Mechanism of Monocyte Priming in Humans - a Feeding Trial
Phase: NA
Conditions: Heart Diseases
Sponsor: Wake Forest University Health Sciences

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IMPACCT: “Investigating Monocyte Priming in Adults in a Caloric Challenge Trial” 
Unique Protocol Identification Number: IRB00083476 
National Clinical Trial (NCT) Identified Number: NCT05348395 
Principal Investigator: Jamy Ard 
IND/IDE Sponsor:  
Funded by: NHLBI 
Draft Number: 0.040 
26 February 2024 
 
 
Summary of Changes from Previous Version: 
 
Affected Section(s) Summary of Revisions Made 
Rationale 
6.4 Study 
intervention 
compliance 
Added verbiage to accommodate 
missed visits 
Improve recruitment and retention by 
planning for missed visits 
1.3 Schedule of 
Activities 
Remove lipids, insulin, CMP blood 
draw at wk4 of experimental phase 
Not needed 
6.1 Study 
Intervention 
Administration 
Revised run-in and experimental 
feeding requirements 
Reduce barriers for participants by 
reducing the number of study visits 
5 Study population Revise BMI range for inclusion 
Accommodate physically fit 
participants that may fall above the 
“normal” range BMI 
1.3 Schedule of 
Activities (SOA) 
Updated to include additional 
details of visit activities 
Improve clarity of activities that will 
occur at visits 
5.1 Inclusion 
Added inclusion for waist 
circumference 
Waist circumference will be helpful to 
determine eligibility, particularly for 
those that have an above “normal” 
range BMI 
6.1 Study 
Intervention 
Administration 
Reduced run-in feeding window 
Reducing number of potential visits 
will improve efficiency for meal 
prepping and reduce participant 
burden  
 
 
 
 

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Table of Contents 
Statement of Compliance .............................................................................................................................................. 4 
1 Protocol Summary ...................................................................................................................................................... 5 
1.1 Synopsis ............................................................................................................................................................... 5 
1.2 Schema ................................................................................................................................................................ 5 
1.3 Schedule of Activities (SoA) ................................................................................................................................. 6 
2 Introduction ................................................................................................................................................................ 8 
2.1 Study Rationale .................................................................................................................................................... 8 
2.2 Background .......................................................................................................................................................... 8 
2.3 Risk/Benefit Assessment...................................................................................................................................... 9 
2.3.1 Known Potential Risks ................................................................................................................................... 9 
2.3.2 Known Potential Benefits ........................................................................................................................... 10 
2.3.3 Assessment of Potential Risks and Benefits ............................................................................................... 10 
3 Objectives and Endpoints ......................................................................................................................................... 11 
4 Study Design ............................................................................................................................................................. 12 
4.1 Overall Design .................................................................................................................................................... 12 
4.2 Scientific Rationale for Study Design ................................................................................................................. 12 
5 Study Population ...................................................................................................................................................... 13 
5.1 Inclusion Criteria ................................................................................................................................................ 13 
5.2 Exclusion Criteria ............................................................................................................................................... 13 
5.3 Lifestyle Considerations ..................................................................................................................................... 13 
5.4 Screen Failures ................................................................................................................................................... 14 
5.5 Strategies for Recruitment and Retention ......................................................................................................... 14 
6 Study Intervention .................................................................................................................................................... 16 
6.1 Study Intervention(s) Administration ................................................................................................................ 16 
6.1.1 Study Intervention Description ................................................................................................................... 16 
6.2 Dietary composition .......................................................................................................................................... 16 
6.2.1 diet formulation- Run in ............................................................................................................................. 16 
6.2.2 diet formulation- experimental .................................................................................................................. 17 
6.4 Study Intervention Compliance ......................................................................................................................... 18 
7 Study Intervention Discontinuation and Participant Discontinuation/Withdrawal ................................................. 19 
7.1 Discontinuation of Study Intervention .............................................................................................................. 19 
7.2 Participant Discontinuation/Withdrawal from the Study .................................................................................. 19 
7.3 Lost to Follow-Up ............................................................................................................................................... 19 
8 Study Assessments and Procedures ......................................................................................................................... 20 
8.1 Efficacy Assessments ......................................................................................................................................... 20 
8.2 Safety and Other Assessments .......................................................................................................................... 20 
8.3 Adverse Events and Serious Adverse Events ..................................................................................................... 20 
8.3.1 Definition of Adverse Events (AE) ............................................................................................................... 20 
8.3.2 Definition of Serious Adverse Events (SAE) ................................................................................................ 21 
8.3.3 Classification of an Adverse Event .............................................................................................................. 21 
8.3.4 Time Period and Frequency for Event Assessment and Follow-Up ............................................................ 22 
8.3.5 Reporting of Pregnancy .............................................................................................................................. 22 
8.4 Unanticipated Problems .................................................................................................................................... 22 
8.4.1 Definition of Unanticipated Problems (UP) ................................................................................................ 22 
8.4.2 Unanticipated Problem Reporting .............................................................................................................. 22 
9 Statistical Considerations ......................................................................................................................................... 24 

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9.1 Statistical Analyses............................................................................................................................................. 24 
9.1.1 General Approach ....................................................................................................................................... 24 
10 Supporting Documentation and Operational Considerations ................................................................................ 25 
10.1 Regulatory, Ethical, and Study Oversight Considerations ................................................................................ 25 
10.1.1 Informed Consent Process ........................................................................................................................ 25 
10.1.2 Confidentiality and Privacy ....................................................................................................................... 25 
10.1.3 Future Use of Stored Specimens and Data ............................................................................................... 25 
10.1.4 Key Roles and Study Governance ............................................................................................................. 26 
10.1.5 Safety Oversight ........................................................................................................................................ 26 
10.1.6 Clinical Monitoring .................................................................................................................................... 26 
10.3 Abbreviations ................................................................................................................................................... 26 
10.4 Protocol Amendment History .......................................................................................................................... 27 
 
 
 

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STATEMENT OF COMPLIANCE 
Provide a statement that the trial will be conducted in compliance with the protocol, International 
Council on Harmonisation Good Clinical Practice (ICH GCP) and applicable state, local and federal 
regulatory requirements. Each engaged institution must have a current Federal-Wide Assurance (FWA) 
issued by the Office for Human Research Protections (OHRP) and must provide this protocol and the 
associated informed consent documents and recruitment materials for review and approval by an 
appropriate Institutional Review Board (IRB) or Ethics Committee (EC) registered with OHRP. Any 
amendments to the protocol or consent materials must also be approved before implementation. Select 
one of the two statements below. If the study is an intramural NIH study, use the second statement 
below: 
1. The trial will be carried out in accordance with International Council on Harmonisation Good 
Clinical Practice (ICH GCP) and the following: 
o 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR Part 
46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812). 
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded clinical trials have 
completed Human Subjects Protection and ICH GCP Training. 
OR 
2. The trial will be conducted in accordance with International Council on Harmonisation Good 
Clinical Practice (ICH GCP), applicable United States (US) Code of Federal Regulations (CFR), and 
the [specify NIH Institute or Center (IC) [ Terms and Conditions of Award. The Principal 
Investigator will assure that no deviation from, or changes to the protocol will take place 
without prior agreement from the funding agency and documented approval from the 
Institutional Review Board (IRB), and the Investigational New Drug (IND) or Investigational 
Device Exemption (IDE) sponsor, if applicable, except where necessary to eliminate an 
immediate hazard(s) to the trial participants. All personnel involved in the conduct of this study 
have completed Human Subjects Protection and ICH GCP Training. 
For either option above, the following paragraph would be included: 
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the IRB for review and approval. Approval of both the protocol and the consent 
form(s) must be obtained before any participant is consented. Any amendment to the protocol 
will require review and approval by the IRB before the changes are implemented to the study. 
All changes to the consent form(s) will be IRB approved; a determination will be made regarding 
whether a new consent needs to be obtained from participants who provided consent, using a 
previously approved consent form. 

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1 PROTOCOL SUMMARY 
1.1 SYNOPSIS 
Title: 
Mechanism of monocyte priming in humans- a feeding trial  
Study Description: 
  
Objectives: 
Primary Objective: 
Determine the molecular mechanisms by which 
high-calorie diets trigger monocyte priming and 
reprogramming in metabolically healthy human 
subjects and whether and to what extent these 
mechanisms differ from mice. 
Secondary 
Objectives: 
To identify which proteins alter their S-
glutathionylation status (and possibly their 
expression levels) in response to HCD-induced 
monocyte priming, and which signaling pathways 
are altered in these cells 
Endpoints: 
Primary Endpoint: 
Monocyte priming will be assessed measuring 
MKP-1 activity 
Unbiased lipidomics 
Secondary Endpoints: Monocyte protein S-glutathionylation state 
Study Population: 
Healthy adults, age 20-45 
Phase: 
  
Description of 
Sites/Facilities Enrolling 
Participants: 
Wake Forest School of Medicine Clinical Research Unit 
Description of Study 
Intervention: 
Feeding study with a run-in period followed by overfeeding on a Western 
diet 
Study Duration: 
Up to 4 weeks of run-in + 8 weeks of experimental diet 
Participant Duration: 
14 weeks 
  
 
 
1.2 SCHEMA 
 
 

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 1.3 SCHEDULE OF ACTIVITIES (SOA) 
Visit Week 
T<-4 
SV1 (<T -2) 
SV2 (T -1) 
Run-in 
week 1
RI-day 5 
RI-2 
RI-3 
RI-4 
EXP-1 
EXP-2 
EXP-3 
EXP-4 
EXP-5 
EXP-6 
EXP-7 
EXP-8 
Study activity 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Self-screen and/or Phone screen 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Consent 
  
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Screen visit (SV) 1 
  
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
History and physical exam 
  
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Screening labs (CMP, lipids, A1c, insulin) 
  
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Past medical history 
  
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Demographics 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Plate waste method orientation and homework 
  
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Review study diet with RD 
  
x 
 x 
  
  
  
  
  
  
  
  
  
  
  
  
  
Body weight 
  
x 
 x 
  
  
  
  
  
  
  
  
  
  
  
  
  
Height 
  
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Blood pressure 
  
x 
 x 
  
  
  
  
  
  
  
  
  
  
  
  
  
Pulse 
  
x 
 x 
  
  
  
  
  
  
  
  
  
  
  
  
  
Waist circumference 
  
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Adverse events 
  
  
x 
 x 
 x 
 x 
 x 
 
x 
 x 
 x 
 
x 
 x 
 x 
 x 
 
x 
 x 
Concomitant medications 
  
  
x 
x  
 x 
 x 
 x 
 
x 
 x 
 x 
 
x 
 x 
 x 
 x 
 
x 
 x 
Pregnancy test5 
  
x 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
Review plate waste homework 
  
  
x 
  
 x 
x  
x 
x 
x 
x 
 
x 
 x 
 x 
 x 
 
x 
 x 
Calculate estimated energy needs6 
  
  
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
Start Run-in feeding 
  
  
  
x 
  
  
  
  
  
  
  
  
  
  
  
  
Establish weight stability target 
  
  
  
  
x 
  
  
  
  
  
  
  
  
  
  
  
Monocyte sample (blood draw) 
  
  
  
  
  
x3 
x
1 
x
1 
x
1 
X
1 
x1 x1 
x
1 
X1
,4 
Body weight 
  
  
  
  
  
  
  
  
x
2 
x
2 
x
2 
x2 
x2 x2 
x
2 
x2 

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Blood pressure 
  
  
  
  
  
  
  
  
x
2 
x
2 
x
2 
x2 
x2 x2 
x
2 
x2 
1- Samples are taken at the end of the week (i.e., 5-7 days of feeding) 
2- Weight and blood pressure will be done once weekly in a fasted state 
3- At least 5 days after establishing weight target, as long as the participant is ±2% of the weight target on the 5th day 
4- Lipids, insulin, and CMP 
5 – A urine pregnancy test will be conducted for all females of child-bearing 
potential 
6 – Physical activity will be assessed in order to get Total Energy Expenditure 
(TEE) to estimate caloric needs 
 
 

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2 INTRODUCTION 
2.1 STUDY RATIONALE 
To determine the mechanism of monocyte priming in humans, we will do a complete feeding trial in 15 
male and 15 female normal weight and metabolically healthy human subjects (20-45 years of age) using 
a western diet (WD), characterized as being high-saturated fat, high-fructose, and high-calorie for 8 
weeks.  
 
 
2.2 BACKGROUND 
Monocytes and macrophages are essential for tissue homeostasis, but in the context of metabolic 
disorders they become dysfunctional and promote chronic inflammatory diseases, including 
atherosclerosis. However, the underlying mechanisms linking metabolism to altered phenotype remain 
poorly understood. We have shown that chronic exposure of blood monocytes to nutrient stress 
induced by a “Western”-style high-calorie diet (HCD) stimulates the formation of reactive oxygen species 
(ROS) and promotes protein thiol oxidation, resulting in monocyte dysfunction and the reprogramming 
of blood monocytes into a pro-inflammatory, pro-atherogenic phenotype, hyper-sensitive to 
chemoattractants. These metabolically “primed” blood monocytes give rise to re-programmed and 
dysfunctional macrophages, sensitive to oxysterol-induced cell death, with defective autophagy and 
dysregulated activation profiles. Monocyte priming by nutrient stress is mediated by the H2O2-
dependent S-glutathionylation, inactivation and degradation of mitogen-activated protein kinase 
phosphatase 1 (MKP-1), a master regulator of both monocyte adhesion and migration and macrophage 
function and plasticity (Fig. 1). However, the source(s) of HCD-induced H2O2 and “oxidative stress” in 
“primed” blood monocytes has not been identified. Identifying this source of H2O2 may open new 
therapeutic avenues and the development of novel drugs for the prevention and treatment of 
cardiovascular diseases. 

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Figure 1: Central Hypothesis: Proposed roles for Mao A and Nox4 in high-calorie diet-induced monocyte 
priming and repro-gramming, macrophage dysfunction and atherogenesis. GSH: glutathione; MKP-1-
SSG; S-glutathionylated MKP-1 
  
Recently, we showed that a HCD also promotes monocyte priming, reprogramming and dysfunction in 
non-human primates. Primed baboon monocytes showed the same hyperchemotactic, proinflammatory 
and proatherogenic phenotype we identified both in HCD-fed mice and, ex vivo, for metabolically 
stressed human monocytes. In baboons, monocyte priming correlated with loss of MKP-1 activity in 
monocytes, validating MKP-1 activity as a sensitive biomarker of monocyte priming and dysfunction. 
More importantly, we identified a highly significant correlation between plasma cholesterol and both 
loss of MKP-1 activity and monocyte priming, suggesting that total plasma cholesterol may be the 
primary driver of monocyte priming, reprogramming and dysfunction. We will test this hypothesis in this 
Aim. 
 
 
2.3 RISK/BENEFIT ASSESSMENT 
2.3.1 KNOWN POTENTIAL RISKS 
There is some minor discomfort and risk of mild bruising during venipuncture.  A breech of participant 
confidentiality is an additional risk for each participant. Because this study involves overfeeding (i.e., 
providing more calories than necessary for maintenance of body weight), participants will be expected 
to see an increase in body weight. The expected weight gain is approximately 0.5 lb per week for an 
average person consuming 2000 kcal/day. We expect some participants will have adverse changes in 
blood lipids and markers of inflammation with the experimental diet and associated weight gain. 

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Discomfort associated with venipuncture is rapidly reversible.  Bruises from venipuncture will heal in 
several days. To deal with the expected weight gain due to overfeeding, we will provide counseling at 
the completion of the trial to help participants lose weight back to baseline. We will provide this 
counseling free of charge through registered dietitians at our Weight Management Center. We will give 
participants guidance on a weight loss plan that includes a healthful dietary pattern that can reverse any 
metabolic changes seen during feeding. 
2.3.2 KNOWN POTENTIAL BENEFITS 
There are no direct benefits to individuals participating in this research trial. 
2.3.3 ASSESSMENT OF POTENTIAL RISKS AND BENEFITS 
Protection against risks: Risks to confidentiality will be protected by coding participant records as 
described above. Additionally, all study participant records will be kept in a locked file cabinet, in a 
locked office (in the Research Interviewer’s office), and in a locked suite of offices that is protected by an 
automatic alarm system. When the Research Interviewer is not in her office, the office door will be kept 
locked. 
All tests are conducted under the supervision of trained personnel.  Since the tests are not inherently 
hazardous, hazard is likely to occur only as the result of impaired subject confidence or sudden 
unwillingness to complete the test.  To avoid such hazards, study personnel thoroughly explain all tests 
to potential subjects before subjects are offered the consent/assent form to sign.  Potential subjects 
have the opportunity to see all equipment and facilities before giving consent or undergoing testing. 
These precautions make it highly unlikely that harm will occur as a result of testing. 
We will monitor the amount of weight gain each participant experiences on a weekly basis. Those 
individuals with much higher rates of weight gain than expected will have a review of their total energy 
expenditure estimates and study prescribed calorie goals. We will adjust the calories to stay within the 
approximate average range of weight gain for the course of the 8 weeks of feeding (i.e., stay within the 
goal of 25% higher calories than total energy expenditure). This should limit the potential for someone 
to gain excessive amounts of weight during the trial. 
Because this trial includes an overfeeding component, and participants would be expected to gain 
weight during the 8 weeks of feeding, we will provide counseling at the completion of the trial to help 
participants lose weight back to baseline. We will provide this counseling free of charge through 
registered dietitians at our Weight Management Center. Participants will have access to counseling on a 
monthly basis for up to 12 months. If additional support is needed beyond this to assist with returning to 
a baseline weight, we will do an individual medical consultation with them and devise additional 
strategies to assist with returning to the baseline weight. Dr. Ard will also oversee this aspect of the trial. 
  
Importance of knowledge to be gained: This study will help us understand how early changes in diet 
and calorie intake affect cells in the body that may ultimately initiate a series of changes that promote 
cardiometabolic disease risk. Diets that promote weight gain may act through a few key pathways to 
reprogram white blood cells and lead to abnormal activity within these cells. If we can identify these 
pathways, and more importantly, the triggers that initiate these events, we can potentially develop 
treatments that block the harmful effects of poor diets. 

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3 OBJECTIVES AND ENDPOINTS 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR ENDPOINTS 
Primary 
Determine the molecular 
mechanisms by which high-calorie 
diets trigger monocyte priming and 
reprogramming in metabolically 
healthy human subjects and 
whether and to what extent these 
mechanisms differ from mice. 
Monocyte priming will be 
assessed measuring MKP-1 
activity 
Unbiased lipidomics 
MKP-1 activity in blood 
monocytes is biomarker of 
metabolic stress-induced 
monocyte “priming” and 
dysfunction. 
We will analyze plasma from all 
subjects at all time points for 
their plasma lipid composition 
using an unbiased lipidomics 
approach in order to identify the 
lipid species responsible for 
monocyte priming 
Secondary 
To identify which proteins alter their 
S-glutathionylation status (and 
possibly their expression levels) in 
response to HCD-induced monocyte 
priming, and which signaling 
pathways are altered in these cells 
Monocyte protein S-
glutathionylation state 
In purified blood monocytes 
isolated from each subject at 
each timepoint . we will isolate 
S-glutathionylated proteins from 
all samples and subject them to 
our redox proteomics approach 
to identify S-glutathionylated 
proteins and top determine the 
extent and directionality of 
modifications on their cysteine 
residues. 
Tertiary/Exploratory 
Determine the genes and pathways 
involved in monocyte priming in 
humans 
RNAseq analysis 
RNAseq will quantify mRNA 
expression of all transcripts in 
expressed monocytes at week 0, 
1, 2, 3, 4 and 8, and any changes 
in their expression over time. 
These measurements will inform 
us on the proteins and pathways 
involved in monocyte “priming”. 

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4 STUDY DESIGN 
4.1 OVERALL DESIGN 
The goal is to challenge metabolically healthy human subjects with a high-calorie, Western diet for 8 
weeks to identify the metabolite(s) responsible for monocyte priming and use redox proteomics, 
RNAseq and Chipseq to determine the genes and pathways involved in monocyte priming in humans. To 
reflect some key components of the typical Western intake, we will compose the diet to be high in 
saturated fat (15% of total energy intake) and fructose (14% of total energy intake) with excess energy 
intake that is 25% higher than estimated total energy expenditure. A total of 30 participants, including 
15 men and 15 women, will complete a run-in phase where weight stability will be achieved on the 
control diet. After weight is stabilized during the run-in period, participants will enter the experimental 
phase of the dietary intervention, where overfeeding will begin with additional calories provided by the 
key nutrients as noted above. Participants will be monitored throughout the feeding trial. After 
completion of the 8-week overfeeding period, individuals will be allowed to return to a normal dietary 
intake. All study participants will be given access to counseling for weight reduction should it be 
needed  following the overfeeding period. 
 
 
4.2 SCIENTIFIC RATIONALE FOR STUDY DESIGN 
Recently, we showed that a HCD also promotes monocyte priming, reprogramming and dysfunction in 
non-human primates. Primed baboon monocytes showed the same hyperchemotactic, proinflammatory 
and proatherogenic phenotype we identified both in HCD-fed mice and, ex vivo, for metabolically 
stressed human monocytes. In baboons, monocyte priming correlated with loss of MKP-1 activity in 
monocytes, validating MKP-1 activity as a sensitive biomarker of monocyte priming and dysfunction. 
More importantly, we identified a highly significant correlation between plasma cholesterol and both 
loss of MKP-1 activity and monocyte priming, suggesting that total plasma cholesterol may be the 
primary driver of monocyte priming, reprogramming and dysfunction.  
  
RNAseq analysis revealed that primed baboon monocytes undergo major reprogramming of their 
transcriptome and their signaling pathways (unpublished data). We also found major changes in the 
acetylation status of lysine residues histone 3 (H3). Acetylation of H3K27 strongly correlated with 
monocyte priming, suggesting that a four-week exposure to a HCD is sufficient to promote the 
transcriptional and epigenetic reprogramming of blood monocytes into a hyperinflammatory, 
proatherogenic phenotype. 
 
 
 
 

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5 STUDY POPULATION 
5.1 INCLUSION CRITERIA 
Age 20-45 
Planning to be available for the entire study period 
Able to speak and read English 
Body mass index 18.5-29.9 kg/m2 
Waist circumference of 88 cm or less 
Able to eat the prescribed diet 
Non-smoker 
 
 
5.2 EXCLUSION CRITERIA 
Excessive alcohol consumption (>2 drinks/day for men, >1 drink/day for women) 
History of chronic cardiometabolic disease or major risk factor, including diabetes, hypertension, 
hyperlipidemia, heart disease 
History of prior surgical procedure for weight control or liposuction 
Use of weight loss medications in previous 6 months 
Recent self-reported weight change (≥ 5% in the last 6 months) 
Severe pulmonary disease requiring supplemental oxygen 
Abnormal renal or liver function 
History of non-skin cancer in the past 5 years 
Regular use of medications (prescribed or over-the-counter) that affect blood pressure, lipids, glucose, 
inflammation, or body weight.  
Works night shifts 
Exercise per week > 420 minutes total for moderate activity or > 210 minutes for vigorous activity 
Any medical or behavioral indication that would make participation unsafe based on the judgement of 
the study physician 
Pregnant or lactating women 
Known or discovered intolerances, allergies or difficulty consuming any of the foods included in the 
study diets  
 
 
5.3 LIFESTYLE CONSIDERATIONS 
During this study, participants are asked to: 
• 
Limit consumption of alcoholic beverages to recommended limits for men of no more than 2 
drinks per day and for women no more than 1 drink per day 

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• 
Refrain from receiving outside treatment for obesity, including other treatment plans, 
medications, or pursuing surgery or other procedures 
• 
Avoid pregnancy by reporting practice of one or more methods of effective contraception 
Additionally, prior to certain study visits, participants will be asked to: 
• 
Abstain from strenuous exercise for  12 hours before each blood collection 
• 
Abstain from eating or drinking anything other than water for 8 hours before blood collection 
for clinical laboratory tests and study measurements. 
 
 
5.4 SCREEN FAILURES 
Screen failures are defined as participants who consent to participate in the clinical trial but are not 
subsequently randomly assigned to the study intervention or entered in the study.  
 
Individuals who do not meet the criteria for participation in this trial (screen failure) because of meeting 
one or more exclusion criteria that are likely to or do change over time may be rescreened. Examples 
include the successful treatment of a previous affective disorder, improvement of blood pressure 
control, and discontinuation of exclusionary medications. Rescreened participants will be assigned the 
same participant number as for the initial screening. 
 
 
5.5 STRATEGIES FOR RECRUITMENT AND RETENTION 
Recruitment 
  
We will recruit 30 adult participants age 20-45, including 15 men and 15 women, for this feeding study. 
We will recruit the volunteers using several strategies proven successful in previous studies by the 
investigative team including general advertising in local media and the Wake Forest Be Involved website. 
Additionally, we will use local media outlets, including print, television, internet, and radio to generate 
interest in this research study, including those that target audiences of interest. 
  
Retention 
  
Participants will receive cash reimbursement of $150 for time lost from other activities to complete 
study related visits and measures. The money will be disbursed over the course of the study (3 x $50). 
  
In addition to the monetary incentive we will provide to assist with retention, the research team will use 
strategies that have been effective in our previous clinical trials to promote retention. These strategies 
include recognizing individual milestones such as birthdays and providing personalized recognition of 
consistent levels of study participations. This type of public recognition encourages and reinforces the 
type of engagement we want to achieve for all volunteers. Our team will also follow up individually with 

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participants who miss appointments or meal times without prior notice to promptly reschedule or 
arrange for a make-up visit. 
 
 

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6 STUDY INTERVENTION 
6.1 STUDY INTERVENTION(S) ADMINISTRATION 
6.1.1 STUDY INTERVENTION DESCRIPTION 
We will use a run-in feeding period of up to 14 days to establish a common baseline and ensure 
weight stability and adequate adherence prior to starting the experimental feeding component of the 
trial. We will provide participants with all meals and snacks during the study. The study diets will include 
a variety of foods and ingredients that are found in a typical American diet, including grains, meat, 
poultry, fish/seafood, fruit, vegetables, dairy, oils/fats, nuts, seeds, and added sugar. Because this menu 
and study protocol are not designed to accommodate special dietary needs, individuals with known food 
allergies, intolerances, or special dietary needs (e.g., religious accommodations) will be excluded from 
participation.  Initial calorie intake will be determined based on estimated total energy expenditure 
using Mifflin St. Jeor with estimated activity factor. For female participants with a regular menstrual 
cycle, the start date will be based on last menstrual period (approximately 12-16 days since the end of 
the last period). The target weight for weight stability will be identified on the fifth day of the run-in 
diet.  Once the participant is weight stable for 5 consecutive days, they will proceed to the experimental 
diet. Weight stability is defined as maintaining a weight within 2% of the weight achieved on the fifth 
day of the run-in diet. The run-in diet phase will be a minimum of 10 days (5 days to identify baseline 
weight + minimum of 5 consecutive days of weight within 2%). Those who have significant variations in 
weight (i.e., >2%) will have adjustment of calorie intake, and weight stabilization will restart. If weight 
stabilization is not in process by day 9 of the run-in diet, the participant will be dismissed. If participants 
demonstrate difficulty with study adherence during the run-in (e.g., unable to consume certain foods, 
missed appointments, incomplete reporting of food intake, etc), they will be excluded as well. The run-in 
diet will be a diet that is low in saturated fat (7% of calories) and fructose (4% of calories) and eucaloric 
to maintain current body weight.   
After the run-in, participants will proceed to the experimental diet for 8 weeks of feeding. The 
experimental diet will provide 15% of calories from saturated fat and 14% of calories from fructose with 
a goal of providing 1.25x total energy needs. We will monitor adherence using direct observation, 
written daily food diaries, and food waste inventory (i.e., participants are trained to estimate portion of 
uneaten foods). Participants will be asked to pick up their meals from the Clinical Research Unit (CRU) 2-
3 weekdays (e.g., Monday, Wednesday, and Friday), at which time they will receive meals and snacks for 
the remainder of that day and enough until the next scheduled visit.  
 
 
6.2 DIETARY COMPOSITION 
6.2.1 DIET FORMULATION- RUN IN 
 The run-in diet will provide adequate nutrition at various calorie levels designed to achieve weight 
maintenance for at least 5 consecutive days after the 5th day of feeding. The macronutrient balance of 

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the diet will include 45% of calories from carbohydrate, 35% from fat, and 20% from protein. This 
balance of macronutrients is consistent with a typical Western dietary pattern. Within the carbohydrate 
distribution of calories, 4% of the energy intake will be from fructose. Among the calories from fat, 7% 
will be provided by saturated fat. Unsaturated fat will be split between monounsaturated fat (14% of 
calories) and polyunsaturated fat (9%). An example of the macronutrient totals and calories provided 
based on sample calorie ranges for the run-in diet is shown below. 
Total 
calories 
Kcal from 
total carbs 
(45%) 
Kcal from 
fructose (4%) 
Kcal from 
non-fructose 
carbs 
Kcal from 
total fat 
(35%) 
Kcal from 
saturated 
fat (7%) 
Kcal from 
unsaturated 
fat 
Kcal from 
total 
protein 
(20%) 
1200 
540 
48 
492 
420 
84 
336 
240 
1600 
720 
64 
656 
560 
112 
448 
320 
1800 
810 
72 
738 
630 
126 
504 
360 
2000 
900 
80 
820 
700 
140 
560 
400 
 
6.2.2 DIET FORMULATION- EXPERIMENTAL 
 The experimental diet will provide adequate nutrition at various calorie levels with an additional 
amount of total energy that is 25% higher than what was required to achieve maintenance of body 
weight during run in. The food groups and general food types used in the run-in diet will serve as the 
basis for the experimental diet. The additional calories will be added through increases in fructose and 
saturated fat primarily. In the experimental diet, fructose and saturated fat will account for 14% and 
15% of total calories, respectively. Within the total carbohydrate and total fat calories, adjustments will 
be made in non-fructose and unsaturated fat calories to accommodate the additional fructose and 
saturated fat to allow for a distribution of macronutrients that is consistent with the run-in diet. An 
example of the calories from macronutrients and the associated changes in non-fructose and 
unsaturated fat calories is shown below. 
 
Participants will gradually increase their consumption to achieve the 25%, so that they have time to 
adjust to the food volume and the amount of fructose in the experimental diet. It is expected that 
participants will be eating 1.25x their calories no later than 4 weeks into the experimental phase. 
1.25x 
calories 
Kcal 
from 
fructose 
(14%) 
Kcal from 
saturated 
fat (15%) 
Kcal 
from 
total 
carbs 
(45%) 
Kcal 
from 
non-
fructose 
carbs 
Change 
in non-
fructose 
carb 
kcals 
Kcal 
from 
total fat 
(35%) 
Kcal from 
unsaturated 
fat 
Change in 
unsaturated 
fat kcals 
Kcal from 
total 
protein 
(20%) 
1500 
210 
225 
675 
465 
-27 
525 
300 
-36 
300 
2000 
280 
300 
900 
620 
-36 
700 
400 
-48 
400 
2250 
315 
337.5 
1012.5 
697.5 
-40.5 
787.5 
450 
-54 
450 
2500 
350 
375 
1125 
775 
-45 
875 
500 
-60 
500 

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6.4 STUDY INTERVENTION COMPLIANCE 
A daily food intake diary will be provided to participants that includes the menu for the day. Participants 
will be asked to notate anytime they consume one of the prepared food items or when they have 
something that is not part of the prepared menus. These diaries will be reviewed at each pickup. We will 
ask participants to estimate the portions of any uneaten food in the food diaries.  
While the gold standard for estimating plate waste is manual weighing, this can be time-consuming and 
expensive. Standardized visualization techniques can take less time and have been used with great 
success in a range of studies.  These visual estimation techniques have been tested for reliability and 
accuracy in many studies, producing results that closely approximate manual weighing methods. 
 
Visual estimation techniques rely on separating food waste into different food components and having 
trained observers, in this case our study participants, estimate the proportion of food left over. One 
such scale, The Visual Comstock Scale, will be used to estimate food waste in this study. Using this 
method, food waste will be separated into its different food components and recorded in different 
values using a six-point scale ranging from 0%, 25%, 50%, 75%, 90% and 100%, where 0% represents 
none consumed and 100% represents all consumed. We may also use digital photography to record food 
waste, which can allow review of estimates of portion sizes by study team members, to ensure 
estimates are accurate and errors minimized.  
 
If participants are having difficulty with any aspects of adherence, study staff will coach them to improve 
adherence. Those who are consistently unable to adhere to the feeding protocol will be dismissed from 
the study. Participants will be advised to maintain the same type of physical activity they did during the 
run-in period and avoid significant changes in overall activity patterns. Individuals with significant 
changes in physical activity levels compared to the run-in period will have adjustment of calorie intake 
to ensure that their calorie intake remains at 25% above total energy expenditure. 
 
To corroborate the metabolic effects of the experimental diet on the study participants, we will 
complete lab work at week8, including a lipid panel, comprehensive metabolic panel, and insulin levels. 
 
While 100% attendance of study visits is ideal, the study team realizes that situations arise where the participant 
may not be able to come in for a visit (i.e. illness or travel). If a participant is sick, traveling, or otherwise unable to 
attend in person visits, up to 1 week of missed visits will be allowed. However, missing visits will not be allowed in 
the first 3 weeks of the experimental phase since these are considered crucial weeks. If the participant is able to 
give advance notice to the study team, arrangements may be made with the CRU to accommodate continued 
feeding while they are away. 

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7 STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL 
7.1 DISCONTINUATION OF STUDY INTERVENTION 
The study intervention will be discontinued at the completion of the 8-week overfeeding period or at an 
early time point if the participant is unable to adhere to the feeding protocol. 
 
 
7.2 PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
Participants are free to withdraw from participation in the study at any time upon request. However, an 
investigator may discontinue or withdraw a participant from the study for the following reasons:   
• 
If any clinical adverse event (AE), laboratory abnormality, or other medical condition 
or situation occurs such that continued participation in the study would not be in 
the best interest of the participant   
• 
Pregnancy  
• 
If a participant has a break in feeding for more than 1 day in any week or more than 
3 days over the course of the 8 week overfeeding period. A break in feeding is 
defined as consuming <50% of the provided food for the day. 
The reason for participant discontinuation or withdrawal from the study will be recorded on the Study 
Withdrawal Case Report Form (CRF).   
 
 
7.3 LOST TO FOLLOW-UP 
A participant will be considered lost to follow-up if he or she fails to return for more than 1 scheduled 
visit and is unable to be contacted by the study site staff. 
 

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8 STUDY ASSESSMENTS AND PROCEDURES 
8.1 EFFICACY ASSESSMENTS 
Body weight will be monitored on a daily basis using a digital body weight scale provided to the 
participant at the beginning of the study. Participants will be asked to weigh at home in light clothing at 
approximately the same time each day. 
  
Blood (20 ml) will be drawn from each subject once after the run-in period and once after each week on 
the experimental diet. Within 2 h after the blood draw, we will bead-isolate by positive selection 
(CD14+) blood monocytes using a RoboSep automated cell separator. We optimized this protocol for 
high yield (>95%) and high purity (>95%). Monocyte priming will be assessed measuring MKP-1 activity. 
To identify which proteins alter their S-glutathionylation status (and possibly their expression levels) in 
response to HCD-induced monocyte priming, and which signaling pathways are altered in these cells, we 
will isolate S-glutathionylated proteins from all samples and subject them to our redox proteomics 
approach. We will also measure Mao A and Grx1 protein expression by scWB analysis and Grx activity 
using [3H]BSA-SSG as a substrate to monitor for possible changes in Grx1 activity in response to the diet 
challenge. In addition, we will conduct RNAseq analysis to determine the genes and pathways involved 
in monocyte priming in humans. 
 
 
8.2 SAFETY AND OTHER ASSESSMENTS 
Our Data and Safety Monitoring Plan consists of several layers including ongoing monitoring by the 
Principal Investigator, Dr. Ard and staff who have contact with the participants, and the Institutional 
Review Board of WFU Health Sciences. First, we will hold bi-weekly meetings of all members of the 
clinical trial team where we will review participants’ status. This meeting will be directed by Dr. Ard. 
Second, Dr. Ard and a designated sub-investigator will be providing medical oversight of the feeding 
study. He will oversee each participant’s progress and medical history. If a participant is observed to be 
in threatening health (regardless of whether it is related to the study or not) or describes a serious 
adverse event, his or her physician will be notified. The participant will be discontinued from the study 
in this situation. Third, all unanticipated problems and serious adverse events will be reported to the 
Institutional Review Board at WFU Health Sciences per institutional policy. A plan for regular 
ascertainment of adverse events will include bi-monthly symptoms forms collected at planned study 
visits. 
 
8.3 ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
8.3.1 DEFINITION OF ADVERSE EVENTS (AE) 
 An adverse event (AE) is defined as any untoward or unfavorable medical occurrence in a human 
subject, including any clinically significant abnormal sign (for example, abnormal physical exam or 

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laboratory finding), symptom, or disease, temporally associated with the subject’s participation in the 
research, whether or not considered related to the subject’s participation in the research. 
8.3.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE) 
 Consistent with NIH guidelines and OHRP policy, SAEs are adverse events that meet any of the following 
criteria: 
• 
fatal or life-threatening, 
• 
result in significant or persistent disability, 
• 
require or prolong hospitalization, 
• 
result in a congenital anomaly/birth defect, or 
• 
are important medical events that investigators judge to represent significant 
hazards or harm to research participants and may require medical or surgical 
intervention to prevent one of the other outcomes listed in this definition (e.g. 
hospitalization, death, persistent disability). 
 
8.3.3 CLASSIFICATION OF AN ADVERSE EVENT 
  
8.3.3.1 SEVERITY OF EVENT 
Once reported, all AEs will first be assessed by the site clinician using a grading system, both in terms of 
severity and duration. Clinicians will categorize each AE as follows: 
• 
Mild or moderate in severity based on its potential to impact the health and well-being of the 
participant. Details of what constitutes a mild vs. moderate severity AE will be outlined in the 
MOP but this will also be at the discretion of the site clinician. All SAEs will be deemed serious. 
• 
Resolved or ongoing at the time of each clinical report. 
• 
Brief (<1 day), Intermediate (1-29 days), or Chronic (≥30 days) based upon duration of reported 
AE. 
  
8.3.3.2 RELATIONSHIP TO STUDY INTERVENTION 
 In addition to categorizing the severity of each AE, the study clinician will also indicate whether each AE 
was likely to be “unrelated” or “possibly related” to the study intervention (e.g. could it be related to the 
dietary intervention?). Relatedness determination will be made based upon several factors: 
• 
The AE is an expected / known effect of the study interventions or a manifestation / symptom of 
an expected / known effect 
• 
The presence of a temporal relationship between the study intervention and the AE 
• 
No other known cause exists that is more likely to be responsible for the AE in question 

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Determination of relatedness should be supported by the data collected by the clinician. In general, the 
study clinician will err on the side of indicating relatedness unless there is a strong argument to the 
contrary (e.g. a sprained ankle sustained in a soccer game). 
8.3.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND FOLLOW-UP 
 At each in-person study visit, including the final study visit, participants will be asked to complete a brief 
screener to ascertain for AEs, as well as potential SAEs. 
 
8.3.5 REPORTING OF PREGNANCY 
 Pregnancy Incidentally Detected at Baseline Visit 
Women found to be pregnant on baseline laboratory examination will not be permitted to enroll in the 
study. 
Pregnancy after Randomization 
Women found to be pregnant after randomization, or those who spontaneously report a new pregnancy 
during study follow-up will have this event documented in their participant record and their 
participation in the study terminated.  
 
 
8.4 UNANTICIPATED PROBLEMS 
8.4.1 DEFINITION OF UNANTICIPATED PROBLEMS (UP) 
 The Office for Human Research Protections (OHRP) considers unanticipated problems involving risks to 
participants or others to include, in general, any incident, experience, or outcome that meets all of the 
following criteria: 
• 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures that are 
described in the protocol-related documents, such as the Institutional Review Board (IRB)-
approved research protocol and informed consent document; and (b) the characteristics of the 
participant population being studied; 
• 
Related or possibly related to participation in the research (“possibly related” means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research); and 
• 
Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or recognized. 
 
8.4.2 UNANTICIPATED PROBLEM REPORTING 
 To satisfy the requirement for prompt reporting, UPs will be reported using the following timeline: 
• 
UPs that are serious adverse events (SAEs) will be reported to the IRB and to the study 
sponsor/funding agency within 10 days of the investigator becoming aware of the event 

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• 
Any other UP will be reported to the IRB and to the study sponsor/funding agency within 15 
days of the investigator becoming aware of the problem 
 
 

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9 STATISTICAL CONSIDERATIONS 
9.1 STATISTICAL ANALYSES 
9.1.1 GENERAL APPROACH 
For the RNAseq analysis, we are underpowered for differential analysis of individual RNA abundance 
values, but we anticipate 80% power to detect differences between integrated transcriptomic pathways 
at α = 0.005 (10 pathways and a mean difference of 1.35 SD) with 15 subjects per group. To obtain a 
more complete picture of the extent of monocyte reprogramming induced by a HCD, we will integrate 
the redox proteomics data with the RNAseq data. We anticipate that the combination of these 
approaches will identify novel signaling pathways dysregulated by HCD and possibly new therapeutic 
targets to prevent monocyte dysfunction and reprogramming. As in our baboon study, we will analyze 
by scWB the acetylation state of H3 lysine residues (H3KnAc). If we identify a similar correlation 
between the acetylation of a specific H3 lysine residue, e.g. H3K27Ac, in these HCD-challenged human 
monocytes as we did in baboons, we will conduct Chipseq targeting specific H3KnAc to determine which 
genes identified by RNAseq are regulated by this epigenetic reprogramming. Finally, to identify the 
metabolite(s) responsible for monocytes priming, we will conduct unbiased metabolomic analyses on 
plasma of these subjects and validate any candidate metabolites for their ability to prime healthy human 
monocytes in our cell culture model of monocyte priming. Quantitative data will be analyzed with 
parametric and nonparametric two-sample and multi-sample tests, such as t-tests or Mann-Whitney, 
correlative analyses, analysis of variance or Kruskall-Wallis, or other linear models, depending on the 
distributional properties of the outcome measures and sample sizes. If the overall test is significant, we 
will select appropriate post-hoc analyses based on the comparisons to be made (all groups or 
interventions versus controls), sample sizes (unequal versus equal), and equal versus unequal variances 
across groups. The overall significance level will be 0.05. 
  
 
 

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10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
10.1.1 INFORMED CONSENT PROCESS 
 Written informed consent will be obtained from all participants during the baseline study visit.  
 
10.1.1.1 CONSENT PROCEDURES AND DOCUMENTATION 
  A trained staff member or investigator will give an overview of the study, describe the intervention 
components and data collection requirements, and encourage potential participants to ask questions. 
All potential participants will be informed of the study design and interventions, the risks and benefits of 
participation, their rights and responsibilities as research participants, and alternatives to participation. 
They will also be informed that participation is voluntary and that they can withdraw from the study if 
their initial or on-going experience makes it oppressive, burdensome, or otherwise uncomfortable. 
Potential participants will be asked to read the informed consent form and to ask questions. The form 
will be written in simple, easy-to-understand language. As a part of the consent process, study staff will 
be required to review all key aspects of the study verbally and will be provided with a structured 
checklist for this purpose. A copy of the signed and dated consent form will be given to participants, and 
the original document will be placed in the participants’ individual study files, which will be stored in a 
secure location at the clinical site.  
10.1.2 CONFIDENTIALITY AND PRIVACY 
 Confidentiality will be protected by collecting only information needed to assess study outcomes, 
minimizing to the fullest extent possible the collection of any information that could directly identify 
subjects, and maintaining all study information in a secure manner.  To help ensure subject privacy and 
confidentiality, only a unique study identifier will appear on the data collection form.  Any collected 
patient identifying information corresponding to the unique study identifier will be maintained on a 
linkage file, store separately from the data.  The linkage file will be kept secure, with access limited to 
designated study personnel.  Following data collection subject identifying information will be destroyed 
by deleting any data files three years after study closure, consistent with data validation and study 
design, producing an anonymous analytical data set.  Data access will be limited to study staff.  Data and 
records will be kept locked and secured, with any computer data password protected.  No reference to 
any individual participant will appear in reports, presentations, or publications that may arise from the 
study. 
 
10.1.3 FUTURE USE OF STORED SPECIMENS AND DATA 
 Participants will provide consent for storage of blood samples will have a sample of blood stored with a 
unique identifier that will not include any identifiable information. The unique identifier will be a 
randomly assigned number and only the principal investigator will have access to the code that links the 
unique identifier to the study participant.  

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10.1.4 KEY ROLES AND STUDY GOVERNANCE 
Staff member 
Role(s) 
Principal Investigator  
Study clinician, study oversight, informed 
consent  
 Study coordinator 
Maintenance of regulatory documents; 
reporting to IRB; informed consent; 
recruitment and screening 
 Investigator team 
Review of data, outcomes, and safety 
events 
Research assistant 
Data collection, data entry, informed 
consent, recruitment and screening 
Study dietitian 
Participant monitoring, dietary adherence 
  
10.1.5 SAFETY OVERSIGHT 
The principal investigator will be responsible for the overall monitoring of the data and safety of study 
participants.  The principal investigator will be assisted by other members of the study staff. 
 
10.1.6 CLINICAL MONITORING 
 Dr. Ard and a designated sub-investigator will be providing medical oversight of the feeding study. He 
will oversee each participant’s progress and medical history. If a participant is observed to be in 
threatening health (regardless of whether it is related to the study or not) or describes a serious adverse 
event, his or her physician will be notified. The participant will be discontinued from the study in this 
situation. 
 
10.3 ABBREVIATIONS 
AE 
Adverse Event 
ANCOVA Analysis of Covariance 
CFR 
Code of Federal Regulations 
CLIA 
Clinical Laboratory Improvement Amendments 
CMP 
Clinical Monitoring Plan 
COC 
Certificate of Confidentiality 
CONSORT Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DSMB 
Data Safety Monitoring Board 
DRE 
Disease-Related Event 
EC 
Ethics Committee 
eCRF 
Electronic Case Report Forms 

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FDA 
Food and Drug Administration 
FDAAA 
Food and Drug Administration Amendments Act of 2007 
FFR 
Federal Financial Report 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practices 
GMP 
Good Manufacturing Practices 
GWAS 
Genome-Wide Association Studies 
HIPAA 
Health Insurance Portability and Accountability Act 
IB 
Investigator’s Brochure 
ICH 
International Conference on Harmonisation 
ICMJE 
International Committee of Medical Journal Editors 
IDE 
Investigational Device Exemption 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
ISO 
International Organization for Standardization 
ITT 
Intention-To-Treat 
LSMEANS Least-squares Means 
MedDRA Medical Dictionary for Regulatory Activities 
MOP 
Manual of Procedures 
MSDS 
Material Safety Data Sheet 
NCT 
National Clinical Trial 
NIH 
National Institutes of Health 
NIH IC 
NIH Institute or Center 
OHRP 
Office for Human Research Protections 
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SMC 
Safety Monitoring Committee 
SOA 
Schedule of Activities 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
UP 
Unanticipated Problem 
US 
United States 
 
 
10.4 PROTOCOL AMENDMENT HISTORY 
Version 
Date 
Description of Change  
Brief Rationale 

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