The Impact of Circadian Misalignment on Colonic Barrier Homeostasis in Ulcerative Colitis
Phase: NA
Conditions: Ulcerative Colitis
Sponsor: Rush University Medical Center

 
 
Title: The Impact of Circadian Misalignment on Colonic Barrier Homeostasis in Ulcerative Colitis 
NCT: NCT05180279 
Last IRB Approval Date: 2/21/2024 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Title:  The Impact of Circadian Misalignment on Colonic Barrier Homeostasis in Ulcerative Colitis
Principal Investigator: Ali Keshavarzian, MD
Affiliations: (1) Department of Internal Medicine, Section of Digestive Diseases, Rush University 
Medical Center
Sponsors: NIH - National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK)
Protocol Version: 5
Date: 2/19/2024
Abstract: Recently there has been compelling evidence that inflammatory bowel disease (IBD) 
subjects, both Crohn’s disease (CD) and Ulcerative Colitis (UC), commonly have disrupted sleep, 
and disrupted sleep correlates with the risk of disease flare.  Sleep/wake cycle, immune 
function, metabolism and multiple biological processes are all orchestrated by circadian 
rhythms. Circadian misalignment between the central circadian clock in the brain and 
environment has been found to contribute to a variety of metabolic and gastrointestinal tract 
(GIT) diseases.  Yet, the prevalence and impact of circadian misalignment on IBD disease activity 
and GIT mucosal inflammation have not been established.  The long-term objective of our 
research is to investigate the hypothesis that circadian malalignment worsens GIT mucosal 
inflammation and disease course in IBD.  To test this hypothesis, we will conduct a prospective 
in lab study with human subjects on the impact of circadian misalignment in left sided mild to 
moderate Ulcerative Colitis.  We will perform two circadian measures phase assessments under 
strictly controlled laboratory conditions at: 1) baseline, and after 2) circadian misalignment 
after three days of simulated night shifts in both inactive UC patients and healthy control 
subjects.   To test the hypothesis that UC patients (compared to healthy subject controls) are 
less resilient to circadian misalignment we will assess: time impact of our protocol on circadian 
rhythms (Aim 1) by the following: phase angle of entrainment [time from dim light melatonin 
onset to sleep onset] (Aim1a);  peripheral circadian rhythms by clock gene expression in colonic 
tissue from a flexible sigmoidoscopy and in subjects peripheral blood mononuclear cells 
(Aim1b); and colonic clock gene expression and Per2::Luc activity over 24 hours utilizing an in-
vitro model of colonic organoids (Aim1c).  Next, we will test the hypothesis that circadian 
misalignment will increase colonic permeability and mucosal inflammation in UC patients (vs. 
Controls) (Aim2) through the following: endoscopy score, stool calprotectin, colonic barrier 
(permeability, AJC proteins), and markers of systemic barrier function and inflammation (Aim 
2a); adversely impacting microbial community structure/function (Aim 2b); and use 2D colonic 
organoid monolayers to explore ex-vivo barrier function by co-culturing with TNF-α/INF-γ (Aim 
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2c).  The results of this innovative proposal will greatly increase our understanding of the 
important role circadian misalignment may have in UC disease activity and colonic 
inflammation, and identify new circadian regulated targets for treatment in UC.
Objective: The long-term objective of our research is to investigate the hypothesis that 
circadian misalignment worsens intestinal mucosal inflammation and disease course in IBD.  
Key evidence supporting this hypothesis include that disruption of circadian rhythms in the host 
impacts the intestinal barrier, microbiota composition, and mucosal/systemic immune function, 
all critical factors in the pathogenesis of IBD.  Data from rodents generated by our group and 
others show that disruption of the light: dark cycle (a model of shift work) or genetic circadian 
rhythm disruption (of a core clock gene) changes the intestinal microbial community, alters the 
production of microbial products like short chain fatty acids (SCFAs) resulting in colonic 
hyperpermeability through disruption of the apical junctional complex (AJC) in colonic IECs, 
increased mucosal inflammation, culminating in a more pro-inflammatory state in the 
intestine.11,24-27  In humans, shift work increases susceptibility of the colonic barrier to an 
injurious agent like alcohol and increases systemic pro-inflammatory cytokines IL-6 and IL-1β28 
and that chronic alterations in light:dark cycles in mouse models of IBD increase colitis severity 
and mortality.29,30 In humans with IBD, our group and others have shown that a later 
chronotype (Fig. 2A) and high variability in rest-activity rhythms (Fig. 2B) (both markers of 
circadian misalignment) are associated with worse IBD disease course, increased colonic 
permeability, alterations in the intestinal microbiota community (dysbiosis), and reduced 
colonic circadian clock gene expression.5,30  Therefore, the immediate goal of this proposal is 
to elucidate the mechanisms of colonic inflammation induced by circadian misalignment in UC.  
This will be the first comprehensive examination of human circadian rhythms in IBD.
Hypothesis: Circadian misalignment worsens intestinal mucosal inflammation and disease 
course in IBD.
Specific Aims:
-
Aim 1: Test the hypothesis that circadian misalignment is more prevalent in UC 
compared to healthy controls and that UC patients are more susceptible
-
Aim 2: Test the hypothesis that circadian misalignment increases colonic permeability 
and mucosal inflammation through disrupting the AJC and adversely impacting microbial 
community structure / function in UC.  
Background: A.1. IBD is Associated with Sleep Disruption.  Crohn’s Disease (CD) and Ulcerative 
Colitis (UC), collectively known as inflammatory bowel disease (IBD), are two devastating 
chronic diseases of the gastrointestinal tract (GIT).  IBD is typically diagnosed at an early age, 
has no known cure, and has a chronic relapsing and remitting disease course that includes 
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disabling symptoms during periodic flare ups.  IBD affects over 1.5 million individuals in the US 
with an estimated direct cost of $14 to 31 billion (31, 32). IBD has a heterogeneous disease 
course that commonly has an aggressive phenotype.  Identifying risk factors that can trigger 
disease flare and aggressive disease course are essential to avoid complications such as 
hospitalizations and surgery and are critical to improve disease course and patient quality of 
life.  Factors that are associated with a more aggressive disease course include the use of 
prednisone or inability to wean off prednisone (steroid dependency), early age of diagnosis, 
hospitalization, need for IBD surgery, and the presence of fistulizing/stricturing perirectal 
disease in CD (33-35). However, these factors cannot fully explain the disease course in many 
patients.  Recently, there has been an increased awareness of the high prevalence of sleep 
disruption in IBD.  Our group was one of the first to report a high prevalence of sleep 
disturbances in IBD, (17, 36) and patients with IBD exhibit disrupted sleep (polysomnography) 
even when their disease is inactive.  A subsequent prospective cohort showed that ileo-cecal 
inflammation in CD correlates with poor sleep.16  In essence, IBD patients in clinical remission 
with poor sleep had higher subclinical disease activity compared to those IBD patients reporting 
good sleep.16  Thus, disrupted sleep/wake cycles, an important output of the circadian clock, 
can be one factor that triggers disease flare and promote progressive disease course; yet the 
impact of circadian rhythm disruption on disease pathogenesis has not been fully evaluated, 
which is one of the goals of this application.
A.2. Circadian Rhythms are Critical to Health.  Sleep, rest-wake activity, and multiple 
biological processes such as metabolism, (37) cell proliferation, (38) and immune activity39 are 
orchestrated by the circadian clock on a roughly 24h cycle.  The discovery of the core molecular 
machinery of the master or central circadian clock in the suprachiasmatic nucleus (SCN)40 
which consists of a transcription and translation feedback loop of positive factors (e.g., Clock, 
Bmal1) and negative repressors (e.g., Per1, Per2, Cry1, Cry2) has led to a number of key 
mechanistic discoveries regarding the circadian clock machinery and its functions that are 
critical for normal physiology and behavior.41-43  Synchronization of internal circadian clock 
with external environmental cues (e.g., light) is critical for good health,44-47 which is not 
surprising as the circadian clock is known to control the expression of 10-20% of the genes in 
the human body.48-50  In addition, peripheral clocks that contain the same core molecular 
machinery as in the SCN are found in cells of every organ in the body, including colonic IECs in 
the GIT.51,52  The GIT exhibits diurnal rhythms in many physiologic functions such as motility, 
intestinal transport, and IEC replication53 all of which are governed by inputs from the SCN as 
well as other signals that entrain the circadian clock in the GIT such as time of food 
consumption.  Circadian misalignment is a mismatch between the environmental cues (e.g., 
light) and the central clock in the SCN or a mismatch between the central and peripheral clocks 
which can occur due to shift work or consumption of a high fat diet, respectively (54, 55). 
Modern 24h society, with increased nighttime light exposure, light pollution, the increasing 
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prevalence of shift work, and nighttime eating have made misalignment of our endogenous 
circadian rhythms more common.  There are multiple causes for human circadian misalignment 
including shift work, long distance travel with jet lag, and perhaps the most common: social jet 
lag, which is changing the midpoint of sleep more than 2h between work and work-free days 
due to social obligations.56  The most well-studied model of circadian misalignment, shift work, 
is now estimated to include 15 million workers in the US.57  Not surprisingly, shift work is 
associated with a number of GIT disorders and diseases including irritable bowel syndrome,22 
peptic ulcer disease,58 and colon cancer.59  Therefore, it is possible that circadian 
misalignment may be one factor that promotes intestinal inflammation and disease flare in UC. 
A.3. IBD is characterized by Intestinal Barrier Dysfunction and Inflammation.  The 
intestinal barrier is the largest interface between the body and the environment and must be 
selectively permeable to restrict pro-inflammatory microbiota and microbial products but at 
the same time allow passage of nutrients, electrolytes, and water.  Thus, integrity of the 
intestinal barrier is critical for maintaining good health.  Indeed, disrupted intestinal barrier 
function (“leaky gut”), endotoxemia (i.e., high levels of systemic lipopolysaccharide (LPS)), and 
subsequent production of pro-inflammatory cytokines and reactive oxygen species are all 
hallmarks of IBD.60  In fact, fluctuations in intestinal barrier function in IBD is an established 
predictor of disease flare in CD61-63 and UC.64,65  Several environmental factors can disrupt 
the intestinal barrier function in IBD like medication,66 alcohol,67,68 and stress69,70 but this 
only occurs in a subset of individuals suggesting that resiliency of the intestine determines the 
impact of these environmental factors in chronic inflammatory disorders like IBD.  The 
intestinal barrier consists of the apical junctional complex (AJC), both tight junction (TJ) and 
adherens junction (AJ) proteins, that links the IECs and seals the paracellular space to prevent 
translocation of pro-inflammatory bacteria and bacterial components such as LPS into the 
intestinal mucosa.71  IECs turnover every 4-5 days,72,73 replenished by a small population of 
intestinal stem cells (ISCs), and IEC regeneration and differentiation are essential to maintain 
barrier homeostasis.74  It has also been established that alterations in gut microbiota are 
another hallmark of IBD, and perturbation of the structure of the microbiome can disrupt the 
intestinal barrier and cause intestinal inflammation in IBD.75  Specifically, in IBD there is a 
decrease in bacterial diversity76,77 and temporal stability78 with a loss of bacteria in the 
Firmicutes phylum79 and increase in Proteobacteria.80  It is not understood if alterations in the 
intestinal microbiota are the cause of or result from increased intestinal permeability and 
inflammation, but IBD is characterized by both barrier dysfunction and dysbiosis.  Disruption of 
central or peripheral circadian rhythms is sufficient to impact both the intestinal barrier and the 
microbiota; therefore, it is important to determine if circadian misalignment is sufficient to 
induce changes in the GIT that promote intestinal inflammation and UC disease flare.  
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A.4. Circadian Rhythms are Critical to Maintain the Intestinal Barrier.  Intestinal barrier 
homeostasis is mediated by cross-talk between IECs and the intestinal microbiota.  This cross-
talk occurs via multiple mechanisms including interactions with toll-like receptors (TLRs) on the 
IECs.81  There are multiple lines of evidence in cell lines, animal models, and human studies 
that support the scientific premise that disruption of circadian rhythms in the host negatively 
impacts intestinal barrier integrity: (1) alcohol (0.2%) increases the expression of the circadian 
clock genes Clock and Per2 in a human IEC line (Caco-2 cells), and knock down of Clock or Per2 
gene expression by siRNA prevents alcohol-induced barrier dysfunction,82 (2) chronic 
disruption of the central clock by either disruption of light:dark cycles or a mutation in the core 
circadian molecular clock (i.e., Clock mutant mice) increases intestinal permeability and 
endotoxemia,26 (3) healthy night workers who consume alcohol (0.5g/kg daily for 7 days) have 
increased colonic permeability compared to healthy day workers consuming alcohol, and 
colonic permeability inversely correlates with decreased plasma melatonin over 24h,28 (4) our 
lab25 and others13,27 show that circadian misalignment in the host causes microbial 
alterations with a decrease in bacteria (Firmicutes) that produce short chain fatty acids (SCFA) 
like butyrate (Bu),83 and (5) the core clock machinery (Bmal1) regulates IEC cell regeneration 
and barrier homeostasis.84,85  Taken together, these studies strongly support the concept that 
circadian misalignment could promote IBD flare via a mechanism involving disrupting the 
colonic barrier and intestinal microbiota dysbiosis.  
A.5. Circadian Rhythms and IBD.  Given that the circadian rhythms of the host can 
impact colonic barrier integrity, it is likely to also impact chronic inflammation in IBD; however, 
the impact of circadian misalignment in humans with IBD has not been investigated.  Prior work 
in animal models of IBD by our group29 and others30 show that chronic disruption of central 
circadian rhythms in the host by shifting light:dark cycles causes significantly worse colitis and 
increases mortality by Dextran Sodium Sulfate (DSS) (Fig. 1) or by Dextran sodium sulfate/2,4,6-
trinitrobenzene sulfonic acid (TNBS).  In addition, we recently found that an intestinal specific 
Bmal1 knock out mice develop worse DSS colitis with increased permeability despite normal 
central circadian behavior (Fig. 1C&D) suggesting that influencing circadian rhythms in the GIT 
are sufficient to impact the barrier and mucosal inflammation.  Furthermore, a Per2 mutant 
mouse with increased Per2 activity had constitutively high levels of the tight junction proteins 
(TJP) occludin and claudin-1 in the colon, is resistant to DSS-induced colitis.86  In humans, 
genetic analysis found an association between polymorphisms in one of the core circadian clock 
genes Period 3 (Per3) and increased incidence of Crohn’s disease.87  Our group has conducted 
several studies in humans with IBD to examine the role of circadian rhythms: 1) We examined 
the association between chronotype (category of circadian timing) and IBD using two validated 
questionnaires – the Owl and Lark and Munich.  We found that a later chronotype is associated 
with a worse IBD quality of life, IBD subjects on aggressive medications (biologics) had a later 
midpoint of sleep, and Crohn’s subjects with increased social jet lag are more likely to have a 
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history of fistulizing or stricturing disease (Fig 2A).88  These data suggest that circadian rhythms 
are associated with IBD disease phenotype.  2) Our examination of endogenous salivary 
melatonin secretion in four subjects with inactive IBD, revealed that two of the subjects had 
low melatonin secretion compared to controls and the one had an arrhythmic profile,89 
indicating circadian abnormalities are present in IBD patients.  3) We used wrist actigraphy to 
identify variations or instability in rest-wake activity that correlate to central circadian rhythms 
in inactive IBD subjects and found that there was less interdaily stability in IBD subjects with an 
aggressive disease course (history of fistulizing/stricturing disease in CD and history of surgery, 
steroid dependence, or biologic use in UC and CD ) and greater interdaily variability correlated 
with increased colonic permeability (Figs. 2B, 2C).  4) Examination of stool microbiota in inactive 
IBD subjects reveals changes in the microbiota that were associated with circadian 
misalignment.  For example, there is a significant decrease in SCFA-producing bacteria (Fig. 2D) 
and bifidobacterium longum which has been used as a treatment in active UC.90,91  Taken 
together, these findings support the hypothesis that circadian rhythms influence the intestinal 
barrier and the intestinal microbiota; however, no human studies have comprehensively 
studied the central or peripheral circadian rhythms in IBD subjects by the gold standard in the 
field (DLMO) while examining subclinical inflammation and colonic barrier function, which is 
one of the goals of our current study. 
A.6. Importance of the Proposed Research.  There is a clear scientific rationale and crucial need 
to take the next step and more comprehensively assess central and peripheral circadian 
rhythms in IBD in order to determine the impact of circadian misalignment in IBD on colonic 
mucosal inflammation, subclinical systemic inflammation, barrier function, and intestinal 
microbiota.  In the proposed study, we will determine if patients with inactive UC have 
circadian misalignment of central and/or peripheral circadian rhythms and if they are more 
susceptible to circadian misalignment after a simulated night shift (Specific Aim 1) and 
determine if circadian misalignment impacts colonic barrier integrity and intestinal microbiota 
(Specific Aim 2).  We selected UC as a model of IBD because: (1) it always affects the 
rectum/sigmoid, thus it is relatively easy to access and obtain the required colonic intestinal 
tissue through sigmoidoscopy, (2) microbiota-directed interventions like fecal microbiota 
transplant (FMT) appear to be more promising in UC than in CD,92,93 and (3) our data support 
that circadian misalignment has a substantial impact on the colonic barrier in UC subjects.  The 
knowledge gained from the proposed studies is likely to lead to the identification of new 
mechanisms of how circadian misalignment promotes chronic inflammation in IBD, and allow 
for the study of new chronobiological therapeutics in IBD that target circadian misalignment 
such as bright light therapy, exogenous melatonin, or other molecular targets associated with 
circadian misalignment induced in IBD.
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Research Design: 40 human subjects will be recruited into this proposed study: 20 Healthy 
Controls and 20 UC subjects with left sided inactive disease (Mayo Score ≤ 2) on stable 
medications. All subjects (UC and HC) will undergo a full medical history and physical 
examination including a blood draw for the screening visit. A urine drug screening will take 
place on the subject’s first day at the sleep laboratory. Subjects who wish to participate will 
have the study explained by the coordinator and will then sign a Rush IRB-approved informed 
consent. All UC subjects will complete a validated questionnaire regarding disease-specific 
symptoms (Mayo Score) to determine they are inactive and may be enrolled in the study.
In addition, subjects will provide basic demographic data, UC history if appropriate, UC 
disease activity status as detailed further above, and the Munich Chronotype questionnaire. All 
subjects will complete a validated 3-month food frequency questionnaire (FFQ) to collect 
dietary information (1). 40 participants (Specific Aims 1-2) will be enrolled for the within-
subject prospective in-laboratory study protocols. Participants will be young (18-50 y), healthy, 
non-diabetic, non-obese, Caucasian/African American/Asian and non-Hispanic/Hispanic in 
ratios consistent with the population of Chicago, and without medication use (excepting oral 
contraceptives) for HC. HC and UC subjects will be matched according to age (   3 years), sex, 
race, and BMI (   3 kg/m2).  UC subjects will be on stable medications with no recent use of 
prednisone or antibiotics for the last 3 months.  After signing an informed consent, the subject 
will fill out questionnaires ), start a food diary, and sleep diary, and have 2 weeks of wrist 
actigraphy before their seven-day in lab session in the Clinical Chronobiology Center (CCC).  
Since participants will be in the sleep lab for 7 days and isolation can cause an increase in 
depression and anxiety, a normal psychological evaluation is part of the inclusion criteria for 
this study. This will be determined based on how participants respond on the BDI and STAI 
questionnaires, which assess depression and anxiety. The study physician will review the 
questionnaire responses and evaluate if the participant will feel comfortable staying in the lab 
for 7 days. .  
Subject Characteristics: We will recruit (1) 20 HC subjects without any medical illnesses (2) 20 
subjects with biopsy proven UC, inactive (Mayo Score ≤ 2) on stable medications and no disease 
flares for > 3 months. HC and UC subject will be matched according to age (   3 years), sex, 
race, and BMI (   3 kg/m2).   
Sample Size Analysis: All power estimates were computed using R package pwr, and we 
assume a Type I error rate of 0.05 and a 2-sided test for all analyses.  Power estimates were 
derived from the data from my prior K grant.  The primary endpoints of this study will be phase 
angle/DLMO (Aim 1) and colonic permeability (13-24h urinary sucralose) (Aim 2).  For Aim 1, we 
found a ~2h phase advance in DLMO in NW compared to DW at -0.2 (SD=2.1) compared to -
4.34 (SD=3.8), respectively.  With a sample size of 20 in each group (10 men/10 women), this 
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provides an 86% power to detect differences in DLMO, but 98% power if sex is pooled.  For Aim 
2, we found a 40% increase in colonic permeability in night shift works compared to day 
workers after moderate alcohol consumption at 0.57 compared to 0.11 (SD=0.20).  With 20 
subjects, this will give us a 84% power to detect a difference in colonic permeability between 
the two groups for each sex and if sex is pooled with give us a 97% power with a two-sided two-
sample t-test at alpha=0.05 level.  These conservative estimates provide a good estimate of the 
power to detect differences in phase angle and colonic permeability between UC patients 
before and after simulated night shifts.  We anticipate we will have nonparametric data and will 
use the Wilcoxon match-pair test for analysis of colonic permeability, inflammatory cytokines, 
and markers of endotoxemia.  Order effects will be used as a covariate in multivariate analysis 
with analysis of variance (ANOVA) and linear regression as indicated.  Microbiota data analysis 
is described above.  Non-parametric analysis of actigraphy data will be analyzed with the 
nparACT package.  In our periodogram analysis of time series data we will use Fast Fourier 
Transform Non-linear Least Squares (FFT-NLLS), a nonlinear least-squares algorithm of a times 
series typically used for luminescence data (149) and analyzed with Biodare software (150). 
Data will be presented as mean ± SEM for variables that can be considered normally distributed 
(or median and range for variables not normally distributed).  All analyses will use R (v.3.2.5), 
Biodare 2, and SPSS (SPSS Inc., Chicago, IL).
Inclusion Criteria:  Study will include individuals that are;
Healthy Controls:
1. M/F, 18-50 y/o, age ± 3y sex, race, and BMI ( 3 kg/m2) match with UC subject
2. No clinical evidence of any medical illness
3. Normal psychological evaluation based on question responses and negative drug screen 
(See Below)
Ulcerative Colitis: 
1. 1. M/F, 18-50 y/o. 
2. 2. Inactive Disease (Mayo Score ≤ 2)
3. 3. Stable medications with no disease flares for the > 3 months
4. 4. Left-sided UC (Montreal E1 or E2)
5. 5. Normal psychological evaluation based on questionnaire responses and negative drug 
screen (See Below)
Exclusion Criteria: Study will not include individuals that are;
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Healthy Controls

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1. History of drug abuse, gastrointestinal (GI) surgery, GI diseases, or systemic diseases 
such as renal (creatinine>1.2 mg/dl), liver, cardiac, or diabetes (Hgb-A1c>8%)
2. Antibiotic use within last 12 weeks
3. Shift work in the last 6 months
4. Use of probiotic supplement except yogurt in last 4 weeks.
5. Atypical American diet with daily fiber ≥ 16 grams or daily saturated fat ≤ 11 grams by 
Food Frequency Questionnaire
6. Chronic use of NSAIDS. A washout period of 3 weeks is needed before the subject could 
be enrolled into the study. 
7. Chronic Alcohol use. A washout period of 3 weeks is needed before the subject could be 
enrolled into the study.
8. Significant Depression (score ≥ 14 BDI)
9. Significant Anxiety (score ≥ 40 STAI)
10. Regular use of medications that affect intestinal permeability, intestinal motility and/or 
endogenous melatonin including metoclopramide, NSAIDs, antibiotics, beta blocker, 
psychotropic medication, hypnotics and exogenous melatonin products during 4 weeks 
prior to the study
11. People who crossed more than 2 time zones in the previous month
12. Inability to sign an informed consent form. 
13. Have children Under 6 Months
Ulcerative Colitis:
1. Patients with other forms of colitis such as Crohn's disease (CD) or indeterminate 
colitis
2. Patient with active UC (Mayo > 2)
3. Pancolonic UC (colitis past the splenic flexure, Montreal E3))
4. Gastrointestinal surgery 
5. Other GI diseases or systemic diseases (cardiac, renal failure, cirrhosis)
6. Shift work in the last 6 months
7. Antibiotic use within last 12 weeks
8. Patients who have used anti-diarrheal agents such as Lomotil or Imodium within 3 
days of the study
9. Prednisone use the last 30 days
10. Significant Depression (score ≥ 14 BDI)
11. Significant Anxiety (score ≥ 40 STAI)
12. Use of probiotic supplement in last 4 weeks except yogurt.
13. Intentional change in diet. 
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14. Chronic use of NSAIDS. A washout period of 3 weeks is needed before the subject 
could be enrolled into the study. Low dose aspirin is allowed.
15. Chronic Alcohol use. A washout period of 3 weeks is needed before the subject 
could be enrolled into the study.
16. Have children under 6 months
Subject Recruitment: All Human UC subjects will be recruited at the RUMC Digestive Disease 
Clinic and the Rush Crohn’s and Colitis Center by the PI or Clinical Coordinator. Healthy control 
subjects will be recruited by established recruiting methods, which we have successfully used 
over the past ten years, including notices to local organizations and internet, newspaper, and 
radio advertising. Based on our experience, of the initial calls made by potential subjects, 15% 
remain interested in the study after they are informed of study procedures. Of these 
individuals, 70% pass an initial telephone screening (in which questions are asked about 
medical condition, current medication use and work and travel history). Of those remaining 
individuals, 33% pass the medical and psychological screening (described below), and of these, 
70% complete the study.
In this study we will recruit 40 Human Subjects in the following two groups:
1) Ulcerative Colitis (UC) (n=20). 
2) Healthy Controls (HC) (n=20).  
Each potential subject will undergo an extensive screening procedure prior to 
participation in the study. The procedure begins with a telephone screening, involving a series 
of questions from a preliminary screening questionnaire and answering any questions potential 
subjects may have about the study. If the original preliminary telephone screening 
questionnaire reveals no grounds for exclusion (see "Inclusion/Exclusion Criteria" below), then 
potential subjects are asked to come to our clinic for a physical examination. These patients will 
all be interviewed by the study Clinical Coordinator and then get a full physical exam by the PI 
who is a licensed Gastroenterologist. Potential subjects will also complete several 
questionnaires to determine their suitability for study.  Each potential subject is given a tour of 
the Clinical Chronobiology Center (CCC) in which they will live during the temporal isolation 
portion of the study. Every attempt will be made to acquaint each prospective subject with all 
of the procedures involved in this study in order to minimize the possible effect of uncertainty 
about the experimental procedures on the results. Written informed consent on an RUMC IRB-
approved form will be obtained from each subject before his/her study begins. Each subject will 
be told that they will be free to discontinue participation in the experiment at any time and that 
the investigators also reserve the right to discontinue the study for medical or other reasons at 
any time.
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Early Withdrawal: Participants may withdraw at any time.
Procedures:  After consenting for the study all subjects: 1) Will be interviewed by a study 
coordinator and fill out detailed questionnaires so that we can collect demographic data, 
medical history, data on disease status and research questionnaires; 2) Will supply blood and 
urine samples (to assess the inclusion/exclusion criteria and to obtain baseline biological 
markers; 3) Will undergo an exam by PI Dr. Ali Keshavarzian to assess the inclusion/exclusion 
criteria and to obtain baseline UC disease characteristics and have initial blood drawn/urine 
sample. 4) After interviews and exam and blood/urine analysis, as part of the screening process, 
eligible patients will be asked to continue participation and will be re-informed about the study.
All experimental procedures will commence after admission to the CCC for a 7 day in-lab 
evaluation.  The sleep episode will be the same as that maintained during the 2 weeks prior to 
the in-lab phase assessment. On Test Day 1 (TD1, Baseline alignment), at 0900h all subjects will 
have a unprepped flexible sigmoidoscopy (FS) with rectal biopsy collection (19 biopsies, ~15 cm 
from the anal verge), after which intestinal permeability will be assessed (24h urine collection 
after ingestion of a sugar cocktail: sucrose, mannitol, lactulose, sucralose103), and every 2 hour 
blood sampling during/surrounding standardized meals (primary and secondary outcomes for 
Specific Aims 1 & 2).  During the last 8h of the phase assessment, subjects will be exposed to a 
background illumination ~3 lux to assess the endogenous circadian phase through hourly 
plasma melatonin (dim light melatonin onset, DLMO).  On Protocol Day 3, subject behavioral 
cycles will be shifted by 12h, and this schedule will be maintained until Protocol Day 7.  
Following the short 4h sleep episode on Day 3, subjects will have 3 “night shift” days (Days 3-5).  
The 12h shift on Day 3 will be achieved by including an 8h wake episode and a 4h sleep 
opportunity, thereby maintaining the same sleep opportunity-to-wake ratio (1:2).  The rationale 
for the 12h inversion of the sleep-wake timing is that we previously found that the influence of 
circadian misalignment on physiological variables are maximal when the sleep/wake and 
fasting/feeding cycle was shifted by ~12h relative to the endogenous circadian timing 
system.19,101  On Test Day 2 (TD2, Circadian misalignment), all subjects will have all the 
identical tests conducted as described on TD1; however, the FS and start of repeated measures 
will be at 2100 h – matched to the sleep/wake cycle and not clock time to isolate the impact of 
circadian misalignment. 24 hours prior to each flexible sigmoidoscopy, stool will be collected 
from subjects. Discharge from the lab will be on day 7 after an 8h sleep period.  We will test for 
order effects (see Statistics).
Blood draw.  Blood (30 cc) will be drawn in endotoxin free tube and serum then 
immediately separated and stored in -80ºC freezer until measurement for Serum endotoxin 
(END). Blood will also be drawn for CBC, comprehensive biochemical profile [CMP that includes 
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liver function test], and PT. At first blood draw on Day 2 and last blood draw on Day 7, an 
additional 10 ml will be collected from patients on biologics, to evaluate antibody levels. 
All subjects will complete an IBDQ, FFQ, BDI, STAI, PSQ, Munich Chronotype, IRLS, 
Demographic Questionnaire, Mayo Score, CSD-C (Consensus Sleep Diary), and Berlin 
Questionnaire. All information collected from subjects will be used for research purposes only.  
All the procedures in the study (blood draw, stool samples, urine samples, sigmoidoscopy with 
biopsies and questionnaires) are well-established and considered routine tests in clinical 
practice (including flexible sigmoidoscopy).
Diet Assessments:  Meal habits will be recorded during screening.  All subjects must eat 
breakfast (>200 kcal) at least 5 days a week, and subjects unusual diets by FFQ (vegan or gluten 
free) will be excluded due to impact on microbiota. For two weeks prior to each inpatient 
laboratory stay, all subjects will undergo ambulatory monitoring of their general activity using a 
small wrist activity monitor for ensuring compliance with the sleep-wake schedule. In addition, 
subjects will be provided meals for the last 3 days before admission and asked to keep regular 
meal schedules. Medical and psychiatric condition summary. Volunteers must be ambulatory 
and have no major visual or auditory handicaps. Medical suitability will be determined by 
clinical history, physical examination, and clinical biochemical screening tests of blood and 
urine, past or present psychopathology. Finally, a clinical interview will be carried out on all 
potential subjects to determine their suitability for the study and their ability to tolerate the 
conditions of the simulated night shifts environment (with regular contact with research staff 
and technicians). All volunteers selected must be drug-free with only moderate or no use of 
caffeine, alcohol, or other compounds by history, which will be verified during the screening 
process by comprehensive toxic analysis. Caffeine, alcohol, or other compounds (including 
herbal medications/remedies) will not be permitted for three weeks immediately prior to 
admission to the Clinical Chronobiology Center (CCC), and a urinary comprehensive toxic 
analysis will be carried out upon admission to ensure compliance. Prescribed medications are 
also prohibited with some exceptions.
Sleep Assessment:  We will use a structured sleep questionnaire, the Pittsburgh Sleep 
Quality index, to access sleep habits.  In addition, a Beck's depression index will be completed 
to evaluate a common confounder in sleep, depression.  2 weeks before participating in the 
circadian lab assessment, subjects completed sleep logs and wear a waterproof wrist actigraphy 
monitor (Actiwatch-L, Spectrum, Bend OR).  
Phlebotomy:  There may be some discomfort or bruising on the initial insertion of the 
catheter into a vein, but wearing the catheter should not be painful. Occasionally,  mild  
discomfort  may  occur from  the catheterin  the  vein.  If this happens, it can  be repositioned 
or removed, asking the subject's permission before any subsequent reinsertion.  To help keep 
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the venipuncture site clean, we may ask permission to shave the forearm hair of the subject 
before insertion of the IV.  There is a rare possibility of developing a small blood clot, 
inflammation, or local infection around the vein where the catheter is inserted, or in rare cases 
a generalized infection spread through the bloodstream as a result of the IV catheter.  
Occasionally, there is a black and blue mark at the site of the IV insertion, which may last a 
couple of weeks; and, rarely, a small scar may remain permanently at the venipuncture site.
Saliva Collection:  Participants will be asked to produce a saliva sample on days 2-6 at 7 
am and 7 pm. They will be provided a conical tube to spit into.  A minimum of 2 ml is needed. 
To minimize contamination, participants will be instructed to not brush their teeth, use 
mouthwash, eat, or drink anything (except water) at least 1 hour prior to sampling.  The tube of 
saliva will then be placed into a centrifuge and then aliquoted into 1.5 mL cryovials. One 
cryovial will have 500ul of saliva + 500ul of Zymo DNA/RNA 2x solution. The second cryovial will 
be 500ul of saliva flash frozen. These samples will be used for analysis of inflammatory 
cytokines and 16S sequencing. Core temperature device: On Day 2 and 5 participants will be 
given a Core device, a non-medical device that will measure their body core temperature. Body 
temperature will be used to measure the central circadian rhythm. The device will be 
programmed with the participant’s study unique ID and it will be secured on their chest via 
medical grade adhesives when they get their catheter inserted on day 2 and 5.  After activation, 
the device automatically collects and transmits core temperature data to our database. Once 
the day is completed the device will be removed from the participant’s chest, the adhesive 
patches will be tossed out and the device will be disinfected.  
Flexible Sigmoidoscopy:  At two points in the study subjects will have an unprepped 
limited flexible sigmoidoscopy by Dr. Keshavarzian and sigmoid mucosal samples will be 
collected:19 endoscopic pinch biopsy samples will be taken and used for: (a) two formalin-fixed 
and paraffin embedded for histology, (b) two OCT fixed for immunohistochemistry of AJC 
proteins, (c) two RNA Later for RT-PCR of AJC protein mRNA expression, (d) five snap frozen in 
liquid nitrogen for Western blot assessment of AJC proteins, (e) two snap frozen in liquid 
nitrogen for microbiota interrogation, and (f) six fresh samples to generate colonoids.   Frozen 
samples will be transferred from liquid nitrogen tank to -80ºC freezer within 8-12 hours.
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Data Collection: All source data will be collected via a paper subject folder and/or a REDCap 
survey. Subject folders will be coded and stored in a locked cabinet within a locked research 
room only study staff will have access to. Data from the paper charts will be entered into the 
REDCap project for the study for electronic storage. The REDCap project is password protected 
on the Rush server and will only be accessible by IRB approved study staff. 
Biospecimen Collection & Storage: All biospecimens collected for the purposes of this study 
will be coded and stored in the Rush Gastroenterology Research Laboratory. Only IRB approved 
study staff will have access to these samples. 
Data Analysis Methods: 40 human subjects will be recruited into this proposed study: 20 
Healthy Controls and 20 left sided UC subjects with inactive disease (Mayo Score ≤ 2) on stable 
mild to moderate medications (aminosalicylates, 5-ASA) . All subjects (UC and HC) will be 
interviewed by the PI and will undergo a full medical history and physical examination including 
blood draw and urine collection.  The PI is a licensed physician Gastroenterologist who 
specializes in the management of inflammatory bowel disease, to determine their eligibility and 
co-morbidities. Subjects who wish to participate will have the study explained by the 
coordinator and PI and will then sign a Rush IRB-approved informed consent. All UC subjects 
will complete a validated questionnaire regarding disease specific symptoms (Mayo Score) to 
determine they are inactive and may be enrolled in the study.
In addition, subjects will provide basic demographic data, UC history if appropriate, UC 
disease activity status as detailed further above, and Munich Chronotype questionnaire. All 
subjects will complete a validated 3 month food frequency questionnaires (FFQ) to collect 
dietary information (149). 40 participants (Specific Aims 1-2) will be enrolled for the within-
subject prospective in-laboratory study protocols. Participants will be young (18-50 y), healthy, 
Fig 3: Circadian Misalignment Protocol 
Ulcerative Colitis Circadian Protocol
7am
11am
3pm
7pm
7am
11pm
3am
Day 2
Day 3
Day 1
Wake; 90lx
Sleep; 0 lx
Wake; 4 lux, serum melatonin (q 1 h)
Day 4
Flexible Sigmoidoscopy and stool collection 
Day 5
Meal
Adaptation
Test Day 1 
(Baseline)
Day 6
Day 7
Discharge
Test Day 2 
24 h permeability test, PBMCs and serum cytokines (q 2 h –12 samples)
In lab seven day protocol includes an adaptation period, followed by a  baseline assessment of primary outcomes 
dim light melatonin onset and  intestinal permeability with other secondary outcomes (Test Day 1).  This is followed by 
three days of simulated night shifts and then a repeat assessment of the primary and secondary outcomes (Test Day 2).
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non-diabetic, non-obese, Caucasian/African American/Asian and non-Hispanic/Hispanic in 
ratios consistent with the population of Chicago, and without medication use (excepting oral 
contraceptives) for HC. HC and UC subject will be matched according to age (   3 years), sex, 
race, and BMI (   3 kg/m2).  UC subjects will have only left sided disease, be on stable 
medications with no recent use of prednisone or antibiotics for the last 3 months.  After signing 
an informed consent, subjects will fill out questionnaires start a food diary, and have 3 weeks of 
wrist actigraphy prior to their seven day in lab session in the Clinical Chronobiology Center 
(CCC).  Since the participants will be in the sleep lab for 7 days, the questionnaire responses will 
be assessed for evidence of psychopathology, which is part of the exclusion criteria. Dr. Sharon 
Jedel, Ph.D., Associate Psychologist, will review the questionnaire responses before the 
participant finishes the screening. Participants who show some evidence of psychopathology on 
the questionnaires will undergo a structured interview (SCID-R) (~30 min) to confirm if they are 
ineligible.
Subject Characteristics: We will recruit (1) 20 HC subjects without any medical illnesses 
(2) 20 subjects with biopsy proven left sided UC, inactive (Mayo Score ≤ 2) on stable 
medications and no disease flares for > 3 months. HC and UC subject will be matched according 
to age (   3 years), sex, race, and BMI (   3 kg/m2).   
Study Outcomes: 
Primary outcome measures:
-
Aim 1 – Circadian Misalignment of central clock: Phase Angle will be measured as the 
difference between circadian clock (dim light melatonin onset) and behavior cycle (sleep 
onset)
-
Aim 2 – Barrier Function: Mayo Endoscopic Score and Intestinal permeability will be 
measured including 12-24-h urinary sucralose (primarily representing colonic 
permeability)
Secondary outcome measures:
-
Aim 1 – Circadian Misalignment of peripheral clocks: Clock gene expression (CLOCK, 
BMAL) in PBMCs during each phase assessment and in colonic biopsies
-
Aim 2 – Barrier Function: Colonic mucosal AJC proteins (E-cadherin, occludin, 
claudin2,3,4), stool calprotectin and mucosal and stool microbiota composition and 
function. Studies will include serum markers of intestinal permeability and bacterial 
translocation [lipopolysaccharide (LPS) and LPS-binding protein (LBP)]; serum zonulin 
and intestinal fatty acid protein; immune changes (serum interleukin-6 as 
proinflammatory marker and soluble CD14 as marker of macrophage/monocyte 
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activation); and stool and serum metabolomics: short-chain fatty acid (SCFA) and 
Trimethylamine N-oxide (TMAO) and TMA.
Exploratory Outcome Measures:
•
Aim 1 – Colonic clock gene expression and Per2::Luc activity in colonic organoids over 24 
hours in both groups and each condition
•
Aim 2 – RNA seq of colonic organoids every 2 hours for over 24 hours to identify key 
molecular and inflammatory pathways under circadian regulation
•
Aim 3:  Use body temperature to measure central circadian rhythm to see if it correlates 
with melatonin levels at baseline and after misalignment
Figure 1. Outcome Measures
Aims
Outcome 
Measure
Test
Assessment
Statistical Test
Baseline
Primary 
Phase Angle of entrainment 
(DLMO – sleep onset)
 
Misalignment
ANOVA
Baseline
Secondary 
Clock gene expression in 
PBMCs
Misalignment
Cosinor Analysis, 
Mixed Model 
ANOVA
Baseline
Secondary 
Urinary Melatonin
Misalignment
Wilcoxon signed-
rank test
Baseline
Aim 1
Exploratory
Clock gene expression and 
Per2::LUC in Colonic 
Organoids
Misalignment
Cosinor Analysis, 
FFT-NLLS
Baseline
Primary 
Intestinal Permeability  (i.e. 
12-24 h Urinary Sucralose)
Misalignment
Mann-Whitney U 
test
Baseline
Secondary
Mayo Score (Endoscopy)
Misalignment
Wilcoxon signed-
rank test
Baseline
Secondary 
Stool Calprotectin
Misalignment
Wilcoxon signed-
rank test
Baseline
Secondary 
Serum cytokines 
(IL-6, TNF-α)
Misalignment
Repeated 
measures ANOVA
Baseline
Secondary 
Fecal Microbiota
Misalignment
Principal 
Coordinate 
Analysis 
Baseline
Secondary 
Serum LBP, LPS, zonulin, 
sCD14, metabolomics
Misalignment
Repeated 
measures ANOVA
Aim 2
Exploratory
TER in 2D Colonic 
Baseline
ANOVA
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Organoids with ex-vivo TNF-
α/INF-γ 
Misalignment
Baseline
Exploratory
RNA seq of Colonic 
Organoids 
Misalignment
JTK Cycle
Safety and Adverse Events: We have identified five key potential risks to human subjects in this 
study:
1. Risk to Confidentiality and Record-Keeping of patient personal health information (PHI).
2. Risk of potential side effects associated with Phlebotomy (blood draw).
3. Risk of potential side effects associated with Sigmoidoscopy and Colonic Biopsy.
4. Risk of potential side effects associated with completion of the Questionnaires.
5. Risk of potential side effects associated with in lab study
Minimizing Risk:
1. Maintenance of Confidentiality and Record-Keeping. Subject confidentiality will be 
protected by maintaining all records of participation in this research project under a 
double lock and kept confidential as required by law. Electronic records are within the 
RUMC firewall and password protected. Data of subjects will only be associated with a 
coded identification number, having had all identifying information removed. Patient 
PHI information will be available only to the PI and clinical coordinators.
2. Minimize the risk of potential side effects associated with Phlebotomy. Blood will be 
drawn over two 24 hour periods.  The PI has significant experience with this protocol 
from previous studies. There may be some discomfort or bruising on initial insertion of 
the catheter into a vein, but wearing the catheter should not be painful.  Occasionally,  
mild  discomfort  may  occur  from  the  tube  in  the  vein.  If this  happens,  it  can  be 
repositioned or removed, asking the subject's permission before any subsequent 
reinsertion.  To help keep the venipuncture site clean, we may ask permission to shave 
the forearm hair of the subject prior to insertion of the IV.  There is a rare possibility of 
developing a small blood clot, inflammation, or local infection around the vein where 
the catheter is inserted, or in rare cases a generalized infection spread through the 
bloodstream as a result of the IV catheter.  Occasionally, there is a black and blue mark 
at the site of the IV insertion, which may last a couple of weeks; and, rarely, a small scar 
may remain permanently at the venipuncture site.  A standard laboratory protocol for 
surveillance for heparin-induced thrombocytopenia will be followed during the protocol. 
This consists of monitoring the subject’s platelet levels at baseline and every 2 days, 
when signs of any heparin-induced thrombocytopenia would be expected to appear. 
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There has not yet been a case of this in our laboratory in over 8 years of operation.  
Prior to insertion of the IV catheter, the skin around the insertion site will be washed 
with a Betadine solution followed by alcohol.  An anti-bacterial ointment will be applied 
to the IV catheter insertion site prior to dressing the site.  Hypoallergenic tape will be 
used on the IV dressing to minimize the development of contact dermatitis.  The IV 
catheter insertion site will be checked every 24-36 hours for signs of infection by 
removing the dressing and cleaning the site with alcohol. The IV catheter dressing, the 
IV bag/bottle, the IV tubing and manifold and the antibacterial ointment will be changed 
every 24-36 hours to minimize the potential for infection.  The IV catheter insertion will 
be done while the subject is supine, and a staff member will remain in the suite with the 
subject for at least 10 minutes after the insertion to ensure that the subject does not 
feel light-headed or dizzy. The subject’s vital signs will be checked at least once per day. 
This will include pulse rate, respiratory rate, systolic and diastolic blood pressure.
3. Minimize the risk of potential side effects associated with Sigmoidoscopy. The primary 
risk for most subjects undergoing sigmoidoscopy and biopsy is minimal discomfort 
during sigmoidoscopic examination. Risks include discomfort from air being added into 
the colon, bloating much like gas pain, possible irritation and a small amount of blood 
loss.  On extremely rare occasions (1 in 17,000), the procedure can cause a tear or hole 
in the lining of the colon or significant bleeding.  This may require surgery to repair.  The 
PI (GS), who will perform all sigmoidoscopic examinations, is an experienced 
gastroenterologist with extensive experience in endoscopy over the last 10+ years. He 
has never experienced any subject complications while performing thousands of 
sigmoidoscopies. Furthermore, these sigmoidoscopic examinations will be limited to the 
most distal 15 to 20cm of the rectum and sigmoid rather than the typical, routine 
examination that requires advancing the scope to 45 to 60 cm (this shorter 
advancement of the scope is adequate to determine whether there is active disease and 
to obtain biopsies). Thus, discomfort and risk will be markedly lower than routine 
testing.  Thus, unlike in routine sigmoidoscopy, we will do the procedure without any 
colon preparation (eliminate discomfort associated with laxative) and to a more limited 
extent. This limited procedure is not associated with any significant discomfort (no need 
to negotiate colon flexures that is the main source of discomfort associated with 
endoscopic procedures) and significantly decreases (and in fact eliminates) the tiny risk 
of perforation (< 1/10,000) associated with routine sigmoidoscopy. This opinion is based 
on our lab’s 30+ years of experience performing limited sigmoidoscopy in research 
subjects in our sections and also based on personal experience—the PI (GS) has been a 
“healthy control” subject in several prior studies and has undergone this limited 
sigmoidoscopy and biopsy.  The biopsy procedure is routine and painless with a tiny risk 
(< 1/10,000) of significant bleeding. The PI has obtained pinch mucosal biopsy 
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specimens from the colon from thousands of subjects including as many as 18-24 
biopsies on many occasions.  He has not had any complications or bleeding in any of his 
research subjects over the past 10+ years.  The expertise and experience of the PI and 
research team should significantly minimize the potential complications including 
bleeding and perforation. Plan to deal with complications. Subjects with possible 
complications will be seen by the PI immediately (within a few hours), regardless of the 
time of day. The PI has full access to the GI Suite 24/7.  These subjects will be evaluated, 
and if necessary, will undergo additional testing, such as CBC and KUB.  If there is 
evidence of bleeding or perforation, the subject will be admitted to the hospital for 
observation and additional therapy. As stated above, we do not expect any 
complications such as bleeding or perforation. Subjects will have blood tests, including 
prothrombin time and platelet count, to avoid biopsy procedures in those with a 
bleeding disorder.  
4. Minimize the risk of potential side effects associated with completion of the 
Questionnaires.  We have taken steps to reduce the risk of discomfort associated with 
answering medical/personal questions by keeping an individual’s responses completely 
confidential (including not sharing an individual’s responses with their physician), 
allowing participants to leave blank any questions that may make them uncomfortable 
on any of the forms, and providing brief assessment/counseling by the Division clinical 
psychologist or PI for any subject who becomes distressed during their participation in 
the study. If a participant appears to have difficulty reading, they can choose to have the 
questionnaires read to them.  
5. Minimize the risk of potential side effects associated with in lab stay.   The subject may 
become sleepy during some segments of the study. The subject will be asked to remain 
awake during the entirety of their scheduled wake times. Should the subject feel that 
he/she is unable to remain awake, he/she is free to withdraw his/her consent to 
participate in this experiment and then go to sleep.  At the end of the study, the subject 
may have difficulty sleeping and waking at their usual times. It may take several days to 
readjust to their regular routine. This experience is similar to jet lag, and may be 
associated with upset stomach, insomnia, irritability, or excessive daytime sleepiness. 
The protocol is designed so that changes in circadian phase are minimized. Subjects may 
not sleep as well in the laboratory as they do when at home. This may be exacerbated 
during the simulated night shift when subjects remain continuously awake. 
Nevertheless, short term partial sleep loss has never been shown to be directly 
deleterious to health although sleep loss can cause drowsiness and increase the risk of 
accidents. While dangerous accidents are unlikely in the controlled environment of the 
laboratory, we have less control when subjects leave the laboratory. Thus, we will 
ensure that the subject fully appreciates this risk and they will be given the opportunity 
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to have recovery sleep after completion of the protocol before leaving the laboratory. 
Taxi rides are arranged so that subjects can return home safely.
6. Minimize the risk of potential psychological stress and suitability of in lab stay.  
Subjects with any psychiatric history will be excluded from the study.  This includes 
individuals with a history of psychiatric illnesses or psychiatric disorders will be excluded 
such as alcoholism, drug dependency, major mood disorders such as major depression 
and manic depressive illness, schizophrenic disorders, anxiety disorders including panic 
disorder, generalized anxiety disorder, post-traumatic stress disorder, obsessive 
compulsive disorder, agoraphobia, claustrophobia, paranoid personality disorder, 
schizoid personality disorder, schizotypal personality disorder, borderline personality 
disorder, and antisocial personality disorder. Participants will complete questionnaires 
prior to their in-person stay in our sleep lab, which the study physician will review. If a 
participant feels distress during their stay, the study physician will be called. Participants 
may leave at any point if they feel psychological stress. 
Safety Monitoring Board: Although the study physician and coordinators will evaluate a 
participant’s comfort level prior to the participant staying in the sleep lab and during their in-
person stay, the study will have a Data Safety and Monitoring Board. The board will convene 
every six months to evaluate the study data for participant safety, study conduct, and progress, 
and make recommendations if applicable. This will ensure that the study participant’s safety is 
always being evaluated and improved. 
Cost to Participants: There is no additional cost to participants in this study. All study activities 
will be covered by the study grant. 
Payment to Participants: Participants may receive up to a total reimbursement of $2,000 for 
completing all study activities. Each participant will receive up to $1500 reimbursement for the 
cost of travel and time invested in the study along with an additional $500 compensation 
incentive for completing both in-laboratory protocols. Participants will receive $700 after 
completion of the Day 2 study activities, $500 after completion of Day 3 and Day 5 activities, 
and the remaining $800 once all study activities have been completed and the actigraphy watch 
has been returned to study staff. 
Data Management: Data and Biospecimens will be securely stored on-site at Rush University 
Medical Center for up to 6 years post study closure. Participants will be offered to participate in 
the department’s tissue repository long-term storage of their data and biospecimens for the 
purposes of future use. For any participants who do not consent to the tissue repository, their 
biospecimens will be destroyed and their data deleted 6 years post-study closure. 
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Confidentiality: Subject confidentiality will be protected by maintaining all records of 
participation in this research project under a double lock and kept confidential as required by 
law. Electronic records are within the RUMC firewall and password protected. Data of subjects 
will only be associated with a coded identification number, having had all identifying 
information removed. Patient PHI information will be available only to the PI and clinical 
coordinators.
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