Racial differences across pregnancy in maternal pro-inflammatory immune responsivity and its regulation by glucocorticoids.

BACKGROUND: The distribution of adverse pregnancy, birth and subsequent child developmental and health outcomes in the U.S. is characterized by pronounced racial (particularly Black-white) disparities. In this context, chronic stress exposure represents a variable of considerable importance, and the immune/inflammatory system represents a leading candidate biological pathway of interest. Previous pregnancy studies examining racial disparities in immune processes have largely utilized circulating cytokine levels, and have yielded null or mixed results. Circulating cytokines primarily represent basal secretion and do not necessarily represent functional features of immune responsivity and regulation. Thus, in order to conduct a more in-depth characterization of racial differences in functional immune properties during pregnancy, we utilized an ex vivo stimulation assay, a dynamic measure of immune function at the cellular level, to investigate Black-white racial differences in in mid- and late-gestation in i) pro-inflammatory (IL-6) responsivity of leukocytes to antigen [lipopolysaccharide (LPS)] challenge, and ii) regulation (dampening) of this pro-inflammatory response by glucocorticoids. METHOD: 177 women (N = 42 Black (24%), n = 135 white (76%)) with a singleton, intrauterine pregnancy provided 20 mL venous blood in mid- (16.6 ± 2.4 wks) and late (33.3 ± 1.1 wks) pregnancy. Maternal pro-inflammatory responsivity of leukocytes was quantified by assessing the release of the pro-inflammatory cytokine IL-6 in response to LPS stimulation, and regulation of the pro-inflammatory response was quantified by assessing the suppression of the stimulated IL-6 response after co-incubation with progressively increasing levels of dexamethasone [10-7, 10-6, 10-5 M] (i.e., glucocorticoid receptor resistance (GRR)). A priori model covariates included maternal age, parity, SES (socioeconomic status), and pre-pregnancy BMI. RESULTS: Maternal pro-inflammatory responsivity (LPS-stimulated IL-6) and GRR increased significantly across mid- and late gestation (adjusted ß = 0.157, p = 0.007; ß = 0.627, p < 0.001, respectively). Across both time points in pregnancy Black women exhibited significantly higher LPS-stimulated IL-6 release and reduced glucocorticoid regulation of the IL-6 response (i.e., higher GRR) relative to white women, before and after adjusting for covariates (ß = 0.381, p = 0.0030; ß = 0.391, p = 0.0075, respectively). There was no racial difference in the concentrations of circulating IL-6 (p = 0.9199). CONCLUSION: Our findings support the hypothesis postulating significant racial (Black-white) differences in key functional properties of the maternal immune system in pregnancy, which were not apparent using circulating cytokine measures. These data elucidate a potentially important physiological mechanism underlying the transduction of environmental conditions into racial disparities in reproductive and subsequent child health outcomes, and the use of these ex vivo measures should be considered in future studies.

Survival and reproductive costs of repeated acute glucocorticoid elevations in a captive, wild animal.

Organisms are continuously encountering both predictable and unpredictable ecological stressors within their environment. The activation of the hypothalamic-pituitaryadrenal (stress) axis is a fundamental process allowing animals to cope with and respond to such encounters. A main consequence of HPA axis activation is the release of glucocorticoid hormones. Although short-term glucocorticoid elevations lead to changes in physiological and behavioral processes that are often adaptive, our understanding of fitness consequences of repeated acute elevations in glucocorticoid hormones over a longer time period is largely lacking. This is of particular current importance as animals are facing a significant increase in exposure to stressors including those associated with human-induced rapid environmental change. Here, we test fitness-relevant consequences of repeated exposure to glucocorticoids in the absence of natural challenges, by treating wild-caught gravid female eastern fence lizards (Sceloporus undulatus) with a daily transdermal dose of a glucocorticoid hormone until laying. This treatment causes an increase in plasma glucocorticoids that mimics the natural response lizards have when they encounter a stressor in the wild, without confounding effects associated with the encounter itself. This treatment reduced females' reproductive success (hatching success) and survival. Further, glucocorticoid-induced reductions in reproductive success were greater when females had experienced higher temperatures the previous winter. This demonstrates the potential significant consequences of repeated exposure to acute elevations in glucocorticoid hormones. Additionally, the costs of repeated glucocorticoid elevation may be further exaggerated by an individual's previous experience, such as the potential compounding effects of winter warming increasing animals' vulnerability to increased glucocorticoid levels during spring breeding.

Do physical habitat complexity and predator cues influence the baseline and stress-induced glucocorticoid levels of a mangrove-associated fish?

As human populations continue to expand, increases in coastal development have led to the alteration of much of the world's mangrove habitat, creating problems for the multitude of species that inhabit these unique ecosystems. Habitat alteration often leads to changes in habitat complexity and predation risk, which may serve as additional stressors for those species that rely on mangroves for protection from predators. However, few studies have been conducted to date to assess the effects of these specific stressors on glucocorticoid (GC) stress hormone levels in wild fish populations. Using the checkered puffer as a model, our study sought to examine the effects of physical habitat complexity and predator environment on baseline and acute stress-induced GC levels. This was accomplished by examining changes in glucose and cortisol concentrations of fish placed in artificial environments for short periods (several hours) where substrate type and the presence of mangrove roots and predator cues were manipulated. Our results suggest that baseline and stress-induced GC levels are not significantly influenced by changes in physical habitat complexity or the predator environment using the experimental protocol that we applied. Although more research is required, the current study suggests that checkered puffers may be capable of withstanding changes in habitat complexity and increases in predation risk without experiencing adverse GC-mediated physiological effects, possibly as a result of the puffers' unique morphological and chemical defenses that help them to avoid predation in the wild.

Assessment of pregnancy status of Asian elephants (Elephas maximus) by measurement of progestagen and glucocorticoid and their metabolite concentrations in serum and feces, using enzyme immunoassay (EIA).

The study was to find patterns of progestagen (progesterone and its metabolite) and glucocorticoid and their metabolite concentrations in serum and feces of pregnant Asian elephants (Elephas maximus). The 5 female Asian domestic elephants were naturally mated until pregnancy. After that, blood and feces samples were collected monthly during pregnancy for progestagen, glucocorticoid and their metabolites analysis by enzyme immunoassay (EIA). The results showed the serum progestagen concentration during gestation was 2.11 ± 0.60 to 18.44 ± 2.28 ng/ml. Overall, serum progestagen concentration rose from the 1st month to reach peak in the 11th month, after which it declined to its lowest level in the 22nd month of pregnancy. Fecal progestagen concentration varied from 1.18 ± 0.54 to 3.35 ± 0.45 µg/g during pregnancy. In general, fecal progestagen concentration increased from the 1st month to its highest level in the 12th month. After this, it declined reaching its lowest point in the 22nd month of pregnancy. Glucocorticoid hormones and their metabolite concentrations both in serum and feces fluctuated from low to medium throughout almost the entire pregnancy period and then rapidly increased around the last week before calving. Our study suggests that this profile of progestagen and glucocorticoid hormones and their metabolite concentration levels in serum and feces can be used to assess the pregnancy status of Asian elephants. If serum and fecal progestagen concentrations were found in very low levels and glucocorticoid and their metabolite concentrations were found in very high levels, it was indicated that the cow elephant would calve within 7 days.

Quantification of 6 glucocorticoids in human plasma by liquid chromatography tandem mass spectrometry: method development, validation, and assessment of matrix effects.

BACKGROUND: Glucocorticoids are a group of steroid hormones with immunosuppressive and anti-inflammatory properties. In this article, we report the development and the validation of a liquid chromatography tandem mass spectrometry method for the simultaneous quantification of prednisolone, prednisone, cortisol, cortisone, methylprednisolone, and dexamethasone in human plasma. Furthermore, matrix effects were assessed qualitatively and quantitatively. METHODS: Plasma protein precipitation was performed with acetonitrile containing internal standards. Liquid-liquid extraction with dichloromethane and evaporation were used for cleanup and enrichment. The glucocorticoids were analyzed using reversed-phase chromatography and multiple reaction monitoring of positive ions. RESULTS: The mean extraction recovery was in the range 66.5%-104.8%, whereas the lower limits of quantification ranged from 1.5 to 4.0 µg/L. The intraday and interday accuracies of all the analytes were within 89.4%-116.6%, and imprecision was <15.6%. Ion suppression ranged from 15.3% to 27.3%. However, the matrix effects did not compromise the assay performance, with mean deviations in calculated concentrations of -4.8% to 2.1% between methanol and matrix. Short-term stability was acceptable for 5 of the analytes, with deviations from baseline between -3.4% and 8.7% after 24 hours at 4°C, although methylprednisolone was stable for 6 hours with a degradation of 10.2%. Deviations from baseline in controls stored at -20°C for 6 months ranged from -22.3% to 6.3%. All analytes were stable after 3 repetitive freeze-thaw cycles, with a maximum degradation of 5.5%. In terms of postpreparative stability, the analytes were stable after 24 and 48 hours at 4°C, with maximum degradation of 6.1% and 9.4%, respectively. CONCLUSIONS: A validated, sensitive, selective, and reproducible method for quantifying the concentrations of 6 glucocorticoids in human plasma by liquid chromatography tandem mass spectrometry is reported.

Variable sensitivity to the glucocorticoid activity of cortisol in patients with primary adrenal insufficiency: assessment with ACTH profiles.

Our aim was to investigate the usefulness of circulating levels of adrenocorticotropic hormone (ACTH) and also salivary cortisol to monitor cortisone substitution in patients with Addison's disease. 13 patients with primary adrenal insufficiency (8 women and 5 men, age 44 ± 11 years) received 12.5 mg cortisone acetate orally at 16:00 h and 25 mg at 07:00 h. Blood samples for cortisol and ACTH analysis were drawn every hour for 24 h, and also every half hour between 07:00 and 12:00 h. Samples for salivary cortisol were collected in parallel. Total ACTH levels showed large inter-individual variations and a diurnal rhythm with a nadir in the early evening at 19:00 (median 19 ng/l, range 2-434 ng/l) and high levels in the early morning, with a peak around 07:30 (median 844 ng/l, range 45-2,249 ng/l). Plasma cortisol concentrations showed 2 peaks distinct in time, but variable in height, 1-2 h after intake of cortisone. Plasma cortisol correlated significantly with ln(ACTH) at 17:00 h (r=-0.56), at 10:00 h (r=-0.51), and at 10.30 h (r=-0.57). When tested at different time points, ln(ACTH) at 10:00 to 12:00 h was negatively correlated with plasma cortisol between 08:30 and 12:00 h. Plasma cortisol was highly correlated to ln(salivary cortisol) most of the time points measured, but 30-60 min after intake of cortisone acetate the correlation disappeared. In conclusion, the large interindividual variation in ACTH levels most likely indicates varying sensitivity to cortisol with a need for individualized dosing schemes. Furthermore ACTH-determinations may be useful for dose titration of cortisol.

Reproductive conflict and the costs of social status in cooperatively breeding vertebrates.

Conflict over reproduction is an inherent part of group living. In many social vertebrates, conflict may be reflected as allostatic load, or the costs of social status and dominance rank, which may be quantified by measuring glucocorticoid stress hormones. Here, we develop the first quantitative model of allostatic load based on the tug-of-war model of reproductive skew to generate insights into the mechanisms underlying reproductive conflict in cooperative breeders and to determine whether glucocorticoids can be used to assess conflict levels in group-living vertebrates. It predicts that subordinates have higher allostatic loads than dominants under most conditions, but when body condition is lower in dominants than in subordinates, dominants experience higher allostatic load. Group structure is also important, as dominants generally have higher allostatic loads than subordinates when there is a large number of subordinates in the group, but this cost can be reduced by increasing the number of dominants, as in plural breeding societies. Using glucocorticoid data from cooperatively breeding superb starlings Lamprotornis superbus, we found empirical support for both predictions. Our model is useful for understanding how the costs of social status influence reproductive sharing, and it suggests that glucocorticoids can be used to examine reproductive conflict and cooperation in social species.

Social regulation of leukocyte homeostasis: the role of glucocorticoid sensitivity.

Recent small-scale genomics analyses suggest that physiologic regulation of pro-inflammatory gene expression by endogenous glucocorticoids may be compromised in individuals who experience chronic social isolation. The present study assessed the relationship between leukocyte distributional sensitivity to glucocorticoid regulation and subjective social isolation in a large population-based sample of older adults. Initial analyses confirmed that circulating neutrophil percentages were elevated, and circulating lymphocyte and monocyte percentages were suppressed, in direct proportion to circulating cortisol levels. However, leukocyte distributional sensitivity to endogenous glucocorticoids was abrogated in individuals reporting either occasional or frequent experiences of subjective social isolation. This finding held in both non-parametric univariate analyses and in multivariate linear models controlling for a variety of biological, social, behavioral, and psychological confounders. The present results suggest that social factors may alter immune cell sensitivity to physiologic regulation by the hypothalamic-pituitary-adrenal axis in ways that could ultimately contribute to the increased physical health risks associated with social isolation.

Simultaneous determination of cortisol, dexamethasone, methylprednisolone, prednisone, prednisolone, mycophenolic acid and mycophenolic acid glucuronide in human plasma utilizing liquid chromatography-tandem mass spectrometry.

Chronic combination immunosuppressive regimens are commonly prescribed to renal transplant recipients. To develop an assay method for pharmacokinetic studies and therapeutic drug monitoring of multiple immunosuppressives, a liquid chromatography-tandem mass spectrometry (LC/MS/MS) approach for the simultaneous analysis of several glucocorticoids, mycophenolic acid (MPA) and mycophenolic acid glucuronide (MPAG) was investigated. The resultant method utilized a gradient reverse phase separation over a Symmetry C18 column using an ammonium acetate-methanol mobile phase at pH 3.5. The analytes were detected by coupling the chromatography system via electrospray to a triple quadrupole mass spectrometer. Multiple-reaction monitoring in the negative mode ion (MH-/product) was employed selecting MPA at 319.1/190.9, MPAG at 495.1/191.0, dexamethasone at 391.0/361.0, hydrocortisone at 361.1/331.1, methylprednisolone at 373.1/343.1, prednisone at 357.1/327.2, and prednisolone at 359.1/329.1. The calibration curve concentrations ranged from 3.60 ng/mL to 50 microg/mL with the lowest limit of quantitation for corticosteroids being 3.60-7.20 ng/mL and 0.656-6.75 microg/mL for MPA and MPAG, respectively. The relative standard deviation for quality control intraday variation and interday variation was between 0.76% and 9.57% for all analytes. This assay offers a versatile, unique method for multi-analyte immunosuppressive determinations during combination immunosuppression.

Pharmacokinetic and pharmacodynamic assessment of bioavailability for two prodrugs of methylprednisolone.

AIMS: The aim of this study was to establish whether pharmacokinetic differences between two pro-drugs of methylprednisolone (MP) are likely to be of clinical significance. METHODS: This study was a single-blind, randomized, crossover design comparing the bioequivalence of MP released from the pro-drugs Promedrol (MP suleptanate) and Solu-Medrol (MP succinate) after a single 250 mg (MP equivalent) intramuscular injection to 20 healthy male volunteers. Bioequivalence was assessed by conventional pharmacokinetic analysis, by measuring pharmacodynamic responses plus a novel approach using pharmacokinetic/pharmacodynamic modeling. The main measure of pharmacodynamic response was whole blood histamine (WBH), a measure of basophil numbers. RESULTS: The MP Cmax was less for MP suleptanate due to a longer absorption halflife of the prodrug from the intramuscular injection site. The bioavailability of MP was equivalent when based on AUC with a MP suleptanate median 108% of the MP succinate value (90% CI: 102-114%). For Cmax the MP suleptanate median was 81% of the MP succinate value (90% CI: 75-88%). The tmax for MP from MP suleptanate was delayed relative to MP succinate. The median difference was 200% (90% non-parametric CI: 141-283%). The area under the WBH effect-time curve (AUEC) and the maximum response (Emax) were found to be equivalent (90% CI: 98-113% and 93-109% respectively). The maximum changes in other white blood cell counts, blood glucose concentration and the parameters of the pharmacodynamic sigmoid Emax model (EC50, Emax and gamma) were also not significantly different between prodrugs. CONCLUSIONS: MP suleptanate is an acceptable pharmaceutical alternative to MP succinate. The use of both pharmacokinetic and pharmacodynamic response data together gives greater confidence in the conclusions compared with those based only on conventional pharmacokinetic bioequivalence analysis.

Assessment of the systemic effects of inhaled glucocorticosteroids: the influence of blood sampling technique and frequency on plasma cortisol and leucocytes.

Twelve healthy males (mean age 27.6 y, range 23-35 y) took part in a randomized, double-blind, cross-over study of the effect of blood sampling technique (separate isolated venepunctures vs use of an IV cannula) and frequency (overnight vs morning) on plasma cortisol and white blood cell count after inhalation of a single dose of budesonide 3.2 mg or placebo, in order to establish the more sensitive method for future use. Sampling technique and frequency affected neither leucocytes nor plasma or urinary cortisol. Budesonide suppressed both plasma and urine free cortisol and delayed the nocturnal rise due to the circadian rhythm, thus reducing the AUC of plasma cortisol vs time. Lymphocytes, eosinophils and monocytes were decreased and neutrophils and total white blood cells were increased by the high dose of budesonide used. Lymphocytes and neutrophils showed significant changes earlier than eosinophils and cortisol and may be the variables of choice under certain conditions. Frequent sampling gave more complete information about the systemic effect of the drug than single morning samples.

[Significance of hormonal studies in the assessment of the health status of children and adolescents]. / Znachenie gormonal'nykh issledovanii v otsenke sostoianiia zdorov'ia detei i podrostkov.

Hormonal studies into adrenal function were carried out in 500 children and adolescents. Comparatively high correlation ratios attest to the relationship between androgenous and glucocorticoid functions of the adrenals and indicators of the physical, sexual development and osseous age. In the muscular type of constitution, the reserve possibilities of the adrenals appeared to be the most powerful as compared to those seen in the thoracal and digestive types, which is conductive to the least development of the lymphoid and throat ring and the least disease incidence. In children and adolescents engaged in sports swimming, favourable reactions of the adrenals were demonstrable in 50% of cases, pointing to the presence of the reserve possibilities of androgenous and glucocorticoid functions which should be taken into consideration on exercise indication. As influenced by the total school load throughout the entire school year, glucocorticoid function of the adrenals deteriorated 1.5-fold, this is accompanied by the astheno-neurotic manifestations and hypotension. All these facts should be taken into account in the assessment of the health status and undertaking preventive measures.