ACTH Response Research Articles

Altered Stress-Induced Regulation of Genes in Monocytes in Adults with a History of Childhood Adversity.

Exposure to serious or traumatic events early in life can lead to persistent alterations in physiological stress response systems, including enhanced cross talk between the neuroendocrine and immune system. These programming effects may be mechanistically involved in mediating the effects of adverse childhood experience on disease risk in adulthood. We investigated hormonal and genome-wide mRNA expression responses in monocytes to acute stress exposure, in a sample of healthy adults (n=30) with a history of early childhood adversity, and a control group (n=30) without trauma experience. The early adversity group showed altered hypothalamus-pituitary-adrenal axis responses to stress, evidenced by lower ACTH and cortisol responses. Analyses of gene expression patterns showed that stress-responsive transcripts were enriched for genes involved in cytokine activity, cytokine-cytokine receptor interaction, chemokine activity, and G-protein coupled receptor binding. Differences between groups in stress-induced regulation of gene transcription were observed for genes involved in steroid binding, hormone activity, and G-protein coupled receptor binding. Transcription factor binding motif analysis showed an increased activity of pro-inflammatory upstream signaling in the early adversity group. We also identified transcripts that were differentially correlated with stress-induced cortisol increases between the groups, enriched for genes involved in cytokine-cytokine receptor interaction and glutamate receptor signaling. We suggest that childhood adversity leads to persistent alterations in transcriptional control of stress-responsive pathways, which-when chronically or repeatedly activated-might predispose individuals to stress-related psychopathology.

Sensitization of the Hypothalamic-Pituitary-Adrenal Axis in a Male Rat Chronic Stress Model.

Stress activation of the hypothalamic-pituitary-adrenal (HPA) axis is regulated by rapid glucocorticoid negative feedback. Chronic unpredictable stress animal models recapitulate certain aspects of major depression in humans, which have been attributed to impaired glucocorticoid negative feedback. We tested for an attenuated HPA sensitivity to fast glucocorticoid feedback inhibition in male rats exposed to a chronic variable stress (CVS) paradigm. In vitro, parvocellular neuroendocrine cells of the hypothalamic paraventricular nucleus recorded in slices from CVS rats showed an increase in basal excitatory synaptic inputs and a decrease in basal inhibitory synaptic inputs compared with neurons from control rats. There was no difference between control and CVS-treated rats in the rapid glucocorticoid suppression of excitatory synaptic inputs, a fast feedback mechanism. In vivo, CVS-treated rats showed an increase in ACTH secretion at baseline and after both iv CRH and acute stress and no impairment of the corticosterone suppression of the ACTH response, compared with controls. In an in vitro pituitary preparation, an increase in basal ACTH release, a small increase in CRH-induced ACTH release, and no decrement in the glucocorticoid suppression of ACTH release were seen in pituitaries from CVS rats. Thus, CVS does not suppress rapid glucocorticoid negative feedback at the hypothalamus or pituitary, but increases the synaptic excitability of paraventricular nucleus CRH neurons and the CRH sensitivity of the pituitary. Therefore, increased HPA activity in chronically stressed male rats is due to sensitization of the HPA axis, rather than to desensitization to rapid glucocorticoid feedback.

Sympathetic nervous system contributes to enhanced corticosterone levels following chronic stress

Exposure to chronic stress often elevates basal circulating glucocorticoids during the circadian nadir and leads to exaggerated glucocorticoid production following exposure to subsequent stressors. While glucocorticoid production is primarily mediated by the hypothalamic–pituitary–adrenal (HPA) axis, there is evidence that the sympathetic nervous system can affect diurnal glucocorticoid production by direct actions at the adrenal gland. Experiments here were designed to examine the role of the HPA and sympathetic nervous system in enhancing corticosterone production following chronic stress. Rats were exposed to a four-day stress paradigm or control conditions then exposed to acute restraint stress on the fifth day to examine corticosterone and ACTH responses. Repeated stressor exposure resulted in a small increase in corticosterone, but not ACTH, during the circadian nadir, and also resulted in exaggerated corticosterone production 5, 10, and 20min following restraint stress. While circulating ACTH levels increased after 5min of restraint, levels were not greater in chronic stress animals compared to controls until following 20min. Administration of astressin (a CRH antagonist) prior to restraint stress significantly reduced ACTH responses but did not prevent the sensitized corticosterone response in chronic stress animals. In contrast, administration of chlorisondamine (a ganglionic blocker) returned basal corticosterone levels in chronic stress animals to normal levels and reduced early corticosterone production following restraint (up to 10min) but did not block the exaggerated corticosterone response in chronic stress animals at 20min. These data indicate that increased sympathetic nervous system tone contributes to elevated basal and rapid glucocorticoid production following chronic stress, but HPA responses likely mediate peak corticosterone responses to stressors of longer duration.

Pituitary-Adrenal Axis Responses to Routine Management Procedures in Sheep (Ovis aries)

Background: Shearing is considered as a stressful factor in sheep, capable of causing some behavioural changes including variations of body condition and hormonal and haematochemical parameters. On these basis, circulating ACTH and cortisol concentrations were determined in sheep before and after exposure to aversive stimuli in order to evaluate the potential use of these hormones to objectively measure physical and mental stress responses to isolation, restraint and shearing.Materials, Methods & Results: Forty Pinzirita sheep, used as experimental group, were evaluated in baseline conditions, 24 h before isolation, restraint and shearing (T0), after isolation (T1), after restraint and shearing (T2), by taking into account the influence of previous shearing experience (just shorn). A control group of forty sheep (intact) was evaluated in baseline condition (T0). According to previous experience the experimental subjects were divided in two groups, respectively group A represented by 20 sheep (10 males and 10 females), aged 1-2 years, without previous shearing experience (intact); and group B represented by 20 sheep (10 males and 10 females), aged older than 2 years, previously exposed to shearing (just shorn). A control group C of 40 sheep splitted into two sub-groups represented by 10 intact males and 10 intact females, aged 1-2 years, and 10 males and 10 females just shorn, aged older than 2 years, was evaluated in baseline conditions (T0). Blood samples were collected 24 h before experimental practices (T0: 08:00 h), after 1 h after of isolation (T1) and after restraint and shearing, around an average of 5 min (T2). All samples were taken in quiet conditions by the same operator. Serum ACTH concentrations were assayed by a commercial radioimmunoassay (RIA). Serum cortisol concentrations were assayed by amplified Enzyme Immunoassay (EIA). Two-way RM ANOVA showed a significant effect of husbandry procedures on ACTH changes, with higher ACTH and cortisol concentrations of just shorn than intact males (F = 15.76; P < 0.0001) and females (F = 17.34; P < 0.0005), and on cortisol changes, with higher cortisol concentrations of just shorn than intact males (F = 6.54; P < 0.01) and females (F = 22.66; P < 0.0001).Discussion: The highest ACTH and cortisol values after isolation, with an appreciable increase especially after restraint and shearing in just shorn sheep, showed that the previous shearing exposure could not induce a habituation to the stimulus, probably promoting a larger release of ACTH and cortisol hormones, so inducing an early and marked coping response to stress in just shorn specimens. Thus, it would appear that different ACTH and cortisol responses of sheep with or without previous shearing experience over the study period resulted in some significant differences when exposed to isolation, restraint and shearing, probably due to changes in pituitary-adrenal axis reactivity. Nevertheless, it is possible to interpret the increase in ACTH and cortisol concentrations related to intrinsic endocrine control mechanisms as an increase due to a rise in the stressful stimulus or coping response. The magnitude of ACTH and cortisol changes following isolation, restraint and shearing suggests that measurements of both hormones are useful in assessing husbandry procedures stress in sheep, with marked elevations in just shorn sheep, indicating that experience can induce an early much larger coping response to stressful practice of shearing.

Once-nightly (QD) dual CYP17-Lyase (L) inhibitor / androgen receptor (AR) antagonist VT-464 in patients with CRPC.

343 Background: VT-464 is an oral, CYP17-L inhibitor and non-clinically an antagonist of the AR and its variants associated with clinical resistance to enzalutamide (ENZ) and abiraterone (AA). The safety, tolerability and initial PD effects (tumor responses, PSA and testosterone (T)-decreases) of QD VT-464 were evaluated in this Phase (Ph) 1/2 study (INO-VT-464-CL-004; NCT02361086). Methods: Chemotherapy-naïve (n = 20) M0 (n = 1) and M1 (n = 19) patients with CRPC prior to (TN, treatment-naïve; n = 9), or after AA (n = 6),ENZ (n = 3) or both (n = 2) were enrolled in sequential dose-cohorts of 600 mg QD with dose titration (DT; 300 mg QD q2w), and 600 and 750 mg QD without DT in 28-day continuous dosing cycles. All doses except the first were given at night with dinner. Patient results through the 750 mg QD cohort are reported (data cut off 17 Aug 2015). Results: Patients received VT-464 without steroids at 600 mg QD+DT (n = 3), 600 mg QD (n = 8) or 750 mg QD (n = 9). Eight patients are on study in cycles 3-12. Most adverse events (AEs) were Grade (Gr) 1 or 2. Fourteen Gr 3 AEs were reported with 7 considered unrelated and 7 ≥ possibly related; 5 of the latter were in the 600 mg QD+DT cohort (bradycardia, hypotension, syncope, hypertension and hyponatremia) with 2 in non-DT cohorts (hypotension, bilateral lower leg weakness). No AEs ≥ Gr 4 were reported. One DLT occurred in the 600 mg QD+DT cohort (syncope). No mineralocorticoid excess syndrome, ACTH response changes, or increased LFTs were observed. Plasma T was below the LOQ (0.03nM) in 67% of patients, with progesterone and cortisol unchanged. Cmax was similar in all cohorts to 450 mg bid (de Bono et al, GU ASCO 2015) but AUC was decreased. All 4 TN patients had PSA declines at 600 mg QD including one 30% and one 50% response; there was one 50% response in the 600 mg QD+DT cohort. Both TN patients treated at 750 mg QD had > 50% response. Of 8 patients with ≥ 1 soft tissue target lesion and ≥ 1 post-baseline scan, 5 had stable disease and 1 had a partial response at Cycle 5. Conclusions: The VT-464 750 mg QD regimen demonstrated potent and selective CYP17 L inhibition and has been selected for all Ph 2 clinical studies based upon improved tolerability, PK and efficacy compared to bid+DT dosing (de Bono et al, GU ASCO 2015). Clinical trial information: NCT02361086.

Making pituitary hormone-producing cells in a dish [Review

The hypothalamic-pituitary system is essential for maintaining life and controlling systemic homeostasis. The functional disorder makes patients suffer from various symptoms all their lives. Pluripotent stem cells, such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, differentiate into neuroectodermal progenitors when cultured as floating aggregates under serum-free conditions. Recent results have shown that strict removal of exogenous patterning factors during the early differentiation period induces rostral hypothalamic-like progenitors from mouse ES cells. The use of growth factor-free, chemically defined medium was critical for this induction. The ES cell-derived hypothalamic-like progenitors generated rostral-dorsal hypothalamic neurons, in particular magnocellular vasopressinergic neurons. We subsequently reported self-formation of adenohypophysis in three-dimensional floating cultures of mouse ES cells. The ES cell aggregates were stimulated to differentiate into both non-neural head ectoderm and hypothalamic neuroectoderm in adjacent layers. Self-organization of Rathke's pouch-like structures occurred at the interface of the two epithelia in vitro. Various pituitary endocrine cells including corticotrophs and somatotrophs were subsequently produced from the Rathke's pouch-like structures. The induced corticotrophs efficiently secreted ACTH in response to CRH. Furthermore, when engrafted in vivo, these cells rescued systemic glucocorticoid levels in hypopituitary mice. Our latest study aimed to prepare hypothalamic and pituitary tissues from human pluripotent stem cells. We succeeded in establishing the differentiation method using human ES/iPS cells. The culture method is characterized by replication of stepwise embryonic differentiation. Therefore, these methods could potentially be used as developmental and disease models, as well as for future regenerative medicine.

Chlorella vulgaris reduces the impact of stress on hypothalamic–pituitary–adrenal axis and brain c-fos expression

Predominantly emotional stressors activate a wide range of brain areas, as revealed by the expression of immediate early genes, such as c-fos. Chlorella vulgaris (CV) is considered a biological response modifier, as demonstrated by its protective activities against infections, tumors and stress. We evaluated the effect of acute pretreatment with CV on the peripheral and central responses to forced swimming stress in adult male rats. Pretreatment with CV produced a significant reduction of stress-related hypothalamic–pituitary–adrenal activation, demonstrated by decreased corticotrophin releasing factor gene expression in the hypothalamic paraventricular nucleus (PVN) and lower ACTH response. Hyperglycemia induced by the stressor was similarly reduced. This attenuated neuroendocrine response to stress occurred in parallel with a diminished c-fos expression in most evaluated areas, including the PVN. The data presented in this study reinforce the usefulness of CV to diminish the impact of stressors, by reducing the HPA response. Although our results suggest a central effect of CV, further studies are necessary to understand the precise mechanisms underpinning this effect.

H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness.

The H295R adrenocortical cell line is widely used for molecular analysis of adrenal functions but is known to have only modest ACTH responsiveness. The lack of ACTH response was linked to a low expression of its receptor, melanocortin 2 receptor (MC2R). We hypothesized that increasing the MC2R accessory protein (MRAP), which is required to traffic MC2R from the endoplasmic reticulum to the cell surface, would increase ACTH responsiveness. Lentiviral particles containing human MRAP-open reading frame were generated and transduced in H295R cells. Using antibiotic resistance, 18 clones were isolated for characterization. The most ACTH-responsive steroidogenic clone, H295RA, was used for further experiments. Successful induction of MRAP and increased expression of MC2R in H295RA cells was confirmed by quantitative real-time RT-PCR and protein analysis. Treatment with ACTH significantly increased aldosterone, cortisol, and dehydroepiandrosterone production in H295RA cells. ACTH also significantly increased transcript levels for all of the steroidogenic enzymes required to produce aldosterone, cortisol, and dehydroepiandrosterone, as well as MC2R mRNA. Using liquid chromatography/tandem mass spectrometry, we further revealed that the main unconjugated steroids produced in H295RA cells were 11-deoxycortisol, cortisol, and androstenedione. Treatment of H295RA cells with ACTH also acutely increased cAMP production and cellular protein levels for total and phosphorylated steroidogenic acute regulatory protein. In summary, through genetic manipulation, we have developed an ACTH-responsive human adrenocortical cell line. The cell line will provide a powerful in vitro tool for molecular analysis of physiologic and pathologic conditions involving the hypothalamic-pituitary-adrenal axis.

Response of GH, ACTH, TSH and IGF-I to Gonadotropin Releasing Hormone Stimulation in Ovariohysterectomized Rats

Response of GH, ACTH, TSH and IGF-I to Gonadotropin Releasing Hormone Stimulation in Ovariohysterectomized Rats

Hypothalamic Nuclear Glucocorticoid Receptors: Acute Stress and Rapid Actions.

Stress, be it physical or psychological, causes the activation of a neuroendocrine cascade that involves both central and peripheral mechanisms. In the simplest terms, central release of CRH from the paraventricular nucleus of thehypothalamus (PVN)andsubsequent releaseofACTH from the pituitary result in the peripheral release of glucocorticoids (cortisol in primates and corticosterone in many rodent species) from the adrenal gland (1). Glucocorticoids then act via negative feedback both centrally and peripherally through the glucocorticoid receptor (GR) to inhibit the further release of CRH from the PVN and ACTH from the pituitary (2). This process is a delicate balance that helps restore homeostasis, but perturbation of these responses can lead to neurobehavioral and physiological dysfunctions, including mood disorders and obesity (3–5). Hence, understanding the basic mechanisms that mediate this neuroendocrine loop has broad implications for both health and disease. Two companion papers in this issue by Solomon et al (6) and Nahar et al (7) have helped to further reveal the mechanisms through which glucocorticoids influence hypothalamic-pituitary-adrenal (HPA) function. Both reports use a powerful mouse model in which Sim1-cre recombinase transgenic mice are bred with homozygous floxed GR mice. Sim1 is a transcription factor specifically expressed in the PVN, supraoptic nucleus, and periventricular nucleus (8) and is responsible for the proper maturation of neurosecretory cells in the PVN (9). As Solomon et al report, theoffspring fromthis cross showsignificantly reduced GR levels in the PVN while leaving GR levels intact in the pituitary and other brain regions important in glucocorticoid-dependent negative feedback (6), thus resulting in a relatively anatomically restricted GR reduction. Using these PVN GR knockdown (KD) mice, Solomon et al (6) find sex-specific and stressor-dependent effects on HPA reactivity. In particular, they show GR KD mice respond to a brief 30-minute session of restraint stress with significantly increased and prolonged ACTH and corticosterone responses compared with their littermate controls, suggesting reduced negative feedback on the HPA axis (6). Notably, however, these effects of GR KD were observed only in males because female GR KD mice show reduced restraint-induced ACTH and no differences in stress-induced corticosterone responsiveness. In response to chronic variable stress, neither male nor female GR KD mice show any differences in HPA reactivity. Hence, the authors conclude that there are significant sex differences in the role that PVN GR plays in response to acute stress and that changes in HPA reactivity after chronic stress in male and female mice are independent of GR in the PVN (6). Complimenting this line of research, the report by Nahar et al (7) investigated rapid nongenomic actions of glucocorticoids in modulating excitatory and inhibitory transmission in the mouse hypothalamus. They show that hypothalamic magnocellular neurons respond to dexamethasone (DEX), a synthetic glucocorticoid, with a rapid suppression of excitatory postsynaptic inputs (ie, glutamatergic synapses). The authors further demonstrate that this effect of DEX is mediated by postsynaptic membrane associated receptors and G protein signaling (7), similar to their seminal work in rats (10–12). The authors also show that DEX leads to a rapid nitric oxide-dependent increase in inhibitory postsynaptic inputs [ie, -aminobutyric acid

Abnormal Responsiveness to Dexamethasone-Suppressed CRH Test in Patients With Bilateral Adrenal Incidentalomas.

The bilateral formation of nodules indicates that the pathogenesis of bilateral adrenal incidentalomas (AI) may differ from that of unilateral AI. A possible role of hypothalamo-pituitary-adrenal (HPA) axis dysregulation in their formation has not been investigated. The objective of the study was to evaluate the presence of altered feedback regulation of HPA axis in patients with bilateral AI. The dexamethasone (DEX) suppression-CRH test was used to assess ACTH and cortisol responses in controls and patients with unilateral and bilateral AI. The study was conducted at endocrine departments of two tertiary centers. We studied 24 controls and 39 patients with unilateral and 46 with bilateral AI. All subjects underwent standard low-dose dexamethasone suppression followed by iv bolus administration of human CRH (100 μg). Bilateral AI had higher levels of ACTH and cortisol after the DEX-CRH challenge compared with both controls (P < .01 for ACTH and P < .001 for cortisol) and unilateral AI (P < .01 for ACTH and cortisol). A positive response, defined as peak ACTH greater than 10 pg/mL at 15 and/or 30 minutes followed by a significant rise in cortisol levels, was noted in 41.3% of bilateral vs 2.6% in unilateral AI (P < .001). Bilateral responders did not differ from nonresponders in demographic or hormonal characteristics, but they had larger total adrenal size compared with nonresponders. A significant proportion of patients with bilateral AI demonstrate positive responses to the DEX-CRH test compared with unilateral AI, providing ground for potential involvement of HPA axis dysregulation in the pathogenesis, in at least a subgroup, of bilateral AI patients.

Neuroendocrine Function After Hypothalamic Depletion of Glucocorticoid Receptors in Male and Female Mice.

Glucocorticoids act rapidly at the paraventricular nucleus (PVN) to inhibit stress-excitatory neurons and limit excessive glucocorticoid secretion. The signaling mechanism underlying rapid feedback inhibition remains to be determined. The present study was designed to test the hypothesis that the canonical glucocorticoid receptors (GRs) is required for appropriate hypothalamic-pituitary-adrenal (HPA) axis regulation. Local PVN GR knockdown (KD) was achieved by breeding homozygous floxed GR mice with Sim1-cre recombinase transgenic mice. This genetic approach created mice with a KD of GR primarily confined to hypothalamic cell groups, including the PVN, sparing GR expression in other HPA axis limbic regulatory regions, and the pituitary. There were no differences in circadian nadir and peak corticosterone concentrations between male PVN GR KD mice and male littermate controls. However, reduction of PVN GR increased ACTH and corticosterone responses to acute, but not chronic stress, indicating that PVN GR is critical for limiting neuroendocrine responses to acute stress in males. Loss of PVN GR induced an opposite neuroendocrine phenotype in females, characterized by increased circadian nadir corticosterone levels and suppressed ACTH responses to acute restraint stress, without a concomitant change in corticosterone responses under acute or chronic stress conditions. PVN GR deletion had no effect on depression-like behavior in either sex in the forced swim test. Overall, these findings reveal pronounced sex differences in the PVN GR dependence of acute stress feedback regulation of HPA axis function. In addition, these data further indicate that glucocorticoid control of HPA axis responses after chronic stress operates via a PVN-independent mechanism.

ACTH after 15 min distinguishes between Cushing's disease and ectopic Cushing's syndrome: a proposal for a short and simple CRH test.

The aim of the present study was to validate criteria of corticotropin-releasing hormone (CRH) stimulation and 8 mg dexamethasone suppression (high-dose dexamethasone suppression, HDDS) to distinguish the etiology of ACTH-dependent Cushing's syndrome. We retrospectively analyzed cortisol and ACTH after the injection of 100 μg human CRH in confirmed Cushing's disease (CD, n=78) and confirmed ectopic Cushing's syndrome (ECS, n=18). Cortisol and ACTH increase (in percentage above basal (%B)) at each time point, maximal increase (Δmax %B), and area under the curve (AUC %B) were analyzed using receiver operator characteristics (ROC) curve analyses. Cortisol suppression (%B) after 8 mg of dexamethasone was evaluated as a supplementary criterion. An increase in ACTH of ≥ 43%B at 15 min after CRH was the strongest predictor of CD, with a positive likelihood ratio of 14.0, a sensitivity of 83%, a specificity of 94%, a positive predictive value of 98% and a negative predictive value of 58%. All of the other criteria of stimulated ACTH and cortisol levels were not superior in predicting CD in response to CRH injection. The addition of cortisol suppression by dexamethasone did not increase the discriminatory power. However, the combination of a positive ACTH response at 15 min and a positive HDDS test excluded ECS in all cases. The present findings support the use of plasma ACTH levels 15 min after the injection of human CRH as a response criterion for distinguishing between CD and ECS. The addition of the HDDS test is helpful for excluding ECS when both tests are positive.

Diabetes Mellitus-Associated Functional Hypercortisolism Impairs Sexual Function in Male Late-Onset Hypogonadism.

Functional hypercortisolism is generated by conditions able to chronically activate hypothalamic-pituitary-adrenal axis and has been proven to have a negative role in several complications. However, no study has evaluated the possible influence of diabetes mellitus-associated functional hypercortisolism on male hypogonadism and sexual function. We aimed to identify any association of hypothalamic-pituitary-adrenal axis dysregulation measures with testosterone and sexual function in men simultaneously affected by diabetes mellitus and late-onset hypogonadism. Fifteen diabetes mellitus and late-onset hypogonadism subjects suffering from functional hypercortisolism and fifteen diabetes mellitus and late-onset hypogonadism subjects who were free of functional hypercortisolism were retrospectively reviewed. Clinical, hormonal, and sexual parameters were considered. Hypercortisolemic subjects showed higher values of body mass index, waist, and glycated hemoglobin and lower ones of testosterone compared to normocortisolemic ones. All sexual parameters, except for orgasmic function, were significantly worse in hypercortisolemic than in normocortisolemic subjects. Hypercortisolemic patients showed higher values of cortisol after dexamethasone and urinary free cortisol as well as a lesser ACTH response after corticotropin releasing hormone test (ACTH area under curve) compared to normocortisolemic ones. No significant association was found at Poisson regression analysis between hormonal and sexual variables in normocortisolemic patients. In hypercortisolemic subjects, negative and significant associations of cortisol response after corticotropin releasing hormone (cortisol area under curve) with erectile function (β: -0.0008; p: 0.015) and total international index of erectile function score (β: -0.0006; p: 0.001) were evident. This study suggests for the first time the impairing influence of the dysregulated hypothalamic-pituitary-adrenal axis on sexual function in diabetes mellitus-associated late-onset hypogonadism.

Evaluation of a thyrotropin-releasing hormone solution stored at room temperature for pituitary pars intermedia dysfunction testing in horses

To determine whether plasma ACTH concentrations vary following administration of a thyrotropin-releasing hormone (TRH) solution prepared for research purposes and stored at -20°C (rTRH) or prepared by a compounding pharmacy and stored at room temperature (approx 22°C; cTRH). 34 adult horses. The study consisted of 2 experiments. In experiment 1, each horse underwent 2 TRH stimulation tests separated by 24 hours; 10 horses were administered cTRH for the first test and rTRH for the second test (group 1), 10 horses were administered rTRH for the first test and cTRH for the second test (group 2), and 10 horses were administered rTRH for both tests (group 3). Plasma ACTH concentrations were measured at 0 (baseline) and 30 minutes after TRH administration and the delta ACTH responses (change in ACTH concentration after TRH administration) were calculated. In experiment 2, the design was the same as that for experiment 1 except there were 14 days between tests, ACTH was measured at 0 and 10 minutes after TRH administration, and 11, 9, and 10 horses were assigned to groups 1, 2, and 3, respectively. Adverse effects associated with TRH administration included transient coughing and yawning. In experiment 1, the median delta ACTH response for the second test was significantly lower than that for the first test for all groups. In experiment 2, the median delta ACTH response did not differ significantly between the first and second tests for any group, ACTH concentrations after rTRH administration were positively correlated (rs = 0.95) with those after cTRH administration, and the mean ± SD bias in post-TRH ACTH concentration between rTRH and cTRH was 2.9 ± 12.4 pg/mL. Results indicated that the TRH stimulation test should not be repeated within 24 hours, and cTRH solution stored at room temperature could be used to effectively perform TRH stimulation testing in horses.

Exaggerated and additive ACTH responses following the combined ghrelin+CRH test in patients with Cushing's disease

Exaggerated and additive ACTH responses following the combined ghrelin+CRH test in patients with Cushing's disease

The Defecation Index as a Measure of Emotionality: Questions Raised by HPA Axis and Prolactin Response to Stress in the Maudsley Model.

The Maudsley Reactive and Maudsley Non-Reactive strains have been selectively bred for differences in open-field defecation (OFD), a putative index of stress. We investigated whether variations in the hypothalamic-pituitary-adrenal (HPA) axis are correlated with strain differences in OFD in the Maudsley model. Exposure to the open-field test did not result in increases in ACTH in male rats of either strain and there were no strain differences in the large increases in ACTH and corticosteroid that occurred in response to intermittent footshock. Parallel studies of prolactin showed that Maudsley Reactive rats had greater response to the open-field and to footshock than Maudsley Non-Reactive rats. The lack of correlation between strain differences in OFD and reactivity of the HPA axis is consistent with the idea that HPA response to stress and OFD reflect the output of different neural systems and that individual differences in emotionality, as indexed by OFD do not influence other measures of stress-reactivity in a simple manner, if at all. The reactivity of the prolactin system to the open-field test and lack of response of ACTH to the same situation is consistent with the idea that the prolactin system is sensitive to lower levels of stress than the HPA axis, a finding at variance with the presumed extreme sensitivity of the latter system. Earlier comparisons of the HPA axis in these strains implicate local factors such as neuropeptide-Y peptide in the adrenal in attenuating the response of the adrenal cortex to ACTH and hints at the complexity of regulation of the HPA axis.

245 Evidence for a New Mechanism Underlying Persistent Visceral Hypersensitivity and Increased Permeability in a Model of Post-Infectious IBS

and cortisol responses to stimulation with CRH and ACTH: 1) differ between IBS patients and healthy controls (HCs), 2) are affected by sex and EALs, 3) are associated with GR mRNA expression. Methods: Male and female Rome III+ IBS patients and HCs underwent CRH (1μg/kg ovine) and ACTH (250μg) stimulation tests with serial plasma ACTH and cortisol levels measured. GR mRNA levels were measured from peripheral blood mononuclear cells using real-time PCR. Presence of EALs <18 yrs of age was measured with the Trauma History questionnaire. Linear mixed effects models were used to compare ACTH and cortisol response measured across time between groups with a knot at the peak of the curve to model the rates of change from baseline to peak (rise) and from peak to the last time point (decline). Area under the curve (AUC) was also measured. Results: 60 IBS patients (65%F, mean age 33yrs) and 56 HCs (52%F, mean age 31yrs) underwent hormone stimulation studies. 74 IBS (65%F, mean age 33yrs) and 69 HCs (52%F, mean age 33yrs) had GR mRNA expression levels. A subset (37 IBS, 28 HCs) had both hormone stimulation and GR mRNA studies. 34 (60%) IBS patients and 27 (48%) HCs had +EAL. CRH stimulation test: IBS patients showed a slower rate of decline from peak ACTH levels than HCs (p<0.05). Women with IBS had a slower rate of decline of ACTH (p=0.01) and cortisol (p=0.001) than men with IBS. ACTH stimulation test: Cortisol responses were similar in IBS and HCs, likely due to a significant interaction between IBS and sex (p<0.001). Within women, IBS had a lower cortisol response (AUC) than HCs. In contrast, in men, IBS had a greater cortisol response than HCs. GR mRNA expression: Levels were lower in IBS vs HCs (p= 0.03), which was mainly seen within men. Lower GR mRNA levels were associated with faster rise in ACTH and cortisol responses (p=0.01-0.03). EAL did not have a significant effect on hormone responses. Conclusion: HPA axis response was dysregulated in IBS. The enhanced HPA axis response was associated with downregulation of the GR mediated negative feedback system. Sex differences in the stress response may play a role in the female predominance of IBS.

The oral CYP17-Lyase (L) inhibitor VT-464 in patients with CRPC.

187 Background: VT-464 is an oral, non-steroidal inhibitor of CYP17-L and an antagonist of androgen receptor (AR) variants F876L and T877A which are associated with resistance to enzalutamide (ENZ) and abiraterone/prednisone (AA), respectively. INO-VT-464-CL-001 is a Phase (Ph) 1/2 study of VT-464 in treatment-naïve (TN) and treatment-failure (TF) patients with CRPC. Results are reported for Ph 1 (TN patients) and early results from Ph 2 patients. Methods: Phase 1: safety, tolerability, PK and initial efficacy (PSA and steroid concentrations) in M0 and M1 TN patients in escalated dose-cohorts from 50 to 600 mg bid in 28-day continuous dosing cycles. Phase 2: preliminary efficacy in M0 and M1 TN and TF (post-AA, -ENZ, and/or CHEMO) patients at 450 mg bid. Results: In Ph 1, 26 TN patients received VT-464 at 50 mg bid (n=1), 100 mg bid (n=1), 200 mg bid (n=4), 300 mg bid (n=8), 450 mg bid (n=9) and 600 mg bid (n=3) with 2 patients currently on study (15 and 29 months). Another 10 TN patients received VT-464 in Ph 2 at 450 mg bid. Most Ph 1 adverse events (AEs) were grade 1 or 2 and not considered drug related. There were 18 grade 3 AEs and 1 grade 5 AE considered not related to study drug, plus 4 grade 3 AEs (vasovagal/syncope) considered at least possibly related. For a single oral 450 mg dose, t½ was 6.8±0.8h (mean±SEM), Cmax was 8.6±1.2uM and Tmax was 3.4±0.7h. 19 of 26 Ph 1 and Ph 2 TN patients who received from 300 to 600 mg bid had PSA responses (6 PSA30%, 2 PSA50% and 1 PSA90% response). Preliminary PSA responses in Ph 2 TF patients included a PSA90% response in a prior-ENZ pre-chemo patient and a PSA50% response in a prior-ENZ and -chemo patient. No mineralocorticoid excess syndrome (MES) or changes in ACTH response or LFTs were observed. Plasma testosterone concentrations were reduced to near or below the limit of quantification in most patients at ≥ 450 mg bid, with normal progesterone and cortisol concentrations. No supplemental steroids were used. Conclusions: Clinical results at the doses evaluated support VT-464 CYP17 L-selectivity. The PSA50% and PSA90% responses in 2 of 7 prior Xtandi patients, combined with a lack of MES despite no supplemental steroids, warrants further investigation in the ongoing Ph 2 study, particularly in prior ENZ and AA patients. Clinical trial information: NCT02012920.

Ketamine Attenuates the ACTH Response to Hypoxia in Late-Gestation Ovine Fetus

Background: Ketamine, a noncompetitive N-Methyl-<smlcap>D</smlcap>-aspartate (NMDA) receptor antagonist, is a commonly used dissociative anesthetic in neonatology. We have proposed that ketamine reduces fetal stress responsiveness to stimuli that involve reduced oxygen supply to the fetal brain. Previously, we have shown that ketamine inhibits plasma ACTH levels in late-gestation fetal sheep subjected to brachiocephalic artery occlusion (BCO), an ischemic hypoxia model that might activate some of the same direct and reflex responses as hypoxia. Objectives: We performed the current study to test the hypothesis that ketamine pretreatment will reduce fetal ACTH responses to hypoxic hypoxia (HH). Methods: Fetal sheep were chronically catheterized at least 5 days prior to study. Ketamine (3 mg/kg) was administered intravenously to the fetus 10 min prior to normoxia or a period of hypoxia induced by administration of nitrogen gas to the maternal trachea for 30 min. Results: Hypoxia significantly increased both fetal ACTH and cortisol levels in both the control and ketamine groups (p < 0.0005, interaction effect of time·stimulus in two-way ANOVA), and the ACTH response was blunted by ketamine (p < 0.005). Conclusions: Ketamine reduced fetal ACTH responses to HH, possibly due to antagonism of the NMDA receptors in the fetal brain. Interestingly, in contrast to the responses to BCO, ACTH responses to HH were only partially inhibited, suggesting that multiple neurotransmitter pathways mediate the ACTH response to HH.