or your insurance provider.

You will be responsible for any deductible, co-insurance, or co-payments. Some insurance companies may not cover costs associated with research studies or research-related injuries. Please contact your insurance company for additional information.

The Principal Investigator will determine whether your injury is related to your participation in this study.

No additional compensation is offered. The Principal Investigator and others involved in this study may be University of Florida employees. As employees of the University, they are protected under state law, which limits financial recovery for negligence.

Please contact David J. Clark, SeD at (352) 376-1611 x5244 (office) or (352) 443-0655 (cell) or William Marena, Aging and Rehabilitation Research Center (ARRC) Manager at (352) 273-9212 (office) if you experience an injury or have questions about any discomforts that you experience while participating in this study.

For testing that will be performed at the Malcom Randall VA Medical Center, separate rules may apply in the event of injury. Please refer to the Informed Consent Form for VA research.

17. How will your health information be collected, used and shared?

If you agree to participate in this study, the Principal Investigator will create, collect, and use private information about you and your health. This information is called protected health information or PHI. In order to do this, the Principal Investigator needs your authorization. The following section describes what PHI will be collected, used and shared, how it will be collected, used, and shared, who will collect, use or share it, who will have access to it, how it will be secured, and what your rights are to revoke this authorization.

Your protected health information may be collected, used, and shared with others to determine if you can participate in the study, and then as part of your participation in the study. This information can be gathered from you or your past, current or future health records, from procedures such as physical examinations, x-rays, blood or urine

IRB Project#: 212-2012 IRB Version: 10/19/2011


Page 6 of 9

tests or from other procedures or tests. This information will be created by receiving study treatments or participating in study procedures, or from your study visits and telephone calls. More specifically, the following information may be collected, used, and shared with others:

• Information about your medical history • Information about your physical function • Information about your cognitive function • Information about your body composition • Information will be collected from your participation in the study

This information will be stored in locked filing cabinets or on computer servers with secure passwords, or encrypted electronic storage devices.

All participants are assigned an alphanumeric code and data are only "attached" to that code. If we refer to your. data we will use only that code and not your name.

Some of the information collected could be included in a "limited data set" to be used for other research purposes. If so, the limited data set will only include information that does not directly identify you. For example, the limited data set cannot include your name, address, telephone number, social security number, photographs, or other codes that link you to the information in the limited data set. If limited data sets are created and used, agreements between the parties creating and receiving the limited data set are required in order to protect your identity and confidentiality and privacy.

18. For what study-related purposes will your prote.cted health information be collected, used, and shared with others?

Your PHI may be collected, used, and shared with others to make sure you can participate in the research, through your participation in the research, and to evaluate the results of the research study. More specifically, your PHI may be collected, used, and shared with others for the following study-related purpose(s):

To understand why some older people develop problems with walking.

Once this information is collected, it becomes part of the research record for this study.

19. Who will be allowed to collect, use, and share your protected health information?

Only certain people have the legal right to collect, use and share your research records, and they will protect the privacy and security of these records to the extent the law allows. These people include:

• the study Principal Investigator (listed in question 3 of this form) and research staff associated with this project


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• other professionals at the University of Florida or Shands Hospital that provide study-related treatment or procedures

• the University of Florida Institutional Review Board (IRB; an IRB is a group of people who are responsible for looking after the rights and welfare of people taking part in research).

20. Once collected or used, who may your protected health information be shared with?

Your PHI may be shared with:

• the study sponsors, National Institute of Health and University of Florida Claude D. Pepper Older Americans Independence Center

• United States and foreign governmental agencies who are responsible for overseeing research, such as the Food and Drug Administration, the Department of Health and Human Services, and the Office of Human Research Protections.

• Government agencies who are responsible for overseeing public health concerns such as the Centers for Disease Control and federal, state and local health departments.

• North Florida/South Georgia Veterans Health System

Otherwise, your research records will not be released without your permission unless required by law or a court order. It is possible that once this information is shared with authorized persons, it could be shared by the persons or agencies who receive it and it would no longer be protected by the federal medical privacy law.

21. If you agree to take part in this research study, how long will your protected health information be used and shared with others?

Your PHI will be used and shared with others until the end of the study.

You are not required to sign this consent and authorization or allow researchers to collect, use and share your PHI. Your refusal to sign will not affect your treatment, payment, enrollment, or eligibility for any benefits outside this research study. However, you cannot participate in this research unless you allow the collection, use and sharing of your protected health information by signing this consent and authorization.

You have the right to review and copy your protected health information. However, we can make this available only after the study is completed.

You can revoke your authorization at any time before, during, or after your participation in this study. If you revoke it, no new information will be collected about you. However, information that was already collected may still be used and shared with others if the researchers have relied on it to complete the research. You can


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revoke your authorization by giving a written request with your signature on it to the Principal Investigator.

SIGNATURES

As an investigator or the investigator's representative, I have explained to the participant the purpose, the procedures, the possible benefits, and the risks of this research study; the alternative to being in the study; and how the participant's protected health information will be collected, used, and shared with others:

Signature of Person Obtaining Consent and Authorization

Date

You have been informed about this study's purpose, procedures, possible benefits, and risks; the alternatives to being in the study; and how your protected health information will be collected, used and shared with others. You have received a copy of this Form. You have been given the opportunity to ask questions before you sign, and you have been told that you can ask questions at any time.

You voluntarily agree to participate in this study. You hereby authorize the collection, use and sharing of your protected health information as described in sections 17-21 above. By signing this form, you are not waiving any of your legal rights.

Signature of Person Consenting and Authorizing




Date

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Page 1 of 4




Protocol Title Cortical control of walking: assessment, mechanisms and functional implications

PI David J. Clark, ScD IRB # 212-2012

Most recent IRB approval date 07/10/2012 (Expires 07/09/2013)

Target Enrollment 30 (20 age 65-85, 10 age <35) Anticipated Study Completion Date 3/31/14

ABSTRACT OF STUDY (Hypothesis and Design):

Walking is a complex sensorimotor task requiring coordination of numerous muscles, yet healthy adults easily control steady state walking with little direct cortical control of movement details. This “automatic” control is facilitated by sub-cortical networks that coordinate walking. Sub-cortical control is beneficial because the cortex remains relatively unencumbered, and thus available for other important information-processing such as ambulating safely in a community setting. We propose that any disruption to this sub-cortical control will necessitate a compensatory increase in the role of the cortex to successfully control walking. An abundance of evidence indicates that older adults have an increased reliance on the cortex for control of walking. Classically, this increased cortical demand has been shown by marked decrements in performance on dual-task paradigms, in which a secondary attention-demanding task is combined with walking. Furthermore, increased cortical activation has been observed during walking and during other coordinated tasks in older adults. The reasons for increased cortical demand are poorly understood, though increased cortical activity in regions associated with sensorimotor monitoring of task performance suggest aberrant processing of sensory information by sub-cortical centers. Sensory information is known to be a critical input to the sub-cortical networks that coordinate walking. Peripheral sensory impairments are common in older adults and have been implicated as a determinant of walking deficits. Based on this cumulative evidence, our overarching hypothesis is that peripheral sensory impairments in older adults disrupt sub-cortical control of walking, leading to increased cortical demand of walking and concomitant deficits in mobility function. Specific Aims: Specific Aim 1a: To determine if peripheral sensory impairment increases cortical demand of walking. Specific Aim 1b: To determine if increased cortical demand of walking reduces mobility function. Specific Aim 2: Test the hypothesis that electromyographic (EMG) measures are more sensitive than gait parameters to determine cortical demand using a dual-tasking paradigm in older adults.

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Study Design: Volunteers who respond to our recruitment ads/flyers will undergo a preliminary screening by telephone. Volunteers who meet the prescreening criteria will be invited to our study site for further screening and assessment. At visit 1 we consent and enroll participants, who will then undergo further screening and assessment. Twenty individuals meeting the eligibility criteria and ten healthy young adults (<35 years old) with good functional capability will be invited to participate in a second assessment visit at the VA Brain Rehabilitation Research Center. At visit 2, the following will be assessed:

• Peripheral sensory testing and functional assessments • Neuromechanical assessment of walking

INCLUSION / EXCLUSION CRITERIA (Please list in box below) Inclusion Criteria:

• age 65-85 • body mass index from 19-32 (based on self-reported weight and height) • no use of cane/walker (based on self-report) • report physical fatigue when walking a quarter mile, climbing two flights of stairs or

performing household chores • no substantial involuntary weight gain/loss in the past six months • blood pressure below 160/95 (based on self report) • no broken/fractured bones in past year • no central nervous system injury • no terminal illness • no pain when walking • body mass index from approximately 19-32 • blood pressure below 160/95 • walking speed < 1.0 m/s, • half of the participants will have peripheral sensory impairments (see next section) • Mini Mental State Exam score ≥ 21 • self-report of some difficulty/fatigue during daily tasks. • Berg Balance Scale ≥41

Current Enrollment Please place an “X” the box for each:


Recruitment X Intervention X

Participant Contact X *If you have marked complete for each row, this will be the last report for the DSMB.


Active Completed Withdrawn or Lost to Follow-up Total Enrolled

7 3 0 10

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Participants Who Have Withdrawn Consent or Lost to Follow Up (List in order of most recent to oldest since start of study.

PID:

Date:


N/A Total Number of Protocol Deviations: __1__ (List in order of most recent to oldest since start of study) Date:

Explanation:

11/16/12 Subject T0001 did not complete MMSE at the screening visit. AE INFORMATION: Please use table below to report any Adverse Events that are related to the study (Possibly, Probably, or Definitely). They do not have to be reportable to IRB to be included (i.e. they also do not need to be unexpected or related). If blinded, you do not need to complete intervention arm. If there is not more than one intervention it may be left blank. TOTAL Number of AEs____0____ (List in order of most recent to oldest since start of study)

Date of Onset

Date Ended PID Acrostic Event

Date Reported to Staff

Intervention arm

Relationship with

intervention Explanation

If related

SAE INFORMATION: Please use table below to report all events that are Serious since the start of the study. They do not have to be reportable to IRB to be included (i.e. they also do not need to be unexpected or related). If blinded, you do not need to complete intervention arm. If there is not more than one intervention it may be left blank. TOTAL Number of SAEs__0______ (List in order of most recent to oldest since start of study)

Date of Onset

Date Ended PID Acrostic Event

Date Reported to Staff

Intervention arm

Relationship with

intervention Explanation

If related

Page 4 of 4

PROTOCOL CHANGES: Include all changes, since last DSMB review. Explain if safety related.


Explanation of Safety

Relation

2/6/13 Revision to Inclusion Criteria • Change MMSE score criteria from ≥ 26 to ≥ 21. • Change Berg Balance Scale from ≥ 50 to ≥ 41. • Delete criteria for SPPB score ≤ 9.

No impact on participant safety

8/30/12 Addition of walking trials in which textured plastic insoles are worn in the participant’s regular shoes

No impact on participant safety

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Protocol

1. Project Title: Establishment of Age-Specific Induced Pluripotent Stem (iPS) Cells from Dermal Fibroblasts of Young and Elderly Individuals: Potential Model of Aging (Aging iPS Cell Study)

2. Investigator(s):

Principal Investigator: Anna-Maria Joseph, PhD Assistant Research Scientist

Department of Aging and Geriatrics, College of Medicine

Co-Principal Investigator Christiaan Leeuwenburgh, PhD

University of Florida, Professor Department of Aging and Geriatric Research, College of Medicine Chief, Division of Biology of Aging

Sub-Investigators: Nao Terada, MD, PhD Professor Department of Pathology, College of Medicine

Peter J. Adhihetty, Ph.D. Assistant Professor Department of Applied Physiology and Kinesiology

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3. Abstract:

Aging in humans is characterized by an overall decline in functional capacity and responsiveness of tissues and organs to physiological stimuli. Postmitotic tissue such as skeletal muscle, is particularly affected due to its limited regenerative capacity, and undergoes a progressive loss in mass (sarcopenia) that ultimately contributes to reduced physical function in the elderly. The underlying mechanisms contributing to age-related dysfunction such as sarcopenia are currently under investigation but detailed molecular and cellular studies in humans are somewhat limited due to the lack of available models that adequately reflect the pathophysiology of aging in humans.

Recently, the development of induced pluripotent stem (iPS) cells has shown great promise to circumvent this problem in the aging field. iPS cells are adult cells that are genetically reprogrammed to an embryonic stem (ES) cell-like state by expressing a cocktail of factors (Oct3, Sox2, Klf4, and c-Myc) that are important for maintaining the properties of ES cells (1). These cells exhibit important characteristics of ES cells including morphology, proliferation, and expression of stem cell markers. Importantly, iPS cells retain the pluripotency of ES cells which allows them to form tissues of all three major lineages both in vitro, as well as in vivo, following transplantation. Thus, iPS cells are capable of differentiating into numerous tissues including skeletal muscle, opening important opportunities to study the pathogenic mechanisms of a disease or condition in the affected tissue. Moreover, human iPS cells have less ethical concerns and immunocompatibility issues than ES cells. The ability of human iPS cells to retain the disease-specific properties of a patient has made them an attractive system to model human disease and opens the opportunity for providing “patient-specific” treatment with respect to clinical medicine. In fact, patient-specific iPS cells have already been utilized extensively for various pathological conditions (2).

We hypothesize that human iPS cells, and iPS-derived muscle cells derived from elderly individuals, will retain an aging phenotype and will provide a highly innovative model to study aging in humans. Additionally, an extremely exciting possibility is that these cells can be utilized for the high-throughput screening of drugs in aging research. Opportunely, the subjects for this proposed study will be recruited from an ongoing study at the Institute on Aging at the University of Florida minimizing both time and recruitment costs. Thus, iPS cells will be derived from dermal fibroblasts of elderly individuals to investigate the underlying pathogenesis of aging, with the goal of developing treatment paradigms to attenuate and delay age-related diseases.

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4. Background:

The number of elderly individuals (65+) has grown significantly in the past decade and is estimated to make up 19.3% of the U.S. population by the year 2030 (3). Associated with these projections is a functionally dependent population with a higher incidence of injuries and age-related diseases including cancer, diagnosed arthritis, heart disease and diabetes. The culmination of this growing population is higher health care costs in the United States. While there are several factors contributing to the loss of functional independence in the elderly population, a decline in muscle mass and function seems to be one of the leading culprits in reduced physical function with age. The sarcopenic phenotype is characterized by lower muscle strength, increased muscle fatigability, and reduced endurance capacity that may contribute to increased frailty and a greater risk of falls and other disabilities (4). Thus, the urgency to understand the pathogenesis of muscle mass loss with aging and develop therapeutic strategies to delay the aging process could potentially provide a dramatic reduction on escalating health care costs and improve the quality of life of our growing elderly population.

The regenerative potential of tissues is dependent on resident progenitor cells known as stem cells and dysfunction in these cells can contribute to the decline in homeostatic capacity with aging. Age-related changes in stem cells within skeletal muscle tissue (known as satellite cells) are of particular importance because of the direct consequence that reduced muscle cell regeneration has on muscle mass loss and physical function. While a number of factors have been associated with the aging phenotype in muscle, the primary mechanisms remain elusive. One of the leading hypotheses is the deleterious effects caused by the accumulation of free radicals (also described as oxidants) and other stressors during chronological aging that can impair satellite cell regeneration (5, 6). Additionally, work in our laboratory and others have shown the effect of age-induced oxidative damage on mitochondria, an occurrence that is particularly relevant to high energy demanding tissues such as skeletal muscle, in both animals and humans (7-9). Therefore, the free radical theory of aging is based on the premise that cumulative damage to mitochondrial DNA (mtDNA) and other macronutrients caused by oxidants can activate mitochondrially-mediated cell death pathways (apoptosis), inducing the release of proteins into the cytosol, and the fragmentation of DNA (8, 10). Furthermore, increased production of oxidants can impair satellite cell function and reduce the regenerative potential of tissue (8, 10, 11). Collectively these age-related changes lead to cell loss and muscle fiber atrophy, and ultimately to reduced physical function in the elderly. The importance of mitochondria in maintaining genomic integrity is inferred by mtDNA mutator mice that express a proofreading deficient mtDNA polymerase gamma. These mice have increased mtDNA mutations and display age-related phenotypes, including severe sarcopenia (12, 13). Similar findings of increased mtDNA damage and reduced mitochondrial enzyme activities have been reported in human skeletal muscle with age (14). Taken together these observations suggest that mitochondrial dysfunction due to chronic DNA damage

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may contribute to aging through the induction of cell senescence and cell death pathways, as well as the diminished replicative capacity and regenerative potential of adult muscle cells. However, the importance of mitochondria in stem cell function remains enigmatic.

Embryonic stem (ES) cells are derived from the inner cell mass of mammalian blastocysts and are primarily distinguished by their pluripotency, which refers to their ability to differentiate into all of the three primary germ layers: ectoderm, endoderm, and mesoderm (15, 16). However, while ES cells show great potential for understanding disease mechanisms and screening the efficacy of drugs, the use of human embryos, as well as rejection issues following transplantation have proven difficult issues to overcome. In 2007, Takahashi and Yamanaka (17) published a study with an alternative approach to generating human ES cells. They found that human fibroblasts could be directly reprogrammed to induce pluripotent status using four transcription factors; Oct4, Sox2, Klf4 and c-Myc (Fig. 1, next page). These nuclear reprogrammed adult somatic cells, known as induced pluripotent stem (iPS) cells, highly resemble ES cells with regards to morphology, proliferation, gene expression and epigenetic profile. In addition, human iPS cells formed germline chimeras giving rise to numerous tissues both in cell culture, as well as in vivo following transplantation (17, 18). Since the first characterization of iPS cells, “patient-specific” iPS cells have been generated for a number of pathological conditions including metabolic disorders, neurodegenerative diseases, as well as cardiac pathologies (2). Even more exciting is the finding that these patient-specific iPS cells differentiate into the disease-relevant tissue that also exhibits the disease phenotype (19-21). The potential use of iPS cells for stem cell therapy was recently demonstrated in a mouse model of Duchenne Muscular Dystrophy. iPS cells transplanted into the muscle of these mice resulted in the expression of both myogenic and satellite cell markers that differentiated into healthy myofibers with a high regenerative capacity (22). Altogether these animal and human studies support our current study proposing the use of iPS cells and in vitro differentiated muscle cells to model age-related conditions/diseases and highlight their potential for regenerative stem cell therapy.

Figure 1. Establishment of Transcription Factor-Induced Pluripotent Stem (iPS) Cells. Adult fibroblasts obtained from a skin biopsy are exposed to retroviral vectors expressing four genes encoding the human factors (Oct 4, Sox2, Klf4, and c-Myc) and grown under human embryonic stem (ES) cell culture conditions. Following transduction (~15 days), human iPS colonies are isolated and tested for embryonic stem cell properties using surface-marker expression, gene expression profiling, and in vivo and in vitro pluripotency tests (23).

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With regards to the establishment of iPS cells derived from donors of older age not much is known. While some studies report cellular rejuvenation during iPS cell reprogramming (24-26) others show that the phenotype of the donor cells are retained during this process (27, 28). Moreover, the involvement of mitochondria in iPS reprogramming is not well understood. Nuclear reprogramming of somatic cells into iPS cells requires a shift in energy metabolism towards a more glycolytic source suggesting that proper mitochondrial bioenergetics is important for maintaining the high proliferative and reprogramming potential of these cells (28, 29). Thus, despite emerging details describing the reprogramming of iPS cells from elderly individuals, the extent to which these cells retain an aging phenotype, as well as their long-term functionality and use in stem cell therapeutics remains controversial and an understudied area of research. SIGNIFICANCE

Aging studies in humans are currently limited by the number of available cell and/or animal models that adequately reflect human biology, as well as the amount of sample and the number of analyses that can be performed for each subject. These limitations have made it difficult for translational research in the aging field. The establishment of age-specific human iPS cells would circumvent these problems and provide a model to study aging in the laboratory. Thus, these studies are essential to understanding the age-related mechanisms leading to the functional decline in tissues. Moreover, they will allow the development of clinical strategies to improve tissue function in the elderly while addressing the current challenges faced by stem cell therapy for age-associated diseases. 5. Specific Aims:

Specific Aim 1: Establishment and characterization of human iPS cells for ES cell properties including stem cell markers, self-renewal, and pluripotency (in vitro induction of lineage differentiation, and in vivo teratoma formation). iPS cells will be generated from dermal fibroblasts of young healthy adults (20-35 years) and healthy elderly individuals (>70 years), and characterized to confirm stem cell properties and pluripotency using morphological studies, surface-marker expression, in vitro formation of embryoid bodies and teratoma formation in vivo. Once characterized, human iPS cell lines will be propagated, frozen, and stored for future use. We hypothesize that established iPS cells will retain the aging characteristics of the subject. Specific Aim 2: Determine the mitochondrial phenotype of iPS cells derived from individuals of disparate ages. Mitochondria are critical for providing bioenergetic support to cells. In contrast, dysfunction in these organelles has been implicated in the age-related decline of most tissues and organs. The involvement of mitochondria and their role during iPS reprogramming and differentiation is currently unclear. Therefore, we will assess specific mitochondrial parameters related to bioenergetics including oxygen consumption, mitochondrial membrane potential, and morphology in established iPS cells. We hypothesize that this data will provide us with an indication of their cellular age,

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and offer insight as to how mitochondrial dysfunction contributes to a decline in the regenerative capacity of tissue with age. Exploratory Aim:

Specific Aim 3: In vitro differentiation of muscle cells from human iPS cells. Human iPS cells will be differentiated in vitro into muscle cells. Few studies have established human iPS-derived muscle cells and therefore this aim will be exploratory because of its highly innovative nature. Muscle cell lines will be characterized based on morphology, expression of myogenic markers, and myogenic potential. This aim adds an extremely exciting yet somewhat risky dimension to our proposal but has the possibility of providing a new model to study the mechanisms of age-related muscle conditions including sarcopenia. We expect that these iPS-derived muscle cells will display the phenotype of aging muscle.

Human iPS cells can potentially be employed to study aging in humans and provide a model system to identify mechanisms responsible for the age-related functional decline in tissues. Additionally, these cells could provide a fast tracking screening tool for drug discovery. The long-term goal of this project includes their potential use for skeletal muscle stem cell therapeutics whereby patient-specific iPS-derived muscle cells are utilized for transplantation to increase the functionality of muscle in the elderly. 6. Research Plan:

Study participants for this study will be in part recruited from the Developmental Project funded by the University of Florida Claude D. Pepper Older Americans Independence Center.

Briefly, the Aging iPS Cell study is a single-site cohort study with subjects separated into two age groups, young (20-35 years) and elderly (age > 70 years). We will enroll 10 individuals in total (5 subjects/group), with up to an additional 6 being recruited if a skin biopsy cannot be obtained for any of the subjects. Subjects recruited from the Developmental Project fit into these two categories and baseline measurements have already been completed and subjects will start returning for a four year follow-up in 2012. The Clinical Recruitment Core of the Department of Aging has already performed all screening, a basic questionnaire regarding participant’s health history, and completed all recruitment of research participants, minimizing time and cost for the proposed iPS cell study.

Inclusion criteria The following inclusion criteria will be adopted to select the study participants:

• males and females aged 20-35 and 70-99 years • sedentary lifestyle (i.e., the subject has spent less than 20 minutes per

week in the past 2 month performing structured physical activity, such as exercising at a gym and/or weight training)

• willing and able to give informed consent Exclusion criteria:

• history of smoking in the prior 12 months

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• active treatment for cancer or history of cancer in the past 3 years • congestive heart failure NYHA Class III or IV • previous stroke with upper and/or lower extremities involvement within the

last 6 months • peripheral vascular disease Fontaine Class III/IV • History of life-threatening cardiac arrhythmias, stroke, severe Parkinson’s

disease or severe neurological disorders likely to interfere with physical function

• cognitive impairment (i.e., MMSE ≤ 23) • renal disease requiring dialysis • lung disease requiring steroids • lower extremity amputation • severe osteoarthritis that interferes with physical function • Complicated diabetes • inflammatory diseases such as active rheumatoid arthritis, vasculitis,

autoimmune disorders, and inflammatory bowel disease • life-threatening illnesses with an estimated life expectancy less than 1

year • history of drug or alcohol abuse • taking GH and/or estrogen replacement therapy • Testosterone medication • Anticoagulant therapy • involved in active weight loss > 5 kg in prior 3 months • pregnancy (determined by a pregnancy test) • SPPB score of 8-10 • Lidocaine allergy


• recent bacterial infection (< 2 weeks) • acute febrile illness in previous 2 months • high blood pressure (i.e., BP ≥ 180/110 mm Hg) at the visit (subject will be

referred to his/her physician and reevaluated after appropriated therapy being instituted)

• major surgery or hip/knee replacement in the past 6 months • Assessment of disability. The uniform measure proposed to use for the

assessment of disability will be a self-report disability questionnaire. This questionnaire was developed for the Fitness Arthritis in Seniors Trial (FAST) and since then has been widely used in intervention and observational studies (e.g. ADAPT, REACT-1, OASIS, CHAMP). The questionnaire is not specific to a certain disease and asks about perceived difficulties in general activities of daily living during the last month. Respondents answer for each of the items whether they experience 1) no difficulty, 2) a little difficulty, 3) some difficulty, 4) a lot of difficulty, 5) unable to do or, 6) did not do for other reasons. Answers can be averaged across the items in order to receive an indication of the overall perceived disability burden by a person.

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Collection of Tissue Samples

a) Skin Biopsy

Skin biopsy will be obtained from the vastus lateralis by punch biopsy technique (11). After cleaning and shaving of the biopsy site, the site will be extensively scrubbed with betadine swabs. Under local anesthesia (1% Lidocaine), skin biopsy device (6 mm in diameter) will be applied. After ascertaining hemostasis by applying pressure to bleeding sites, the skin will be approximated by subcutaneous 1-2 absorbable sutures.

b) Establishment of iPS cells: the following procedure will be conducted in labs as previously described (17, 31).

Preparation of Human Dermal Fibroblast

Biopsy specimen will be placed in sterile HBSS supplemented with penicillin/streptomycin on ice for transport to the lab. Soak the specimen in 70% ethanol for 30s to reduce contamination. All the following procedures will be performed in a tissue-culture hood. • Wash the specimen with sterile HBSS supplemented with

penicillin/streptomycin twice.

• Cut the skin biopsy into 1-2mm3 pieces with sterile forceps and dissecting

scissors. • Digest skin with 0.1 dispase at 4°C overnight to remove epidermal layers. • Further digest remaining dermal parts with 0.1% collagenase I at 37°C for 4

hours. • Centrifuge the digested cells, and then re-suspend in 2ml of FP medium

• Seed the cells in 6-well plates at 1X103 cells/cm2 and maintain at 37 with

5% CO2.

• Renew FP medium to remove residual non-adherent cell, after 1 day. • Culture until confluency (within 7 days) • Harvest, pool the cells and plate to a T-25 flask (passage 2). • Subculture the cells when the fibroblast cells have reached 80% confluency

(about 4-6 days). Passage 3 will be used for induction. A portion of the cell will be used for DNA extraction and genetic confirmation. Freeze the rest fibroblast cells.

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c) Preparation of Packaging Cells (Plat-A cells) for Retrovirus Production

• Culture of Plat-A cells and transfection of retroviral vector (encoding Oct 4, Sox2, Klf4 and c-Myc) will be performed according to standard protocol (31).

• Collect medium containing virus packaged with vectors, filter and mix equal portion of four viruses to be ready for transduction.

d) Establishment of iPS Cells

• Culture human fibroblast cells to ~90% confluency in 25-T flask.

• Pass cells to 6-well plate (8X104 per well) and incubate overnight.

• Transduction of four defined factors (hOct4, hSox2, hKlf4, hc-Myc) will be performed according to a recent published protocol (31).

• Cells will be replated into 60 mm-Matrigel coated plate and culture will be maintained under hES cell medium.

• Colonies are expected to appear by day 15. Mechanically pass the colonies clumps on freshly plated Matrigel and deposit one colony per well of a 24-well plate.

• When colony becomes large enough, mechanically passage the colony to 6-well plate.

• Once line is well established in culture, iPS cells will be treated like hES cells.

e) Characterization of iPS cells (17)

• Immunofluorescence staining for stem cell marker: SSEA3, SSEA4, Oct4, Nanog, Tra-1-60, Tra-1-80. Alkaline phosphatase staining and karyotyping.

• In vitro pluripotency test: will be evaluated by Embryoid Body-mediated differentiation analysis and lineage-directed differentiation by reported methods for hESC.

• In vivo pluripotency test: Teratoma formation by s.c. injection into nude mice will be conducted to demonstrate the developmental potential to give rise to differentiated derivatives of all three primary germ layers.

f) Propagation and freezing of iPS cells

• Propagation and freezing of iPS cells will be performed according to published protocol (31, 32)

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g) Establishment of iPS-derived muscle cells

• Differentiation of human iPS cells will be performed by using embryoid bodies (EBs) and based on a previously established protocol (34). Following the attachment of EBs to the bottom of the coated dishes, spindle-shaped fibers will start to appear and with time, they will begin to fuse and form thick multinucleated fibers resembling muscle fibers. These muscle cells will then be frozen.

h) Assessing mitochondrial properties in human iPS cells and iPS-derived muscle cells

• Mitochondrial respiration will be measured in cells as previously described

(33). Additionally, confocal microscopy will be employed to examine mitochondrial morphology, as well as mitochondrial membrane potential as previously described (25).

i) Registration of iPS cells and iPS-derived muscle cells to NIH (http://stemcells.nih.gov/research/registry)

Future studies: pathogenic mechanism, expansion stability, drug development, gene correction and cell replacement therapy. Resource Sharing Plans

NIH mandates to share materials, reagents, cell lines and animal disease models developed under NIH funding. We will generate iPS cells according to the NIH requirements. Ownership of samples Derived from the Study Subject

Samples, skin fibroblasts, and cell line information derived from the study subject will be stored at the PI’s laboratory with limited access for authorized personnel only. All samples will be identified by a code that can be led to the subject’s identity only by the reference information file, which will be stored separately on the secure server with limited access and password protected. If the subject wishes to destroy the samples, we will destroy the samples. iPS cells established from this study will be maintained in the PI’s laboratory, and will not be destroyed, and intellectual property rights for these resources will belong to the inventor(s) and University of Florida. Data Safety Monitoring Plan

Based on assessments of the risk-to benefit ratio, the risk level associated with the study is low. All risks have been described in the consent form.

http://stemcells.nih.gov/research/registry

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Plan for Safety Review:

Monitoring procedure for safety, efficacy, eligibility, data management, and protocol compliance are delineated in the protocol. The PI will adhere to the protocol as described. Only subjects who meet the protocol inclusion criteria will be enrolled.

Plan for adverse event reporting:

Monitoring for documentation of an adverse event, whether anticipated or unanticipated, is the responsibility of the principal investigator who will maintain oversight but may delegate collection of information related to this function to other study team member. In term of SAFETY monitoring, all adverse events spontaneously reported, elicited, or observed by the investigators will be recorded.

All Serious Adverse Events, should they occur, whether study-related or expected, will be documented on a Case Report Form, under Adverse Event section in the Patient’s binder, reported by the principal investigator to the IRB within five (5) working days.

In addition, the principal investigator will follow the reporting requirements for serious and unexpected adverse events outlined in the UF IRB Adverse Event Evaluation and Reporting Guide. All unanticipated, serious, fatal and/or life-threatening adverse events will be reported to the UF IRB. Aggregate reports of adverse events will be prepared on an annual basis or at the end of the study, whichever may occur earlier and forwarded to the IRB at annual review.

Plan for data management:

Case Report Form (CRF) will be used for each subject. To protect the participant’s right of privacy, the Patient Identification Number will identify subjects individual records related to the study and will be stored in locked cabinets with limited access; electronic files will be kept in secured database in which only the patient identification number will be used. The database will then be shared with statistician for analysis. During the study, data will be analyzed as it becomes available by Dr. Christiaan Leeuwenburgh. No early closure is planned because of the limited scope and projected low risk of the study.

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7. Possible Discomforts and Risks:

Skin biopsy: Skin biopsy will cause minor pain when local anesthesia is injected in the skin of the biopsy site. An allergic reaction may occur to the local anesthetics. Minor pain may be experienced during the twitching of punch biopsy device. After the procedure, pain may be felt at the biopsy site. There will be less than 1cm scar, and if the biopsy site is infected or the incision forms keloid, the scar may become more pronounced than usual. Hematoma formation and infection may occur at the biopsy site. Local allergic skin reaction may occur if patients are allergic to adhesive tapes.

Breach of Confidentiality of the Study Subject’s Health Information: Although protective measures (see below) should minimize this risk, the risk of inappropriate or accidental disclosure of health information, including genetic information cannot be totally eliminated. Depending on the nature of the information, the subject may suffer from psychological, financial, social and legal complications of such a disclosure.

Strategies to Minimize Risks: Sterile technique and appropriate hemostatic techniques will be used to minimize the risk of infections and hematoma formation associated with biopsies. The subject will be asked to withhold aspirin and other anti-platelet agents, warfarin, heparin, non-steroidal anti-inflammatory agents and other medications which increase bleeding (if it is medically safe to stop them) for 72 hr prior to the biopsy appointment. Identifiable electronic data of study subjects will be protected by password and limited access. Hard-copy data that could lead to identification of research subjects will be stored in a locked cabinet in a room to which access is limited to authorized personnel. If these preventive measures do not work and the subject experiences an adverse event, the subject will contact the P.I. or the study team member. Appropriate actions will be taken to take care of the adverse event. Adverse events will be reported to the IRB.

8. Possible Benefits:

There is no prospect of direct benefit to individual subject. Nevertheless, the study may advance the following fields: • Understanding the pathogenic mechanism of nucleotide repeats expansion-

related neurodegenerative disorders in general • Creating a platform for drug screening • Designing gene correction strategy and cell replacement therapy

Such understanding is deemed essential for development of rational treatment strategies.

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9. Conflict of Interest:

Publications that may result from such research can enhance the reputation of the investigators.

References: 1. Takahashi, K., Yamanaka, S. (2006) Induction of pluripotent stem cells from mouse

embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676. 2. Unternaehrer, J. J., Daley, G. Q. (2011) Induced pluripotent stem cells for

modelling human diseases. Philos.Trans.R.Soc.Lond B Biol Sci 366, 2274-2285. 3. Olshansky, S. J., Goldman, D. P., Zheng, Y., Rowe, J. W. (2009) Aging in America

in the twenty-first century: demographic forecasts from the MacArthur Foundation Research Network on an Aging Society. Milbank Q. 87, 842-862.

4. Marzetti, E., Calvani, R., Bernabei, R., Leeuwenburgh, C. (2011) Apoptosis in Skeletal Myocytes: A Potential Target for Interventions against Sarcopenia and Physical Frailty - A Mini-Review. Gerontology, 1-8.

5. Brack, A. S., Rando, T. A. (2007) Intrinsic changes and extrinsic influences of myogenic stem cell function during aging. Stem Cell Rev. 3, 226-237.

6. Renault, V., Thornell, L. E., Butler-Browne, G., Mouly, V. (2002) Human skeletal muscle satellite cells: aging, oxidative stress and the mitotic clock. Exp.Gerontol 37, 1229-1236.

7. Conley, K. E., Jubrias, S. A., Esselman, P. C. (2000) Oxidative capacity and ageing in human muscle. J Physiol 526 Pt 1, 203-210.

8. Dirks, A., Leeuwenburgh, C. (2002) Apoptosis in skeletal muscle with aging. Am J Physiol Regul Integr Comp Physiol 282, R519-R527.

9. Hagen, J. L., Krause, D. J., Baker, D. J., Fu, M. H., Tarnopolsky, M. A., Hepple, R. T. (2004) Skeletal muscle aging in F344BN F1-hybrid rats: I. Mitochondrial dysfunction contributes to the age-associated reduction in VO2max. J Gerontol A Biol Sci Med Sci 59, 1099-1110.

10. Whitman, S. A., Wacker, M. J., Richmond, S. R., Godard, M. P. (2005) Contributions of the ubiquitin-proteasome pathway and apoptosis to human skeletal muscle wasting with age. Pflugers Arch. 450, 437-446.

11. Park, I. H., Lerou, P. H., Zhao, R., Huo, H., Daley, G. Q. (2008) Generation of human-induced pluripotent stem cells. Nat.Protoc. 3, 1180-1186.

12. Kujoth, G. C., Hiona, A., Pugh, T. D., Someya, S., Panzer, K., Wohlgemuth, S. E., Hofer, T., Seo, A. Y., Sullivan, R., Jobling, W. A., Morrow, J. D., Van, R. H., Sedivy, J. M., Yamasoba, T., Tanokura, M., Weindruch, R., Leeuwenburgh, C., Prolla, T. A. (2005) Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science 309, 481-484.

13. Trifunovic, A., Wredenberg, A., Falkenberg, M., Spelbrink, J. N., Rovio, A. T., Bruder, C. E., Bohlooly, Y., Gidlof, S., Oldfors, A., Wibom, R., Tornell, J., Jacobs, H. T., Larsson, N. G. (2004) Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 429, 417-423.

14. Lezza, A. M., Boffoli, D., Scacco, S., Cantatore, P., Gadaleta, M. N. (1994) Correlation between mitochondrial DNA 4977-bp deletion and respiratory chain enzyme activities in aging human skeletal muscles.Biochem.Biophys.Res.Commun. 205,772-779.

15. Evans, M. J., Kaufman, M. H. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292,154-156.

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16. Martin, G. R. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc.Natl.Acad.Sci U.S.A 78, 7634-7638.

17. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., Yamanaka, S. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872.

18. Yu, J., Vodyanik, M. A., Smuga-Otto, K., ntosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I., Thomson, J. A. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-192019.

19. Ebert, A. D., Yu, J., Rose, F. F., Jr., Mattis, V. B., Lorson, C. L., Thomson, J. A., Svendsen, C. N. (2009) Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 457, 277-280.

20. Moretti, A., Bellin, M., Welling, A., Jung, C. B., Lam, J. T., Bott-Flugel, L., Dorn, T., Goedel, A., Hohnke, C., Hofmann, F., Seyfarth, M., Sinnecker, D., Schomig, A., Laugwitz, K. L. (2010) Patient-specific induced pluripotent stem-cell models for long-QT syndrome. N.Engl.J Med 363, 1397-1409.

21. Yazawa, M., Hsueh, B., Jia, X., Pasca, A. M., Bernstein, J. A., Hallmayer, J., Dolmetsch, R. E. (2011) Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Nature 471,230-234.

22. Mizuno, Y., Chang, H., Umeda, K., Niwa, A., Iwasa, T., Awaya, T., Fukada, S., Yamamoto, H., Yamanaka, S., Nakahata, T., Heike, T. (2010) Generation of skeletal muscle stem/progenitor cells from murine induced pluripotent stem cells. FASEB J 24, 2245-2253.

23. Zaehres, H., Scholer, H. R. (2007) Induction of pluripotency: from mouse to human. Cell 131, 834-835.

24. Lapasset, L., Milhavet, O., Prieur, A., Besnard, E., Babled, A., it-Hamou, N., Leschik, J., Pellestor, F., Ramirez, J. M., De, V. J., Lehmann, S., Lemaitre, J. M. (2011) Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state. Genes Dev. 25, 2248-2253.

25. Suhr, S. T., Chang, E. A., Rodriguez, R. M., Wang, K., Ross, P. J., Beyhan, Z., Murthy, S., Cibelli, J. B. (2009) Telomere dynamics in human cells reprogrammed to pluripotency. PLoS.One. 4, e8124.

26. Suhr, S. T., Chang, E. A., Tjong, J., Alcasid, N., Perkins, G. A., Goissis, M. D., Ellisman, M. H., Perez, G. I., Cibelli, J. B. (2010) Mitochondrial rejuvenation after induced pluripotency. PLoS.One. 5, e14095.

27. Armstrong, L., Tilgner, K., Saretzki, G., Atkinson, S. P., Stojkovic, M., Moreno, R., Przyborski, S., Lako, M. (2010) Human induced pluripotent stem cell lines show stress defense mechanisms and mitochondrial regulation similar to those of human embryonic stem cells. Stem Cells 28, 661-673.

28. Prigione, A., Fauler, B., Lurz, R., Lehrach, H., Adjaye, J. (2010) The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells. Stem Cells 28,721-733.

29. Chen, C. T., Hsu, S. H., Wei, Y. H. (2010) Upregulation of mitochondrial function and antioxidant defense in the differentiation of stem cells. Biochim.Biophys.Acta 1800, 257-263.

30. Buford, T. W., Lott, D. J., Marzetti, E., Wohlgemuth, S. E., Vandenborne, K., Pahor, M., Leeuwenburgh, C., Manini, T. M. (2011) Age-related differences in lower extremity tissue compartments and associations with physical function in older adults. Exp.Gerontol Epub Ahead of Print.

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31. Baharvand, H., Salekdeh, G. H., Taei, A., Mollamohammadi, S. (2010) An efficient and easy-to-use cryopreservation protocol for human ES and iPS cells. Nat.Protoc. 5, 588-594.

32. Mollamohammadi, S., Taei, A., Pakzad, M., Totonchi, M., Seifinejad, A., Masoudi, N., Baharvand, H. (2009) A simple and efficient cryopreservation method for feeder-free dissociated human induced pluripotent stem cells and human embryonic stem cells. Hum.Reprod. 24, 2468-2476.

33. Joseph, A. M., Rungi, A. A., Robinson, B. H., Hood, D. A. (2004) Compensatory responses of protein import and transcription factor expression in mitochondrial DNA defects. Am J Physiol Cell Physiol 286, C867-C875.

34. Chang, H., Yoshimoto, M., Umeda, K., Iwasa, T., Mizuno, Y., Fukada, S., Yamamoto, H., Motohashi, N., Miyagoe-Suzuki, Y., Takeda, S., Heike, T., Nakahata, T. (2009) Generation of transplantable, functional satellite-like cells from mouse embryonic stem cells. FASEB J 23, 1907-1919.

1

Newly Supported Pepper Studies Data Safety Monitoring Plan

Protocol title: Establishment of Age-Specific Induced Pluripotent Stem (iPS) Cells from Dermal Fibroblasts of Young and Elderly Individuals: Potential Model of Aging (Aging iPS Cell Study) PI: Anna-Maria Joseph, PhD Please attach latest stamped informed consent. Describe the plan to monitor participant safety by addressing the following elements. If any of these do not apply, explain why. 1. What is the risk level for your study (check only 1 box)? No Greater than Minimal Risk

The probability and magnitude of harm or discomfort anticipated in the research are not greater than those ordinarily encountered in daily life or during the performance of routine physical and psychological examinations or tests. This category includes protocols that pose “no greater than minimal risk” according to federal regulations. Requires Minimal-Intensity Monitoring.

Minor Increase over Minimal Risk Research involves a minor increase over minimal risk. There is an increased probability of a low-severity event that is reversible (e.g., muscle/joint soreness or sprain from exercising) or the likelihood of serious harm occurring is extremely rare (e.g., fatal anaphylaxis from skin testing). Requires Low-Intensity Monitoring.

Moderate Risk Risks are greater than minimal, but are not considered high. There is an increased probability of a moderate-severity event that is reversible (e.g., hypoglycemic episode, bronchoconstriction or infection), but there is adequate surveillance and protections to identify adverse events (AEs) promptly and to keep their effects minimal. The likelihood of serious harm is extremely rare to very rare. Requires Moderate- Intensity Monitoring.

High Risk The study risk is greater than a moderate-risk study due to the increased probability for generating SAEs. There is an increased probability for the occurrence of a study related event that is serious and prolonged or permanent, or there is significant uncertainty about the nature or likelihood of adverse events. Requires High-Intensity Monitoring.

2. What level of safety monitoring will be used for your study (check only 1 box)?

Minimal-Intensity Monitoring -The PI will monitor the study (interpreting test results, determining levels of severity and association for adverse events), with prompt reporting of AEs and other study related information to the IRB, GCRC, sponsor, and other agencies as appropriate. Other information to be reported includes continuing annual renewal updates, any protocol deviations, changes to the protocol or consent form, etc.

Low-Intensity Monitoring -The PI will monitor the study as noted under “Minimal-Intensity Monitoring.” Team meetings by the PI and his/her staff will be conducted on a routine basis when appropriate. The IRB and the GCRC will be promptly notified if the risk/benefit ratio changes.

Moderate-Intensity Monitoring - The PI will monitor the study on a day-to-day basis. This includes all monitoring activities described above in “Low-Intensity Monitoring.” In addition, most drug trials will require well-described criteria for dose escalation, criteria defining maximum tolerated dose (MTD), and/or criteria for stopping the trial or discontinuing involvement of a study subject. A Safety Monitor or a Data Monitoring Committee must also be utilized to review adverse events.

High-Intensity Monitoring - The PI monitors the study on a day-to-day basis. This includes all monitoring activities described above in “Moderate-Intensity Monitoring.” Most high-risk protocols will also require a Data Monitoring Committee (DMC). High-risk, double-blinded trials must have an independent DMC. An independent DMC is also required by NIH Guidelines for all NIH-funded multicenter Phase III clinical trials involving greater than minimal risk of harm or gene transfer or

2

3. Who in addition to the Pepper Center DSMB will be reviewing the accumulating study wide

safety data (across all study participants) for your study? PI only Safety monitor(s) – provide names / affiliations below Study affiliated DMC* Independent DMC* * If checked, please remember to forward these reports along with your study report for the

Pepper DSMB to review.

4. How frequently will the accumulating study wide safety data (across all study participants) be

reviewed by the above noted individual(s) or committee (e.g., quarterly, every 6 months)? Given that this is a small clinical trial, the safety data for this study will be reviewed every 6 months.

5. Study Risks and Safety

a. List all expected risks (physical, psychological, social, risks to confidentiality, etc.) associated with the study intervention(s) (e.g., drug, procedures, device) and/or the study procedures/tests. Quantify risks whenever possible (e.g., tremors: 5% of subjects; tachycardia: common vs. rare). All risks must be included in the study consent form.

Skin biopsy (physical)

• Minor pain due to local anesthesia injected in skin of biopsy site • Allergic reaction may occur due to the local anesthetic • Bleeding experienced following the procedure • Pain experienced following the procedure • Infection of biopsy site and/or hematoma • Allergic reaction (skin allergy) to adhesive tape/bandage placed on the wound following

the procedure • Round scar remaining following procedure

b. How will the expected risks described in 5a above be minimized?

• The subjects blood pressure, pulse, respiration rate and temperature is taken prior to the procedure

• Sterile technique and appropriate hemostatic techniques will be used to minimize risk of infections and hematoma formation associated with the skin biopsy

• Subjects will be asked to withhold aspirin and other anti-platelet agents etc. which can increase bleeding (if medically safe to do so) for 72 hr prior to biopsy appointment

• Subjects will be asked in the initial phone screen for enrollment into the study whether they have any known skin allergies including an allergy to local anesthetics, as well as to adhesive tapes/bandages

• Subjects are monitored following the skin biopsy via phone calls (3 phone calls following the procedure at 1 d, 3-5 d and 2 weeks post skin biopsy) to ensure proper healing of the skin biopsy. If the subject experiences any of the events listed in 6a they will be asked to seek the advice of a medical professional

gene therapy. If no other DMC is in place for a single-institution clinical trial, the PI may use the GCRC Human Subject Protection Committee as an independent board to fulfill this function.

3

c. How will the expected risks described in 5a above be treated, should they occur?

• If the subject experiences an allergic reaction during the procedure due to the local anesthetic, the procedure will be stopped immediately and the subject monitored by the physician performing the skin biopsy

• If the subject experiences an allergic reaction (i.e. skin allergy) following the procedure or any time during the 3 follow up calls the subject will be asked to see a medical professional

• If the subject experiences pain, infection of biopsy site or hematoma, they will be advised to see a medical professional

d. What tests/measurements (and their frequencies) will be performed to monitor participant

safety (e.g., labs, x-rays/scans, PE, questionnaires, performance measures, ECG, pathology)?

• Prior to the commencement of the skin biopsy procedure, subjects vital signs will be obtained (i.e. blood pressure, pulse, respiration rate etc.)

• Subjects are monitored by a study coordinator during the skin biopsy procedure to ensure their comfortability. Additionally, the subjects will be monitored following the skin biopsy by phone (3 phone calls following the procedure at 1 d, 3-5 d and 2 weeks post-skin biopsy). If the subject experiences any of the events listed in 6a they will be asked to seek the advice of a medical professional.

e. Once subjects are enrolled in the study, what pre-specified lab alert values or other clinical criteria would lead to discontinuation or alteration of treatment (e.g., ALT>3x ULN: decrease dose by 50%)?

• Blood pressure ≥ 180 mmHg systolic or ≥ 110 mmHg diastolic taken prior to the

procedure would not allow for the subject to have the skin biopsy performed.

UF I Institutional Review Board UNIVERSITY of FLORIDA

INFORMED CONSENT FORM to Participate in Research, and

AUTHORIZATION to Collect, Use, and Disclose Protected

Health Information (PHI)

University of Florida

Health Center

Institutional Review Board

APPROVED FOR USE

From_:s/;?jp.o tb Through aft, �oLS �

INTRODUCTION

Name of person seeking your consent: _ ________________ _

Place of employment & position: ___________ ________ _

Please read this form which describes the study in some detail. A member of the research team will describe this study to you and answer all of your questions. Your participation is entirely voluntary. If you choose to participate you can change your mind at any time and withdraw from the study. You will not be penalized in any way or lose any benefits to which you would otherwise be entitled if you choose not to participate in this study or to withdraw. If you have questions about your rights as a research subject, please call the University of Florida Institutional Review Board (IRB) office at (352) 273-9600.

GENERAL INFORMATION ABOUT THIS STUDY

1. Name of Participant ("Study Subject")

2. What is the Title of this research study?

Establishment of Age-Specific Induced Pluripotent Stem (iPS) Cells from Dermal Fibroblasts of Young and Elderly Individuals: Potential Model of Aging (Aging iPS Cells Study)


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3. Who do you call if you have questions about this research study?

Principal Investigator: Anna-Maria Joseph, PhD at 352-273-6879

Co-Principal Investigator: Christiaan Leeuwenburgh, PhD at 352-273-6796 or

William Marena, Aging Research and Rehabilitation Center (ARRC) Manager at 352-

273-9212.

4. Who is paying for this research study?

The sponsor of this study is National Institute of Health (NIH) through Claude D. Pepper Older American's Independence Center.

5. Why is this research study being done?

Human aging is associated with a lower physical function of the body. Muscles are also affected by aging and we lose muscle mass as we get older. The decrease in muscle mass with aging can lead to physical decline and loss of independence in elderly individuals.

The purpose of this research study is to establish cells that will allow us to study aging in the laboratory and learn about the process of aging. To do this, we will take samples from your skin that contains cells which we will grow in a dish. These cells will have similar features as found in your body and are called "induced pluripotent stem (iPS) cells". We will use these cells for a variety of experiments to better understand the aging process. Additionally, these cells will be stored for future experiments and may be shared with other researchers to speed up the research of aging in humans.

You are being asked to be in this research study because you are a healthy young or older individual.

WHAT CAN YOU EXPECT IF YOU PARTICIPATE IN THIS STUDY?

6. What will be done as part of your normal clinical care {even if you did not participate in this research study)?

Nothing, your normal clinical care will not change because you are in this study.

No aspects of this study are related to your normal health care.



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7. What will be done only because you are in this research study?

If you decide to participate in this study, you will have 1 visit for the study that will include a skin biopsy. Upon arrival to the research center, we will explain to you the purpose of this study and what it entails. We will at this time ask you to consent to the skin biopsy. You will have the opportunity to ask questions and the research staff will answer your questions. If you decide to participate in this study, a skin biopsy will be performed during this time, upon you signing the consent form.

During this visit in addition to performing the skin biopsy, you will be asked about your medical history and medications you take, your vital signs, including blood pressure, pulse, and temperature will also be recorded. Also, you will be weighed and a short physical examination will be performed. If you are fe!llale and of childbearing potential, we will collect your urine to perform a pregnancy test prior to the skin biopsy to exclude the possibility that you are pregnant.

The skin biopsy will be done under local anesthesia and a small patch of skin the size of a pencil eraser will be taken from the thigh region of the leg. 1 or 2 self-absorbable stitches may or may not be needed to close the wound. These procedures will be done at the Aging and Rehabilitation Research Center (ARRC) in Gainesville, Florida. The skin will be further processed in Drs. Terada's, Joseph's and Leeuwenburgh's laboratories at the University of Florida, for the establishment of iPS cells. These cells will be stored for future research to study the process of aging. Several experiments will be conducted on these cells which will provide us with data that we will use to learn more about the aging process.

You will be instructed to leave the dressing untouched for 48 hours after the biopsy. You should avoid bathing (showering is preferred) during this time period. If you see signs of redness, heat, or a discharge after removing the dressing, or if you have pain or discomfort please contact one of the research team members listed in question 3 of this form who will refer you to a medical staff member of our study team to determine the severity of the situation and instruct you on how to proceed. As a follow-up to your visit, you will also be contacted by phone on three separate times to make sure that you biopsy site heals without any complications Phone calls will be placed 1 day, 3-5

days and approximately 2 weeks after biopsy was performed.

The table below shows what will happen during the study visit.

Procedure

Informed Consent

Medical History

Review Study's Inclusion/Exclusion Criteria

Vital signs: blood pressure, pulse, respiration rate, temperature, weight

IRS Project#: 381-2012

IRS Version: 10/19/2011


Study Visit

X

X

X

X

Follow Up

-

-

-

-

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Procedure Study Visit Follow Up

Physical Examination X -

Skin biopsy X -

Phone calls - X

If you have any questions now or at any time during the study, please contact one of the research team members listed in question 3 of this form.

8. How long will you be in this research study?

You will be required to be in. the research facility for 1 study visit. Your study visit will last for up to 3 hours. No follow up visit will be necessary. You will receive follow up phone calls during 2 weeks after the procedure.

9. How many people are expected to take part in this research study?

We plan to establish one cell line from each individual. The total number of participants will be 10 (5 young and 5 elderly), with up to an additional 6 participants being recruited in case if a skin biopsy cannot be obtained from any of the first 10

participants . The study may be extended if additional funding becomes available.

WHAT ARE THE RISKS AND BENEFITS OF THIS STUDY AND

WHAT ARE YOUR OPTIONS?

10. What are the possible discomforts and risks from taking part in this research study?

Biopsy:

• Skin biopsy will cause minor pain when local anesthesia is injected in the skin of the biopsy site.

•An allergic reaction may occur to the local anesthesia medication (Lidocaine).

•After the biopsy, we will make sure there is no active bleeding. However, bleeding may develop later. Usually this can be stopped by putting pressure over the dressing.



Page 4 of 11

• The biopsy site may become red and painful to pressure. These may be signs of infection, especially if there is also heat, and green or yellow discharges. If not treated, it may develop into more serious infections.

• Local allergic skin reaction may occur if you are allergic to adhesive tapes.

• After the biopsy, pain may be felt at the biopsy site.

• There will be a round scar, and if the surgical site is infected or the incision forms scar tissue, the scar may become more pronounced than usual.

To prevent the above mentioned risks, the skin biopsy will be performed by a health care professional that has been well trained to perform this procedure and an aseptic method will be employed throughout the procedure.

Other possible risks to you may include:

• Researchers will take appropriate steps to protect any information they collect about you. However, there is a slight risk that information about you could be revealed inappropriately or accidentally. Depending on the nature of the information, such a release could upset or embarrass you, or possibly affect your insurability or employability. Questions 17-21 in this form discuss what information about you will be collected, used, protected, and shared.

• This study may include risks that are unknown at this time.

• Participation in more than one research study or project may further increase the risks to you. If you are already enrolled in another research study, please inform one of the research team members listed in question 3 of this form or the person reviewing this consent with you before enrolling in this or any other research study or project.

• A Federal law, called the Genetic Information Nondiscrimination Act (GINA), makes it illegal for health insurance companies, group health plans, and most employers to discriminate against you based on your genetic information. Additional information can be obtained at: http://irb.ufl.edu/gina.html or call1-800-669-3362 . If you think this law has been violated, it will be up to you to pursue any compensation from the offending insurance company and/or employer.

Throughout the study, the researchers will notify you of new information that may become available and might affect your decision to remain in the study.

If you wish to discuss the information above or any discomforts you may experience, please ask questions now or call one of the research team members listed in question 3 in this form.




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11a. What are the potential benefits to you for taking part in this research study?

There is no direct benefit to you for participating in this research study.

11 b. How could others possibly benefit from this study?

While you will not benefit directly from participating in this study, your participation may help researchers to understand the mechanisms contributing to aging and agerelated diseases including muscle loss. Such understanding is essential for development of treatments or interventions that can delay or prevent the consequences of aging and the physical decline that occurs with aging.

11 c. How could the researchers benefit from this study?

In general, presenting research results helps the career of a scientist. Therefore, the Principal Investigators listed in question 3 of this form may benefit if the results of this study are presented at scientific meetings or in scientific journals

12. What other choices do you have if you do not want to be in this study?

The other option to taking part in this study is doing nothing. If you do not want to take part in this study, do not sign this Informed Consent Form.

13a. Can you withdraw from this study?

You are free to withdraw your consent and to stop participating in this study at any time. If you do withdraw your consent, you will not be penalized in any way and you will not lose any benefits to which you are entitled. If you decide to withdraw your consent to participate in this study for any reason, please contact one of the research team members listed in question 3 of this form. They will tell you how to stop your participation safely.

If you have any questions regarding your rights as a research subject, please call the Institutional Review Board (IRB) office at (352) 273-9600.

13b.lf you withdraw, can information about you still be used and/or collected?

The data obtained by the time of your withdrawal may be used in the data analyses. However, this data will continue to be identified by letter and/or number codes only and all personally identifiable information (e.g. name, medical history etc.) will remain separate (i.e. paper copy and electronic files). Only authorized personnel will have access to this information. Furthermore, any cells established from your skin sample will be destroyed upon receipt of your written request of withdrawal.




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13c. Can the Principal Investigator withdraw you from this study?

You may be withdrawn from the study without your consent for the following reasons:

- Unsuccessful establishment of iPS cell line.

WHAT ARE THE FINANCIAL ISSUES IF YOU PARTICIPATE?

14. If you choose to take part in this research study, will it cost you anything?

The Sponsor will provide all medical services required as part of your participation in this study. There will be no cost to you. If you receive a bill related to this study, please contact the PI Anna-Maria Joseph, PhD at 352-273-6879 or the ARRC Manager William Marena at 352-273-9212.

Any other medical services provided to you that are not directly related to the study will be billed to you or your insurance company. You will be responsible for paying any deductible, co-insurance, and/or co-payments for these services, and any noncovered or out-of-network services.

Some insurance companies may not cover costs associated with studies. Please contact your insurance company for additional information.

15. Will you be paid for taking part in this study?

Yes, you will be paid with a $50 gift card for taking part in this study upon completion of your study visit.

If you are paid more than $25 for taking part in this study, your name and social security number will be reported to the appropriate University employees for purposes of making and recording the payment as required by law. You are responsible for paying income taxes on any payments provided by the study. If the payments total $600 or more or you are a nonresident alien, payment will be processed through the University of Florida Accounts Payable department and the University must report the amount you received to the Internal Revenue Service (IRS). If you have questions about the collection and use of your Social Security Number, please visit: http://privacv.ufl.edu/SSNPrivacv.html

IRB Project.#: 381-2012



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16. What if you are injured because of the study?

If you are injured as a direct result of your participation in this study, only the professional services that you receive from any University of Florida Health Science Center healthcare provider will be provided without charge. These healthcare providers include physicians, physician assistants, nurse practitioners, dentists or psychologists. Any other expenses, including Shands hospital expenses, will be billed to you or your insurance provider.

You will be responsible for any deductible, co-insurance, or co-payments. Some insurance companies may not cover costs associated with research studies or research-related injuries. Please contact your insurance company for additional information.

The Principal Investigator will determine whether your injury is related to your participation in this study.

No additional compensation is offered. The Principal Investigator and others involved in this study may be University of Florida employees. As employees of the University, they are protected· under state law, which limits financial recovery for negligence.

Please contact the PI Anna-Maria Joseph, PhD at 352-273-6879, the ARRC Manager William Marena at 352-273-9212, or Bhanu Sandesara, MD, by pager number 352-413-0592 (24-hour number) if you experience an injury or have questions about any discomforts that you experience while participating in this study.

17. How will your health information be collected, used and shared?

If you agree to participate in this study, the Principal Investigators will create, collect, and use private information about you and your health. This information is called protected health information or PHI. In order to do this, the Principal Investigator needs your authorization The following section describes what PHI will be collected, used and shared, how it will be collected, used, and shared, who will collect, use or share it, who will have access to it, how it will be secured, and what your rights are to revoke this authorization.

Your protected health information may be collected, used, and shared with others to determine if you can participate in the study, and then as part of your participation in the study. This information can be gathered from you or your past, current or future health records, from procedures such as physical examinations, x-rays, blood or urine tests or from other procedures or tests. This information will be created by receiving study treatments or participating in study procedures, or from your study visits and telephone calls. More specifically, the following information may be collected, used, and shared with others:




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• Name

• Age

• Date of Birth

• Gender

• Address

• Telephone number • Social security number for compensation purposes • Current and past medical history and records of physical exams to determine

eligibility criteria.

This information will be stored in locked filing cabinets or on computer servers with secure passwords, or encrypted electronic storage devices.

Some of the information collected could be included in a "limited data set" to be used for other research purposes. If so, the limited data set will only include information that does not directly identify you. For example, the limited data set cannot include your name, address, telephone number, social security number, photographs, or other codes that link you to the information in the limited data set. If limited data sets are created and used, agreements between the parties creating and receiving the limited data set are required in order to protect your identity and confidentiality and privacy.

18. For what study-related purposes will your protected health information be collected, used, and shared with others?

Your PHI may be collected, used, and shared with others to make sure you can participate in the research, through your participation in the research, and to evaluate the results of the research study. More specifically, your PHI may be collected, used, and shared with others for the following study-related purpose(s):

• To determine the causes or effects of the study condition - more specifically, to assess the medical or health conditions that may alter the mechanisms of aging and age-related diseases.

Once this information is collected, it becomes part of the research record for this study.

19. Who will be allowed to collect, use, and share your protected health information?

Only certain people have the legal right to collect, use and share your research records, and they will protect the privacy and security of these records to the extent the law allows. These people include:

• the study Principal Investigators (listed in question 3 of this form) and research staff associated with this project.


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• other professionals at the University of Florida or Shands Hospital that provide study-related treatment or procedures.

• the University of Florida Institutional Review Board (IRB; an IRB is a group of people who are responsible for looking after the rights and welfare of people taking part in research).

20. Once collected or used, who may your protected health information be shared with?

Your PHI may be shared with:

• the study sponsor (listed in Question 4 of this form).

• United States and foreign governmental agencies who are responsible for overseeing research, such as the Food and Drug Administration, the Department of Health and Human Services, and the Office of Human Research Protections.

• Government agencies who are responsible for overseeing public health concerns such as the Centers for Disease Control and federal, state and local health departments.

Otherwise, your research records will not be released without your permission unless required by law or a court order. It is possible that once this information is shared with authorized persons, it could be shared by the persons or agencies who receive it and it would no longer be protected by the federal medical privacy law.

21. If you agree to take part in this research study, how long will your protected health information be used and shared with others?

Your PHI will be used and shared with others until the end of the study.

You are not required to sign this consent and authorization or allow researchers to collect, use and share your PHI. Your refusal to sign will not affect your treatment, payment, enrollment, or eligibility for any benefits outside this research study. However, you cannot participate in this research unless you allow the collection, use and sharing of your protected health information by signing this consent and authorization.

You have the right to review and copy your protected health information. However, we can make this available only after the study is finished.

You can revoke your authorization at any time, before, during, or after your participation in this study. If you revoke it, no new information will be collected about you. However, information that was already collected may still be used and shared with others if the researchers have relied on it to complete the research. You can revoke your authorization by giving a written request with your signature on it to the Principal Investigator.




Page 10 of 11

SIGNATURES

As an investigator or the investigator's representative, I have explained to the participant the purpose, the procedures, the possible benefits, and the risks of this research study; the alternative to being in the study; and how the participant's protected health information will be collected, used, and shared with others:

Signature of Person Obtaining Consent and Authorization

Date

You have been informed about this study's purpose, procedures, possible benefits, and risks; the alternatives to being in the study; and how your protected health information will be collected, used and shared with others. You have received a copy of this Form. You have been given the opportunity to ask questions before you sign, and you have been told that you can ask questions at any time.

You voluntarily agree to participate in this study. You hereby authorize the collection, use and sharing of your protected health information as described in sections 17-21 above. By signing this form, you are not waiving any of your legal rights.

Signature of Person Consenting and Authorizing

IRS Project#: 381-2012



Date

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Protocol Title Establishment of Age-Specific Induced Pluripotent Stem (iPS) Cells from Dermal Fibroblasts of Young and Elderly Individuals: Potential Model of Aging (Aging iPS Cell Study)

PI Anna-Marie Joseph, PhD IRB # 381-2012

Most recent IRB approval date 8/17/12 (expires 8/16/13)

Target Enrollment 10 (N=5 ages 20-35; N=5 ages 70-99) Anticipated Study Completion Date June, 2014

ABSTRACT OF STUDY (Hypothesis and Design):

Aging in humans is characterized by an overall decline in functional capacity and responsiveness of tissues and organs to physiological stimuli. Postmitotic tissue such as skeletal muscle, is particularly affected due to its limited regenerative capacity, and undergoes a progressive loss in mass (sarcopenia) that ultimately contributes to reduced physical function in the elderly. The underlying mechanisms contributing to age-related dysfunction such as sarcopenia are currently under investigation but detailed molecular and cellular studies in humans are somewhat limited due to the lack of available models that adequately reflect the pathophysiology of aging in humans.

Recently, the development of induced pluripotent stem (iPS) cells has shown great promise to circumvent this problem in the aging field. iPS cells are adult cells that are genetically reprogrammed to an embryonic stem (ES) cell-like state by expressing a cocktail of factors (Oct3, Sox2, Klf4, and c-Myc) that are important for maintaining the properties of ES cells (1). These cells exhibit important characteristics of ES cells including morphology, proliferation, and expression of stem cell markers. Importantly, iPS cells retain the pluripotency of ES cells which allows them to form tissues of all three major lineages both in vitro, as well as in vivo, following transplantation. Thus, iPS cells are capable of differentiating into numerous tissues including skeletal muscle, opening important opportunities to study the pathogenic mechanisms of a disease or condition in the affected tissue. Moreover, human iPS cells have less ethical concerns and immunocompatibility issues than ES cells. The ability of human iPS cells to retain the disease-specific properties of a patient has made them an attractive system to model human disease and opens the opportunity for providing “patient-specific” treatment with respect to clinical medicine. In fact, patient-specific iPS cells have already been utilized extensively for various pathological conditions (2).

We hypothesize that human iPS cells, and iPS-derived muscle cells derived from elderly individuals, will retain an aging phenotype and will provide a highly innovative model to study aging in humans. Additionally, an extremely exciting possibility is that these cells can be utilized for the high-throughput screening of drugs in aging research. Opportunely, the subjects for this proposed study will be recruited from an ongoing study at the Institute on Aging at the University of Florida minimizing both time and recruitment costs. Thus, iPS cells will be derived from dermal fibroblasts of elderly individuals to investigate the underlying

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pathogenesis of aging, with the goal of developing treatment paradigms to attenuate and delay age-related diseases. Research Plan:

Study participants for this study will be in part recruited from the Developmental Project funded by the University of Florida Claude D. Pepper Older Americans Independence Center.

Briefly, the Aging iPS Cell study is a single-site cohort study with subjects separated into two age groups, young (20-35 years) and elderly (age > 70 years). We will enroll 10 individuals in total (5 subjects/group), with up to an additional 6 being recruited if a skin biopsy cannot be obtained for any of the subjects. Subjects recruited from the Developmental Project fit into these two categories and baseline measurements have already been completed and subjects will start returning for a four year follow-up in 2012. The Clinical Recruitment Core of the Department of Aging has already performed all screening, a basic questionnaire regarding participant’s health history, and completed all recruitment of research participants, minimizing time and cost for the proposed iPS cell study. INCLUSION / EXCLUSION CRITERIA (Please list in box below) Inclusion criteria The following inclusion criteria will be adopted to select the study participants:

• males and females aged 20-35 and 70-99 years • sedentary lifestyle (i.e., the subject has spent less than 20 minutes per week in the past

2 month performing structured physical activity, such as exercising at a gym and/or weight training)

• willing and able to give informed consent Exclusion criteria:

• history of smoking in the prior 12 months • active treatment for cancer or history of cancer in the past 3 years • congestive heart failure NYHA Class III or IV • previous stroke with upper and/or lower extremities involvement within the last 6 months • peripheral vascular disease Fontaine Class III/IV • History of life-threatening cardiac arrhythmias, stroke, severe Parkinson’s disease or

severe neurological disorders likely to interfere with physical function • cognitive impairment (i.e., MMSE ≤ 23) • renal disease requiring dialysis • lung disease requiring steroids • lower extremity amputation • severe osteoarthritis that interferes with physical function • Complicated diabetes • inflammatory diseases such as active rheumatoid arthritis, vasculitis, autoimmune

disorders, and inflammatory bowel disease • life-threatening illnesses with an estimated life expectancy less than 1 year • history of drug or alcohol abuse • taking GH and/or estrogen replacement therapy • Testosterone medication • Anticoagulant therapy • involved in active weight loss > 5 kg in prior 3 months • pregnancy (determined by a pregnancy test) • SPPB score of 8-10 • Lidocaine allergy

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• recent bacterial infection (< 2 weeks) • acute febrile illness in previous 2 months • high blood pressure (i.e., BP ≥ 180/110 mm Hg) at the visit (subject will be referred to

his/her physician and reevaluated after appropriated therapy being instituted) • major surgery or hip/knee replacement in the past 6 months

Current Enrollment Please place an “X” the box for each:


Recruitment X Intervention X

Participant Contact X *If you have marked complete for each row, this will be the last report for the DSMB.


Active Completed Withdrawn or Lost to Follow-up Total Enrolled

0 4 0 4

Total Number of Protocol Deviations: __4__ (List in order of most recent to oldest since start of study)

Date:

PID:

Explanation:

11/29/12 381-2012-0004 (Elderly cohort)

Participant did not meet inclusion/exclusion criteria of sedentary lifestyle and testosterone treatment and was not screened for criteria of cognitive impairment, osteoarthritis, anticoagulant treatment, and surgical history. Please refer to corrective action described below in the Protocol Changes section.

11/16/12 381-2012-0003 (Young cohort)

Participant did not meet inclusion/exclusion criteria of sedentary lifestyle and was not screened for the criteria of cognitive impairment, osteoarthritis, testosterone or anticoagulant treatment, and surgical history. Please refer to corrective action described below in the Protocol Change section.

11/8/12 381-2012-0002 (Young cohort)


Participants Who Have Withdrawn Consent or Lost to Follow Up (List in order of most recent to oldest since start of study.

Date:

PID:


N/A

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11/2/12 381-2012-0001 (Young cohort)


AE INFORMATION: Please use table below to report any Adverse Events that are related to the study (Possibly, Probably, or Definitely). They do not have to be reportable to IRB to be included (i.e. they also do not need to be unexpected or related). If blinded, you do not need to complete intervention arm. If there is not more than one intervention it may be left blank. TOTAL Number of AEs___2_____ (List in order of most recent to oldest since start of study) Date of Onset

Date Ended PID Event Relationship


If related 12/3/12 12/5/12 381-2012-0004 Rash Yes Rash resolved. 11/16/12 12/5/12 831-2012-0003 Rash Yes Participant treated with

prescribed ointment. Rash resolved.

SAE INFORMATION: Please use table below to report all events that are Serious since the start of the study. They do not have to be reportable to IRB to be included (i.e. they also do not need to be unexpected or related). If blinded, you do not need to complete intervention arm. If there is not more than one intervention it may be left blank. TOTAL Number of SAEs__0______ (List in order of most recent to oldest since start of study) Date of Onset

Date Ended PID Event Relationship


If related PROTOCOL CHANGES: Include all changes, since last DSMB review. Explain if safety related.


Explanation of Safety Relation

Pending IRB approval

Initially this protocol was designed as an ancillary study to the Developmental Apoptosis Study (IRB# 429-2005) and as an ancillary study, its draft protocol included inclusion and exclusion criteria to match that of the parent study. It was recommended later during the IRB submission preparation that it would be more practical to instead have the Aging iPS Cells Study as an independent study. However due to Investigator error, the inclusion/ exclusion criteria was not updated to reflect the specific goals of the Aging iPS Cells Study, which are less stringent than that of the Developmental Apoptosis Study. Therefore, the first four participants enrolled in the Aging iPS Cells Study were inappropriately enrolled under the inclusion/exclusion criteria as it is stated in the approved protocol. The specific criteria that each participant did not meet are detailed in the Protocol Deviations section above.

This protocol amendment does not impact the safety of the study.

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An updated study protocol has been submitted the IRB and is pending review. This submission includes updates to the inclusion/exclusion criteria in the study protocol (see updated criteria below). Additionally, the telephone screening questionnaire was updated to ensure that potential participants are assessed of the updated entry criteria. Additional recruitment in this study will not commence until after this IRB submission is approved and the revised form has been implemented. Proposed updated Inclusion/Exclusion Criteria as submitted for IRB approval: Inclusion criteria The following inclusion criteria will be adopted to select the study participants:

• males and females aged 20-35 and 65-99 years • willing and able to give informed consent

Exclusion criteria:

• active treatment for cancer or history of cancer in the past 3 years

• congestive heart failure NYHA Class III or IV • previous stroke with upper and/or lower

extremities involvement within the last 6 months • peripheral vascular disease Fontaine Class III/IV • History of life-threatening cardiac arrhythmias,

stroke, severe Parkinson’s disease or severe neurological disorders likely to interfere with physical function

• renal disease requiring dialysis • lung disease requiring steroids • lower extremity amputation • Complicated diabetes • life-threatening illnesses with an estimated life

expectancy less than 1 year • Anticoagulant therapy • involved in active weight loss > 5 kg in prior 3

months • pregnancy (determined by a pregnancy test) • Lidocaine allergy


• recent bacterial infection (< 2 weeks) • acute febrile illness in previous 2 months • high blood pressure (i.e., BP ≥ 180/110 mm Hg)

at the visit (subject will be referred to his/her physician and reevaluated after appropriated therapy being instituted)

Lifestyle Interventions and Independence for Elders (LIFE) Data and Safety Monitoring Board (DSMB) Teleconference

November 30, 2012

DSMB: Gerald Beck, PhD (Chair); Patricia Dubbert, PhD; Jay Magaziner, PhD, MS, Hyg; David Buchner, MD, MPH; William Hazzard, MD; J. Richard Landis, PhD. (absent), Steve Hulley, MD, MPH NIH: Barbara Radziszewska, MD (NHLBI) Recommendations: 1. The DSMB congratulated investigators on the excellent detailed report and study progress. 2. Proposed ancillary studies:

a. AS12-04 Nadkarni – needs further information on actual sample size achievable, the power that goes with this, and the impact on the main trial of any SAEs that occur during the additional testing.

b. AS12-08 Strotmeyer – approved

c. AS12-11 Cohen - approved

2. The DSMB expressed no concerns about the LIFE study and unanimously recommended continuation of the trial. 3. The Board requested several modifications or additions to tables or figures in the open and closed session reports (to be conveyed separately). 4. The time to adjudicate SAEs should be reduced so that as the trial nears the end, there is not a large lag (currently about 6 months, Figure 8b closed report) in being able to report final results.

LIFE DSMB Recommended Further Analyses Open Session Report: Pages 29-30, make the y-axis on the same scale for these plots. Pages 57, 59 – to remove in-patient hospitalizations from the all SAE figures by epoch. That is, present both “all” and then separate figures for “all “ without in-patient hospitalizations, and in-patient hospitalizations alone (as already done on pages 58 and 60). Closed Session Report: Page 228plus – present in future reports “broader” categories of event classification (like you just sent in November monthly report).

Page 262 – also show Table 6A.6 classified by gender. Page 280plus, Section 6B – As far as ER visits, consider if you can classify persons as far as “time at center” or # times visited center to see if those “more familiar” with being at the medical center is associated with increased likelihood of going to ER. Page 299plus, Section 6C - Look at what types of events are occurring on site at clinical centers Page 330plus, Section 9 - Look at 400m walk failure by site

The Testosterone Trial Data and Safety Monitoring Board (DSMB) Teleconference

May 24, 2012

T Trial DSMB recommendations 1 May 24, 2012

Study Staff: Peter J. Snyder, M.D., Principal Investigator; J. Richard Landis, Ph.D., Susan Ellenberg, Ph.D.; Laura Fluharty, M.P.H.,

DSMB: J. Philip Miller, Ph.D., Richard Chappell, Ph.D., George A. Kuchel, M.D., Wayne Meikle, M.D., Wayne Hellstrom, M.D., Manuel Cerqueria, M.D., Michael Thorner, M.D. Absent: Eric Klein M.D. E. Magnus Ohman, M.D.

NIA: Sergei Romashkan, M.D.

NHLBI: Diane Bild, M.D., M.P.H., Nakela Cook, M.D.

Recommendations:

1. The DSMB congratulated investigators on the excellent study progress.

2. The Board expressed no concerns about the safety of the participants enrolled in the T Trial and recommended continuation of the study.

3. The DSMB requested the following changes to the format and content of the DSMB reports:

a. Develop a table showing changes in doses of important medications during the study. This table should be included in the closed session report and show changes by a blinded treatment arm.

i. The changes should be shown as “no change,” “dose increased” and “dose decreased.”

ii. Medications to report include, but are not limited to antidiabetic, statins and other drugs that could affect the outcomes measured in the trials. Complete list of medications to monitor should be developed by the investigators and agreed upon with the DSMB.

b. The closed session report should include a table presenting distribution of serum testosterone levels in the treatment and control groups by study visit and blinded arm (A and B). The report should show mean, median, minimal and maximum values as well as proportion of men with testosterone levels within the study defined normal range, those with the testosterone levels > 800 ng/dl and < 400 ng/dl.

4. The Board expressed significant concerns about the progress and scientific value of the

CT angiography component of the cardiovascular study and deferred its recommendations on continuation of this study until review of additional information, which should be provided to the DSMB within two weeks after investigators receiving the Board’s recommendations. The following should be provided to the DSMB:

The Testosterone Trial Data and Safety Monitoring Board (DSMB) Teleconference

May 24, 2012

T Trial DSMB recommendations 2 May 24, 2012

a. Amended cardiovascular study protocol showing the number of participants that would be enrolled in the cardiovascular study by the end of enrollment in the parent T Trial.

i. The protocol should include power calculations for the CT angiography outcome for the new N and discussion of the scientific value of the measurement at the expected power.

ii. The protocol should specify investigators’ expectations for changes in artery plaque volume in the control group.

b. Data showing the variability of repeat CT angiography measurements. c. Data supporting the accuracy of measuring coronary artery plaque volume in the

presence of a large amount of calcifications in this group of men.

ASPREE DSMB MEETING

10/22/2012

Attendance: ASPREE PI/Co-Is: AUS John McNeil, Robin Woods, Christopher Reid; US: Richard Grimm, Anne Murray, and Brenda Kirpach DSMB Members: J.P. Mohr, Garnet Anderson, JoAnn Manson, Mary Sano, Sean Morrison, Lawrence Friedman, and Stuart Connolly

NIA: Barbara Radziszewska, Evan Hadley, Judy Hannah, and Sergei Romashkan

Action Items from the ASPREE DSMB Meeting:

1. There are no safety concerns and the study should continue.

2. The data reports are not complete and confusing. The following modifications to reports going

forward should be made:

A. Graphs showing actual and expected rates according to the design assumptions, by

treatment group and by age group, and overall, for the following:

cumulative randomization

off study drug status (crossovers)

loss-to-follow-up

B. Table showing endpoint adjudication rates (you can use the existing table)

C. Table showing annual visit completion rates (you can use the existing table). Also please

provide your best estimates of the number of person-years of follow-up observed overall

and by age-group.

D. Cumulative data on event rates, by treatment group and overall, by age groups, for the

following:

The 3 components of the composite primary outcome:

o Death

o Dementia

o Physical disability

Composite primary outcome

Cancer (by major type, e.g., melanoma, breast cancer)

CVD (by major type, including MI, stroke, arrhythmia, bypass surgery)

Clinically significant bleeding

Hospitalization for other reason

Please present the above in tabular form (example of a shell table provided below) and in a graphic

form. For the latter, please provide Kaplan-Meyer graphs showing cumulative incidence of the

events discussed above. Please prepare the graphs by blinded treatment group A and B. In addition,

please add an “assumed” (or “expected”) curve for the placebo group to each graph, which would

serve as a reference. Such a curve must be estimated based upon characteristics of the actual

ASPREE population, but using known event rates data from similar populations in other trials (e.g.,

MRFIT, Physicians Health Study). A comparison of the overall “expected” rate to the rates used in

the original power calculations would also be useful.

Example of a shell table:

Cumulative incidence of death, by treatment group and by age strata where annualized % provides

an estimate of the annual event rate per 100 person-years of follow-up.

Event: Death Treatment Group

Total

Age Group

A N (annualized %)

B N (annualized %)

65-69

70-74

75-79

80-84

85+

Overall

3. ASPREE should provide updated power calculations based on current data using age

distribution, follow-up time, and other factors.

4. The ASPREE CC needs to generate additional analyses to address questions regarding

recruitment. For now the decision on opening Caucasian recruitment in the US should be

deferred.

5. The ASPREE IMAGE Pilot Study was not approved by the DSMB because it could change the

enrollment of the study population.

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