Clinical trial NCT04233164
Conditions: Systemic Lupus Erythematous (SLE)
Keywords: Immunosuppressive Drugs Methylprednisolone PREDNISONE Steroids Autoimmunity
Sponsor: National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Start date: 2020-03-04; Completion date: 2022-05-03
Enrollment: 47 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Background:

The immune system is the body's defense against bacteria and other harmful invaders. In people with systemic lupus erythematosus (SLE), the immune system becomes overactive and attacks healthy cells by mistake. Many people use glucocorticoids (GCs) to treat their SLE. GCs can calm down an overactive immune system by changing how the body reads genes. But GCs have side effects that can increase over time. Researchers want to learn more about how GCs work. This may help to develop new and better drugs for treating SLE without the side effects GCs have.

Objective:

To better understand how GCs affect the immune system in people with SLE.

Eligibility:

People age 18-80 with SLE.

Design:

Participants will be screened with a physical exam. They will have a health and medical history. They will have blood and urine tests. They will have an electrocardiogram to measure heart activity. For this, sticky pads are put on their chest, arms, and legs.

Participants will have a methylprednisolone infusion for about 30 minutes. It will be given through a needle in a vein.

Blood will be collected immediately before, 2 hours after, and 4 hours after the start of the infusion. Blood pressure and heart activity will be monitored. Participants will repeat some of the screening tests.

Participants will be contacted twice in the week after the infusion visit. They will discuss any health problems they are having.

Detailed description:
Study Design:

This is a study of the acute effects of glucocorticoids on the immune system of patients with SLE. Participants will undergo baseline blood collection prior to receiving a single intravenous (IV) dose of methylprednisolone sodium succinate. Participants will be randomized into 1 of 2 dose groups: 1 mg/kg or 250 mg. Blood will be collected again at 2 and 4 hours after the methylprednisolone infusion. Individual participation requires 2 visits to the NIH CC and 2 follow-up phone calls. Total length of individual subject participation including screening is 1-12 weeks. Blood samples will be processed for isolation of hematopoietic cell sub-populations (eg, neutrophils, B cells, plasmacytoid dendritic cells, CD4+ T cells, CD8+ T cells, monocytes, and natural killer cells). Laboratory studies will be performed on the purified cells, with the goal of understanding the human response to glucocorticoids in vivo at the level of circulating cell populations (eg, flow cytometry, mass cytometry), RNA (eg, RNA sequencing \[RNA-seq\], small-RNA-seq, real-time polymerase chain reaction \[PCR\]), DNA (eg, chromatin immunoprecipitation sequencing \[ChIP-seq\], methylation analysis, DNA sequencing, genotyping), and protein (eg, flow cytometry, mass spectrometry). At each time point, serum methylprednisolone levels will be measured.

Study Agent/ Intervention Description:

A single IV infusion of methylprednisolone sodium succinate at either 1 mg/kg or 250 mg.

Primary Objective:

To understand the cellular and molecular response to glucocorticoids in individuals with SLE.

Glucocorticoid Genomics in SLE

Secondary Objectives:

1. To identify candidate targets for therapeutic interventions that could mimic the action of glucocorticoids in patients with SLE, without the significant toxicity caused by the broad range of glucocorticoid actions.
2. To test whether the transcriptional response to glucocorticoids differs between the two doses being studied.
3. To identify similarities and differences, at baseline and in response to glucocorticoids, between the transcriptome of cells from patients and those from the previously studied healthy subjects.

Primary Endpoint:

A list of human protein-coding genes and non-coding RNAs that are differentially expressed in response to glucocorticoids in patients with SLE, for each of the studied cell types and doses.

Secondary Endpoints:

1. A comparison of the transcriptional response to glucocorticoids between the two dose groups.
2. 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.
3. Validation of the targets identified by functional studies.
4. For each cell type, a list of protein-coding or non-coding transcripts that are shared and a list of transcripts that are different, between patients with SLE and the previously studied healthy controls, at baseline or in response to glucocorticoids.

Arms: arm 1: Participants with Systemic Lupus Erythematosus (SLE) received a single intravenous dose of methylprednisolone 1 mg/kg and blood was collected two hours and four hours after the start of the infusion. arm 2: Participants with Systemic Lupus Erythematosus (SLE) received a single intravenous dose of 250 mg of methylprednisolone and blood was collected two hours and four hours after the start of the infusion.
Interventions: intervention 1: Solu-Medrol 1mg/kg intervention 2: Solu-Medrol 250 mg
Intervention descriptions: intervention 1: Methylprednisolone sodium succinate for injection, United States Pharmacopeia grade (USP), sold as SOLU-MEDROL sterile powder by Pfizer, Inc, is an anti-inflammatory glucocorticoid that occurs as a white, or nearly white, odorless hygroscopic, amorphous solid. It is very soluble in water and in alcohol, but is insoluble in chloroform and is very slightly soluble in acetone. The study agent solution will be administered as an IV infusion over 30 minutes, for a total single dose of 1 mg/kg of methylprednisolone. intervention 2: Methylprednisolone sodium succinate for injection, United States Pharmacopeia grade (USP), sold as SOLU-MEDROL sterile powder by Pfizer, Inc, is an anti-inflammatory glucocorticoid that occurs as a white, or nearly white, odorless hygroscopic, amorphous solid. It is very soluble in water and in alcohol, but is insoluble in chloroform and is very slightly soluble in acetone. The study agent solution will be administered as an IV infusion over 30 minutes, for a total single dose of 250 mg of methylprednisolone.

Protocol documents:
Prot_SAP_000.pdf: 
Genomic Effects of Glucocorticoids in Patients with Systemic Lupus 
Erythematosus
Protocol Number: 20AR0032
Latest IRB Approval Date of Protocol : 30 November 2021
Current Protocol Version and Date: 6th Amendment/F_V.7  05/11/2022
Principal Investigator:
Luis M. Franco, MD
National Institute of Allergy and Infectious Diseases (NIAID)
and
Lupus Clinical Trials Unit
National Institute of Arthritis and Musculoskeletal and Skin 
Diseases (NIAMS)
National Institutes of Health (NIH)
301-827-2461
luis.franco@nih.gov 
Conducted by:
NIAMS, NIH


Glucocorticoid Genomics in SLE
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Table of Contents
Table of Contents.............................................................................................................................2
List of Abbreviations .......................................................................................................................4
Protocol Summary ...........................................................................................................................5
Précis................................................................................................................................................7
1
Background Information and Scientific Rationale ..................................................................8
1.1
Scientific Background and Preliminary Data....................................................................8
1.2
Scientific Rationale.........................................................................................................12
1.3
Choice of a Glucocorticoid, Route of Administration, and Dose ...................................13
2
Study Objectives....................................................................................................................14
2.1
Primary Objective ...........................................................................................................14
2.2
Secondary Objectives......................................................................................................14
3
Study Design..........................................................................................................................14
3.1
Description of the Study Design .....................................................................................14
3.2
Study Endpoints ..............................................................................................................15
3.2.1
Primary Endpoint...................................................................................................15
3.2.2
Secondary Endpoints .............................................................................................15
4
Study Population....................................................................................................................15
4.1
Recruitment Plan.............................................................................................................15
4.2
Inclusion Criteria.............................................................................................................15
4.3
Exclusion Criteria ...........................................................................................................16
4.4
Justification for Exclusion of Special Populations .........................................................18
5
Study Agent ...........................................................................................................................18
5.1
Disposition, Dispensation, and Accountability...............................................................18
5.2
Formulation, Packaging, and Labeling ...........................................................................18
5.3
Study Agent Storage and Stability..................................................................................18
5.4
Randomization ................................................................................................................19
5.5
Preparation, Administration, and Dosage of Study Agent..............................................19
5.6
Concomitant Medications and Procedures......................................................................19
5.7
Prohibited Medications and Procedures..........................................................................19
5.8
Investigational New Drug Exemption.............................................................................20
6
Study Schedule ......................................................................................................................20
6.1
Screening (day −75 to −1)...............................................................................................20
6.1.1 Management of Patients who are Experiencing a Lupus Flare at the Time of the 
Screening Visit.......................................................................................................................21
6.2
Glucocorticoid Taper ......................................................................................................22
6.3
Infusion Visit (day 0) ......................................................................................................24
6.4
Follow-up Phone Calls....................................................................................................24
7
Study Procedures/Evaluations ...............................................................................................25
7.1
Return of Research Results.............................................................................................25
8
Potential Risks and Benefits ..................................................................................................26
8.1
Potential Risks.................................................................................................................26
8.2
Potential Benefits ............................................................................................................28
9
Research Use of Stored Human Samples, Specimens, and Data...........................................28
10
Data Sharing Plan ..................................................................................................................29


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11
Remuneration Plan for Participants.......................................................................................29
12
Assessment of Safety.............................................................................................................30
12.1
Toxicity Scale..............................................................................................................30
12.2
Causality......................................................................................................................30
12.3
Recording/Documentation...........................................................................................31
12.4
Expected Events ..........................................................................................................31
12.5
Type and Duration of the Follow-up of Participants after Adverse Events ................32
12.6
Halting Rules for the Protocol.....................................................................................32
12.6.1
Reporting a Study Halt ..........................................................................................32
12.6.2
Resumption of a Halted Study...............................................................................32
12.7
Study Discontinuation .................................................................................................32
12.8
Premature Withdrawal of a Participant .......................................................................33
12.9
Replacement of Withdrawn Participants or Participants Who Discontinue Study 
Treatment...................................................................................................................................33
13
Reporting Procedures.............................................................................................................33
13.1
Reporting to the NIH IRB ...........................................................................................33
13.2
Reporting to the NIAMS Clinical Director .................................................................33
14
Clinical Monitoring Structure................................................................................................33
14.1
Quality Management Plan...........................................................................................34
14.2
Safety Monitoring Plan ...............................................................................................34
15
Statistical Considerations.......................................................................................................34
15.1
Definition of the Study Endpoints...............................................................................34
15.2
Bioinformatics Pipeline and Data Handling................................................................35
15.3
Identification of Glucocorticoid-responsive Genes.....................................................35
15.4
Analysis of Flow Cytometry and Cytokine Data ........................................................36
15.5
Summary Statistics for Demographic and Clinical Data.............................................36
15.6
Adaptive Sample Size Determination .........................................................................36
16
Ethics/Protection of Human Subjects ....................................................................................38
16.1
Informed Consent Process...........................................................................................38
16.2
Participant Confidentiality ..........................................................................................38
16.3
Participation of NIH Employees .................................................................................39
17
Data Handling and Record Keeping ......................................................................................39
17.1
Data Capture and Management ...................................................................................39
17.2
Record Retention.........................................................................................................40
18
Scientific References .............................................................................................................40


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List of Abbreviations
AE
Adverse event
BCR
B-cell receptor
BMI
Body mass index
BTRIS
Biomedical Translational Research Information System
CC
Clinical Center
CFR
Code of Federal Regulations
ChIP-seq
Chromatin immunoprecipitation sequencing
CPK
Creatine kinase
CRIMSON
Clinical Research Information Management System of the NIAID
CTCAE
Common Terminology Criteria for Adverse Events
CYP
Cytochrome P450 isozyme
DEXA
Dual energy X-ray absorptiometry
DNA
Deoxyribonucleic acid
dsDNA
Double-stranded DNA
DSMB
Data and safety monitoring board
ECG
Electrocardiogram
ESR
Erythrocyte sedimentation rate
FDA
Food and Drug Administration
FDR
False-discovery rate
FRAX
Fracture risk assessment tool
GR
Glucocorticoid receptor
GRE
Glucocorticoid response element
GRIS
Genomic Research Integration System
HIV
Human immunodeficiency virus
HPA
Hypothalamic-pituitary-adrenal
HRPP
Human Research Protections Program
hsCRP
High-sensitivity C-reactive protein
IGRA
Interferon gamma release assay
IRB
Institutional review board
IV
Intravenous(ly)
LCTU
Lupus Clinical Trials Unit
NIAID
National Institute of Allergy and Infectious Diseases 
NIAMS
National Institute of Arthritis and Musculoskeletal Diseases
NIH
National Institutes of Health
PBMC
Peripheral blood mononuclear cell
PCR
Polymerase chain reaction
PT/PTT
Prothrombin/partial thromboplastin time
RNA
Ribonucleic acid
RNA-seq
RNA sequencing
SAE
Serious adverse event
SELENA/SLEDAI
Safety of Estrogens in Lupus Erythematosus National Assessment SLE Disease 
Activity Index
SLE
Systemic lupus erythematosus
TLR
Toll-like receptor
UP
Unanticipated problem
USP
United States Pharmacopeia


Glucocorticoid Genomics in SLE
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Protocol Summary
Short Title:
Glucocorticoid Genomics in SLE
Sample Size:
N = 40 (20 participants per dose group). The final sample size will 
be determined by an adaptive study design after the initial 
recruitment of 20 participants per group.
Accrual Ceiling:
N = 100
Study Population:
Individuals aged 18 to 80 years with systemic lupus erythematous 
(SLE).
Accrual Period:
48 months
Study Design:
This is a study of the acute effects of glucocorticoids on the 
immune system of patients with SLE. Participants will undergo 
baseline blood collection prior to receiving a single intravenous 
(IV) dose of methylprednisolone sodium succinate. Participants 
will be randomized into 1 of 2 dose groups: 1 mg/kg or 250 mg. 
Blood will be collected again at 2 and 4 hours after the 
methylprednisolone infusion. Individual participation requires 
2 visits to the NIH CC and 2 follow-up phone calls. Total length of 
individual subject participation including screening is 1-12 weeks.
Blood samples will be processed for isolation of hematopoietic cell 
sub-populations (eg, neutrophils, B cells, plasmacytoid dendritic 
cells, CD4+ T cells, CD8+ T cells, monocytes, and natural killer 
cells). Laboratory studies will be performed on the purified cells, 
with the goal of understanding the human response to 
glucocorticoids in vivo at the level of circulating cell populations 
(eg, flow cytometry, mass cytometry), RNA (eg, RNA sequencing 
[RNA-seq], small-RNA-seq, real-time polymerase chain reaction 
[PCR]), DNA (eg, chromatin immunoprecipitation sequencing 
[ChIP-seq], methylation analysis, DNA sequencing, genotyping), 
and protein (eg, flow cytometry, mass spectrometry). At each time 
point, serum methylprednisolone levels will be measured. 
Study Agent/
Intervention Description:
A single IV infusion of methylprednisolone sodium succinate at 
either 1 mg/kg or 250 mg.
Primary Objective:
To understand the cellular and molecular response to 
glucocorticoids in individuals with SLE.


Glucocorticoid Genomics in SLE
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Secondary Objectives:
1. To identify candidate targets for therapeutic interventions that 
could mimic the action of glucocorticoids in patients with SLE, 
without the significant toxicity caused by the broad range of 
glucocorticoid actions.
2. To test whether the transcriptional response to glucocorticoids 
differs between the two doses being studied.
3. To identify similarities and differences, at baseline and in 
response to glucocorticoids, between the transcriptome of cells 
from patients and those from the previously studied healthy 
subjects.
Primary Endpoint:
A list of human protein-coding genes and non-coding RNAs that 
are differentially expressed in response to glucocorticoids in 
patients with SLE, for each of the studied cell types and doses.
Secondary Endpoints:
1. A comparison of the transcriptional response to glucocorticoids 
between the two dose groups.
2. 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.
3. Validation of the targets identified by functional studies.
4. For each cell type, a list of protein-coding or non-coding 
transcripts that are shared and a list of transcripts that are different, 
between patients with SLE and the previously studied healthy 
controls, at baseline or in response to glucocorticoids.


Glucocorticoid Genomics in SLE
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Précis
Systemic lupus erythematosus (SLE) is a systemic autoimmune syndrome that causes pervasive 
immune dysregulation in all cells of the innate and adaptive immune system. Most patients with 
SLE require glucocorticoid treatment at some point of the disease course, but the specific effects 
of glucocorticoids that lead to therapeutic benefit in this condition are not well understood. In 
addition, the glucocorticoid dosing regimens that are administered to patients with SLE in 
clinical practice are arbitrary and not based on detailed knowledge of the effects of 
glucocorticoids at different doses on cells of the immune system. This study aims to improve our 
understanding of the effects of glucocorticoids on the immune system of patients with SLE, and 
to test for possible differences in these effects at a range of clinically relevant doses.
A consistent outcome of glucocorticoid exposure is a significant reprogramming of a cell’s 
transcriptional state. The exact changes, however, vary substantially across cell types. Studies of 
specific cell populations, therefore, are necessary to gain a realistic view of the transcriptional 
effects of glucocorticoids. Given that cell composition and molecular behavior are known to 
differ among pathological states, the study of patients with SLE is necessary to identify the 
effects of glucocorticoids that are most likely to be responsible for their clinically beneficial 
effects in this condition.
In this study, participants with SLE, aged 18-80 years, will be randomized to receive a single 
intravenous infusion of 1 of 2 doses of the glucocorticoid methylprednisolone (1 mg/kg or 
250 mg). Blood will be collected within 75 days before infusion, immediately before the 
infusion, and again at 2 and 4 hours after the start of the infusion. Blood samples will be 
processed for isolation of hematopoietic cell subsets, including neutrophils, B cells, 
CD4+ T cells, CD8+ T cells, monocytes, natural killer cells, and plasmacytoid dendritic cells. 
Laboratory studies will be performed on the purified cells, with the goal of understanding the 
human response to glucocorticoids in vivo at the level of RNA (eg, RNA sequencing [RNA-seq], 
small-RNA-seq, real-time polymerase chain reaction), DNA (eg, chromatin immunoprecipitation 
sequencing, methylation analysis, DNA sequencing, genotyping), and protein (eg, flow 
cytometry, mass spectrometry). At each time point, serum methylprednisolone levels will be 
measured and flow cytometry for standard cell-lineage markers will be performed.


Glucocorticoid Genomics in SLE
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1
Background Information and Scientific Rationale
1.1
Scientific Background and Preliminary Data
The goal of this study is to improve our understanding of the effects of glucocorticoids on the 
immune system of people with SLE.
SLE is a systemic autoimmune syndrome of unclear etiology that primarily affects young 
women. It is characterized by substantial clinical heterogeneity and unpredictable responses to 
various forms of immunosuppressive treatment. Immune dysregulation is pervasive in SLE and 
affects all cells of the innate and adaptive immune system. Most patients with SLE require 
glucocorticoid use at some point of the disease course, but the specific effects of glucocorticoids 
that lead to therapeutic benefit in this condition are unclear. 
Glucocorticoids were first introduced to clinical practice in the late 1940s.1 They remain 
extensively used today due to their rapid and potent immunosuppressive and anti-inflammatory 
effects. However, even short courses of glucocorticoid therapy are associated with severe, 
dose-limiting side effects that can affect every organ system. Despite nearly seven decades of 
intense clinical use, it has been pointed out recently that there are still important gaps in our 
understanding of glucocorticoid-induced immunoregulation.2 Two of the most salient knowledge 
gaps involve disease-specific effects and cell type–dependent effects. This study aims to directly 
address these two gaps, by analyzing the effects of in vivo glucocorticoid administration on 
specific cell types in individuals with SLE.
The glucocorticoid dosing regimens that are administered to patients with SLE in current clinical 
practice are arbitrary and not based on detailed knowledge of the effects of glucocorticoids at 
different doses on cells of the immune system. This unfortunately means that there may be 
unnecessary harm in current clinical practice. While repeated exposure to very high doses of 
glucocorticoids can lead to debilitating side effects, the extent to which such doses have effects 
on cells of the immune system that differ substantially from those achieved with lower doses 
remains unclear. Because the level of disease activity in patients with SLE tends to fluctuate over 
time, and higher doses are generally used to treat patients with higher disease activity, this 
patient population is particularly susceptible to the potential harm of arbitrary glucocorticoid 
dosing regimens. Therefore, a secondary aim of this study is to analyze the transcriptional 
response to in vivo administration of glucocorticoids at a range of clinically used doses. 
Glucocorticoids act primarily by binding in the cytosol to the glucocorticoid receptor (GR), a 
nuclear receptor of the steroid/thyroid hormone receptor superfamily.3 The ligand-bound GR 
translocates into the nucleus and can directly bind DNA at specific recognition sequences known 
as glucocorticoid response elements (GREs), increasing or decreasing transcription rates 
depending on the type of GRE.4,5 In addition, ligand-bound GR can be recruited to specific 
genomic sites without directly binding DNA, via protein-protein interactions with other 


Glucocorticoid Genomics in SLE
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DNA-bound transcription factors.6 Genomic sites of direct GR binding represent 
glucocorticoid-induced enhancers, and genomic sites of indirect (tethered) GR binding appear to 
cluster around and amplify the activity of direct binding sites.7 Composite sites of direct and 
tethered interactions with DNA have also been described.6 Beyond the direct or tethered 
recruitment of ligand-bound GR to specific genomic sites, a key component of the mechanism of 
action of glucocorticoids involves interference with the activity of other transcription factors and 
signaling molecules, most notably NF-κB. This form of interference can be mediated by direct 
protein-protein interactions between the ligand-bound GR and other transcription factors,8 but 
also by indirect effects via inhibitory long non-coding RNAs,9 proteins that dissociate from the 
GR-chaperone complex upon glucocorticoid binding,10 or competition for nuclear co-activators. 
Finally, some of the most rapid effects of glucocorticoids may occur independently of the 
cytosolic GR. These include alterations in ion transport across membranes,11,12 which have been 
hypothesized to result from intercalation of glucocorticoid molecules into the membrane.13 They 
also include interactions with membrane-bound forms of GR.14-16
While the mechanisms are diverse, a consistent outcome of glucocorticoid exposure is a 
significant reprogramming of a cell’s transcriptional state.17,18 The genomic locations of GR 
binding have been shown to vary widely across cell types,19-21 a phenomenon that is explained at 
least in part by differences in chromatin accessibility and expression differences of GR 
cofactors.19,22,23 This, in turn, suggests that the transcriptional response to glucocorticoids could 
vary significantly across cell types. In this context, studies of specific cell populations in the 
species of interest are necessary to gain a realistic view of the genomic effects of glucocorticoids 
in any system. Immortalized and tumor-derived cell lines have been valuable tools for the study 
of the molecular biology of GR signaling. However, their genomic composition and chromatin 
landscape are known to differ substantially from those of human primary cells. Similarly, 
complex cell mixtures, such as whole blood and peripheral blood mononuclear cells (PBMCs), 
have offered an initial glimpse of the genes and pathways affected by a glucocorticoid stimulus 
in primary human cells,17,18 but they are limited in their ability to discern cell-specific effects. 
To develop a greater understanding of how pharmacologic doses of glucocorticoids regulate 
immunity and the extent to which they differentially affect distinct cell subsets, we have studied 
the genome-wide transcriptional response to glucocorticoids in 9 primary human hematopoietic 
and non-hematopoietic cell types from healthy human volunteers.24 For each cell type, we 
performed total RNA-seq before, 2 hours after, and 6 hours after in vitro stimulation with 
methylprednisolone or vehicle. We found that the number of genes that responded to the 
glucocorticoid stimulus differed substantially among cell types. In addition, the specific 
transcripts that responded to the glucocorticoid stimulus were very different in each cell type. Of 
the 10,665 glucocorticoid-responsive transcripts identified, 5184 (48.6%) were significantly 
differentially expressed after glucocorticoid treatment in only 1 of the 9 cell types, and only 
23 (0.2%) were significantly differentially expressed after treatment in all cell types. In principle, 
the observed differences in the transcriptional response to glucocorticoids among cell types could 


Glucocorticoid Genomics in SLE
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simply result from differences in the baseline transcriptome of each cell type. However, our 
results suggest that both the magnitude and the direction of transcriptional regulation by 
glucocorticoids can differ across cell types, even for genes that are similarly expressed at 
baseline. This suggests that classifying genes as glucocorticoid-induced or 
glucocorticoid-repressed, as is common in the literature, should only be done in the context of a 
specific cell type. This also suggests that baseline expression, chromatin accessibility, and 
glucocorticoid receptor binding can only partially explain how a gene responds to a 
glucocorticoid stimulus. 
An important advantage of genome-wide approaches is that they allow an unbiased view of 
biological processes, which can reveal previously unrecognized effects. Integrating the 
transcriptome datasets from the 9 primary human cell types exposed in vitro to glucocorticoids, 
we generated a pathway-level map of the effects of glucocorticoids.24 We observed substantial 
differences in the way that glucocorticoids affect specific molecular pathways in individual cell 
types, and in hematopoietic versus non-hematopoietic cells. 
This pathway-level map of glucocorticoid actions has served as a tool for generating hypotheses 
about glucocorticoid actions in individual cell types, which can be tested experimentally. We 
have demonstrated the utility of this approach in uncovering functional effects of glucocorticoids 
by focusing on B cells, which play a key role in antibody-mediated autoimmune diseases. 
Cross-talk between the B-cell receptor (BCR) and toll-like receptor (TLR) signaling pathways 
plays a central role in the integration of functional B-cell responses25 and in the development of 
autoreactive B cells.26 Our pathway-level map suggested that pharmacologic doses of 
glucocorticoids had strong transcriptional effects on key genes in both pathways. Based on this, 
we hypothesized that glucocorticoids could functionally impair BCR and intracellular TLR 
signaling in human B cells.
To determine whether the in vitro findings on BCR signaling genes were consistent with 
transcriptional changes in B cells exposed to glucocorticoids in vivo, we measured the 
expression of key BCR signaling genes over time in a cohort of 20 unrelated healthy volunteers 
who received a single IV dose of methylprednisolone as part of our clinical protocol, 16-I-0126, 
“Genomic Responses of Human Immune and Non-Immune Cells to Glucocorticoids” (principal 
investigator: Franco). Consistent with the in vitro data, we observed a significant drop in the 
expression of CD79B, which encodes Igβ, a protein required for BCR assembly and for signal 
initiation after antigen stimulation.27,28 In contrast, expression of CD79A, which encodes Igα, 
was not significantly affected. The gene CR2, which encodes a key component of the B-cell 
co-receptor complex that enhances BCR-mediated signaling,29 was also significantly reduced in 
expression in response to glucocorticoid.
We also observed decreased expression of BTK, which encodes a key tyrosine kinase 
immediately downstream of the BCR complex, and of BLNK, which encodes a B-cell adaptor 


Glucocorticoid Genomics in SLE
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protein central to BCR signaling. Interestingly, the gene that encodes the key upstream kinase 
LYN was significantly reduced in expression after glucocorticoid administration in vivo, 
although it had not reached statistical significance in vitro, and the opposite was true of the 
upstream kinase SYK, which emphasizes the importance of studying these phenomena in vivo 
and in multiple unrelated individuals. 
Consistent with our observation of a glucocorticoid-induced drop in transcriptional output from 
the immunoglobulin and CD79B loci, in vitro exposure of human B cells to glucocorticoid led to 
a significant drop in surface expression of the BCR when compared to vehicle-treated cells. We 
found, however, that within 4 hours of glucocorticoid exposure, human B cells still had sufficient 
levels of BCR on the cell surface to allow stimulation with anti-IgM antibodies, with the goal of 
functionally assessing signaling downstream of the BCR. We then measured phosphorylation of 
CD79A (an upstream signaling component) and of PLCγ2 (a downstream signaling component) 
in response to BCR stimulation with anti-IgM, in the presence of glucocorticoid or vehicle. We 
measured signaling by phospho-flow cytometry to simultaneously assess the response of total, 
naïve, and unswitched memory B cells. We found that glucocorticoids impair IgM-BCR 
signaling. Interestingly, for both readouts, the observed reduction in signaling in total B cells 
appeared to be primarily driven by a stronger and significant effect on unswitched memory 
cells.24
With respect to TLR signaling, our in vitro work revealed a significant glucocorticoid-induced 
reduction in expression of the gene encoding the intracellular receptor TLR7 in human B cells. 
TLR7 is involved in RNA-associated antigen recognition and autoantibody production and is 
believed to play a role in the pathogenesis of human autoimmune diseases.26 Our clinical data 
confirmed a significant drop in TLR7 expression by human B cells after in vivo glucocorticoid 
administration. To test whether this transcriptional change had a functional effect, we then 
purified circulating human B cells, stimulated them with the TLR7-specific ligand imiquimod, 
and measured phosphorylation of p38 MAP kinase as a functional readout of TLR7 activation. 
As a negative control, we also stimulated the cells with the TLR8-specific ligand motolimod. 
TLR8 signals similarly to TLR7, but its expression is not affected by glucocorticoids. At 
saturating or higher ligand concentrations, the proportion of cells that responded to the TLR7 
stimulus, and the intensity of the phospho-p38 MAPK signal, were both significantly lower in 
glucocorticoid-treated than in vehicle-treated cells. In contrast, glucocorticoids did not 
significantly alter the response to the TLR8 stimulus. To extend these findings, we measured 
expression of CCL3 and CCL4, two genes that are responsive to TLR7 and TLR8 stimulation but 
whose expression is unaffected by glucocorticoid treatment of B cells. Consistent with our prior 
observations, glucocorticoids significantly decreased the transcriptional response of CCL3 and 
CCL4 after TLR7 stimulation, whereas no significant difference was observed after TLR8 
stimulation.


Glucocorticoid Genomics in SLE
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1.2
Scientific Rationale
In summary, our in vitro and in vivo work to date suggests that:
a.
A genomics approach can serve as an entry point for the unbiased study of glucocorticoid 
responses in primary human cells and it can uncover specific mechanisms of immune 
regulation that can be further characterized by functional studies.
b. The response to glucocorticoids is highly cell type–dependent. Therefore, information at 
the level of individual cell types and the study of patients with specific diseases will be 
necessary to identify the effects of glucocorticoids that are most likely to be responsible 
for their clinically beneficial effects.
These observations are the basis for the present study. We will analyze the genomic response to 
in vivo glucocorticoid administration in patients with SLE by serial sampling of individual 
immune cell populations before and after glucocorticoid administration (Figure 1). This will 
allow us to study, in a clinically realistic setting, the effects of glucocorticoids at different doses 
on the immune system of patients with SLE; the level of interindividual variation in 
glucocorticoid responses at the molecular level; and the differences at baseline and in response to 
glucocorticoids between cells from patients and those from the previously studied healthy 
subjects.
Figure 1. Study design. Patients with SLE will be enrolled. Each participant will be randomized to one of 2 dose 
groups and will receive a single IV dose of the glucocorticoid (GC) methylprednisolone. Blood will be sampled 
serially for purification of hematopoietic cell subsets. Total RNA will be purified from each cell subset and total 
RNA-seq and small RNA-seq will be performed. The transcriptome-level changes in response to GC for each cell 
type will serve as the basis for the identification of the key molecular pathways and cellular functions that are 
affected by GC in each cell type. Important observations will be followed up by functional analyses.
Glucocorticoids remain a mainstay of therapy in a broad range of conditions, despite their 
well-recognized adverse effects and a large and rapidly growing number of other drugs with 


Glucocorticoid Genomics in SLE
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immunotherapeutic potential. Identifying the drug targets that, alone or in combination, could 
best mimic the immunosuppressive effects of glucocorticoids on relevant cell types while 
limiting off-target actions will be facilitated by greater insight into the mechanisms underlying 
desirable versus untoward actions of glucocorticoids in distinct human cell types and in relevant 
disease states. We believe this work will be an important step towards achieving that goal.
1.3
Choice of a Glucocorticoid, Route of Administration, and Dose
Chemical modifications to the 21-carbon corticosteroid backbone (eg, introduction of a 
carbon 1-2 double bond, methylation at carbon 6, and 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 
and on the availability of an IV formulation (to reduce potential interindividual variation in rates 
of absorption). 
Methylprednisolone is widely used in clinical practice, with indications in the management of 
diverse conditions including lupus nephritis,30 dermatomyositis,31 acute asthma exacerbations,32 
and relapses of multiple sclerosis.33 IV methylprednisolone has also been used in multiple studies 
of healthy volunteers since the early 1970s, including our own,24,34-36 without reports of serious 
adverse events (SAEs) in those studies. Sodium succinate and phosphate esters are available for 
IV use. The pharmacokinetics of IV methylprednisolone are also dose-linear,35,36 which 
facilitates correlation between the concentrations studied in vitro and the equivalent doses 
in vivo. Therefore, we have identified methylprednisolone sodium succinate as the 
glucocorticoid of choice for our study. 
An important clinical question with respect to glucocorticoid use is that of the minimum dose 
that can effectively suppress the immune system. The dosing regimens for glucocorticoids that 
are commonly used in clinical medicine are mostly arbitrary and based on historical precedent 
rather than on an objective assessment of their effects on the immune system.3,13 While the 
glucocorticoid receptor is nearly saturated at prednisone-equivalent doses in the high-dose range 
(30-100 mg/day),3 much higher doses are often used in the clinic, despite limited objective 
evidence that they are clinically better than a lower dose. To determine the extent to which the 
transcriptional response to a single dose of glucocorticoid varies by dose, we will randomize 
study participants into 1 of 2 dose groups: 1 mg/kg or 250 mg.3 The 1-mg/kg dose was selected 
because it is commonly used as a starting dose in multiple clinical settings, including in patients 
with moderate SLE disease activity. The 250-mg dose was selected because that was the dose 
used in our clinical study of healthy volunteers; administering the same dose to patients with 


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SLE will facilitate the use of data from the healthy volunteer study in the planned comparison of 
the genomic response to glucocorticoids between patients with SLE and unaffected controls. 
2
Study Objectives
2.1
Primary Objective
To understand the cellular and molecular response to glucocorticoids in individuals with SLE.
2.2
Secondary Objectives
1. To identify candidate targets for therapeutic interventions that could mimic the action of 
glucocorticoids in patients with SLE, without the significant toxicity caused by the broad 
range of glucocorticoid actions.
2. To test whether the transcriptional response to glucocorticoids differs between the two 
doses being studied.
3. To identify similarities and differences, at baseline and in response to glucocorticoids, 
between the transcriptome of cells from patients and those from the previously studied 
healthy subjects.
3
Study Design
3.1
Description of the Study Design
Potential participants will present to the NIH CC for a screening visit. Objective documentation 
of the diagnosis of SLE, the specific clinical manifestations, and the level of disease activity will 
be established. A baseline whole blood sample will be obtained at screening. Each enrolled 
participant will be randomly assigned to 1 of 2 methylprednisolone dose groups: 1 mg/kg or 
250 mg. 
All enrolled participants will return for a day-long study visit (“infusion visit”) within 75 days of 
the screening visit. Following collection of a pre-infusion whole blood sample, participants will 
receive a single IV dose of methylprednisolone (based on the dose group assignment), infused 
over 30 minutes. Whole blood samples will then be obtained serially, 2 and 4 hours after the start 
of infusion.
Participants will be discharged after collection of the 4-hour blood sample, measurement of vital 
signs, and documentation of any adverse events (AEs). Two follow-up phone calls will be made 
to document any AEs related to the methylprednisolone. If any AEs that may require treatment 
are documented on the follow-up phone calls, then participants will be asked to return to the NIH 
CC’s outpatient clinic for evaluation by the principal investigator.


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We propose an adaptive study design, in which we will recruit an initial cohort of 20 participants 
into each of the 2 dosing groups. After processing the samples from these participants, we will 
perform differential expression analysis to determine if recruitment of additional participants (up 
to the accrual ceiling of 100) would affect the biological interpretation of the data. If additional 
data would be unlikely to change the study outcome, then no further recruitment will be pursued.
3.2
Study Endpoints
3.2.1
Primary Endpoint
A list of human protein-coding genes and non-coding RNAs that are differentially expressed in 
response to glucocorticoids in patients with SLE, for each of the studied cell types and doses.
3.2.2
Secondary Endpoints
1. A comparison of the transcriptional response to glucocorticoids between the 2 dose 
groups.
2.  A list of protein-coding or non-coding transcripts, their corresponding proteins, and the 
molecular pathways representing the best candidates for targeted therapeutic alternatives 
to glucocorticoids.
3. Validation of the targets identified by functional studies.
4. For each cell type, a list of protein-coding or non-coding transcripts that are shared and a 
list of transcripts that are different, between patients with SLE and the previously studied 
healthy controls, at baseline or in response to glucocorticoids.
4
Study Population
4.1
Recruitment Plan
Participants will primarily be recruited from NIH study 94-AR-0066, “Studies of the 
Pathogenesis and Natural History of Systemic Lupus Erythematosus” (principal investigator: 
Hasni). Because there are NIH employees enrolled in 94-AR-0066, employees may also enroll in 
this study.
Participants may also self-refer, be referred by outside physicians, or be referred by other 
investigators at the NIH CC. Any individuals who self-refer must first be enrolled onto study 
94-AR-0066 to be eligible for this study.
4.2
Inclusion Criteria
Individuals must meet all of the following criteria to be eligible for study participation:
1. Aged 18-80 years.


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2. Has a diagnosis of SLE based on the 1997 Update of the 1982 American College of 
Rheumatology Revised Criteria for Classification of SLE. 
3. Currently enrolled in study 94-AR-0066.
4. Able to provide informed consent.
5. Willing to allow genetic testing.
6. Has a primary care physician or other healthcare provider who will manage all health 
conditions related or unrelated to the study objectives.
7. If receiving immunosuppressive therapies other than glucocorticoids for SLE, then on a 
stable dose (defined as no increases in dose for the 60 days prior to screening). 
8.
A. If receiving glucocorticoid therapy and not experiencing a lupus flare at the 
time of the screening visit, then potential participants must be on a daily 
prednisone or prednisone-equivalent dose ≤ 10 mg/day and agree to undergo a 
glucocorticoid taper (sections 6.1 and 6.2).
OR
B. If receiving glucocorticoid therapy experiencing a lupus flare at the time of 
the screening visit, then potential participants must be on a daily prednisone or 
prednisone-equivalent dose ≤ 15mg/day. A glucocorticoid taper will not be 
performed.
4.3
Exclusion Criteria
Individuals meeting any of the following criteria will be excluded from study participation:
1. Body mass index (BMI) < 18 or > 40.
2. History of a severe allergic reaction to glucocorticoids.
3. History of a severe adverse cardiovascular or psychiatric event related to glucocorticoid 
administration.
4. Use of a glucocorticoid at a prednisone-equivalent dose ˃ 10 mg/day in the 15 days prior 
to screening (> 15 mg/day if experiencing a flare on the day of the screening visit).
5. A previously established diagnosis of an active solid or hematologic malignancy.
6. A previously established diagnosis of untreated osteoporosis. For the purpose of this 
study, osteoporosis is defined as a dual-energy X-ray absorptiometry (DEXA) scan 
obtained within 2 years of screening demonstrating a hip or spine bone mineral density 
T score ≤ −2.5 in men  ≥ 50 years of age or in postmenopausal women.
7. A previously established diagnosis of untreated osteopenia with a high fracture risk. For 
the purpose of this study, osteopenia is defined as a DEXA scan obtained within 2 years 
of screening demonstrating a hip or spine bone mineral density T score < −1 and > −2.5 
in men  ≥ 50 years of age or in postmenopausal women. For the purpose of this study, a 


Glucocorticoid Genomics in SLE
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high fracture risk is defined as a fracture risk assessment tool (FRAX) 10-year risk of 
major osteoporotic fracture > 20%, or a FRAX 10-year risk of hip fracture > 3%.
8. A previously established diagnosis of diabetes mellitus or a fasting blood glucose level ≥ 
125 mg/dL at the time of screening. For patients without a previously established 
diagnosis of diabetes mellitus and a fasting blood glucose level < 125 mg/dL at the time 
of screening, no additional screening tests (e.g., HbA1c or oral glucose tolerance test) 
will be performed. A history of glucocorticoid-induced hyperglycemia that is not present 
at the time of screening in the absence of treatment will not be considered an exclusion 
criterion.
9. Cancer chemotherapy within the 5 years prior to screening.
10. Surgery requiring general anesthesia in the 8 weeks prior to screening.
11. History of an infection requiring IV antibiotics in the 30 days prior to screening.
12. A positive test for HIV or hepatitis A, B, or C virus infection.
13. A positive or indeterminate test for active or latent tuberculosis (interferon gamma 
release assay [IGRA]) without evidence of prior treatment.
14. History of chronic or possible latent untreated parasitic, amebic, fungal, or mycobacterial 
infections.
15. Use of desmopressin in the 30 days prior to screening.
16. Use of one of the following cytochrome P450 isozyme (CYP) 3A4 inducers in the 
30 days prior to screening: nafcillin, rifabutin, rifampin, St. John’s wort, or troglitazone.
17. Use of one of the following CYP3A4 inhibitors in the 30 days prior to screening: 
clarithromycin, itraconazole, or ketoconazole.
18. Use of belimumab or rituximab within the past 180 days.
19. Vaccination within the past 30 days.
20. Pregnancy, current or in the 90 days prior to screening.
21. Currently breastfeeding.
22. Any electrocardiogram (ECG) abnormality that is clinically significant. 
23. Any condition that, in the opinion of the investigator, contraindicates participation in this 
study.
Laboratory evaluations that will be used to establish eligibility are listed in sections 6 and 7, 
below. For exclusion criteria that involve previously established diagnoses, such as active 
malignancy, osteoporosis, or diabetes mellitus, any previously obtained diagnostic studies will be 
considered, but diagnostic testing for the sole purpose of establishing eligibility will not be 
performed.
Co-enrollment guidelines: Co-enrollment in other studies is permitted. Study staff should be 
notified of co-enrollment on any other protocol to ensure the schedules are compatible and that 
the volume of blood drawn for research is within NIH CC limits.


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4.4
Justification for Exclusion of Special Populations
Pregnancy is an exclusion criterion because there are no adequate and well-controlled studies of 
glucocorticoids in pregnant people. Glucocorticoids should be used during pregnancy only if the 
potential benefit justifies the potential risk to the fetus. Additionally, pregnancy itself may alter 
the immune system, and thus may affect study results. Breastfeeding is an exclusion criterion 
because of the potential for serious adverse reactions in nursing infants from glucocorticoids. 
People younger than 18 years are excluded from this study because there is the potential for 
greater than minimal risk and no prospect for direct benefit. 
5
Study Agent
5.1
Disposition, Dispensation, and Accountability
The study agent, methylprednisolone sodium succinate, will be distributed and accounted for by 
the NIH pharmacy according to standard pharmacy procedures.
5.2
Formulation, Packaging, and Labeling
Methylprednisolone sodium succinate for injection, United States Pharmacopeia grade (USP), 
sold as SOLU-MEDROL sterile powder by Pfizer, Inc, is an anti-inflammatory glucocorticoid 
that occurs as a white, or nearly white, odorless hygroscopic, amorphous solid.37 It is very 
soluble in water and in alcohol but 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.
The study agent is packaged in the preservative-free Act-O-Vial System, which is a single-use 
vial. 
Each vial will be individually labeled with the patient identification number, dosing instructions, 
recommended storage conditions, the name and address of the manufacturer, and a statement that 
the agent should be kept out of reach of children.
5.3
Study Agent Storage and Stability
The study agent must be protected from light. Unreconstituted product and solution are stored at 
a controlled room temperature of 20°C to 25°C (68°F to 77°F). Solution must be used within 
48 hours after mixing.


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5.4
Randomization
Study participants will be randomized evenly to 2 dose groups. The purpose of randomization in 
this setting is to produce balanced dose groups where participants are comparable between 
groups in terms of possible confounding factors such as age, sex, or concomitant medication use. 
To further ensure that participants are similar in terms of SLE severity between the groups, we 
will adopt stratified randomization according to their disease activity using the Safety of 
Estrogens in Lupus Erythematosus National Assessment SLE Disease Activity Index 
(SELENA/SLEDAI) score on the day of enrollment. Participants will be classified into 1 of 
3 disease-severity categories: low (SELENA/SLEDAI score = 0-4), intermediate 
(SELENA/SLEDAI score = 5-8), or high (SELENA/SLEDAI score > 9).  Randomization will be 
carried out within each category. This stratified randomization method ensures the proportion of 
participants with high disease activity is the same between the dose groups.
The study statistician will design the randomization scheme. The randomization code list will be 
maintained by the study statistician and the study coordinator.  
5.5
Preparation, Administration, and Dosage of Study Agent
Methylprednisolone will be prepared according to the package insert and depending on the dose 
group to which each participant has been assigned.37 The study agent solution will be 
administered as an IV infusion over 30 minutes, for a total single dose of 1 mg/kg or 250 mg of 
methylprednisolone. Pulse oximetry will be followed during the infusion for safety monitoring.
5.6
Concomitant Medications and Procedures
All concomitant prescription and nonprescription (including over-the-counter) medications taken 
during study participation will be recorded. For this protocol, a prescription medication is 
defined as a medication that can be prescribed only by a properly authorized/licensed clinician.
5.7
Prohibited Medications and Procedures
Treatment with the following drugs/medications will not be permitted during participation in this 
study:

Desmopressin.

CYP3A4 inducers nafcillin, rifabutin, rifampin, St. John’s wort, or troglitazone.

CYP3A4 inhibitors clarithromycin, itraconazole, or ketoconazole.

Belimumab or rituximab.

Receipt of any vaccine.


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5.8
Investigational New Drug Exemption
Use of methylprednisolone sodium succinate in this study is exempt from an investigational new 
drug application to the Food and Drug Administration (FDA) because its use meets the following 
criteria, per Title 21 of the United States Code of Federal Regulations (CFR) Part 312.2(b)38:
1. The drug product is lawfully marketed in the United States.
2. The investigation is not intended to be reported to FDA as a well-controlled study in 
support of a new indication and there is no intent to use it to support any other significant 
change in the labeling of the drug.
3. In the case of a prescription drug, the investigation is not intended to support a significant 
change in the advertising for the drug.
4. The investigation does not involve a route of administration, dose, patient population, or 
other factor that significantly increases the risk (or decreases the acceptability of the risk) 
associated with the use of the drug product.
5. The investigation is conducted in compliance with the requirements for review by an IRB 
(21 CFR 56) and with the requirements for informed consent (21 CFR 50).
6. The investigation is conducted in compliance with the requirements of 21 CFR 312.7 
(ie, the investigation is not intended to promote or commercialize the drug product).
6
Study Schedule
This study will involve 2 study visits at the NIH CC (screening and infusion visits) and 
2 follow-up phone calls. Only those participants found to be eligible after the screening 
procedures will return to continue the study. Participants will be referred to their primary care 
physician or other healthcare provider if any test results need further follow-up.
6.1
Screening (day −75 to −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.

SELENA/SLEDAI score.

Urine collection for the following:
o
Urinalysis with microscopic examination.
o
Spot urine protein, creatinine, and protein-to-creatinine ratio.

Blood draw for the following:
o
Complete blood count with differential.
o
Acute care panel.
o
Calcium and magnesium levels.


Glucocorticoid Genomics in SLE
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o
Random morning serum cortisol level.
o
Liver function tests (hepatic panel).
o
Complement levels: C3 and C4.
o
Anti–double-stranded DNA (dsDNA) antibodies.
o
Erythrocyte sedimentation rate (ESR).
o
High-sensitivity C-reactive protein (hsCRP).
o
Lipid panel.
o
Serum creatine kinase (CPK).
o
Serum pregnancy test (for participants capable of becoming pregnant).
o
Prothrombin/partial thromboplastin time (PT/PTT).
o
HIV and hepatitis A, B and C (viral markers screen test, which includes HBsAg, 
anti-HCV IgG, anti-HAV IgM). Test results obtained within the past 30 days will be 
accepted.
o
Interferon gamma release assay.
o
Baseline purification of hematopoietic cell sub-types.
o
Baseline flow cytometry.
o
Banking of baseline plasma, peripheral blood mononuclear cells, and DNA.
With the exception of the serum pregnancy test, test results obtained within 30 days of the 
screening visit under protocol 94-AR-0066 will be accepted for screening.
After eligibility is confirmed, participants who are receiving glucocorticoid therapy may be 
directed to undergo a taper before the infusion visit, as described in section 6.2, below.
6.1.1
Management of Patients who are Experiencing a Lupus Flare at the Time of the 
Screening Visit
Patients who are eligible for enrollment based on the study’s inclusion and exclusion criteria, but 
who are experiencing a lupus flare at the time of their screening visit, will be considered for 
enrollment only when the following two criteria are met:
a.
The infusion visit can be scheduled for a date that is within 7 days of enrollment, and 
b. The severity of the flare does not require administration of glucocorticoids prior to 
the anticipated date of the infusion visit.
The above criteria will apply irrespective of whether the patients are on glucocorticoid therapy at 
the time of their screening visit, but patients who are experiencing a lupus flare while on 
glucocorticoid therapy will not be deemed eligible for a glucocorticoid taper. For patients on 
glucocorticoid therapy who are experiencing a lupus flare at the time of their screening visit, the 
maximum prednisone or prednisone-equivalent dose will be ≤ 15 mg/day. Patients on higher 
doses of chronic glucocorticoid therapy who are experiencing a flare are deemed likely to require 
an infusion earlier than 7 days after evaluation and will thus be excluded.


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If a patient who is experiencing a lupus flare on the day of the screening visit is enrolled, but the 
infusion visit is later delayed beyond 7 days after enrollment or the severity of the flare increases 
to the point where glucocorticoids must be administered before the infusion visit can be 
scheduled, glucocorticoid treatment will be administered as clinically indicated and the patient 
will be withdrawn from the study.
6.2
Glucocorticoid Taper
Given the known toxicity of glucocorticoid therapy, it is standard clinical practice to attempt to 
reduce the lifetime exposure of patients with SLE to glucocorticoids, by periodically tapering 
their glucocorticoid to the minimum effective dose. An ideal tapering regimen, or a reliable way 
to identify patients on chronic glucocorticoid therapy in whom the dose can be tapered, have not 
been defined. Similarly, the biological changes that take place upon glucocorticoid tapering, or 
the glucocorticoid-responsive genes that help maintain patients in clinical remission, are very 
poorly understood. In this protocol, we will enroll patients who are in clinical remission and 
receiving chronic glucocorticoid treatment. In those patients, a glucocorticoid taper will be 
performed. These are patients who would be good clinical candidates for tapering their 
glucocorticoid dose under standard clinical practice. In the setting of this study, the rationale for 
tapering is two-fold:
i. Patients in whom the dose of glucocorticoid can be tapered to zero can be 
studied in the same context as those who were not receiving glucocorticoids 
prior to the study.
ii. Patients in whom the dose of glucocorticoid is tapered, but their disease 
activity increases during the taper, can provide very valuable information. 
Identifying the genes and molecular pathways that differ between a chronic 
stable dose, a disease flare at a lower dose, and the subsequent response to a 
glucocorticoid infusion, can help identify the genes that glucocorticoids 
regulate chronically to sustain clinical remission.
To maximize the safety of glucocorticoid tapering in this study, enrolled participants will be 
considered eligible for a glucocorticoid taper if they are on a stable prednisone or prednisone-
equivalent dose ≤ 10 mg/day and have not had escalation of other immunosuppressive therapies 
for SLE, as indicated in inclusion criteria 7 and 8, and in exclusion criterion 4.
The taper will be managed by the clinical staff of the NIAMS Lupus Clinical Trials Unit 
(LCTU). The LCTU staff are actively involved in the care of patients in the study of the natural 
history of SLE (94-AR-0066), in which all participants in this study will also be enrolled. The 
tapering protocol is described in Error! Not a valid bookmark self-reference..


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Table 1. Glucocorticoid Tapering Protocol
Stable glucocorticoid dose
(prednisone-equivalent)
Tapering regimen
7.5-10 mg/day
Decrease dose by 2.5 mg/week until a dose of 5 mg/day is 
reached. Then, decrease dose by 1 mg/week until the taper is 
complete.
≤ 5 mg/day
Decrease dose by 1 mg/week until the taper is complete
The taper will be considered complete when a participant reaches one of two endpoints:
1. The participant is no longer receiving a glucocorticoid, or
2. Upon clinical evidence of a disease flare.
For participants whose taper ends because they are no longer receiving a glucocorticoid, the 
infusion visit will be performed approximately 10 calendar days after the last dose of 
glucocorticoid. After the infusion visit, participants will be followed up by the LCTU team 
and/or their outside rheumatologist for further management of the disease and decision to restart 
glucocorticoid medication.
For participants whose taper ends due to clinical evidence of a disease flare, or if a disease flare 
occurs between the end of the taper and the infusion visit, then the infusion visit will be 
scheduled within 7 days after the documentation of the flare. After the infusion visit, participants 
will be followed by the LCTU team and/or their outside rheumatologist for further management 
of the disease and decision to restart glucocorticoid medication. If the severity of the flare 
requires intervention prior to a scheduled infusion visit, then the participant will be withdrawn 
from the study and will continue to be managed by the LCTU team and/or their outside 
rheumatologist, as appropriate. 
Of note, exogenous glucocorticoid administration suppresses the hypothalamic-pituitary-adrenal 
(HPA) axis, and the time following a glucocorticoid taper that it takes for the HPA axis to return 
to normal function can vary among individuals. Formal documentation of the function of the 
HPA axis by a cosyntropin stimulation test is not part of routine clinical practice after 
glucocorticoids are tapered, unless a person is experiencing signs or symptoms of adrenal 
insufficiency. Therefore, cosyntropin stimulation testing will not be part of this study. It is 
unclear whether the baseline function of the HPA axis influences the transcriptional response by 
cells of the immune system to high doses of glucocorticoids. Although this study is not designed 
to address that question, the glucocorticoid panel that will be obtained before and after 
medication administration on the day of infusion (Table 2) includes measurement of plasma 
cortisol. Morning cortisol levels > 15 µg/dL are predictive of a normal result on a short 
cosyntropin stimulation test in nearly all patients, and values < 5 µg/dL predict an abnormal 
result in nearly all patients.39,40 Therefore, depending on the distribution of values in this group 


Glucocorticoid Genomics in SLE
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of participants, these data could eventually be used to stratify the participants by baseline level of 
function of the HPA axis, to assess whether this leads to measurable differences in the response 
to exogenous glucocorticoids.
6.3
Infusion Visit (day 0)

Targeted physical exam with vital signs (on initial encounter and prior to discharge).

Acute care panel (results will be obtained before infusion).

IV catheter insertion.

Serum pregnancy test (for those capable of becoming pregnant; results will be obtained 
before infusion). For participants tested within the past 24 hours, the test will not be 
repeated.

Interim medical history and medication review.

Pre-infusion blood draw.

IV infusion of methylprednisolone sodium succinate (30 minutes).

Blood pressure monitoring every 30 minutes from the time of infusion until the time of 
collection of the last blood sample.

Pulse oximetry monitoring during the infusion. 

Nursing staff will monitor participants for the presence of behavioral abnormalities 
(altered mood, hyperactivity, disorientation, psychosis).

Blood draws at 2 and 4 hours (± 10 minutes) after the start of infusion.
In participants whose glucocorticoid taper ends due to clinical evidence of a disease flare, the 
following additional procedures will be performed on the day of the infusion visit:

SELENA-SLEDAI score.

Urine collection for the following:
o
Urinalysis with microscopic examination.
o
Spot urine protein, creatinine, and protein-to-creatinine ratio.

Blood draw for the following:
o
Complete blood count with differential.
o
Complement levels: C3 and C4.
o
Anti–double-stranded DNA (dsDNA) antibodies.
6.4
Follow-up Phone Calls
A follow-up phone call will be made between days 1 and 3 after the infusion visit to document 
the presence or absence of side effects related to the study drug. A second follow-up phone call 
will be made 5 days (± 2 days) after the infusion visit. If any AEs that may require treatment are 
documented on the follow-up phone calls, then the participant will be asked to come to the NIH 
CC’s outpatient clinic for evaluation by the principal investigator. Participation in this study will 
be complete after the second phone call or, if applicable, after any follow-up visits.


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7
Study Procedures/Evaluations
Methylprednisolone sodium succinate infusion: Participants will receive a single IV dose of 
methylprednisolone sodium succinate (based on the dose group to which they are assigned) 
administered through an IV catheter over a period of 30 minutes.
Blood draw: Blood will be drawn at screening, immediately prior to the study agent infusion, 
and 2 hours (± 10 minutes) and 4 hours (± 10 minutes) after the start of the infusion. Blood will 
be used for laboratory and research 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 
participants by the NIH CC (Medical Administrative Series Policy M95-9, Guidelines for Limits 
of Blood Drawn for Research Purposes in the Clinical Center: http://cc-
internal.cc.nih.gov/policies/PDF/M95-9.pdf).
ECG: 12-lead ECG will be performed at screening to rule out electrical cardiac abnormalities 
and ensure eligibility to receive the study agent.
SELENA/SLEDAI: The SELENA/SLEDAI index is a tool for measuring disease activity in 
patients with SLE. The score is determined by points assigned to symptoms and laboratory 
values the patient had within 28 days of evaluation.41,42 A higher score represents a more 
significant degree of disease activity.43 The score will be calculated by a qualified practitioner.
7.1
Return of Research Results
Any clinically relevant test results will be shared with the participant throughout the study. 
Unexpected or incidental medical findings unrelated to the study may occur and may require the 
participant to follow up with an appropriate physician/healthcare provider for standard medical 
care or follow-up.
The genetic research in this study is 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 finding related to a 
participant’s health, we will share the information with the participant. Genetic counseling and 
advice is available from the NIH to help participants understand the implications of incidental 
findings, if necessary.


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Table 2. Blood Volumes and Research Uses
Visit
Blood sample
Initial processing
Downstream use
60 mL:b Isolation of hematopoietic cell sub-types (eg, 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
8 mL: Hepatitis A, B and C
N/A
6 mL: HIV 
N/A
5 mL: DNA purification
Genome-wide genotyping 
and/or DNA sequencing
4 mL: Acute care panel, C3 and C4 complement levels, calcium, CPK, 
hsCRP, LFTs, lipid panel, magnesium, random morning serum 
cortisol, serum pregnancy test
N/A
4 mL: Anti-dsDNA
N/A
4 mL: IGRA
N/A
3 mL: PT/PTT
NA
3 mL: CBC/diff
N/A
Screening 
visit
Baseline whole 
blood sample
(116 mL)a
1 mL: ESR
N/A
60 mL: Isolation of hematopoietic cell sub-types (eg, 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: Serum
Glucocorticoid panel
4 mL: Acute care panel, serum pregnancy test, C3 and C4 
complement levelsc
N/A
4 mL: Anti-dsDNAc
N/A
Pre-infusion whole 
blood sample
(90 mL)
3 mL: CBC/diff
N/A
60 mL: Isolation of hematopoietic cell sub-types (eg, 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: Serum
Glucocorticoid panel
4 mL: Acute care panel
N/A
Infusion 
visit
Serial post-infusion 
whole blood 
samples
(90 mL × 2)
3 mL: CBC/diff
N/A
CBC/diff = complete blood count with differential; CPK = creatine kinase; dsDNA = double-stranded DNA; ESR = erythrocyte sedimentation 
rate; hsCRP = high sensitivity C-reactive protein; IGRA = interferon gamma release assay; LFT = liver function test; N/A = not applicable; 
NK = natural killer; PBMC = peripheral blood mononuclear cell; PT/PTT = prothrombin/partial thromboplastin time.
a
Baseline blood will be collected at screening from all potential participants and will be processed as described even if the participant is 
determined to not be eligible for the study.
b
The purpose of this initial sample will be to assess the level of variation at baseline.
C      C3 and C4 complement levels and anti-ds DNA antibodies will only be ordered on the day of infusion in patients whose    
glucocorticoid taper ended due to clinical evidence of a disease flare.
8
Potential Risks and Benefits
8.1
Potential Risks
Methylprednisolone sodium succinate infusion: Side effects from a one-time IV 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, weight gain, temporary increase in blood glucose, temporary increase in blood 
pressure, changes in white blood cell counts (eg, leukocytosis , neutrophilia, monocytopenia, or 
eosinopenia), mood disturbance or unusual behavior, electrolyte imbalance, muscle cramps, back 
pain, headaches, flushing, and frequent urination lasting 1-2 weeks after infusion.37


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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 duration and frequency of treatment and we do not 
expect that they will occur with a single dose.
Glucocorticoids 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 
glucocorticoids alone or in combination with other immunosuppressive agents. Glucocorticoids 
may reactivate or exacerbate latent fungal, bacterial, or viral infections. Individuals with known 
or suspected active or latent infections will be excluded from study participation. In addition, 
participants 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 
IV methylprednisolone have been reported in patients with underlying disease. 
There are reports of cardiac arrhythmias and/or cardiac arrest following the rapid administration 
of large IV doses of methylprednisolone (greater than 0.5 g 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. 
Glucocorticoid taper: For individuals who are eligible for the study and undergo a 
glucocorticoid taper, the progressive reduction in the dose of glucocorticoid may result in 
destabilization of SLE, potentially leading to a disease flare.
Blood draw and IV placement: The risks of drawing blood include pain, bruising, bleeding, 
fainting, and, rarely, infection.
ECG: ECGs are of minimal risk to the participants and cause no significant side effects. The 
electrodes may feel cold when applied; in rare cases, some people will develop a rash or skin 
irritation where the patches are placed. This type of irritation usually resolves by itself or 
occasionally with topical medication.
RNA-seq, DNA sequencing, and genome-wide genotyping: Studies of RNA molecules will be 
performed to identify changes in gene expression in response to the study agent. Studies of DNA 
molecules (genome-wide genotyping) will be performed to identify variants in the genome that 


Glucocorticoid Genomics in SLE
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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. Incidental findings from genetic studies may 
have psychological implications for participants and family members. Genetic counseling and 
advice is available from the NIH to help participants 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 (eg, 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.
8.2
Potential Benefits
This research is not designed to benefit study participants. This research is designed to provide 
generalizable knowledge about SLE pathogenesis and the mechanisms of action of 
glucocorticoids in SLE. The study may also inform the development of future anti-inflammatory 
and immunosuppressive medications.
9
Research Use of Stored Human Samples, Specimens, and Data
Intended Use: Samples, specimens, and data collected under this protocol may be used to study 
the body’s response to glucocorticoids. Genetic testing will be performed.
Storage: Samples are stored in a secure facility with limited access. Samples and data will be 
de-identified prior to storage using a code assigned by the investigators. All data, including the 
database linking the code with a specific individual, will be kept in password-protected 
computers. Only investigators or their designees will have access to the samples and data.
Tracking: Samples and data acquired under this protocol will be tracked using the Clinical 
Research Information Management System of the NIAID (CRIMSON). After de-identification, 
laboratory specimens will be tracked using the Biological Specimen Inventory system, under a 
NIAID-wide contract for laboratory specimen banking and tracking.
Disposition:

In the future, other investigators (at NIH or outside) may wish to study these samples 
and/or data. If the planned research falls within the category of “human subjects 
research” on the part of the NIH researchers, IRB review and approval will be obtained. 
This includes the NIH researchers sending out coded or linked samples or data, or getting 
results that they can link back to their participants.

At the time of protocol completion, samples will be either destroyed or, after IRB 
approval, transferred to another existing protocol. Data will be archived by the study 


Glucocorticoid Genomics in SLE
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team in compliance with requirements for retention of research records; alternatively, 
after IRB approval, the data may be transferred to another repository.
Loss or Destruction:

Any loss or unanticipated destruction of samples (for example, due to freezer 
malfunction) or data (for example, misplacing a printout of data with identifiers) that 
meets the definition of a protocol deviation, unanticipated problem (UP), and/or 
compromises the scientific integrity of the data collected for the study, will be reported to 
the NIH IRB.

Additionally, participants 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 participant and to the IRB. Participants who revoke permission to 
have their samples stored will be withdrawn from the protocol. However, this decision 
will not affect the individual’s participation in any other protocols at NIH.
10 Data Sharing Plan
Human data generated in this study will be shared for future research as follows:

De-identified data in an NIH-funded or approved public repository. Gene expression data 
will be made available through the Gene Expression Omnibus and Sequence Read 
Archive repositories.

De-identified data in public repositories.  

Identifiable data in the Biomedical Translational Research Information System (BTRIS, 
automatic for activities in the CC) or the Genomic Research Integration System (GRIS).

De-identified or identifiable data with approved outside collaborators under appropriate 
agreements.

In publications and/or public presentations.
Data may be shared before publication, at the time of publication, or shortly thereafter.
Study participants will be offered co-enrollment in the NIAID Centralized Sequencing Program 
(protocol 17-I-0122). For participants who choose to enroll in that protocol, genome sequencing 
and microarray analysis will be performed as part of that protocol. Data generated as part of the 
Centralized Sequencing Program is shared with the referring investigators via the NIH Genomic 
Research Integration System (GRIS).
11 Remuneration Plan for Participants
Participants will be compensated for their time and inconvenience according to the following 
schedule:


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
Screening visit: $60 

Infusion visit: $180 

Total = $240
In the event that additional study-related visits need to be scheduled after the infusion visit, as 
detailed in section 12.5 below, each additional visit will be compensated at $60. Travel costs 
may be covered as per the current NIH/NIAMS policy.
12 Assessment of Safety
AEs and other reportable events are defined in NIH Human Research Protections Program 
(HRPP) Policy 801.
12.1 Toxicity Scale
The Investigator will grade the severity of each AE according to the “Common Terminology 
Criteria for Adverse Events (CTCAE)” (v 5.0) which can be found at 
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm 
12.2 Causality
Causality (likelihood that the event is caused by the study agent) 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


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Unlikely Related

does not have a reasonable temporal relationship
OR

good evidence for a more likely alternative etiology
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.
12.3 Recording/Documentation
At each contact with the participant, information regarding AEs will be elicited by appropriate 
questioning and examinations. All events, both expected/unexpected and related/unrelated will 
be recorded on a source document. Source documents may include, but not limited to, progress 
notes, laboratory reports, consult notes, and phone call summaries. Source documents will be 
reviewed in a timely manner by the research team. All reportable AEs that are identified will be 
recorded in CRIMSON. The start date, the stop date, the severity of each reportable event, and 
the principal investigator’s judgment of the AE’s relationship and expectedness to the study 
agent will also be recorded in CRIMSON.
12.4 Expected Events
Some 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 reported 
unless they reach a grade of 3 or higher (as defined in the CTCAE, v 5.0), are deemed a risk to 
the participant’s rights or wellbeing, or are deemed appropriate to report under specific 
circumstances by the principal investigator. Examples include:

Hyperglycemia

Leukocytosis

Neutrophilia

Monocytopenia

Eosinopenia

Flushing


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
Change in appetite

Bitter taste in mouth

Metallic taste in mouth

Anxiety

Changes in mood

Insomnia

Lymphopenia (only reported if grade 4 or above)
12.5 Type and Duration of the Follow-up of Participants after Adverse Events
AEs related to research procedures will be followed through resolution or until the investigator 
determines the event has stabilized and no additional follow-up is required. AEs related to the 
methylprednisolone infusion that have not resolved by the end of study participation will be 
followed until they have returned to baseline values. If it is not possible to obtain a final outcome 
for an AE (eg, the participant is lost to follow-up), then the reason a final outcome could not be 
obtained will be recorded by the investigator in CRIMSON.
12.6 Halting Rules for the Protocol
Halting the study requires immediate discontinuation of study agent administered for all 
participants 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 participants experience the same or similar SAEs that are possibly, probably, or 
definitely related to the study agent;
OR

any safety issue that the principal investigator determines should halt the study.
The principal investigator will determine if the study should be halted. The data and safety 
monitoring board (DSMB) may also recommend a halt.
12.6.1 Reporting a Study Halt
If a halting rule is met, then a description of the AE(s) or safety issue must be reported by the 
principal investigator within 1 business day to the IRB. The principal investigator will also notify 
the DSMB.


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12.6.2 Resumption of a Halted Study
The principal investigator, in collaboration with the DSMB, 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.
12.7 Study Discontinuation
The IRB, the NIAID, and other oversight bodies as applicable, as part of their duties to ensure 
that research participants are protected, may discontinue the study at any time. Subsequent 
review of serious, unexpected, and related AEs by the IRB may also result in suspension of 
enrollment and further administration of study agent.
12.8 Premature Withdrawal of a Participant
An individual participant will be withdrawn for any of the following:

An individual participant’s decision. (The investigator should attempt to determine the 
reason for the participant’s decision.)

Non-compliance with study procedures to the extent that it is potentially harmful to the 
participant or to the integrity of the study data.

A participant becomes pregnant between screening and the infusion visit.

A participant experiences a disease flare during tapering of current glucocorticoid therapy 
that requires intervention before the infusion visit.

A participant experiences an anaphylactoid reaction, anaphylaxis, or angioedema 
requiring medical therapy during the glucocorticoid infusion.

The investigator determines that continued participation in the study would not be in the 
best interest of the participant.

A participant who has a flare at screening visit and not scheduled for infusion within 7 
days or needs to receive glucocorticoids before the scheduled infusion visit. 
12.9 Replacement of Withdrawn Participants or Participants Who Discontinue Study 
Treatment
Participants who withdraw or are withdrawn from the study prior to the infusion visit will be 
replaced. 
13
Reporting Procedures
13.1
Reporting to the NIH IRB
UPs, non-compliance, and other reportable events will be reported to the NIH IRB according to 
HRPP Policy 801.


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13.2 Reporting to the NIAMS Clinical Director
The principal investigator will report UPs, major protocol deviations, and deaths to the NIAMS 
clinical director according to institutional timelines.
14 Clinical Monitoring Structure
To help ensure that NIH Office of Research Support and Compliance procedures and Good 
Clinical Practices are being carried out, a Clinical Trials Management designee within the Office 
of Clinical Research Policy and Regulatory Operations, Regulatory Compliance and Human 
Subjects Protection Program will conduct a study initiation visit before study enrollment begins. 
The purpose of this meeting is to review with the principal investigator and study team designees 
the roles and responsibilities concerning their commitment to adhere to the requirements of the 
protocol, especially in terms of NIH Office of Human Subjects Research Protections reporting 
requirements for reportable events. In addition, the quality management and data management 
plan for the study will be reviewed.
14.1 Quality Management Plan
During the study, the principal investigator and study team will be responsible for implementing 
a quality management plan. Additionally, the study team will be responsible for completing and 
submitting a summary report on the quality plan to the NIAID clinical director or designee at 
least annually as detailed in the quality management plan. A courtesy copy will also be sent to 
Clinical Trials Management.
14.2 Safety Monitoring Plan
The NIAID intramural DSMB includes independent experts that do not have direct involvement 
in the conduct of the study and have no significant conflicts of interest as defined by NIAID 
policy. The DSMB will review the study protocol and informed consent document prior to 
initiation and twice a year thereafter, or as may be determined by the DSMB. 
The DSMB may convene additional reviews as necessary. The DSMB will review the study data 
as needed to evaluate the safety, efficacy, study progress, and conduct of the study. 
All deaths, SAEs, and UPs will be reported to the DSMB at the same time they are submitted to 
the IRB unless otherwise specified herein. 
The principal investigator will notify the DSMB at the time halting criteria are met and obtain a 
recommendation concerning continuation, modification, or termination of the study. The 
principal investigator will submit the written DSMB summary reports with recommendations to 
the IRB.


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15 Statistical Considerations
15.1 Definition of the Study Endpoints
The primary endpoint of this study is a list of human protein-coding genes and non-coding RNAs 
(small RNAs and long non-coding RNAs) that are differentially expressed in response to 
glucocorticoids in patients with SLE, for each of the studied cell types and doses.
The secondary endpoints of this study are:
1. A comparison of the transcriptional response to glucocorticoids between the 2 dose 
groups.
2. A list of protein-coding and non-coding transcripts, their corresponding proteins, and 
molecular pathways representing the best candidates for targeted therapeutic alternatives 
to glucocorticoids.
3. Validation of the targets identified by functional studies.
4. For each cell type, a list of protein-coding or non-coding transcripts that are shared and a 
list of transcripts that are different, between patients with SLE and the previously studied 
healthy controls, at baseline or in response to glucocorticoids.
15.2 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-seq data will be aligned to the most recent 
human genome assembly using the software program STAR.44 The resulting BAM files and the 
most recent version of the GENCODE will be used to obtain raw read counts for each transcript, 
with the featureCounts program of the package Subread.45 
15.3 Identification of Glucocorticoid-responsive Genes
To identify glucocorticoid-responsive genes, differential expression analysis for the RNA-seq 
and small-RNA-seq data will be performed in R, with the package DESeq2.46
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. For participants who undergo a glucocorticoid 
taper, this analysis will contrast the pre- and post-taper patterns of gene expression. We will then 
perform differential analysis comparing each of the two post-infusion time points (2 hours and 
4 hours) independently against the baseline. DESeq2 (section 15.6) adopts the method of 
Benjamini-Hochberg to adjust for multiplicity. We will draw conclusions based on the 
Benjamini-Hochberg adjusted p values, which control for the family-wise false discovery rate 
(FDR) in the presence of multiple comparisons. We will employ a cutoff FDR47 value of < 5% to 


Glucocorticoid Genomics in SLE
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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. 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. 
A separate analysis will compare data obtained at the same time point (2 hours or 4 hours) 
between the high-dose and low-dose groups, to test whether there are significant differences in 
the transcriptional effect of glucocorticoids at the two doses. Finally, we will identify differences 
and similarities between the baseline transcriptome and the transcriptional response to 
glucocorticoids in patients with SLE recruited in this study and in healthy volunteers recruited in 
our previous study. Since the healthy and the SLE participants come from two different studies, 
they are not directly comparable in many aspects. Therefore, these results will be derived from 
the two studies separately and no formal comparison will be made across the two studies. We 
will present the baseline distribution of gene expression, the distribution of gene expression 
changes, and the subset of differentially expressed genes. For each cell type, we will generate a 
list of protein-coding or non-coding transcripts that are shared and a list of transcripts that are 
different between SLE patients and healthy controls.
Sequencing library preparation and sequencing batches will be carefully designed such that 
samples from the same participant and cell type are run in the same batch and samples from 
different participants are evenly distributed across batches. This will keep batch effects to a 
minimum in the identification of differentially expressed genes. For analyses across different 
participants, such as comparisons among different cell types, linear regression will be adopted 
with batch as a 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, then the R 
package SVA will be used.
15.4 Analysis of Flow Cytometry and Cytokine Data
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.


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15.5 Summary Statistics for Demographic and Clinical Data
Demographic characteristics and medical history will be summarized, eg, 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.
15.6 Adaptive Sample Size Determination
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. The primary endpoint of the 
study is a list of human protein-coding genes and non-coding RNAs that are differentially 
expressed in response to glucocorticoids in patients with SLE, for each of the studied cell types 
and doses. With a sample size of 20 in each dose group, there would be 90% power to detect a 
change of effect size 0.72 before and after glucocorticoid by the standard paired t-test. That 
means, within each dose arm and for each cell type, we could detect a differentially expressed 
gene with 90% power if the gene expression difference before and after glucocorticoid was no 
less than 0.72 SD.  
Due to the specific properties of RNA-seq data, an advanced mixed-effects negative binomial 
modeling approach will be adopted for evaluating gene expression change and the significance 
of the change. This will be achieved with the R package DESeq2, a widely adopted tool for 
differential expression analysis on sequencing data, which targets the issue of large number of 
genes and small number of samples. In DESeq2, data from all genes are shared and utilized in 
assessing the differential expression of each gene. This leads to more stable estimation of 
differential expression and its variability, and consequently to better power for detecting 
differentially expressed genes.
Based on the data from our previous study of healthy volunteers (protocol 16-I-0126) and a 
bootstrap procedure, with a sample size of 20 we can detect differentially expressed genes from 
0 to 2 hours after glucocorticoid infusion with a median of 0.45 log2 fold change at a 
significance level of 0.05 after adjusting for multiplicity, and 0.62 log2 fold change from 0 to 
4 hours. In SLE patients, there may be higher variability in the data than what we observed in 
healthy volunteers. Meanwhile, the magnitude of gene expression changes is also expected to be 
higher, given the state of activation of many immune cells in SLE compared to healthy controls. 
Therefore, we estimate that the proposed initial sample size in this study will be sufficient to 
detect the genes associated with the effect of glucocorticoids in SLE patients. 
To empirically evaluate whether this estimate is correct, we propose an adaptive study design 
similar to the one we performed on the study with healthy volunteers. Specifically, we will 


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recruit an initial cohort of 20 participants in each dose group. Based on the observed data from 
20 participants, we will then assess the gain in discovery with up to 30 additional participants per 
group. We will perform a bootstrap procedure, sampling with replacement up to 50 units from 
the 20 observed participants in each group to evaluate the improvement in gene detection. For 
example, if the subset of genes that can be detected as differentially expressed over time and the 
subset of genes that can be detected as differentially expressed over the 2 doses do not change 
significantly above a number, then we will stop at that number. If there is change in the gene 
subsets, but the pathways and the biological interpretation of the data remain unchanged, then 
additional participants 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 again use a 
nominal significance level α < 0.05.
16 Ethics/Protection of Human Subjects
16.1 Informed Consent Process
Informed consent is a process where information is presented to enable persons to voluntarily 
decide whether or not to participate in research. It is an ongoing conversation between the human 
research participant and the researchers that begins before consent is given and continues until 
the end of the participant’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. Participants will be given the opportunity to ask 
questions and have them answered.
The participants will sign the informed consent document prior to undergoing any research 
procedures. The participants may withdraw consent at any time throughout the course of the trial. 
A copy of the informed consent document will be given to the participants for their records. The 
researcher will document the signing of the consent form in the participant’s medical record. The 
rights and welfare of the participants 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.
We anticipate the enrollment of Spanish-speaking participants in this study. The English 
language consent document will be translated in full into Spanish by the NIH Library. For 
Spanish-speaking participants, the Spanish language consent form will be presented by the 
investigator through an interpreter, unless the investigator is fluent in Spanish.
Two Step Consenting Process
In certain cases, when prospective participants would like to learn additional details about the 
study before coming to the NIH (e.g., due to geographical distance or other logistical and 
personal reasons), we would like to do a two-step informed consent process. In the first step, we 
will either mail or email the informed consent document to the patient; this will be followed by a 


Glucocorticoid Genomics in SLE
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telephone call or videoconference, to go over the informed consent form document orally. For 
patients who agree to participate at that point, the second step will involve scheduling a 
screening visit at the NIH Clinical Center. As with any other screening visit, a member of the 
study team will then answer any additional questions and obtain the patient’s written signature in 
the informed consent form. This informed consent process will then be documented in the 
subject’s medical records.
16.2 Participant Confidentiality
All records will be kept confidential to the extent provided by federal, state, and local law. The 
study monitors and other authorized individuals 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 data will be coded. Any personally identifiable information maintained for 
this study will be kept on restricted-access computers and networks. Personally identifiable 
information will only be shared with individuals authorized to receive it under this protocol. 
Individuals not authorized to receive personally identifiable information will be provided with 
coded information only, as needed. Clinical information will not be released without written 
permission of the participant, except as necessary for monitoring by the IRB, NIAID, and the 
Office for Human Research Protections.
16.3 Participation of NIH Employees
NIH employees may participate in this protocol. We will follow the Guidelines for the Inclusion 
of Employees in NIH Research Studies and will give each employee a copy of the “NIH 
Information Sheet on Employee Research Participation.”
For NIH employees:

NIH staff may be a vulnerable class of participants.

Neither participation nor refusal to participate will have an effect, either beneficial or 
adverse, on the participant’s employment or work situation.

The employee participant’s privacy and confidentiality will be preserved in accordance 
with NIH CC policies, which define the scope and limitations of the protections.

For NIH employee participants, consent will be obtained by an individual independent of 
the employee’s team. Those in a supervisory position to any employee and co-workers of 
the employee will not obtain consent.

The importance of maintaining confidentiality when obtaining potentially sensitive and 
private information from co-workers or subordinates will be reviewed with the study staff 
at least annually and more often if warranted.


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17 Data Handling and Record Keeping
17.1 Data Capture and Management 
Study data will be maintained in CRIMSON and collected directly from participants during 
study visits and telephone calls, or will be abstracted from participants’ 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.
17.2 Record Retention
The investigator is responsible for retaining all essential documents listed in the International 
Council on Harmonisation Good Clinical Practice guidelines. Study records will be maintained 
by the principal investigator for a minimum of 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.
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