Comparing vaccines: a systematic review of the use of the non-inferiority margin in vaccine trials.

BACKGROUND: Non-inferiority (NI) randomized controlled trials (RCTs) aim to demonstrate that a new treatment is no worse than a comparator that has already shown its efficacy over placebo within a pre-specified margin. However, clear guidelines on how the NI margin should be determined are lacking for vaccine trials. A difference (seroprevalence/risk) of 10% or a geometric mean titre/concentration (GMT) ratio of 1.5 or 2.0 in antibody levels is implicitly recommended for vaccine trials. We aimed to explore which NI margins were used in vaccine RCTs and how they were determined. METHODS: A systematic search for NI vaccine RCTs yielded 177 eligible articles. Data were extracted from these articles using a standardized form and included general characteristics and characteristics specific for NI trials. Relations between the study characteristics and the NI margin used were explored. RESULTS: Among the 143 studies using an NI margin based on difference (n=136 on immunogenicity, n=2 on efficacy and n=5 on safety), 66% used a margin of 10%, 23% used margins lower than 10% (range 1-7.5%) and 11% used margins larger than 10% (range 11.5-25%). Of the 103 studies using a NI margin based on the GMT ratio, 50% used a margin of 0.67/1.5 and 49% used 0.5/2.0. As observed, 85% of the studies did not discuss the method of margin determination; and 19% of the studies lacked a confidence interval or p-value for non-inferiority. CONCLUSION: Most NI vaccine RCTs used an NI margin of 10% for difference or a GMT ratio of 1.5 or 2.0 without a clear rationale. Most articles presented enough information for the reader to make a judgement about the NI margin used and the conclusions. The reporting on the design, margins used and results of NI vaccine trials could be improved; more explicit guidelines may help to achieve this end.

Hepatitis B vaccination strategies tailored to different endemicity levels: some considerations.

Hepatitis B is a serious public health problem. Worldwide three different levels of hepatitis B endemicity (high, intermediate and low) can be distinguished. Areas with different levels of endemicity require tailored vaccination strategies to fit the needs for individuals at risk and/or countries, depending on the infection risk per age group, vaccination rate, duration of protection after vaccination, cost effectiveness of vaccination strategies and ease of implementation in the national immunization schedules.This opinion paper evaluates these factors and proposes a combination of infant risk group and universal adolescent vaccination for low endemic countries thus targeting the different groups at risk. A universal infant vaccination schedule starting with a newborn vaccination within 24h after birth is more appropriate in intermediate- and high-endemic regions.

Induced differentiation of U937 cells by 1,25-dihydroxyvitamin D3 involves cell cycle arrest in G1 that is preceded by a transient proliferative burst and an increase in cyclin expression.

The hormonal form of vitamin D, 1,25-dihydroxyvitamin D3 [1, 25(OH)2D3], is a potent inhibitor of cellular proliferation as well as an inducer of differentiation of myeloid leukemic cells to macrophages. We have previously reported that a number of genes are upregulated by 1,25(OH)2D3 during myeloid differentiation, including the cyclin-dependent kinase (CDK) inhibitors p21, p27, 15, and p18, suggesting that cell cycle arrest and differentiation are tightly linked processes. We further explore here the relationship between growth inhibition and differentiation. We report that, upon 1, 25(OH)2D3 treatment, U937 cells exhibited an early proliferative burst followed by growth inhibition and subsequent differentiation. Although CDK levels remain constant throughout, this transient increase in proliferation was accompanied by increases in cyclin A, D1, and E protein levels. p21 and p27 levels were also elevated during both the proliferative burst and subsequent inhibition of cell growth. Ectopic overexpression of p21 and/or p27 in U937 cells, in the absence of hormone, resulted in an induction of the expression of monocyte/macrophage-specific markers, whereas overexpression of p15 and p18 had no effect, suggesting that a subset of CDK inhibitors are important for both growth arrest and differentiation and that an early increase in proliferation is somehow a prerequisite for subsequent differentiation. However, no such biphasic behavior was detected in cells that are growth inhibited by 1,25(OH)2D3 but do not differentiate, such as MCF-7 cells. Taken together, these results indicate that both growth stimulation and subsequent inhibition precede differentiation and involve induction of both cyclins and p21 and p27, whereas cell cycle arrest of differentiated cells can be achieved simply by elevations in CDK inhibitors.

A differential screen for ligand-regulated genes: identification of HoxA10 as a target of vitamin D3 induction in myeloid leukemic cells.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the hormonal ligand for vitamin D3, is a potent inducer of myeloid-leukemic-cell differentiation. Such cells differentiate exclusively into monocytes/macrophages in response to this ligand. Since 1,25(OH)2D3 transduces its hormone signal through the vitamin D3 receptor (VDR), a ligand-modulated transcription factor and member of the nuclear hormone receptor superfamily, we sought to identify direct VDR target genes induced during this differentiation process. To do so, we applied a modified differential screen with a nascent-RNA purification strategy using biases for immediate-early-response genes induced by 1,25(OH)2D3 in the myelomonocytic cell line U937. Using this screen, we had previously identified p21Waf1/Cip1 as a gene transcriptionally induced by 1,25(OH)2D3 and demonstrated that this induction facilitates the differentiation of U937 cells into monocytes/macrophages (24). Here, we describe in detail our differential screen strategy and the identification and isolation of 20 1,25(OH)2D3-inducible genes or unknown cDNAs by means of this screen. One gene newly identified as a target of VDR regulation in myeloid cells is the homeobox HoxA10 gene. HoxA10 protein may act as a general regulator of cell growth, since overexpression of HoxA10 facilitated the differentiation of U937 cells into monocytes/macrophages independent of 1,25(OH)2D3 and acted to strongly inhibit the growth of the breast cancer cell line MCF-7 by arresting these cells in G1.

Neonatal maternally deprived rats have as adults elevated basal pituitary-adrenal activity and enhanced susceptibility to apomorphine.

Maternal deprivation of neonatal rats for 24 h enhances the adrenocortical response to stress and/or adrenocorticotropin hormone (ACTH) stimulation during the stress hyporesponsive period (SHRP). The present study tests the hypothesis that such maternally deprived neonatal male rats show altered hypothalamic-pituitary-adrenal (HPA) regulation not only immediately after deprivation but also in later life. In addition, we found previously that neonatal changes in HPA activity preceded modulation of nigrostriatal dopamine function. Therefore, we also measured dopamine responsiveness in adult rats which were deprived of their mother during infancy. Neonatal male rats were maternally deprived for 24 h at the age of 3 days, whereas rats of the control group were left undisturbed. At 60 days of age deprived and non-deprived rats were decapitated and brain, adrenal glands and thymus were removed. Trunk blood was collected for determination of plasma ACTH, corticosterone and prolactin concentrations using radioimmunoassay procedures. mRNA levels of mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs), corticotropin-releasing hormone (CRH) mRNA and tyrosine hydroxylase (TH) mRNA were measured in brain sections with in situ hybridization. In a second group of male deprived and non-deprived rats apomorphine-induced stereotypic gnawing behaviour was examined at 60 days of age as a measure for functional activity of the dopamine system. Deprived neonatal rats showed the following characteristics as compared with non-deprived rats: (i) lower basal CRH mRNA concentration in parvocellular neurons of the paraventricular nucleus of the hypothalamus (PVN), while basal plasma ACTH and corticosterone concentrations were significantly elevated. Basal prolactin levels were not different. (ii) Similar hippocampal MR and GR mRNA levels. (iii) Significantly reduced GR mRNA levels in PVN and anterior pituitary. (iv) Significantly enhanced apomorphine-induced stereotypic gnawing behaviour and (v) higher TH mRNA levels in substantia nigra, while no changes were found in the ventral tegmental area (VTA). It is concluded that maternally deprived neonatal male rats display as young adults elevated basal pituitary-adrenal activity and enhanced apomorphine susceptibility.

Brain-corticosteroid hormone dialogue: slow and persistent.

1. The stress response system is shaped by genetic factors and life experiences, of which the effect of a neonatal life event is among the most persistent. Here we report studies focused on the "nature-nurture" question using rat lines genetically selected for extreme differences in dopamine phenotype as well as rats exposed as infants to the traumatic experience of maternal deprivation. 2. As key to the endocrine and behavioural adaptations occurring in these two animal models the hormone corticosterone (CORT) is considered. The stress hormone exerts slow and persistent genomic control over neuronal activity underlying the stress response system via high affinity mineralocorticoid (MR) and glucocorticoid receptors (GR). This action is exerted in a coordinate manner and involves after stress due to the rising CORT levels progressive activation of both receptor types. 3. Short periods of maternal separation (neonatal handling) trigger subsequently enhanced maternal care and sensory stimulation. However, a prolonged period (24 h) of depriving the infant of maternal care disrupts the stress hyporesponsive period (SHRP) and causes an inappropriate rise in CORT. During development exposure to CORT and to sensory stimulation has longlasting consequences for organization of the stress response system. 4. We find that these factors embodied by mother-pup interaction are critical for dopamine phenotype, CORT receptor dynamics and neuroendocrine regulation in adult life. The findings provide a conceptual framework to study dopamine-related psychopathology against a background of genetic predisposition, early life events, stress hormones and brain development.

Divergent prolactin and pituitary-adrenal activity in rats selectively bred for different dopamine responsiveness.

The present study explores the significance of brain dopamine phenotype for individual variation in the neuroendocrine stress response of the rat. For this purpose, we used two Wistar rat lines previously selected for high or low responsiveness of the dopamine system to apomorphine using the gnawing response as the selection criterion. Systemic administration of the drug evoked in apomorphine-susceptible (apo-sus) rats a vigorous gnawing response, whereas apomorphine-unsusceptible (apo-unsus) rats did not gnaw under these conditions. These two rat lines represent individuals displaying extreme differences in gnawing behavior that otherwise coexist in a normal Wistar population. In this study basal and stress-induced hypothalamic-pituitary-adrenal activity and PRL release were measured in chronically cannulated, freely moving rats that endured a conditioned emotional response. Tyrosine hydroxylase messenger RNA (mRNA), corticosteroid receptor mRNA, and in vivo retention of [3H]corticosterone were measured in rat brain sections using in situ hybridization and in vivo autoradiography. The result show that 1) apo-sus rats had a markedly reduced PRL response to stress compared to apo-unsus animals, whereas basal levels were not significantly different. A12 dopaminergic neurons in the arcuate nucleus expressed significantly higher levels of tyrosine hydroxylase mRNA in apo-sus rats, suggesting that the reduced stress-induced PRL release could be due to an increased inhibitory control by dopaminergic neurons; 2) in apo-sus rats, stress resulted in a sustained elevation of ACTH and free corticosterone levels, whereas the total corticosterone levels were not different between the two rat lines; 3) under basal morning conditions, apo-sus rats had significantly higher plasma ACTH, but, in contrast, lower free corticosterone than apo-unsus rats; total plasma corticosterone levels were not different; 4) the basal evening ACTH level was elevated in apo-sus rats; after removal of the adrenals in the morning, this increased ACTH level in apo-sus rats persisted into the afternoon 6 h postadrenalectomy; and 5) hippocampal mineralocorticoid (MR), but not glucocorticoid (GR), receptor capacity for the ligand comparable between the groups; the MR of apo-sus rats displayed an increased retention of [3H]corticosterone in all hippocampal cell fields measured 24 h adrenalectomy; MR and GR mRNA in hippocampus as well as GR mRNA in the paraventricular nucleus were not significantly different in the two rat lines. In conclusion, the data suggest a common genetic background for individual variation in stress responsiveness and dopamine phenotype. High dopamine reactivity is linked to a reduced PRL and an increased ACTH response after stress. These high dopamine responders display a hyporesponsive adrenal cortex and corticosteroid feedback resistance associated with altered brain corticosteroid receptor properties.

Development of divergence in dopamine responsiveness in genetically selected rat lines is preceded by changes in pituitary-adrenal activity.

Two pharmacogenetically selected Wistar rat lines have been used as a model for individual variability in behavioral and neuroendocrine responses. As a selection criterion the behavioral responsiveness for the dopamine agonist apomorphine was used, giving rise to the apomorphine-susceptible (apo-sus) and apomorphine-unsusceptible (apo-unsus) rat lines. This selection has been maintained over 16 generations. Recent studies have shown that adult rats of these selection lines also show pronounced differences in responsiveness of the hypothalamic-pituitary-adrenal (HPA) system. In this study we analyzed to what extent the divergence in dopamine phenotype and HPA responsiveness, as observed in adult rats, are linked to possible differences, within both systems, during early postnatal development. Therefore, we measured in neonatal female rats of 10 and 18 days of age several parameters of the dopamine and HPA system which show significant differences in adult rats. These include tyrosine hydroxylase (TH) and dopamine D1 and D2 receptor mRNA levels, which were determined within the nigrostriatal system since this system shows the most pronounced differences between adult rats of both selection lines. As indices of HPA activity we measured CRH mRNA, ACTH and total and free corticosterone plasma concentrations under basal conditions in the morning. Transcripts of the two types of corticosteroid receptors, mineralocorticoid (MR) and glucocorticoid (GR) receptor were measured in hippocampus and paraventricular nucleus. In 10-day-old rats all dopamine and HPA parameters were similar in rats of the two selection lines, except for GR mRNA in the parvocellular neurons of the paraventricular nucleus of the hypothalamus (PVN) of apo-sus rats, which was significantly higher than in apo-unsus rats. Eighteen-day-old apo-sus rats, however, showed significantly higher ACTH, comparable total corticosterone and a trend towards lower free corticosterone plasma levels. This HPA profile resembles the situation in adult apo-sus rats as compared with adult apo-unsus rats. Hippocampal GR mRNA expression and thymus weight were also higher in apo-sus rats. In addition, these rats showed an age-related increase in hippocampal MR mRNA expression, while in apo-unsus rats MR mRNA levels did not change between pnd 10 and 18. The measures of the nigrostriatal dopamine system at day 18 were still similar in rats of both lines. In conclusion, divergence in the dopamine systems of the two pharmacogenetically selected rat lines emerges subsequent to divergence in pituitary-adrenal activity.

Rats bred for enhanced apomorphine susceptibility have elevated tyrosine hydroxylase mRNA and dopamine D2-receptor binding sites in nigrostriatal and tuberoinfundibular dopamine systems.

From a Wistar population two rat lines were generated using as criterion the behavioral response to the dopamine agonist apomorphine. Rats of the apomorphine-susceptible (apo-sus) line revealed a vigorous gnawing response to apomorphine administration while the other rat line, the apomorphine-unsusceptible (apo-unsus) line, was selected for lack of response to the drug. In the present study using the 12th and 13th generation of these genetically selected lines, we have investigated whether this difference in apomorphine responsiveness was correlated with changes in dopamine neurochemistry. Therefore, we measured tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis, as well as dopamine D1 and D2 receptor mRNA levels in discrete brain regions by in situ hybridization. Dopamine (D2/D3) receptor binding was assessed with [125I]iodosulpride in a membrane binding assay and by quantitative autoradiography on tissue sections. [3H]SCH 23390 was used to analyze D1 receptor binding. Apo-sus rats displayed significantly higher TH mRNA levels in the A9 cell group of the substantia nigra pars compacta and in the A12 cell group of the arcuate nucleus. No difference was found in the A10 cell group of the VTA and the A6 cell group of the locus coeruleus. The density of D2/3 binding sites as well as D1 receptor mRNA levels in the striatal projection area of the A9 substantia nigra neurons, were significantly elevated in apo-sus rats. Dopamine D2 receptor mRNA and D1 receptor binding levels in caudate putamen and nucleus accumbens, however, were similar in rats of both lines. In conclusion, high apomorphine susceptibility is related to a potentially enhanced dopamine responsiveness selective for the nigrostriatal and tuberoinfundibular pathways.

Corticosteroid feedback resistance in rats genetically selected for increased dopamine responsiveness.

Pharmacogenetically selected Wistar rat lines were used to investigate the implication of either high or low responsiveness of the dopamine system for the activity of the hypothalamus-pituitary-adrenal (HPA) axis. As selection criterion the gnawing response induced by the dopamine agonist apomorphine was used. This criterion allows to distinguish apomorphine susceptible (apo-sus) rats which show a vigorous gnawing response from apomorphine unsusceptible (apo-unsus) rats. The present study, using male animals of the 9-12th generation of the two rat lines, revealed the following characteristics of the stress response system: (i) in apo-sus rats under basal conditions corticotrophin-releasing hormone (CRH) mRNA level in the paraventricular nucleus (PVN) and plasma adrenocorticotropin (ACTH) concentration were significantly higher; total corticosterone (B) plasma level was similar but free B level was lower; (ii) exposure to a novel environment resulted in a higher and prolonged plasma ACTH and total B response in the apo-sus rats. Moreover, the elevated free B level was also prolonged; (iii) apo-sus rats had increased CRH-induced pituitary ACTH release and B secretion was also increased, but not as prolonged as during novelty. (iv) In dexamethasone-pretreated rats an intravenous ACTH1-24 injection resulted in a similar plasma B response in rats of both lines; (v) In vitro, ACTH1-24 produced a significantly higher B secretion by adrenocortical cells of apo-sus rats reflecting the higher in vivo ACTH priming of the adrenal glands in these animals. (vi) apo-sus rats had higher body and thymic weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain mineralocorticoid receptor diversity: functional implications.

Mineralocorticoid receptors (MRs) in neurons of the anterior hypothalamus and the periventricular brain regions mediate aldosterone-selective actions on sodium homeostasis, salt appetite and cardiovascular regulation. Corticosterone is not effective in these neurons, possibly because it is enzymatically inactivated. However, MRs in limbic brain regions, notably in the hippocampal neurons, do already respond to very low concentrations of both corticosterone and aldosterone. The MR-mediated effects stabilize neuronal transmission and appear critical for neuronal integrity of a sub-region of the hippocampus: the dentate gyrus. Higher concentrations of corticosterone induced by stress and the circadian rise progressively activate the lower affinity glucocorticoid receptors (GRs), which in coordination with MR-mediated actions then facilitate adaptive processes required for recovery of homeostasis. It is postulated that this balanced MR- and GR-mediated action of corticosterone is of critical importance for regulation of the stress response and behavioural adaptation.

Bimodal shape of individual variation in behavior of Wistar rats: the overall outcome of a fundamentally different make-up and reactivity of the brain, the endocrinological and the immunological system.

An overview of the most important features of the two distinct types of individuals which normally co-occur in an unselected population of Wistar rats is given. It reveals that the overall make-up and reactivity of the brain, as well as the endocrinological and immunological systems differ completely between the two types of individuals. Each of these types of individuals has its own individual-specific hardware and software to cope with challenges from the internal or external environment, requires its own optimal niche, and is vulnerable for its own set of stressors.

Corticosteroid receptor plasticity in the central nervous system of various rat models.

The action of corticosteroids in the central nervous system (CNS) is mediated by two distinct corticosteroid receptors: the mineralocorticoid and glucocorticoid receptors (MR and GR respectively). Using an established in vitro binding assay system, MR and GR binding parameters were determined in the hippocampal, hypothalamic and pituitary cytosol of various rat models. In the (pharmaco-)genetically selected rat lines, the apomorphine susceptible (APO-SUS) rats showed a significant increase in the hippocampal and pituitary MR binding capacities (but not affinity) compared to those in the apomorphine-unsusceptible (APO-UNSUS) rats. In immunologically-altered Lewis (LEW/N) rats and in spontaneously hypertensive rats (SHR), increased hippocampal MR capacity (but not affinity) and hypothalamic MR capacity were observed compared to their respective control, Wistar (WIST) and Wistar Kyoto (WKY) rats. In addition, compared to WKY rats, SHR rats also showed a much greater pituitary but lesser hypothalamic GR binding capacity. In rats subjected to alteration in environmental conditions, the long-term effects of a short inescapable stress resulted in a significant increase in both hippocampal MR and GR while the pituitary and hypothalamic MR and GR do not differ in the stress and control groups. In rats subjected to a defeat test, a decrease in hippocampal MR and GR was observed 3 weeks (but not 1 week) later.

The effect of aging on stress responsiveness and central corticosteroid receptors in the brown Norway rat.

The present study examined the stress responsiveness of the hypothalamic-pituitary-adrenal axis in relation to the properties of corticosteroid receptors in the brain and pituitary of old (30 months) and young (3 months) male Brown Norway rats. Adrenocorticotropin hormone (ACTH) and corticosterone (B) were measured following exposure to novelty and to a conditioned emotional stimulus in blood samples sequentially obtained from chronically cannulated animals. Mineralocorticoid (MR) and glucocorticoid (GR) receptors were quantified by radioligand binding assay and in situ hybridization. The receptor binding constants were determined in tissue of rats that were adrenalectomized 24 hours previously, whereas gene expression was measured in the brain of intact animals. Aged Brown Norway rats showed a small but significant elevation in basal circulating ACTH level. The conditioned emotional stimulus, rather than the exposure to novelty, triggered a more than two-times higher ACTH response in the aged compared to the young rat. The termination of the stress-induced ACTH response seemed to proceed more efficiently in the aged rat. Basal and stress-induced total plasma B level did not differ in the young and old rats. The latter showed a 65% lower binding capacity of corticosteroid-binding globulin (CBG). Interestingly, in the aged rat the stress-induced rise in free circulating plasma B level was not elevated, but only prolonged. The hippocampus of aged rats displayed a decrease of maximally 44% in the apparent Bmax of MR, but no change in GR number. The Bmax of GR showed a 40% reduction in the hypothalamus and a 50% reduction in the anterior pituitary. GR affinity was considerably increased in the anterior pituitary, but was unchanged in the hippocampus and hypothalamus. Old age affected MR and GR gene expression differentially. GR mRNA was significantly reduced in cell field CA3 (-42%), CA4 (-41%) and the dentate gyrus (-26%) of the dorsal hippocampus, but did not change either in hippocampal cell field CA1 or in the hypothalamic paraventricular nucleus (PVN) of the old rat. There was no significant difference in MR mRNA between young and aged rats in the different cell fields of the hippocampus. The aged rat, therefore, is characterized by site- and receptor-specific changes in binding constants as well as by changes in receptor transcription and translation. The data demonstrate that in the old Brown Norway rats, a conditioned emotional stimulus results in enhanced pituitary ACTH release.(ABSTRACT TRUNCATED AT 400 WORDS)

Brain corticosteroid receptor gene expression and neuroendocrine dynamics during aging.

The present study examined the stress responsiveness of the hypothalamic-pituitary-adrenal axis in relation to the properties of corticosteroid receptors in the brain and pituitary in old (30 months) and young (3 months) male Brown Norway rats. The data demonstrate that circulating ACTH rather than the corticosteroid plasma level was elevated under basal conditions and following stress. Furthermore, a reduction of mineralocorticoid receptor (MR) number in the hippocampus and of glucocorticoid receptor (GR) number in the hypothalamus and the pituitary correspond to increased neuroendocrine responsiveness and negative feedback following stress. The changes in receptor binding do not parallel the changes in the amount of MR and GR mRNA measured with in situ hybridization. This suggests that the processing rather than the receptor gene expression is affected in senescence.

Enhanced Neurohypophyseal Vasopressin Release is Associated with Increased Opioid Inhibition of Oxytocin Release.

Abstract We tested the hypothesis of a cross-inhibition of oxytocin (OT) release by endogenous opioid peptides co-released with vasopressin (VP). This opioid cross-inhibition resulted in a selective block of OT release and hence in preferential release of VP. The effects of the opiate receptor antagonist naloxone were tested on neurohypophyseal VP release during dehydration, ethanol administration and sulphated cholecystokinin octapeptide (CCK-8S) application, assuming that the inhibition of pituitary OT release by endogenous opioids increases as neurohypophyseal VP output increases. A high VP output was found to coincide with increased inhibition of OT release: Subcutaneous injection of graded doses of naloxone (30 min prior to decapitation), augmented OT plasma levels significantly more in 24 h water-deprived male rats than in normally hydrated rats. Naloxone had no effect on VP release. Ethanol (10% in saline) administered intragastrically 50 min prior to decapitation and 20 min before subcutaneous naloxone (5 mg/kg) resulted in the inhibition of VP output. The ethanol treatment resulted in a rise in plasma OT levels that was additional to the effect of naloxone. These features were present in normally hydrated as well as in 24 h water-deprived animals, but were more pronounced in the latter group. Peripheral CCK-8S administration induces an abrupt and selective secretion of OT. Blocking the opioid inhibition of OT release with naloxone resulted in a significant rise of OT compared to that with CCK-8S alone. The magnitude of the opioid inhibition coincided with the activity of the VP system, and a higher dose of naloxone was needed to potentiate the CCK-8S effect on OT release in the water-deprived group than in euhydrated rats. No effect of CCK-8S and/or naloxone was found on VP plasma levels. The data indicate that opioid peptides co-released with VP (like dynorphin) may be responsible for cross-inhibition of OT release during dehydration. This suggests that dynorphin acts in a paracrine way, making it a strong candidate for this role.