Objective: Description of the clinical, biochemical and genetic features of a Polish patient with familial glucocorticoid deficiency. Methods: Detailed clinical investigation, hormonal analysis and sequencing of the coding region of the melanocortin 2 receptor (MC2R) gene in this patient. Results: We report on a 3-month-old boy with familial glucocorticoid deficiency who presented at the age of 3 months with skin hyperpigmentation, muscle weakness, mild jaundice and constipation. Hormonal analyses revealed high ACTH and TSH serum concentrations, low serum cortisol concentration along with normal blood electrolytes. On hydrocortisone supplementation, the disease symptoms disappeared and the child recovered completely. His physical and mental development progresses normally. Genetic analysis disclosed a novel compound heterozygous MC2R mutation p.Leu46fs and p.Val49Met. Conclusion: The heterozygous p.Leu46fs mutation adds to the small number of MC2R nonsense mutations and is the first frameshift mutation within the first transmembrane domain of the receptor. According to molecular modeling the Val49Met mutation results in a structural change of the first transmembrane domain and in a potential novel interaction of the transmembrane domains I and VII. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

These studies present a series of novel roles for glucocorticoid signaling in the developing zebrafish embryo. The best-characterized roles of cortisol, the primary circulating corticosteroid in teleost fish, are known to occur by the activation of the glucocorticoid receptor (GR) in the post-hatch animal to mobilize energy reserves for response and recovery from stressful situations. For the first time, evidence is presented that GR and cortisol are key developmental regulators in the pre-hatch zebrafish embryo and that glucocorticoid signaling modulates multiple critical developmental pathways and affects embryogenesis in diverse ways. Prior to these experiments, very little was known regarding the developmental role of glucocorticoids in lower vertebrates. In mammalian models, there has been extensive study of the action of these steroids in late-stage organ maturation, and they have a variety of clinical and biomedical applications. However, in fish, there was a relative dearth of information regarding the basic dynamics and potential functional roles of cortisol and GR in embryogenesis. Zebrafish are a popular model for developmental study, with optically transparent embryos that allow for reliable observation. Additionally, the zebrafish genome is fully sequenced and extensively annotated, and a variety of molecular biology techniques are well-established in the existing literature. The zebrafish is also now recognized as an advantageous model for endocrine and stress axis studies, as it expresses a single GR gene, unique among teleosts but comparable to mammals. Preliminary studies published in the literature described cortisol and GR as deposited in the zebrafish embryo prior to fertilization, and showed their expression declining prior to hatch, then rising significantly as larvae approach the stage of first feeding. This dynamic expression of both ligand and receptor during embryogenesis, combined with knowledge from mammalian models, led to the hypothesis that glucocorticoid signaling may be functionally relevant in zebrafish development. A variety of techniques were used to examine the roles of cortisol and GR in the zebrafish embryo. Morpholino oligonucleotides were injected into one-cell embryos to block GR protein translation, allowing for the identification of GR-responsive developmental events and putative GR target genes. High-density microarray analysis of GR morphants presented numerous novel genes and pathways that are modulated by glucocorticoid signaling in the embryo. The ability to microinject molecules into a newly-fertilized zygote also allowed for other manipulations, including the addition of exogenous cortisol or the use of a cortisol-specific antibody to sequester maternally deposited cortisol. These studies provided the first evidence regarding the functional importance of the maternal cortisol deposition in the zebrafish oocyte prior to fertilization. The detailed temporal and spatial expression of GR mRNA and protein in the developing embryo has been characterized for the first time. GR expression is widespread, especially in developing mesoderm, and de novo GR transcription in the zebrafish embryo begins within 12 hours post fertilization. Lack of GR protein expression in the developing zebrafish embryo causes reduced growth, delayed somitogenesis, altered myogenesis, and severely reduces post-hatch survival. Additionally, GR modulates the expression of bone morphogenetic proteins, a family of morphogens that are involved in major developmental events including dorsoventral patterning, somitogenesis, myogenesis, and organogenesis. Reduction in GR protein content in the developing embryo is also linked to other major developmental processes including neurogenesis, eye formation, cellular adhesion, and development and function of the hypothalamic-pituitary-interrenal (HPI) axis. Cortisol in the early embryo, which is contributed entirely by maternal deposition prior to fertilization, is an important regulator of cardiogenesis and development of the HPI axis. Modulation of cortisol content in the early embryo causes an impairment of the post-hatch response to a physical stressor, as larvae exposed to increased cortisol during embryogenesis displayed an inability to increase heart rate in response to an acute physical stress, and did not display the classical increase in cortisol that follows a stressor challenge. Embryos that experience lowered glucocorticoid signaling in development tend to have a heightened post-hatch response to stress, further supporting the conclusion that HPI axis development is regulated by glucocorticoid signaling. These studies have identified key cardiogenic and HPI axis genes that are GR-responsive, providing mechanistic explanations for these phenotypic changes. Together these findings indicate that maternal deposition of cortisol in the embryo can pattern the post-hatch larva and has definitive impacts on performance as the offspring begin locomotion and approach feeding stages. In total, these studies demonstrate that glucocorticoid signaling is critically important to zebrafish embryogenesis, defining novel roles that are completely independent of the classical vertebrate stress response. These functions have significant effects on diverse developmental pathways and processes, and with the potential applicability of the zebrafish model to studies in higher vertebrates, may have important biomedical applications.

[Truncated abstract] There is a large body of evidence describing the benefits and risks, to the human fetus, of antenatal glucocorticoid treatment, but no published research on the effects on lactation. The withdrawal of progesterone, in the presence of high levels of endogenous glucocorticoids and prolactin, triggers the onset of copious milk secretion (lactogenesis II) at the end of pregnancy. The alteration of lactogenesis II by exogenous glucocorticoids could potentially have adverse impacts on postnatal nutrition in both term and preterm infants. I aimed to determine the effects of maternal antenatal glucocorticoid treatment on lactogenesis II in both ewes and women. I found profound adverse effects on lactation in ewes, and similar but more subtle effects on lactation in women . . . This thesis represents the first investigation of the effects of antenatal glucocorticoid treatment on lactogenesis II in both ewes and women. I found that, in ewes, antenatal glucocorticoid treatment stimulated premature lactogenesis II, and this was caused by disruptions to hormonal regulation during pregnancy. This event was followed by profound delays in lactogenesis II after term parturition. More subtle effects in women suggest that antenatal glucocorticoid treatment did not have a major, prolonged impact on postnatal lactogenesis II. Very preterm gestational age strongly predicted delays in lactogenesis II stressing the importance of assistance for these mothers when they are establishing lactation.

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / "July 1999." Typescript. Vita. Includes bibliographical references (leaves 128-129). Also available on the Internet.

Human epidemiological studies have provided compelling evidence that prenatal environmental events are associated with significantly increased risks of developing neuropsychiatric disorders in adulthood. This phenomenon has been studied and known as ‘fetal programming of adult diseases’. According to the theory, environmental insults occurring at critical periods of pregnancy can permanently alter the structure and physiology of the developing organs, and may lead to adverse functional consequences that manifest later in life. It has been suggested that increased maternal glucocorticoids may be one common mechanism through which various environmental insults can affect on the developing fetus. Therefore, in this thesis, I studied the long-term prenatal glucocorticoid overexposure effects on the functioning of the brain and investigated possible molecular mechanisms underlying the programmed effects. By using a rat model of prenatal dexamethasone (100μg/kg per day) administered to pregnant dams during the last week of gestation, I found that prenatal dexamethasone overexposure significantly reduced birth weight in both male and female offspring. I also assessed the consequences of prenatal dexamethasone treatment on stress response and cognition in the adult male offspring by using a number of physiological and behavioural measures. Overall I found no evidence of prenatal dexamethasone treatment effect on the hypothalamic-pituitaryadrenal (HPA) axis activity and behavioural responsivities to an acute stress in adult male offspring; however, prenatal dexamethasone exposure affected hippocampus and prefrontal cortex related cognition in the adult offspring, including contextual fear conditioning and behavioural flexibility on water maze tasks. I further explored possible molecular mechanisms that underlie the behavioural phenotypes of the prenatal dexamethasone rat model, and found altered brain gene expression with possible implications in synaptic plasticity and retrotransposon activities. Collectively, my current study suggests that prenatal dexamethasone exposure induces long-term programming effects on adult behaviour, associated with altered gene expression profile of the brain in the rat offspring. These results provide further evidence that prenatal glucocorticoid exposure may affect the development of the brain and its influence may endure into adulthood.

Background: Nephrotic syndrome (NS) occurs when the glomerular filtration barrier becomes abnormally permeable, leading to the clinical triad of proteinuria, massive oedema, and hypoalbuminaemia. Historically, NS has been thought to result from dysregulation of the immune system, although recent evidence suggests the glomerular podocyte plays a central role in disease pathogenesis. Children with NS are generally treated with an empiric course of glucocorticoid (Gc) therapy; a class of steroids which are activating ligands for the glucocorticoid receptor (GR) transcription factor. A major factor limiting the clinical utility of these agents is the marked variation observed in response to treatment. Although Gc-therapy has been the cornerstone of NS management for several decades, the mechanism of action, and target cell, remain poorly understood. Hypothesis and aims: The central hypothesis for this thesis states that glucocorticoids act directly on the podocyte to produce clinically useful effects without involvement of the immune system. Findings: Using a wild-type human podocyte cell line, I demonstrated that the basic GR-signalling mechanism is intact in the podocyte, and that glucocorticoids produce a direct, protective effect on the podocyte without immune cell involvement, by using electrical resistance across a podocyte monolayer as a surrogate marker for barrier integrity. To understand potential mechanisms underpinning this direct effect I defined the podocyte GR cistrome (using a combination of chromatin immunoprecipitation followed by massively parallel DNA sequencing and transcriptomic analysis) as well as total cell proteomics. Subsequent gene ontology analysis revealed that Gc treatment had prominent effects on podocyte motility, and these findings were validated with live-cell imaging. To gain mechanistic insight, I investigated the role of the pro-migratory small GTPase regulator Rac1, and demonstrated that treatment with Gc reduced Rac1 activity. Furthermore, the Rac1 inhibitor EHT 1864 had a direct, protective effect on the podocyte. To create a model to study the role of podocyte GR in vivo I generated a mouse line with a podocyte-specific GR deletion. Impact: Gc exposure produces potentially clinically-relevant effects directly on the podocyte, and Gc-induced podocyte hypomobility may underlie the clinical efficacy of these agents. Future animal studies investigating the consequences of GR deletion in the podocyte and the anti-proteinuric effects of Rac1 inhibition are warranted.

Chronic Obstructive Pulmonary Disease (COPD) is a chronic, inflammatory condition, characterised by airflow limitation. The use of glucocorticoids (GC) as an anti-inflammatory treatment in COPD has limited clinical benefits, and as such, new treatments are needed. Identifying key pathways involved in the inflammatory response in COPD may enable the development of novel treatments. The aims of this thesis were to examine the steroid sensitivity of an in vitro mixed sputum culture cell model, comparing COPD cells to smoking and non-smoking controls, examine expression of the intracellular signalling molecule p38 Mitogen Activated Protein Kinase (MAPK) in COPD lungs compared with controls, examine the GC and p38 MAPK inhibitor and dual therapy sensitivity of a bronchial epithelial cell line and finally, to understand the mechanisms by which a p38 MAPK inhibitor in combination with a GC synergistically inhibit pro-inflammatory mediator production in a bronchial epithelial cell line. Dexamethasone inhibits mixed sputum cell pro-inflammatory mediator release, with no differences in sensitivity observed between COPD and control cells. Isolated sputum neutrophils demonstrate modest sensitivity to dexamethasone, which is in contrast to blood neutrophils. There are increased numbers of cells positive for activated p38 MAPK in COPD lungs compared with controls, specifically localised to follicular B and CD8+ T cells, bronchial epithelial cells and alveolar and sputum macrophages. Lung and sputum neutrophils are devoid of activated p38 MAPK, and a pharmacological p38 MAPK inhibitor has no effect on pro-inflammatory mediator production from these cells. This is in contrast to blood neutrophils, whereby p38 MAPK activation can be induced following LPS stimulation and in vitro cell culture, and pro-inflammatory mediator release is inhibited by a p38 MAPK inhibitor. Dexamethasone and birb 796 inhibit stimulated pro-inflammatory mediator release from a bronchial epithelial cell line in a dose-dependent manner. Sensitivity to either drug is dependent on stimuli and the pro-inflammatory mediator analysed. There is additive and synergistic inhibition of pro-inflammatory mediator production when combination therapy comprising dexamethasone and birb 796 is used compared with either drug alone. This may be due to Birb 796 enhancing dexamethasone-mediated nuclear translocation of the glucocorticoid receptor, which may enhance the GC-mediated anti-inflammatory effects. Combination therapy may therefore be a useful therapeutic in the treatment of COPD.

Glucocorticoids (Gc) are essential for life. Clinically, Gcs are potent anti-inflammatory agents, prescribed as first line treatment for a range of inflammatory disorders, including rheumatoid arthritis and asthma. In this thesis I present two studies that advance our understanding of the diverse actions of Gcs by using bioinformatics approaches. I have identified the functional network of two glucocorticoid receptor (GR) isoforms and characterised the effects of Gcs on cell function. Study 1 Systems analysis of GR function: Gcs regulate a diverse range of biological processes through a single receptor. How this is achieved is unclear, but it is thought at least in part to be due to the tissue specific expression of GR isoforms. The constitutive splice variant, GRγ is conserved through mammalian evolution, suggesting a gain of function, but currently no clear biological role has been identified. GRγ differs from the most abundant GR isoform, GRα by a single arginine inserted in the DNA binding domain (DBD) of GRγ, and may therefore affect the transcriptome profile and protein interactions of the receptor. Indeed, marked differences between the GRα and GRγ interactomes were revealed by proteomic analysis, identifying a potential association of GRγ with the mitochondria. These differences in the protein interactomes were accompanied by altered intracellular distributions. A clear tangible result of these differences is an observed delay in both the translocation kinetics and transactivation potential of GRγ. Analysis of the GRγ regulated transcriptome revealed a clear distinction in the regulation of a subset of target genes. Gene ontology and gene enrichment analysis identified oxidative phosphorylation protein degradation and cell morphology as potential GRγ specific functions. These findings suggest a distinct biological role is conferred by the additional arginine in the level arm of the DBD.Study 2 Mathematical modelling of GR function: Cell migration is a fundamental biological process. Clinically, Gcs inhibit wound healing, yet the mechanisms by which Gcs regulate migration remain unclear. The impact of Gcs on cellular motility was initially characterised through traditional migration assays. However, as cell populations are heterogeneous, live cell microscopy and mathematical modelling were employed to monitor the response of single cells. Dynamic tracking of migration in individual cells revealed that the movement of A549 cells is modelled by an alpha stable distribution. Gcs changes the parameters of the distribution, without altering the nature of the walk statistics. Gcs reduce the overall displacement of a cell, by causing a significant shift in step length selection, resulting in a reduction of the large steps, and replacement with short steps. Changes in migration following treatment with Gcs were seen within hours, which is much faster than previously reported. To identify a potential mechanism a panel of actin cytoskeleton regulators were screened, but prolonged exposure of Gcs were required to see a response, implying that these markers are modified secondary to the shift in migration. As a more dynamic readout, I tracked microtubule reorganisation and found stabilisation of the microtubule network rapidly following Gc exposure. I identified acetylation of α-tubulin (within 10 minutes) as the earliest change following Gc treatment, which implicated the inactivation of HDAC6 as a candidate mechanism. Indeed, overexpression of HDAC6 was able to restore cell motility characteristics to the Gc-free state. I have used a combination of systems level, chemical biology and mathematical approaches to better understand how Gc work in vivo. Research of this kind will aid new drug development for more specific targeting of Gc action.

Venous thromboembolism (VTE) which includes pulmonary embolism (PE), or deep venous thrombosis (DVT) poses an important disease burden, however, much remains unknown about the risk factors that cause it. In recent years, more attention has focused on medications that play a role in the development of drug-induced venous thrombosis. The three studies in this dissertation explore the risk of VTE in relation to the use of specific pharmacologic agents; glucocorticoids, 5ARIs and statins in three distinct patient populations with; asthma, benign prostatic hyperplasia (BPH) and hypercholesterolemia, using different approaches to address the inevitable confounding present in etiologic pharmacoepidemiologic research. Using a nested case-control design for the first two studies and cohort design for the last, we assessed the risk of VTE in relation to timing of drug exposure, duration of use, number, and dose of prescriptions. The base populations comprised subjects who received at least one prescription for any of the pharmacologic agents of interest (during 1995–2015 for study 1 and 2 and 1995–2018 for study 3) in the UK-based Clinical Practice Research Datalink. We used descriptive analyses as well as conditional logistic regression and Poisson regression models to evaluate the relationship between these drugs and the risk of VTE. Study 1 examined the relationship between glucocorticoid use and venous thromboembolism among asthma patients age 20–59. We found that current and systemic glucocorticoid use was associated with an increased risk of VTE, with a dose-response relationship. Study 2 explored the relationship between VTE and 5ARI use compared to alpha blocker use among patients, age 40–79, with BPH. We observed that cumulatively high doses of 5ARI+/-AB increases the risk of incident VTE. In study 3, we examined the relationship between VTE and statin use compared to fibrate use among 40–79-year-old patients with hypercholesterolemia. We observed that statin use decreases the risk of incident VTE. These three studies in a large population-based database of high quality, efficiently evaluate the safety/unintended benefit of glucocorticoids, 5ARIS and statins, with the hope to guide the development of guidelines for their use in prolonged care of asthma, BPH and hypercholesterolemia, respectively. / 2023-08-25T00:00:00Z

Cyclosporine is a commonly used immunosuppressive drug in dogs, but dosing is often empirical and based primarily on clinical response. Pharmacokinetic monitoring of blood drug concentrations can be performed, but target blood concentrations for various disease states in dogs are not well described. Pharmacodynamic assays measuring the effects of cyclosporine on target cells are being used to evaluate immunosuppressive effectiveness in humans, but have been minimally explored in veterinary medicine. This dissertation describes the development of pharmacodynamic assays for measuring the effects of cyclosporine on canine T cell cytokine production and surface antigen expression. Incubation with cyclosporine in vitro caused significant suppression of activated T cell production of interleukin-2 (IL-2), IL-4, interferon-gamma (IFN-gamma), CD25, and CD95 measured in peripheral blood mononuclear cells using flow cytometry. IL-2 and IFN-gamma were then evaluated using flow cytometry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) in whole blood incubated with cyclosporine and dexamethasone in vitro. Cyclosporine caused concentration-dependent inhibition of both cytokines, and a greater degree of suppression was noted with qRT-PCR than flow cytometry. Dexamethasone caused concentration-dependent inhibition of IFN-gamma with both methods, but IL-2 reduction was only significant for qRT-PCR. Both methods were then used to evaluate IL-2 and IFN-gamma after administration of high dose oral cyclosporine to dogs. Both qRT-PCR and flow cytometry identified marked cytokine suppression after cyclosporine dosing, but qRT-PCR was uniformly suppressed across the 12-hour dosing interval, while flow cytometry results were significantly higher at trough blood drug concentrations than at peak blood concentrations and subsequent post-dosing time points. Both flow cytometry and qRT-PCR are valid methods for evaluation of T cell cytokine expression in dogs. Further study at lower drug doses is needed to correlate pharmacodynamic results with pharmacokinetic drug concentrations, and to confirm the best method for cytokine monitoring. Studies in clinic patients are also needed to determine the level of cytokine suppression associated with clinical effectiveness in different disease states. Pharmacodynamic evaluation of cyclosporine’s effects shows promise, and may allow for more individualized dosing of cyclosporine in dogs.