Monitoring Glucocorticoid Receptor in Plasma-derived Extracellular Vesicles as a Marker of Resistance to Androgen Receptor Signaling Inhibition in Prostate Cancer.

Disease progression following androgen ablation was shown to be associated with upregulation of the glucocorticoid receptor (GR). Longitudinal monitoring of GR expression in circulating extracellular vesicles (EV) may reflect changes in the tumor cell and facilitates detection of acquired resistance. We utilized LNCaP, LREX cells and a patient-derived xenograft, MDA PDX 322-2-6a, for in vitro and in vivo experiments. Plasma-derived EVs were isolated from patients with localized high-risk prostate cancer undergoing androgen ablation. The mRNA levels of GR in EVs and their responsive genes were detected by transcriptome analysis, qRT-PCR and the protein levels by Western blot analysis. We detected changes in GR expression at mRNA and protein levels in EVs derived from LNCaP and LREX cells in in vitro studies. In in vivo experiments, LNCaP and the PDX MDA 322-2-6a-bearing mice were treated with enzalutamide. GR levels in plasma-derived EVs were increased only in those tumors that did not respond to enzalutamide. Treatment of mice bearing enzalutamide-resistant tumors with a GR inhibitor in combination with enzalutamide led to a transient pause in tumor growth in a subset of tumors and decreased GR levels intracellular and in plasma-derived EVs. In a subgroup of patients with high-risk localized prostate cancer treated with androgen signaling inhibition, GR was found upregulated in matching tissue and plasma EVs. These analyses showed that GR levels in plasma-derived EVs may be used for monitoring the transition of GR expression allowing for early detection of resistance to androgen ablation treatment. SIGNIFICANCE: Longitudinal monitoring of GR expression in plasma-derived EVs from patients with prostate cancer treated with androgen signaling inhibitors facilitates early detection of acquisition of resistance to androgen receptor signaling inhibition in individual patients.

Plasma glucocorticogenic activity, race/ethnicity and alcohol intake among San Francisco Bay Area women.

Racial and ethnic minorities are at higher risk for a variety of diseases. While sociodemographic and lifestyle factors contribute to racial/ethnic health disparities, the biological processes underlying these associations remain poorly understood. Stress and its biological consequences through the glucocorticoid receptor (GR) have been hypothesized to mediate adverse disease outcomes. In fasting morning samples of 503 control women from the San Francisco Bay Area Breast Cancer Study, we used a sensitive Chemical-Activated LUciferase gene eXpression (CALUX) assay to examine the association of sociodemographic and lifestyle factors with plasma glucocorticogenic (G) activity in three racial/ethnic groups. The G activity is a sensitive measure that reflects biological activity of total plasma glucocorticoids including cortisol and glucocorticoid-like compounds. Associations between G activity and sociodemographic and lifestyle factors were examined using multivariable linear regression models. Latina and non-Latina Black (NLB) women had 9% (P = 0.053) and 14% (P = 0.008) lower morning G activity than non-Latina White (NLW) women, respectively. Additionally, we replicated a previously reported association between G activity and alcohol intake (women who drank >10gms had 19% higher G activity than non-drinkers, P = 0.004) in Latina and NLB women. Further research should assess the association between G activity and health outcomes in a prospective cohort so as to characterize the relationship between total plasma G activity in pre-disease state and disease outcomes across different racial/ethnic populations.

Mechanisms of glucocorticoid resistance in hypereosinophilic syndromes.

BACKGROUND: Glucocorticoids (GC) are considered first-line therapy for most patients with hypereosinophilic syndrome (HES). Although response rates are generally high, many patients require moderate to high doses for control of eosinophilia and symptoms, and up to 15% of patients do not respond at all. Despite this, little is known about the mechanisms of GC resistance in patients with HES. OBJECTIVE: To explore the aetiology of GC resistance in HES. METHODS: Clinical data and samples from 26 patients with HES enrolled on a prospective study of GC responsiveness and 23 patients with HES enrolled on a natural history study of eosinophilia for whom response to GC was known were analysed retrospectively. Expression of GC receptor isoforms was assessed by quantitative RT-PCR in purified eosinophils. Serum cytokine levels were quantified by suspension array assay in multiplex. RESULTS: Despite an impaired eosinophil response to GC after 7 days of treatment, the expected rise in absolute neutrophil count was seen in 7/7 GC-resistant patients, suggesting that GC resistance in HES is not a global phenomenon. Eosinophil mRNA expression of glucocorticoid receptor (GR) isoforms (α, ß, and P) was similar between GC-sensitive (n = 20) and GC-resistant (n = 9) patients with HES. Whereas geometric mean serum levels were also comparable between GC-r (n = 11) and GC-s (n = 19) for all cytokines tested, serum IL-5 levels were >100 pg/mL only in GC-r patients. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggest that the mechanism of GC resistance in HES is not due to a global phenomenon affecting all lineages, but may be due, at least in some patients, to impairment of eosinophil apoptosis by increased levels of IL-5.

Glucocorticoids Regulate Bone Marrow B Lymphopoiesis After Stroke.

RATIONALE: After a stroke, patients frequently experience altered systemic immunity resulting in peripheral immunosuppression and higher susceptibility to infections, which is at least partly attributed to lymphopenia. The mechanisms that profoundly change the systemic leukocyte repertoire after stroke are incompletely understood. Emerging evidence indicates that stroke alters hematopoietic output of the bone marrow. OBJECTIVE: To explore the mechanisms that lead to defects of B lymphopoiesis after ischemic stroke. METHODS AND RESULTS: We here report that ischemic stroke triggers brain-bone marrow communication via hormonal long-range signals that regulate hematopoietic B lineage decisions. Bone marrow fluorescence-activated cell sorter analyses and serial intravital microscopy indicate that transient middle cerebral artery occlusion in mice arrests B-cell development beginning at the pro-B-cell stage. This phenotype was not rescued in Myd88-/- and TLR4-/- mice with disrupted TLR (Toll-like receptor) signaling or after blockage of peripheral sympathetic nerves. Mechanistically, we identified stroke-induced glucocorticoid release as the main instigator of B lymphopoiesis defects. B-cell lineage-specific deletion of the GR (glucocorticoid receptor) in CD19-Cre loxP Nr3c1 mice attenuated lymphocytopenia after transient middle cerebral artery. In 20 patients with acute stroke, increased cortisol levels inversely correlated with blood lymphocyte numbers. CONCLUSIONS: Our data demonstrate that the hypothalamic-pituitary-adrenal axis mediates B lymphopoiesis defects after ischemic stroke.

Characterization of the Glucocorticoid Receptor in Children Undergoing Cardiac Surgery.

OBJECTIVES: Postoperative administration of corticosteroids is common practice for managing catecholamine refractory low cardiac output syndrome. Since corticosteroid activity is dependent on the glucocorticoid receptor, we sought to characterize glucocorticoid receptor levels in children undergoing cardiac surgery and examined the association between glucocorticoid receptor levels and cardiovascular dysfunction. DESIGN: Prospective observational cohort study. SETTING: Large, tertiary pediatric cardiac center. SUBJECTS: Children undergoing corrective or palliative cardiac surgery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A prospective observational cohort study was conducted in 83 children with congenital heart disease. Total glucocorticoid receptor levels were measured in the peripheral WBCs using flow cytometry. In addition, blood samples were collected for total cortisol levels. The primary outcome studied was the time to being inotrope free. An increase in glucocorticoid receptor level from postoperative day 1 to postoperative day 3 was associated with a longer time to being inotrope free (hazard ratio, 0.49 [0.29-0.81]; p = 0.01) in the univariate analysis. This association remained significant after adjusting for age, weight, cardiopulmonary bypass time, cross clamp time, Risk Adjustment for Congenital Heart Surgery-1 score, and postoperative steroid use (hazard ratio, 0.53 [0.29-0.99]; p = 0.05). Postoperative day 3 glucocorticoid receptor level showed a trend to have longer time to being inotrope free (hazard ratio, 0.66 [0.42-1.02]; p = 0.0.06). The cortisol levels minimally increased during the study duration and did not correlate with glucocorticoid receptor levels. CONCLUSIONS: Increasing glucocorticoid receptor levels in peripheral WBCs of children undergoing cardiac surgery are associated with a longer time to being inotrope free. Cortisol levels minimally increased during the study duration. These results suggest that exposure to high-dose perioperative corticosteroids may suppress the hypothalamic-pituitary-adrenal axis leading to increase in glucocorticoid receptor levels in response to a low cortisol environment. Further studies are required to better delineate the interplay between glucocorticoid receptor levels, cortisol levels, corticosteroid exposure, and postoperative inotropic requirements.

Glucocorticoid receptor DNA methylation and childhood trauma in chronic fatigue syndrome patients.

OBJECTIVE: Although the precise mechanisms are not yet understood, previous studies have suggested that chronic fatigue syndrome (CFS) is associated with hypothalamic-pituitary-adrenal (HPA) axis dysregulation and trauma in early childhood. Consistent with findings suggesting that early life stress-induced DNA methylation changes may underlie dysregulation of the HPA axis, we previously found evidence for the involvement of glucocorticoid receptor (GR) gene (NR3C1) methylation in whole blood of CFS patients. METHODS: In the current study, we assessed NR3C1-1F region DNA methylation status in peripheral blood from a new and independent sample of 80 female CFS patients and 91 female controls. In CFS patients, history of childhood trauma subtypes was evaluated using the Childhood Trauma Questionnaire short form (CTQ-SF). RESULTS: Although absolute methylation differences were small, the present study confirms our previous findings of NR3C1-1F DNA hypomethylation at several CpG sites in CFS patients as compared to controls. Following multiple testing correction, only CpG_8 remained significant (DNA methylation difference: 1.3% versus 1.5%, p<0.001). In addition, we found associations between DNA methylation and severity of fatigue as well as with childhood emotional abuse in CFS patients, although these findings were not significant after correction for multiple testing. CONCLUSIONS: In conclusion, we replicated findings of NR3C1-1F DNA hypomethylation in CFS patients versus controls. Our results support the hypothesis of HPA axis dysregulation and enhanced GR sensitivity in CFS.

miR-30c is specifically repressed in patients with active pulmonary tuberculosis.

Tuberculous pleurisy (PLTB) is a common form of extrapulmonary tuberculosis. It often resolves without chemotherapy being hence considered a rather benign manifestation of the disease. Patients with PLTB mount an effective anti-mycobacterial response, unlike those with active pulmonary TB (pTB) that were shown to present an imbalance in plasma immune and endocrine mediators. In this work, we explored whether expression of the active isoform of the glucocorticoid receptor (hGRα) in the context of the inflammatory-anti-inflammatory responses of TB patients may be associated to microRNA levels. As expected, the inflammatory response triggered in patients coexists with increased circulating cortisol and altered hGRα levels in the peripheral blood mononuclear cells. However, while hGRα expression is significantly downregulated in PLTB, its levels in pTB patients are higher within the control values. These results point out to the existence of an additional mechanism tending to preserve hGRα levels probably to deal with the chronic inflammation observed in pTB. In this regard, we found that miR-30c is strongly downregulated in mononuclear cells of pTB patients compared to PLTB cases, showing an expression profile opposite to that seen with hGRα. Interestingly, low levels of miR-30c are specific for this active form of TB, as its expression is not altered in mononuclear cells from either healthy controls or patients with tuberculous or non-tuberculous pleurisy. Moreover, miR-30c and hGRα also showed an inverse expression pattern in M. tuberculosis-stimulated THP-1 macrophage cultures. In sum, our studies identify miR-30c as a specific correlate of pulmonary manifestations of TB, potentially involved in the altered glucocorticoid sensitivity observed in these patients.

Peripheral blood mRNA expressions of stress biomarkers in manic episode and subsequent remission.

Theoretical models of the neuroprogressive nature of bipolar disorder (BD) are based on the hypothesis that it is an accelerated aging disease, with the allostatic load playing a major role. Glucocorticoids, oxidative stress markers, inflammatory cytokines and neurotrophins play important roles in BD. The messenger ribonucleic acid (mRNA) expressions of brain-derived neurotrophic factor (BDNF), tissue plasminogen activator (tPA), glucocorticoid receptor (GR), heat shock protein 70 (HSP70), tumour necrosis factor-alpha (TNF-α) were examined in the peripheral blood of 20 adult male, drug-free BD patients during manic and remission periods and in 20 adult male, healthy controls. mRNA expression was measured using the quantitative real-time polymerase chain reaction (qRT-PCR). Compared to the controls, the expressions of BDNF and tPA mRNA were down-regulated in mania. In remission, BNDF and tPA mRNA levels increased, but they were still lower than those of the controls. Between mania and remission periods, only the change in mRNA levels of BDNF reached statistical significance. The results suggest that BDNF and tPA may be biomarkers of BD and that proteolytic conversion of BDNF may be important in the pathophysiology of BD. The change in BDNF levels between mania and remission could be adaptive and used to follow the progression of BD.

The effect of fermented red ginseng on depression is mediated by lipids.

OBJECTIVES: The cortico-limbic hypothalamic-pituitary-adrenal axis has emerged as an important area for the cause and treatment of depression. The primary aim of this study was to test the hypothesis that hormones, energy sources, and minerals have a causal relationship with depression. The secondary aim was to test whether consumption of fermented red ginseng (FRG) would influence that causal relationship. METHODS: For this study, 93 postmenopausal women were randomly divided into two groups. One group (49 women) was supplied with FRG capsules, and the other group (44 women) with placebo capsules, for 2 weeks. Both before and after the study, the participants filled out the Beck depression inventory questionnaire, and then blood samples were collected. The structural regression model was established. The causative latent variables were hormone (adrenocorticotropic hormone and cortisol), energy (low-density lipoprotein (LDL) cholesterol, total cholesterol, and blood glucose), mineral 1 (potassium, sodium, chloride, and iron), and mineral 2 (magnesium, calcium), and the resultant latent variables were cognitive depression (CD) and somatic depression. The goodness-of-fit statistics of the final model were good (root mean square error of approximation =0.033, comparative fit index =0.877, and Tucker-Lewis index =0.870). RESULTS: The structural regression path of the energy factor on CD showed a significant difference between the FRG group (0.259) and the placebo group (-0.201). The factor loadings of total cholesterol (1.236) and LDL cholesterol (1.000) on the energy factor were much higher than that of glucose (0.166). CONCLUSION: Based on the analysis used in this model, the effect of FRG consumption on CD occurred via the energy factor, which is mainly attributable to cholesterol.

Increased expression of the glucocorticoid receptor ß in infants with RSV bronchiolitis.

OBJECTIVES: The majority of studies on glucocorticoid treatment in respiratory syncytial virus (RSV) bronchiolitis concluded that there are no beneficial effects. We hypothesized that RSV-infected patients may have an increased glucocorticoid receptor (GR) ß expression, the isoform that is unable to bind cortisol and exert an antiinflammatory action. METHODS: By using real-time polymerase chain reaction, we studied the expression of α and ß GR in the peripheral blood mononuclear cells obtained from 49 RSV-infected infants (<1 year of age) with severe (n = 29) and mild to moderate (n = 20) illness. In plasma, we analyzed the level of cortisol by radioimmunoassay and inflammatory cytokines interleukin (IL)-10, IL-6, tumor necrosis factor-α, IL-1ß, IL-8, IL-12p70, IL-2, IL-4, IL-5, interferon-γ, and IL-17 by cytometric beads assay. Statistical analysis was performed by nonparametric analysis of variance. RESULTS: We found a significant increase of ß GR expression in patients with severe illness compared with those with mild disease (P < .001) and with a group of healthy controls (P < .01). The α:ß GR ratio decreased significantly in infants with severe disease compared with those with mild illness (P < .01) and with normal controls (P < .001). The expression of ß GR was positively correlated with the clinical score of severity (r = .54; P < .0001). CONCLUSIONS: The decrease of the α:ß GR ratio by an increase of ß receptors expression is related to illness severity and may partly explain the insensitivity to corticoid treatment in RSV-infected infants. The increased expression of ß GR could be a marker of disease severity.

Glucocorticoid receptor density correlates with health risk factors and insulin resistance in Caucasian and African American subjects.

Activation of the hypothalamic-pituitary-adrenal axis leads to secretion of cortisol, which binds to peripheral glucocorticoid receptor and mediates a complex series of metabolic and immune effects. Cortisol also binds to receptors in the hypothalamus and pituitary, and inhibits further secretion of adrenocorticotropic hormone thus preventing an excessive response. Excess glucocorticoid effect is seen in Cushings disease, adrenal adenomas/carcinomas and in glucocorticoid resistance. Within such pathology there are health consequences of excessive glucocorticoid action, including obesity, hypertension, and glucose intolerance or diabetes. We hypothesized that increased glucocorticoid receptor in peripheral tissue might mediate an excess glucocorticoid effect in the absence of increased cortisol secretion. The objective of the study was to investigate the relationship between glucocorticoid receptor density in leukocytes and health risk indices relevant to obesity and diabetes in a sample of Caucasian and African American subjects. Comparison of glucocorticoid receptor concentration with subject body mass index, percentage body fat, waist circumference, insulin resistance, plasma cortisol levels, gender, and lipid profiles were conducted. Increased glucocorticoid receptor density significantly correlated with body mass index, percentage body fat, waist circumference, and insulin resistance. No significant correlation was observed for glucocorticoid receptor density with lipid profiles. Furthermore, no significant differences were observed in glucocorticoid receptor density between Caucasian and African American subjects or male and female participants. Our results show that high risk health conditions, such as obesity and type-2 diabetes, may be associated with a form of hypothalamic-pituitary-adrenal axis dysfunction, characterized by localized leukocyte glucocorticoid receptor over-expression.

Hormonal determinants and effect of ER22/23EK glucocorticoid receptor gene polymorphism on health status deterioration in the participants of the Mataró Ageing Study.

The purpose of this study is to assess the potential relationships of circulating IGF-I, adrenal and gonadal steroids, and polymorphism ER22/23EK of the glucocorticoid receptor (GC-R) gene with nutritional, functional and cognitive deterioration in a group of elderly people living independently. This is a population-based prospective study with 313 individuals (160 women and 153 men, 76.7 ± 7 years) who participated. A physical exam, evaluation of functional capacity (Barthel scale), cognitive function (mini-mental state examination-MMSE), geriatric depression scale (GDS), mininutritional assessment (MNA-SF) and cardiometabolic status were performed at basal time point and at 2 years of follow-up. Biological measurements included cortisol, dehydroepiandrosterone (DHEA), DHEA sulphate, testosterone, estradiol, IGF-I and polymorphism ER22/23EK of the GC-R gene. Estradiol was associated with MNA-SF decrease over time (p < 0.01, adjusted for age and gender, beta = -0.17, p = 0.03). Weight loss was related to testosterone in men (8.6 vs 12.1 pg/ml in no losers; p = 0.03), and in women with GDS (13.0% with depression vs 3.3% with no depression; p = 0.05) and MMSE (22.2% with cognitive deterioration vs 4.8% with no cognitive deterioration; p = 0.049). Barthel decrease was associated with testosterone (p = 0.02, after adjusting for age and gender, beta = -0.520, p < 0.001), and SHBG (p < 0.01, adjusted for age and gender, beta = 0.18, p < 0.01). DHEA was associated with deterioration in the MMSE (p = 0.01, after adjusting for age, gender, GDS scale and academic status, beta = -0.26, p = 0.01). Frailty development was related only in men with testosterone levels at the beginning of the study (p = 0.017). ER22/23EK was found in 3% of the subjects and carriers had a lower prevalence of hypertension. Adrenal and gonadal steroids are associated to impairment of the ageing health condition in elderly individuals living independently in Spain. ER22/23EK polymorphism of the GC-R gene has a low prevalence in our population.

Regulation of mRNA expression encoding chaperone and co-chaperone proteins of the glucocorticoid receptor in peripheral blood: association with depressive symptoms during pregnancy.

BACKGROUND: Major depressive disorder during pregnancy associates with potentially detrimental consequences for mother and child. The current study examined peripheral blood gene expression as a potential biomarker for prenatal depressive symptoms. METHOD: Maternal RNA from whole blood, plasma and the Beck Depression Inventory were collected longitudinally from preconception through the third trimester of pregnancy in 106 women with a lifetime history of mood or anxiety disorders. The expression of 16 genes in whole blood involved in glucorticoid receptor (GR) signaling was assessed using real-time polymerase chain reaction. In parallel, plasma concentrations of progesterone, estradiol and cortisol were measured. Finally, we assessed ex vivo GR sensitivity in peripheral blood cells from a subset of 29 women. RESULTS: mRNA expression of a number of GR-complex regulating genes was up-regulated over pregnancy. Women with depressive symptoms showed significantly smaller increases in mRNA expression of four of these genes - FKBP5, BAG1, NCOA1 and PPID. Ex vivo stimulation assays showed that GR sensitivity diminished with progression of pregnancy and increasing maternal depressive symptoms. Plasma concentrations of gonadal steroids and cortisol did not differ over pregnancy between women with and without clinically relevant depressive symptoms. CONCLUSIONS: The presence of prenatal depressive symptoms appears to be associated with altered regulation of GR sensitivity. Peripheral expression of GR co-chaperone genes may serve as a biomarker for risk of developing depressive symptoms during pregnancy. The presence of such biomarkers, if confirmed, could be utilized in treatment planning for women with a psychiatric history.

Age, body mass index, and serum level of DHEA-S can predict glucocorticoid receptor function in women with polycystic ovary syndrome.

Glucocorticoid receptor (GR) transduces the glucocorticoid (GC) signal that could lead to metabolic derangements depending on the tissue responsiveness to GC. We aimed to investigate possible causative relation of the GR functional properties in peripheral blood mononuclear cells of women with polycystic ovary syndrome (PCOS), with their clinical and biochemical characteristics. Thirty women with PCOS [mean age: 26.5 ± 5.1 years, mean body mass index (BMI) 24.5 ± 5 kg/m(2)], and thirty respective controls were analyzed for the number of GR sites per cell (B (max)), apparent equilibrium dissociation constant (K (d)), and binding potency (GR potency). A strong association between B (max) and K (d) (r = 0.70, P < 0.0001), and GR potency with age (r = 0.49, P = 0.009) was observed in PCOS women. The multiple regression analyses within the PCOS group revealed that independent predictors for K (d) were BMI, total cholesterol, and dehydroepiandrosterone-sulfate (DHEA-S) (r = 0.58, P = 0.038), while for GR potency (r = 0.687, P = 0.013) were age, BMI, DHEA-S, and basal cortisol concentration. The results suggest that PCOS pathophysiology may be related to alterations of a cross stalk between glucocorticoid signaling, age, and metabolic parameters. These findings should be further explored in studies on the role of GR in PCOS-related metabolic derangements.

Antidepressants, but not antipsychotics, modulate GR function in human whole blood: an insight into molecular mechanisms.

Clinical studies have demonstrated an impairment of glucocorticoid receptor (GR)-mediated negative feedback on the hypothalamic-pituitary-adrenal (HPA) axis in patients with major depression (GR resistance), and its resolution by antidepressant treatment. Recently, we showed that this impairment is indeed due to a dysfunction of GR in depressed patients (Carvalho et al., 2009), and that the ability of the antidepressant clomipramine to decrease GR function in peripheral blood cells is impaired in patients with major depression who are clinically resistant to treatment (Carvalho et al. 2008). To further investigate the effect of antidepressants on GR function in humans, we have compared the effect of the antidepressants clomipramine, amytriptiline, sertraline, paroxetine and venlafaxine, and of the antipsychotics, haloperidol and risperidone, on GR function in peripheral blood cells from healthy volunteers (n=33). GR function was measured by glucocorticoid inhibition of lypopolysaccharide (LPS)-stimulated interleukin-6 (IL-6) levels. Compared to vehicle-treated cells, all antidepressants inhibited dexamethasone (DEX, 10-100nM) inhibition of LPS-stimulated IL-6 levels (p values ranging from 0.007 to 0.1). This effect was specific to antidepressants, as antipsychotics had no effect on DEX-inhibition of LPS-stimulated IL-6 levels. The phosphodiesterase (PDE) type 4 inhibitor, rolipram, potentiated the effect of antidepressants on GR function, while the GR antagonist, RU-486, inhibited the effect of antidepressants on GR function. These findings indicate that the effect of antidepressants on GR function are specific for this class of psychotropic drugs, and involve second messenger pathways relevant to GR function and inflammation. Furthermore, it also points towards a possible mechanism by which one maybe able to overcome treatment-resistant depression. Research in this field will lead to new insights into the pathophysiology and treatment of affective disorders.

Cortisol and its action on the glucocorticoid receptor in malnutrition and acute infection.

Severe malnutrition alone is believed to cause hypercortisolemia. Cortisol's effects are mediated through the glucocorticoid receptor, which binds the hormone in the cytosol, translocates to the nucleus, and promotes gene transcription. This observational study in marasmic children with and without acute infection tested the hypothesis that marasmus is associated with hypercortisolemia, less glucocorticoid receptor, and less receptor translocation to the nucleus. Twenty-eight Malawian children participated; 14 with marasmus and infection, 6 with marasmus without infection, and 8 well nourished with infection. Free serum cortisol, interleukin 6 and tumor necrosis factor alpha, leucine derived from whole-body proteolysis, and the amount of whole-cell and nuclear leukocyte glucocorticoid receptor were measured upon admission. Free serum cortisol concentration was increased in marasmic and well-nourished children with infection compared with uninfected children with marasmus (14.2 [8.5, 16.3], 24.4 [15.0, 39.2], 5.1 [3.5, 7.0] microg/L, median [25th, 75th percentiles]; P < .05 by Kruskal-Wallis test). The amount of whole-cell leukocyte glucocorticoid receptor was similar in all children (0.48 +/- 0.33 signal units), but the amount in the nucleus was greatest in marasmic children with infection, followed by the amount in uninfected marasmic children, and then in well-nourished infected children (0.54 +/- 0.58, 0.19 +/- 0.13, 0.02 +/- 0.5 signal units [mean +/- SD]; P < .05 for all comparisons by analysis of variance). These findings suggest that hypercortisolemia is not associated with malnutrition alone, but does occur appropriately with acute infection. The increased nuclear glucocorticoid receptor abundance in marasmus demonstrates that nutritional status modulates glucocorticoid receptor action by mechanisms in addition to circulating glucocorticoid concentrations.

Expression of the human glucocorticoid receptor splice variants alpha, beta, and P in peripheral blood mononuclear leukocytes in healthy controls and in patients with hyper- and hypocortisolism.

CONTEXT: The effects of cortisol are mediated by the alpha-isoform of the glucocorticoid receptor (GR). GR-alpha levels and activity are modulated by alternative splicing of the common pre-mRNA into mRNAs for the GR-beta and GR-P isoforms. OBJECTIVE: The objective of this study was to investigate whether chronic hypercortisolism, chronic hypocortisolism, or acute, relative hypocortisolism influences the expression levels of the GR splice variants in mononuclear leukocytes. DESIGN: This was a case-control study. SETTING: The study was performed at a university hospital. PARTICIPANTS: Eighteen patients with Cushing's syndrome, five patients with hypocortisolemia, seven patients undergoing metyrapone testing, and 14 controls were studied. MAIN OUTCOME MEASURES: The main outcome measures were mRNA levels, GR affinity, and number per cell. RESULTS: All three GR mRNA isoforms were detected in participants from all groups at relative levels of alpha/P/beta = 1:0.25:0.001. There was a significant correlation between the expression levels of the three splice variants and between the mRNA levels and the number of receptors per cell. The GR in Cushing patients had an increased Kd (P < 0.05) preoperatively. GR number was not significantly different. Postoperatively, the Kd decreased. GR-beta mRNA expression was increased compared with controls (P < 0.05) and was decreased after surgery (P < 0.05). In patients with chronic hypocortisolism, GR-alpha mRNA expression was increased, and receptor numbers were increased (P < 0.05), whereas GR affinity was normal. No changes were observed in patients undergoing a metyrapone test. CONCLUSIONS: Cushing's syndrome is accompanied by a reversible decrease in GR affinity, possibly related to an increased GR-beta expression, which may be a compensatory mechanism to GC excess. In chronic hypocortisolism, adaptive changes in GR status seem to occur at the level of GR number.

High levels of glucocorticoid receptors in patients with active Crohn's disease may predict steroid resistance.

Up to 20% of Crohn's disease (CD) patients respond poorly to glucocorticoids (GC). A product of an alternative splicing of the glucocorticoid receptor (GR) premRNA, GRbeta, may play a role as a dominant inhibitor of the glucocorticoid response. Increasing evidence suggests that inflammatory cytokines such as interleukin (IL)-18 alternate the splicing of the primary transcript between the two isoforms GRbeta and GRalpha in hGR gene of CD patients. The aim of this study is to assess the expression of GRalpha and GRbeta in patients with CD and to look for a possible correlation between these receptors and the response to glucocorticoid treatment. Forty-two CD patients and 17 healthy volunteers were studied. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed using real-time PCR techniques. Serum IL-18 protein levels were measured by enzyme-linked immunosorbent assay (ELISA). The amount of hGRalpha-mRNA in patients in remission was significantly lower than in controls (P < 0.05). The amount of hGRbeta-mRNA was significantly higher in GC-resistant patients in the active stage of disease compared with all other groups (P < 0.05). Patients in the active stage of the disease had higher levels of IL-18 than patients in remission and both had higher levels than controls (P < 0.05). The amounts of IL-18 were directly correlated with the amount of hGRbeta mRNA in GC-resistant patients with an active disease. High levels of hGRbeta might be connected to GC resistance. IL-18 might participate in the alternative splicing of the hGR preliminary mRNA of CD patients. The results support the theory that augmented hGRbeta mRNA expression level in PBMC is connected with GC-resistance of CD patients.

Chronic treatment with the glutamate receptor antagonist MK-801 alters periodontal disease susceptibility.

OBJECTIVE: Previous experiments in rats suggest that hypothalamic-pituitary-adrenal (HPA) axis over-responsiveness, which leads to increased secretion of immunoregulatory glucocorticoid hormones, increases periodontal disease susceptibility, whereas HPA axis under-responsiveness is associated with increased resistance to the disease. The present study was designed to investigate whether MK-801 (dizocilipine malate), an antagonist of the glutamate receptor N-methyl-D-aspartate (NMDA) in the brain, which has been found to play an important role in the regulation of the HPA axis, would influence the outcome of experimental ligature-induced periodontal disease in a rat model. METHODS: Experimental periodontal disease was induced in periodontal disease susceptible and HPA axis high-responding Fischer 344 rats 2 days before chronic treatment with MK-801(1 mg/kg intraperitoneally). The periodontal breakdown was assessed after the ligatures had been in place for 23 days. Following intraperitoneal Gram-negative bacterial lipopolysaccharide stimulation (Escherichia coli, 250 microg/kg), concentrations of glucocorticoid receptors (GRs) in the hippocampus, and levels of the cytokine tumour necrosis factor alpha (TNF-alpha), as well as the HPA axis-derived hormone corticosterone, were measured in serum. RESULTS: Compared to vehicle-treated controls, MK-801-treated rats had significantly increased periodontal tissue destruction (p < 0.01). MK-801-treated rats also showed significantly increased expression of GRs in the hippocampus (p < 0.05), elevated levels of corticosterone (p < 0.001) and reduced levels of TNF-alpha (p < 0.01) in serum 2 h after lipopolysaccharide stimulation. CONCLUSION: These findings may implicate glutamate receptor-dependent mechanisms in periodontal disease, and support the concept of a bidirectional immune-brain-immune regulatory network with importance for periodontal health and disease.