Longitudinal Study of Pro-inflammatory Cytokines as

Depression Indicators in Relatives of Homicide Victims

Keelan Tobia - Bridges 2015

Grief vs. Depression

Cytokine

Cytokine Theories & Research

Objectives & HypothesisMaterials & Methods

Study Design

Budget

Works Cited

overview

Depression

Grief is the normal response to bereavement

Major depression is a common and sometimes fatal mood disorder

Sadness decreases over time Feelings of sadness worsen

CytokineCytokines communicate with cells to trigger the protective defenses of the immune system.

Pro-inflammatory Cytokines• Small nonstructural proteins that are released during infection, immune

responses, inflammation and trauma

Reason for studying Cytokines

Can’t we just use anti-depressants?• Although these drugs have greatly improved since their arrival, they

appear to have hit a plateau (30% TRD)• Past decade, inflammation has been revisited as factor of mood

disorders• patients with increased inflammatory cytokines before treatment

have been reported to be less responsive to antidepressant treatment

• Understanding the mechanisms by which inflammation effects brain function to induce mood disorders may lead to new forms of treatment

Controversy

Theories on Physiologic Pathways

Potential mechanisms• Inflammatory cytokines are increased during depression and are

produced in the gut, in adipose tissue. • Once produced, the cytokines can access the brain and activate local

central nervous system inflammatory networks to produce alterations in neurotransmitter function, leading to further behavioral alterations.

Theories on Physiologic Pathways

Pre-clinical evidence suggests that increased cytokines induce mood symptoms by

– decreasing serotonin levels– activating the hypothalamic-pituitary-adrenal (HPA) axis to induce

high levels of glucocorticoid neuron cell death– activating microglial cells to cause pathological synaptic pruning

and induce structural brain changes depression

Potential Support

In the treatment of hepatitis C, IFN is commonly used to boost the immune system to clear the viral infection• High doses 50% , 90%

Aspirin significantly improved depressive behaviors in fluoxetine treatmentResistant depressiverats

Objectives• Likelihood of developing MDD after experiencing traumatic event based on

specified factors. • Further investigate the idea of a pertinent subtype of immune-based depression.• Test validity of the findings that claim pro-inflammatory cytokines are related to

depression• Identify threshold that precedes MDD relative to baseline measurements.• If depression and cytokine are linked, determine effectiveness of anti-

inflammatory treatmentAddress • Do people who develop MDD have increased cytokine activity, while those who

share depressive symptoms during bereavement have a reduction in cytokine activity over time?

Hypothesis

1. Pro-inflammatory cytokines can be used as a valid predictor for the onset of major depression in relatives of homicide victims

2. Aspirin may be an effective additional treatment for those experiencing immune-based depression

Warrant for the Study

• Potential confounders are relatively unaccounted • Further research, especially longitudinal studies of cytokine

activity as a valid biomarker is needed to produce definitive results

• Research involves studies on animals• Human studies typically compare pre-diagnosed patients with

healthy patients.– Or patients with chronic diseases who are induced with cytokines as

treatment

Participating Organizations

Collaboration with research-based institutions

• University of Michigan Ann-arbor– Ann Arbor, Michigan

• Johns Hopkins University– Baltimore, Maryland

• UC Berkeley – Berkeley, California

Materials and MethodsRecruitment

Location • Close proximity to participating institutions• Near and in cities with high homicide rates

Advertising (in-person and online)• Grief share programs• Primary care offices• Hospitals• County assistance offices (financial assistance)• Court houses• Law offices• Police stations • Churches• Mortuary and cemetery offices• Victims advocacy agencies (NOVA & NOPMC)

Materials and Methods

Homicide: unexpected reckless or intentional taking of another human life by an individual

• 13-55 (Male and Female)• RHVs must meet criteria • Multiple nuclear family members • Recruitment of 1 year• Reimbursement

Materials and Methods

Screening Process overt inflammation screen (evaluation of medical history)

– To exclude patients who have health disorder known to cause obvious inflammation• cardiovascular disease• Cancer

• Patients with pre-existing depression will also be excluded from the study– The Beck’s Depression Inventory-II (>30)

• Liver disease• Autoimmune disease• Immunodeficiency virus

Materials and Methods

Baseline Assessments• Blood Analysis for cytokine activity • Depression Severity & Diagnosis

– BDI-II & Psychiatric AssessmentOther Initial Assessments • Health Questionnaire (Potential Confounders)

– BMI (Obesity = BMI> 30)– Medication use– Sleeping habits– Family history of depression– Lifestyle factors (smoking, alcohol consumption, diet, exercise)

Materials and Methods

Blood Analysis• collect, date, and label blood samples (6 mL collection tubes)• Centrifuge for 15min at 3300 rpm w/ Nanopure water• Place plasma supernatant in 2 mL centrifuge tubes Eppendorf mini

centrifuge 10 min at 2.8 rpm• Plasma cryotubes stored at -150° C • Once samples thaw slowly to room temp undergrad RAs determine

cytokine levels using Instant ELISA kit (eBioscience®)• Follow recommendations for biomarkers• Final reading using SpectraMax Plus plate reader

TNFR1, IL-6, CRP, TNFRa, IFN

The Beck Depression Inventory (BDI-II)

1-10_______considered normal11-16______Mild mod disturbance17-20______Borderline clinical dep.21-30______Moderate depression31-40______Severe depression>40________Extreme depression

Depression Severity Assessment• Used alongside psychiatric

assessments to determine diagnosis

SPSS

All statistical analysis using Statistical Package for Social Sciences – Continuous data – Correlational– Categorical data – Cross-sectional analysis– Onset of depression between variables

Relative of Homicide Victim Questionnaire(as best described)Evaluation of closeness to victim• How were you informed of the homicide?• Age of victim• Biological or non-biological.• Age difference• Ability to understand event• Cause of Death (may or may not be known at time of interview)• Knowledge of Gang affiliation• Known criminal offenses (violent and non-violent crimes)• Instant death or prolonged death

• Severity of crime – Alcohol related car crash, shot, stabbed, rape-involved murder,

torture, dismemberment, abandoned• Witness• Relationship of the RHV to the murder victim• Last time RHV had contact with victim• Level of regret in relationship between victim• Closure

– Suspect arrested or convicted– If arrested, status of trial

Coping Systems section• Religiosity• Support systems available• primary coping mechanisms

– (asked every six months)

Materials and Methods

Demographic Questionnaire • Age• Gender• Ethnicity• Education • Current marital status• Professional or Employment

Status• Household income• Best describes occupation

Personality Questionnaire (1-5)(Resilience and SAS)• Am a bad loser• Not easily affected by my

emotions• Can stand criticism• Believe that events in my life are

determined only by me• Can handle opposition• Respond well to change

Timeline

• Once a participant has given consent for study, initial overt inflammation screening will be given and assessments will begin1st year Every month Health Assessment, ELISA test, BDI (12 times) Psychiatric Evaluation/ Diagnosis (6 times) Relative Homicide Victim Questionnaire Personality assessment Demographic Questionnaire 2nd year Health Assessment, ELISA test, BDI (12 times) Psychiatric Evaluation/ Diagnosis (6 times)

Timeline

3rd year Health Assessment, ELISA test, BDI (12 times) Psychiatric Evaluation/ Diagnosis (6 times)Next 3 months (Experiment) New baseline Health Assessment, ELISA test, BDI (3 times) Psychiatric Evaluation/ Diagnosis (3 times) Fluoxetine Aspirin

Design

Design

Treatment

Budget$226,800.00 Instant® ELISA kits by eBioscience

$2,000.00 Advertisements

$30,000.00 My salary

$40,000.00 CO Principal Investigators salaries

$64,800.00 undergraduate researchers salaries

$54,000.00 Participant reimbursement

$108.00 Cotton balls

$1,476 Becton-Dickinson hypodermic single-use needles and syringes

Budget$385.00 3 HpmTM Reusable Tourniquet Cuffs (128.50 x 3)

$3063.96 3,600 6ml BD Vacutainer Plus Venous Blood Collection Tubes

$340.20 Alcohol wipes

$500.00 Undergraduate computer programmer salary

$14,850.00 3 SpectraMax Plus plate reader (Molecular Devices)

$103.52 8 rolls of 3MTM Medical Cloth Adhesive Tape 12.94 x 4

$1800 Fluoxetine (SRI) dosage based on

$1125 Bayer Aspirin Low Dose 81mg Child/ Adult

$555,000 Psychiatrist salaries for diagnosis & prescriptions

$991,352 Total Cost

Elisa; 226,800

Advertisements; 2,000

my salary; 25,000

CO PI; 40,000

und research; 64,800

Participants ; 54,000

syringes & needles; 1,476

tourniquets; 385blood collection tubes; 3,064

Alcohol wipes; 340Comp pro; 5003 Used Spec; 14,850 8 rolls med tape;

104

Psychiatrists; 555,000

cotton balls; 108 aspirin; 1,125 fluoxetine; 1,800

Works cited

Al-Hakeim, H. Al-Rammahi, D., & Al-Dujaili, A. (2015). IL-6, IL-18, sIL-2R, and TNFa proinflammatory markers in depression and schizophrenia patients who are free of overt inflammation. Journal of Affective Disorders, 106-114.

Charles, L. R., Capuron, L., & Andrew, H. M. (2006). Evolutionary imperatives for the depression-inflammation link. Trends in Immunology, 27(1), 24-31

Felger, J., & Lotrich, F. (2013). Inflammatory cytokines in depression: Neurobiological mechanisms and therapeutic implications. Neuroscience, 199-229

Jo, W., Zhang, Y., Emrich, H. ,& Dietrich, D. (2015). Glia in the cytokine-mediated onset of depression: Fine tuining the immune response. Frontiers in Cellulare Neuroscience, 9,

Kelber, O., Okpanyi, S. , Abdel-Aziz, H., & Khayyal, M. (2014). Brain, joint, gut: inflammation as link between depression, rheumatism and irritable bowel syndrome. Planta Medica, 80(16), 1361.

Rosenblat, J., Cha, D., Mansur, R., & Mcintyre, R. (2014). Inflamed moods: A review of interactions between inflammation and mood disorders. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 23-34

Sayers, J. (2001). The world health report 2001; mental health: New understanding, new hope. (books & electronic media). Bulletin of the World Health Organization, 79(11), 1085

Wang, Y., Yang, F., Liu, Y., Gao, F., & Jiang, W. (2011). Acetylsalicylic acid as an augmentation agent in fluoxetine treatment resistant depressive rats. Neuroscience Letters, 74-79