From an evolutionary perspective, stress is an adaptive system that is necessary togenerate appropriate responses to stochastic and unpredictable events, and copeaccordingly with the environment.... Show moreFrom an evolutionary perspective, stress is an adaptive system that is necessary togenerate appropriate responses to stochastic and unpredictable events, and copeaccordingly with the environment. The physiological response to stress has beenremarkably conserved in vertebrate evolution. However, the threats to ourinternal “equilibrium” have changed between our ancestral environments and ourcurrent modern societies, and the demands for survival have evolved. Theglucocorticoid receptor (GR) is a timeless component of stress adaptation, as it is atthe intersection between the environmental stressors (i.e., physical, or psychosocial)and the genome. Therefore, the GR represents a valuable therapeutic target instress- and glucocorticoid-related disorders. This thesis provides new insightsinto the molecular mechanisms underlying GR signaling in metabolic diseases andbrain function and highlights the promise and importance of selectivity in novel GRtargeting treatments. Show less
The research described in this thesis has, using the zebrafish as a model system, shed new light on the intricate relationship between TB and DM2, in particular on the role of leptin, SHP-1 and... Show moreThe research described in this thesis has, using the zebrafish as a model system, shed new light on the intricate relationship between TB and DM2, in particular on the role of leptin, SHP-1 and glucocorticoids.Leptin plays an important role during TB infection and has a huge impact on insulin sensitivity in zebrafish larvae. Similarly to what has been observed in the murine model, leptin deficiency in zebrafish increased the bacterial burden and mortality during the infection, leading to hyperglycemia and the development of insulin resistance. In addition, a novel SHP-1/SHP-2 inhibitor, NSC-87877, was shown to represent a promising anti-diabetic drug that can be used for further DM2 research, as it is able to rescue the phenotype of the leptin-deficient zebrafish and to restore glucose transport to the tissues. In contrast to metformin, NSC-87877 can act at very early developmental stages and inhibits the function of SHP-1 and factors that underlay impaired glucose metabolism, whereas metformin is mostly known to improve insulin sensitivity. Additionally, treatment with the glucocorticoid beclomethasone attenuates the metabolic changes associated with the infection, and transcriptional alterations induced by beclomethasone treatment suggest that genes involved in glucose metabolism, insulin and leptin signaling all play an important role in the modulation of the metabolism.Our data show that zebrafish larvae represent an interesting model system to investigate the complex pathology of TB, and the studies described in this thesis in which this model has been used have provided novel insights into the molecular mechanisms underlying wasting syndrome and the possibilities for adjunctive glucocorticoid therapy to alleviate this metabolic state. Show less
Stress, obesity and (stress-related) mood disorders such as depression and anxiety disorders are highly prevalent in modern society. These diseases often coincide. This may be because they trigger... Show moreStress, obesity and (stress-related) mood disorders such as depression and anxiety disorders are highly prevalent in modern society. These diseases often coincide. This may be because they trigger and reinforce each other, creating a vicious cycle. Social and psychological factors play a role in this cycle, but in this thesis we focused on the underlying biological mechanisms. We identified new obesity-related factors that likely affect fear and discovered new sites of action in the brain for factors that are already known to regulate fear. We also applied three novel drug treatment strategies. Each of these target (different) components of the stress system and were shown to alleviate stress- or diet-induced metabolic disease in mice. Certain strategies reduced obesity, while others seemed to prevent or even cure fatty liver disease in mice. This thesis thus represents a step towards breaking the vicious cycle between stress, obesity and (stress-related) mood disorders. Show less
Glucocorticoids (GCs) are widely prescribed as anti-inflammatory drugs due to their well-established immunosuppressive effects. However, their utilization is severely limited by the occurrence of... Show moreGlucocorticoids (GCs) are widely prescribed as anti-inflammatory drugs due to their well-established immunosuppressive effects. However, their utilization is severely limited by the occurrence of side effects and drug resistance. Therefore, there is still a major need to investigate the molecular and cellular mechanisms underlying the effects of GCs. Zebrafish are increasingly used as an in vivo model system for studying the immune system, in particular the inflammatory response. In Chapter 2, an overview is provided of the available inflammation models in zebrafish, and how they are used to unravel molecular mechanisms underlying the inflammatory response and for testing of potential novel anti-inflammatory drugs, in particular GCs. In this thesis, we have used zebrafish model system to study molecular and cellular mechanisms of GC action on the immune system and to develop a model for in vivo screening of the anti-inflammatory effects as well as possible adverse effects of novel GC therapies. For this purpose, we have studied the effect of GCs on leukocyte migration and differentiation during an inflammatory response (Chapter 3), how GCs modulate the immune response to a mycobacterial infection (Chapter 4), and we have investigated targeting of GCs to inflamed tissue by liposomal delivery (Chapter 5). Show less
The aim of this thesis is to identify emerging risk factors for VTE. To achieve this goal, we describe the supposedly causal role of statin and glucocorticoid use (i.e. two drugs that can influence... Show moreThe aim of this thesis is to identify emerging risk factors for VTE. To achieve this goal, we describe the supposedly causal role of statin and glucocorticoid use (i.e. two drugs that can influence inflammation) with changes in hemostasis and VTE risk. Systemic glucocorticoid use increases the relative risk of first VTE by more than three-fold and confers an 5% absolute risk of recurrent VTE per year. On the other hand, rosuvastatin use may reduce the risk of first VTE by 40%. Although the mechanisms behind this association are not fully elucidated, this thesis shows that rosuvastatin is capable of decreasing the thrombin generation potential by 10% in patients with a prior VTE. This thesis has shown that both statins and systemic glucocorticoids can affect the risk of VTE, improving the knowledge on the influence of these two commonly prescribed drugs on VTE pathophysiology. These findings have the potential to further refine the assessment of VTE risk since they highlight that the use of these drugs should be considered when evaluating the risk of VTE. Finally, this thesis provides insight into new therapeutic approaches since the results underscore that treatment strategies on VTE prevention in patients already taken statins, which may be sufficient for VTE prevention, are lacking. Treatment strategies to prevent glucocorticoid-associated VTE are also needed. Show less
Throughout evolution, humans have lived in synchrony with the natural light-dark cycle. Our bodies were used to going to sleep a few hours after dark, and waking up just before dawn. However, in... Show moreThroughout evolution, humans have lived in synchrony with the natural light-dark cycle. Our bodies were used to going to sleep a few hours after dark, and waking up just before dawn. However, in modern society the unambiguous availability of artificial light has desynchronized our biological clock from the naturally occurring day and night, with large consequences for metabolic health. This thesis sheds light on the negative health consequences of a disturbed biological clock, and elucidates novel approaches to prevent disease associated with chronic rhythm disruption, as occurs in shift work. We have identified important mechanisms through which rhythm disruption contributes to (cardio)metabolic disease, namely by exacerbating vascular inflammation and by deregulating rhythm in glucocorticoid hormone, thereby affecting the metabolic activity of tissues such as brown fat and bone. We continued by investigating two main approaches to prevent diseases associated with circadian disturbances: (1) by limiting disruption of the circadian timing system, and (2) by directly targeting the affected tissues. We found that timed feeding (1) and stimulation of the metabolic activity of brown fat (2) are both promising strategies to prevent and/or reduce (cardio)metabolic disease risk in the ever-increasing population of individuals who suffer from circadian disturbances. Show less
The first part describes a non-genomic glucocorticoid action that inhibits T cell activation which was used to develop GC receptor ligands with safer profiles. The 2nd part focuses on repurposing... Show moreThe first part describes a non-genomic glucocorticoid action that inhibits T cell activation which was used to develop GC receptor ligands with safer profiles. The 2nd part focuses on repurposing of miltefosine as an anti-inflammatory drug in IBD. Show less
Pharmacotherapy plays an essential role in an allogeneic hematopoietic stem cell transplantation (allo-HSCT) procedure. Busulfan and treosulfan are two alkylating agents often applied in the... Show morePharmacotherapy plays an essential role in an allogeneic hematopoietic stem cell transplantation (allo-HSCT) procedure. Busulfan and treosulfan are two alkylating agents often applied in the conditioning regimen administered prior to the allo-HSCT. Finding the right balance between efficacious conditioning and toxicity remains the challenge for both agents. A majore One of the major complications after allo-HSCT is Acute graft-versus-host disease (aGvHD). When patients are diagnosed with grade 2 or higher aGvHD, systemic treatment with high-dose glucocorticoids is started as first line treatment. However, only half of the patients respond adequately to this therapy and also in other diseases non-responsiveness to glucocorticoids has been observed. Optimization of current drug therapies in allo-HSCT holds the potential to improve the outcome and safety. The goal of this thesis is to optimize busulfan- and treosulfan-based conditioning regimens and acute GvHD treatment with glucocorticoids by applying pharmacokinetic and pharmacogenetic profiling. Studies are performed in adults and pediatric patients. Show less
Today__s Western society and work promotes a sedentary lifestyle. This, coupled with high caloric food availability has increased obesity followed by an increased prevalence of the metabolic... Show moreToday__s Western society and work promotes a sedentary lifestyle. This, coupled with high caloric food availability has increased obesity followed by an increased prevalence of the metabolic syndrome (MetS), type 2 diabetes (T2D) and cardiovascular diseases (CVD). Epidemiological data show a clear association between increased cortisol plasma levels, CVD and obesity, but animal models of the MetS are inconclusive with respect to whether the hypothalamus-pituitary-adrenal (HPA) axis is activated or not. It has remained unknown if, and to what extent HPA axis activity plays a pathogenic role in MetS development, and if the development of MetS changes it. In this thesis, we investigate high fat diet (HFD)-effect on the basal HPA axis activity and the effect of chronically elevated, exogenuously administered, corticosterone on MetS features as well as atherosclerosis development in spesific mouse models. From these studies we concluded that HFD reduces HPA axis activity where supraphysiological corticosterone levels worsen the features of MetS and the metabolic adaptations to a period of corticosterone excess in mice are long-lasting and may not completely recover to restore homeostasis. Futher more corticosteroid excess has both beneficial and harmful effects on cardiovascular risk factors that can be explained by their immunosuppressive and metabolic properties. Show less
Synthetic glucocorticoids such as dexamethasone are frequently used to enhance pulmonary development in preterm ventilator-dependent infants. In contrast to the short-term benefit on survival and... Show moreSynthetic glucocorticoids such as dexamethasone are frequently used to enhance pulmonary development in preterm ventilator-dependent infants. In contrast to the short-term benefit on survival and lung maturation, early glucocorticoid exposure has been shown to adversely affect neurodevelopmental processes. Both human and animal studies have reported acute and long-lasting impairments, including shortening of the lifespan in rodents. Therefore, the objective of the studies described in this thesis was to investigate, using an animal model: 1) the short- and long-term consequences of neonatal dexamethasone treatment and 2) the possibility to prevent these effects using pharmacological and behavioural intervention strategies. We reported that systemic dexamethasone treatment acutely affects brain development by suppressing cell proliferation and glial activity. These acute effects on the brain can be partially prevented by central glucocorticoid receptor antagonist pre-treatment, which might serve as a protective strategy against the adverse effects of dexamethasone treatment on the developing brain. Although neonatal dexamethasone exposure clearly affects the developmental trajectory, we did not observe the frequently described detrimental long-lasting consequences of this treatment. We showed that daily handling of the neonate, which was an inevitable component of our experimental design and leads to enhanced levels of maternal care towards the offspring, may compensate for some of the adverse effects of dexamethasone treatment. We conclude that the impact of neonatal glucocorticoid exposure highly depends on interactions with other components of the early environment and is therefore susceptible to pharmacological and behavioural intervention strategies. Show less
Glucocorticoid receptor (GR) signaling is versatile and plays an essential role in the survival and well-being of organisms. Glucocorticoids are also widely used clinically in order to combat... Show moreGlucocorticoid receptor (GR) signaling is versatile and plays an essential role in the survival and well-being of organisms. Glucocorticoids are also widely used clinically in order to combat inflammatory medical conditions. In the present thesis we aimed at studying the GR pathway by means of stimulation with synthetic GCs and genetic manipulation. Since the GR is a transcription factor, our main readout for GR function in most of our experimental settings was transcriptome analysis. By gathering whole transcriptome information, we aimed at unraveling the molecular pathways affected by GR signaling in different physiological conditions, thus exploring its functional role. As a model organism we employed the zebrafish, since it allows fine genetic, molecular and cellular experimental approaches and its GR pathway closely resembles that of humans. Our aim was also to further characterize the function of this versatile signaling cascade in zebrafish, in order to establish this animal model as a valid system for detailed as well as high throughput research on GR, enabling us to test hypotheses and complement results obtained from other well-established experimental animal models such as rodents. Show less
Schizophrenia is a devastating mental disorder characterized by a hyperactive dopamine system and deregulated stress system. Human studies have suggested that the schizophrenia symptoms precipitate... Show moreSchizophrenia is a devastating mental disorder characterized by a hyperactive dopamine system and deregulated stress system. Human studies have suggested that the schizophrenia symptoms precipitate if a hyperactive dopaminergic genotype interacts with adverse life experiences that activate the stress system. To examine this gene-by-environment interaction, we exposed rats genetically-selected for enhanced apomorphine susceptibility to two stress-provoking life events, poor maternal care early-in-life, and isolation rearing later-in-life. This promoted the development of schizophrenia endophenotypes. Our experiments involved two complementary steps: First, we focused on the immediate endocrine adaptations to maternal separation in common rats. It is known that a single episode of prolonged maternal separation slowly increases corticosterone levels in the neonate rat. We discovered that if the pups had been previously exposed to maternal separation, this rise in corticosterone was abolished, suggesting that the pups had learned to predict the return of the dam. While readily adapting to repeated maternal absence, the pups, surprisingly, stayed alert and displayed a rapid response to an acute stressor. We then investigated whether pup__s stress responsiveness was influenced by the context of maternal separation. It appeared that the experience of being kept in isolation in a novel environment during repeated maternal separation, rather than the maternal absence per se, caused priming of the amygdala fear pathway, with lasting consequences for the responsiveness of the neuroendocrine and behavioral stress system. These endocrine and behavioral alterations, caused by early-life stress experience, consisted of schizophrenia-like phenotypes. Second, we sought to investigate the interplay of such early-life stress experience with schizophrenia genetic predisposition and/or later-life social stress experience. Thus, we were able to test the three-hit (cumulative stress) and the developmental mismatch hypotheses. The former states that exposure to earlylife adversity and later-life psychosocial stressors, superimposed on genetic susceptibility, result in a severe schizophrenia-like phenotype. The latter proposes that experiences early-in-life program the developing brain in preparation for the future. In the case of genetically-predisposed apomorphine susceptible rats (schizophrenia-susceptible), we provide strong evidence for the three-hit hypothesis. In the case of the nongenetically selected Wistar rats, the mismatch hypothesis is supported since the outcome of early-life stress often negatively interacted with the pre-puberty social context. In agreement with the three-hit hypothesis of schizophrenia, we conclude from the current experiments that early-life stress experience in interaction with highly reactive dopaminergic alleles, leads to amygdala priming that, together with additional stressors, precipitate schizophrenia. Show less
Pronounced ultradian and circadian rhythms in the hormones of the hypothalamic-pituitary-adrenal (HPA) axis (i.e. glucocorticoids), one of the body__s major neuroendocrine axes, were already... Show morePronounced ultradian and circadian rhythms in the hormones of the hypothalamic-pituitary-adrenal (HPA) axis (i.e. glucocorticoids), one of the body__s major neuroendocrine axes, were already demonstrated several decades ago. Until now, the clinical relevance of the pulsatile nature of glucocorticoids was poorly understood or sometimes even regarded as not important. Its evolutionary conservation across many species however implies biological significance. Indeed, glucocorticoids have been proven to be crucial for a plethora of bodily functions, e.g. emotion, cognition and the central mechanism underlying the adaptation to stress. Furthermore, disturbances in the characteristic temporal pattern of glucocorticoid exposure have often been described in stress-related pathology. However, the significance of glucocorticoids secretory patterns for physiology, stress responsiveness and nuclear receptor signalling is still largely unexplored and is accordingly addressed in this thesis. A new concept in the endocrinology of glucocorticoids has evolved from the data presented here showing that pulsatile release of glucocorticoids is a major determinant in __resilience__ of glucocorticoid signalling in neuronal cells and stress responsiveness. Moreover, we show that particularly the glucocorticoid receptor is affected after disrupting glucocorticoid pulsatility and could thus provide an excellent target for therapy to normalise the downstream effects of disturbances in glucocorticoid rhythms in stress-related disease. Show less
Currently, the raising awareness of the role of glucocorticoids in the onset of numerous (neuro)-pathologies constitutes the increasing necessity of understanding the mechanisms of action of... Show moreCurrently, the raising awareness of the role of glucocorticoids in the onset of numerous (neuro)-pathologies constitutes the increasing necessity of understanding the mechanisms of action of glucocorticoids in bodily processes and brain functioning. Glucocorticoids mediate their effects by binding to intracellular receptors which act as transcription factors. A remarkable and yet unexplained phenomenon described more than two decades ago, is the cell-specific effects glucocorticoids bring about on gene expression in brain. For example, while glucocorticoids suppress corticotrophin-releasing hormone (CRH) synthesis in the hypothalamus, production of CRH in the central nucleus of the amygdala (CeA) is stimulated by increased hormone levels. Inasmuch as the neuroanatomical distribution of the corticosteroid receptors does not satisfactorily explain these effects, it is of interest to decipher the role of recently discovered coregu lator proteins that modulate the direction and the magnitude of steroid receptor-driven transcription. Therefore, in the current thesis the expression and function of central coregulators was studied: the coactivators SRC1a and SRC1e along with the corepressors N-CoR and SMRT were found to be expressed in brain and involved in regulation of CRH gene expression. Finally, a method that allows detection of coregulator recruitment by steroid receptors in brain tissue was developed. Show less
Peripheral and autonomous neuropathies are well-known and devastating complications of type 1 diabetes (T1D). However, T1D can also impact the integrity of the central nervous system (CNS), and the... Show morePeripheral and autonomous neuropathies are well-known and devastating complications of type 1 diabetes (T1D). However, T1D can also impact the integrity of the central nervous system (CNS), and the reason why T1D affects CNS integrity remains to be elucidated. Studies on diabetic patients demonstrated mild to moderate slowing of mental speed and diminished mental flexibility. Although the alterations in cognitive functions under normal conditions are not severe, mild cognitive defects can influence everyday activities in more demanding situations. Indeed, in 2004 Sandeep et al reported that hypercortisolism in diabetic patients may contribute to their hippocampal dysfunction. To investigate disease initiation, progression, and treatments without exposing humans to unnecessary and potentially unethical risks, animal models have been developed. The physiology of mice, rats, and other animals is remarkably conserved in comparison to the human condition, and over the last 40 years several animal models have become available. We have used two animal models, i.e. a pharmacological model, the streptozotocin (STZ) treated mouse, and a genetic model the NOD mouse, which spontaneously develops type 1 diabetes. As type 1 diabetic patients, these animal models show high circulating glucocorticoid levels, increased sensitivity to stress, and morphological alteration in various brain areas. In the present study these models were used to test the hypothesis that the onset of diabetes induces first dysregulation of the hypothalamus-pituitary-adrenal (HPA) axis and subsequently hypersecretion of glucocorticoids which then renders the brain more vulnerable to metabolic insults causing damage and concomitant cognitive disturbances. The NOD model revealed a surge in ACTH release, which likely preceded the onset and progression of diabetes marked by adrenal hyperreponsiveness and hypersecretion of corticosterone. To our surprise we found in the STZ model that not the initial ACTH surge was the most proximal cause of hypercorticism in diabetes, but rather the induction of adrenocortical ACTH receptors per se. At no time-point after STZ administration ACTH levels did rise reinforcing the notion that hyperresponsiveness of the adrenals to ACTH may occur independent of the mitogenic activity of the peptide. In the same model, excess glucocorticoids rather than glycemia and insulin appeared causal to cerebral damage and mild cognitive impairment. These deficits in hippocampal function induced by high glucocorticoid concentrations were readily ameliorated by a brief treatment with the glucocorticoid receptor antagonist mifepristone. These findings make the GRs a suitable target for new therapeutic strategies aimed to normalize the disturbed hippocampal functions characteristic for diabetes neuropathology. Show less
Not everyone who experiments with cocaine acquires compulsive drug use. The mechanism underlying this individual difference in susceptibility to addiction is poorly understood. Recent studies have... Show moreNot everyone who experiments with cocaine acquires compulsive drug use. The mechanism underlying this individual difference in susceptibility to addiction is poorly understood. Recent studies have identified genes and adverse life events (stress) as risk factors. The objective of this thesis is to investigate the contribution of the adrenal stress hormones glucocorticoids and epinephrine to the psychostimulant effects of cocaine in the inbred DBA/2 and C57BL/6 mouse strains. Behavioural sensitisation, measured as an enhanced locomotor response to repeated cocaine exposure, was used as a model for the long-term neural adaptations underlying aspects of drug addiction. The results demonstrate that adrenal hormones play a critical role in cocaine sensitivity, which depends on genetic background because surgical removal of the adrenals (__adrenalectomy__) prevented cocaine sensitisation in DBA/2, but not C57BL/6 mice. The impact of genetic background was further emphasised by strain-specific changes in the midbrain dopamine system that mediates the rewarding effects of drugs. The effects of adrenalectomy could only be fully reversed by co-administration of glucocorticoids and epinephrine. These findings show that, depending on genetic background, adrenal stress hormones are important risk factors for vulnerability to cocaine, suggesting that pharmacological intervention in stress hormone action has therapeutic potential in drug addiction. Show less
Glucocorticoid hormones exert modulatory effects on neural function in a delayed genomic fashion. The two receptor types that can bind glucocorticoids, the mineralocorticoid receptor (MR) and the... Show moreGlucocorticoid hormones exert modulatory effects on neural function in a delayed genomic fashion. The two receptor types that can bind glucocorticoids, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), are ligand-inducible transcription factors. Therefore, changes in gene expression most likely underlie glucocorticoid-mediated genomic effects on neural function. In this thesis, the central aim was to gain more insight into the transcriptional changes that mediate the effects of acutely activated GRs on neural function. Two different biological substrates i.e. ex vivo hippocampal slices and neuronal catecholaminergic PC12 cells, were used to measure the transcriptional response after GR-activation. Using microarrays, an interesting time-dependent pattern of gene transcription was observed, shifting from exclusively downregulated genes 1 hour after GR-activation to both up and downregulated genes 3 hours afterwards. This pattern suggests that the fast genomic effects of glucocorticoids may be realized via transrepression, preceding a later wave of transactivation. Additionally, many new candidate genes were found that could potentially underlie (part of) the effects glucocorticoids mediate on hippocampal and catecholaminergic neuronal function. Hence, these candidate genes can be used to formulate new hypotheses on how glucocorticoids affect neural function and future research should therefore focus on testing these hypotheses. Show less
The ultimate goal in the transplantation field is the induction and maintenance of donor specific tolerance. Treg cells that control immune responses to alloantigens give opportunities for... Show moreThe ultimate goal in the transplantation field is the induction and maintenance of donor specific tolerance. Treg cells that control immune responses to alloantigens give opportunities for tolerogenic therapies in transplantation. However, it is important to investigate the mechanisms of tolerance induction in order to use the optimal strategy. Therefore, we exploded both natural towards NIMA that can be induced during fetal life and induced tolerance by modulation of DC. Naturrally induced tolerance towards NIMA can have an influence on transplant outcome later in life. In this thesis we explored the influence of NIMA on the alloreactive T cells repetoire in healthy individuals and additionally we focused on the NIMA effect in patients transplanted with a NIMA haplotype mismatched kidney graft. In order to actively induce tolerance, we modulated DC to generate Treg cells, since this may be of clinical relevance in the future for patients that are on the waiting list for transplantation. In this thesis we explored the possibility of using modulated DC for the induction of transplantation tolerance in a fully allogeneic setting in mice. Furthermore we describe an in vitro system for the use of human modulated DC to induce Treg cells. We show that two differentially modulated human DC can lead to different types of Treg cells. Finally, we examined the possibility to use in vitro tools to measure a possible tolerant state in patients. Monitoring of e.g. Treg cells and/or cytokines may give an indication which patients are at risk for rejection and which patients are more predisposed to tolerance. We describe the Elispot technique as a possible tool to monitor patients that received a renal allograft. In conclusion , this thesis contributes to the fundamental understanding of both natural and induced tolerance in transplantation and gives a handhold for future research. As donor-specific tolerance is still far away from the clinic, the in vitro monitoring tool described in this thesis may contribute to the optimalization of immunosuppressive therapies in transplant recipients. Show less
