Genomic Response of Human Immune and Non-Immune Cells to Glucocorticoids
Phase: PHASE1
Conditions: Normal Physiology
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

Genomic Response to GCs 
Protocol Version 9.0 
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Genomic Response of Human Immune and Non-Immune Cells to Glucocorticoids 
 
NIH Protocol Number: 
16-I-0126 
Version Number: 
 
9.0 
Date: 
30 March 2021 
Investigational New Drug 
(IND) Number for  
Advantan Emulsion:  
131527 
IND Sponsor: 
Office of Clinical Research Policy and Regulatory Operations 
(OCRPRO), Division of Clinical Research, National Institute of 
Allergy and Infectious Diseases (NIAID), National Institutes of 
Health (NIH) 
Sponsor Medical Monitor: Shirley Jankelevich, M.D. 
Clinical Monitoring Research Program 
Frederick National Laboratory for Cancer Research  
Leidos Biomedical Research, Inc.  
5705 Industry Lane 
Frederick, MD 21704 
Phone: 301-846-7322 
Fax: 301-846-6224 
Cell: 301-401-3426 
Email: shirley.jankelevich@nih.gov 
Principal Investigator: 
Luis M. Franco, M.D. 
Systemic Autoimmunity Branch 
National Institute of Arthritis and Musculoskeletal and Skin 
Diseases (NIAMS) 
National Institutes of Health 
9000 Rockville Pike 
Building 10, Room 13C101A 
Bethesda, MD 20892 
Phone: 301-827-2461 
Email: luis.franco@nih.gov 
Conducted by:  
Systemic Autoimmunity Branch, NIAMS, NIH  

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Table of Contents 
Table of Contents ............................................................................................................................ 2 
List of Tables .................................................................................................................................. 3 
List of Figures ................................................................................................................................. 3 
Statement of Compliance ................................................................................................................ 5 
List of Abbreviations ...................................................................................................................... 6 
Protocol Summary .......................................................................................................................... 7 
Précis ............................................................................................................................................... 9 
1 
Background Information and Scientific Rationale................................................................ 10 
 
Scientific Background and Preliminary Data ................................................................. 10 
 
Rationale for Performing the Study in Healthy Subjects ............................................... 13 
 
Choice of a Glucocorticoid and Routes of Administration ............................................ 14 
 
Choice of Dose of Glucocorticoid .................................................................................. 15 
2 
Study Objectives and Endpoints ........................................................................................... 16 
3 
Study Design ......................................................................................................................... 16 
4 
Study Population ................................................................................................................... 18 
 
Recruitment Plan ............................................................................................................ 18 
 
Inclusion Criteria ............................................................................................................ 18 
 
Exclusion Criteria ........................................................................................................... 18 
 
Justification for Exclusion of Special Populations ......................................................... 19 
5 
Study Agents ......................................................................................................................... 20 
 
Methylprednisolone sodium succinate for injection ...................................................... 20 
 
Disposition and Dispensation ................................................................................. 20 
 
Formulation ............................................................................................................. 20 
 
Dosage and Administration..................................................................................... 21 
 
Storage .................................................................................................................... 21 
 
Topical methylprednisolone ........................................................................................... 21 
 
Disposition and Dispensation ................................................................................. 21 
 
Formulation ............................................................................................................. 21 
 
Dosage and Administration..................................................................................... 21 
 
Storage .................................................................................................................... 21 
6 
Study Schedule ...................................................................................................................... 21 
 
Screening visit (Day -30 to Day -1) ............................................................................... 21 
 
Infusion visit (Day 0) ..................................................................................................... 22 
 
Follow-up Phone Calls (Day 1 and Day 5) .................................................................... 22 
7 
Study Procedures/Evaluations............................................................................................... 23 
8 
Potential Risks and Benefits ................................................................................................. 23 
 
Risks ............................................................................................................................... 23 
 
Benefits........................................................................................................................... 26 
9 
Research Use of Stored Human Samples, Specimens, or Data ............................................ 26 
10 Remuneration Plan for Volunteers ........................................................................................ 27 
11 Assessment of Safety ............................................................................................................ 27 
 
Definitions .................................................................................................................. 27 
 
Documenting, Recording, and Reporting Adverse Events ......................................... 29 
 
Follow-up of reportable abnormal laboratory values .............................................. 30 
 
Investigator Assessment of Adverse Events ............................................................... 31 

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Severity ................................................................................................................... 31 
 
Causality ................................................................................................................. 31 
 
Investigator Reporting Responsibilities to the Sponsor ............................................. 32 
 
Adverse Events ....................................................................................................... 32 
 
Serious Adverse Events .......................................................................................... 32 
 
Unanticipated Problems .......................................................................................... 33 
 
Sponsor’s Reporting Responsibilities ......................................................................... 33 
 
Halting Rules for the Protocol .................................................................................... 33 
 
Reporting a Study Halt............................................................................................ 33 
 
Resumption of a Halted Study ................................................................................ 33 
 
Withdrawal Criteria for an Individual Subject ........................................................... 34 
 
Replacement of Withdrawn Subjects or Subjects Who Discontinue Study Agent . 34 
 
Safety Oversight ......................................................................................................... 34 
 
Safety Review and Communications Plan .............................................................. 34 
 
Sponsor Medical Monitor ....................................................................................... 34 
12 Reporting Procedures ............................................................................................................ 34 
 
Reporting to the NIH IRB .......................................................................................... 34 
 
NIH Definitions of Protocol Deviation and Non-compliance ................................ 35 
 
Reporting to the NIAID Clinical Director .................................................................. 35 
13 Site Monitoring Plan ............................................................................................................. 35 
14 Statistical Considerations ...................................................................................................... 36 
15 Ethics/Protection of Human Subjects ................................................................................... 39 
 
Informed Consent Process .......................................................................................... 39 
 
Considerations for Consent of NIH Staff................................................................ 40 
 
Subject Confidentiality ............................................................................................... 40 
16 Data Handling and Record Keeping ..................................................................................... 41 
 
Data Capture and Management .................................................................................. 41 
 
Record Retention ........................................................................................................ 41 
 
List of Tables 
Table 1:  Approximately 16% of the transcripts that are differentially expressed in immune but 
not in non-immune cells after a glucocorticoid stimulus correspond to long non-coding RNAs. 
FDR=False Discovery Rate .......................................................................................................... 13 
Table 2.  Processing and use of samples from healthy volunteers ................................................ 17 
 
 
List of Figures 
Figure 1. (A) Genome-wide view of the transcriptional response 2 hours after in vitro stimulation 
with 1 µM dexamethasone in lymphoblasts (outer circle) and fibroblasts (inner circle). Each dot 
corresponds to a gene.. .................................................................................................................. 10 
Figure 2. Immune genes that are differentially expressed in lymphoblasts but not in fibroblasts 
include upregulated and downregulated genes. ............................................................................ 11 

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Figure 3. Many of the immune genes that are differentially expressed in lymphoblasts at 2 and 6 
hours are not expressed at all in fibroblasts. ................................................................................. 12 
 
 

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Statement of Compliance 
The trial will be carried out in accordance with International Council for Harmonisation of 
Technical Requirements for Pharmaceuticals for Human Use (ICH) Good Clinical Practice 
(GCP) and the following:  
• United States 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)  
 
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. 
 
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (IRB) for review and approval.  Approval of both 
the protocol and the consent form must be obtained before any participant is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study.  In addition, all changes to the consent form will be IRB-approved; an 
IRB 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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List of Abbreviations 
AE 
Adverse event/adverse experience 
AR 
Adverse reaction 
BMI 
Body Mass Index 
CBC 
Complete Blood Count 
CC 
Clinical Center 
CFR 
Code of Federal Regulations 
CRIMSON 
Clinical Research Information Management System of the NIAID 
CSO 
Clinical Safety Office 
ECG 
Electrocardiogram 
GC 
Glucocorticoid 
GCP 
Good Clinical Practice 
GRE 
Glucocorticoid Response Element 
HAV 
Hepatitis A virus 
HBsAg 
Hepatitis B surface antigen 
HCV 
Hepatitis C virus 
HRPP 
Human Research Protection Program 
ICH 
International Council for Harmonisation of Technical Requirements for 
Pharmaceuticals for Human Use 
IND 
Investigational new drug 
iPS 
Induced Pluripotent Stem Cells 
IRB 
Institutional Review Board 
NIAID  
National Institute of Allergy and Infectious Diseases  
NIAMS 
National Institute of Arthritis and Musculoskeletal and Skin Diseases 
NIH 
National Institutes of Health 
NK 
Natural Killer (immune cells) 
OCRPRO 
Office of Clinical Research Policy and Regulatory Operations 
OHRP 
Office for Human Research Protections 
PBMC 
Peripheral Blood Mononuclear Cells 
RNAi 
RNA interference 
SAE 
Serious adverse event/serious adverse experience 
SAR 
Suspected adverse reaction 
siRNA 
Small Interfering RNA 
SUSAR 
Serious and unexpected suspected adverse reaction 
UP 
Unanticipated Problem 
UPnonAE 
Unanticipated Problem that is not an Adverse Event 
 
 

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Protocol Summary 
 
Full Title: 
Genomic Response of Human Immune and Non-Immune Cells to 
Glucocorticoids 
Short Title: 
Genomic response to GCs 
Conducted by: 
 
Systemic Autoimmunity Branch, NIAMS, NIH  
Principal Investigator: 
Luis M. Franco, M.D. 
Sample Size: 
N=50 maximum. The required sample size will be determined by 
an adaptive study design after an initial recruitment of 20 subjects 
Accrual Ceiling: 
60 
Study Population: 
 
Healthy volunteers ages 18-64 years 
Accrual Period: 
 
60 months 
Study Design: 
This is a study of glucocorticoid action at the genomic level. 
Healthy volunteers will undergo baseline blood collection, then 
receive a single intravenous dose of 250 mg of 
methylprednisolone.  Blood will be collected in one of two 
regimens: 1 and 2 hours or 2 and 4 hours after the start of the 
infusion.  A skin punch biopsy may be obtained before healthy 
volunteers receive IV methylprednisolone.  If so, topical 
methylprednisolone will be applied to a limited area of skin, 
contralateral to the site of the initial skin biopsy, and an additional 
skin biopsy will be obtained (from the area of topical 
methylprednisolone application) at the 4-hour time point. 
Individual participation requires 2 visits to the NIH Clinical Center 
(CC) and 2 follow-up calls. Total participation time is 1-5 weeks. 
Blood samples will be processed for isolation of hematopoietic cell 
sub-populations (e.g., neutrophils, B cells, CD4+ T cells, CD8+ T 
cells, monocytes, and natural killer [NK] cells). Laboratory studies 
will be performed in the purified cells, with the goal of 
understanding the human response to glucocorticoids in vivo at the 
level of RNA (e.g., RNA sequencing, small-RNA-sequencing, 
real-time PCR), DNA (e.g., ChIP-seq, methylation analysis, DNA 
sequencing, genotyping), and protein (e.g., flow cytometry, mass 
spectrometry).  At each time point, plasma methylprednisolone 
levels will be measured and flow cytometry for standard lineage 
markers will be performed. Skin biopsies will be subjected to RNA 

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isolation for RNA sequencing and small-RNA sequencing. A 
fragment of each skin biopsy will undergo fibroblast isolation and 
culture for in vitro exposure to glucocorticoids and for the 
generation of induced pluripotent stem (iPS) cells 
Study Duration: 
 
Start Date: October 2016; End Date: October 2022  
Primary Objective:  
To elucidate fundamental differences in the genomic response of 
immune and non-immune cells to glucocorticoids 
Primary Endpoint:  
A list of human genes and non-coding RNAs that are differentially 
expressed and regulated in response to glucocorticoids between 
immune and non-immune cells 
Secondary Objective: 
To identify potential targets for small-molecule or nanoparticle- 
facilitated RNA interference interventions that reproduce the 
therapeutic action of glucocorticoids while avoiding harmful 
effects 
Secondary Endpoints: 
  A list of protein-coding and non-coding transcripts, their 
corresponding proteins, and the molecular pathways representing 
the best candidates for targeted therapeutic alternatives to 
glucocorticoids 
• Validation of the targets identified by functional studies 

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Précis 
Glucocorticoids are among the most frequently prescribed immunosuppressive and anti-
inflammatory medications worldwide. Long-term use, however, is complicated by serious non-
immunologic side effects. Ongoing in vitro experiments with human primary cells in our 
laboratory suggest that there are indeed fundamental differences in the genomic response of 
immune and non-immune cells to glucocorticoids. These and other aspects of drug action at the 
genomic level have not been completely characterized. This study will attempt to generate a list 
of human genes and non-coding RNAs that are differentially expressed and regulated in response 
to glucocorticoids between immune and non-immune cells. These data will be used to identify 
transcripts, their corresponding proteins, and the molecular pathways that are best candidates for 
targeted intervention. Potential targets could be validated with small interfering RNA (siRNA) 
libraries, with the long-term goal of developing small-molecule or nanoparticle-facilitated RNA 
interference (RNAi) interventions that reproduce the therapeutic action of glucocorticoids in 
immune cells while avoiding their harmful side effects on other tissues. 
 
Healthy volunteers will undergo baseline blood collection prior to receiving a single intravenous 
dose of 250 milligrams of methylprednisolone sodium succinate. Blood will be collected in one 
of two regimens: 1 and 2 hours or 2 and 4 hours after the start of the infusion.  A skin punch 
biopsy may be obtained before healthy volunteers receive IV methylprednisolone.  If so, topical 
methylprednisolone will be applied to a limited area of skin, contralateral to the site of the 
baseline skin biopsy, and an additional skin biopsy will be obtained 4 hours after drug 
administration, from the area where topical methylprednisolone was applied. Follow-up phone 
calls 1 and 5 days after discharge will document any adverse effects related to the drug or skin 
biopsy. Total length of individual study participation is 1-5 weeks. 
 
Blood samples will be processed for isolation of hematopoietic cell sub-populations (e.g. 
neutrophils, B cells, CD4+ T cells, CD8+ T cells, monocytes, and natural killer [NK] cells).  
Laboratory studies will be performed in the purified cells, with the goal of understanding the 
human response to glucocorticoids in vivo at the level of RNA (e.g., RNA sequencing, small-
RNA-sequencing, real-time PCR), DNA (e.g., ChIP-seq, methylation analysis, DNA sequencing, 
genotyping), and protein (e.g., flow cytometry, mass spectrometry). At each time point, serum 
methylprednisolone levels will be measured and flow cytometry for standard lineage markers 
will be performed. Skin biopsies will be subjected to RNA isolation for RNA sequencing and 
small-RNA sequencing. A fragment of each skin biopsy will undergo fibroblast isolation and 
culture for in vitro exposure to glucocorticoids and for the generation of induced pluripotent stem 
(iPS) cells. 
 
 

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1 
Background Information and Scientific Rationale 
 
 
Scientific Background and Preliminary Data 
 
Glucocorticoids are among the most frequently prescribed immunosuppressive and anti-
inflammatory medications worldwide (1, 2). Long-term use, however, is complicated by serious 
non-immunologic side effects. A large gap of knowledge about the differences between the 
actions of this class of drugs on immune and non-immune cells has prevented the development 
of more targeted therapies or the use of drug combinations that would preserve the desired 
immunological effects while avoiding unwanted systemic activities. Glucocorticoids are ligands 
for a nuclear receptor and their mechanism of action involves broad effects on gene expression 
via binding to the well-recognized mammalian glucocorticoid response element (GRE). Recent  
 
Figure 1. (A) Genome-wide view of the transcriptional response 2 hours after in vitro stimulation with 1 µM 
dexamethasone in lymphoblasts (outer circle) and fibroblasts (inner circle). Each dot corresponds to a gene. Genes 
that are differentially expressed (dexamethasone versus vehicle) will appear higher in the plot. The black line 
represents a differential expression q-value of 0.01 (-log10 q-value = 2). Genes marked in red are differentially 
expressed in lymphoblasts but not in fibroblasts, as seen in the close-up of chromosome 4 (B). 

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discoveries, including that of a negative GRE (3, 4) and of a glucocorticoid-responsive long non-
coding RNAs (5) suggest that other important aspects of drug action at the genome level remain 
incompletely characterized. Ongoing in vitro experiments with human cells in our laboratory 
suggest that there are indeed fundamental differences in the genomic response of immune and 
non-immune cells to glucocorticoids (Figure 1). 
 
We have identified genes that are known to be involved in immune function and are 
differentially expressed in immune but not in non-immune cells after in vitro exposure to 
glucocorticoids. Within this group, we have observed genes that increase and genes that decrease 
in expression after glucocorticoid stimulus (Figure 2). 
 
This includes genes that had been previously observed to 
respond to glucocorticoids (e.g., decreased expression of the 
cytokines IL1B and TNF), as well as genes that were 
previously not known to be affected by glucocorticoids (e.g., 
increased expression of KLF2, a transcription factor involved 
in T cell quiescence, regulation of T cell migration(6), 
negative regulation of B cell clonal expansion (7), and 
regulation of acute and chronic inflammation (8)). In addition, 
the genome-wide view of the transcriptional response 
provided by RNA sequencing has given us a more complete 
picture of the effects of glucocorticoids on hematopoietic and 
non-hematopoietic cell types than was previously available. 
Among genes that are differentially expressed in 
hematopoietic but not in non-hematopoietic cells after a 
glucocorticoid stimulus, we have observed a strong 
representation of TNF signaling molecules (decreased 
expression of TNFSF11, TNFSF14, TNF, LTA, and LTB), 
TLR signaling molecules (decreased expression of TLR1, 
TLR6, CCL3, CD80, and CCL4), and genes known to be 
involved in leukocyte migration and margination (CXCR4, 
CXCR5, KLF2).       
 
 
 
Figure 2. Immune genes that are differentially expressed in lymphoblasts but not in fibroblasts include upregulated 
and downregulated genes. 
 

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Importantly, a number of genes known to be involved in immune function and differentially 
expressed in lypmhoblastoid cells are not expressed at all in fibroblasts (Figure 3), providing 
support for the hypothesis that there are potential targets for mimicking the immunologic effects 
of glucocorticoids without direct adverse consequences on non-immune cells.    
Figure 3. Many of the immune genes that are differentially expressed in lymphoblasts at 2 and 6 hours are not 
expressed at all in fibroblasts. Expression levels are displayed in fragments per kilobase of transcript per million 
mapped reads (FPKM) in an RNA sequencing experiment 2 hours after stimulation with 1 µM dexamethasone. The 
3 columns on the left are technical triplicates of a fibroblast culture, and the 3 columns on the right are technical 
triplicates of a lymphoblast culture. 
 
Finally, our in vitro data suggests that there is a significant and differential response of the non-
coding genome to the glucocorticoid stimulus: Approximately 16% of the transcripts that are 
differentially expressed in lymphoblasts but not in fibroblasts after a glucocorticoid stimulus 
correspond to long-non-coding RNAs (Table 1). 
 
 
 
 
 

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Table 1.  Approximately 16% of the transcripts that are differentially expressed in immune but not in non-immune 
cells after a glucocorticoid stimulus correspond to long non-coding RNAs. FDR=False Discovery Rate 
At the completion of this study, we expect to generate a list of human genes and non-coding 
RNAs that are differentially expressed and regulated in response to glucocorticoids between 
immune and non-immune cells in vivo. These data will be used to identify transcripts, their 
corresponding proteins, and the molecular pathways that are best candidates for targeted 
intervention. Potential targets could then be validated with siRNA libraries, with the long-term 
goal of developing small-molecule (9, 10) or nanoparticle-facilitated-RNAi (11, 12) 
interventions that reproduce the therapeutic action of glucocorticoids in immune cells while 
avoiding the harmful side effects of this class of drugs on other tissues. 
 
 
Rationale for Performing the Study in Healthy Subjects 
 
While the long-term goal of this line of research is to allow the development of targeted 
therapeutic interventions for people with inflammatory and autoimmune disorders, the 
immediate obstacle to accomplishing that goal is a lack of understanding of the human genomic 
response to glucocorticoids in immune and non-immune cells. The immediate goal of this study 
is to understand this response. For that purpose, one could start with either patients or healthy 
volunteers. However, patients who require glucocorticoids, even with a common clinical 
indication, show great variability in terms of disease state, co-morbidities, and the use of other 
immunosuppressive and anti-inflammatory drugs. For an initial study, these strong confounders 
would likely obscure the identification of the specific effects of glucocorticoids, and would likely 
require a large number of subjects to reach an answer that could potentially be obtained by 
studying a smaller number of less clinically variable subjects, such as healthy volunteers. 
Similarly, the risk of side effects from an experimental administration of glucocorticoids would 

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likely be higher in patients with significant co-morbidities and additional immunosuppressive 
drugs than in a well-selected cohort of healthy volunteers. Therefore, the safest and most 
scientifically rational approach to the question of how the human genome responds to 
glucocorticoids is to perform an initial study in a cohort of healthy volunteers, and to use the 
results obtained in this cohort as the basis for designing and interpreting future studies in patients 
with inflammatory and autoimmune disorders. 
 
 
Choice of a Glucocorticoid and Routes of Administration 
 
Chemical modifications to the 21-carbon corticosteroid backbone (e.g. introduction of a carbon 
1-2 double bond, methylation at carbon 6, fluorination at carbon 9), have led to the diverse set of 
glucocorticoids that is currently available for clinical use. These modifications have mostly 
altered the pharmacokinetic properties of the drugs, and the relative proportion of 
mineralocorticoid and glucocorticoid activity, but they do not appear to have significantly altered 
the mechanism of action or the side effect profile of glucocorticoids. Therefore, the choice of a 
specific glucocorticoid for our study was based on the frequency of its use in clinical practice, 
the availability of reports of safe use in studies of healthy human volunteers, and the availability 
of an intravenous formulation (to reduce potential inter-individual variation in rates of 
absorption). Methylprednisolone is widely used in clinical practice, with indications in the 
management of diverse conditions including dermatomyositis (13), lupus nephritis (14), acute 
asthma exacerbations (15), and relapses of multiple sclerosis (16). Intravenous 
methylprednisolone has been used in multiple studies of healthy volunteers since the early 1970s 
(17-19), without reports of serious adverse events (SAEs) in those studies. Sodium succinate and 
phosphate esters are available for intravenous use. Therefore, we have identified 
methylprednisolone sodium succinate as the glucocorticoid of choice for our study. Because our 
study involves a comparison of the effect of glucocorticoids in immune and non-immune cells, in 
addition to blood we will serially sample a non-hematopoietic tissue. Skin is composed mostly of 
non-hematopoietic cells and is easily accessible, so we have selected the skin as a non-
hematopoietic tissue for sampling. It is unclear from existing literature whether the 
concentrations of methylprednisolone in skin after intravenous administration are sufficient to 
induce gene expression changes after short-term administration. However, high concentrations of 
methylprednisolone in the skin can be achieved with topical administration. Although topical 
methylprednisolone is widely used in clinical practice around the world, an IND application will 
be required for topical methylprednisolone use in our study given that topical 
methylprednisolone is not currently marketed in the United States.         
 

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Choice of Dose of Glucocorticoid 
 
Clinically, the dosing of glucocorticoids has been described in categorical terms based on 
prednisone equivalents per day (20). These categories are low dose (prednisone-equivalent dose 
> 0 and ≤ 7.5 mg/day), medium dose (prednisone-equivalent dose >7.5 and ≤ 30 mg/day), high 
dose (prednisone-equivalent dose > 30 and ≤ 100 mg/day), very high dose (prednisone-
equivalent dose > 100 and ≤ 250 mg/day), and pulse dose (prednisone-equivalent dose >250 
mg/day). Complete saturation of the cytosolic glucocorticoid receptor is achieved with very high 
and pulse doses of glucocorticoids (21). Importantly, however, some of the pharmacologic 
effects of glucocorticoids that are not mediated directly through their interaction with the 
glucocorticoid receptor appear to be higher at the pulse dose range when compared to the very 
high dose range (20). Although the effects that are not mediated directly through binding of the 
glucocorticoid molecule to its cytosolic receptor have been termed “non-genomic effects”, it is 
clear that some of those effects ultimately lead to changes in gene expression, via direct or 
indirect interactions with other transcription factors (including NFκB, AP-1, and STAT5) or 
competition for nuclear co-activators. Therefore, in order to examine the full range of the human 
transcriptional response to glucocorticoids, the pulse dose range is the most scientifically sound. 
From a clinical point of view, pulse doses of methylprednisolone sodium succinate are routinely 
employed when a maximum anti-inflammatory effect is desired, as is the case in 
dermatomyositis (13), lupus nephritis (14), and relapses of multiple sclerosis (16). Although the 
most significant clinically observable effects on inflammation are obtained at pulse doses of 
glucocorticoids, long-term administration of these doses is not possible without severe, dose-
limiting side effects. Therefore, most of the clinical use of pulse doses of glucocorticoids 
involves short courses of 3 to 10 days. Our study involves a single intravenous dose. It is 
important, in this context, to make an objective assessment of the potential risks of short-term 
administration of pulse doses of glucocorticoids, based on the available literature. There have 
been isolated case reports of serious cardiovascular events following the administration of 
methylprednisolone at a dose of 1 gram (22), but a causal relationship is difficult to establish. A 
retrospective study of adverse events (AEs) in 84 patients with rheumatic diseases who received 
a combined total of 275 treatments with 1 gram IV methylprednisolone daily for 3 consecutive 
days found a low rate of AEs. Again, the events that were reported were difficult to attribute to 
the methylprednisolone infusion due to the concomitant presence of medical illness and the use 
of other drugs (23). We were unable to identify any studies that directly assessed the potential 
long-term effects of a single dose of methylprednisolone, likely because the drug is clinically 
used in courses of 3 to 10 days. The use of doses of methylprednisolone up to 30 mg/kg IV in 
healthy volunteers was initially reported in 1970 (17). In this small study (6 subjects received the 
highest dose and 6 subjects received placebo), no short-term toxic effects were identified even at 
a high rate of infusion (30 mg/kg infused over 10 minutes). Subsequent studies have 
administered methylprednisolone phosphate at doses up to 1 gram IV to small groups (n = 6 and 
n = 12) of healthy volunteers (18, 19), again without reports of serious short-term AEs. 

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While a dose of 1 gram of methylprednisolone IV is likely to be safe based on the existing data, 
we have selected a lower dose, near the lower end of the pulse-dose range. The dosage forms of 
methylprednisolone sodium succinate that are clinically available include vials of 40, 125, 500, 
and 1000 mg. Therefore, we have selected a dose of 250 mg, equivalent to 312.5 mg of 
prednisone.  
            
2 
Study Objectives and Endpoints 
Primary Objective: To elucidate fundamental differences in the genomic response of immune 
and non-immune cells to glucocorticoids 
Primary Endpoint: A list of human genes and non-coding RNAs that are differentially 
expressed and regulated in response to glucocorticoids between immune and non-immune cells 
Secondary Objective: To identify potential targets for small-molecule or nanoparticle- 
facilitated-RNAi interventions that reproduce the therapeutic action of glucocorticoids while 
avoiding harmful effects 
Secondary Endpoints: 
• A list of transcripts and molecular pathways representing the best candidates for targeted 
therapeutic alternatives to glucocorticoids 
• Validation of such targets by functional studies 
3 
Study Design 
Healthy volunteers will present to the NIH CC for a brief screening visit. A baseline whole-blood 
sample will be obtained on the screening day from volunteers who are eligible for the study and 
choose to enroll. Enrolled volunteers will return for a day-long study visit (“infusion visit”): 
following a pre-infusion whole-blood sample, volunteers will receive a single intravenous dose 
of 250 milligrams (mg) of methylprednisolone sodium succinate infused over 30 minutes.  
Whole-blood samples will then be obtained serially, in one of two regimens: 1 and 2 hours or 2 
and 4 hours after the start of drug administration.  A 3-mm skin punch biopsy sample may be 
obtained prior to IV methylprednisolone administration.  If so, a single application of topical 
methylprednisolone 0.1% will be applied to a small area (2 x 2 cm) of the skin contralateral to 
the baseline biopsy site post IV methylprednisolone administration, and a second skin punch 
biopsy will be performed 4 hours after the start of drug administration, in the area of skin where 
topical methylprednisolone was applied. Samples will be processed as follows: 
 

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Table 2.  Processing and use of samples from healthy volunteers 
Visit 
Volunteer 
sample 
Initial processing 
Downstream use 
Screening 
visit 
 Baseline whole-
blood sample 
(103 mL) 
10 mL: Complete blood count, acute care panel, prothrombin 
time (PT) & partial thromboplastin time (PTT), interferon 
gamma release assay (IGRA), and serum pregnancy test 
NA 
10 mL: HIV and hepatitis A, B and C testing 
NA 
5 mL: DNA purification 
Genome-wide genotyping 
and/or DNA sequencing 
8 mL: Plasma purification 
Plasma banking 
60 mL: Isolation of hematopoietic cell sub-types (e.g. 
neutrophils, B cells, CD4+ T cells, CD8+ T cells, monocytes, 
NK cells)a 
RNA-level, DNA-level,  
protein-level, and/or 
functional assays 
10 mL: PBMC isolation 
PBMC banking 
Infusion 
visit 
Pre-infusion and 
4-hour skin 
biopsies 
(up to 3 mm x 2) 
Fibroblast isolation and establishment of a fibroblast cell line 
In vitro exposure of 
fibroblasts to glucocorticoids 
 
iPS generation for in vitro 
study of other non-
hematopoietic cell types 
RNA isolation 
RNA sequencing 
Small-RNA sequencing 
Pre-infusion 
whole-blood 
sample 
(88 mL) 
60 mL: Isolation of hematopoietic cell sub-types (e.g. 
neutrophils, B cells, CD4+ T cells, CD8+ T cells, monocytes, 
NK cells) 
RNA-level, DNA-level,  
protein-level, and/or 
functional assays 
10 mL: PBMC isolation 
PBMC banking 
8 mL: Plasma purification 
Plasma banking 
5 mL: Complete blood count, acute care panel and serum 
pregnancy test 
NA 
5 mL: Serum 
Concentration of 
methylprednisolone 
Serial post-
infusion whole-
blood samples 
(88 mL x 2) 
60 mL: Isolation of hematopoietic cell sub-types (e.g. 
neutrophils, B cells, CD4+ T cells, CD8+ T cells, monocytes, 
NK cells)  
RNA-level, DNA-level,  
protein-level, and/or 
functional assays 
10 mL: PBMC isolation 
PBMC banking 
8 mL: Plasma purification 
Plasma banking 
5 mL: Complete blood count and acute care panel  
NA 
5 mL: Serum  
Concentration of 
methylprednisolone 
 
aThe purpose of this initial sample will be to assess the level of variation in the baseline. 
 
Abbreviations: iPS induced pluripotent stem (cells), NA not applicable, NK natural killer, PBMC: Peripheral blood 
mononuclear cell, TBNK: Flow cytometry panel for T lymphocytes, B lymphocytes, and NK cells 
 

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Volunteers will be discharged after collection of the 2-hour or 4-hour blood and biopsy samples 
(depending on the regimen of blood collection), measurement of vital signs, and documentation 
of any AEs. Follow-up phone calls 1 day and 5 days after discharge will document any adverse 
effects related to the drug or skin biopsy. If any AEs that may require treatment are documented 
on the follow-up phone calls, volunteers will be asked to come to the NIH CC’s outpatient clinic 
for evaluation by the Principal Investigator. 
 
We propose an adaptive study design, in which we will recruit an initial cohort of 20 subjects. 
After processing the samples from these subjects, we will perform differential expression 
analysis to determine if recruitment of additional subjects (up to a maximum of 50) would 
impact the biological interpretation of the data. If additional data would be unlikely to change the 
study outcome, then no further recruitment will be pursued.  
4 
Study Population 
 
Recruitment Plan 
Healthy volunteers (N=50 maximum) may be recruited through the NIH Clinical Research 
Volunteer Program, the Patient Recruitment and Public Liaison Office, or through the posting of 
advertisements. All flyers and advertisements will be submitted to the NIH IRB for approval. 
 
Inclusion Criteria 
1. Age 18 to 64 years 
2. Willingness to have samples stored for future research 
3. Willingness to undergo genetic testing 
 
Exclusion Criteria 
1. Body Mass Index < 18 or > 35 
2. Difficult peripheral venous access (as determined by study staff at screening) 
3. History of severe allergic reaction to glucocorticoids 
4. History of autoimmune or autoinflammatory disease 
5. Active solid or hematologic malignancy 
6. History of a skin condition (such as psoriasis, pemphigus, or atopic dermatitis) that could 
affect the results of the transcriptional analysis of the skin biopsy samples 
7. Diabetes mellitus 
8. Cancer chemotherapy within the past 5 years 
9. Surgery within the past 8 weeks 
10. History of recent (within the past 30 days) infection 

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11. A positive test for human immunodeficiency virus, or hepatitis A, B or C virus infection 
(viral markers hepatitis screen, which includes HBsAg, anti-HCV IgG, anti-HAV IgM)  
12. A positive or indeterminate test for latent tuberculosis (interferon gamma release assay) 
13. History of parasitic, amebic, fungal or mycobacterial infections, or other possible latent 
infections 
14. Coagulation test (PT and PTT) results outside of normal range 
15. History of a bleeding disorder 
16. Use of a glucocorticoid (including topical or inhaled), a nonsteroidal anti-inflammatory 
drug (including aspirin and selective COX-2 inhibitors), an anti-epileptic drug, an 
anticoagulant, a statin, a selective serotonin reuptake inhibitor, a macrolide, an azole, 
diltiazem, troglitazone, rifabutin, ranitidine, rifampin, quinine, quinidine, cyclosporine, 
amiodarone, or St. John’s wort, within the past 30 days. A list of prohibited medications, 
including specific examples for each class, is provided as an attachment. 
17. Vaccination within the past 30 days  
18. Receipt of an immunosuppressant or immunomodulatory drug within the past 30 days  
19. Pregnancy, current or within the past 90 days, or trying to become pregnant during the 
study 
20. Current breastfeeding 
21. Complete blood count (CBC) and/or acute care panel values are both outside of the NIH 
Department of Laboratory Medicine normal reference range and deemed clinically 
significant by the principal investigator 
22. Any electrocardiogram (ECG) abnormality that is clinically significant  
23. Any condition that, in the investigator’s opinion, may put the participant at undue risk or 
compromise the study’s scientific objectives  
24. Participation in a clinical protocol which includes an intervention that, in the opinion of 
the investigator, may affect the results of the current study 
 
Justification for Exclusion of Special Populations 
Children: Minors will be excluded because there is the potential for greater than minimal risk 
and no prospect for direct benefit. 
 
Adults who lack capacity to consent: Because this study has no prospect for direct benefit, 
adults who lack decision-making capacity to provide informed consent will be excluded at 
screening. Enrolled participants who permanently lose capacity to consent during the study will 
be withdrawn (section 11.7), in accordance with NIH Human Research Protection Program 
(HRPP) Policy 403. 
 

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Pregnant and breastfeeding individuals: Pregnant volunteers will be excluded because there 
are no adequate and well-controlled studies of glucocorticoids in pregnant women. 
Glucocorticoids should be used during pregnancy only if the potential benefit justifies the 
potential risk to the fetus. Breastfeeding volunteers will be excluded due to the potential for 
serious adverse reactions in nursing infants from glucocorticoids.  
 
NIH staff: NIH staff and family members of study team members may be enrolled in this study 
as this population meets the study entry criteria.  Neither participation nor refusal to participate 
as a subject in the research will have an effect, either beneficial or adverse, on the participant’s 
employment or position at NIH. Every effort will be made to protect participant information, but 
such information may be available in medical records and may be available to authorized users 
outside of the study team in both an identifiable an unidentifiable manner. 
 
The NIH investigator will provide and request that the NIH staff member review the Frequently 
Asked Questions (FAQs) for Staff Who are Considering Participation in NIH Research and the 
Leave Policy for NIH Employees Participating in NIH Medical Research Studies (NIH Policy 
Manual 2300-630-3). Please see section 15.1.1 for consent of NIH staff. 
5 
 Study Agents 
 
Methylprednisolone sodium succinate for injection 
 Disposition and Dispensation 
Study agent will be distributed via the NIH CC Pharmacy according to standard pharmacy 
procedures. 
 Formulation 
Methylprednisolone sodium succinate for injection, USP (SOLU-MEDROL sterile powder, 
Pfizer, Inc.) is an anti-inflammatory glucocorticoid which occurs as a white, or nearly white, 
odorless hygroscopic, amorphous solid. It is very soluble in water and in alcohol; it is insoluble 
in chloroform and is very slightly soluble in acetone. 
Methylprednisolone sodium succinate has the same metabolic and anti-inflammatory actions as 
methylprednisolone. When given parenterally and in equimolar quantities, the two compounds 
are equivalent in biologic activity. 
125-milligram Act-O-Vial System: Each 2 mL (when mixed) contains methylprednisolone 
sodium succinate equivalent to 125 milligrams methylprednisolone; also 1.6 mg monobasic 
sodium phosphate anhydrous; and 17.4 mg dibasic sodium phosphate dried.  

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 Dosage and Administration 
Study volunteers will receive 250 milligrams administered intravenously over 30 minutes. 
 Storage 
Protect from light. Store unreconstituted product and solution at a controlled room temperature 
of 20° to 25°C (68° to 77°F). Use solution within 48 hours after mixing. 
 
Topical methylprednisolone 
 Disposition and Dispensation 
Study agent will be distributed via the NIH CC Pharmacy according to standard pharmacy 
procedures. 
 Formulation 
1g Advantan emulsion 0.1% (Bayer) contains methylprednisolone aceponate (21-acetoxy-
11beta-hydroxy-6alpha-methyl-17-propionyloxy-1,4-pregnadiene-3,20-dione) 1 mg, as the active 
ingredient. It is an oil-in-water emulsion containing medium chain triglycerides, caprylic-capric-
stearic triglyceride, polyoxyethylene alcohol 2-stearylether, polyoxyethylene alcohol-21-
stearylether, benzyl alcohol, disodium edetate, glycerol, and purified water.  
 Dosage and Administration 
Advantan emulsion 0.1% will be applied thinly to the skin in a 2 x 2 cm area. This is a one-time 
application. 
 Storage 
Store below 25°C (77°F). 
6 
Study Schedule 
This study will involve 2 study visits at the NIH CC and 2 follow-up phone calls. Only those 
volunteers found to be eligible after the screening procedures will return to continue the study. 
Volunteers will be referred to their Primary Care Physician if any screening tests need further 
follow-up. 
 
Screening visit (Day -30 to Day -1) 
The following tests will be conducted after informed consent is obtained: 
• Physical exam (including BMI assessment) with vital signs 
• Medical history and medication review 
• ECG 

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• Blood draw for the following: 
o CBC with differential 
o Acute care panel 
o Serum pregnancy test (for volunteers capable of becoming pregnant) 
o PT and PTT 
o Screening for HIV and hepatitis A, B and C. Test results obtained within the past 
30 days will be accepted. 
o Screening for latent tuberculosis with interferon gamma release assay (IGRA) 
o Baseline purification of six hematopoietic cell sub-types (following confirmation 
of volunteer eligibility) 
o Baseline flow cytometry 
o Banking of baseline plasma, peripheral blood mononuclear cells (PBMCs), and 
DNA 
 
Infusion visit (Day 0) 
• Targeted physical exam 
• Vital signs on first encounter, every 30 minutes from the time of infusion until the time of 
collection of the four-hour blood sample, and discharge. 
• Serum pregnancy test (for volunteers capable of becoming pregnant). For volunteers 
screened within the past 24 hours, the test will not be repeated. 
• Interim medical history and medication review 
• Pre-infusion blood draw (for analyses listed in Table 2) 
• Optional: Pre-infusion skin biopsy from one arm 
• Optional: Application of topical methylprednisolone to the contralateral arm 
• Intravenous infusion of methylprednisolone sodium succinate (30 minutes) 
• Nursing staff will monitor study subjects for the presence of behavioral abnormalities 
(altered mood, hyperactivity, disorientation, psychosis) 
• Blood draws at 1 and 2 hours or 2 and 4 hours (+/-10 minutes) after the start of infusion (for 
analyses listed in Table 2) 
• Optional: Skin biopsy (at the site where topical methylprednisolone was applied) 4 hours 
after the start of infusion 
 
Follow-up Phone Calls (Day 1 and Day 5) 
Follow-up phone calls will be made one day (+/- 0 days) and five days (+/- 2 days) after 
discharge to document the presence or absence of side effects related to the skin biopsy or study 
drug.  

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7 
Study Procedures/Evaluations 
Methylprednisolone sodium succinate infusion: Volunteers will receive a single intravenous 
dose of methylprednisolone sodium succinate administered through an intravenous catheter over 
a period of 30 minutes. 
 
Blood draw: Blood will be drawn at screening, immediately prior to the drug infusion, and in 
one of two regimens: 1 hour and 2 hours (+/- 10 minutes) after the infusion, or 2 hours and 4 
hours (+/-10 minutes) after the start of the infusion. Blood will be used for laboratory evaluations 
and storage as indicated in Table 2. The amount of blood drawn for research purposes will be 
within the limits allowed for adult research subjects by the NIH CC (Medical Administrative 
Policy 95-9, Guidelines for Limits of Blood Drawn for Research Purposes in the Clinical Center: 
http://cc-internal.cc.nih.gov/policies/PDF/M95-9.pdf). 
 
Punch biopsy of the skin, with application of methylprednisolone emulsion: A full-thickness 
skin sample will be taken from the posterior aspect of the upper third of one arm at baseline for 
some volunteers. The sample will be obtained with a 3-mm skin biopsy punch, following 
intradermal anesthesia with 2% lidocaine injected with a 30-gauge needle. Prior to insertion of 
the biopsy punch, the skin will be stretched along the long axis of the arm, perpendicular to the 
lines of least tension. After collection of the sample, the elliptical wound will be closed with gel 
foam or steri-strips and covered with a dressing. Methylprednisolone emulsion will be applied to 
the same location (upper third of the posterior aspect) on the contralateral arm, which will be 
biopsied 4 hours after the start of infusion, following the same procedure. The skin biopsy 
sample will be divided into two fragments: The most superficial fragment (which is expected to 
contain the epidermis and dermis) will be used for RNA purification, and the deeper fragment 
(which is expected to contain sub-dermal connective tissue) will be employed to generate a 
fibroblast culture for future testing of in vitro exposure to glucocorticoids and for future 
generation of iPS cells to study the response of other cell types to glucocorticoids. 
 
ECG (12-lead) will be performed to rule out electrical cardiac abnormalities and ensure 
eligibility to receive the study agent.  
8 
Potential Risks and Benefits 
 
Risks 
Methylprednisolone sodium succinate infusion: Side effects from a one-time, intravenous 
dose of methylprednisolone may include metallic or bitter taste in the mouth during and possibly 
after infusion, upset stomach, sleep disturbance (insomnia, sleepiness/fatigue), increased 
appetite, water retention, swelling in hands/ankles/feet, weight gain, temporary increase in blood 
glucose, temporary increase in blood pressure, mood disturbance or unusual behavior, electrolyte 

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imbalance, muscle cramps, back pain, headaches, and frequent urination lasting 1-2 weeks after 
infusion. 
 
Individuals who received methylprednisolone to treat illness have had skin problems such as rash 
or dry skin, easy bruising, growth of body hair, irregular menstrual cycles, abnormal results of 
liver enzyme tests, enlargement of the liver, inflammation of the pancreas, numbness in the feet 
and hands, eye problems, and bone problems such as osteoporosis and damage to the hip bone. 
Many of these effects were dependent on the length of treatment and we do not expect that they 
will occur with a single dose. 
 
Corticosteroids may decrease resistance to infection and the ability to localize infection. 
Infections with any pathogen in any location of the body may be associated with the use of 
corticosteroids alone or in combination with other immunosuppressive agents. Corticosteroids 
may reactivate or exacerbate latent fungal, bacterial, or viral infections. Individuals with known 
or suspected active or latent infections, as well as those using immunosuppressive agents, will be 
excluded from study participation. In addition, subjects will be advised to limit travel for 4 
weeks following the methylprednisolone infusion. 
 
Allergic reactions including anaphylactoid reaction, anaphylaxis and angioedema have been 
reported in patients receiving methylprednisolone. 
 
Serious cardiac, musculoskeletal, neurologic/psychiatric and other side effects of intravenous 
methylprednisolone have been reported in patients with underlying disease. Such effects are not 
expected in this study involving administration of a single dose in healthy volunteers.  
 
There are reports of cardiac arrhythmias and/or cardiac arrest following the rapid administration 
of large IV doses of methylprednisolone (greater than 0.5 gram administered over a period of 
less than 10 minutes). Bradycardia has been reported during or after the administration of large 
doses of methylprednisolone sodium succinate, and may be unrelated to the speed or duration of 
infusion.  
 
Methylprednisolone emulsion: The most common side effects are itching, burning and redness 
of skin. Allergic skin reactions are rare. The following side effects may occur: vesiculation 
folliculitis, hypertrichosis, skin thinning, telangiectasia, and perioral dermatitis. Systemic 
absorption of topically applied corticosteroids may occur, particularly under the following 
conditions: when large quantities are used, or when application is made to wide areas of the 
body, or to damaged skin, when potent topical corticosteroids are used, and when the occlusive 
dressing technique is applied. Depression of the hypothalamic-pituitary-adrenal axis with 
consequent suppression of the adrenal gland may occur. Benign increased intracranial pressure 

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has been rarely reported. Methylprednisolone emulsion must not be applied to the face or to skin 
crease areas, and should not come in contact with the eyes. 
 
Blood draw: The risks of drawing blood include pain, bruising, bleeding, and, rarely, fainting or 
infection. 
 
Skin biopsy: The risks of skin biopsy include local pain, bleeding, infection, and potential scar 
and keloid formation. The use of local and systemic glucocorticoids can increase the risk of 
infection and poor healing of the biopsy site. A local anesthetic administered prior to biopsy can 
cause a mild burning sensation upon injection, skin irritation and rarely allergic reaction. Oral 
analgesics will be used to manage pain. 
 
ECG: An ECG is of minimal risk to the subjects and causes no significant side effects. 
Unexpected or incidental medical findings unrelated to the study may occur and may require the 
subject to follow up with an appropriate physician for standard medical care or follow up. The 
electrodes may feel cold when applied; in rare cases some people will develop a rash or skin 
irritations where the patches are placed. This type of irritation usually resolves by itself or 
occasionally with topical medication. 
 
RNA sequencing, DNA sequencing, and genome-wide genotyping: Studies of RNA 
molecules (RNA sequencing) will be performed to identify changes in gene expression in 
response to the study drug. Studies of DNA molecules (genome-wide genotyping) will be 
performed to identify variants in the genome that may influence gene expression. These studies 
will involve assays of specific regions of the genome. Depending on the findings, sequencing of 
DNA (either specific regions or the entire DNA molecule) may be performed in the future. These 
studies are not designed or intended to produce data regarding health risks, incurable conditions, 
or information contradictory to stated biological relationships between individuals. Therefore, it 
is not expected that the results from these studies will be returned to volunteers. In the rare event 
of detecting an incidental DNA finding related to a volunteer’s health, we will share the 
information with the volunteer, after confirming the result on an additional sample (collected 
prospectively or previously stored) in a laboratory certified by the Clinical Laboratory 
Improvement Amendments program.  The confirmed result would be placed in the NIH medical 
record.  Such information may have psychological implications; genetic counseling and advice is 
available from the NIH to help volunteers understand the implications of incidental findings, if 
necessary. Following genetic testing, data will be shared in a controlled-access public database in 
accordance with NIH guidelines, for other investigators to benefit from the information (e.g., 
dbGaP, the Database of Genotypes and Phenotypes). However, no personal, identifiable 
information will be shared in this process, as the results will only be shared with a code. 

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Benefits 
This research is not designed to benefit study volunteers. Knowledge gained from this research 
may inform the development of future anti-inflammatory and immunosuppressive medications. 
9 
Research Use of Stored Human Samples, Specimens, or Data 
Intended Use: Samples and data collected under this protocol may be used to study the body’s 
response to glucocorticoids. Genetic testing will be performed on some of these samples. A 
separate signed informed consent document will be obtained for any other research not described 
in this protocol. 
 
Storage: Samples will be stored in a secure location with limited access. Samples and data will 
be coded prior to storage using barcodes assigned by the investigators. The database linking a 
barcode with a specific individual will be kept in password-protected computers. Only 
investigators will have access to the samples and data.  
 
Tracking: Data acquired under this protocol will be tracked using the Clinical Research 
Information Management System of the NIAID (CRIMSON). After coding, laboratory 
specimens will be tracked using the Biological Specimen Inventory (BSI) system, under a 
NIAID-wide contract for laboratory specimen banking and tracking. 
 
Disposition at the completion of the protocol: 
• In the future, other investigators (both at NIH and outside) may wish to use these samples 
and/or data for research purposes. If the planned research falls within the category of “human 
subjects research” on the part of the NIH researchers, NIH IRB review and approval will be 
obtained. This includes the NIH researchers sending out coded and linked samples or data 
and getting results that they can link back to their subjects.  
 
Reporting the loss or destruction of samples/specimens/data to the IRB: 
• Any loss or unanticipated destruction of samples or data (for example, due to freezer 
malfunction) that meets the definition of a reportable event will be reported to the NIH IRB 
according to NIH HRPP Policy 801.  
• Subjects may decide at any point not to have their samples stored. In this case, the principal 
investigator will destroy all known remaining samples and report what was done to both the 
subject and to the IRB.  
 
Handling of specimens/data from subjects who must be removed from the study: Subjects 
who are enrolled may need to be removed from the study after the screening visit, if the 
laboratory and/or ECG results obtained on that visit show an abnormality that meets the study’s 
exclusion criteria. Subjects may also need to be removed from the study if they develop a 

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condition or side effect that makes them ineligible to continue participation, if the study is 
cancelled or stopped, or if they fail to comply with the study requirements. In any of these cases, 
the subject will be notified by the Study Coordinator. Any blood or skin samples collected prior 
to removal of a subject will be processed in the same way as those obtained from subjects who 
are not excluded from the study. Data obtained from these samples may be used for the purpose 
of establishing the level of variation of the baseline measurements under different circumstances, 
which can be informative. At the time of notification of removal from the study, subjects will be 
reminded of their right, as detailed in the consent form, to request for their samples to be 
destroyed. The purpose of this provision is to ensure that research samples of potential use are 
not unnecessarily destroyed, while effectively respecting the research subjects’ right to request 
destruction of their samples.  
10 Remuneration Plan for Volunteers 
Volunteers will be compensated for their time and inconvenience according to the following 
schedule: 
Screening visit: $20 for the 1st hour plus $10 for each additional hour (20+10[3]) = $50 
Screening blood draw: $20 
Infusion visit: $20 for the 1st hour plus $10 for each additional hour (20+10[6]) = $80 
Pre- and post-infusion blood draws: $20 (x3) = $60 
Pre- and post-infusion punch biopsies of the skin: $50 (x2) = $100 
Total with biopsy = $310 
Total without biopsy = $210 
 
In the event that follow-up blood draws need to be scheduled after the infusion visit, as detailed 
in Section 11.2.1 below, each additional visit will be compensated at $20. 
 
Volunteers will receive compensation by check, direct deposit, or Automated Clearing House 
(ACH) payment after their participation in the study is complete. 
 
Travel-related expenses (e.g., transportation, hotel, meals) will not be paid for or reimbursed 
under this protocol. 
11 Assessment of Safety 
 Definitions 
The NIAID Clinical Safety Office (CSO) is responsible for sponsor safety oversight of this 
study, and the definitions below comply with CSO requirements. 
 
Adverse Event (AE): An AE is any untoward or unfavorable medical occurrence in a human 
subject, including any abnormal sign (eg, abnormal physical exam or laboratory finding), 

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symptom, or disease, temporally associated with the subject’s participation in the research, 
whether or not considered related to the research. 
 
Adverse Reaction (AR): An AE that is caused by an investigational agent (drug or biologic). 
 
Suspected Adverse Reaction (SAR): An AE for which there is a reasonable possibility that the 
investigational agent caused the AE. ‘Reasonable possibility’ means that there is evidence to 
suggest a causal relationship between the drug and the AE. An SAR implies a lesser degree of 
certainty about causality than AR, which implies a high degree of certainty. 
 
Serious Adverse Event (SAE): An SAE is an AE that results in one or more of the following 
outcomes: 
• death 
• a life-threatening event (places the subject at immediate risk of death from the event as it 
occurred) 
• an inpatient hospitalization or prolongation of an existing hospitalization 
• a persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
• a congenital anomaly/birth defect 
• a medically important event* 
 
*Medical and scientific judgment should be exercised in deciding events that may not be 
immediately life threatening or result in death or hospitalization but may jeopardize the subject 
or may require intervention to prevent one of the other outcomes listed above. 
 
Unexpected Adverse Event: An AE is unexpected if it is not listed in the Investigator’s 
Brochure or Package Insert (for marketed products) or is not listed at the specificity or severity 
that has been observed. It is the responsibility of the IND sponsor to make this determination. 
 
Serious and Unexpected Suspected Adverse Reaction (SUSAR): A SUSAR is an SAR that is 
both serious and unexpected. 
 
Unanticipated Problem (UP): A UP is any event, incident, experience, or outcome that is 
1. unexpected in terms of nature, severity, or frequency in relation to 
a. the research risks that are described in the IRB-approved research protocol and 
informed consent document, Investigator’s Brochure, or other study documents; 
and 
b. the characteristics of the subject population being studied; and 

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2. possibly, probably, or definitely related to participation in the research; and 
3. places subjects or others at a greater risk of harm (including physical, psychological, 
economic, or social harm) than was previously known or recognized. (Per the IND 
sponsor, an AE with a serious outcome will be considered increased risk.) 
 
Serious Unanticipated Problem (UP): A UP that meets the definition of a Serious Adverse 
Event or compromises the safety, welfare or rights of subjects or others. 
 
Unanticipated Problem that is not an Adverse Event (UPnonAE): A UP that does not fit the 
definition of an AE, but which may, in the opinion of the investigator, involve risk to the subject, 
affect others in the research study, or significantly impact the integrity of research data. Such 
events would be considered non-serious UPs. For example, we will report occurrences of 
breaches of confidentiality, accidental destruction of study records, or unaccounted-for study 
drug. 
 Documenting, Recording, and Reporting Adverse Events 
AEs occurring from the time the informed consent is signed through study day 5 will be 
documented, recorded, and reported to the CSO. 
 
The following signs or symptoms are induced by or associated with Methylprednisolone sodium 
succinate, and any combination of these is expected to occur.  These events will not be recorded 
or reported to the CSO unless they reach a Grade of 3 or higher, are deemed a risk to the 
participants’ rights or well-being, or are deemed appropriate to report under specific 
circumstances by the Principal Investigator: 
 
• Hyperglycemia 
• Leukocytosis 
• Neutrophilia 
• Monocytopenia 
• Eosinopenia 
• Flushing 
• Increased appetite 
• Bitter taste in mouth 
• Metallic taste in mouth 
• Anxiety 
 
The following signs or symptoms are induced by or associated with Methylprednisolone sodium 
succinate. These events will not be recorded or reported to the CSO unless they reach a Grade of 

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4 or higher, are deemed a risk to the participants’ rights or well-being, or are deemed appropriate 
to report under specific circumstances by the Principal Investigator: 
 
• Lymphopenia (Lymphocyte Count Decreased) 
 
The following signs or symptoms are induced by or associated with Methylprednisolone 
emulsion 1%, and any combination of these is expected to occur.  These events will not be 
recorded or reported to the CSO, unless they reach a Grade of 3 or higher, are deemed a risk to 
the participants’ rights or well-being, or are deemed appropriate to report under specific 
circumstances by the Principal Investigator: 
 
• Application site burning 
• Application site itching 
• Application site redness 
 
At each contact with the subject, information regarding AEs will be elicited by appropriate 
questioning and examinations and will be: 
• immediately documented in the subject’s medical record/source document, 
• recorded in CRIMSON, and 
• reported as outlined below (eg, IND sponsor, IRB, and Food and Drug Administration 
[FDA]). 
 
If a diagnosis is clinically evident (or subsequently determined), the diagnosis rather than the 
individual signs and symptoms or lab abnormalities will be recorded as the AE. 
 
All abnormal laboratory findings will be reviewed on a routine basis by the principal investigator 
to identify potential safety signals. An abnormal lab not included on the toxicity table should be 
assessed in a similar fashion to the criteria above. 
 Follow-up of reportable abnormal laboratory values 
Abnormal laboratory values noted during the infusion visit that require immediate medical 
intervention will be managed by the study team at the NIH CC, and followed until resolution. 
 
Abnormal laboratory values that do not require immediate medical intervention, but that will be 
recorded and reported based on the criteria listed in Section 11.2, will be followed until 
resolution. Laboratory values that remain within the reportable range at the final (4-hour) time 
point on the day of the infusion visit, will be repeated on an outpatient basis at the NIH CC, until 
they have returned to the non-reportable range. The first outpatient follow-up blood draw will be 
scheduled within 72 hours of discharge from the Day Hospital. In the unlikely event that 

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subsequent follow-up blood draws are necessary, the follow-up intervals will be at the discretion 
of the Principal Investigator.     
 Investigator Assessment of Adverse Events 
The investigator will assess all AEs with respect to seriousness (criteria listed above), severity 
(intensity or grade), and causality (relationship to study agent and relationship to research) 
according to the following guidelines. 
 Severity 
The Investigator will grade the severity of each AE according to the “Common Terminology 
Criteria for Adverse Events (CTCAE)” (v 4.0) which can be found at: 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm  
 Causality 
Causality (likelihood that the event is caused by the study agent(s)) will be assessed considering 
the factors listed under the following categories: 
 
Definitely Related 
• reasonable temporal relationship 
• follows a known response pattern 
• clear evidence to suggest a causal relationship 
• there is no alternative etiology 
 
Probably Related 
• reasonable temporal relationship 
• follows a suspected response pattern (based on similar agents) 
• no evidence of a more likely alternative etiology 
 
Possibly Related 
• reasonable temporal relationship 
• little evidence for a more likely alternative etiology 
 
Unlikely Related 
• does not have a reasonable temporal relationship 
OR 
• good evidence for a more likely alternative etiology 
 

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Not Related 
• does not have a temporal relationship 
OR 
• definitely due to an alternative etiology 
 
Note: Other factors should also be considered for each causality category when appropriate. 
Causality assessment is based on available information at the time of the assessment of the AE. 
The investigator may revise the causality assessment as additional information becomes 
available. 
 Investigator Reporting Responsibilities to the Sponsor 
 Adverse Events 
AE data will be submitted to the IND sponsor when requested for periodic safety assessments, 
review of IND annual reports, review of IND safety reports, and preparation of final study 
reports. 
 Serious Adverse Events 
All SAEs (regardless of relationship and whether or not they are also UPs) must be reported on 
the Safety Expedited Report Form (SERF) and sent to the Clinical Safety Office (CSO) by fax or 
e-mail attachment. Deaths and immediately life threatening SAEs must be reported to the CSO 
within 1 business day after the site becomes aware of the event. All other SAEs must be reported 
within 3 business days of site awareness. 
 
CSO CONTACT INFORMATION: 
Clinical Safety Office 
5705 Industry Lane 
Frederick, MD 21704 
Phone: 301-846-5301 
Fax: 301-846-6224 
E-mail: rchspsafety@mail.nih.gov  
 
SAEs that have not resolved by the end of the follow-up period are followed until final outcome 
is known. If it is not possible to obtain a final outcome for an SAE (eg, the subject is lost to 
follow-up), the reason a final outcome could not be obtained will be recorded by the investigator 
in CRIMSON and on the SERF. 
 
SAEs that occur after study day 5 that are reported to and are assessed by the investigator to be 
possibly, probably, or definitely related to study drug must be reported to the CSO. 

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 Unanticipated Problems 
UPs that are also AEs must be reported to the CSO by fax or e-mail attachment using the NIH 
Problem Report Form no later than 7 calendar days of site awareness of the event. UPs that are 
not AEs are not reported to the CSO. 
 Sponsor’s Reporting Responsibilities 
SUSARs, as defined in 21 CFR 312.32 and determined by the IND sponsor, will be reported to 
FDA and all participating investigators as IND Safety Reports. 
 
The IND sponsor will also submit an IND Annual Report of the progress of the investigation to 
the FDA as defined in 21 CFR 312.33. 
 Halting Rules for the Protocol 
Halting the study requires immediate discontinuation of study agent administered for all subjects 
and suspension of enrollment until a decision is made whether or not to continue enrollment and 
study agent administration. 
 
The halting rules are: 
• 2 or more subjects experience the same or similar SAEs that are possibly, probably, or 
definitely related to the study agent; 
OR 
• 2 or more of the same or similar AE in different subjects that are grade 3 or above and are 
possibly, probably, or definitely related to the study agent; 
OR 
• any safety issue that the principal investigator and/or the CSO determines should halt the 
study. 
 
The principal investigator and/or CSO will determine if the study should be halted. In addition, 
the FDA may halt the study at any time following review of any safety concerns. 
 Reporting a Study Halt 
If a halting rule is met, a description of the AE(s) or safety issue must be reported by the 
principal investigator within 1 business day to the CSO and IRB by fax or email. 
 Resumption of a Halted Study 
The IND sponsor, in collaboration with the principal investigator, will determine if it is safe to 
resume the study. The principal investigator will notify the IRB of the decision on resumption of 
the study. 

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 Withdrawal Criteria for an Individual Subject 
An individual subject will be withdrawn for any of the following: 
• An individual subject’s decision. The investigator will attempt to determine the reason 
for the subject’s decision. 
• The subject permanently loses decision-making capacity to provide informed consent. 
• The investigator determines that continued participation in the study would not be in the 
best interest of the subject. 
 
Subjects who withdraw or are withdrawn from the study at any point after the pre-infusion skin 
biopsy will, if willing, continue to receive follow-up phone calls for safety assessments. 
 Replacement of Withdrawn Subjects or Subjects Who Discontinue Study Agent 
Subjects who withdraw or are withdrawn prior to the pre-infusion skin biopsy will be replaced. If 
a subject is replaced, all the data collected from that subject will still be included for the safety 
assessment. 
 Safety Oversight 
 Safety Review and Communications Plan 
A Safety Review and Communications Plan (SRCP) has been developed for the protocol. The 
SRCP is an internal communications document between the principal investigator and the CSO, 
which delineates the safety oversight responsibilities of the principal investigator, the CSO, and 
other stakeholders. The SRCP also includes the overall plan for conducting periodic safety 
surveillance assessments. 
 Sponsor Medical Monitor 
A medical monitor, representing the IND sponsor (OCRPRO), has been appointed for oversight 
of safety in this clinical study. The Sponsor Medical Monitor will be responsible for performing 
safety assessments as outlined in a Safety Review and Communications Plan (SRCP). 
12 Reporting Procedures 
 Reporting to the NIH IRB 
Reportable events will be tracked and submitted to the IRB as outlined in Policy 801. 

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NIH Definitions of Protocol Deviation and Non-compliance 
Protocol Deviation: Any change, divergence, or departure from the IRB-approved research 
protocol.  
1. Major Deviation – Deviation from the IRB-approved protocol that have, or may have the 
potential to, negatively impact, the rights, welfare or safety of the subject, or to 
substantially negatively impact the scientific integrity or validity of the study. 
2. Minor Deviation – A Deviation that does not have the potential to negatively impact the 
rights, safety, or welfare of subjects or others, or the scientific integrity or validity of the 
study. 
 
Non-compliance: Failure of an investigator to follow the applicable laws, regulations, or 
institutional policies governing the protection of human subjects in research, or the requirements 
or determinations of the IRB, whether the failure is intentional or not. 
• Continuing Non-compliance – A pattern of recurring non-compliance that either has 
resulted, or, if continued, may result in harm to subjects or otherwise materially 
compromise the rights, welfare and/or safety of subjects, affect the scientific integrity of 
the study or validity of the results. The pattern may comprise repetition of the same non-
compliant action(s), or different non-compliant events. Such non-compliance may be 
unintentional (e.g. due to lack of understanding, knowledge, or commitment), or 
intentional (e.g. due to deliberate choice to ignore or compromise the requirements of any 
applicable regulation, organizational policy, or determination of the IRB). 
• Serious Non-compliance – Non-compliance, whether intentional or not, that results in 
harm or otherwise materially compromises the rights, welfare and/or safety of the subject. 
Non-compliance that materially affects the scientific integrity or validity of the research 
may be considered serious non-compliance, even if it does not result in direct harm to 
research subjects. 
 
 Reporting to the NIAID Clinical Director 
The principal investigator will report UPs, major protocol deviations, and deaths to the NIAID 
clinical director according to institutional timelines. 
13 Site Monitoring Plan 
According to the ICH GCP guidelines, section 5.18, and FDA 21 CFR 312.50, clinical protocols 
are required to be adequately monitored by the study sponsor. This study monitoring will be 
conducted according to the “NIAID Intramural Clinical Monitoring Guidelines.” Monitors under 
contract to the NIAID/OCRPRO will visit the clinical research site to monitor aspects of the 
study in accordance with the appropriate regulations and the approved protocol. The objectives 
of a monitoring visit will be: 1) to verify the existence of signed informed consent documents 

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and documentation of the consent process for each monitored subject; 2) to verify the prompt 
and accurate recording of all monitored data points, and prompt reporting of all SAEs; 3) to 
compare CRIMSON data abstracts with individual subjects’ records and source documents 
(subjects’ charts, laboratory analyses and test results, physicians’ progress notes, nurses’ notes, 
and any other relevant original subject information); and 4) to help ensure investigators are in 
compliance with the protocol. The monitors also will inspect the clinical site regulatory files to 
ensure that regulatory requirements (Office for Human Research Protections [OHRP]), FDA, and 
applicable guidelines (ICH GCP) are being followed. During the monitoring visits, the 
investigator (and/or designee) and other study personnel will be available to discuss the study 
progress and monitoring visit. 
 
The investigator (and/or designee) will make study documents (eg, consent forms, CRIMSON 
data abstracts) and pertinent hospital or clinical records readily available for inspection by the 
local IRB, the FDA, the site monitors, and the NIAID staff for confirmation of the study data.  
 
A specific protocol monitoring plan will be discussed with the principal investigator and study 
staff prior to enrollment. The plan will outline the frequency of monitoring visits based on such 
factors as study enrollment, data collection status, and regulatory obligations. 
14 Statistical Considerations 
Definition of the Study Endpoints 
• The primary endpoint of this study is a set of lists of human genes and non-coding RNAs 
(small RNAs and lncRNAs) that are differentially expressed in vivo after a glucocorticoid 
stimulus in each of six immune cell types (B cells, CD4+ T cells CD8+ T cells, NK cells, 
neutrophils, and monocytes) and one non-immune tissue (skin).  
• The secondary endpoint is a list of the genes and molecular pathways that represent the 
best candidates for targeted therapeutic alternatives to glucocorticoids. 
Bioinformatics Pipeline and Data Handling 
The output of the RNA and small-RNA sequencing runs (bcl files) will be converted to FASTQ 
format with the software program bcl2fastq. Quality control of the resulting FASTQ files will be 
performed with the software program FASTQC.  
 
RNA sequencing data will be aligned to the most recent human genome assembly using the 
software program TopHat (24). The resulting BAM files, the most recent version of the 
GENCODE human genome annotation, and a mask file of ribosomal and mitochondrial RNA 
annotations will be used to generate a transcriptome assembly with the software program 
Cufflinks (25), which models expression at the level of spliced transcripts, and not only at the 
level of genes or exons.  A merged transcriptome file with data from all subjects in an analysis 
will then be generated with the software program Cuffmerge (26). Transcript abundance will be 

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estimated with the software program Cuffquant (26), which outputs expression values for each 
transcript normalized by the length of the transcript and by the number of mapped reads for a 
particular sample (fragments per kilobase of transcript per million mapped reads, or FPKM).  
 
Small RNA sequencing data will be aligned to the most recent human genome assembly using 
the software program STAR (27). The resulting BAM files and the most recent version of the 
GENCODE human genome annotation will be used to annotate and quantify small RNAs with 
the software program ShortStack (28, 29). 
 
Visualization and gating for the flow cytometry data will be performed with the software 
program FlowJo. Analysis of flow cytometry and high-throughput cytokine measurement data 
will be performed in R using custom scripts.  
 
Differential Expression Analysis: 
Differential expression analysis for the RNA-seq data will be performed with the software 
program Cuffdiff (25). In short, the Cuffdiff algorithm models how variability in the measured 
fragment counts for a transcript depends on its expression level as well as its splicing structure. 
The algorithm captures uncertainty in a transcript's fragment count as a beta distribution and the 
overdispersion in this count (determined by globally fitting the observed variance in fragment 
counts as a function of the mean across replicates) with a negative binomial distribution. Under 
the resulting beta negative binomial model of fragment count variability, it estimates count 
variances for each transcript in each sample. These variance estimates are used for statistical 
testing to report significantly differentially expressed genes and transcripts. Raw p-values and 
False Discover Rate (FDR)-based q-values (30) are reported. 
 
Differential expression analysis for the small-RNA-seq data will be performed in R, with the 
package DESeq (31). In short, the DESeq algorithm models fragment count data with a negative 
binomial distribution and a shrinkage estimator for the distribution’s variance. Our choice of this 
algorithm for the small-RNA-seq data is based on the fact that most human small RNA 
molecules are not spliced, and DESeq uses raw fragment counts, without the assumption of 
spliced transcripts employed by the Cufflinks/Cuffdiff algorithms. 
 
For each cell type, we will assess the level of variation in the baseline measurements by 
comparing the expression values on the day of the screening visit to the 0-hour (pre-infusion) 
values on the day of the glucocorticoid infusion. We will then perform differential analysis 
comparing each of the two post-infusion time points (2 hours and 4 hours), independently, 
against the baseline. We will employ a cutoff value of < 5% FDR to select the transcripts that 
will be considered differentially expressed in each cell type at each time point. The resulting 
gene lists will be contrasted to determine, for example, which genes are uniquely differentially 
expressed in a specific cell type, or common to a specific hematopoietic lineage (myeloid or 

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lymphoid). They will also be used for pathway enrichment analysis, to determine the molecular 
pathways and biological functions that are most responsive to the glucocorticoid stimulus in each 
cell type. The genes and pathways that are found to be modified by the glucocorticoid stimulus 
in immune cells but not in non-immune cells (skin cells from this in vivo study and the primary 
human cells we have studied in vitro), will be selected as the top candidates for targeted 
therapeutic intervention, which we expect will form the basis of future studies.   
 
Sequencing library preparation and sequencing batches will be carefully designed such that 
samples from the same subject and cell type are run in the same batch and samples from different 
subjects are evenly distributed across batches. This will keep batch effects to a minimum in the 
identification of differentially expressed genes. For analyses across different subjects, such as 
comparisons among different cell types, linear regression will be adopted with batch as 
regression variable to adjust for batch difference. While prior experience suggests that library 
preparation is the greatest source of batch-to-batch variation in RNA-seq experiments, if the 
source of batch effects is unclear or cannot be adequately modeled, the R package SVA will be 
used.  
 
Summary Statistics for Demographic and Clinical Data 
Demographic characteristics and medical history will be summarized, e.g., proportion, mean, 
median, standard deviation, and minimum and maximum values. Means and standard deviations 
will be reported for all continuous variables; percent will be reported for categorical variables. 
 
Sample Size Justification 
Given the high-throughput data generation strategy of next-generation sequencing, the use of 
different starting materials for sequencing (mRNA, small RNA, DNA), and the use of serial 
sampling, standard sample-size calculation methods are not appropriate in this case and an 
adaptive study design is a more realistic approach. Sample size consideration will be based 
primarily on the power to identify differentially expressed genes. A recent pilot study of 
glucocorticoid action performed by the NIH Center for Human Immunology (CHI) (32) 
administered intravenous hydrocortisone at doses of 50 or 250 mg to a group of 20 healthy 
volunteers, where 10 subjects received each dose. They performed transcriptional analysis by 
expression microarrays in PBMCs before and at six time points after the infusion. They used the 
R package limma for analysis, which implements linear models and empirical Bayes methods for 
assessing differential expression in microarray experiments (33). P-values underwent multiple-
testing correction using the method of Benjamini and Hochberg to estimate the false-discovery 
rate (FDR). Using cut-off values of < 5% FDR-adjusted p-value and > 0.5 absolute average log2-
fold-change, they found evidence of differential expression in over 400 genes at the lower dose 
and over 600 genes at the higher dose, with a maximum number of differentially expressed genes 
4 hours after the infusion for both doses. This was accomplished with only 10 subjects per dose, 

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despite technical difficulties (notably hemolysis), and the use of hydrocortisone (which is 
approximately five times less potent than methylprednisolone, the glucocorticoid we plan to use 
in our study). In addition, the data from the study by Olnes et al. used bulk PBMCs, which are 
likely to give a more variable expression signal than the purified cell sub-types that we plan to 
employ in our study. Based on this information, we estimate that an initial recruitment of at least 
20 subjects is likely to reveal the changes in gene expression over time that our study aims to 
define in individual cell sub-types. We propose an adaptive study design, in which we will 
recruit an initial cohort of 20 subjects. Based on the observed data from 20 subjects, we will then 
assess the conditional power that can be obtained with up to 30 additional subjects.  We will also 
perform a bootstrap procedure, sampling with replacement up to 50 units from the 20 observed 
subjects, to evaluate the improvement in gene detection. For example, if the number of genes that 
can be detected reaches a plateau after 30 subjects, we will stop at that number. However, if the 
number of detected genes is seen to rise with a sample size of 30, but the pathways and the 
biological interpretation of the data remain unchanged, additional subjects will be unlikely to 
change the outcome of the study and no further recruitment will be pursued.  Due to the adaptive 
sample size determination, we will use a nominal significance level α <0.05.  
15 Ethics/Protection of Human Subjects 
 Informed Consent Process 
Informed consent is a process where information is presented to enable a person to voluntarily 
decide whether or not to participate as a research subject. It is an ongoing conversation between 
the human research subject and the researchers which begins before consent is given and 
continues until the end of the subject's involvement in the research. Discussions about the 
research will provide essential information about the study and include: purpose, duration, 
experimental procedures, alternatives, risks, and benefits. Subjects will be given the opportunity 
to ask questions and have them answered. 
 
Informed consent will be obtained in person in a private setting or remotely via telephone or 
video conference, by a study team member authorized to obtain consent. The subjects will sign 
the informed consent document prior to undergoing any research procedures. The signature may 
be obtained in ink or digitally using a mouse, stylus, or finger. The subjects may withdraw 
consent at any time throughout the course of the trial. A copy of the informed consent document 
will be given to the subjects for their records. The researcher will document the signing of the 
consent form in the subject’s medical record. The rights and welfare of the subjects will be 
protected by emphasizing to them that the quality of their medical care will not be adversely 
affected if they decline to participate in this study. 
 
Remote Consent Process: The informed consent document will be sent via secure email or file 
transfer to the potential participant. An explanation of the study will be provided over the 

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telephone or an NIH-approved videoconferencing platform (e.g., Microsoft Teams) after the 
participant has had the opportunity to read the consent form. The participant can print the 
appropriate form to sign and date in ink, or they can sign and date electronically with a finger, 
stylus, or mouse.  
 
The participant will return the signed and dated consent form to the consenting investigator, who 
will sign and date it with the date it was received. The consent form can either be printed and 
signed and dated in ink, or signed and dated digitally with a finger, stylus, or mouse. A fully 
executed copy will be sent to the participant for their records. 
 
The informed consent process will be documented on a progress note by the consenting 
investigator. The investigator will confirm that written consent has been obtained prior to 
initiating any study interventions. 
 Considerations for Consent of NIH Staff 
Consent for NIH staff will be obtained as detailed above and will comply with the requirements 
of NIH HRPP Policy 404 Research Involving NIH Staff as Subjects.  
 
Consent from NIH staff for whom this research is taking place within their own work unit or is 
conducted by any of their supervisors will, when possible, be obtained by an individual in a non-
supervisory relationship with that staff member. When consent of that staff member is 
conducted, a third party will be present to observe the consent process in order to minimize the 
risk of undue pressure on the staff member.  
 Subject Confidentiality 
All records will be kept confidential to the extent provided by federal, state, and local law. The 
study monitors and other authorized representatives of the sponsor may inspect all documents 
and records required to be maintained by the investigator, including but not limited to, medical 
records. Records will be kept locked and all computer entry and networking programs will be 
done with coded numbers only. Clinical information will not be released without written 
permission of the subject, except as necessary for monitoring by the IRB, NIAID, or the OHRP. 
 
To further protect the privacy of study participants, a Certificate of Confidentiality has been 
issued by the NIH. This certificate protects identifiable research information from forced 
disclosure. It allows the investigator and others who have access to research records to refuse to 
disclose identifying information on research participation in any civil, criminal, administrative, 
legislative, or other proceeding, whether at the federal, state, or local level. By protecting 
researchers and institutions from being compelled to disclose information that would identify 
research participants, Certificates of Confidentiality help achieve the research objectives and 
promote participation in studies by helping assure confidentiality and privacy to participants. 

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16 Data Handling and Record Keeping 
 Data Capture and Management  
Study data will be maintained in CRIMSON and collected directly from subjects during study 
visits and telephone calls, or will be abstracted from subjects’ medical records. Source 
documents include all recordings of observations or notations of clinical activities and all reports 
and records necessary to confirm the data abstracted for this study. Data entry into CRIMSON 
will be performed by authorized individuals. The investigator is responsible for assuring that the 
data collected are complete, accurate, and recorded in a timely manner. 
 Record Retention 
The investigator is responsible for retaining all essential documents listed in the ICH GCP 
guideline. Study records will be maintained by the principal investigator for a minimum of 5 to 7 
years and in compliance with institutional, IRB, state, and federal medical records retention 
requirements, whichever is longest. All stored records will be kept confidential to the extent 
required by federal, state, and local law. 
 
Should the investigator wish to assign the study records to another party and/or move them to 
another location, the investigator will provide written notification of such intent to 
OCRPRO/NIAID with the name of the person who will accept responsibility for the transferred 
records and/or their new location. Destruction or relocation of research records will not proceed 
without written permission from OCRPRO/NIAID. 

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