Immune-mediated inflammatory diseases are a health burden for approximately seven percent of the population of Western nations. Preliminary data suggest variations in ethnic identity and/or geography influence discrimination experiences and inflammatory response trends. This study investigates how geography, ethnicity, and laboratory manipulation of discrimination experiences affect immune cell function and genomic regulation. Flow cytometry and immune cell stimulation will test monocytes collected from peripheral blood for functional effects. Next-generation transcriptomics and epigenomics will assess genomic and epigenetic mechanisms. The hypothesis is that geography, self-identified race, and ethnicity, interacting with laboratory discrimination conditions during the virtual ballgame Cyberball™, significantly affect immune cell function through genomic and epigenetic mechanisms, with perceived discrimination as a moderating factor on the immune outcomes. The transdisciplinary nature of the proposed study aims to provide valuable insights into differential susceptibility to immune-mediated inflammatory diseases across diverse populations. Uncovering these insights will better inform population-relevant interventions for immune-mediated inflammatory diseases.
More than a third of the residents of the United States suffer from a chronic disease, with almost half involving dysregulated immune processes. Immune-mediated inflammatory diseases pose a public health burden in the United States. Preliminary data from previous work suggest that variations in ethnic identity and geography might influence discrimination experiences and inflammatory response trends. To investigate the functional implications of these findings, a multi-institutional study is proposed, examining how social experiences and demographic factors predict the regulation and activity of immune cells. Specifically, the hypothesis is that geography, self-identified race, and ethnicity, interacting with laboratory discrimination conditions, significantly affect immune cell function through genomic and epigenetic mechanisms, with perceived discrimination moderating the immune outcomes. Participants will provide saliva samples, complete psychosocial and demographic questionnaires, and play the virtual social exclusion game Cyberball™ in a randomly assigned block order. Acute discrimination experiences are manipulated by conditions during Cyberball. Virtual players, who appear to be of a different race from the participant, exclude them by not passing the participant the ball in the race-based exclusion condition. In the inclusion condition, the participant receives the ball regardless of participant and virtual player race. In the general social exclusion (not race-based), the players and participant races are similar to the inclusion condition, except that the participant does not receive the ball. The block of Cyberball and blood draws randomly assigned are either: 1) race-based social exclusion and inclusion first, a blood draw, then non-race-based social exclusion, inclusion, and blood draw, or 2) non-race-based social exclusion and inclusion, a blood draw and then the race-based social exclusion and inclusion and a blood draw. Baseline and post-first block inflammatory responses in saliva will be measured using enzyme-linked immunosorbent assays (ELISAs) to determine the concentration of cytokines like C-reactive protein, interleukin-6, and tumor necrosis factor-alpha. Flow cytometry and immune cell stimulation with toxin will test monocytes purified and sorted from the participant's blood for functional effects. Next-generation transcriptomics and epigenomics will assess differentially expressed RNA and methylation enrichment, emphasizing genes involved in inflammation signaling pathways. The data will be statistically analyzed using regression analysis and structural equation modeling to determine the relationship between discrimination, geography, immune cell function, and regulation while controlling for other socio-demographic factors. The findings could inform public health initiatives and interventions to reduce health disparities and improve outcomes for marginalized communities.
Study Type
INTERVENTIONAL
Allocation
RANDOMIZED
Purpose
BASIC_SCIENCE
Masking
DOUBLE
Enrollment
480
Cyberball™ is a virtual social exclusion game that simulates social interactions and acute discrimination experiences in a controlled laboratory setting. The game involves passing a virtual ball between players represented by images. In this study, only the participant is a real player, while the other "players" are pre-programmed virtual avatars controlled by the experimenter using Cyberball software. The ethnicity of these virtual players can be manipulated by changing the stock photos used to represent them. Participants in the Race-Based Social Exclusion condition play Cyberball with virtual players who appear to be of a different ethnicity from the participant. In the Non-Race-Based Social Exclusion, the participant plays with virtual players who do not have any obvious ethnic differences from the participant, and social inclusion is a washout condition where the participant receives the ball from the virtual players, regardless of the ethnicity of the avatars.
Morgan State University
Baltimore, Maryland, United States
RECRUITINGTexas Christian University
Fort Worth, Texas, United States
RECRUITINGFrequency of Perceived Discrimination Experiences
Before the game, participants will be asked to complete the "Everyday Discrimination Scale" designed to measure their perceptions of discrimination encountered in their everyday lives. This instrument prompts respondents to rate the frequency of discriminatory events in their daily lives, such as "being treated with less courtesy than others." Everyday Discrimination Scale Participants provide their responses on a scale that ranges from "0" for "never" to "5" for "almost every day." The summed responses provide a measure of how frequently they experienced discrimination. The summed score can range from 0 to 50; the higher the score, the more frequently participants perceived that they experienced discrimination. Additionally, participants specify whether these experiences are due to various factors, including race and gender.
Time frame: Survey response collected before the first Cyberball Sequence within 30 minutes of arriving at the lab) and score calculated during data analysis
Type, Timing and Frequency of Lifetime Discrimination Experiences
The "Experiences of Discrimination Scale" measures the type, timing, and frequency of discrimination. Participants answer "yes" or "no" to 9 types of unfair treatment, such as, "Have you ever been unfairly fired?" The sum of "yes" responses ranges from 0 to 9, with a score above 6 indicating many unfair treatments. Participants also specify if the unfair treatment was due to race, skin color, or other characteristics. The sum of responses related to ancestry, race, ethnicity, or skin color indicates the frequency of racial discrimination. Additionally, participants report the last time they experienced unfair treatment due to race or ethnicity, with responses from 1 ("past week") to 4 ("more than a year"). Lower scores indicate more recent occurrences.
Time frame: Survey response collected within 30 minutes after completing the first Cyberball Sequence and score calculated during data analysis]
Recent Discrimination and Perceived Ethnic Discrimination Score
The sum of response scores for the Recent Discrimination (a) and Ethnic Discrimination (b) subscale items of the "General Ethnic Discrimination Scale"provides the frequency of recent discrimination experience and includes a manipulation check for the validity of the recent Cyberball condition Recent Discrimination is calculated as the sum of responses to all (a) items ranging from a choice of 1 for "never" to 6 for "almost all the time. A summed minimum score of 18 corresponds to no lifetime discrimination, and a summed maximum score of 108 corresponds to the most frequent experience of perceived lifetime discrimination. Lifetime Discrimination is calculated as the sum of all (b) item responses ranging from a choice of 1 for "never" to 6 for "almost all the time. A summed minimum score of 18 corresponds to no lifetime discrimination, and a summed maximum score of 108 corresponds to the most frequent experience of perceived lifetime discrimination.
Time frame: Survey responses collected within 30 minutes after completing the Second Cyberball Sequence and score calculated during data analysis]
Recent and Lifetime Discrimination Stress Score
The Cumulative response score from the Stress from Discrimination (c) items on the General Ethnic Discrimination Scale measures a person's perception of the stress of recent and lifetime unfair treatment due to a participant's racial/ethnic identity. Participants are asked to respond to the statement "How stressful was this for you?" for each discrimination experience. Response choices range from 1 for "not at all stressful" to 6 for "extremely stressful." Discrimination Stress is calculated as the sum of all (c) items on the General Ethnic Discrimination (GED) scale, where a summed score of 17 indicates little to no stress, and 102 indicates the highest amount of stress experienced due to recent and lifetime discrimination experiences.
Time frame: Survey responses collected within 30 minutes after completing the Second Cyberball Sequence and score calculated during data analysis
Relationship between salivary and serum immune responses
Multiplex enzyme-linked immunosorbent assays (ELISA) will allow assessment of the pg/mL concentration of inflammatory cytokines in saliva and serum. These data will be used to calculate correlation coefficients that depict the magnitude and direction of the relationship between saliva and serum inflammatory cytokine concentration. The cytokines to be measured are Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α, and c-Reactive Protein (CRP).
Time frame: Samples used in assay are saliva collected at baseline (30 mins before) and 40 minutes after the first Cyberball sequence, and serum is purified from whole blood collected 30 minutes after the first and second Cyberball sequence.
Distribution and Quantity of Immune Cells in Whole blood
To determine the quantity of each type of peripheral blood mononuclear cells (PBMCs) available after the different Cyberball sequences, the PBMCs will be sorted and quantified from whole blood collected 30 minutes after the first and 30 minutes after the second Cyberball sequence. A Sony SH800 cell sorter is used to perform Fluorescence-Activated Cell Sorting (FACS) with appropriate antibodies to identify T cells (CD3+), B cells (CD19+), NK cells (CD56+), and monocytes (CD14+CD16-, CD14+CD16+) and counts provided of viable cells with the cell counter.
Time frame: These data will be from whole blood samples collected 30 minutes after the first and 30 minutes after the second Cyberball sequence. The sequences are between 90 to 120 minutes apart separated by a washout period.
Stimulated PBMC Cytokine Release
Cellular Cytokine release from (PBMCs) purified from the participant's whole blood is assessed in the presence or absence of stimulation with lipopolysaccharide (LPS) toxin. The LPS incubation time will vary in length, and ELISA will measure pg/ml concentration of IL-6, TNF-α, IL-1β, CRP
Time frame: PMBCs purified from whole blood collected collected 30 minutes after the first and 30 minutes after the second Cyberball sequence are compared at 0, 2, 24 and 26 hours after incubation in LPS
Gene expression levels
RNA expression counts and distribution will be measured in PBMCs purified from participant whole blood collected participants after completing the first and second Cyberball sequences (collection tines separated by up 2 hours)
Time frame: PMBCs purified from whole blood collected collected 30 minutes after the first and 30 minutes after the second Cyberball sequence
Magnitude of Race/Ethnic Residential Segregation (Dissimilarity Index)
The Dissimilarity Index or DI measures Race/Ethnic Residential Segregation. We aim to measure the DI between non-Hispanic Black and non-Hispanic White, Hispanic and non-Hispanic White, and Hispanic and non-Hispanic Black populations. Census tract data from the participant's childhood zip code will be used to calculate the three childhood dissimilarity indices. The 2020 census tract data for the current zip code will be used in the equation for calculating this index. The equation for the index is DI=100×0.5× SUM(PxiPx-PyiPy) where Pxi and Pyi are the populations of groups x and y in census tract i, and Px and Py are the total populations of groups x and y in the overall geographic area. The DI ranges from 0 to 100, with scores less than 30 indicating low segregation, 30-60 indicating moderate segregation, and greater than 60 indicating high segregation.
Time frame: Questionnaire responses to the zip code items are collected within first 30 minutes in the lab and Index calculated during data analysis
Average Community Risk Protective Factors
"The Communities That Care Youth Survey" assesses Community risk and protective factors. Calculated average risk scores are based on responses to risk items, and calculated average protection scores are based on the protective items, with minimum and maximum average scores equaling 1 and 4, respectively. The average community risk is determined by calculating the average responses to items based on the five risk factors: Drug availability, norms for drug use, availability of handguns, neighborhood attachment, and community disorganization. A sample Community Risk item states:"If I wanted to get some marijuana, how easy would it be for me to get some?" response choices range from 1 for "Very hard to 4 for "Very easy." Average scores of less than 2 for the five factors indicate low community risk, and 3 or greater indicates high community risk.
Time frame: Survey responses are collected within 30 minutes after completing the first Cyberball sequence and score calculated during data analysis
Average Community Protective Factors
"The Communities That Care Youth Survey" assesses Community risk and protective factors. Calculated average protective factors scores are based on responses to protective items, with minimum and maximum average scores equaling 1 and 4, respectively. The Average Community Protection is determined by calculating the average responses to items on two protective factors: Opportunities and Rewards for prosocial involvement. A sample Community Protective item states: "My neighborhood offers lots of opportunities to participate in positive activities." With response choices ranging from 1 for "Strongly disagree" to 4 for "Strongly agree" Average scores lower than 2 indicate low community protection and a score of 3 or greater indicates high community protective factors.
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Time frame: Survey responses are collected within 30 minutes after completing the first Cyberball sequence and score calculated during data analysis
Resilience
The Brief Resilience Scale consists of six items that assess participants' resilience or buffer against stressful experiences. A sample item states, "I tend to bounce back quickly after hard times," with response choices ranging from 1 for "Strongly Disagree" to 5 for "Strongly Agree." The participant's Resilience Score is based on a calculated average of the response scores to the six items on the scale. The minimum possible average score is 1, and the maximum is 6. Scores below 3 indicate low resilience and scores above 4.31 indicate high resilience.
Time frame: Survey responses are collected within 30 minutes after completing the second Cyberball sequence and the average response score calculated during data analysis
Frequency of recent stress
The frequency of recent stress is calculated by summing the response scores on the ten-item "Perceived Stress Scale." The response options for each feeling or thought indicate the frequency with which it occurred, ranging from 0 for "Never" to 4 for "Very Often." Scores can be made by summing across all 10 items. Scores can range from 0 to 40, with higher scores indicating greater stress.
Time frame: Survey responses are collected within 30 minutes after completing the first Cyberball sequence and Perceived Stress score calculated during data analysis
Degree of Morbidity
The sum of response items on the "Comorbidity Questionnaire" provides a single measure of the magnitude of chronic medical conditions faced by participants. The questionnaire can have up to fifteen items with twelve listed illnesses and options for participants to list an additional three items. One point if given each is given for the presence of the condition, with an additional point for receiving treatment for the condition, and another if the condition affects daily function. The Degree of Morbidity is calculated by summing the responses to the three components for each listed illness. The minimum score that can be earned is 0, and the maximum score is 45, with higher scores indicating greater morbidity.
Time frame: Survey responses are collected within 30 minutes after completing the second Cyberball sequence and the sum of response scores calculated during data analysis
Methylation Score
The proportion of methylated to unmethylated cytosines at CpG sites around regulatory transcripts in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway will be assessed in purified PBMCs
Time frame: Methylsequencing of PMBCs purified from whole blood collected collected 30 minutes after the first and 30 minutes after the second Cyberball sequence