Presence Of Glucocorticoids Research Articles

The histone methyltransferase KMT2D maintains cellular glucocorticoid responsiveness by shielding the glucocorticoid receptor from degradation

Because of their ability to induce lymphocyte apoptosis, glucocorticoids (GC) are widely used to treat hematological malignancies such as lymphomas and multiple myeloma. Their effectiveness is often limited, however, due to the development of glucocorticoid resistance by a variety of molecular mechanisms. Here we performed an unbiased genome-wide CRISPR screen with the human T cell leukemia cell line Jurkat to find previously unidentified genes required for GC-induced apoptosis. One such gene was KMT2D (also known as MLL2 or MLL4), which encodes a histone lysine methyltransferase whose mutations are associated with a variety of cancers, blood malignancies in particular, and are considered markers of poor prognosis. Knockout of KMT2D by CRISPR/Cas9 gene editing in Jurkat and several multiple myeloma cell lines downregulated GR protein expression. Surprisingly, this was not due to a reduction in GR transcripts, but rather to a decrease in the protein's half-life, primarily due to proteasomal degradation. Reconstitution of KMT2D expression restored GR levels. In contrast to the known ability of KMT2D to control gene transcription through covalent histone methylation, KMT2D-mediated upregulation of GR levels did not require its methyltransferase activity. Co-immunoprecipitation and proximity ligation assays found constitutive binding of KMT2D to the GR, which was enhanced in the presence of GC. These observations reveal KMT2D to be essential for stabilization of cellular GR levels, and suggest a possible mechanism by which KMT2D mutations may lead to GC resistance in some malignancies.

SAT328 Severe Exogenous Cushing Syndrome Caused By A Potent Steroid Concealed In Arthritis Supplement

Abstract Disclosure: S. Maraqa: None. L. Bushnaq: None. R. Singh, PhD: None. N. Genere: None. Cushing syndrome (CS) is a rare disorder caused by excessive systemic glucocorticoids and associated with increased mortality and worsening quality of life. CS most commonly occurs from exogenous glucocorticoid use, while Cushing disease causes most of endogenous cases. Here, we present a case of a 64-year-old woman with rapidly progressive CS due to an over-the-counter (OTC) arthritis supplement with unlisted dexamethasone. A 64-year-old woman with reported CS who was evaluated in our Endocrinology center after she moved to the United States. She was previously diagnosed with pituitary CS in Mexico following a 6 months history of rapid 50-pound weight gain, profound proximal muscle weakness requiring assistive device, and unprovoked vertebral fractures; laboratory evaluation found elevated 24-hour urinary cortisol 272 mcg/24h, 1mg dexamethasone suppression test total cortisol 5.6 mcg/dl (< 1.8), and adrenocorticotropic hormone (ACTH) 16 pg/mL. Pituitary MRI was unremarkable. Past medical history included primary hypothyroidism and hypertension. Physical exam was notable for blood pressure 160/108 mmHg, heart rate 96, BMI 33 kg/m², moon face, supraclavicular fat pads, central obesity, skin bruises, and proximal muscle weakness. Unexpectedly, re-evaluation showed undetectable serum ACTH and cortisol. A thorough review of medications identified no recent exposure to glucocorticoids. However, a synthetic glucocorticoid screen showed markedly elevated serum dexamethasone concentration. Follow-up discussion revealed that she had started an arthritis supplement “Artri-King” 6 months prior to these symptoms. We collaborated with Mayo Clinic Labs to evaluate dexamethasone contents in this drug; each tablet was found to contain 0.9 mg of dexamethasone, which was unreported in the ingredient list. This supplement was stopped, and the patient was started on physiologic hydrocortisone. Two months later, AM cortisol was 5.6 mcg/dL and ACTH 23 pg/ml. Eight months later, she continues to have adrenal insufficiency but has had a substantial clinical improvement.Exogenous CS is readily diagnosed in patients taking prescribed glucocorticoids; however, diagnosis can be more challenging in cases where the presence of glucocorticoids is concealed. The US Food and Drug Administration follows more limited regulatory guidelines for dietary supplements compared to prescribed drugs. A study by Tucker.et.al. reported that 20% of dietary supplements have unapproved ingredients.1 This is especially concerning, considering that over half of the US population admits to taking dietary supplements.2 On April 2022, the FDA published a public notice for “Artri-King”, declaring the presence of unlisted dexamethasone.3 Should such agents prove to be the culprit of CS, they must be tapered off gradually or in the presence of physiologic hydrocortisone to avoid the risk of adrenal crisis. Presentation: Saturday, June 17, 2023

Self-Declared and Measured Prevalence of Glucocorticoid Use in Polish Athletes

Glucocorticoids (GCs) are included in the list of prohibited substances and methods in sport published annually by the World Anti-Doping Agency (WADA). In its 2022 update, the WADA list prohibits all injectable routes of administration of GCs for use during in-competition periods. Previously, GCs were prohibited in-competition when administered by oral, intravenous, intramuscular, or rectal routes, but local injections (in addition to topical applications) were allowed. This study first investigated the prevalence of GC use by athletes in Poland, declared in 2130 doping control forms, and the related 2130 urine samples analysed at the Polish Anti-Doping Laboratory. Second, the validity of the analytical methodology to detect GCs was evaluated with the updated WADA requirement for substance-specific minimum reporting levels and considering the proposed washout periods. Despite the new regulation in place, the use of 30 different GC preparations were declared in a total of 162 occurrences (8% of the tests) with therapeutic purposes. Laboratory analyses resulted in the presence of GCs in 16 occurrences with only two samples with a concentration triggering an adverse analytical finding. Our study allowed us to confirm that the applied methodology for the determination of GCs in urine samples (ultra-high-performance liquid chromatography–tandem mass spectrometry) remains fully valid after the latter regulation change while the challenge to assess the timing and administration route for GCs persists.

Determination of selected glucocorticoids in healthy foods by ultra-performance convergence chromatography–triple quadrupole mass spectrometry

The presence of glucocorticoids in healthy foods has recently become a topic of concern because of their side effects. In this study, we developed a method based on ultra-performance convergence chromatography–triple quadrupole mass spectrometry (UPC2–MS/MS) to detect 63 glucocorticoids in healthy foods. The analysis conditions were optimized, and the method was validated. We further compared the results of this method with those of the RPLC–MS/MS method. Glucocorticoids were separated on an Acquity Torus 2-picolylamine column (100 mm × 3.0 mm, 1.7 µm) and detected via MS/MS. CO2 and methanol (containing 0.1% formic acid) were used as mobile phases. The method demonstrated good linear relationships between 1 and 200 µg·L−1 (R2 ≥ 0.996). The limits of detection in different types of samples were 0.3–1.5 µg·kg−1 (S/N = 3). The average recoveries (n = 9) and RSDs in different types of samples were 76.6–118.2% and 1.1–13.1%, respectively. The matrix effect, calculated as the ratio between calibration curves built in matrix and pure solvent, was less than 0.21 for both a fish oil and a protein powder. This method exhibited better selectivity and resolution than RPLC–MS/MS method. Lastly, it could realize the baseline separation of 31 isomers of 13 groups, including four groups of eight epimers. This study provides new technical support for assessing the risk of exposure to glucocorticoids in healthy foods.

Glucocorticoids Inhibit EGFR Signaling Activation in Podocytes in Anti-GBM Crescentic Glomerulonephritis.

Glucocorticoids are commonly used to treat anti-GBM crescentic glomerulonephritis, however, the mechanism underlying its therapeutic effectiveness is not completely understood. Since podocyte EGFR/STAT3 signaling is known to mediate the development of anti-GBM glomerulonephritis, we investigated the effect of glucocorticoids on EGFR/STAT3 signaling in podocytes. We found that the levels of phosphorylated (activated) EGFR and STAT3 in podocytes were markedly elevated in anti-GBM patients without glucocorticoids treatment, but were normalized in patients with glucocorticoids treatment. In a rat model of anti-GBM glomerulonephritis, glucocorticoids treatment significantly attenuated the proteinuria, crescent formation, parietal epithelial cell (PEC) activation and proliferation, accompanied by elimination of podocyte EGFR/STAT3 signaling activation. In cultured podocytes, glucocorticoids were found to inhibit HB-EGF-induced EGFR and STAT3 activation. The conditioned medium from podocytes treated with HB-EGF in the absence but not presence of glucocorticoids was capable of activating Notch signaling (which is known to be involved in PEC proliferation and crescent formation) and enhancing proliferative activity in primary PECs, suggesting that glucocorticoids prevent podocytes from producing secreted factors that cause PEC proliferation and crescent formation. Furthermore, we found that glucocorticoids can downregulate the expression of EGFR ligands, EGF and HB-EGF, while upregulate the expression of EGFR inhibitor, Gene 33, explaining how glucocorticoids suppress EGFR signaling. Taken together, glucocorticoids exert therapeutic effect on anti-GBM crescentic glomerulonephritis through inhibiting podocyte EGFR/STAT3 signaling and the downstream pathway that leads to PEC proliferation and crescent formation.

The effect of Moringa oleifera polysaccharides on the regulation of glucocorticoid-induced femoral head necrosis: In vitro and in vivo

One of the common challenges in using glucocorticoid in the long term is the development of femoral head necrosis. To address this challenge, the use of glucocorticoid suppressors like plant polysaccharides has been considered. In this study, Moringa oleifera polysaccharide was isolated through hot water–ethanol precipitation method and purified by DEAE-Sepharose fast flow column. Then, they were characterized by FT-IR, NMR, methylation, and chromatography assays. The polysaccharide biocompatibility was investigated by MTT assay and its effect on osteoblasts was evaluated by controlling gene expression. Also, the effect of polysaccharide on dexamethasone-induced femoral head necrosis in rats was assessed by hydroxyproline, hexosamine and morphometric parameters. The results show that 2 Da molecular weight polysaccharide is mainly composed of Rha, Ara, Fru, Xyl, Man and Gal in the molar ratio of 1.7:2.1:3.4:5.9:5.8:1.3. Meanwhile, MTT results on osteoblasts cells showed polysaccharide biocompatibility, while significantly reducing the negative effects of glucocorticoid. Likewise, polysaccharide significantly reduced the levels of apoptosis and intracellular ROS of glucocorticoid-induced femoral necrosis. Moreover, the results of gene expression indicated a decrease in the expression of TNF-α and IL-6 genes using polysaccharide, which is very effective in preventing apoptotic activity. Also, Polysaccharide increased bone density, bone volume per tissue volume, trabecular thickness, and the hexosamine to hydroxyproline ratio in the rat serum in the presence of glucocorticoids, which are very effective in the process of femoral head necrosis. Furthermore, polysaccharide significantly increases the OCN, RUNX2 and COL-1 genes expression in cartilage tissue, which is in line with the result of morphometric parameters. Overall, this study suggests that the use of polysaccharide could result in the treatment of femoral head necrosis.

Glucocorticoid imprints a low glucose metabolism onto CD8 T cells and induces the persistent suppression of the immune response

Glucocorticoids (GCs), immunosuppressive, and anti-inflammatory agents have various effects on T cells. However, the long-term influence of GCs on the T cell-mediated immune response remain to be elucidated. We demonstrated that the administration of GC during the TCR-mediated activation phase induced long-lasting suppression of glycolysis, even after the withdrawal of GC. The acquisition of the effector functions was inhibited, while the expression of PD-1 was increased in CD8 T cells activated in the presence of GC. Furthermore, adoptive transfer experiments revealed that GC-treated CD8 T cells reduced memory T cell formation and anti-tumor activity. These findings reveal that GCs have long-lasting influence on the T cell-mediated immune response via modulation of T cell metabolism.

Efficacy of the herbal pair, Radix Achyranthis Bidentatae and Eucommiae Cortex, in preventing glucocorticoid-induced osteoporosis in the zebrafish model

ObjectiveIn traditional Chinese medicine, the herbal pair, Radix Achyranthis Bidentatae (RAB) and Eucommiae Cortex (EC), is widely used to treat osteoporosis. Herein, we determined whether this herbal pair can be used to ameliorate glucocorticoid (GC)-induced osteoporosis (GIOP) and find its optimal dosage in zebrafish. MethodsThe characteristics of the aqueous extract of RAB and EC were separately characterized using high-performance liquid chromatography. Osteoporosis was induced in 5-day post-fertilization zebrafish larvae by exposing them to 10 μmol/L dexamethasone (Dex) for 96 h. Seven combinations of different ratios of RAB and EC were co-administered. Treatment efficacy was determined by calculating zebrafish vertebral area and sum brightness, via alizarin red staining, and by detecting alkaline phosphatase (ALP) activity. Multiple regression analysis was conducted to test the optimal dosage ratio. ResultsAccording to the Chinese Pharmacopoeia (2015), β-ecdysone (β-Ecd) is a major bioactive marker in RAB extract, while pinoresinol diglucoside (PDG) is the major marker in EC extract. Both of β-Ecd and PDG content values aligned with the Chinese Pharmacopoeia standards. Treatment with 10 μmol/L Dex reduced zebrafish vertebral area, sum brightness, and ALP activity, but RAB and EC attenuated these effects. Combining 50 µg/mL RAB and 50 µg/mL EC was optimal for preventing GIOP in zebrafish. Reverse transcription-quantitative polymerase chain reaction was used to evaluate the mRNA expression of osteogenesis-related genes. A treatment of 10 μmol/L Dex decreased runt-related transcription factor 2 (Runx2), osteogenic protein-1 (OP-1), bone γ-carboxyglutamic acid-containing protein (BGLAP), and β-catenin levels. This effect was counteracted by RAB and EC co-treatment (P < 0.05). Additionally, the effect of using the two herbal extracts together was better than single-herb treatments separately. These results demonstrated that RAB and EC preserve osteoblast function in the presence of GC. The best mass ratio was 1:1. ConclusionRAB and EC herbal pair could ameliorate GC-induced effects in zebrafish, with 1:1 as the optimal dosage ratio.

The glucocorticoids prednisone and dexamethasone differentially modulate T cell function in response to anti-PD-1 and anti-CTLA-4 immune checkpoint blockade.

On-treatment steroids for countering immune checkpoint inhibitor-induced inflammatory responses (irAEs) are a hallmark of cancer immunotherapy. However, the suppressive nature of steroids has raised questions regarding their ability to compromise the function of the 'proliferative burst' of effector T cells induced by immune checkpoint antibodies. We investigated the effector functions and the co-inhibitory receptor profile of stimulated peripheral blood mononuclear cells (PBMCs) pre-treated with prednisone and dexamethasone alone or in the presence of anti-PD-1/CTLA-4 antibodies. Also, clinical analysis of a patient who exhibited irAEs following combination (anti-PD-1/CTLA-4) in the presence of glucocorticoids was done. We found that prednisone in contrast to dexamethasone did not compromise T cell cytokine production (IL-2, IFN-γ and TNF-α) and proliferation in the absence or presence of anti-PD-1/CTLA-4 antibodies, when a physiological concentration was used. Neither single prednisone treatment nor co-treatment with checkpoint inhibitors impacted the expression of co-inhibitory receptors PD-1, CTLA-4, TIM-3 and LAG-3. In contrast, dexamethasone treatment promoted downregulation of LAG-3 expression by T cells. In addition, co-treatment of PD-1 + Jurkat cells with prednisone and/or dexamethasone with anti-PD-1 before stimulation significantly reduced SHP-2 phosphorylation, indicative of increased T cell function. Our findings hereby demonstrate a differential steroid effect on T cell function, which should be taken into consideration for patients undergoing immunotherapy. Also, the clinical analysis of a patient who exhibited irAEs following combination (anti-PD-1/CTLA-4) therapy indicated complete metabolic response in the presence of glucocorticoids. Therefore, concomitant use of prednisone does not appear to interfere with the function of immune checkpoint blockade.

Dexamethasone exhibits its anti-inflammatory effects in S. aureus induced microglial inflammation via modulating TLR-2 and glucocorticoid receptor expression

Microglial inflammation plays crucial role in the pathogenesis of CNS infections including brain abscesses. Staphylococcus aureus (S. aureus) is considered as one of the major causative agents of brain abscesses. Due to the emergence of multidrug resistant bacteria the available treatment options including conventional antibiotics and steroid therapy become ineffective in terms of inflammation regulation which warrants further investigation to resolve this health issue. Microglial TLR-2 plays important roles in the bacterial recognition as well as induction of inflammation whereas glucocorticoid receptor (GR) triggers anti-inflammatory pathways in presence of glucocorticoids (GCs). The main objective of this study was to figure out the interdependency between TLR-2 and GR in presence of exogenous dexamethasone during microglial inflammation as an alternative therapeutic approach. Experiments were done either in TLR-2 neutralized condition or GR blocked condition in presence of dexamethasone. Free radicals production, arginase, superoxide dismutase (SOD), catalase enzyme activities and corticosterone concentration were measured along with Western blot analysis of TLR-2, GR and other inflammatory molecules. The results suggested that dexamethasone pre-treatment in TLR-2 neutralized condition efficiently reduces the inflammatory consequences of S. aureus induced microglial inflammation through up regulating GR expression. During TLR-2 blocking dexamethasone exerted its potent anti-inflammatory activities via suppressing reactive oxygen species (ROS), NO production and up regulating arginase, SOD and catalase activities at the time point of 90 min. Further in-vivo experiments are needed to conclude that dexamethasone could resolve brain inflammation possibly through microglial phenotypic switching from pro-inflammatory M1 to anti-inflammatory M2.

FGF-2 targets sclerostin in bone and myostatin in skeletal muscle to mitigate the deleterious effects of glucocorticoid on musculoskeletal degradation

AimMyokines are associated with regulation of bone and muscle mass. However, limited information is available regarding the impact of myokines on glucocorticoid (GC) mediated adverse effects on the musculoskeletal system. This study investigates the role of myokine fibroblast growth factor-2 (FGF-2) in regulating GC-induced deleterious effects on bone and skeletal muscle. MethodsPrimary osteoblast cells and C2C12 myoblast cell line were treated with FGF-2 and then exposed to dexamethasone (GC). FGF-2 mediated attenuation of the inhibitory effect of GC on osteoblast and myoblast differentiation and muscle atrophy was assessed through quantitative PCR and western blot analysis. Further, FGF-2 was administered subcutaneously to dexamethasone treated mice to collect bone and skeletal muscle tissue for in vivo analysis of bone microarchitecture, mechanical strength, histomorphometry and for histological alterations in treated tissue samples. Key findingsFGF-2 abrogated the dexamethasone induced inhibitory effect on osteoblast differentiation by modulating BMP-2 pathway and inhibiting Wnt antagonist sclerostin. Further, dexamethasone induced atrophy in C2C12 cells was mitigated by FGF-2 as evident from down regulation of atrogenes expression. FGF-2 prevented GC-induced impairment of mineral density, biomechanical strength, trabecular bone volume, cortical thickness and bone formation rate in mice. Additionally, skeletal muscle tissue from GC treated mice displayed weak myostatin immunostaining and reduced expression of atrogenes following FGF-2 treatment. SignificanceFGF-2 mitigated GC induced effects through inhibition of sclerostin and myostatin expression in bone and muscle respectively. Taken together, this study exhibited the role of exogenous FGF-2 in sustaining osteoblastogenesis and inhibiting muscle atrophy in presence of glucocorticoid.

Endoribonucleolytic Cleavage of m6A-Containing RNAs by RNase P/MRP Complex.

N6-methyladenosine (m6A) is the most abundant internal modification in RNAs and plays regulatory roles in a variety of biological and physiological processes. Despite its important roles, the molecular mechanism underlying m6A-mediated gene regulation is poorly understood. Here, we show that m6A-containing RNAs are subject to endoribonucleolytic cleavage via YTHDF2 (m6A reader protein), HRSP12 (adaptor protein), and RNase P/MRP (endoribonucleases). We demonstrate that HRSP12 functions as an adaptor to bridge YTHDF2 and RNase P/MRP, eliciting rapid degradation of YTHDF2-bound RNAs. Transcriptome-wide analyses show that m6A RNAs that are preferentially targeted for endoribonucleolytic cleavage have an HRSP12-binding site and a RNase P/MRP-directed cleavage site upstream and downstream of the YTHDF2-binding site, respectively. We also find that a subset of m6A-containing circular RNAs associates with YTHDF2 in an HRSP12-dependent manner and is selectively downregulated by RNase P/MRP. Thus, our data expand the known functions of RNase P/MRP to endoribonucleolytic cleavage of m6A RNAs.

Tumor Lysis Syndrome in the Course of Burkitt Lymphoma Revealed by Medullar Compression

Background: Tumor lysis syndrome is unknown to many neurosurgical teams. A dreaded complication, especially in the course of hematological malignancies, like it was the case in two of our patients operated for spinal cord compression by Burkitt’s lymphoma. Objectives: To describe the clinical and biological presentation of this potentially fatal complication, which can be improved by early preventive or curative treatments. Case Reports: Patient 1: A 49-year-old woman was admitted for vertebral and intercostal pain and paraparesis. Myelography revealed epidural medullary compression at the 7th thoracic vertebrae (Th7) level. Patient 2: A 67-year-old woman was admitted for cervico-thoracic pain and paraparesis. MRI showed posterior epidural medullary compression between the 1st thoracic vertebrae (Th1) and 5th thoracic vertebrae (Th5). In both cases, an urgent decompressive laminectomy was performed. They had also received postoperative corticosteroid therapy. The multi-visceral failure occurred on the 1st day (D1) and 3rd day (D3) postoperatively, respectively. In the absence of adequate management, the outcome was fatal for the two patients respectively at D3 and D5 postoperatively. This syndrome was retained in the presence of glucocorticoid (triggering factor) and the histological result in favor of Burkitt lymphoma (target disease). Conclusion: Malignant hemopathies is a common cause of spinal cord compressions. From these two cases, the importance of biological and clinical surveillance emerges, with patients requiring urgent management for radiculo-medullar tumor compression.

Establishment of a conditional TALEN system using the translational enhancer dMac3 and an inducible promoter activated by glucocorticoid treatment to increase the frequency of targeted mutagenesis in plants.

Transcription activator-like effector nuclease (TALEN) is an artificial nuclease that causes DNA cleavage at the target site and induces few off-target reactions because of its high sequence specificity. Powerful and variable tools using TALENs can be used in practical applications and may facilitate the molecular breeding of many plant species. We have developed a convenient construction system for a plant TALEN vector named the Emerald Gateway TALEN system. In this study, we added new properties to this system, which led to an increase in the efficiency of targeted mutagenesis. Rice dMac3 is a translational enhancer that highly increases the efficiency of translation of the downstream ORF. We inserted dMac3 into the 5' untranslated region of the TALEN gene. In the cultured rice cells to which the TALEN gene was introduced, the frequency of targeted mutagenesis was highly increased compared with those altered using the conventional system. Next, the promoter for the TALEN gene was replaced with iPromoter, and its expression was stringently controlled by a GVG transcription factor that was activated in the presence of glucocorticoid. This conditional expression system worked effectively and led to a higher frequency of targeted mutagenesis than that by the constitutive expression system, while no mutagenesis was detected without glucocorticoid treatment. These results suggest that our system can be applied to genome editing to create the desired mutation.

Roles of 14-3-3β and γ in regulation of the glucocorticoid receptor transcriptional activation and hepatic gluconeogenesis

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that mediates the effects of glucocorticoids, and plays a crucial role in cell growth, development, inflammation, and gluconeogenesis. The 14-3-3 proteins bind to target proteins via phosphorylation, and influence many cellular events by altering their subcellular localization or by acting as chaperones. However, the mechanisms by which 14-3-3 proteins regulate GR transactivation and their involvement in gluconeogenesis remain uncharacterized. We found that 14-3-3β and γ increased GR transcriptional activity and the promoter activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase in the presence of glucocorticoids. Inhibition of the endogenous 14-3-3β and γ decreased dexamethasone- and cAMP-stimulated PEPCK expression. Further, both 14-3-3β and γ increased glucose production in response to glucocorticoids. Our findings suggest that 14-3-3β and γ function as positive regulators of GR transactivation and glucocorticoid-mediated hepatic gluconeogenesis.

SDF-1 improves wound healing ability of glucocorticoid-treated adipose tissue-derived mesenchymal stem cells

Glucocorticoids cause the delayed wound healing by suppressing inflammation that is required for wound healing process. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) play an important role for wound healing by their cytokine productions including stromal derived factor 1 (SDF-1). However, it has not been clear how glucocorticoids affect the wound healing ability of AT-MSCs.In this study, we found that glucocorticoid downregulated SDF-1 expression in AT-MSCs. In addition, glucocorticoid-treated AT-MSCs induced less migration of inflammatory cells and impaired wound healing capacity compared with glucocorticoid-untreated AT-MSCs. Of note, prostaglandin E2 (PGE2) synthesis-related gene expression was downregulated by glucocorticoid and PGE2 treatment rescued not only SDF-1 expression in the presence of glucocorticoid but also their wound healing capacity in vivo. Furthermore, we found SDF-1-overexpressed AT-MSCs restored wound healing capacity even after treatment of glucocorticoid. Consistent with the results obtained from glucocorticoid-treated AT-MSCs, we found that AT-MSCs isolated from steroidal osteonecrosis donors (sAT-MSCs) who received chronic glucocorticoid therapy showed less SDF-1 expression and impaired wound healing capacity compared with traumatic osteonecrosis donor-derived AT-MSCs (nAT-MSCs). Moreover, the SDF-1 level was also reduced in plasma derived from steroidal osteonecrosis donors compared with traumatic osteonecrosis donors.These results provide the evidence that concomitant application of AT-MSCs with glucocorticoid shows impaired biological modulatory effects that induce impaired wound healing.

Abstract B068: Hydrocortisone, prednisolone, methylprednisolone, and dexamethasone inhibit CetuGEX™-mediated ADCC in vitro

Abstract Introduction: Antibody dependent cellular cytotoxicity is a key mode of action of therapeutic antibodies. ADCC enhancement as well as a broader coverage of F/V allotypes leads to maximal utilization of the anti-tumor effect. Glycooptimization provides the best available opportunity to maximize ADCC activity of monoclonal antibodies. Utilizing the GEX™ platform allows the combination of minimizing fucosylation, maximizing galactosylation and branching, which maximally increases the ADCC activity. The GEX™ platform comprises a comprehensive portfolio of proprietary glycoengineered human suspension cell lines, which allow for the high yield production of proteins with tailored glycosylation patterns. Productivities of 22g/L perfusion bioreactor volume have been achieved for ADCC optimized monoclonal antibodies. Cancer therapies with monoclonal antibodies including CetuGEX™ are often accompanied by treatment with glucocorticoids in order to prevent or counteract side effects like infusion reactions. However, the immunosuppressive character of glucocorticoids may also have the potential to inhibit the ADCC effect and immune cells mediating ADCC, respectively. In this study, we analyzed the influence of different glucocorticoids on ADCC mediated by CetuGEX™. Methods: By using a europium release assay with an EGFR-expressing cancer cell line and PBMCs as effector cells we tested the ability of hydrocortisone, prednisolone, methylprednisolone and dexamethasone to inhibit ADCC mediated by CetuGEX™. In addition, we analyzed the direct effect of glucocorticoids on effector and target cells using flow cytometry. Results: All tested glucocorticoids inhibited the CetuGEX™-mediated ADCC. This effect was not caused by a decreased expression of EGFR on the target cells. NK cells which are key effector cells mediating ADCC were found to be less activated in presence of glucocorticoids and CetuGEX™ compared to CetuGEX™ alone. Conclusions: Glucocorticoids have a negative influence on ADCC mediated by NK cells which is one of the key modes of actions of CetuGEX™ and other ADCC enhanced antibodies. Therefore, the use of glucocorticoids during may impair clinical the efficacy ADCC-enhanced antibodies and should be avoided or at least minimized. Citation Format: Christoph Goletz, Johanna Rühmann, Beate Habel, Antje Danielczyk, Steffen Goletz. Hydrocortisone, prednisolone, methylprednisolone, and dexamethasone inhibit CetuGEX™-mediated ADCC in vitro [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B068.

Healthy glucocorticoid receptor N363S carriers dysregulate gene expression associated with metabolic syndrome

The glucocorticoid receptor single-nucleotide polymorphism (SNP) N363S has been reported to be associated with metabolic syndrome, type 2 diabetes, and cardiovascular disease. Our aim was to determine how the N363S SNP modifies glucocorticoid receptor signaling in a healthy population of individuals prior to the onset of disease. We examined the function of the N363S SNP in a cohort of subjects from the general population of North Carolina. Eighteen N363S heterozygous carriers and 36 noncarrier, control subjects were examined for clinical and biochemical parameters followed by a low-dose dexamethasone suppression test to evaluate glucocorticoid responsiveness. Serum insulin measurements revealed that N363S carriers have higher levels of insulin, although not statistically significant, compared with controls. Glucocorticoid receptor protein levels evaluated in peripheral blood mononuclear cells from each clinical subject showed no difference between N363S and control. However, investigation of gene expression profiles in macrophages isolated from controls and N363S carriers using microarray, quantitative RT-PCR, and NanoString analyses revealed that the N363S SNP had an altered profile compared with control. These changes in gene expression occurred in both the absence and the presence of glucocorticoids. Thus, our observed difference in gene regulation between normal N363S SNP carriers and noncarrier controls may underlie the emergence of metabolic syndrome, type 2 diabetes, and cardiovascular disease associated with the N363S polymorphism.

Glucocorticoid-independent modulation of GR activity: Implications for immunotherapy.

Pharmacological doses of glucocorticoids (GCs), acting via the glucocorticoid receptor (GR) to repress inflammation and immune function, remain the most effective therapy in the treatment of inflammatory and immune diseases. Since many patients on GC therapy exhibit GC resistance and severe side-effects, much research is focused on developing more selective GCs and combination therapies, with greater anti-inflammatory potency. GCs mediate their classical genomic transcriptional effects by binding to the cytoplasmic GR, followed by nuclear translocation and modulation of transcription of target genes by direct DNA binding of the GR or its tethering to other transcription factors. Recent evidence suggests, however, that the responses mediated by the GR are much more complex and involve multiple parallel mechanisms integrating simultaneous signals from other receptors, both in the absence and presence of GCs, to shift the sensitivity of a target cell to GCs. The level of cellular stress, immune activation status, or the cell cycle phase may be crucial for determining GC sensitivity and GC responsiveness as well as subcellular localization of the GR and GR levels. Central to the development of new drugs that target GR signaling alone or as add-on therapies, is an in-depth understanding of the molecular mechanisms of GC-independent GR desensitization, priming and activation of the unliganded GR, as well as synergy and cross-talk with other signaling pathways. This review will discuss the information currently available on these topics and their relevance to immunotherapy, as well as identify unanswered questions and future areas of research.

Vitamin K2 Ameliorates Damage of Blood Vessels by Glucocorticoid: a Potential Mechanism for Its Protective Effects in Glucocorticoid-induced Osteonecrosis of the Femoral Head in a Rat Model

Glucocorticoid has been reported to decrease blood vessel number and harm the blood supply in the femoral head, which is recognized to be an important mechanism of glucocorticoid-induced osteonecrosis of the femoral head (ONFH). To prevent glucocorticoid-induced ONFH, medication that promotes both bone formation and angiogenesis would be ideal. Vitamin K2 has been revealed to play an important role in bone metabolism; however, few studies have focused on the effect of Vitamin K2 on new vascular formation. Thus, this study aimed to investigate whether Vitamin K2 promoted new blood vessel formation in the presence of glucocorticoids, both in vitro and in vivo. The effect of Vitamin K2 on viability, migration, in vitro tube formation, and VEGF, vWF, CD31, KDR, Flt and PDGFB in EAhy926 incubated with or without dexamethasone were elucidated. VEGF, TGF-β and BMP-2, angiogenesis-related proteins secreted by osteoblasts, were also detected in the osteoblast-like cell line of MG63. In addition, blood vessels of the femoral head in rats administered with or without methylprednisolone and Vitamin K2 were evaluated using angiography and CD31 staining. In vitro studies showed that Vitamin K2 significantly protected endothelial cells from dexamethasone-induced apoptosis, promoted endothelial cell migration and in vitro tube formation. Angiogenesis-related proteins both in EAhy926 and MG63 were also upregulated by Vitamin K2 when cotreated with dexamethasone. In vivo studies showed enhanced blood vessel volume and CD31-positive staining cells in rats cotreated with VK2 and methylprednisolone compared to rats treated with methylprednisolone only. Collectively, Vitamin K2 has the ability to promote angiogenesis in vitro and to ameliorate vessels of the femoral head in glucocorticoid-treated rats in vivo, indicating that Vitamin K2 is a promising drug that may be used to prevent steroid-induced ONFH.