Impact of Poor Sleep on Inflammation and the Adenosine Signaling Pathway in HIV Infection
Phase: NA
Conditions: HIV-1-infection, Sleep Deprivation, Inflammation
Sponsor: University of Pittsburgh

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Study Protocol  
  
  
  
  
  
Official Title: Poor Sleep and Inflammation in HIV-Infected Adults 
(SASH)  
  
  
  
ClinicalTrials.gov ID (NCT number): NCT03848325  
  
  
  
Protocol Date: 7/13/2023  
  
 
 
 
 
 
 
 
 
 
 
 
 
  
  
 
 
 
 
 

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Scientific Background   
  
Background:  
 
Although antiretroviral therapy (ART) has significantly increased the life expectancy of HIV-
infected individuals, HIV+ individuals are still at an increased risk of non-AIDS associated chronic 
diseases such as cardiovascular and pulmonary disease. This increased risk has been associated 
with persistent immune activation and increased levels of systemic inflammation. Indeed, people 
living with HIV (PLWH) with higher levels of inflammatory biomarkers have higher hazards of 
long-term mortality. Furthermore, this chronic inflammation and immune activation has been 
associated with premature cardiovascular disease and obstructive pulmonary disease. Even with 
virologic suppression and complete immune reconstitution on ART, PLWH have greater levels of 
immune activation and persistent inflammation compared to the HIV- population. Poor sleep, a 
common comorbidity observed among PLWH, may worsen the burden of long-term 
cardiovascular and pulmonary morbidity since it is also associated with both inflammation and 
inflammation-related long-term morbidity among HIV- individuals. 
 
We propose a novel mechanistic framework by which acute, sub-acute and chronic sleep 
disruption may synergistically interact with HIV infection to increase systemic inflammation 
leading to an increased risk of cardiopulmonary dysfunction. Both, poor sleep acting via reduced 
expression of adenosine receptors and HIV infection with reduced frequencies of T cells 
expressing the rate-limiting ectonucleotidase CD73 can lead to reduced adenosine signaling. This 
loss of the normal immunosuppressive actions of adenosine results in an unregulated inflammatory 
output predisposing to the long-term cardiovascular and pulmonary complications associated with 
HIV. The disrupted adenosine signaling may also feedback to perpetuate chronic sleep disruption.  
  
 Significance:  
 
Given the high prevalence of poor sleep among PLWH and the growing understanding of the 
adverse consequences of poor sleep, obtaining a better biological understanding of how poor sleep 
may interact with HIV infection not only will provide the rationale to develop public health 
interventions to improve sleep in this high-risk population, but also will inform the identification 
of novel interventions that might minimize the adverse effects of poor sleep and improve non-
infectious outcomes in PLWH. Drugs that modulate adenosine signaling (such as dipyridamole 
which increases extracellular adenosine levels) are already clinically available and could be readily 
utilized to improve outcomes if our hypotheses are proven accurate.  
This proposal is highly innovative in being the first to test the impact of poor sleep on 
adenosine signaling in lymphocytes and monocytes, a pathway already established to be impaired 
in the setting of HIV infection and hypothesized to contribute to the persistent inflammation and 
long-term cardiopulmonary morbidity observed in PLWH. Further, this proposal will be the first 
to directly test the hypothesis that poor sleep and HIV infection have a synergistic impact on 
inflammation through the adenosine pathway.  
  
From a methodologic standpoint, this will be one of the first large-scale assessments of sleep 
in an HIV+ population to use objective assessments of sleep. In addition, we will use a multi-
dimensional framework to comprehensively assess sleep. Finally, we will separately assess both 
long-term effects of chronically poor sleep as well as short-term effects of acute and sub-acute 

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sleep loss, providing a comprehensive understanding of the interaction between sleep, 
inflammation, and adenosine signaling that can lead to the development of intervention strategies 
to decrease the levels of inflammation and subsequently the risk for cardiopulmonary 
comorbidities in PLWH.  
  
Study Objectives  
 
In this proposal we seek to test the hypothesis that one mechanism by which poor sleep may 
impact inflammation and cardiopulmonary risk in people living with HIV is via effects on 
peripheral adenosine signaling. We will do this by performing a cross-sectional evaluation to test 
Hypotheses 1-2 and an interventional study to perform a within-subject analysis to test Hypothesis 
3.  
  
Hypothesis 1: Objectively measured poor sleep is associated with greater levels of inflammation 
and markers of cardiopulmonary disease among antiretroviral therapy (ART)-treated people living 
with HIV (PLWH) compared to those without HIV.  
  
Hypothesis 2: Among ART-treated PLWH, downregulation of the adenosine pathway is associated 
with objectively measured markers of poor sleep.  
  
Hypothesis 3: Acute sleep deprivation in ART-treated PLWH does not produce a compensatory 
increase in extracellular adenosine generation and signaling, leading to increased inflammation 
and endothelial dysfunction.  
  
Study Design & Methods  
  
Research Activities:  
 
Recruitment  
Individuals with and without HIV will be recruited through advertising in local longitudinal 
research cohorts studying PLWH, by advertising through clinics that care for PLWH and patients 
at high risk for developing HIV, and through platforms developed for recruiting people in the 
community interested in participating in research. Telephone screening will be performed to assess 
eligibility and review the study protocol. Individuals who meet eligibility criteria and are interested 
in participating will be scheduled for an in-person study visit.   
  
Cross-Sectional Protocol  
   
Visit 1  
A point-of-care urine pregnancy test will be given to women of child-bearing potential. If the 
pregnancy test is positive, the participant will be ineligible for the study and procedures will be 
discontinued. The participant will then be asked to complete a battery of interviewer-administered 
questionnaires. This battery consists of questions on demographics, education, work schedule, 
caffeine use, nicotine use, and the reduced Morningness Eveningness Questionnaire (rMEQ) to 
assess circadian preference. Participants will have a wrist actigraph (Respironics Spectrum) placed 
on the non-dominant wrist and instructed to wear for two weeks. They will be given instructions 

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on pressing the event monitor whenever getting into or out of bed and also asked to complete a 
sleep diary each morning on awakening. Participants will also be given a wrist device for home 
sleep apnea testing (Itamar Medical WatchPAT) to place on their dominant wrist to wear for one 
night while they are sleeping. They will be given instructions for activating and wearing the device 
as well as proper placement of the fingertip sensor cap. Participants will be asked to return the 
WatchPAT device at Visit 2 where the data will be downloaded and reviewed. One week after the 
visit, a staff member will call participants to remind them to continue wearing the actigraph and 
completing the sleep diary.  
  
Visit 2  
Visit 2 will take place as soon as possible at least two weeks after Visit 1. Participants will be 
instructed to continue to wear the actigraph until this visit and to bring the completed sleep diaries 
with them to this visit. Actigraphy data will be downloaded and reviewed as will the sleep diaries. 
If data are incomplete (< one week of complete data), participants will be asked to repeat sleep 
diary and actigraphy testing. They will be issued a new actigraph and diaries to wear for an 
additional two weeks and be rescheduled to complete Visit 2 activities described below. Urine will 
be collected and frozen for adenosine analysis. Phlebotomy will be performed by research staff to 
obtain 100cc of blood. One tube will be prepared with a solution that specifically blocks adenosine 
metabolism and the rest will be standard Na-EDTA tubes. A battery of interviewer-administered 
questionnaires will be administered including: the Pittsburgh Sleep Quality Index, Epworth 
Sleepiness Scale, Insomnia Severity Index, Munich Chronotype Questionnaire, PROMIS 8a sleep 
disturbance scale, PROMIS 8b sleep-related impairment scale, eight-item Patient Health 
Questionnaire, seven-item Generalized Anxiety Disorder screener, Alcohol Use Disorders 
Identification Test, and the ten-item Drug Abuse Screen Test. At the end of Visit 2, staff will ask 
permission from HIV+ participants to be contacted at a later date to present the interventional 
study and discuss possible enrollment.  
  
Interventional Protocol  
  
Upon detailed analysis of actigraphy data from the cross-sectional study, HIV+ participants 
who may be eligible for the interventional study will be contacted by telephone. Staff will review 
the protocol with the participant, and if they are interested, screen them to confirm that they still 
meet inclusion criteria. They will also be screened for any changes in their medical history or 
medications since they participated in the cross-sectional study as these changes might make them 
ineligible based on exclusion criteria for Aim 3.  
  
Visit 3  
At Visit 3, eligibility for the interventional protocol will be evaluated by administering a 
pregnancy test to women of childbearing potential followed by administration of number of 
questionnaires. Participants will be asked about caffeine and nicotine use as well as the Mood 
Disorder Questionnaire and Ask Suicide-Screening Questions to identify those at elevated risk for 
adverse effects from sleep deprivation (bipolar disorder episode and suicide risk, respectively). 
The PSQI (used in Visit 2) will be repeated to help confirm that the participant has had a regular 
sleep schedule in the month prior to the study. A medical history and physical examination 
(including an evaluation of drug/alcohol use) will be completed by a study physician. The protocol 
will be reviewed and informed consent obtained. The participant will then be given a wrist 

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actigraph, sleep diaries, and asked to maintain a regular sleep schedule (both regular bedtime and 
regular duration of at least seven hours of sleep per night) until Visit 4.  
   
Visit 4  
Participants will be asked to maintain their normal sleep patterns during the week prior to this 
visit. They will arrive in the sleep lab at 6 p.m., and the actigraph will be removed and data 
reviewed to confirm the participant maintained a regular sleep schedule. In addition, the sleep 
midpoint will be calculated from the actigraphy data to define the participant’s normal 8-hour 
period of sleep (lights-off and lights-on) and determine the timing of all study procedures in 
relation to it. In other words, those with a sleep midpoint of 3 a.m. lights-out for the sleep study 
will be 11 p.m. and lights-on will be 7 a.m. For those with a sleep midpoint of 2 a.m., lights-out 
would be 10 p.m. with lights-on at 6 a.m., and for those with a sleep midpoint of 4 a.m., it would 
be 12 a.m. and 8 a.m. The single-item Karolinska Sleepiness Scale (KSS) and the 10-minute 
Psychomotor Vigilance Task (PVT) will be administered prior to sleep. The participant will then 
be prepared for full polysomnography with application of EEG, EOG, ECG, chin EMG, and 
pretibial EMG electrodes. A nasal cannula will measure nasal airflow, a thermistor will measure 
body temperature and a pulse oximeter will be placed on the finger to measure oxygen. Respiratory 
effort belts will be placed around the chest and the abdomen. Four hours after the participant’s 
calculated sleep midpoint, the lights will be turned on and the participant awoken. The KSS and 
the PVT will again be administered. After this, the participant will be sent home. Individuals found 
to have sleep-disordered breathing (AHI>15 events/hr) on overnight polysomnography will be 
excluded from further testing.  
  
Visit 5  
The timing of all procedures of Visit 5 is relative to the participant’s sleep midpoint. The 
descriptions in the Figure below are based on a hypothetical midpoint of 3 a.m. and will be adjusted 
accordingly to maintain the same amount of time between each procedure and the participant’s 
actual sleep midpoint. For those participants who are eligible to complete this protocol, they will 
need to abstain use of caffeine, alcohol, nicotine or any illicit drugs for the entirety of their two-
day visit.  
 
 
  
  
  
 

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Day 1  
Participants will arrive to the sleep lab by 6 p.m. The KSS is administered upon arrival and 
then every hour until lights-out, which will occur four hours before the participant’s sleep 
midpoint. The PVT is administered upon arrival and then every two hours until lights-out. The 
Positive and Negative Affect Schedule (PANAS) is administered upon arrival and then every four 
hours until lights-out. Overnight polysomnography with a limited montage using only EEG, EOG, 
and chin EMG will be performed in order to assess sleep/wake state and sleep staging. Lights-on 
will be four hours after sleep midpoint. Upon awakening in the morning, participants will provide 
a urine sample and have a 100cc blood sample drawn. The KSS, PVT, and PANAS will again be 
administered every hour, every two hours, and every four hours respectively, beginning one hour 
after lights-on. Vascular ultrasound for brachial artery reactivity will be performed as described 
below. In addition to abstaining from caffeine, alcohol, nicotine, and illicit substances for the entire 
visit, participants will need to fast (except water) from the time of lights out through the end of 
vascular testing. Morning antihypertensive doses will be delayed until after vascular testing is 
completed. Breakfast will be served after this vascular testing. Participants will stay onsite all day, 
and staff will remain with them at all times (except bathroom breaks) to ensure their safety and to 
make sure that they stay awake with no daytime naps.  
  
Vascular Testing  
After 10 minutes of lying quietly, brachial artery diameter and flow velocities will be measured 
with high-resolution ultrasound equipped with an 11-L linear transducer. Image acquisition will 
be ECG-gated, so all measurements are done at end diastole. The brachial artery will be identified 
in the longitudinal plane above the antecubital fossa and a segment with clear anterior and posterior 
intimal interfaces between the lumen and vessel wall will be selected for continuous 2D-grayscale 
imaging. After obtaining baseline images, blood pressure cuff on the arm will be inflated to 50 
mm Hg above resting systolic blood pressure (up to 300 mm Hg) for five minutes. After deflation, 
images will be obtained continuously and the maximal diameter between 30 seconds and two 
minutes used for calculating change. Endothelial function will be quantified as the ratio of the 
maximum diameter identified following cuff release to baseline diameter.  
  
Day 2  
Participants will stay awake for the remainder of procedures. Study staff will remain with the 
participants at all times (except for during bathroom trips) during sleep deprivation to monitor 
mood and to keep them awake through conversations, playing board games, and watching 
television. The KSS, PVT, and PANAS will be administered every hour, every two hours, and 
every four hours, respectively. The participants will again be prepared with limited montage 
polysomnography equipment described above and will need to remain fasting (except water) 
starting four hours prior to sleep midpoint until vascular testing is completed (the same time frame 
as the prior day). In the morning, the participant will provide a urine sample and have 100cc of 
blood drawn at the same time as the previous day. Vascular testing will be performed as described 
above, after which breakfast will be served. The KSS, PVT, and PANAS will continue to be 
administered until seven hours after the participant’s normal sleep midpoint time. At this point the 
participant will be provided the opportunity to have eight hours of recovery sleep, and staff will 
encourage them to do so for their safety before returning home. They will be allowed to leave 
without recovery sleep if another adult is able to drive them home.  
  

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Eligibility Criteria  
  
Aims 1-2  
Inclusion- HIV+  
- 18-75 years old  
- On contiguous anti-retroviral therapy (ART) regimen for at least 48 weeks  
- CD4+ cell count > 200 cells/mm3  
Inclusion- HIV- (Aim 1 only)  
- 18-75 years old  
*Note: HIV- participants are only eligible for participation in Aim 1.  
Exclusion- all  
- Presence of a significant sleep disorder (restless legs syndrome, narcolepsy, sleep disordered 
breathing on active treatment)  
- Presence of an active chronic inflammatory or autoimmune disease (e.g., scleroderma, Crohn’s 
disease)  
- Regular use of medications which would impact assessment of inflammatory markers (systemic 
glucocorticoids, interleukins, anti-TNF drugs)  
- Use of medications impacting adenosine function (theophylline or dipyridamole)  
- Any initiation of an investigational/experimental therapy in the prior 30 days  
- Current pregnancy  
  
Aim 3  
Inclusion 
- HIV+  
- Completion of Aim 1 with at least 5 days of valid actigraphy data  
- On continuous anti-retroviral therapy (ART) regimen for at least 48 weeks  
- CD4+ cell count > 200 cells/mm3  
Exclusion  
- Atypical sleep patterns (mean sleep duration < 6 hours, sleep efficiency < 85%, standard 
deviation of sleep duration > 60 minutes, sleep midpoint < 2 a.m., sleep midpoint > 5 a.m.)  
- Elevated risk of adverse health effects from sleep deprivation (bipolar disorder, epilepsy, history 
of suicidal ideation in the past 6 months)  
- Heavy caffeine use (>3 caffeinated beverages per day)  
- Alcohol abuse  
- Drug abuse  
- Daily use of medications for treatment of insomnia  
- Sleep disordered breathing (AHI >15 on in-lab sleep study)  
- Current pregnancy  
  
Statistical Analysis Plan  
  
Hypothesis 1:   
The primary exposures will be four domains of sleep health: sleep duration (mean hrs/night 
during the main rest period), sleep fragmentation index (mean across main rest periods), sleep 
regularity (interdaily stability over a 14-consecutive day period), and sleep apnea severity (pAHI). 
Secondary measures will be mean sleep midpoint, mean diary-reported sleep quality, and 

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proportion of days with naps. The primary measures of inflammation will be serum IL-6 and 
sCD14 levels. Secondary measures will include the other 12 cytokines both inflammatory and anti-
inflammatory on the Luminex platform including TNFα, CRP, IL-10, and sCD163. All 
inflammatory biomarkers will be transformed as needed to approximate a normal distribution. 
Given that some sleep exposures (particularly sleep duration) have U-shaped associations with 
cardiovascular outcomes, we will initially model sleep exposures in quintiles to assess for non-
linear relationships with inflammation measures. If a linear association is confirmed, we will then 
model sleep exposures continuously to maximize power and use multivariable linear regression to 
regress IL-6 and sCD14 levels on each of the three sleep exposures among HIV+ subjects. Initial 
analyses will adjust for age and sex. Alternatively, we will use fractional polynomial regression to 
test the nonlinear relationship between sleep measures and inflammatory markers. In all models, 
we will further adjust for CD4+ T cell count and viral load to determine if sleep exposures are 
associated with inflammation in HIV+ subjects independent of severity of infection. Subsequently, 
we will generate models including all four sleep exposures simultaneously to understand the 
independent effects of each sleep domain. If needed, we will apply dimension reduction methods 
(e.g., principal components analysis) to overcome collinearity in sleep domains. Secondary 
analyses will utilize structural equation modelling to evaluate the extent to which identified 
associations represent effects mediated by sleep directly versus effects specific to depression. 
Furthermore, we will stratify by sex to separately evaluate the association between sleep and 
inflammation in men and women. Formal testing of effect modification will be performed by 
including an interaction term in the multivariable models. We will repeat models in HIV- subjects 
and then test whether HIV infection moderates the association between sleep and inflammation by 
modeling data from those with and without HIV infection in one model with a sleep by HIV 
interaction term, using a two-sided p<0.05 as the criterion for rejecting the null hypothesis of no 
effect modification.  
  
Hypothesis 2:  
We will compare the frequencies of immune cells expressing ectonucleotiinterdases (CD39+ 
T cells, CD73+ T cells, and CD39+ CD73+ T cells), monocyte adenosine receptor expression 
(A1R and A2AR), ADO and inosine levels, and measures of T cell activation between those HIV+ 
subjects with healthy and poor sleep. Ectonucleotidase expression on B cells and monocytes, 
additional purine pathway metabolites, and frequencies of CD14+ monocytes expressing IL-6 
and/or TNFα will also be compared in secondary analyses. Analyses will initially use t-tests for 
normally distributed variables or Mann Whitney tests for non-normal distributions. Subsequent 
analyses will consider potential confounders (age, sex, CD4+ T cell count, viral load, habitual 
caffeine use, chronotype, sleep midpoint) and adjust in multivariable linear regression for the set 
of variables that differ between the healthy and poor sleep groups at p<0.10. Sex-related 
differences will be tested by evaluating the statistical significance of a sex * sleep interaction term.  
  
Hypothesis 3:   
The primary outcome measures are levels of soluble CD14, soluble CD163, and circulating 
IL-6 levels. Secondary outcome measures are flow mediated dilation (percent change in brachial 
artery diameter to reactive hyperemia), monocyte expression of IL-6, monocyte expression of 
TNFα, frequency of CD4+ T cells expressing HLA-DR and CD38, and frequency of CD8+ T cells 
expressing HLA-DR and CD38. Additional pre-specified outcomes include plasma adenosine and 
inosine levels, urinary 3’5’-cAMP, and frequencies of CD4+ and CD8+ T cells expressing CD39 

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and CD73. The impact of sleep deprivation will be assessed by performing paired t-tests of each 
of these outcomes comparing baseline to post-sleep deprivation values. For variables with non-
normal distributions, we will either transform variables or use Wilcoxon signed-rank tests. The 
correlation between the changes in each domain (expressed as percent change from baseline) will 
be calculated to assess the extent to which change in endothelial function may be predicted by 
change in inflammation or ADO signaling and change in inflammation by change in ADO 
signaling. Secondary analyses will consider the extent to which sex, CD4+ T cell count, and viral 
load impact sensitivity to sleep deprivation by regressing each of these potential effect modifiers 
on the percent change in each of the primary outcome measures for inflammation, endothelial 
function, and ADO signaling.