Comprehensive observational study evaluating the enduring effectiveness of 4CMenB, the meningococcal B vaccine against gonococcal infections in the Northern Territory and South Australia, Australia: study protocol.

INTRODUCTION: The effectiveness of antibiotics for treating gonococcal infections is compromised due to escalating antibiotic resistance; and the development of an effective gonococcal vaccine has been challenging. Emerging evidence suggests that the licensed meningococcal B (MenB) vaccine, 4CMenB is effective against gonococcal infections due to cross-reacting antibodies and 95% genetic homology between the two bacteria, Neisseria meningitidis and Neisseria gonorrhoeae, that cause the diseases. This project aims to undertake epidemiological and genomic surveillance to evaluate the long-term protection of the 4CMenB vaccine against gonococcal infections in the Northern Territory (NT) and South Australia (SA), and to determine the potential benefit of a booster vaccine doses to provide longer-term protection against gonococcal infections. METHODS AND ANALYSES: This observational study will provide long-term evaluation results of the effectiveness of the 4CMenB vaccine against gonococcal infections at 4-7 years post 4CMenB programme implementation. Routine notifiable disease notifications will be the basis for assessing the impact of the vaccine on gonococcal infections. Pathology laboratories will provide data on the number and percentage of N. gonorrhoeae positive tests relative to all tests administered and will coordinate molecular sequencing for isolates. Genome sequencing results will be provided by SA Pathology and Territory Pathology/New South Wales Health Pathology, and linked with notification data by SA Health and NT Health. There are limitations in observational studies including the potential for confounding. Confounders will be analysed separately for each outcome/comparison. ETHICS AND DISSEMINATION: The protocol and all study documents have been reviewed and approved by the SA Department for Health and Well-being Human Research Ethics Committee (HREC/2022/HRE00308), and the evaluation will commence in the NT on receipt of approval from the NT Health and Menzies School of Health Research Human Research Ethics Committee. Results will be published in peer-reviewed journals and presented at scientific meetings and public forums.

Preliminary evidence of increased alcohol use associated with ethinyl estradiol levels in women using oral contraceptives.

Alcohol use is highly prevalent in young adult women and rates of alcohol use disorder are rising rapidly in this population. Further, emerging evidence suggests that circulating levels of ovarian hormones influence alcohol consumption, with increased consumption associated with higher estradiol and lower progesterone levels. However, less is known about the influence of synthetic hormones (contained in oral contraceptive (OC) pills) on alcohol use. The current study examined the influence of OC pill phase, ethinyl estradiol (EE) levels, and progestin levels on self-reported alcohol consumption in healthy female drinkers. Young adult female drinkers using OCs (N = 21) reported alcohol use across one OC pill pack using the Timeline Followback and provided blood samples during both pill phases to measure synthetic hormone levels. We compared alcohol use between OC pill phases (active vs. inactive) using linear mixed effects models for repeated measures and examined correlations between alcohol use and EE and progestin levels. Results showed that women with higher EE levels reported increased alcohol consumption (r = 0.56, p = 0.01) and binge drinking (r = 0.45, p = 0.04) in the active pill phase. Progestin levels and pill phase were not significantly associated with alcohol consumption. These findings provide preliminary data suggesting increased levels of EE from OC pills are associated with excessive alcohol consumption in women. Further research is needed to determine if EE plays a causal role in increased alcohol consumption. This line of research could inform female-specific AUD prevention and treatment strategies among the large subpopulation of women using hormonal contraceptives.

Alcohol conditioned contexts enhance positive subjective alcohol effects and consumption.

Associations between alcohol and the places it is consumed are important at all stages of alcohol abuse and addiction. However, it is not clear how the associations are formed in humans or how they influence drinking, and there are few effective strategies to prevent their pathological effects on alcohol use. We used a human laboratory model to study the effects of alcohol environments on alcohol consumption. Healthy regular binge drinkers completed conditioned place preference (CPP) with 0 vs. 80 mg/100 mL alcohol (Paired Group). Control participants (Unpaired Group) completed sessions without explicit alcohol-room pairings. After conditioning, participants completed alcohol self-administration in either the alcohol- or no alcohol-paired room. Paired group participants reported greater subjective stimulation and euphoria, and consumed more alcohol in the alcohol-paired room in comparison to the no alcohol-paired room, and controls tested in either room. Moreover, the strength of conditioning significantly predicted drinking; participants who exhibited the strongest CPP consumed the most alcohol in the alcohol-paired room. This is the first empirical evidence that laboratory-conditioned alcohol environments directly influence drinking. The results also confirm the viability of the model to examine the mechanisms by which alcohol environments stimulate drinking and to test strategies to counteract their influence on behavior.

The first 2 months of COVID-19 contact tracing in the Northern Territory of Australia, March-April 2020.

The Northern Territory (NT) Centre for Disease Control (CDC) undertook contact tracing of all notified cases of coronavirus disease 2019 (COVID-19) within the Territory. There were 28 cases of COVID-19 notified in the NT between 1 March and 30 April 2020. In total 527 people were identified as close contacts over the same period; 493 were successfully contacted; 445 were located in the NT and were subsequently quarantined and monitored for disease symptoms daily for 14 days after contact with a confirmed COVID-19 case. Of these 445 close contacts, 4 tested positive for COVID-19 after developing symptoms; 2/46 contacts who were cruise ship passengers (4.3%, 95% CI 0.5-14.8%) and 2/51 household contacts (3.9%, 95% CI 0.5-13.5%). None of the 326 aircraft passengers or 4 healthcare workers who were being monitored in the NT as close contacts became cases.

Quantitative meta-analysis of heart rate variability finds reduced parasympathetic cardiac tone in women compared to men during laboratory-based social stress.

Heart rate variability (HRV) is the inter-beat interval variation between consecutive heartbeats and an autonomic reflection of emotional regulatory abilities to flexibly respond to challenges, such as psychosocial stress. Whereas there are known sex differences in stress-induced hormonal and emotional responses, we identified a gap in our understanding of sex-specific autonomic cardiac control during stress. Thus, we assessed HRV prior to, during and after administration of a public speech task in healthy participants (n = 929) according to sex. Our meta-analysis found that during stress, women had lower HRV than men, with an overall Hedges' g of 0.29 (p < 0.0001) and 0.29 (p = 0.0003) for fixed and random effects models, respectively. We did not find significant heterogeneity or evidence of publication bias. Analyses of additional timepoints showed no baseline difference and marginally lower HRV in women during anticipation and recovery. Findings of the present meta-analysis confirm sex differences in stress-induced hyperarousal and form a justification for implementation of mechanistic studies evaluating gonadal hormones, their potent metabolites and pro-inflammatory cytokines as mediators of this relationship.

Amygdala-orbitofrontal functional connectivity mediates the relationship between sensation seeking and alcohol use among binge-drinking adults.

BACKGROUND: Decreased amygdala-orbitofrontal cortex (OFC) neural functional connectivity (FC) positively predicts alcohol use among adolescents. Low amygdala-OFC FC is also associated with poor emotion regulation, a trait robustly linked to alcohol use. Thus, decreased amygdala-OFC connectivity may represent a risk factor for the development of alcohol use disorder (AUD) via impaired emotion regulation or reward processing. In this study, we examined amygdala-OFC FC among young adult binge drinkers at high risk for AUD. We also tested if amygdala-OFC FC mediates the relationship between externalizing personality traits and alcohol use. METHODS: Healthy male and female (n = 39) binge drinkers completed a resting state fMRI scan and the Eysenck Impulsive Personality questionnaire. We utilized seed-based connectivity of the left and right amygdala to prefrontal regions as well as mediation analysis. RESULTS: Individuals with higher weekly alcohol use displayed decreased right amygdala-OFC FC. Furthermore, high trait venturesomeness, but not impulsivness, was associated with decreased right amygdala-OFC FC. Finally, right amygdala-OFC FC mediated the relationship between trait venturesomeness and weekly drinking; individuals with high trait venturesomeness displayed decreased right amygdala-OFC FC, which in turn predicted greater weekly drinking. CONCLUSIONS: Our findings corroborate and extend the adolescent literature by showing that decreased amygdala-OFC FC is associated with higher alcohol consumption among adults at elevated risk for AUD. This study also demonstrates for the first time that this neural profile reflects a tendency to sensation seeking. In sum, our findings suggest that amygdala-OFC FC may be an objective neural target for alcohol use prevention and intervention.

Striatal activity correlates with stimulant-like effects of alcohol in healthy volunteers.

Individuals who experience greater stimulation and less sedation from alcohol are at increased risk for alcohol-related problems. However, little is known regarding the neurobiological mechanisms underlying subjective response to alcohol. The current study examined the degree to which alcohol-induced brain activation correlates with ratings of stimulation and sedation, using a within-subjects, double-blind, placebo-controlled design. Participants (N = 34 healthy adults with no history of alcohol use disorder) completed three sessions: a calibration session to determine the duration of infusion needed to bring the breath alcohol to 80 mg/dl for each subject, and two counterbalanced fMRI sessions with placebo and alcohol administration. During the fMRI sessions, participants underwent 50 min scans, which included a 10 min baseline period, the IV infusion period needed to bring breath alcohol concentration (BrAC) to a peak 80 mg/dl (on the alcohol session), followed by a post-peak decline period. Participants rated their subjective stimulation and sedation at regular intervals throughout the scan. A priori VOI analyses showed that the time course of stimulation correlated with BOLD signal in the striatum. The time course of sedation did not correlate with BOLD signal in any VOIs. There were no correlations in primary visual cortex, which served as a control. These findings are the first to show that alcohol effects in the striatum are linked to the positive, stimulant-like effects of the drug and advance our understanding of the neurobiological mechanisms underlying individual differences in subjective responses to alcohol, and more broadly, risk for alcohol use disorders.

Acute calming effects of alcohol are associated with disruption of the salience network.

The mood-altering properties of alcohol are a key motivation for drinking, and people commonly report that they drink alcohol to alleviate stress or to relax. To date, the neural processes associated with the self-reported calming effects of alcohol are not well understood. Existing data imply that alcohol may target and disrupt activity within anterior insula (aINS) and amygdala-based neural networks, which are regions implicated in threat detection and anxious responding. The aims of the current study were (1) to examine the acute effect of alcohol upon functional connectivity within aINS and amygdala circuits and (2) to assess relationships between alcohol effects on functional connectivity and self-reported subjective mood. Healthy men and women (N = 39) who reported regular binge drinking completed a within-subjects, double-blind, placebo-controlled pharmacological functional magnetic resonance imaging experiment with i.v. infusions of either alcohol or placebo. Infusion profiles were personalized for each participant and raised breath alcohol concentration to 80 mg percent. Before, during and after infusions, participants rated their subjective mood (stimulation, sedation and calm). Results showed that alcohol dampened functional connectivity between bilateral aINS seed-regions-of-interest and the dorsal anterior cingulate cortex (dACC), key nodes of the salience network. Additionally, the more that alcohol reduced right aINS-dACC functional connectivity, the calmer participants felt during alcohol administration. Alcohol had no effect on amygdala functional connectivity. These findings suggest that alcohol disrupts aINS-dACC functional connectivity, which may impair detection and appraisal of emotionally salient information and relate to acute relaxing effects of the drug.

Dose-related effects of delta-9-THC on emotional responses to acute psychosocial stress.

OBJECTIVES: Cannabis smokers often report that they use the drug to relax or to relieve emotional stress. However, few clinical studies have shown evidence of the stress-relieving effects of cannabis or cannabinoid agonists. In this study, we sought to assess the influence of delta-9-tetrahydrocannabinol (THC), a main active ingredient of cannabis, upon emotional responses to an acute psychosocial stressor among healthy young adults. METHODS: Healthy volunteers (N=42) participated in two experimental sessions, one with psychosocial stress (Trier Social Stress Test, TSST) and another with a non-stressful task, after receiving 0 (N=13), 7.5mg (N=14) or 12.5mg (N=15) oral THC. Capsules were administered under randomized, double blind conditions, 2.5h before the tasks began. We measured subjective mood and drug effects, vital signs and salivary cortisol before and at repeated times after the capsule and tasks. Subjects also appraised the tasks, before and after completion. RESULTS: In comparison to placebo, 7.5mg THC significantly reduced self-reported subjective distress after the TSST and attenuated post-task appraisals of the TSST as threatening and challenging. By contrast, 12.5mg THC increased negative mood overall i.e., both before and throughout the tasks, and pre-task ratings of the TSST as threatening and challenging. It also impaired TSST performance and attenuated blood pressure reactivity to the stressor. CONCLUSIONS: Our findings suggest that a low dose of THC produces subjective stress-relieving effects in line with those commonly reported among cannabis users, but that higher doses may non-specifically increase negative mood.

Virtual reality conditioned place preference using monetary reward.

Computerized tasks based on conditioned place preference (CPP) methodology offer the opportunity to study learning mechanisms involved in conditioned reward in humans. In this study, we examined acquisition and extinction of a CPP for virtual environments associated with monetary reward ($). Healthy men and women (N=57) completed a computerized CPP task in which they controlled an avatar within a virtual environment. On day 1, subjects completed 6 conditioning trials in which one room was paired with high $ and another with low $. Acquisition of place conditioning was assessed by measuring the time spent in each room during an exploration test of the virtual environments and using self-reported ratings of room liking and preference. Twenty-four hours later, retention and extinction of CPP were assessed during 4 successive exploration tests of the virtual environments. Participants exhibited a place preference for (spent significantly more time in) the virtual room paired with high $ over the one paired with low $ (p=0.015). They also reported that they preferred the high $ room (p<0.001) and liked it significantly more than the low $ room (p<0.001). However, these preferences were short-lived: 24h later subjects did not exhibit a behavioral or subjective preference for the high $ room. These findings show that individuals exhibit transient behavioral and subjective preferences for a virtual environment paired with monetary reward. Variations on this task may be useful to study mechanisms and brain substrates involved in conditioned reward and to examine the influence of drugs upon appetitive conditioning.

Test-retest reliability of the underlying latent factor structure of alcohol subjective response.

RATIONALE: Alcohol subjective experiences are multi-dimensional and demonstrate wide inter-individual variability. Recent efforts have sought to establish a clearer understanding of subjective alcohol responses by identifying core constructs derived from multiple measurement instruments. OBJECTIVE: The aim of this study was to evaluate the temporal stability of this approach to conceptualizing alcohol subjective experiences across successive alcohol administrations in the same individuals. METHODS: Healthy moderate alcohol drinkers (n = 104) completed six experimental sessions each, three with alcohol (0.8 g/kg), and three with a non-alcoholic control beverage. Participants reported subjective mood and drug effects using standardized questionnaires before and at repeated times after beverage consumption. We explored the underlying latent structure of subjective responses for all alcohol administrations using exploratory factor analysis and then tested measurement invariance over the three successive administrations using multi-group confirmatory factor analyses. RESULTS: Exploratory factor analyses on responses to alcohol across all administrations yielded four factors representing "Positive mood," "Sedation," "Stimulation/Euphoria," and "Drug effects and Urges." A confirmatory factor analysis on the separate administrations indicated acceptable configural and metric invariance and moderate scalar invariance. CONCLUSIONS: In this study, we demonstrate temporal stability of the underlying constructs of subjective alcohol responses derived from factor analysis. These findings strengthen the utility of this approach to conceptualizing subjective alcohol responses especially for use in prospective and longitudinal alcohol challenge studies relating subjective response to alcohol use disorder risk.

Alcohol-induced place conditioning in moderate social drinkers.

AIMS: To test whether non-dependent drinkers show place preference for a location paired with alcohol, and to test if the amount of time spent in the alcohol-paired location is related to self-reported subjective alcohol effects experienced in that environment. DESIGN: Two groups of subjects completed six conditioning sessions: three with alcohol (0.8 g/kg) and three without alcohol. Individuals were assigned randomly to two groups, paired and unpaired, in a 2 : 1 ratio. The paired group (n = 78) received alcohol in one testing room and no-alcohol in another testing room (biased assignment). The unpaired group (n = 30) received alcohol and no-alcohol in each testing room. SETTING: Human Behavioral Pharmacology Laboratory, University of Chicago, Chicago, IL, USA (single site). PARTICIPANTS: Healthy male and female social drinkers (n = 108) aged 21-40 years participated in the study (consisting of 10 separate laboratory visits) between March 2012 and August 2014 (an average of 36 separate subject visits per month). MEASUREMENTS: The primary outcome measure was the pre- to post-conditioning change in the percentage of time spent in the least preferred room (obtained during drug-free exploration tests conducted at separate visits before and after the six conditioning sessions were completed). Secondary measures included self-reported subjective mood and drug effects obtained during the conditioning sessions. FINDINGS: The groups differed in the change in the percentage of time spent in the initially least preferred room, from pre- to post-conditioning; paired group = 11.0%, unpaired group = -1.4%, mean difference = 12.4%, 95% confidence interval = 1.9-23.0, P = 0.02. The change in the percentage of time spent in the least-preferred room was related to the self-reported sedative effects of alcohol during conditioning sessions among paired group participants only. CONCLUSIONS: Non-dependent consumers of alcohol appear to develop a behavioral preference for locations paired with alcohol consumption, more so for those who experience sedative effects from alcohol in those locations.

Effects of d-amphetamine upon psychosocial stress responses.

Psychostimulant drugs alter the salience of stimuli in both laboratory animals and humans. In animals, stimulants increase rates of responding to conditioned incentive stimuli, and in humans, amphetamine increases positive ratings of emotional images. However, the effects of stimulants on real-life emotional events have not been studied in humans. In this study, we examined the effect of d-amphetamine on responses to acute psychosocial stress using a public speaking task. Healthy volunteers (N=56) participated in two experimental sessions, one with a psychosocial stressor (the Trier Social Stress Test) and one with a non-stressful control task. They were randomly assigned to receive d-amphetamine (5 mg n=18, 10 mg n=20) or placebo (n=18) on both sessions under double blind conditions. Salivary cortisol, subjective mood, and vital signs were measured at regular intervals during the session. Subjects also provided cognitive appraisals of the tasks before and after their performances. Amphetamine produced its expected mood and physiological effects, and the Trier Social Stress Test produced its expected effects on cortisol and mood. Although neither dose of amphetamine altered cardiovascular or hormonal responses to stress, amphetamine (10 mg) increased participants' pre-task appraisals of how challenging the task would be, and it increased post-task ratings of self-efficacy. Paradoxically, it also increased ratings of how stressful the task was, and prolonged aversive emotional responses. These findings suggest that amphetamine differentially affects stress response components: it may increase participants' appraisals of self-efficacy without dampening the direct emotional or physiological responses to the stress.

Opioid partial agonist buprenorphine dampens responses to psychosocial stress in humans.

Pre-clinical and clinical evidence indicates that opioid drugs have stress-dampening effects. In animal models, opioid analgesics attenuate responses to isolation distress, and in humans, opioids reduce stress related to anticipation of physical pain. The stress-reducing effects of opioid drugs may contribute to their abuse potential. Despite this evidence in laboratory animals, the effects of opioids on responses to psychosocial stress have not been determined in humans. Here we examined the effects of buprenorphine, a µ-opioid partial agonist used to treat opioid dependence and pain, on subjective and physiological responses to a stressful public speaking task in healthy adults. We hypothesized that buprenorphine would reduce subjective and physiological stress responses. Healthy adult volunteers (N=48) were randomly assigned to receive placebo, 0.2mg sublingual buprenorphine, or 0.4mg sublingual buprenorphine in a two-session study with a stressful speaking task (Trier Social Stress Test; TSST) and a non-stressful control task. During the sessions, the participants reported on their mood states, provided subjective appraisals of the task, and measures of salivary cortisol, heart rate, and blood pressure at regular intervals. Stress produced its expected effects, increasing heart rate, blood pressure, salivary cortisol, and subjective ratings of anxiety and negative mood. In line with our hypothesis, both doses of buprenorphine significantly dampened salivary cortisol responses to stress. On self-report ratings, buprenorphine reduced how threatening participants found the tasks. These results suggest that enhanced opioid signaling dampens responses to social stress in humans, as it does in laboratory animals. This stress-dampening effect of buprenorphine may contribute to the non-medical use of opioid drugs.

Influence of energy drink ingredients on mood and cognitive performance.

Sales of energy products have grown enormously in recent years. Manufacturers claim that the products, in the form of drinks, shots, supplements, and gels, enhance physical and cognitive performance, while users believe the products promote concentration, alertness, and fun. Most of these products contain caffeine, a mild psychostimulant, as their foremost active ingredient. However, they also contain additional ingredients, e.g., carbohydrates, amino acids, herbal extracts, vitamins, and minerals, often in unspecified amounts and labeled as an "energy blend." It is not clear whether these additional ingredients provide any physical or cognitive enhancement beyond that provided by caffeine alone. This article reviews the available empirical data on the interactive effects of these ingredients and caffeine on sleep and cognitive performance and suggests objectives for future study.

Personality traits modulate emotional and physiological responses to stress.

An individual's susceptibility to psychological and physical disorders associated with chronic stress exposure, for example, cardiovascular and infectious disease, may also be predicted by their reactivity to acute stress. One factor associated with both stress resilience and health outcomes is personality. An understanding of how personality influences responses to acute stress may shed light upon individual differences in susceptibility to chronic stress-linked disease. This study examined the relationships between personality and acute responses to stress in 125 healthy adults, using hierarchical linear regression. We assessed personality traits using the Multidimensional Personality Questionnaire (MPQ-BF), and responses to acute stress (cortisol, heart rate, blood pressure, mood) using a standardized laboratory psychosocial stress task, the Trier Social Stress Test. Individuals with high Negative Emotionality exhibited greater emotional distress and lower blood pressure responses to the Trier Social Stress Test. Individuals with high agentic Positive Emotionality exhibited prolonged heart rate responses to stress, whereas those with high communal Positive Emotionality exhibited smaller cortisol and blood pressure responses. Separate personality traits differentially predicted emotional, cardiovascular, and cortisol responses to a psychosocial stressor in healthy volunteers. Future research investigating the association of personality with chronic stress-related disease may provide further clues to the relationship between acute stress reactivity and susceptibility to disease.

Regular exercise is associated with emotional resilience to acute stress in healthy adults.

Physical activity has long been considered beneficial to health and regular exercise is purported to relieve stress. However empirical evidence demonstrating these effects is limited. In this study, we compared psychophysiological responses to an acute psychosocial stressor between individuals who did, or did not, report regular physical exercise. Healthy men and women (N = 111) participated in two experimental sessions, one with the Trier Social Stress Test (TSST) and one with a non-stressful control task. We measured heart rate, blood pressure, cortisol, and self-reported mood before and at repeated times after the tasks. Individuals who reported physical exercise at least once per week exhibited lower heart rate at rest than non-exercisers, but the groups did not differ in their cardiovascular responses to the TSST. Level of habitual exercise did not influence self-reported mood before the tasks, but non-exercisers reported a greater decline in positive affect after the TSST in comparison to exercisers. These findings provide modest support for claims that regular exercise protects against the negative emotional consequences of stress, and suggest that exercise has beneficial effects in healthy individuals. These findings are limited by their correlational nature, and future prospective controlled studies on the effects of regular exercise on response to acute stress are needed.

Conditioned preference to a methamphetamine-associated contextual cue in humans.

Classical conditioning is widely used to study motivational properties of addictive drugs in animals, but has rarely been used in humans. We established a procedure suitable for studying the neurobiology and individual determinants of classical conditioning in humans. Healthy volunteers were randomly assigned to four groups that received methamphetamine or placebo in the presence of distinctive environmental cues under paired or unpaired conditions. During each session, subjects performed tasks known to activate the ventral striatum. Tasks were performed in the presence of a distinctive context, consisting of a screen background image of a beach or mountains, accompanied by corresponding sounds. Separate groups of subjects carried out the tasks under high ($35-50) or low ($5-20) reward conditions. Within each of the two reward conditions, one group (paired) received methamphetamine (20 mg, oral) or placebo consistently associated with one of the contexts, while the other (unpaired) received drug or placebo unrelated to context. A fifth group (paired) performed the tasks with contextual cues but in the absence of monetary incentives. Before and after conditioning, participants carried out a series of forced choice tasks for the contextual cues, and change of preference over time was analyzed. All paired groups showed a significant increase in preference for the drug-associated context, with a linear trend for increase across the levels of reward. Preference was unrelated to subjective drug effects, and did not change in the unpaired group. These data support the translational utility of our conditioning procedure for studies of reward mechanisms in humans.

Psychopharmacology of theobromine in healthy volunteers.

BACKGROUND: Theobromine, a methylxanthine related to caffeine and present in high levels in cocoa, may contribute to the appeal of chocolate. However, current evidence for this is limited. OBJECTIVES: We conducted a within-subjects placebo-controlled study of a wide range of oral theobromine doses (250, 500, and 1,000 mg) using an active control dose of caffeine (200 mg) in 80 healthy participants. RESULTS: Caffeine had the expected effects on mood including feelings of alertness and cardiovascular parameters. Theobromine responses differed according to dose; it showed limited subjective effects at 250 mg and negative mood effects at higher doses. It also dose-dependently increased heart rate. In secondary analyses, we also examined individual differences in the drug's effects in relation to genes related to their target receptors, but few associations were detected. CONCLUSIONS: This study represents the highest dose of theobromine studied in humans. We conclude that theobromine at normal intake ranges may contribute to the positive effects of chocolate, but at higher intakes, effects become negative.