Neuroinflammatory Mechanisms Linking Chronic Stress to Motivational Deficits
Phase: PHASE4
Conditions: Stress, Psychological, Motivation
Sponsor: University of North Carolina, Chapel Hill

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Neuroinflammatory Mechanisms Linking Chronic Stress to Motivational 
Deficits Study Protocol, including Statistical Analysis Plan
NCT number NCT06044090
  Document Date   09/13/2023 

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Master Protocol Document 
 
Title 
Neuroinflammatory Mechanisms Linking Chronic Stress to Motivational 
Deficits  
Sub-Title 
 
The Brain and Stress Study 
 
Principal Investigator 
Gabriella M. Alvarez, Department of Psychology & Neuroscience 
 
Co-Investigators 
 
Keely A. Muscatell, Department of Psychology & Neuroscience 
Mary C. Kimmel, Department of Psychiatry 
Zev Nakamura, Department of Psychiatry 
Elizabeth “Ellie” Richardson, Department of Psychiatry 
Version  
13 September 2023    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Statement of Confidentiality and Nondisclosure     This document is a confidential communication.  Acceptance of this document 
constitutes agreement by the recipient that no unpublished information contained herein will be published or disclosed without 
prior written approval of the Principal Investigator or other participating study leadership, except that this document may be 
disclosed in any medium to appropriate investigators, Institutional Review Boards, Scientific Review Committees, and others who 
are directly involved in the study specified herein under the condition that they keep the information confidential. 
 
 

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Table of Contents 
Statement of Compliance ............................................................................................................................... 3 
Table of Abbreviations ................................................................................................................................... 4 
1. 
Protocol Synopsis ............................................................................................................................... 5 
2. 
Introduction: Background and Rationale ............................................................................................ 7 
2.1. 
Background Information ................................................................................................................. 7 
2.2. 
Scientific Rationale .......................................................................................................................... 9 
3. 
Specific Aims ...................................................................................................................................... 9 
4. 
Investigational Plan .......................................................................................................................... 10 
5. 
Study Participants ............................................................................................................................ 12 
5.1. 
Numbers of Participants ................................................................................................................ 12 
5.2. 
Eligibility Criteria ........................................................................................................................... 12 
5.3. 
Randomization and Blinding .......................................................................................................... 13 
5.4. 
Recruitment and Retention ........................................................................................................... 14 
6. 
Schedule of Activities and Procedures .............................................................................................. 14 
6.1. 
Table of Events.............................................................................................................................. 14 
6.2. 
Screening ...................................................................................................................................... 14 
6.3. 
Enrollment .................................................................................................................................... 16 
6.4. 
Study Visit Procedures .................................................................................................................. 16 
6.5. 
Final Debriefing Visit .................................................................................................................. 17 
6.6. 
Phone Contacts .......................................................................................................................... 17 
6.7. 
Assessing Medication Compliance ........................................................................................... 17 
7. 
Statistical Analysis Plans ........................................................................................................... 18 
8. 
Sample Size Rationale ................................................................................................................. 20 
9. 
Data Collection and Management .................................................................................................... 20 
10. 
Safety Monitoring and Management ............................................................................................... 21 
10.1. 
Risk / Benefit Assessment ............................................................................................................. 21 
10.2. 
Safety Monitoring ......................................................................................................................... 22 
10.3. 
Adverse Events and Serious Adverse Events Reporting Procedure ................................................. 23 
11. 
Regulatory, Ethical, and Study Oversight Specifications ................................................................... 25 
11.1. 
Informed Consent Process ............................................................................................................ 25 
11.2. 
Confidentiality and Privacy ............................................................................................................ 25 
11.3. 
Key Roles and Study Governance .................................................................................................. 26 
12. 
Appendices ....................................................................................................................................... 26 
13. 
References ........................................................................................................................................ 27 
 

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Statement of Compliance 
This study will be conducted as specified in the protocol and in accordance with the International Conference on 
Harmonisation Guidelines for Good Clinical Practice (ICH E6) and the Code of Federal Regulations on the Protection 
of Human Subjects (45 CFR Part 46).  
The protocol, informed consent form(s), recruitment materials, and all participant materials will be submitted to 
the Institutional Review Board (IRB) for review and approval.  Approval of both the protocol and the consent form 
must be obtained before any subject is enrolled.  Any amendment to the protocol will require review and 
approval by the IRB before the changes are implemented to the study.  In addition, all changes to the consent 
form will be IRB-approved; a determination will be made regarding whether a new consent needs to be obtained 
from subjects who provided consent, using a previously approved consent form. 
If required by the IRB, the master protocol document, informed consent form(s), recruitment materials, and all 
subject materials will be submitted to the Scientific Review Committee (SRC) prior to IRB review 
(research.unc.edu/clinical-trials/src). 
The statistical analysis plans will be consistent with guidance in CONSORT Statement [1] or STROBE Statement [2], 
ICMJE recommendations [3], the 2016 and 2019 statements of the American Statistical Association [4,5], and 
recommendations in Nature [6,7].*  
All personnel involved in the conduct of this study have completed human subjects protection training. 
 
 
 
 
 
 
 
 
 
 
*  [1]   www.consort-statement.org  
   [2]  www.strobe-statement.org  
   [3]  www.icmje.org  
   [4]  Wasserstein RL, et al. (2016), The ASA's Statement on p-Values, The American Statistician, 70:2, 129-133  
   [5]  Wasserstein RL, et al. (2019), Moving to a World Beyond p < 0.05, The American Statistician, 73:sup1, 1-19  
   [6]  Amrhein, et al. (2019) Scientists rise up against statistical significance, Nature 567, 305-307  
   [7]  Editorial (2019) It’s time to talk about ditching statistical significance: Looking beyond a much used and abused measure 
          would make science harder, but better. Nature 567, 283-283. 

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Table of Abbreviations 
AE/SAE 
Adverse Events/ Serious Adverse 
Events 
CI 
Confidence Intervals 
CNS 
Central Nervous System 
HIV 
Human Immunodeficiency Virus 
HRF 
Hemodynamic Response Function 
IDS 
Investigational Drug Service  
NSAID 
Non-steroidal anti-inflammatory drugs 
OHRE 
Office of Human Research Ethics  
PET 
Positron Emission Tomography 
PI 
Principal Investigator 
SE 
Standard Error 
SHAPS 
Snaith Hamilton Pleasure Scale  
TBI 
Traumatic Brain Injury 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1. Protocol Synopsis 
 
Title 
Neuroinflammatory Mechanisms Linking Chronic Stress to 
Motivational Deficits 
Study Description 
Motivational deficits such as anhedonia are core to several 
psychiatric disorders and underlie significant functional 
impairment. This double-blind, placebo-controlled crossover trial of 
minocycline, an anti-[neuro]inflammatory agent, examines links 
between chronic stress and responses to a reward-related 
motivation task. It will evaluate the effects of pharmacologically 
attenuating neuroinflammation on behavioral responses to a 
reward-related motivation task in individuals experiencing 
unemployment. Understanding the effects of neuroinflammation on 
reward function among individuals experiencing chronic stress 
represents a critical first step in identifying novel neuroimmune 
targets for future clinical trials. 
Specific Aims 
(objectives) 
Aim 1: To examine differences in performance on a pure 
motivation task following minocycline (vs placebo).  
Aim 2: To examine differences on self-report measures of 
motivation following minocycline (vs placebo).  
Target Population 
Inclusion criteria:  
• 
25-60 years old  
• 
Unemployed (working less than 20 hours per week)  
• 
Seeking employment  
• 
Having trouble finding job (i.e., actively seeking and 
applying for jobs but not successful in landing a job)  
• 
Reports greater than 5 points on Job Stress Items  
• 
Regular access to a mobile phone   
Exclusion Criteria:  
• 
Currently enrolled as a student 
• 
Prescribed medication for the following physical illnesses: 
diabetes, cardiovascular diseases, high blood pressure, 
autoimmune disease, neurological conditions (e.g., TBI, 
stroke)  
• 
Diagnosis of a severe mental illness (i.e., disorders evincing 
psychotic symptoms, severe major depression, bipolar 
disorder)  
• 
Pregnant or breastfeeding  
• 
Daily psychotropic medications including mood stabilizers, 
stimulants, antipsychotics.  
• 
Daily antidepressants use that has been recently added or 
modified in fewer than 3 months.   
• 
Drug or alcohol dependence 
• 
Known allergies or hypersensitivities to tetracycline 
antibiotics, aspirin or other NSAIDs  
• 
Current antibiotic use  
• 
Regular use of steroidal or non-steroidal anti-inflammatory 
medications (i.e., 2 or more times a week) 
Numbers of Enrollees 
A total of N = 54 eligible individuals will be enrolled. We anticipate 
that at least n = 50 of the enrollees will complete all aspects of the 
protocol and have complete data.                       

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Interventions   
In this crossover design, all participants will take a 5-day course of 
200mg of minocycline and 5 days of a placebo-control pill. 
Sessions will be separated by a minimum washout period of 14 
days. 
Outcome Measures 
For Aim 1.   Mean difference in accuracy and reaction time to 
Progressive Reward Task during reward receipt after minocycline 
course and placebo course. 
For Aim 2.   Mean difference in cognitive performance after 
minocycline course versus placebo course 
Statistical Analysis Plans  
for Each Aim  
Aim 1 Plans. A paired samples t-test will be conducted to assess whether 
taking minocycline was associated with significantly better performance on the 
PRT as compared to when participants are taking a placebo.  
Aim 2 Plans. A paired samples t-test will be conducted to assess whether 
taking minocycline was associated with differences on two self-report measures 
of motivation following minocycline (vs placebo). 
 
All outcome variables of interest are continuous measures. All 
statistical estimates of population parameters and corresponding 
confidence intervals (CIs) will be computed to convey levels of 
precision. 
Study Duration 
The entire study is expected to last 12 months, depending on how 
quickly a participant can be scheduled for the study sessions. 
Ideally, both sessions will be completed at least 32 days after the 
initial phone screen.  
Participation Duration 
Participation includes:  
1) a screening session for assessing inclusion/exclusion eligibility,  
2) a baseline onboarding visit and receipt of arm 1 of intervention 
(~1.5 hr) 
3) 5-day course of minocycline (or placebo depending on 
randomization) 
4) behavioral study session 1 with computer tasks (~1.5 hours) 
5) 5-day course of placebo (or minocycline depending on 
randomization) 
6) behavioral study session 2 with computer tasks (~1.5 hours) 
 
Minimum enrollment period for participants is 32 days. 
 
 
 

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2. Introduction: Background and Rationale 
2.1. Background Information 
Motivational deficits such as anhedonia are common to several psychiatric conditions, including 
depression1 and schizophrenia 2. Moreover, these deficits are notoriously difficult to treat and predict 
poorer treatment response 3, greater chronicity of illness 4, significant functional impairment 2, and 
suicidality 5. The lack of efficacy for improving motivational deficits in current psychiatric treatments is 
mainly due to a lack of understanding of the mechanisms that give rise to these transdiagnostic symptoms. 
One significant risk factor that promotes decreases in motivation is chronic stress 6. In fact, many preclinical 
models of anhedonia rely on chronic stress paradigms to induce reductions in motivation and elicit 
depression-like symptoms 7. This preclinical work has demonstrated that a critical pathophysiological 
model linking chronic stress to deficits in motivation is via neuroinflammatory processes 8. Considering 
these findings, there is a paucity of work examining the mechanisms linking stress, neuroinflammation, 
and the neurobehavioral underpinnings of motivation in humans. Translational research aiming to replicate 
and extend preclinical research in a clinical sample is critical for elucidating the brain mechanisms 
underlying the reward-related motivational deficits implicated across several disorders. Thus, this project 
incorporates an experimental therapeutics approach and computational neuroscience techniques to 
generate information about the neuroinflammatory mechanisms contributing to stress-related motivational 
deficits. This mechanistic clinical trial will utilize minocycline to reduce inflammation in the brain in a sample 
of chronically stressed individuals to examine molecular to neural alterations implicated in motivational 
deficits. Results from the proposed study will shed light on neuroimmune targets for future prevention and 
intervention efforts.  
Evidence linking chronic stress to anhedonia via neuroinflammatory processes. Although less work 
has examined the effects of stress on anhedonia in humans, evidence across rodents and humans 
converges to suggest that chronic stressors contribute to the development of motivational deficits and a 
lack of responsiveness to rewards6,9. In rodents, chronic stress causes anhedonia-like behavioral 
changes10 (e.g., prolonged immobility, reduced preference for sweetness, and reduced exploratory 
activity); and in humans, acute and chronic stressors are associated with motivational deficits (e.g., motor 
slowing 11, reductions in pleasure12, and reduced ability for participants’ to modulate behavior as a function 
of rewards 13,14) as well. Additionally, several studies have found that stress-induced remodeling occurs in 
the corticostriatal circuit (i.e., medial prefrontal cortex, basal ganglia) to attenuate the reward-seeking and 
goal-directed behaviors involved in motivation15–18. However, despite the evidence linking stress to 
motivational deficits, more research is needed to understand the stress-induced molecular mechanisms 
driving corticostriatal alterations in humans. 
While multiple neurobiological pathways are likely relevant for understanding the influence of chronic 
stress on motivational processes, preclinical work identifies neuroinflammation as a key molecular 
mechanism underlying anhedonia. Indeed, chronic stress leads to changes in the central nervous system 
(CNS) innate immune cells19, including microglia, astrocytes, and other CNS-infiltrating macrophages. As 
the brain’s resident immune cells, microglia are studied as markers of neuroinflammation20,21 and are 
responsible for actively surveying the environment while also maintaining homeostasis to ensure a healthy 
nervous system. Functionally, neuroimmune cells such as microglia are critical for defending against 
infectious and stress derived-agents as well as purging damaged or unnecessary neurons and synapses19. 
Consequently, chronic stressors can damage the CNS by modifying microglial threshold of activation, 
ultimately giving rise to a maladaptive brain immune response22,23.  
The clinical work examining the role of neuroinflammatory processes underlying anhedonia is in its 
infancy. Hence, an understanding of the neuroinflammatory disruptions to network functioning and 
communication that may contribute to anhedonia in humans is severely lacking. Nevertheless, there is 

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some evidence that neuroinflammation is an issue in patients experiencing conditions characterized by 
motivational deficits. Specifically, post-mortem and PET imaging studies have found that 
neuroinflammation is elevated in patients with depression and schizophrenia 24–30. While these studies 
provide a critical first step in demonstrating the association between neuroinflammation and motivational 
deficits in patient populations, studies that examine the links between precursors and consequences of 
neuroinflammation in these living patient populations are necessary. 
Although a link between chronic stress and microglia has been established, what lacks understanding 
is the relationship between alterations in these neuroimmune cells and neural functioning relevant to 
motivated behavior in humans. In other words, how exactly does chronic stress engender motivational 
deficits through alterations in neuroinflammatory processes? Limited preclinical evidence demonstrates 
that alterations in microglial cell function due to chronic stress may be associated with structural and 
functional changes in the brain 32 and regions within the corticostriatal circuit 33. Most relevant to the current 
proposal, experimental evidence in rodents document that neuroinflammatory processes driven by 
microglia can result in motivational deficits via alterations in neuron-to-glia communication 34–36, 
neurogenesis 37–39, neuroplasticity 40–43, long term potentiation 44, and neurotransmitter systems 45–47. For 
instance, microglial activation has been associated with the selective degeneration of dopamine-producing 
nerve cells 48. This is interesting considering that dopamine has been implicated as a critical mediator 
between neuroinflammation and social behavior 49. Certainly, microglia produce disturbances in striatal 
dopamine function and reward-related behavior 50–52. These findings indicate that more clarity regarding 
the neuroimmune to neuron interface can be critical for understanding the neurobiology of motivational 
deficits. A clinical extension of this work, then, should examine how altering levels of neuroinflammation in 
humans may influence corticostriatal activity while engaging in a motivation task. 
What is minocycline? Because neuroinflammation, especially microglia, is implicated in anhedonia, 
pharmacological strategies to suppress microglial activity or blunt neuroinflammatory processes have been 
explored as therapies for depression and schizophrenia. One such treatment is minocycline, a tetracycline 
antibiotic commonly used to study neuroinflammatory mechanisms in preclinical work 53–56. Specifically, 
minocycline has been found to attenuate neuroinflammation’s deleterious effects on neurogenesis 57, long-
term potentiation 58, and neuronal survival 59. Minocycline’s anti-inflammatory mechanism of action is 
thought to involve an ability to inhibit microglia functioning 53,54. Unlike most anti-inflammatory agents, 
minocycline’s lipid-solubility allows for its penetration into the CNS 60. Indeed, minocycline has been shown 
to only reduce pro-inflammatory levels of the cytokine IL-1β in the brain and not plasma levels of the same 
cytokine in the periphery 61, validating that minocycline specifically alters neuroinflammatory processes. 
This relationship was further substantiated in a mechanistic study, which showed that minocycline reduced 
experimentally induced neuroinflammation and anhedonia-like behaviors in rodents by limiting microglial 
functioning 61. The authors also reported that minocycline consistently attenuated the microglial expression 
of pro-inflammatory response genes across four independent experiments. Although minocycline trials in 
clinical samples are significantly limited, there is some evidence that minocycline also attenuates microglial 
functioning in humans62 and has been shown to reduce negative symptoms scores (including anhedonia) 
among individuals with schizophrenia 63,64. Thus, minocycline would be a candidate drug to map 
neuroinflammatory effects on neural functioning. The proposed minocycline mechanistic trial would be an 
initial yet necessary step in understanding the molecular and neural alterations underlying motivational 
deficits. The results obtained here may have implications for identifying the circuit elements that can be 
modulated to reverse or compensate for neuroinflammation induced alterations. Notably, while minocycline 
has been used in the preclinical literature to study mechanisms involved in neuroinflammation, there are 
several other biological actions that minocycline can exert, including anti-apoptotic and antioxidant 
properties65. 
Unemployment as a translational model of chronic stress. Conceptualizing unemployment stress as a 
translational examination of chronic stress can help to bridge preclinical and clinical understandings of 

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inflammation and motivation. In animal models, chronic unpredictable stress paradigms have been pivotal 
for understanding inflammation-related dysregulation underlying anhedonia, depression, and general 
psychophysiological health. Because these stress paradigms are unethical to administer to clinical 
samples, the reproducibility and translation of preclinical work in this area limit the findings’ external validity. 
However, unemployment stress is a natural, ecologically valid model of chronic unpredictable stress such 
that unemployment stress can beget psychological distress over extended periods66,67, increase exposure 
to unpredictable and uncontrollable events68,69, and has been linked with long-term physiological effects70-
73. Additionally, unemployment stress has been associated with alterations in immune functioning such 
that those experiencing longer durations of unemployment are more likely to exhibit higher levels of 
immune system activation74. For example, in one study, those experiencing unemployment were five times 
more likely to have elevated levels of inflammation75. 
Summary and Significance of the Proposal. This proposal seeks to conduct translational work that 
extends rich preclinical findings to the clinical domain to validate whether neuroinflammatory dysregulation 
is strongly tied to anhedonia. This project addresses critical gaps in the scientific literature by recruiting a 
chronically stressed sample of individuals—employment seeking individuals who report significant stress-
- and will use an experimental therapeutics approach to attenuate neuroinflammation and assess neural 
activity underlying motivational deficits.  
2.2. Scientific Rationale 
One major obstacle in understanding how neuroinflammation influences human corticostriatal activity 
involves technological challenges such that conventional approaches are invasive, expensive, and/or 
lacking specificity. Although static levels of neuroinflammation in humans have been measured, capturing 
neural dynamics during behavioral tasks has been difficult to image in vivo. This obstacle limits the ability 
to develop a more precise understanding of how neuroinflammation causes circuit-level dysregulation in 
humans. The proposed project will employ a mechanistic clinical trial of the anti-[neuro]inflammatory agent, 
minocycline, to address these limitations. In animal models, minocycline has attenuated the deleterious 
effects of neuroinflammation on neurogenesis, long-term potentiation, and neuronal survival. This study 
will extend research to humans to examine whether links between neuroinflammation and neural 
responses to a reward-related motivation task differ among chronically stressed individuals taking 
minocycline and the placebo control. The proposed project will provide the first evidence of neural 
alterations that have relevance for motivational deficits due to neuroinflammation.  
 
3. Specific Aims 
 
Aim 1: To examine differences in performance on a pure motivation task following minocycline (vs 
placebo). We hypothesize that objective performance on a motivation task will be better when individuals 
are taking minocycline compared to performance after taking placebo. 
Aim 2: To examine differences in performance on a task measuring the influence of motivation on 
cognitive performance following minocycline (vs placebo). We hypothesize that objective cognitive 
performance will be better after individuals are taking minocycline compared to performance after taking 
placebo. 

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4. Investigational Plan 
Study Design: This study is a randomized, double-blind, placebo-controlled, crossover trial.  
Brief overview of study events: Potential participants will complete eligibility screening for the study. 
Participants meeting eligibility criteria will take a 5-day course of minocycline (or placebo) and then 
complete a reward processing task. They will also complete a self-reported motivational deficits scale 
(i.e., Snaith Hamilton Pleasure scale76). Participants will then complete a 14-day washout period before 
beginning the other arm of the trial. Again, participants will complete a 5-day course and then complete a 
reward processing task as well as the Snaith Hamilton Pleasure scale. Participants will be monitored for 
any adverse events throughout study participation. See Figure 1 for visualization of study procedures. 
 
Study Duration: 12 months  
Target Number of Participants: 54 chronically stressed individuals 
Minocycline Dosage: A 200mg/day dosage was selected based on prior studies examining the efficacy 
of minocycline for psychiatric symptoms 78-82. As described in the prescribing information leaflet (see 
Appendix 1), the serum half-life in normal participants ranged from 11-22 hours (M=15.5 hours).  It takes 
4 days for minocycline to achieve a steady state and approximately 4 days for it to be eliminated. 
Because a 5-day course of 200 mg/day dose has been found to have neuroprotective effects in response 
to brain injury (i.e., stroke)83-86, we plan to assess whether a similar 5-day course can potentially 
decrease neuroinflammation and ameliorate motivational deficits among individuals with chronic stress. 
To follow standards for other neuroactive agent crossover trials, a 14-day washout period will be 
implemented. Because we expect that the washout period is sufficiently long to eliminate the potential for 
carryover effects, we assume that there will be no carryover effects with our design. 
Timing of Visits: Participants will first complete a screening session for assessing inclusion/exclusion 
eligibility and can occur at -30 to -1 days. At day 0, participants will complete a baseline onboarding visit 
(~1.5 hr) and receive the first arm 1 of intervention. The 5-day course of minocycline (or placebo 
depending on randomization) will begin between day 1 and up to day 30 of enrollment. This will be 
decided based on participant availability. Behavioral session 1 will be scheduled 6 days after beginning 
the treatment (~day 6 to day 36). This session is approximately 1.5 hours and participants will receive 
the second treatment during this visit. Following the visit, a 2-week washout period will commence. The 
second behavioral visit will determine when the second treatment will begin and will not occur any earlier 
than 20 days after behavioral session 1. This 20+ day window accounts for 2 weeks of washout and 5 
days of the second treatment.  The 5-day course of placebo (or minocycline depending on 
randomization) can begin as early as day 26 or as late as is needed. Behavioral session 2 will be 
scheduled to occur 6 days after beginning the second treatment (day 32 or later). After the second visit, 
participants will be debriefed and receive the remaining payment.  The minimum enrollment period for 
participants is 32 days. 
 
Figure 1.  Crossover randomized trial schematic.  
 
  
Recruitment 
and Screening 
 
 
 
 
Verification of Eligibility 
and Enrollment 
 
Obtain informed consent.   
Obtain screening data and baseline data. 
Apply inclusion and exclusion criteria.  
Enrollment of Eligible Patients 
N=54 
N participantsN participants

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Behavioral Visit 1 
(6 days after  
the Start of  
regimen # 1) 
 
 
 
 
 
 
 
 
 
Behavioral Visit 2 
(6 days after  
the Start of  
regimen # 2) 
 
 
 
 
  
 
 
 
 
Baseline assessment & 
Enrollment Visit 
Post-minocycline 
tasks + 
assessments 
Post-minocycline 
tasks + 
assessments 
14-day washout, then begin other treatment regimen 
Minocycline 
N = 27 
Placebo 
N = 27 
Debriefing and study termination. 
Participation completed.  
Post-placebo tasks 
+ assessments  
Post-placebo tasks 
+ assessments  

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5. Study Participants 
5.1. Numbers of Participants 
A total of N = 54 eligible individuals will be enrolled. We anticipate that at least n = 50 of the enrollees will 
complete all aspects of the protocol and have complete data.  
 
5.2. Eligibility Criteria 
The following criteria were selected to minimize confounding effects and optimize signal detection for 
effects of chronic stress in this preliminary study. Individuals will be excluded from the study if they 
endorse any of the following, which are known to influence inflammation 87 or interfere/confound with 
minocycline effects 88: regular nicotine or recreational drug use; neurological conditions (e.g., history of 
stroke, TBI); chronic diseases or infections that significantly impact inflammatory markers (i.e., diabetes, 
cardiovascular diseases, high blood pressure, inflammatory bowel diseases, autoimmune disease); 
known allergies or hypersensitivities to tetracycline antibiotics, aspirin or other NSAIDs; antibiotics; 
regular use of steroidal or non-steroidal anti-inflammatory medications; and have any liver or kidney 
problems. Individuals will also be excluded if they are pregnant or breastfeeding or meet diagnostic 
criteria for a severe mental illness (i.e., disorders evincing psychotic symptoms, severe major 
depression, bipolar disorder). Finally, adults aged 25-60 will be eligible to participate.  
 
Several decisions regarding the stress and unemployment inclusion criteria were defined based on a 
prior study that examined links between inflammation and neural activity among a chronically stressed 
sample of unemployed individuals92. For that study, researchers included participants who were: 
• 
Unemployed (working less than 20 hours per week) 
• 
Seeking employment 
• 
Having trouble finding job 
• 
Stressed: greater than 5 points on Job Stress Items 
The Job Seeking Stress Items were adapted to screen participants for reports of stress due to 
unemployment in the Creswell et al. study92. The items are described in further detail in section 6.2. 
Finally, given recommendations and best practices for stress measurement93,94, a situation should be 
ongoing for at least six months to be considered a chronic stressor.  
 
 
5.2.1. ￼Inclusion Criteria  
 
In order to be eligible to participate in this study, an individual must meet all of the following criteria: 
• 
25-60 years old  
• 
Unemployed (working less than 20 hours per week)  
• 
Seeking employment  
• 
Having trouble finding job (i.e., actively seeking and applying for jobs but not successful in 
landing a job)  
• 
Reports greater than 5 points on Job Stress Items  
• 
Regular access to a mobile phone   
 
Having trouble finding a job includes actively seeking jobs and not being successful. Active search 
methods are defined as those that have the potential to result in a job offer without any further action on 
the part of the job seeker. Examples of active job search methods include: 
• 
contacting an employer directly about a job 
• 
having a job interview 
• 
submitting a resume or application to an employer or to a job website 

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• 
using a public or private employment agency, job service, placement firm, or university 
employment center 
• 
contacting a job recruiter  
• 
seeking assistance from friends, relatives, or via social networks; for example, asking friends and 
family for job leads or indicating one's job seeking status on social media 
• 
placing or answering a job advertisement 
• 
checking union or professional registers 
 
 
5.2.1.  Exclusion Criteria  
 
Any individual who meets one or more of the following criteria will be excluded from participation: 
• 
Currently enrolled as a student 
• 
Prescribed medication for the following physical illnesses: diabetes, cardiovascular diseases, high 
blood pressure, autoimmune disease, neurological conditions (e.g., TBI, stroke)  
• 
Diagnosis of a severe mental illness (i.e., disorders evincing psychotic symptoms, severe major 
depression, bipolar disorder)  
• 
Pregnant or breastfeeding  
• 
Daily psychotropic medications including mood stabilizers, stimulants, antipsychotics.  
• 
Daily antidepressants use that has been recently added or modified in fewer than 3 months.   
• 
Drug or alcohol dependence 
• 
Known allergies or hypersensitivities to tetracycline antibiotics, aspirin or other NSAIDs  
• 
Current antibiotic use  
• 
Regular use of steroidal or non-steroidal anti-inflammatory medications (i.e., 2 or more times a 
week) 
5.3. Randomization and Blinding 
The UNC Investigational Drug Service (IDS) will randomly assign participants to start the experimental 
regimen (minocycline) or the placebo regimen. A randomized block approach by gender will be 
implemented such that there are equal numbers of male and female participants in each arm of the 
study. Research staff who have direct contact with the participant will be blind to the condition of the 
participant. The psychiatrist who is monitoring safety and possible adverse events while the participants 
are on minocycline or placebo will be unblinded during the study and therefore able to identify which pill 
was taken if there are any significant adverse effects felt by the participant after taking the course of 
medication. The psychiatrist will not have direct contact with participants during the experimental visits 
unless the participant is experiencing adverse side-effects. Thus, no study team member who interacts 
with participants during visits will know whether a participant is on placebo or minocycline. After data 
collection for the study is completed, the pharmacist who made order assignments will reveal when 
participants were in the minocycline vs. placebo phase so appropriate analyses can be conducted.   
In order to maintain randomization and blinding for participants, the minocycline and placebo pills will be 
enclosed in the same capsules as to preclude any noticeable differences between the pills. The pills will 
be placed in the same containers and be administered to the participants following the same procedures 
during both conditions. 
5.4. Recruitment and Retention 
Participants will be recruited via online registries of research studies at UNC (e.g., Join the Conquest; 
emails to UNC staff listservs), flyers posted in the Chapel Hill, Durham, and Raleigh communities (e.g., 
community centers, grocery stores), targeted ads on social media (e.g., Facebook), and ads on buses 
and in local publications. To ensure participation from individuals experiencing stress due to 
unemployment, we will offer vouchers to cover transportation to UNC for the study session, free on-site 

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parking, and evening/weekend sessions in an effort to lower barriers to participation. To facilitate 
retention, we will provide bonus compensation to those who complete the follow-up assessments. 
 
6. Schedule of Activities and Procedures 
6.1. Table of Events 
 
 
Table 3.   Schedule of activities and procedures  
Procedure 
Visit 0 
screening 
Visit 1 
baseline 
Pre-V2 
 
Visit 2 
 
Pre-V3 
 
Visit 3 
Debrief 
Early 
Stop 
Recruit a Sample of 
Patients 
Informed consent 
 
X 
 
 
 
 
 
 
Eligibility assessments 
X 
X 
X 
 
X 
 
 
 
Enrollment and 
randomization 
 
X 
 
 
 
 
 
 
Treatment 
Administer regimens 
 
 
X 
 
X 
 
 
 
Measure Compliance 
 
 
X 
X 
X 
X 
 
X 
Safety 
Monitoring 
Review concomitant meds 
 
X 
X 
X 
X 
X 
 
 
Medical history  
 
X 
 
X 
 
X 
 
 
Psychological surveys 
X 
X 
X 
X 
X 
X 
 
 
Assessment of AEs 
 
X 
 
X 
 
X 
X 
X 
Reported Outcomes 
Motivation Scale 
 
X 
X 
X 
X 
 
 
X 
6.2. Screening 
Prospective participants who email the Social Neuroscience and Health Lab regarding participation will 
first complete a pre-screening questionnaire that collects information about the individuals. The 
prescreen questionnaire will be administered via Qualtrics and will ask about age, gender identity, 
pregnancy status, employment status, job seeking stress screening items, and gather name and contact 
info. 
 
Participants will be eligible for inclusion if they indicate that: 
• 
25-60 years old  
• 
Unemployed (working less than 20 hours per week)  
• 
Seeking employment  
• 
Having trouble finding job (i.e., actively seeking and applying for jobs but not successful in 
landing a job)  
• 
Reports greater than 5 points on Job Stress Items  
• 
Regular access to a mobile phone   
 
Job Seeking Stress Screening Items92: Participants will be asked the following questions and be asked to 
rate them on a scale of 0 (never) to 4 (very often). To be eligible for participation, participants will need to 
score greater than 5 points on the scale. Items 2 and 3 are reversed scored. 
1. In the last month, how often have you felt that you were unable to control the important things in 
your life? 
2. In the last month, how often have you felt confident about your ability to handle your job-related 
problems? This could include your efforts at finding a job or paying your bills. 
3. In the last month, how often have you felt that things were going your way in finding a job? 
4. In the last month, how often have you felt difficulties were piling up too high that you could not 
overcome them? 
 

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After completing the prescreen questions, participants will then be screened via telephone by a trained 
research assistant in the lab to assess whether they meet additional eligibility requirements. On this call, 
participants will be asked about exclusion criteria including:  
• 
Currently enrolled as a student 
• 
Prescribed medication for the following physical illnesses: diabetes, cardiovascular diseases, high 
blood pressure, autoimmune disease, neurological conditions (e.g., TBI, stroke)  
• 
Diagnosis of a severe mental illness (i.e., disorders evincing psychotic symptoms, severe major 
depression, bipolar disorder)  
• 
Pregnant or breastfeeding  
• 
Daily psychotropic medications including mood stabilizers, stimulants, antipsychotics.  
• 
Daily antidepressants use that has been recently added or modified in fewer than 3 months.   
• 
Drug or alcohol dependence 
• 
Known allergies or hypersensitivities to tetracycline antibiotics, aspirin or other NSAIDs  
• 
Current antibiotic use  
• 
Regular use of steroidal or non-steroidal anti-inflammatory medications (i.e., 2 or more times a 
week) 
 
Additionally, a Mini-International Neuropsychiatric Interview (M.I.N.I.)95 will be administered to assess 
whether participants meet the criteria for a severe mental illness via phone. The MINI is a brief diagnostic 
structured interview for major psychiatric disorders and takes approximately 15 minutes to administer. 
The researcher will complete the MINI screening questions first. If the participant responds positively to 
any of the screening prompts, follow-up questions will be asked to assess the possibility of a diagnosis. 
Participants who meet eligibility for severe mental illnesses will be excluded. 
 
If eligible, participants will learn more information about the study and be asked whether they would be 
interested in participating. If so, they will be scheduled for all the following study visits. 
 
Eligible participants will be scheduled for the Baseline in-person visit. There, the informed consent 
process will begin, and confirmatory tests/evaluations will be performed to verify eligibility criteria. During 
this visit: 
 
• 
The study coordinator will review the consent forms and answer any questions the participant 
may have 
• 
Participant will be given time to review materials and then sign the consent form 
• 
Participants will meet with the study physician to complete a brief health screening, collect vital 
signs information, and to carefully discuss the risks/benefits of participating in the study 
• 
Complete questionnaires about employment stress and their levels of motivation 
• 
Begin enrollment procedures outlined below 
• 
Participant will be thanked for their time and reminded of their next in-person appointment   
 
The names, phone numbers and emails of participants who do not meet eligibility criteria or who decline 
participation will be retained on a secure password protected network for the study term (5 years) to 
ensure that the same participant is not contacted more than once regarding participation. All other 
identifiable information about these individuals will be deleted as soon as they decline participation. 
Participants who endorse one or more exclusionary criteria will also be asked if they would like to be 
contacted for future studies in our lab.  
6.3. Enrollment  
During the baseline screening and enrollment visit, participants and research team will: 

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• 
verify inclusion/exclusion criteria,  
• 
review study procedures and schedule and briefly describe the purposes of the study 
• 
provide first course of medication 
• 
explain minocycline and its known side effects 
• 
explain that individuals will be asked about AEs during each study visit and will be monitored 
• 
develop plan and help participant set an alarm for when to take the medication 
• 
explain the text reminder system where they are reminded to take the medication around a 
specific mealtime and then asked to take a picture of medication in palm to verify 
• 
explain the payment schedule for participation in the study  
Before both study visits, inflammation-related drug use and employment status will be reassessed at least 
24 hours before the appointment. If the individual has recently taken an antibiotic or anti-inflammatory 
medication, the visit will be cancelled.   
6.4. Study Visit Procedures 
During study visits 1 and 2, the schedule of events will be nearly identical. First, participants will provide 
dried blood spots to measure levels of pro-inflammatory cytokines. Then, participants would complete the 
Progressive Reward Task. Next, participants will complete a few questionnaires including measures of 
anhedonia, stress, and an assessment of medication adherence. Finally, participants will be given the next 
course of medication in preparation for behavioral visit 2.  
 
The following measures will be collected at study visits 1 and 2: 
• 
Resting Heart Rate Variability (HRV) will be collected for 5 minutes while participants complete the 
following questionnaires. Sensors will be placed to collect autonomic nervous system reactivity. 
• 
The Snaith–Hamilton Pleasure Scale (SHAPS) is a tool to assess symptoms of reduced motivation. 
The SHAPS uses 14 questions, each rated on a Likert scale of 1-4. The total score on the scale 
ranges from 14-56, with lower scores reflecting lower motivation. Scores will be compared across 
conditions to determine whether motivation changes in the minocycline condition as compared to 
the placebo. 
• 
The Motivation and Pleasure Scale (MAP) will be used to capture self-reported aspects of reduced 
motivation. The scale uses 18 questions, each rated on a Likert scale of 0-4. The total score on the 
scale ranges from 0-72, with lower scores reflecting lower motivation. Scores will be compared 
across conditions to determine whether motivation changes in the minocycline condition as 
compared to the placebo. 
• 
Job Seeking Stress Items as discussed in section 6.2. 
• 
Given that unemployment is linked to greater levels of stress generally, participants will also 
complete the Perceived Stress Scale (PSS)96. The PSS is the most commonly used and well-
validated measure of stress. The PSS is a 10-item scale that asks how often individuals have 
experienced irritations or lack of control in one’s life. Participants completing the PSS will be 
instructed to respond based on their experiences since the last visit. 
• 
The Patient Health Questionnaire (PHQ-9)97 will be administered to measure symptoms of 
depression. The PHQ-9 is a 9-item scale that is used for diagnostic purposes to assess the severity 
of depression an individual is experiencing. Participants completing the PHQ-9 will be instructed to 
respond based on their experiences since the last visit. Scores on this scale will be used to assess 
whether Aims 1 and 2 are moderated by depression severity. This scale will also be used to assess 
the safety of each participant. See section 10 for more information about safety plans. 
• 
A trained research assistant will administer a Dried Blood Spot (DBS) test. To complete this test, 
participants' skin will be punctured with a single-use safety lancet, which is the same procedure 
diabetic individuals use on a daily basis. This may be momentarily uncomfortable or painful for 
some participants, but we expect that it will be a mild experience. 
• 
The Health Information Form is used to assess current medications, illness, or other covariates that 
could affect inflammation and thereby eligibility. 

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• 
Participants will be verbally instructed on the Progressive Reward Task and able to follow along 
with written instructions. They will be informed that the goal of the task is to win as much money as 
possible by correctly identifying the presence of a short versus long mouth on a cartoon face. 
Further, they are informed that not all correct responses will receive reward feedback. They will 
then complete a brief practice trial. Each trial of the task begins with a fixation cross in the center 
of the screen for 500 ms. Then, a mouthless cartoon face will be presented. With a 500 ms delay, 
either a short mouth (11.5 mm) or a long mouth (13 mm) is presented on the face for 100 ms. The 
participants must identify whether they saw a short or long mouth by pressing a key on the 
keyboard. The mouthless cartoon remains present until the participant responds. The short and 
long mouths are presented equally across the trials. However, the number of reinforcers presented 
for short VS long mouths is not balanced. The task aims to produce a response bias toward the 
mouth length that is more often positively reinforced. 
 
At the end of both study visits, the research assistant will contact the study physician to report whether the 
participants reported any AEs.  
 
6.5. 
Final Debriefing Visit 
 
After completing both randomized conditions, participants will be asked to stay for a 30-minute debriefing 
session with the study coordinator. During this visit, participants will be unblinded, learn more about the 
purpose of the study (i.e., links between stress, inflammation, and motivation), have any concerns or 
questions participants may have about the study answered and addressed. Participants will also receive 
final compensation for participation in the study during this time.  
6.6. 
Phone Contacts  
Considering the complexity of activities, there will be frequent communication by the research team to 
the participants to remind them about study visits and to take the medication as directed.  
 
• 
Study visit reminders will be sent once a day starting 2 days before the study visit via text 
messaging. 
• 
Medication reminders will be sent once a day, during the medication period, at the participant’s 
earliest mealtime (e.g., breakfast, lunch). If participants elect to be reminded at their next 
mealtime, they will be texted 4 hours later.  
• 
To verify that participants have taken the medication, participants will be called each evening to 
ask about compliance and if they experienced any adverse events. 
 
6.7. 
Assessing Medication Compliance 
During the enrollment visits, participants will be asked what time of the day would be best to contact 
them for daily medication check-ins when the medication period begins. Participants will be given the 
option to receive daily reminder texts to take the medication and can confirm taking them in response to 
that text. Whether participants indicate evening, morning, or afternoon, all participants will be contacted 
by study staff to assess compliance for the previous day and whether the individual experienced any 
AEs. If a participant is unable to answer or call us back within 24 hours, it will be marked in a log for 
follow-up on the following visit. The following daily calls will proceed until the visit. To compensate 
participants for their time completing the daily check-ins, they will be entered in a raffle to win an extra 
prize at the end of the study. 

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During study visits, medication compliance during the treatment period will be assessed via a 
questionnaire in order to decrease pressure to respond in a socially desirable way. The questionnaire 
prompt will state “Taking pills is difficult for a lot of people. It is not uncommon for people to miss doses 
from time to time. These items/questions ask you about doses you took and doses you missed. Please 
try to remember as best you can what actually happened and not what you intended to have happen or 
what you think that other people want you to report. By answering these questions accurately, you are 
making a big contribution to this research.”  
Participants will be asked to return the pill bottle and extra pills at the end of the study. The extra pills will 
be returned to IDS for disposal. Together, the pill bottle, questionnaire responses, and daily check-ins will 
be used to assess medication adherence. 
7. Statistical Analysis Plans 
 
Statistical procedures will be performed in R. The study was designed to reduce the possibility of period, 
carryover, and sequence effects and we assume that these effects will not occur with this data. 
Specifically, we will employ a blocked randomized design to prevent period and sequence effects and 
allow for the opportunity to observe if the effects will occur. Additionally, we have extended our washout 
period to decrease the possibility of carryover effects.  
Aim 1: To examine differences in performance on a pure motivation task following minocycline (vs 
placebo). 
Aim 1 analysis plan: A paired samples t-test will be conducted to assess whether taking minocycline was 
associated with a change in response bias on the PRT as compared to when participants are taking a 
placebo. 
Aim 2: To examine differences on self-report measures of motivation following minocycline (vs placebo). 
Aim 2 analysis plan: A paired samples t-test will be conducted to assess whether taking minocycline was 
associated with a change in SHAPS scores as compared to when participants are taking a placebo. 
Another paired samples t-test will be conducted to assess whether taking minocycline was associated 
with a change in MAPS scores as compared to when participants are taking a placebo. 
 
8. Sample Size Rationale 
We conducted a power analysis to determine the appropriate sample size for this study. Although a prior 
study exploring the effects of minocycline on a memory task demonstrated a large effect size (Berens et 
al., 2020), we conducted a power analysis that assumed a small-medium effect size given that 
associations with our task types have not been explored in the literature before. Power analyses were 
conducted assuming a small-medium effect size Dz of 0.4 for a paired-sample t-test examining the 
change in accuracy on both tasks from post-minocycline to post-placebo. Given an effect size of Dz=0.4 
for a paired-sample t-test and an alpha of .05, we would need 52 participants to achieve 80% power. As 
such, we aim to recruit 54 participants to account for any potential issues with participant retention.   
9. Data Collection and Management 
 
Questionnaire and Symptom Data Collection: Research material obtained from all of participants will 
include self-report data and responses on behavioral tasks. These assessments will be conducted via in-

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person assessment sessions. Self-report measures will be collected using Qualtrics survey software 
implemented behind the HIPAA-compliant firewall at UNC-Chapel Hill. Qualtrics is encrypted and requires 
ONYEN credentials and Duo two-step authentication by authorized study personnel to access. Each 
session will last between 1 and 2.5 hours and breaks will be provided throughout the session as needed. 
Responses on self-report and behavioral measures will exist in digital form on computer disk and on secure 
servers at UNC.  
 
Data Quality and Monitoring: All data are collected by trained bachelor's-level or Master’s level study staff. 
Data quality and safety will be monitored by the PIs and research team members. Any issues will be 
discussed with the PI, and as needed, the scientific advisory team. The study PI assumes responsibility for 
ensuring study staff is complying with the investigational plan and IRB regulations, as well as ensuring any 
changes to the protocol have received IRB approval and have been reported to the sponsor, and that 
accurate, complete, and timely reports are made to the IRB. A weekly study team meeting will be conducted 
to discuss research procedures.  
• 
All identifiable data will be recorded in the database using alphanumeric identifiers that are unrelated 
to any personal identifying information about the participant. All participant data are coded by subject 
number only on password-protected secure servers of which only the research team has access.   
• 
All sources of electronic data (i.e., computerized symptom data collected via UNC Qualtrics) are 
saved on a password-protected file on a secure UNC server, to which only the research team 
members will have access.   
• 
All sources of non-electronic research data will be coded by a unique subject number and stored in 
a dedicated locked file cabinet. Materials identifying participants by name (e.g., signed consent 
forms) will be stored in a separate locked file cabinet. The computer file matching participant names 
with code numbers will be saved in a double password-protected file on a secure UNC server 
directory only accessible by the PI and key research team. All data will be entered into the dataset 
using the subject identification number only. Participants are not identified by name in any analysis 
of these data, or any presentation or publication resulting from the analysis of these data. 
10. Safety Monitoring and Management 
10.1. 
Risk / Benefit Assessment 
Potential Risks:  
• 
Minocycline: The most common side effects are gastrointestinal (i.e., nausea) and mild central 
nervous system effects (e.g., vertigo, light-headedness). Like other tetracycline-class antibiotics, 
minocycline may cause fetal harm when administered to pregnant women. Pregnant woman 
(measured by self-report during prescreen) will be excluded from participation due to the potential 
risks of minocycline to a fetus. The use of minocycline during pregnancy may affect tooth 
development and cause permanent discoloration of teeth. If any tetracycline is used during 
pregnancy or if the patient becomes pregnant while taking these drugs, the participant will be 
apprised of the potential hazard to the fetus. Most adverse effects occur early after administration 
and disappear when the medication is discontinued. Participants will also be advised to use a 
second method of contraception as minocycline may increase the chances of an unintended 
pregnancy. 
• 
Questionnaires / Behavioral Measurements: There are minimal risks involved in the collection of 
questionnaires and behavioral data. Although it is unlikely, it is possible that participants may 
become bored or frustrated by completing the tasks. Individuals may become distressed while 
reporting on difficult emotional experiences.  
 
Potential Benefits: Beyond monetary compensation for participation, there are no direct benefits to 
individuals for participating in the study. However, participants may derive satisfaction, knowing that 
their participation may help advance knowledge about chronic stress-related neuroinflammation. The 

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proposed study is similar to other experiments conducted in the lab. The adverse experiences (e.g., 
dropping out of a study due to discomfort with study procedures) have been minor and rare. Thus, we 
maintain that participants’ risks are minimal and less than the benefit they gain from participating in 
the study. 
10.2. 
Safety Monitoring 
Personnel: The present study does not raise any obvious health concern issues. Nonetheless, during 
the course of individual study sessions with participants, researchers will frequently verbally check in with 
participants to make sure they are comfortable and not experiencing any physical or psychological 
discomfort. Participants can withdraw participation at any point in the study. All research staff will be 
trained on recognizing and reporting adverse events prior to engaging in research activity. If a serious, 
unexpected adverse event occurs, the PI will report the event to the UNC IRB in accordance with their 
regulations. The PI will review the adverse event report and gather other information as needed to 
investigate the event and determine the need for subsequent action. Any subsequent action will be 
documented and reported to the UNC IRB. All study team personnel will obtain research training 
certification before working in the lab as required by NIH. Confidentiality will be protected to the fullest 
extent permitted by law. All research personnel will have completed CITI training and HIPPA training for 
researchers. Consent forms will be stored in locked file cabinets only accessible to those on the study 
team. Computerized data (behavioral task data and questionnaire data entered into the computer) will be 
stored on secured, password-protected servers behind a UNC-Chapel Hill and lab-specific firewall. 
Minocycline: The 200mg dose of minocycline to be given in this study is expected to be well-tolerated in 
this young, healthy population. The greatest risk to participants taking this medication is allergic reaction, 
and we will assess all medication allergies prior to study enrollment. Minocycline rarely causes side 
effects, especially when used for a short duration like this study. However, like with most antibiotics, 
participants will be counseled about the possibility of diarrhea and gastrointestinal upset as well as signs 
of an allergic reaction. They will also be alerted to the possibility of photosensitivity and be encouraged to 
wear sunscreen. The study physician will be on-call to assess any side effects and direct appropriate 
management as needed. If a participant begins to notice signs of an allergic reaction when taking the 
medication at home (e.g., difficulty breathing, swelling) they will be advised to call 911 and seek medical 
attention immediately. While we will encourage participants to alert the study team to any issues that 
may arise, we will also contact participants daily to assess potential adverse reactions during the 
medication duration via phone and surveys. Once contact with the participants is established, the study 
psychiatrist will follow-up to assess the participants wellbeing and treatment plan. Participants will be 
contacted daily during the at-home medication period to assess any potential adverse reactions to 
minocycline. During the daily calls, participants will be asked about any symptoms of allergy, 
gastrointestinal upset such as nausea and diarrhea, as well as any feelings of dizziness or fatigue. 
Questionnaires / Behavioral Measurements: To minimize the risk of a participant becoming 
uncomfortable, the study team will make every effort to maintain a personally supportive relationship with 
each participant. Participants will be informed at the start of the study what to expect and how long the 
study visit will be. All data will be coded with identification numbers and will not contain personally 
identifying information.  
Protocol for Assessing the Emergence of Severe Depression or Suicidality: Participants will be 
recruited to be at low risk for severe depression and suicide attempt following the baseline assessments 
with the study psychiatrist. However, the PHQ-997 will be administered at all study visits to track risk for 
psychological crises. If a participant screens positive to question 9 on the questionnaire, selecting 
anything other than zero for the item “Thoughts that you would be better off dead or of hurting yourself in 
some way”, the study psychiatrist will assess suicide risk with the Ask Suicide-Screening Questions 
(ASQ). See Appendix 4 for the ASQ. If the psychiatrist identifies potential positive risk, they will be 
offered information for immediate crisis services including Orange County mobile crisis units and the 
Emergency Department. The same resources will be shared with participants who score higher than 15 
on the PHQ-9, indicating severe depression. 

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10.3. 
Adverse Events and Serious Adverse Events Reporting Procedure 
An adverse event (AE) is generally defined as any unfavorable and unintended diagnosis, 
symptom, sign (including an abnormal laboratory finding), syndrome or disease which either 
occurs during the study, having been absent at baseline, or if present at baseline, appears to 
worsen. Adverse events are to be recorded regardless of their relationship to the study 
intervention.  
A serious adverse event (SAE) is generally defined as any untoward medical occurrence 
that results in death, is life threatening, requires inpatient hospitalization or prolongation of 
existing hospitalization, results in persistent or significant disability/incapacity, or is a 
congenital anomaly. 
The most common side effects from this medicine are gastrointestinal symptoms, dizziness 
and fatigue. Minocycline may increase sensitivity to sunlight, resulting in more frequent 
sunburns or the development of rashes following sun exposure. We will recommend that 
patients apply sunscreen (SPF 15 or greater) before outdoor activities or avoid prolonged 
exposure to the sun while taking minocycline. Minocycline-associated SAEs are very rare, 
and the number needed to cause one additional case has been estimated at 1/11,364 from a 
UK database of over 97,000 patients treated for acne103. No neurological side effects such as 
extra pyramidal symptoms have been reported. 
Each participant will be asked about the presence and absence of adverse events at every 
study visit. The research team will record the event, severity, the start and end date of the 
event, the relationship to the study (e.g., whether related or unrelated to the study 
procedures) and the final outcome. Data will be collected using a form (see Appendix 2 for 
examples). The study physician will be consulted when events are unexpected and/or of 
greater than mild severity. The study physician will speak to the participant within 24 hours to 
more thoroughly assess the next steps to determine the best course of action.  If deemed 
necessary, the physician will refer the participant to a follow-up. The occurrence of adverse 
events will be immediately reported by study staff (i.e., study coordinator, graduate student 
research assistants, research nurses at the CTRC) to the PI and the study physician. The PI 
will then report the adverse event to the UNC Office of Human Research Ethics (OHRE). 
OHRE standard operating procedures specify that the PI must notify information previously 
unknown to the IRB involving new or increased risk to subjects (i.e., New Safety Information) 
to OHRE within seven calendar days of the investigator becoming aware of the information. 
New Safety Information is communicated to OHRE through the online IRB management 
system, IRBIS. Reports to the IRBIS will include Adverse Events, Serious Adverse Events, 
and Unanticipated Problems. 
The following grading scale will be used to identify the intensity of an Adverse Event:  
• 
Mild: Awareness of a sign or symptom that does not interfere with the participants 
usual activity or is transient, resolved without treatment and with no sequelae;  
• 
Moderate: Interferes with the participants usual activity, but the patient is still able to 
function;  
• 
Severe: Events that interrupt a patient’s usual daily activity and generally require a 
systemic drug therapy or other treatment.  
All AEs/SAEs will be evaluated as to its expected occurrence, or lack of expected occurrence:  
• 
Expected: An adverse event is expected when the specificity and severity of the event 
is consistent with the study procedures and Standard of Care, (e.g., the participant 
fainted in response to having his blood drawn) or underlying disease state, (the 
participant’s depressive symptoms worsened).  

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• 
Unexpected: An adverse event is unexpected when the specificity or severity of an 
adverse event is not consistent with the study procedures or Standards of Care, (e.g., 
the phlebotomist used the same blood collection needle on multiple participants, 
resulting in a participant acquiring Hepatitis C), or underlying disease state (e.g., the 
participant experienced a manic episode during the study). Unexpected, as defined 
above, refers to an adverse event that has not been observed before.  
The following conditions will be used to define the relatedness of any study procedure to an 
AE/SAE.  
• 
Not related: The PI has determined that the event is not related to any study 
procedure. 
• 
Possible: The PI has determined that the event possibly has a reasonable relationship 
to the study procedures.  
• 
Probable: The PI has determined that the event probably has a reasonable 
relationship to the study procedures. Definite: The PI has determined that the event is 
related to study procedures.  
• 
Unknown: The PI has determined it is impossible to determine the relationship of the 
event to the study procedures.  
The clinical outcome of the AE/SAE will be characterized as follows:  
• 
Recovered/Resolved: The patient recovered from the AE/SAE.  
• 
Not Recovered/Not resolved: Patient did not recover, and symptoms continue.  
• 
Recovered/Resolved with Sequelae: The patient has recovered but with clinical 
sequelae from the event.  
• 
Fatal: The event resulted in death. The SAE, Death, and Exit forms must be 
completed for this outcome.  
• 
Unknown: The clinical outcome of the patient remains unknown at the time of the 
report, or the patient was lost to follow-up.  
All AEs/SAEs will be followed until resolution or until the end of participation in the protocol. 
Participants may report AEs freely or in response to general questioning or through patient or 
clinician assessments. If it is determined that an AE has occurred, the PI will be notified.  
11. Regulatory, Ethical, and Study Oversight Specifications 
 
11.1. 
Informed Consent Process 
 
Consent forms will be Institutional Review Board (IRB)-approved and the participant will be asked to read 
and review the document. The investigator will explain the research study to the participant and answer 
any questions that may arise. A verbal explanation will be provided in terms suited to the participant’s 
comprehension of the purposes, procedures, and potential risks of the study and of their rights as 
research participants.  Participants will have the opportunity to carefully review the written consent form 
and ask questions prior to signing. The participants should have the opportunity to discuss the study with 
their family or surrogates or think about it prior to agreeing to participate. The participant will sign the 
informed consent document prior to any procedures being done specifically for the study. Participants will 
be informed that participation is voluntary and that they may withdraw from the study at any time, without 
penalty. A copy of the informed consent document will be given to the participants for their records. The 
informed consent process will be conducted, and the form signed, before the participant undergoes any 
study-specific procedures. 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. 

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11.2. 
Confidentiality and Privacy 
We will safeguard against breaches of confidentiality by refraining from collecting personal identifying 
information and instead coding participant data by assigning alphanumeric ID numbers on digital data 
and hardcopy forms. The key linking the alphanumeric ID and participant’s identity will be maintained on 
a separate drive that is double password protected and to which only the PI and key study personnel 
have access. The only exception to alphanumeric ID coding will be for information, including names, 
telephone numbers, dates, mailing and electronic address, provided on the Contact Form, which will only 
be used to contact participants to schedule and confirm study visits. Contact Forms will be kept 
separately from study data under double locks only available to the PI and key research team. Clinical 
records will be kept confidential with access granted only to those medical and research professionals 
directly involved with the study. If any scientific paper based on the data collected for this study is 
published, no information that could be linked to any single participant will be reported. Confidentiality will 
be protected to the fullest extent permitted by law. All research personnel must complete HIPPA training 
for researchers and CITI training. 
 
11.3. 
Key Roles and Study Governance 
 
Principal Investigator Graduate Student 
 
Principal Investigator Faculty Advisor 
Gabriella M Alvarez, MA 
 
Keely A Muscatell, PhD 
(954) 610-9003 
 
(919) 843-9113 
alvarezg@unc.edu 
 
kmuscatell@unc.edu 
 
Study Psychiatrist & Medical Contact 
 
 
Mary Kimmel, MD 
 
 
(984) 974-5217 
 
 
mary_kimmel@med.unc.edu 
 
 
 
 
12. Appendices 
 
Appendix 1:  Prescribing Information Leaflet for Minocycline 
 
Appendix 2:  Example Adverse Events Form 
 
Appendix 3: Minocycline Care Notes for Participants  
 
Appendix 4: Suicide Risk Screening Tool  
 
 
 
 
 
 
 

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