Replicability in measures of attentional set-shifting task performance predicting chronic heavy drinking in rhesus monkeys.

This study was designed to replicate and extend a previous report that the increase in performance of an attentional set-shifting task (ASST) in rhesus monkeys predicted their future alcohol drinking status as a heavy drinker (HD) or non-heavy drinker (NHD). A cohort of 6 young adult male monkeys was trained and tested under the same ASST and then underwent a alcohol self-administration protocol that maintained open-access (22 hours/day) choice of alcohol or water 7 days/week for approximately 6 months. The average improvement in performance in the ASST, as measured by a performance index, was replicated in the cohort of 6 monkeys when compared to the increase in the task performance in a previous cohort of 9 male monkeys. The alcohol self-administration protocol was then used to determine the drinking status (HD: n = 4 or NHD: n = 2) of the replicate cohort, which was accurately predicted by the performance on the ASST. Finally, individuals from both cohorts could be combined based on future drinking status of HD (n = 8) or NHD (n = 7), and the association with pre-alcohol ASST performance remained. Specifically, monkeys that had lower rates of PI improvement were more likely to become HDs. To our knowledge, this is the first study to replicate that deficits in the set-shifting performance can predict chronic heavy alcohol drinking in primates.

Labeled oxytocin administered via the intranasal route reaches the brain in rhesus macaques.

Oxytocin may have promise as a treatment for neuropsychiatric disorders. Its therapeutic effect may depend on its ability to enter the brain and bind to the oxytocin receptor. To date, the brain tissue penetrance of intranasal oxytocin has not been demonstrated. In this nonhuman primate study, we administer deuterated oxytocin intranasally and intravenously to rhesus macaques and measure, with mass spectrometry, concentrations of labeled (exogenously administered) and endogenous oxytocin in 12 brain regions two hours after oxytocin administration. Labeled oxytocin is quantified after intranasal (not intravenous) administration in brain regions (orbitofrontal cortex, striatum, brainstem, and thalamus) that lie in the trajectories of the olfactory and trigeminal nerves. These results suggest that intranasal administration bypasses the blood-brain barrier, delivering oxytocin to specific brain regions, such as the striatum, where oxytocin acts to impact motivated behaviors. Further, high concentrations of endogenous oxytocin are in regions that overlap with projection fields of oxytocinergic neurons.

Human foodborne listeriosis in England and Wales, 1981 to 2015.

Almost all cases of human listeriosis are foodborne, however the proportion where specific exposures are identified is small. Between 1981 and 2015, 5252 human listeriosis cases were reported in England and Wales. The purpose of this study was to summarise data where consumption of specific foods was identified with transmission and these comprised 11 sporadic cases and 17 outbreaks. There was a single outbreak in the community of 378 cases (7% of the total) which was associated with pâté consumption and 112 cases (2% of the total) attributed to specific foods in all the other incidents. The proportion of food-attributed cases increased during this study with improvements in typing methods for Listeria monocytogenes. Ten incidents (one sporadic case and nine outbreaks of 2-9 cases over 4 days to 32 months) occurred in hospitals: all were associated with the consumption of pre-prepared sandwiches. The 18 community incidents comprised eight outbreaks (seven of between 3 and 17 cases) and 10 sporadic cases: food of animal origin was implicated in 16 of the incidents (sliced or potted meats, pork pies, pâté, liver, chicken, crab-meat, butter and soft cheese) and food of non-animal origin in the remaining two (olives and vegetable rennet).

Synaptic adaptations in the central amygdala and hypothalamic paraventricular nucleus associated with protracted ethanol abstinence in male rhesus monkeys.

Alcohol use disorder is a significant global burden. Stress has been identified as an etiological factor in the initiation and continuation of ethanol consumption. Understanding adaptations within stress circuitry is an important step toward novel treatment strategies. The effects of protracted abstinence following long-term ethanol self-administration on the central nucleus of the amygdala (CeA) and the hypothalamic paraventricular nucleus (PVN) were evaluated in male rhesus monkeys. Using whole-cell patch-clamp electrophysiology, inhibitory GABAergic transmission in the CeA and excitatory glutamatergic transmission in the PVN were measured. CeA neurons from abstinent drinkers displayed an elevated baseline spontaneous inhibitory postsynaptic current (sIPSC) frequency compared with controls, indicating increased presynaptic GABA release. Application of acute ethanol significantly increased the frequency of sIPSCs in controls, but not in abstinent drinkers, suggesting a tolerance to ethanol-enhanced GABA release in abstinent rhesus monkeys with a history of chronic ethanol self-administration and repeated abstinence. In the PVN, the frequency of spontaneous excitatory postsynaptic currents (sEPSC) was elevated in abstinent drinkers compared with controls, indicating increased presynaptic glutamate release. Notably, acute ethanol decreased presynaptic glutamate release onto parvocellular PVN neurons in both controls and abstinent drinkers, suggesting a lack of tolerance to acute ethanol among PVN neurons. These results are the first to demonstrate distinct synaptic adaptations and ethanol sensitivity in both the extrahypothalamic and hypothalamic stress circuits in abstinent rhesus males. Importantly, our findings describe adaptations in stress circuitry present in the brain at a state during abstinence, just prior to relapse to ethanol drinking.

A severe 2017 influenza season dominated by influenza A(H3N2), Victoria, Australia.

Surveillance for influenza-like illness (ILI) and laboratory-confirmed influenza in Victoria, Australia is undertaken jointly by the Victorian Infectious Diseases Reference Laboratory and the Victorian Government Department of Health and Human Services from May to October each year. Surveillance data comprise notifiable laboratory-confirmed influenza and ILI reporting from from two sources - a general practice sentinel surveillance programme and a locum service. The magnitude of the 2017 influenza season was high in Victoria with widespread circulation of influenza type A(H3N2), which peaked in September. A record number of laboratory-confirmed influenza cases were notified, and the proportion of ILI cases to total consultations from both the general practice and locum service were higher than previous years. Notified cases of influenza A were older than influenza B cases with 25% compared to 17% aged more than 65 years, respectively. The proportion of swabs that were positive for influenza peaked at 58%. Antigenic characterization suggested a good match between the circulating and vaccine strains of influenza A(H3N2). Most of the increases observed in notified cases of laboratory-confirmed influenza in recent years in Victoria have been attributed to increases in testing. However, that cases of ILI also increased in Victoria in 2017 is suggestive that 2017 was a relatively severe season. The dominance of influenza type A(H3N2), the extended duration of elevated activity, and a potential phylogenetic mismatch of vaccine to circulating strains are likely to have contributed to the relative severity of the 2017 season. Victoria is Australia's second most populous state and is the mainland's southernmost state. It has a temperate climate with an influenza season usually occurring in the cooler months between May and October. The Victorian Infectious Diseases Reference Laboratory (VIDRL), in partnership with the Victorian Government Department of Health and Human Services (DHHS), coordinates influenza-like illness (ILI) and laboratory-confirmed influenza surveillance in Victoria. There are three data sources included in the influenza surveillance system.

A relationship between the aldosterone-mineralocorticoid receptor pathway and alcohol drinking: preliminary translational findings across rats, monkeys and humans.

Aldosterone regulates electrolyte and fluid homeostasis through binding to the mineralocorticoid receptors (MRs). Previous work provides evidence for a role of aldosterone in alcohol use disorders (AUDs). We tested the hypothesis that high functional activity of the mineralocorticoid endocrine pathway contributes to vulnerability for AUDs. In Study 1, we investigated the relationship between plasma aldosterone levels, ethanol self-administration and the expression of CYP11B2 and MR (NR3C2) genes in the prefrontal cortex area (PFC) and central nucleus of the amygdala (CeA) in monkeys. Aldosterone significantly increased after 6- and 12-month ethanol self-administration. NR3C2 expression in the CeA was negatively correlated to average ethanol intake during the 12 months. In Study 2, we measured Nr3c2 mRNA levels in the PFC and CeA of dependent and nondependent rats and the correlates with ethanol drinking during acute withdrawal. Low Nr3c2 expression levels in the CeA were significantly associated with increased anxiety-like behavior and compulsive-like drinking in dependent rats. In Study 3, the relationship between plasma aldosterone levels, alcohol drinking and craving was investigated in alcohol-dependent patients. Non-abstinent patients had significantly higher aldosterone levels than abstinent patients. Aldosterone levels positively correlated with the number of drinks consumed, craving and anxiety scores. These findings support a relationship between ethanol drinking and the aldosterone/MR pathway in three different species.

Alcohol-dose-dependent DNA methylation and expression in the nucleus accumbens identifies coordinated regulation of synaptic genes.

Alterations in DNA methylation have been associated with alcohol exposure and proposed to contribute to continued alcohol use; however, the molecular mechanisms involved remain obscure. We investigated the escalating effects of alcohol use on DNA methylation, gene expression and predicted neural effects in the nucleus accumbens of rhesus macaques that self-administered 4% alcohol for over 12 months. Using an exploratory approach to identify CpG-rich regions, followed by bisulfite sequencing, the methylation levels of 2.7 million CpGs were compared between seven low-binge drinkers and nine heavy-very heavy drinking subjects. We identified 17 significant differential methylation regions (DMRs), including 14 with methylation levels that were correlated with average daily alcohol consumption. The size of the DMRs ranged from 29 to 158 bp (mean=63.7), included 4-19 CpGs per DMR (mean=8.06) and spanned a range of average methylation values from 5 to 34%. Eight of the DMRs mapped to genes implicated in modulating synaptic plasticity. Six of the synaptic genes have not previously been linked to alcohol use. Validation studies of these eight DMRs using bisulfite amplicon sequencing and an expanded set of 30 subjects confirmed the significant alcohol-dose-associated methylation of the DMRs. Expression analysis of three of the DMR-associated genes, LRP5, GPR39 and JAKMIP1, revealed significant correlations between DMR methylation and whole-gene or alternative transcript expression, supporting a functional role in regulating gene expression. Together, these studies suggest that alcohol-associated synaptic remodeling may be regulated and coordinated at the level of DNA methylation.

An Outbreak of Human Listeriosis in England between 2010 and 2012 Associated with the Consumption of Pork Pies.

An outbreak of listeriosis in England affecting 14 people between 2010 and 2012 and linked to the consumption of pork pies was investigated. All 14 individuals were older than 55 years, 12 were men, and 10 reported the presence of an underlying condition. All were resident in or had visited either of two English regions and were infected with the same strain of Listeria monocytogenes. In interviews with 12 patients, 9 reported eating pork pies, and individuals that consumed pork pies were significantly more likely to be infected with an outbreak strain than were individuals with sporadic cases of listeriosis infections in England from 2010 to 2012. Pork pies were purchased from seven retailers in South Yorkshire or the East Midlands, and the outbreak strain was recovered from pork pies supplied by only the producer in South Yorkshire. The outbreak strain was also recovered from samples of finished product and from environmental samples collected from the manufacturer. The likely source of contamination was environmental sites within the manufacturing environment, and the contamination was associated with the process of adding gelatin to the pies after cooking. Inadequate temperature control and poor hygienic practices at one of the retailers were also identified as possible contributory factors allowing growth of the pathogen. Following improvements in manufacturing practices and implementation of additional control measures at the retailers' premises, L. monocytogenes was not recovered from subsequent food and environmental samples, and the outbreak strain was not detected in further individuals with listeriosis in England.

Pooled influenza vaccine effectiveness estimates for Australia, 2012-2014.

Data were pooled from three Australian sentinel general practice influenza surveillance networks to estimate Australia-wide influenza vaccine coverage and effectiveness against community presentations for laboratory-confirmed influenza for the 2012, 2013 and 2014 seasons. Patients presenting with influenza-like illness at participating GP practices were swabbed and tested for influenza. The vaccination odds of patients testing positive were compared with patients testing negative to estimate influenza vaccine effectiveness (VE) by logistic regression, adjusting for age group, week of presentation and network. Pooling of data across Australia increased the sample size for estimation from a minimum of 684 to 3,683 in 2012, from 314 to 2,042 in 2013 and from 497 to 3,074 in 2014. Overall VE was 38% [95% confidence interval (CI) 24-49] in 2012, 60% (95% CI 45-70) in 2013 and 44% (95% CI 31-55) in 2014. For A(H1N1)pdm09 VE was 54% (95% CI-28 to 83) in 2012, 59% (95% CI 33-74) in 2013 and 55% (95% CI 39-67) in 2014. For A(H3N2), VE was 30% (95% CI 14-44) in 2012, 67% (95% CI 39-82) in 2013 and 26% (95% CI 1-45) in 2014. For influenza B, VE was stable across years at 56% (95% CI 37-70) in 2012, 57% (95% CI 30-73) in 2013 and 54% (95% CI 21-73) in 2014. Overall VE against influenza was low in 2012 and 2014 when A(H3N2) was the dominant strain and the vaccine was poorly matched. In contrast, overall VE was higher in 2013 when A(H1N1)pdm09 dominated and the vaccine was a better match. Pooling data can increase the sample available and enable more precise subtype- and age group-specific estimates, but limitations remain.

MAOA expression predicts vulnerability for alcohol use.

The role of the monoamines dopamine (DA) and serotonin (5HT) and the monoamine-metabolizing enzyme monoamine oxidase A (MAOA) have been repeatedly implicated in studies of alcohol use and dependence. Genetic investigations of MAOA have yielded conflicting associations between a common polymorphism (MAOA-LPR) and risk for alcohol abuse. The present study provides direct comparison of tissue-specific MAOA expression and the level of alcohol consumption. We analyzed rhesus macaque MAOA (rhMAOA) expression in blood from males before and after 12 months of alcohol self-administration. In addition, nucleus accumbens core (NAc core) and cerebrospinal fluid (CSF) were collected from alcohol access and control (no alcohol access) subjects at the 12-month time point for comparison. The rhMAOA expression level in the blood of alcohol-naive subjects was negatively correlated with subsequent alcohol consumption level. The mRNA expression was independent of rhMAOA-LPR genotype and global promoter methylation. After 12 months of alcohol use, blood rhMAOA expression had decreased in an alcohol dose-dependent manner. Also after 12 months, rhMAOA expression in the NAc core was significantly lower in the heavy drinkers, as compared with control subjects. The CSF measured higher levels of DA and lower DOPAC/DA ratios among the heavy drinkers at the same time point. These results provide novel evidence that blood MAOA expression predicts alcohol consumption and that heavy alcohol use is linked to low MAOA expression in both the blood and NAc core. Together, the findings suggest a mechanistic link between dampened MAOA expression, elevated DA and alcohol abuse.

Investigation of an outbreak of vomiting in nurseries in South East England, May 2012.

On 30 May 2012, Surrey and Sussex Health Protection Unit was called by five nurseries reporting children and staff with sudden onset vomiting approximately an hour after finishing their lunch that day. Over the following 24 h 50 further nurseries supplied by the same company reported cases of vomiting (182 children, 18 staff affected). Epidemiological investigations were undertaken in order to identify the cause of the outbreak and prevent further cases. Investigations demonstrated a nursery-level attack rate of 55 out of 87 nurseries (63·2%, 95% confidence interval 52·2-73·3). Microbiological tests confirmed the presence of Bacillus cereus in food and environmental samples from the catering company and one nursery. This was considered microbiologically and epidemiologically consistent with toxin from this bacterium causing the outbreak. Laboratory investigations showed that the conditions used by the caterer for soaking of pearl haricot beans (known as navy bean in the USA) used in one of the foods supplied to the nurseries prior to cooking, was likely to have provided sufficient growth and toxin production of B. cereus to cause illness. This large outbreak demonstrates the need for careful temperature control in food preparation.

Functional imaging of the nonhuman primate Placenta with endogenous blood oxygen level-dependent contrast.

PURPOSE: To characterize spatial patterns of T2* in the placenta of the rhesus macaque (Macaca mulatta), to correlate these patterns with placental perfusion determined using dynamic contrast-enhanced MRI (DCE-MRI), and to evaluate the potential for using the blood oxygen level-dependent effect to quantify placental perfusion without the use of exogenous contrast reagent. METHODS: MRI was performed on three pregnant rhesus macaques at gestational day 110. Multiecho spoiled gradient echo measurements were used to compute maps of T2*. Spatial maxima in these maps were compared with foci of early enhancement determined by DCE-MRI. RESULTS: Local maxima in T2* maps were strongly correlated with spiral arteries identified by DCE-MRI, with mean spatial separations ranging from 2.34 to 6.11 mm in the three animals studied. Spatial patterns of R2* ( = 1/ T2*) within individual placental lobules can be quantitatively analyzed using a simple model to estimate fetal arterial oxyhemoglobin concentration [Hbo,f] and a parameter viPS/Φ, reflecting oxygen transport to the fetus. Estimated mean values of [Hbo,f] ranged from 4.25 mM to 4.46 mM, whereas viPS/Φ ranged from 2.80 × 105 cm-3 to 1.61 × 106 cm-3 . CONCLUSIONS: Maternal spiral arteries show strong spatial correlation with foci of extended T2* observed in the primate placenta. A simple model of oxygen transport accurately describes the spatial dependence of R2* within placental lobules and enables assessment of placental function and oxygenation without requiring administration of an exogenous contrast reagent. Magn Reson Med 76:1551-1562, 2016. © 2015 International Society for Magnetic Resonance in Medicine.

The utility and public health implications of PCR and whole genome sequencing for the detection and investigation of an outbreak of Shiga toxin-producing Escherichia coli serogroup O26:H11.

Many serogroups of Shiga toxin-producing Escherichia coli (STEC) other than serogroup O157 (non-O157 STEC), for example STEC O26:H11, are highly pathogenic and capable of causing haemolytic uraemic syndrome. A recent increase in non-O157 STEC cases identified in England, resulting from a change in the testing paradigm, prompted a review of the current methods available for detection and typing of non-O157 STEC for surveillance and outbreak investigations. Nineteen STEC O26:H11 strains, including four from a nursery outbreak were selected to assess typing methods. Serotyping and multilocus sequence typing were not able to discriminate between the stx-producing strains in the dataset. However, genome sequencing provided rapid and robust confirmation that isolates of STEC O26:H11 associated with a nursery outbreak were linked at the molecular level, had a common source and were distinct from the other strains analysed. Virulence gene profiling of DNA extracted from a polymerase chain reaction (PCR)-positive/culture-negative faecal specimen from a case that was epidemiologically linked to the STEC O26:H11 nursery outbreak, provided evidence at the molecular level to support that link. During this study, we describe the utility of PCR and the genome sequencing approach in facilitating surveillance and enhancing the response to outbreaks of non-O157 STEC.

Pregnancy-associated listeriosis in England and Wales.

Listeriosis is a rare but severe foodborne disease with low morbidity and high case-fatality rates. Pregnant women, unborn and newborn babies are among the high-risk groups for listeriosis. We examined listeriosis cases reported to the enhanced surveillance system in England and Wales from 1990 to 2010 to identify risk factors influencing outcome. Cases were defined as pregnancy-associated if Listeria monocytogenes was isolated from a pregnant woman or newborn infants aged <28 days. Of the 3088 cases reported, pregnancy-associated listeriosis accounted for 462 (15%) cases and 315 cases resulted in a live birth. Several factors were identified as affecting the severity and outcome of listeriosis in pregnancy in both mother and child including: presence or absence of maternal symptoms, gestational age at onset of symptoms, and clinical presentation in the infant (meningitis or septicaemia). Deprivation, ethnicity and molecular serotype had no effect on outcome.

Quantifying differences in the epidemic curves from three influenza surveillance systems: a nonlinear regression analysis.

Influenza surveillance enables systematic collection of data on spatially and demographically heterogeneous epidemics. Different data collection mechanisms record different aspects of the underlying epidemic with varying bias and noise. We aimed to characterize key differences in weekly incidence data from three influenza surveillance systems in Melbourne, Australia, from 2009 to 2012: laboratory-confirmed influenza notified to the Victorian Department of Health, influenza-like illness (ILI) reported through the Victorian General Practice Sentinel Surveillance scheme, and ILI cases presenting to the Melbourne Medical Deputising Service. Using nonlinear regression, we found that after adjusting for the effects of geographical region and age group, characteristics of the epidemic curve (including season length, timing of peak incidence and constant baseline activity) varied across the systems. We conclude that unmeasured factors endogenous to each surveillance system cause differences in the disease patterns recorded. Future research, particularly data synthesis studies, could benefit from accounting for these differences.

Decreased varicella and increased herpes zoster incidence at a sentinel medical deputising service in a setting of increasing varicella vaccine coverage in Victoria, Australia, 1998 to 2012.

We performed an ecological study using sentinel consultation data from a medical deputising service to assess the impact of increasing coverage with childhood varicella vaccine on the incidence risk of varicella and zoster in the population served by the deputising service in Victoria, Australia from 1998 to 2012. Following a successful vaccination programme, the incidence of varicella in Australia was modelled to decrease and the incidence of zoster to increase, based on a theoretical decrease in boosting of zoster immunity following a decrease in wild varicella virus circulation due to vaccination. Incidence risks (consultation proportions for varicella and zoster) were directly age-standardised to the Melbourne population in 2000, when varicella vaccine was first available. Age-standardised varicella incidence risk peaked in 2000 and halved by 2012. Age-standardised zoster incidence risk remained constant from 1998 to 2002, but had almost doubled by 2012. The increase in zoster consultations largely reflected increases in people younger than 50 years-old. Although causality cannot be inferred from ecological studies, it is generally agreed that the decrease in varicella incidence is due to increasing varicella vaccine coverage. The possible indirect effect of the vaccine on zoster incidence is less clear and ongoing monitoring of zoster is required.

Moderate alcohol consumption enhances vaccine-induced responses in rhesus macaques.

We have recently shown that chronic alcohol consumption in a rhesus macaque model of ethanol self-administration significantly modulates the serum cytokine profile. In this study, we extended these observations by investigating the impact of chronic ethanol exposure on the immune response to Modified Vaccinia Ankara (MVA). All animals were vaccinated with MVA before ethanol exposure to ethanol and then again after 7 months of 22 h/day of "open-access" drinking of 4% (w/v) ethanol. Our results indicate that animals whose blood ethanol concentration (BEC) chronically exceeded 80 mg/dl had lower CD4 and CD8 T cell proliferation as well as IgG responses following MVA booster than control animals. In contrast, relatively moderate drinkers whose BEC remained below 80 mg/ml exhibited more robust MVA-specific IgG and CD8 T cell responses than controls. To begin to uncover mechanisms underlying the differences in MVA-specific responses between the three groups, we analyzed plasma cytokine levels and microRNA expression in peripheral blood mononuclear cells following MVA booster. Our findings suggest that moderate ethanol consumption results in higher levels of antiviral cytokines and an expression profile of microRNAs linked to CD8 T cell differentiation. In summary, moderate alcohol consumption enhances recall vaccine responses, whereas chronic alcohol intoxication suppresses this response.

Retrospective analysis of whole genome sequencing compared to prospective typing data in further informing the epidemiological investigation of an outbreak of Shigella sonnei in the UK.

The aim of this study was to retrospectively assess the value of whole genome sequencing (WGS) compared to conventional typing methods in the investigation and control of an outbreak of Shigella sonnei in the Orthodox Jewish (OJ) community in the UK. The genome sequence analysis showed that the strains implicated in the outbreak formed three phylogenetically distinct clusters. One cluster represented cases associated with recent exposure to a single strain, whereas the other two clusters represented related but distinct strains of S. sonnei circulating in the OJ community across the UK. The WGS data challenged the conclusions drawn during the initial outbreak investigation and allowed cases of dysentery to be implicated or ruled out of the outbreak that were previously misclassified. This study showed that the resolution achieved using WGS would have clearly defined the outbreak, thus facilitating the promotion of infection control measures within local schools and the dissemination of a stronger public health message to the community.

Genetic load is associated with hypothalamic-pituitary-adrenal axis dysregulation in macaques.

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis pathway is associated with several neuropsychiatric disorders, including post-traumatic stress disorder (PTSD), major depressive disorder (MDD), schizophrenia and alcohol abuse. Studies have demonstrated an association between HPA axis dysfunction and gene variants within the cortisol, serotonin and opioid signaling pathways. We characterized polymorphisms in genes linked to these three neurotransmitter pathways and tested their potential interactions with HPA axis activity, as measured by dexamethasone (DEX) suppression response. We determined the percent DEX suppression of adrenocorticotropic hormone (ACTH) and cortisol in 62 unrelated, male rhesus macaques. While DEX suppression of cortisol was robust amongst 87% of the subjects, ACTH suppression levels were broadly distributed from -21% to 66%. Thirty-seven monkeys from the high and low ends of the ACTH suppression distribution (18 'high' and 19 'low' animals) were genotyped at selected polymorphisms in five unlinked genes (rhCRH, rhTPH2, rhMAOA, rhSLC6A4 and rhOPRM). Associations were identified between three variants (rhCRH-2610C>T, rhTPH2 2051A>C and rh5-HTTLPR) and level of DEX suppression of ACTH. In addition, a significant additive effect of the 'risk' genotypes from these three loci was detected, with an increasing number of 'risk' genotypes associated with a blunted ACTH response (P = 0.0009). These findings suggest that assessment of multiple risk alleles in serotonin and cortisol signaling pathway genes may better predict risk for HPA axis dysregulation and associated psychiatric disorders than the evaluation of single gene variants alone.