Hormonal effects on glucose and ketone metabolism in a perfused liver of an elasmobranch, the North Pacific spiny dogfish, Squalus suckleyi.

Hormonal influence on hepatic function is a critical aspect of whole-body energy balance in vertebrates. Catecholamines and corticosteroids both influence hepatic energy balance via metabolite mobilization through glycogenolysis and gluconeogenesis. Elasmobranchs have a metabolic organization that appears to prioritize the mobilization of hepatic lipid as ketone bodies (e.g. 3-hydroxybutyrate [3-HB]), which adds complexity in determining the hormonal impact on hepatic energy balance in this taxon. Here, a liver perfusion was used to investigate catecholamine (epinephrine [E]) and corticosteroid (corticosterone [B] and 11-deoxycorticosterone [DOC]) effects on the regulation of hepatic glucose and 3-HB balance in the North Pacific Spiny dogfish, Squalus suckleyi. Further, hepatic enzyme activity involved in ketogenesis (3-hydroxybutyrate dehydrogenase), glycogenolysis (glycogen phosphorylase), and gluconeogenesis (phosphoenolpyruvate carboxykinase) were assessed in perfused liver tissue following hormonal application to discern effects on hepatic energy flux. mRNA transcript abundance key transporters of glucose (glut1 and glut4) and ketones (mct1 and mct2) and glucocorticoid function (gr, pepck, fkbp5, and 11ßhsd2) were also measured to investigate putative cellular components involved in hepatic responses. There were no changes in the arterial-venous difference of either metabolite in all hormone perfusions. However, perfusion with DOC increased gr transcript abundance and decreased flow rate of perfusions, suggesting a regulatory role for this corticosteroid. Phosphoenolpyruvate carboxykinase activity increased following all hormone treatments, which may suggest gluconeogenic function; E also increased 3-hydroxybutyrate dehydrogenase activity, suggesting a function in ketogenesis, and decreased pepck and fkbp5 transcript abundance, potentially showing some metabolic regulation. Overall, we demonstrate hormonal control of hepatic energy balance using liver perfusions at various levels of biological organization in an elasmobranch.

Preparation of Budesonide-Loaded Liposomal Nanoparticles for Pulmonary Delivery and Their Therapeutic Effect in OVA-Induced Asthma in Mice.

Purpose: Inhaled corticosteroids, including budesonide (BUD), are widely employed for the treatment of asthma. However, the frequent use of corticosteroids is associated with numerous adverse effects and poses challenges to ongoing drug therapy and patient adherence. Budesonide liposomal nanoparticles (BUD-LNPs) were developed to improve the bioavailability of the drug and thereby improve the effectiveness of asthma treatment. Methods: BUD-LNPs were prepared via thin-film hydration, and the characterizations, stability, and in vitro release of BUD-LNPs were studied. In vitro cellular uptake was observed by laser-scanning confocal microscope (LSCM) and flow cytometry. And the in vitro anti-inflammatory activity of BUD-LNPs was evaluated by measuring the expression of pro-inflammatory cytokines in activated macrophages. Besides, the accumulation time in the lung of drugs delivered via liposomal carriers and free drugs was compared in vivo. And the in vivo therapeutic efficacy of BUD-LNPs was assessed in OVA-induced asthmatic mice. Finally, in vivo biosafety assessment was performed. Results: The particle size, PDI, and zeta potential of BUD-LNPs were 127.63±1.33 nm, 0.27±0.02, and 3.33±0.13 mV, respectively. BUD-LNPs exhibited excellent biosafety and anti-inflammatory activity in vitro. Furthermore, compared with the free drugs, the utilization of liposomal nano-vehicles for drugs delivery could effectively extend the duration of drugs accumulation in the pulmonary system. Additionally, treatment with BUD-LNPs alleviated airway hyperresponsiveness, reduced airway mucus secretion, and mitigated pulmonary inflammation in OVA-induced asthmatic mice. And the BUD-LNPs demonstrated superior therapeutic efficacy compared to free BUD. Conclusion: BUD-LNPs was successfully prepared with excellent stability and sustained release for 24 h in vitro. The data of anti-inflammatory activity, asthma therapeutic effects and safety studies indicated that drug delivery mediated by liposomal nano-vehicles was a feasible and desirable strategy for medical strategy and showed great promise in the clinical therapy of asthma.

A phase 2 open-label study of the safety and efficacy of weekly dosing of ATL1102 in patients with non-ambulatory Duchenne muscular dystrophy and pharmacology in mdx mice.

BACKGROUND: ATL1102 is a 2'MOE gapmer antisense oligonucleotide to the CD49d alpha subunit of VLA-4, inhibiting expression of CD49d on lymphocytes, reducing survival, activation and migration to sites of inflammation. Children with DMD have dystrophin deficient muscles susceptible to contraction induced injury, which triggers the immune system, exacerbating muscle damage. CD49d is a biomarker of disease severity in DMD, with increased numbers of high CD49d expressing T cells correlating with more severe and progressive weakess, despite corticosteroid treatment. METHODS: This Phase 2 open label study assessed the safety, efficacy and pharmacokinetic profile of ATL1102 administered as 25 mg weekly by subcutaneous injection for 24 weeks in 9 non-ambulatory boys with DMD aged 10-18 years. The main objective was to assess safety and tolerability of ATL1102. Secondary objectives included the effect of ATL1102 on lymphocyte numbers in the blood, functional changes in upper limb function as assessed by Performance of Upper Limb test (PUL 2.0) and upper limb strength using MyoGrip and MyoPinch compared to baseline. RESULTS: Eight out of nine participants were on a stable dose of corticosteroids. ATL1102 was generally safe and well tolerated. No serious adverse events were reported. There were no participant withdrawals from the study. The most commonly reported adverse events were injection site erythema and skin discoloration. There was no statistically significant change in lymphocyte count from baseline to week 8, 12 or 24 of dosing however, the CD3+CD49d+ T lymphocytes were statistically significantly higher at week 28 compared to week 24, four weeks past the last dose (mean change 0.40x109/L 95%CI 0.05, 0.74; p = 0.030). Functional muscle strength, as measured by the PUL2.0, EK2 and Myoset grip and pinch measures, and MRI fat fraction of the forearm muscles were stable throughout the trial period. CONCLUSION: ATL1102, a novel antisense drug being developed for the treatment of inflammation that exacerbates muscle fibre damage in DMD, appears to be safe and well tolerated in non-ambulant boys with DMD. The apparent stabilisation observed on multiple muscle disease progression parameters assessed over the study duration support the continued development of ATL1102 for the treatment of DMD. TRIAL REGISTRATION: Clinical Trial Registration. Australian New Zealand Clinical Trials Registry Number: ACTRN12618000970246.

Mitochondrial morphology in the mouse adrenal cortex: Influence of chronic psychosocial stress.

Mitochondria within the adrenal cortex play a key role in synthesizing steroid hormones. The adrenal cortex is organized in three functionally specialized zones (glomerulosa, fasciculata, and reticularis) that produce different classes of steroid hormones in response to various stimuli, including psychosocial stress. Given that the functions and morphology of mitochondria are dynamically related and respond to stress, we applied transmission electron microscopy (TEM) to examine potential differences in mitochondrial morphology under basal and chronic psychosocial stress conditions. We used the chronic subordinate colony housing (CSC) paradigm, a murine model of chronic psychosocial stress. Our findings quantitatively define how mitochondrial morphology differs among each of the three adrenal cortex zones under basal conditions, and show that chronic psychosocial stress mainly affected mitochondria in the zona glomerulosa, shifting their morphology towards the more typical glucocorticoid-producing zona fasciculata mitochondrial phenotype. Analysis of adrenocortical lipid droplets that provide cholesterol for steroidogenesis showed that chronic psychosocial stress altered lipid droplet diameter, without affecting droplet number or inter-organellar mitochondria-lipid droplet interactions. Together, our findings support the hypothesis that each adrenal cortex layer is characterized by morphologically distinct mitochondria and that this adrenal zone-specific mitochondrial morphology is sensitive to environmental stimuli, including chronic psychosocial stressors. Further research is needed to define the role of these stress-induced changes in mitochondrial morphology, particularly in the zona glomerulosa, on stress resilience and related behaviors.

Specificity protein 1 (Sp1) and glucocorticoid receptor (GR) stimulate bovine alphaherpesvirus 1 (BoHV-1) replication and cooperatively transactivate the immediate early transcription unit 1 promoter.

Bovine alphaherpesvirus 1 (BoHV-1) infections cause respiratory tract disorders and suppress immune responses, which can culminate in bacterial pneumonia. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons present in trigeminal ganglia (TG) and unknown cells in pharyngeal tonsil. Latently infected calves consistently reactivate from latency after an intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics the effects of stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two key viral transcriptional regulators. The IEtu1 promoter contains two functional glucocorticoid receptor (GR) response elements (GREs), and this promoter is transactivated by GR, DEX, and certain Krüppel transcription factors that interact with GC-rich motifs, including consensus specificity protein 1 (Sp1) binding sites. Based on these observations, we hypothesized that Sp1 stimulates productive infection and transactivates key BoHV-1 promoters. DEX treatment of latently infected calves increased the number of Sp1+ TG neurons and cells in pharyngeal tonsil indicating that Sp1 expression is induced by stress. Silencing Sp1 protein expression with siRNA or mithramycin A, a drug that preferentially binds GC-rich DNA, significantly reduced BoHV-1 replication. Moreover, BoHV-1 infection of permissive cells increased Sp1 steady-state protein levels. In transient transfection studies, GR and Sp1 cooperatively transactivate IEtu1 promoter activity unless both GREs are mutated. Co-immunoprecipitation studies revealed that GR and Sp1 interact in mouse neuroblastoma cells (Neuro-2A) suggesting this interaction stimulates IEtu1 promoter activity. Collectively, these studies suggested that the cellular transcription factor Sp1 enhances productive infection and stress-induced BoHV-1 reactivation from latency.IMPORTANCEFollowing acute infection, bovine alphaherpesvirus 1 (BoHV-1) establishes lifelong latency in sensory neurons in trigeminal ganglia (TG) and pharyngeal tonsil. The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. The number of TG neurons and cells in pharyngeal tonsil expressing the cellular transcription factor specificity protein 1 (Sp1) protein increases during early stages of dexamethasone-induced reactivation from latency. Silencing Sp1 expression impairs BoHV-1 replication in permissive cells. Interestingly, mithramycin A, a neuroprotective antibiotic that preferentially binds GC-rich DNA, impairs Sp1 functions and reduces BoHV-1 replication suggesting that it is a potential antiviral drug. The glucocorticoid receptor (GR) and Sp1 cooperatively transactivate the BoHV-1 immediate early transcript unit 1 (IEtu1) promoter, which drives expression of infected cell protein 0 (bICP0) and bICP4. Mithramycin A also reduced Sp1- and GR-mediated transactivation of the IEtu1 promoter. These studies revealed that GR and Sp1 trigger viral gene expression and replication following stressful stimuli.

Endocrine Disruptors: Focus on the Adrenal Cortex.

Endocrine-disrupting chemicals (EDCs) are exogenous substances known to interfere with endocrine homeostasis and promote adverse health outcomes. Their impact on the adrenal cortex, corticosteroids and their physiological role in the organism has not yet been sufficiently elucidated. In this review, we collect experimental and epidemiological evidence on adrenal disruption by relevant endocrine disruptors. In vitro data suggest significant alterations of gene expression, cell signalling, steroid production, steroid distribution, and action. Additionally, morphological studies revealed disturbances in tissue organization and development, local inflammation, and zone-specific hyperplasia. Finally, endocrine circuits, such as the hypothalamic-pituitary-adrenal axis, might be affected by EDCs. Many questions regarding the detection of steroidogenesis disruption and the effects of combined toxicity remain unanswered. Not only due to the diverse mode of action of adrenal steroids and their implication in many common diseases, there is no doubt that further research on endocrine disruption of the adrenocortical system is needed.

Differential early response of monocyte/macrophage subsets to intra-operative corticosteroid administration in lung transplantation.

Introduction: Lung transplantation often results in primary and/or chronic dysfunctions that are related to early perioperative innate allo-responses where myeloid subsets play a major role. Corticosteroids are administered upon surgery as a standard-of-care but their action on the different myeloid cell subsets in that context is not known. Methods: To address this issue, we used a cross-circulatory platform perfusing an extracorporeal lung coupled to cell mapping in the pig model, that enabled us to study the recruited cells in the allogeneic lung over 10 hours. Results: Myeloid cells, i.e. granulocytes and monocytic cells including classical CD14pos and non-classical/intermediate CD16pos cells, were the dominantly recruited subsets, with the latter upregulating the membrane expression of MHC class II and CD80/86 molecules. Whereas corticosteroids did not reduce the different cell subset recruitment, they potently dampened the MHC class II and CD80/86 expression on monocytic cells and not on alveolar macrophages. Besides, corticosteroids induced a temporary and partial anti-inflammatory gene profile depending on cytokines and monocyte/macrophage subsets. Discussion: This work documents the baseline effects of the standard-of-care corticosteroid treatment for early innate allo-responses. These insights will enable further optimization and improvement of lung transplantation outcomes.

Augmented Placental Protein 13 in Placental-Associated Extracellular Vesicles in Term and Preterm Preeclampsia Is Further Elevated by Corticosteroids.

Placental protein 13 (PP13) is a regulatory protein involved in remodeling the vascular system of the pregnancy and extending the immune tolerance of the mother to the growing fetus. PP13 is localized on the surface of the syncytiotrophoblast. An ex vivo placental model shows that the PP13 is released via placental-associated extracellular vesicles (PEVs) to the maternal uterine vein. This exploratory study aimed to determine PEV-associated PP13 in the maternal circulation as compared to the known soluble fraction since each has a specific communication pathway. Patients admitted to Bnai Zion Medical Center for delivery were recruited, and included 19 preeclampsia (PE) patients (7 preterm PE gestational age < 37 weeks' gestation), 16 preterm delivery (PTD, delivery at GA < 37 weeks' gestation), and 15 matched term delivery controls. Treatment by corticosteroids (Celestone), which is often given to patients with suspected preterm PE and PTD, was recorded. The PEV proteome was purified from the patients' plasma by size exclusion chromatography (SEC) to separate the soluble and PEV-associated PP13. The total level of PP13 (soluble and PEV-associated) was determined using mild detergent that depleted the PEV proteome. PP13 fractions were determined by ELISA with PP13 specific antibodies. ELISA with alkaline phosphatase (PLAP)- and cluster differentiation 63 (CD63)-specific antibodies served to verify the placental origin of the PEVs. SPSS was used for statistical analysis. The patients' medical, pregnancy, and delivery records in all groups were similar except, as expected, that a larger number of PE and PTD patients had smaller babies who were delivered earlier, and the PE patients had hypertension and proteinuria. The SEC analysis detected the presence of PP13 in the cargo of the PEVs and on their surface, in addition to the known soluble fraction. The median soluble PP13 was not significantly different across the PE, PTD, and term delivery control groups. However, after depleting the PEV of their proteome, the total PP13 (soluble and PEV-associated) was augmented in the cases of preterm PE, reaching 2153 pg/mL [IQR 1866-2838] but not in cases of PTD reaching 1576 pg/mL [1011-2014] or term delivery groups reaching 964 pg/mL [875-1636]), p < 0.01. On the surface of the circulating PEV from PTD patients, there was a decrease in PP13. Corticosteroid treatment was accompanied by a massive depletion of PP13 from the PEV, especially in preterm PE patients. This exploratory study is, thus, the first to determine PEV-associated PP13 in maternal circulation, providing a quantitative determination of the soluble and the PEV-associated fractions, and it shows that the latter is the larger. We found an increase in the amount of PP13 carried via the PEV-associated pathway in PE and PTD patients compared to term delivery cases, which was further augmented when the patients were treated with corticosteroids, especially in preterm PE. The signal conveyed by this novel communication pathway warrants further research to investigate these two differential pathways for the liberation of PP13.

Sublethal Hyperthermia Transiently Disrupts Cortisol Steroidogenesis in Adrenocortical Cells.

Primary aldosteronism is the most common cause of secondary hypertension. The first-line treatment adrenalectomy resects adrenal nodules and adjacent normal tissue, limiting suitability to those who present with unilateral disease. Use of thermal ablation represents an emerging approach as a possible minimally invasive therapy for unilateral and bilateral disease, to target and disrupt hypersecreting aldosterone-producing adenomas, while preserving adjacent normal adrenal cortex. To determine the extent of damage to adrenal cells upon exposure to hyperthermia, the steroidogenic adrenocortical cell lines H295R and HAC15 were treated with hyperthermia at temperatures between 37 and 50°C with the effects of hyperthermia on steroidogenesis evaluated following stimulation with forskolin and ANGII. Cell death, protein/mRNA expression of steroidogenic enzymes and damage markers (HSP70/90), and steroid secretion were analyzed immediately and 7 days after treatment. Following treatment with hyperthermia, 42°C and 45°C did not induce cell death and were deemed sublethal doses while ≥50°C caused excess cell death in adrenal cells. Sublethal hyperthermia (45°C) caused a significant reduction in cortisol secretion immediately following treatment while differentially affecting the expression of various steroidogenic enzymes, although recovery of steroidogenesis was evident 7 days after treatment. As such, sublethal hyperthermia, which occurs in the transitional zone during thermal ablation induces a short-lived, unsustained inhibition of cortisol steroidogenesis in adrenocortical cells in vitro.

Concurrent versus delayed exposure to corticosteroids in equine articular tissues cultured with local anesthetic.

OBJECTIVE: To determine the effect of concurrent versus delayed treatment with corticosteroid on equine articular tissues also treated with local anesthetic in vitro in the presence of inflammatory mediators. STUDY DESIGN: Controlled laboratory study. ANIMALS: Five geldings, one mare (aged 3-18 years). METHODS: From each horse, 24 synovial and 12 osteochondral explants were cultured in a 12-well plate (2 wells/group, 2 synovial and 1 osteochondral explant/well, total 216 explants in the study). Explants were stimulated in culture medium with 10 µg/ml recombinant equine interleukin-1ß and 10 µg/ml tumor necrosis factor-α for 48 hours, then randomly assigned to six treatments: unstimulated control, stimulated control, triamcinolone acetonide (TA, 10-6  M), mepivacaine hydrochloride (MH, 4.4 mg/ml), MH + TA (concurrent) and MH + TA (delayed). The delayed group was treated with MH and, 6 days later, treated with TA. Every 3 days for 9 days total, medium levels of lactate dehydrogenase (LDH), prostaglandin E2 (PGE2 ), matrix metalloproteinase 13 (MMP-13) and glycosaminoglycan (GAG) were quantified via ELISA. Data were analyzed with mixed-effects models with Tukey's multiple comparisons. RESULTS: Stimulation increased medium PGE2 and MMP-13 and had no effect on LDH or GAG. Treatment with MH increased LDH and decreased PGE2 and MMP-13. Treatment with TA decreased PGE2 and MMP-13. CONCLUSION: There were no differences in cytotoxicity, inflammation or matrix degradation for delayed or concurrent MH and TA treatment groups up to 9 days in culture. CLINICAL SIGNIFICANCE: The lack of an effect of concurrent versus delayed treatment might indicate that concurrent therapy is acceptable.

Characterization of Transcriptomic and Proteomic Changes in the Skin after Chronic Fluocinolone Acetonide Treatment.

While topical corticosteroid (TCS) treatment is widely used for many skin diseases, it can trigger adverse side effects, and some of such effects can last for a long time after stopping the treatment. However, molecular changes induced by TCS treatment remain largely unexplored, although transient changes in histology and some major ECM components have been documented. Here, we investigated transcriptomic and proteomic changes induced by fluocinolone acetonide (FA) treatment in the mouse skin by conducting RNA-Seq and quantitative proteomics. Chronic FA treatment affected the expression of 4229 genes, where downregulated genes were involved in cell-cycle progression and ECM organization, and upregulated genes were involved in lipid metabolism. The effects of FA on transcriptome and histology of the skin largely returned to normal by two weeks after the treatment. Only a fraction of transcriptomic changes were reflected by proteomic changes, and the expression of 46 proteins was affected one day after chronic FA treatment. A comparable number of proteins were differentially expressed between control and FA-treated skin samples even at 15 and 30 days after stopping chronic FA treatment. Interestingly, proteins affected during and after chronic FA treatment were largely different. Our results provide fundamental information of molecular changes induced by FA treatment in the skin.

Loss of SUMO-specific protease 2 causes isolated glucocorticoid deficiency by blocking adrenal cortex zonal transdifferentiation in mice.

SUMOylation is a dynamic posttranslational modification, that provides fine-tuning of protein function involved in the cellular response to stress, differentiation, and tissue development. In the adrenal cortex, an emblematic endocrine organ that mediates adaptation to physiological demands, the SUMOylation gradient is inversely correlated with the gradient of cellular differentiation raising important questions about its role in functional zonation and the response to stress. Considering that SUMO-specific protease 2 (SENP2), a deSUMOylating enzyme, is upregulated by Adrenocorticotropic Hormone (ACTH)/cAMP-dependent Protein Kinase (PKA) signalling within the zona fasciculata, we generated mice with adrenal-specific Senp2 loss to address these questions. Disruption of SENP2 activity in steroidogenic cells leads to specific hypoplasia of the zona fasciculata, a blunted reponse to ACTH and isolated glucocorticoid deficiency. Mechanistically, overSUMOylation resulting from SENP2 loss shifts the balance between ACTH/PKA and WNT/ß-catenin signalling leading to repression of PKA activity and ectopic activation of ß-catenin. At the cellular level, this blocks transdifferentiation of ß-catenin-positive zona glomerulosa cells into fasciculata cells and sensitises them to premature apoptosis. Our findings indicate that the SUMO pathway is critical for adrenal homeostasis and stress responsiveness.

Topical Losartan and Corticosteroid Additively Inhibit Corneal Stromal Myofibroblast Generation and Scarring Fibrosis After Alkali Burn Injury.

Purpose: To evaluate the efficacy of losartan and prednisolone acetate in inhibiting corneal scarring fibrosis after alkali burn injury in rabbits. Methods: Sixteen New Zealand White rabbits were included. Alkali injuries were produced using 1N sodium hydroxide on a 5-mm diameter Whatman #1 filter paper for 1 minute. Four corneas in each group were treated six times per day for 1 month with 50 µL of (1) 0.8 mg/mL losartan in balanced salt solution (BSS), (2) 1% prednisolone acetate, (3) combined 0.8 mg/mL losartan and 1% prednisolone acetate, or (4) BSS. Area of opacity and total opacity were analyzed in standardized slit-lamp photos with ImageJ. Corneas in both groups were cryofixed in Optimal cutting temperature (OCT) compound at 1 month after surgery, and immunohistochemistry was performed for alpha-smooth muscle actin (α-SMA) and keratocan or transforming growth factor ß1 and collagen type IV with ImageJ quantitation. Results: Combined topical losartan and prednisolone acetate significantly decreased slit-lamp opacity area and intensity, as well as decreased stromal myofibroblast α-SMA area and intensity of staining per section and confined myofibroblasts to only the posterior stroma with repopulation of the anterior and mid-stroma with keratocan-positive keratocytes after 1 month of treatment. Corneal fibroblasts produced collagen type IV not associated with basement membranes, and this production was decreased by topical losartan. Conclusions: Combined topical losartan and prednisolone acetate decreased myofibroblast-associated fibrosis after corneal alkali burns that produced full-thickness injury, including corneal endothelial damage. Increased dosages and duration of treatment may further decrease scarring fibrosis. Translational Relevance: Topical losartan and prednisolone acetate decrease myofibroblast-mediated scarring fibrosis after corneal injury.

Prenatal or postnatal corticosteroids favor clinical, respiratory, metabolic outcomes and oxidative balance of preterm lambs corticotherapy for premature neonatal lambs.

Challenges of the extrauterine environment can be life threatening for a premature fetus with inadequate fetal maturity. Maternal corticosteroids therapy is widely employed to induce fetal pulmonary maturation. Nevertheless, whenever therapeutic pregnancy interruption has to be performed in a time manner insufficient to treat the dam, postnatal corticotherapy can be considered an alternative. However, it is not known if antenatal and postnatal corticotherapy can improve similarly neonatal outcomes and pulmonary function. This research aimed to analyze antenatal and postnatal corticotherapy on premature lambs vitality, pulmonary functioning, metabolic and oxidative status. Lambs were evaluated according to the mode of treatment: Prenatal Corticosteroid Group (8 lambs born after maternal betamethasone treatment 48 h prior to birth), Postnatal Corticosteroid Group (9 lambs subjected to betamethasone treatment 10 min after birth) and Control Group (5 lambs remained untreated). Lambs were medically followed-up from birth to 72 h thereafter through a complete physical examination, as well as lactatemia, glycaemia, blood acid-base balance and antioxidant status. Treated lambs had higher vitality score than the Control Group. Heart rate was higher in postnatal therapy compared to prenatal treatment. Respiratory rate and rectal temperature were higher in treated groups. Treated lambs had hyperglycemia, while the Postnatal Group had higher lactatemia than the Control Group. The Prenatal Group had lower and normal pCO2 from 60 min onwards. The Postnatal Group had higher superoxide dismutase activity than untreated lambs. In conclusion, prenatal and postnatal betamethasone treatments favor neonatal clinical outcome, respiratory function, glucose homeostasis and oxidative balance.

Changes in hormones after apneic hypoxia/hypercapnia - An investigation in voluntary apnea divers.

BACKGROUND: Prolonged apnea is characterized by hypoxia/hypercapnia. Hypoxia can be associated with hormonal dysfunction. We raised the question as to whether steroid hormonal and gonadotropin levels could be influenced by short-term hypoxia/hypercapnia in a model of dry apnea in trained apnea divers. METHODS: Adrenal, sex steroid and pituitary hormones were measured in ten trained voluntary apnea divers before, immediately after, 0.5 h and 4 h after a maximal breath-hold. Apnea was carried out under dry conditions. RESULTS: Corticosterone, progesterone, cortisol, 17-OH-progesterone, dehydroepiandrosterone and androstenedione showed a significant continuous increase with a maximum at 0.5 h after apnea, followed by a decrease back to or below baseline at 4 h after apnea. Testosterone, estradiol, cortisone and dihydrotestosterone showed a decrease 4 h after apnea. Dehydroepiandrosteronesulfate, luteinizing hormone (LH) and follicle stimulating hormone (FSH) showed no significant changes. CONCLUSION: Even a single apnea resulted in two different patterns of hormone response to apnea, with increased adrenal and reduced sex steroid levels, while LH/FSH showed no clear kinetic reaction. Apnea divers might be a suitable clinical model for hypoxic disease.

Development of Human Adrenocortical Adenoma (HAA1) Cell Line from Zona Reticularis.

The human adrenal cortex is composed of distinct zones that are the main source of steroid hormone production. The mechanism of adrenocortical cell differentiation into several functionally organized populations with distinctive identities remains poorly understood. Human adrenal disease has been difficult to study, in part due to the absence of cultured cell lines that faithfully represent adrenal cell precursors in the early stages of transformation. Here, Human Adrenocortical Adenoma (HAA1) cell line derived from a patient's macronodular adrenocortical hyperplasia and was treated with histone deacetylase inhibitors (HDACis) and gene expression was examined. We describe a patient-derived HAA1 cell line derived from the zona reticularis, the innermost zone of the adrenal cortex. The HAA1 cell line is unique in its ability to exit a latent state and respond with steroidogenic gene expression upon treatment with histone deacetylase inhibitors. The gene expression pattern of differentiated HAA1 cells partially recreates the roster of genes in the adrenal layer that they have been derived from. Gene ontology analysis of whole genome RNA-seq corroborated increased expression of steroidogenic genes upon HDAC inhibition. Surprisingly, HDACi treatment induced broad activation of the Tumor Necrosis Factor (TNF) alpha pathway. This novel cell line we developed will hopefully be instrumental in understanding the molecular and biochemical mechanisms controlling adrenocortical differentiation and steroidogenesis.

Glucocorticoid Receptor Antagonist Alters Corticosterone and Receptor-sensitive mRNAs in the Hypoxic Neonatal Rat.

Hypoxia, a common stressor with preterm birth, increases morbidity and mortality associated with prematurity. Glucocorticoids (GCs) are administered to the preterm infant to improve oxygenation; prolonged use of GCs remains controversial. We evaluated a selective glucocorticoid receptor (GR) antagonist (CORT113176) in our neonatal rat model of human prematurity to assess how fasting and hypoxia-induced increases in neonatal corticosterone affects endogenous hormones and endocrine pancreas function. Neonatal rat pups at postnatal day (PD) 2, PD8, and PD15 were pretreated with CORT113176 and, after 60 minutes of separation and fasting, exposed to hypoxia (8% O2) or control (normoxia) for 30 or 60 minutes while fasting was continued. Plasma corticosterone, ACTH, glucose, and insulin were measured and fasting Homeostatic Model Assessment of Insulin Resistance was calculated. Glucocorticoid and insulin receptor-sensitive gene mRNAs were analyzed in liver, muscle, and adipose to evaluate target tissue biomarkers. CORT113176 pretreatment augmented baseline and hypoxia-induced increases in corticosterone and attenuated hypoxia-induced increases in insulin resistance at PD2. Normoxic and hypoxic stress increased the hepatic GR-sensitive gene mRNAs, Gilz and Per1; this was eliminated by pretreatment with CORT113176. CORT113176 pretreatment decreased baseline insulin receptor-sensitive gene mRNAs Akt2, Irs1, Pik3r1, and Srebp1c at PD2. We show that CORT113176 variably augments the stress-induced increases in corticosterone concentrations (attenuation of negative feedback) and that GR is critical for hepatic responses to stress in the hypoxic neonate. We also propose that measurement of Gilz and Per1 mRNA expression may be useful to evaluate the effectiveness of GR antagonism.

A new p65 isoform that bind the glucocorticoid hormone and is expressed in inflammation liver diseases and COVID-19.

Inflammation is a physiological process whose deregulation causes some diseases including cancer. Nuclear Factor kB (NF-kB) is a family of ubiquitous and inducible transcription factors, in which the p65/p50 heterodimer is the most abundant complex, that play critical roles mainly in inflammation. Glucocorticoid Receptor (GR) is a ligand-activated transcription factor and acts as an anti-inflammatory agent and immunosuppressant. Thus, NF-kB and GR are physiological antagonists in the inflammation process. Here we show that in mice and humans there is a spliced variant of p65, named p65 iso5, which binds the corticosteroid hormone dexamethasone amplifying the effect of the glucocorticoid receptor and is expressed in the liver of patients with hepatic cirrhosis and hepatocellular carcinoma (HCC). Furthermore, we have quantified the gene expression level of p65 and p65 iso5 in the PBMC of patients affected by SARS-CoV-2 disease. The results showed that in these patients the p65 and p65 iso5 mRNA levels are higher than in healthy subjects. The ability of p65 iso5 to bind dexamethasone and the regulation of the glucocorticoid (GC) response in the opposite way of the wild type improves our knowledge and understanding of the anti-inflammatory response and identifies it as a new therapeutic target to control inflammation and related diseases.

Corticosteroid insensitivity persists in the absence of STAT1 signaling in severe allergic airway inflammation.

Corticosteroid insensitivity in asthma limits the ability to effectively manage severe asthma, which is characterized by persistent airway inflammation, airway hyperresponsiveness (AHR), and airflow obstruction despite corticosteroid treatment. Recent reports indicate that corticosteroid insensitivity is associated with increased interferon-γ (IFN-γ) levels and T-helper (Th) 1 lymphocyte infiltration in severe asthma. Signal transducer and activator of transcription 1 (STAT1) activation by IFN-γ is a key signaling pathway in Th1 inflammation; however, its role in the context of severe allergic airway inflammation and corticosteroid sensitivity remains unclear. In this study, we challenged wild-type (WT) and Stat1-/- mice with mixed allergens (MA) augmented with c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate], an inducer of Th1 cell infiltration with increased eosinophils, neutrophils, Th1, Th2, and Th17 cells. Compared with WT mice, Stat1-/- had reduced neutrophils, Th1, and Th17 cell infiltration. To evaluate corticosteroid sensitivity, mice were treated with either vehicle, 1 or 3 mg/kg fluticasone propionate (FP). Corticosteroids significantly reduced eosinophil infiltration and cytokine levels in both c-di-GMP + MA-challenged WT and Stat1-/- mice. However, histological and functional analyses show that corticosteroids did not reduce airway inflammation, epithelial mucous cell abundance, airway smooth muscle mass, and AHR in c-di-GMP + MA-challenged WT or Stat1-/- mice. Collectively, our data suggest that increased Th1 inflammation is associated with a decrease in corticosteroid sensitivity. However, increased airway pathology and AHR persist in the absence of STAT1 indicate corticosteroid insensitivity in structural airway cells is a STAT1 independent process.

Neurosteroids and steroid hormones are allosteric modulators of muscarinic receptors.

The membrane cholesterol was found to bind and modulate the function of several G-protein coupled receptors including muscarinic acetylcholine receptors. We investigated the binding of 20 steroidal compounds including neurosteroids and steroid hormones to muscarinic receptors. Corticosterone, progesterone and some neurosteroids bound to muscarinic receptors with the affinity of 100 nM or greater. We established a structure-activity relationship for steroid-based allosteric modulators of muscarinic receptors. Further, we show that corticosterone and progesterone allosterically modulate the functional response of muscarinic receptors to acetylcholine at physiologically relevant concentrations. It can play a role in stress control or in pregnancy, conditions where levels of these hormones dramatically oscillate. Allosteric modulation of muscarinic receptors via the cholesterol-binding site represents a new pharmacological approach at diseases associated with altered cholinergic signalling.