Assessment of adaptive and innate immune function
Relative frequencies of specific cell types are obtained by flow cytometry (BD LSRFortessa™). Peripheral blood mononuclear cells (PBMC) are isolated from whole blood buffy coats by Ficoll density gradient centrifugation. Lymphocyte subsets are characterized based on expression of surface proteins and include: memory and naïve B cells, memory and naïve CD4+ and CD8+ T cells, regulatory T cells, type-1 helper T cells (Th1), type-2 helper T cells (Th2), type-17 helper T cells (Th17). Innate immune cells (monocytes, neutrophils, eosinophils) are enumerated in whole blood and identified by flow cytometry. T cell responses are measured in freshly isolated peripheral blood mononuclear cells cultured with anti-CD3 and anti-CD28 antibodies or isotype control antibodies for 48 h to stimulate production of Th1 (IFN-γ), Th2 (IL-13), Th17 (IL-17A) and regulatory (IL-10) T cell cytokines. Innate immune responses are measured in freshly isolated PBMC cultured with bacterial lipopolysaccharide (LPS) for 24 h to stimulate monocyte cytokine (IL-1β, TNF-α, IL-6, IL-10) production. The concentrations of cytokines in culture supernatants are quantified by electrochemiluminescent multiplex assays [Meso Scale Discovery Sector Imager 2400].
Plasma markers of inflammation including acute phase proteins (C-reactive protein and serum amyloid A), markers of vascular inflammation (ICAM-1 and VCAM-1), chemokines (eotaxin, IP-10, MDC, MCP-1), cytokines (TNF-α, IL-6, IL-10, IL-8, IL-1β), and markers of innate immune activation (neopterin and myeloperoxidase) are measured by ELISA or electrochemiluminescent multiplex assays [Meso Scale Discovery Sector Imager 2400].
Identification of intestinal microbiota and inflammation
The relative abundances of stool bacteria are characterized by sequence analysis of the 16S ribosomal RNA genes. Microbiome analysis of raw DNA sequencing data is performed using Quantitative Insights into Microbial Ecology bioinformatics software. Neopterin and myeloperoxidase in stool samples will be measured by ELISA.
Metabolomic profiles are determined using both quantitative targeted and semi-quantitative untargeted mass spectrometric methods. Endpoints include but are not limited to non-esterified fatty acids, total fatty acids and oxylipins, endocannabinoids, NS-ceramides, sphingoid bases, bile acids, steroids, acylcarnitines, amino acids, biogenic amines, phospholipids, triglycerides, sphingomyelins, and cholesteryl esters. Prior to analyses, samples are enriched with isotopically labeled non-native analytes to be used as analytical surrogates, and samples are randomized across analytical batches, each containing a laboratory reference material to document analytical stability. Samples are randomly replicated at a rate of 5% across the study.
Chronic stress and stress reactivity
Allostatic load is an aggregate value derived from several parameters reflecting stress exposure. Concentrations of cortisol, epinephrine, norepinephrine, and creatinine in a 12-h urine sample are determined by ELISA. Hemoglobin A1c is measured in whole blood using the Cobas® Integra® 400 plus. Dehydroepiandosterone-sulfate concentrations are determined in plasma by electrochemiluminescence using a Cobas® e411 Immunology analyzer. Other parameters included in the allostatic load score were previously described (resting blood pressure, waist: hip circumference ratio, cholesterol level, and high sensitivity C-reactive protein). Acute stress system reactivity is measured by examining autonomic nervous system and endocrine responses to the meal, physical fitness, and emotional challenge tasks.
Endocrine hormone concentrations (cholecystokinin and peptide YY) are quantified by radioimmunoassay and gamma counter (Packard Cobra II Auto Gamma), Meso Scale Discovery multiplex panels (ghrelin, glucagon-like peptide-1, plasminogen activator inhibitor-1, adiponectin and leptin) and with the Cobas® e411 instrument (insulin). Blood specimens are collected in vacutainers containing EDTA plus protease inhibitor additives: DPPIV inhibitor (dipeptidyl-peptidase IV inhibitor (Millipore, St. Charles, MO, USA) 10 μl/ml whole blood) and aprotinin (G-Biosciences, St. Louis, MO, USA) 240 KIU/ml whole blood). In addition, for the ghrelin aliquot, a 1 M HCl (Trace Metal Grade HCl, Fisher-Scientific, Pittsburg, PA, USA) solution is added (100 μl/ml plasma) prior to plasma storage at −80C.
Plasma glucose and lipid concentrations and serum non-esterified fatty acid concentrations are measured using the Cobas® Integra® 400 plus.
Genomics and transcriptomics
Genomic DNA is isolated from whole blood (8 mL) using Qiagen© PAXgene Blood DNA kits and purified genomic DNA is aliquoted and stored at -80 °C until use. Single nucleotide polymorphisms in the genes of interest will be analyzed using TaqMan® SNP Genotyping Assays (ThermoFisher Scientific) performed on a QuantStudio® 7 Flex Real-Time PCR System (ThermoFisher Scientific) according to the manufacturer’s instructions. Allelic discriminations will be performed using Genotyper™ software (ThermoFisher Scientific). All ambiguous genotypes will be repeated in independent PCR experiments.
Transcriptomic profiling of whole blood is planned for a subset of participants to assess systemic immune activation. The Tempus™ blood RNA collection tube and the Tempus™ Spin RNA isolation kit (ThermoFisher Scientific) is used to stabilize and to isolate RNA.
Plasma carotenoid levels are quantified by liquid chromatography-diode array detection. Plasma homocysteine, thiamin, vitamin B6 and red blood cell riboflavin levels are quantified by liquid chromatography-fluorescence detection. Plasma vitamin B12 and folate status are analyzed by electrochemiluminescence immunoassay on Cobas®e411 analyzer.
Concentrations of trace minerals including iron, zinc, and copper are determined by Inductively Coupled Plasma-Optical Emission Spectrophotometry (Vista, Agilent Technologies). Blood is collected using Sarstedt Lithium Heparin polypropylene tubes.
Specimens required for clinical laboratory panels are submitted to the University of California Davis Medical Center clinical laboratory following the specified collection requirements. Comprehensive metabolic panels are determined by automated chemical analyzer. Complete blood count with differential analysis is quantified by Coulter count.
The Wechsler Abbreviated Scale of Intelligence™ Vocabulary and Matrix Reasoning modules are administered to assess cognitive function. A trained interviewer leads the assessments and records the responses given without providing indication of performance. The WASI®-II Stimulus Book is placed directly in front of the respondent to provide visual prompts for each task. The interviewer provides a standard set of instructions and begins when the participant is ready. Responses are scored by the interviewer in real-time according to published instructions for both modules. The T score, intelligence quotient, and 95% confidence interval are calculated according to published instructions.
The Cambridge Neuropsychological Test Automated Battery® is administered to assess cognitive function, including executive function. CANTAB® eclipse™ 5 software by Cambridge Cognition Ltd. is licensed on a touch screen tablet computer to complete each of the following four modules: 1) Motor Screening, 2) One Touch Stockings of Cambridge, 3) Spatial Working Memory, and 4) Stop Signal Task. Trained study personnel will provide instructions and brief demonstrations according to the standard instructions for each module before the participant is tested.
Examination of decision-making is performed using The Iowa Gambling Task™ by Psychological Assessments Resources, Inc. The computer-based game is run using Inquisit 3 Lab software . Trained study personnel read a scripted introductory statement, providing all of the required instructions. Participants are asked to complete the task on their own; study personnel step out of the testing room until the participant indicates that the task is complete.
Eating behaviors and motivations are assessed by questionnaire and food preference activities. The Yale Food Addiction Scale addresses addictive eating behaviors reported over the past 12 months, particularly with high-fat/sugar foods . The Food Choice Questionnaire assesses nine motives related to food choice: 1) health, 2) mood, 3) convenience, 4) sensory appeal, 5) natural content, 6) price, 7) weight, 8) familiarity, and 9) ethical concern . The Three-Factor Eating Questionnaire is used to measure: 1) ‘cognitive restraint of eating’, 2) ‘disinhibition’, and 3) ‘hunger’ .
Implicit and explicit wanting of foods is assessed using a paired foods test, which is based on the Implicit Associations Test . A forced-choice paired comparison paradigm presents paired images of foods side-by-side on a computer screen. Participants are asked to select the food that they most want to eat by pressing either the L or D key on a standard keyboard, corresponding to the food image on the right or left of the screen, respectively. The food preference activity is administered only once, at approximately 1.5 h postprandial, with respect to the timing of consumption of the mixed macronutrient challenge meal. Study personnel provide instructions for the assessment, but are not present as it is being completed. This computerized task is designed to measure implicit liking and wanting of four classifications of foods: high-fat and sweet, high-fat and savory, low-fat and sweet, low-fat and savory. As part of this task, images of single food items are presented individually to determine explicit liking of particular foods. Participants are instructed to indicate how much they like the taste of a single food item by clicking a location along an electronic visual analog scale that is presented below the image of the single food item on the computer screen.
MindWare mobile impedance cardiograph
Heart rate variability, respiration variability, and sympathetic nervous system activity as measured by skin conductance, is collected using the MindWare hardware and software systems by MindWare Technologies LTD. Data is monitored in real-time for the majority of one research visit day by wireless data transmission or captured locally on the collection device worn by the participant. Electrodes are placed in seven identified locations on the torso, five on the front torso and two on the back. Locations are predetermined by the manufacturer. Men are asked to shave the placement sites according to a provided diagram before the study visit. Trained study personnel clean skin at the application site using sterile alcohol prep pads and wipe dry using gauze before placing the torso electrodes. Two additional electrodes are placed on the non-dominant hand using only water to clean the hand locations. Data is acquired using the MindWare system for 15 min before and immediately after the two study challenges: 1) mixed macronutrient dietary challenge and 2) emotional recall challenge. Participants are asked to sit quietly during these dedicated data collection/rest periods without access to mobile devices or reading materials. Data is continuously collected throughout the morning and early afternoon, in addition to the described data collection/rest periods.
The Profile of Mood States questionnaire is administered before and immediately after the anger recall task. Participants are asked to remember, relive, and vividly recall a negative event that occurred within the last six months that makes them feel extremely angry. A paper and pen are provided for taking notes and recording details of the memory for a period of 2 min. The participant is then asked to share the experience aloud with a study staff member for 3 min.
Threshold concentrations of perceived taste of salty, sweet, and bitter flavors are assessed by a forced choice masked taste test. Solutions of varying concentrations of sodium chloride, sucrose, and caffeine, respectively, are prepared using distilled water at room temperature. Sensitivity to salty taste is assessed using five concentration levels: 1) 0.5 g/L, 2) 1.75 g/L, 3) 6 g/L, 4) 21 g/L, and 5) 75 g/L (approximately 10 times the concentration in chicken broth). Perception of sweet taste is assessed using five levels of sucrose concentration: 1) 1.23 g/L, 2) 3.7 g/L, 3) 11 g/L, 4) 33 g/L, and 5) 99 g/L (similar to the concentration found in regular soft drinks). Threshold of detection of bitter taste is assessed using five concentrations of caffeine in distilled water: 1) 0.16 g/L, 2) 0.25 g/L, 3) 0.40 g/L, 4) 0.64 g/L, and 5) 1.01 g/L (similar to the concentration in brewed coffee).
Solutions are presented to study participants in increasing order, starting with the most dilute and increasing in concentration until three consecutive concentrations are correctly identified. Samples are provided in clear, plastic cups labeled with randomly generated code numbers. The participant is instructed to rinse the mouth with distilled water before each round of tastings. Each round consists of three taste cups; one cup contains the tastant (solution) and two identical clear plastic cups that contain fresh distilled water only. The participant is asked to record the unique identifiers on each of the three cups presented at each round and then to select which solution is different from the other two. The participant records the three-digit number identifying the odd cup and if taste distinction is not possible, the participant is instructed to guess which solution is different. Participants may spit the taste solution out or swallow it, depending on preference, and are provided with more sample volume to taste, if requested. Sample cups are removed after each round and study personnel record the identifier of the selected cup behind a wall to determine if the taste solution was correctly identified. The lowest concentration correctly identified in a series of three consecutive correctly identified solutions is determined to be the threshold of detection.
Skin reflectance is assessed using a Konica-Minolta Spectrophotometer CM-2500d as described previously . The instrument is placed flat against the skin in three locations: 1) back of hand, in the area between the index finger and thumb, 2) inside of the upper arm, and 3) upper center of the forehead. Measurements are taken in duplicate and averaged.
Peripheral endothelial vasodilation responses to reactive hyperemia are assessed using an EndoPAT 2000 (endothelial peripheral arterial tone)  as a non-diagnostic indicator of endothelial function. The participant lies supine on a bed with legs uncrossed and must rest for 10–15 min. A blood pressure cuff is placed on the non-dominant upper arm and fit snuggly above the elbow. The wrists and forearms rest on arm supports positioned at a comfortable distance based on arm length. The index finger of each hand is placed in a probe that is positioned in a designated socket of the arm support. The probe covers inflate in order to ensure connectivity between the tip of the finger and the wire probe. A soft anchor is placed on the finger adjacent to the probe finger and the tubing is looped to avoid contact with the hand. The participant must lie still in order to produce a stable baseline period of data for at least 5 min. The upper arm blood pressure cuff is inflated to 200 mmHg and is kept at that pressure to occlude the brachial artery for 5 min. After 5 min, the blood pressure cuff is released and deflates quickly. The participant remains resting for 5 min post-occlusion. The reactive hyperemia index score is calculated as the post-to-pre occlusion PAT signal ratio on the occluded side, normalized to the control side and corrected for baseline vascular tone (Itamar Medical Ltd).