GR Gene Research Articles

Assessing antioxidant responses in C6 and U-87 MG cell lines exposed to high copper levels

Copper excess has been tested as an anticancer therapy, due to its properties to generate oxidative stress resulting in tumoral cell death. Thus, this study aimed to evaluate the impact of copper excess on oxidative stress and antioxidant responses in glioma cells, establishing the antioxidant system as a target of copper toxicity in tumoral cells. C6 and U-87 MG cells were exposed to CuSO4 (0–600 μM) for 24-48 h. SOD, CAT, GPx, GR, and CK activities, protein and non-protein thiol levels (PSH and NPSH), and O2− production were assessed, alongside SOD1, GPx1, and GR gene expression. Results revealed a decrease in GPx, GR, and CAT activity after CuSO4 exposure in both cell lines over 24-48 h, while SOD activity initially increased, then declined after 48 h. CK activity was also decreased in C6 cells. NPSH and PSH levels dropped after 24 h, and O2− production was observed in all CuSO4 concentrations. GR mRNA was reduced in both cell lines, contrasting with increased GPx1 mRNA in C6. U-87 MG cells exhibited higher levels of SOD1 mRNA, while C6 cells displayed lower expression. Our findings suggest that copper excess limits antioxidant enzyme activity and thiol levels, particularly in the C6 cells, likely attributable to oxidative stress or direct copper-enzyme interactions. Moreover, our results imply differences in copper toxicity regarding the cell lineage used, highlighting the importance of analyzing high copper levels effects in different models. Moreover, it could be proposed that the antioxidant system is a target of copper toxicity, contributing to glioma cell death.

Silicon nanoparticles (SiNPs) mediate GABA, SOD and ASA-GSH cycle to improve Cd stress tolerance in Solanum lycopersicum

Contamination of agricultural products with Cadmium (Cd) is a global problem that should be considered for minimizing the risks to human health. Considering the potential effects of SiNPs in decreasing abiotic stress, a study was conducted to investigate the effect of SiNPs in the reduction of Cd stress on Solanum lycopersicum. SiNPs was used at 0, 25, 50 and 100 mg/l and CdCl2 at 0, 100 and 200 µM concentrations. The results showed that Cd stress caused a significant decrease in dry weight, content of GSH, ASA, significently increasing the activity of GR, APX, GST, SOD, as well as content of H2O2, MDA, proline, and GABA in shoots and roots compared to the control. SiNPs significantly increased shoot and root dry weight compared to the control. As a coenzyme, SiNPs induced the activity of antioxidant enzymes and significantly increased GST and GR gene expression compared to the control. SiNPs also caused a substantial increase in the content of ASA, GSH, proline and GABA compared to the control. By inducing the activity of antioxidant enzymes and metabolites of the ascorbate-glutathione (ASA-GSH) cycle, SiNPs removed a large content of H2O2 and significantly reduced the MDA content, and as a result led to the stability of cell membrane under Cd stress. Induction of ASA-GSH, GABA and SOD cycle by SiNPs clearly showed that SiNPs could be a potential tool to alleviate Cd stress in plants cultivated in areas contaminated with this heavy metal.

Humic Acid Mitigates Drought Stress in Tomato

Drought stress, one of the most important abiotic stresses, severely limits global crop production. To increase tolerance to this stress, environmentally friendly practices are emphasised. Humic acid, one of the most important natural biostimulants, has positive effects on plant growth and yield. Recently, it has also been reported to play an important role in resistance to various abiotic stresses. However, many physiological and molecular mechanisms by which humic acid confers drought resistance have not been fully elucidated. Therefore, the effects of humic acid application on different morphological and physiological stress indicators and some antioxidative enzyme gene expressions of tomato seedlings under drought stress conditions were investigated in this study. It was found that drought stress decreased shoot fresh/dry weight, root fresh/dry weight, shoot and root length, chlorophyll and relative water content of plants by 67%, 56%, 31%, 38%, 22%, 20%, 15% and 25%, respectively. Humic acid application significantly increased these parameters, while reducing ion leakage, MDA and proline levels. The antioxidant enzyme gene expression of tomato seedlings under drought conditions showed no significant difference in SOD and APX gene expression, whereas CAT gene expression increased and GR gene expression decreased with humic acid application. Our results showed that humic acid application interacted with stress-related antioxidant enzyme gene expression and may be effective in reducing drought stress.

Environmental Effect of High-voltage Towers on the Cerebellum and Cognitive Impairments in the Monkey.

Today, high-voltage (HV) lines create a pernicious environment for humans living or working in the vicinity and even under these lines. The male rhesus monkey is used to investigate the effects of fields produced by HV towers. This study examines the function and level of impact in rhesus monkeys' brains from the cerebellum's cognitive, biological, and structural perspective. Two monkeys have been used, one as a control and the second as a test. The monkey under test was subjected to a simulated HV electrical field of 3 kV/m, 4 hours a day, for 1 month. Behavioral tests were performed using a device designed and built for this purpose. Concentration analysis of adrenocorticotropic hormones (ACTH) and inspection of glucocorticoid receptor gene's (GR) expression were performed by the reverse transcription polymerase chain reaction method. Changes in cerebellar anatomy were examined with magnetic resonance imaging (MRI). All tests were performed before and after the study period and compared with the control monkey. Cognitive tests showed a significant reduction for the monkey exposed to the HV electrical field in the first week after imposition compared with the same time before. Also, the expression of the GR gene decreased, and the concentration of ACTH hormone in plasma increased. Surveying the level of cerebral MRI images did not show any difference, but hemorrhage was evident in a part of the cerebellum. The tested monkey's cognitive, biological, and MRI results showed a decrease in visual learning and memory indices.

Impact of climate factors and climate-gene interaction on systemic lupus erythematosus patients' response to glucocorticoids therapy.

Glucocorticoids (GCs) were the essential drugs for systemic lupus erythematosus (SLE). However, different patients differ substantially in their response to GCs treatment. Our current study aims at investigating whether climate variability and climate-gene interaction influence SLE patients' response to the therapy of GCs. In total, 778 SLE patients received therapy of GCs for a study of 12-week follow-up. The efficacy of GCs treatment was evaluated using the Systemic Lupus Erythematosus Disease Activity Index. The climatic data were provided by China Meteorological Data Service Center. Additive and multiplicative interactions were examined. Compared with patients with autumn onset, the efficacy of GCs in patients with winter onset is relatively poor (ORadj = 1.805, 95%CIadj : 1.181-3.014, padj = 0.020). High mean relative humidity during treatment decreased the efficacy of GCs (ORadj = 1.033, 95%CIadj : 1.008-1.058, padj = 0.011), especially in female (ORadj = 1.039, 95%CIadj : 1.012-1.067, padj = 0.004). There was a significant interaction between sunshine during treatment and TRAP1 gene rs12597773 on GCs efficacy (Recessive model: AP = 0.770). No evidence of significant interaction was found between climate factors and the GR gene polymorphism on the improved GCs efficacy in the additive model. Multiplicative interaction was found between humidity in the month prior to treatment and GR gene rs4912905 on GCs efficacy (Dominant model: OR = 0.470, 95%CI: 0.244-0.905, p = 0.024). Our findings suggest that climate variability influences SLE patients' response to the therapy of GCs. Interactions between climate and TRAP1/GR gene polymorphisms were related to GCs efficacy. The results guide the individualized treatment of SLE patients.

Дослідження зв'язку фенотипу бронхіальна астма-ожиріння з ER22/23EK і TTH111I поліморфізмами гена глюкокортикоїдного рецептора

(GR) gene with the body mass index (BMI) of patients with bronchial asthma (BA), considering the age of BA onset. Materials and methods. 553 patients with BA and 95 practically healthy persons who previously signed an informed consent to participate in the study were examined. Patients were divided into two clinical groups according to the age of BA onset. Group I included 282 patients with late-onset of asthma (late asthma phenotype), and Group II included 271 patients with early onset (early asthma phenotype). The diagnosis of BA and the severity of the course were established according to the recommendations of GINA-2016 and subsequent versions. Diagnosis of obesity was carried out in accordance with the Order of the Ministry of Health of Ukraine dated August 5, 2009, No. 574, and the European Association for the Study of Obesity (EASO, 2016). The study was approved by the Bioethics Committee of the Medical Institute of Sumy State University. Determination of ER22/23EK (rs 6189/6190) and Tth111I (rs10052957) polymorphisms of the GR gene was performed using the polymerase chain reaction followed by the analysis of restriction fragments. Statistical analysis of the obtained results was carried out using the SPSS-17 program. The results. The analysis of anthropometric parameters showed that among the examined patients with BA, there were 152 (27.5 %) patients with normal body weight (NBW), 206 (37.3 %) were overweight, and 195 (35.2 %) with obesity. Visceral type of obesity was verified among all patients. It was established that among overweight and obese BA patients, there was a higher frequency of GG genotype according to the ER22/23EK polymorphism of the GR gene compared to patients with NBW. Heterozygotes were found 5.6 and 3.5 times more often in patients with normal body weight compared to patients with obesity. Analysis of the ratio of G and A alleles depending on BMI shows a higher frequency of the G allele in obese patients compared to patients with NBW. The distribution of alleles and genotypes according to the Tth111I polymorphism in the examined patients with BA depending on the BMI shows a twice higher frequency of homozygotes for the main C allele in overweight and patients with obesity compared to patients with NBW. Carriers of homozygotes for the minor allele were detected 4.7 times and 2.1 times more often in patients with NBW compared to overweight patients and with obesity. A probable difference in the distribution of alleles and genotypes according to the Tth111I polymorphism of the GR gene was established in patients with early and late BA (p = 0.001). Carriers of homozygotes for the main allele of CC were found more often in patients with early and late BA in the presence of obesity. Conclusions. A higher frequency of the GG genotype according to the ER22/23EK polymorphism of the GR gene and homozygotes according to the main CC allele according to the Tth111I polymorphism of the GR gene in overweight BA patients and with obesity compared to patients with NBW was proven. The protective role of the ER22/23EK polymorphism of the GR gene in relation to the occurrence of obesity in the dominant, superdominant and additive models of inheritance and the Tth111I polymorphism of the GR gene in the superdominant model was established. A higher frequency of carriers of homozygotes for the main C allele in patients with early-onset and late-onset BA in the presence of obesity compared to patients with NBW, a protective role of the Tth111I polymorphism of the GR gene on the risk of developing obesity in patients with late-onset BA in superdominant and recessive models of inheritance was established. Key words: bronchial asthma, obesity, onset, course, ER22/23EK, and Tth111I polymorphisms of the glucocorticoid receptor gene.

LBODP010 A Case of Glucocorticoid Resistance Syndrome

Abstract Case presentation A 55 yr old female presented with persistent mild elevation of cortisol for the previous 7 years. Four years prior to presentation, she was incidentally noted to have a small left sided adrenal nodule on CT scan of the abdomen. Biochemical evaluation of the nodule showed 24 hour urine cortisol of 176(0-50), morning cortisol of 36 ug/dl(6.2-19.4), ACTH 18 pg/ml(7.3-66.3), two out of three salivary samples with elevated cortisol. High dose dexamethasone suppressed ACTH to <1.1 from 13.8, cortisol from 30 to 3.3. She did not have signs and symptoms of Cushing's syndrome, mineralocorticoid excess nor androgen excess. Serum testosterone and DHEA were normal. DEXA revealed normal bone density. MRI brain demonstrated normal pituitary . She had no family history of pituitary or adrenal tumors nor endocrine syndromes. She underwent laparoscopic left adrenalectomy with pathology demonstrating benign adrenal adenoma, although the pathologist was unable to exclude nodular hyperplasia due to fragmented specimen. Elevation of 24 hour urine cortisol, serum cortisol and ACTH have persisted after surgery, without any clinical features of Cushing's. Gene sequencing with copy number analysis revealed heterozygous variant in the NR3C1 gene c.2239A>T(p. Ile747Phe), suspicious for autosomal dominant hereditary primary glucocorticoid resistance syndrome also known as Chrousos syndrome. Discussion Glucocorticoid resistance syndrome is rare, may be familial or sporadic, and is characterized by end organ insensitivity to glucocorticoids. Glucocorticoids are steroid hormones synthesized and secreted by adrenal cortex. The actions of glucocorticoids are mediated by the glucocorticoid receptor, which belongs to nuclear receptor family of ligand-dependent transcription factors. Affected individuals have elevations in circulating cortisol and ACTH, but no clinical evidence of hypercortisolism. Excess ACTH can result in increased mineralocorticoid and/or androgenic activity resulting in HTN and/or hypokalemic alkalosis and acne/hirsutism/infertility/male pattern hair loss. Asymptomatic, normotensive patients with primary glucocorticoid resistance do not require any treatment. Aim of treatment is to suppress excess ACTH to decrease mineralocorticoid and androgen effects. Treatment involves high doses of dexamethasone(1-3 mg/day), which activate the mutated and/or wild type hGR alpha and suppresses endogenous secretion of ACTH. Conclusion Mutations in human GR gene impair one or more molecular mechanisms of glucocorticoid action affecting signal transduction and altering tissue sensitivity to these hormones. Recognition of this condition avoids unnecessary adrenal surgery and informs appropriate treatment and long term follow up of patients and family members. Presentation: No date and time listed

Differential Diagnosis of Major Depressive Disorder and Bipolar Disorder: Genetic and Hormonal Assessment and the Influence of Early-Life Stress.

Few studies have assessed biomarkers for the differentiation of major depressive disorder (MDD) and bipolar disorder (BD). However, some elements of depression such as hormones and receptors of the renin-angiotensin-adrenal system (RAAS), the hypothalamus-pituitary-adrenal (HPA) axis, and history of early-life stress (ELS) could be considered for differential diagnosis. Therefore, this study aimed to assess aldosterone and cortisol levels, MR and GR gene polymorphisms, and ELS as potential biomarkers for differentiating MDD and BD. This study presents a case-control design. Groups comprised samples for genetic, cortisol, and aldosterone analysis: healthy control (HC; n = 113/97/103), MDD (n = 78/69/67) and BD (n = 82/68/65) subjects. Furthermore, all subjects were assessed for diagnostic screening, the severity of depression, and history of ELS by applying MINI-PLUS, GRID-HDRS, and CTQ, respectively. In addition, genotype and allelic frequencies of GR (N363S, R22/23K and BclI) and MR (MI180V and -2G/C) polymorphisms were evaluated via PCR. Our findings demonstrate that basal aldosterone levels may be a biomarker for differentiating BD and MDD. Furthermore, ELS affects the HPA axis in BD, cortisol may be considered a biomarker for distinguishing BD and MDD, but only in the absence of ELS, and, finally, history of ELS and MR-2G/C variant alleles are factors that contribute to the severity of depressive symptoms in MDD and BD.

Impact of maternal physical exercise on inflammatory and hypothalamic-pituitary-adrenal axis markers in the brain and lungs of prenatally stressed neonatal mice.

This study aimed to evaluate the effects of maternal exercise on alterations induced by prenatal stress in markers of the inflammatory process and the hypothalamic-pituitary-adrenal axis in the brain and lungs of neonatal mice. Female Balb/c mice were divided into three groups: control, prenatal restraint stress, prenatal restraint stress and physical exercise before and during the gestational period. On day 0 (PND0) and 10 (PND10), mice were euthanized for brain and lung analyses. The gene expression of GR, MR, IL-6, IL-10, and TNF in the brain and lungs and the protein expression of MMP-2 in the lungs were analyzed. Maternal exercise reduced IL-6 and IL-10 gene expression in the brain of PND0 mice. Prenatal stress and maternal exercise decreased GR, MR, IL-6, and TNF gene expression in the lungs of PND0 mice. In the hippocampus of PND10 females, exercise inhibited the effects of prenatal stress on the expression of MR, IL-6, and IL-10. In the lungs of PND10 females, exercise prevented the decrease in GR expression caused by prenatal stress. In the hippocampus and lungs of PND10 males, prenatal stress decreased GR gene expression. Our findings confirm the effects induced by prenatal stress and demonstrate that physical exercise before and during the gestational period may have a protective role on inflammatory changes.

Association of the Glucocorticoid Receptor Gene Polymorphisms with TGF-β1 mRNA Expression in Childhood Asthma

Background: Glucocorticoid (GC) is a fundamental drug used to treat asthma. GC binds to its corresponding receptor (GR) to formulate a complex that increases the production of anti-inflammatory factors and decreases the amount of pro-inflammatory mediators, covering many cytokines. GR is a nuclear receptor superfamily protein, encoded by NR3C1 gene. Studies suggest that polymorphisms of the NR3C1 gene contribute to a decreased response to GC for the treatment of asthma, even leading to drug-resistance. Also, TGF-β1 plays a central role in airway remodeling, GC significantly inhibits the production of TGF-β1, and TGF-β1 can induce GC resistance. Thus, it is possible that the polymorphisms of the NR3C1 gene can affect the expression of TGF-β1 mRNA and tissue remodeling. Objectives: This study evaluates the effect of polymorphisms (TthIII1, BclI, ER22/23EK, and N363S) of the NR3C1 GR gene on TGF-β1 mRNA expression in children with asthma. Methods: The samples of this study included 52 outpatients (age range: 6 - 14 years) with asthma referred to Huai’an First People’s Hospital, Nanjing Medical University, from January 2018 to June 2019. Meanwhile, 40 healthy volunteers were included as the control group. Results: The polymorphisms of the NR3C1 GR gene were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and TGF-β1 mRNA levels were measured by real-time reverse transcription (RT)-PCR. TthIII1 and TGF-β1 mRNA expression levels had significant (P = 0.011) correlations. But BclI showed no e effect on TGF-β 1 mRNA, N363S, and ER22/23EK had not been examined. Conclusions: According to the results, there was a relationship between single nucleotide polymorphisms (SNPs) of the NR3C1 gene and TGF-β1 mRNA in asthmatic children. TthIII1 CC and CT genotype have the strongest induction effect on the expression of TGF-1. The phenomenon suggests that SNPs may be involved in the asthma pathology.

Role of Non-Coding Regulatory Elements in the Control of GR-Dependent Gene Expression.

The glucocorticoid receptor (GR, also known as NR3C1) coordinates molecular responses to stress. It is a potent transcription activator and repressor that influences hundreds of genes. Enhancers are non-coding DNA regions outside of the core promoters that increase transcriptional activity via long-distance interactions. Active GR binds to pre-existing enhancer sites and recruits further factors, including EP300, a known transcriptional coactivator. However, it is not known how the timing of GR-binding-induced enhancer remodeling relates to transcriptional changes. Here we analyze data from the ENCODE project that provides ChIP-Seq and RNA-Seq data at distinct time points after dexamethasone exposure of human A549 epithelial-like cell line. This study aimed to investigate the temporal interplay between GR binding, enhancer remodeling, and gene expression. By investigating a single distal GR-binding site for each differentially upregulated gene, we show that transcriptional changes follow GR binding, and that the largest enhancer remodeling coincides in time with the highest gene expression changes. A detailed analysis of the time course showed that for upregulated genes, enhancer activation persists after gene expression changes settle. Moreover, genes with the largest change in EP300 binding showed the highest expression dynamics before the peak of EP300 recruitment. Overall, our results show that enhancer remodeling may not directly be driving gene expression dynamics but rather be a consequence of expression activation.

Effects of running before pregnancy on long-term memory and hippocampal alterations induced by prenatal stress

Studies have shown that an adverse environment in utero influences fetal growth and development, leading to several neuroendocrine and behavioral changes in adult life. Nevertheless, the mechanisms involved in the long-term benefits of pregestational exercise are still poorly understood. Thus, this study aimed to evaluate the effects of physical exercise before the gestational period on memory behavior and gene expression in the hippocampus of adult mice submitted to prenatal stress. Female Balb/c mice were divided into three groups: control (CON), prenatal restraint stress (PNS), and exercise before the gestational period plus PNS (EX + PNS). When adults, male and female offspring were submitted to the object recognition test followed by the hippocampal evaluation of BDNF exons I and IV mRNA expression, as well as hypothalamic-pituitary-adrenal axis related genes. Pregestational exercise did not prevent the decreased recognition index, as well as GR and CRHR1 gene expression observed in PNS males. Conversely, prenatal stress did not influence female memory behavior. Moreover, exercise attenuated the effects of prenatal stress on female BDNF IV gene expression. The results indicate that pregestational exercise was able to prevent the effects of maternal stress on hippocampal BDNF IV gene expression in females, although no effects were seen on the stress-induced memory impairment in males.

EFFECT OF LEPR AND GR GENE POLYMORPHISMS ON HEALTH STATUS OF MOTHERS WITH METABOLIC DISORDERS AND THEIR NEWBORNS

The high prevalence of obesity and metabolic syndrome throughout the world over the past two decades provided the grounds to the WHO to consider overweight as a non-infectious pandemic. Among the factors that negatively affect the metabolism, result in metabolic disorders and obesity there are genetic, prenatal, diet-related, somatic, psychogenic and others. Analysis of LEPR and GR gene polymorphisms among mothers and their newborns can be an important component in the prediction of metabolic disorders and obesity. The aim of this study was to analyze the presence of clinical and genetic determinants of metabolic syndrome in mothers and their newborns. Materials and methods. A cross-sectional study of 42 pairs of mothers and newborns was performed; we analyzed their anthropometric, clinical and laboratory parameters and, in particular, 2 types of genetic polymorphisms (LEPR rs1137101 and GR, BClI polymorphism). The main group consisted of 15 mother-child pairs, where the woman was clinically obese (body mass index more than 30), the comparison group included 27 mother-child pairs, where the women were overweight (body mass index more than 25). Results. Obese mothers were significantly more likely to have a miscarriage (p = 0.01). Children from obese mothers had significantly higher percentiles of birth weight compared to the control group (60.9 ± 7.7 and 49.75 ± 4.5, respectively, p = 0.05). The GG genotype of the GR gene was associated with more common indications for hospitalization (p = 0.012), rapid delivery (p = 0.01), umbilical cord entanglement around the neck of the foetus (p = 0.012). Among the children- carriers of the GG BclI genotype GR, cardio-metabolic disorders detected by the auscultation of the heart (arrhythmia, functional noise) were found out as significantly more frequent (p = 0.003). Conclusion. The study did not find the associations between LEPR and GR gene polymorphism in obese mothers and the development of overweight in their children, but the study revealed an adverse effect of polymorphic variants on obstetric and gynaecological status of mothers, labour and adaptation of infants in the early neonatal period.

Synergistic effects of depression and NR3C1 methylation on prognosis of acute coronary syndrome

High levels of methylation in the GR gene (nuclear receptor subfamily 3, group C, member 1; NR3C1) have been associated with depression and cardiovascular risk. This study aimed to investigate whether NR3C1 methylation status was associated with the long-term prognosis of acute coronary syndrome (ACS) considering depression and cardiovascular status at the early phase of ACS. A total of 969 patients with recent ACS were recruited at a tertiary university hospital in Korea. Baseline evaluations were made from 2007 to 2012, including DSM-IV depressive disorder, NR3C1 methylation, and various demographic and clinical characteristics such as cardiovascular risk markers. Over a 5~12 year follow-up after the index ACS, time to major adverse cardiac event (MACE) was investigated using Cox regression models. Higher NR3C1 methylation status was associated with depression and several cardiovascular risk markers at baseline. NR3C1 hypermethylation predicted worse long-term prognosis of ACS only in the presence of depressive disorder with significant synergistic interaction terms and independent of potential confounding factors. Synergistic effects of depressive disorder and NR3C1 hypermethylation on long-term cardiac outcomes in ACS were found. NR3C1 methylation status represents a candidate prognostic biomarker for ACS in combination with a diagnosis of depressive disorder. Further research is needed to ascertain the generalisability of these findings.

Next Generation CRISPR Gene-Edited and Off-the-Shelf Virus-Specific T-Cells for the Immunocompromised Patient

Introduction: A number of Clinical trials have demonstrated the feasibility, safety and efficacy of cell and gene therapy for cancer, autoimmune disorders and infectious disease. Strategies that enhance the function and survival of immune cells are critical for the success of immunotherapy. We have developed a strategy for the ex vivo expansion of off-the-shelf viral-specific T cells (VSRs) from healthy donor buffy coat which have been extremely effective in eradicating refractory cytomegalovirus (CMV), polyomavirus and adenovirus infections in immunocompromised patients. Glucocorticoids commonly used to treat graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are a common cause of iatrogenically-induced immunosuppression and contribute the risk of life-threatening viral-infections. To render VSTs resistant to the lymphocytotoxic effect of glucocorticoids, we have developed a novel strategy to silence the expression of the glucocorticoid receptor using RNA-guided endonucleases CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) 9 gene editing.. Methods: The technique involves the expansion from donor blood of CMV, BKV or adenoviral-specific T cells using peptide libraries from the immunodominant viral proteins followed by CRISPR knockout of exon 2 of the GR gene on chromosome 5 of the human NR3C1 gene. Cells are electroporated with the RNP (Cas9 plus guide RNA) complex (IDT pre-designed alt-R crispr Cas9 platform) using Neon electroporation and the Amaxa 4-D nucleofector system. Results: GR knockout efficiency in ex vivo expanded virus-specific T cells was consistently > 90%. In vitro experiments confirmed the resistance of VSTs to corticosteroid treatment as assessed by annexin V assay. GR KO VSTs maintained potent antiviral activity as assessed by their ability to proliferate and release effector cytokines in response to viral antigens. Conclusions: CRISPR gene-editing to knock-out the glucocorticoid receptor gene in viral-specific T cells can preserve the activity of VSTs in the presence of corticosteroid-induced immunosuppression. Engineering runs using GMP-compliant Cas9 protein and gRNA are underway in anticipation of a clinical trial. Disclosures Champlin: Sanofi-Genzyme: Research Funding; Actinium: Consultancy; Johnson and Johnson: Consultancy.

Nascent transcript analysis of glucocorticoid crosstalk with TNF defines primary and cooperative inflammatory repression.

The glucocorticoid receptor (NR3C1, also known as GR) binds to specific DNA sequences and directly induces transcription of anti-inflammatory genes that contribute to cytokine repression, frequently in cooperation with NF-kB. Whether inflammatory repression also occurs through local interactions between GR and inflammatory gene regulatory elements has been controversial. Here, using global run-on sequencing (GRO-seq) in human airway epithelial cells, we show that glucocorticoid signaling represses transcription within 10 min. Many repressed regulatory regions reside within “hyper-ChIPable” genomic regions that are subject to dynamic, yet nonspecific, interactions with some antibodies. When this artifact was accounted for, we determined that transcriptional repression does not require local GR occupancy. Instead, widespread transcriptional induction through canonical GR binding sites is associated with reciprocal repression of distal TNF-regulated enhancers through a chromatin-dependent process, as evidenced by chromatin accessibility and motif displacement analysis. Simultaneously, transcriptional induction of key anti-inflammatory effectors is decoupled from primary repression through cooperation between GR and NF-kB at a subset of regulatory regions. Thus, glucocorticoids exert bimodal restraints on inflammation characterized by rapid primary transcriptional repression without local GR occupancy and secondary anti-inflammatory effects resulting from transcriptional cooperation between GR and NF-kB.

Non-canonical dimerization of the androgen receptor and other nuclear receptors: implications for human disease.

Nuclear receptors are transcription factors that play critical roles in development, homeostasis and metabolism in all multicellular organisms. An important family of nuclear receptors comprises those members that respond to steroid hormones, and which is subdivided in turn into estrogen receptor (ER) isoforms α and β (NR3A1 and A2, respectively), and a second subfamily of so-called oxosteroid receptors. The latter includes the androgen receptor (AR/NR3C4), the glucocorticoid receptor (GR/NR3C1), the mineralocorticoid receptor (MR/NR3C2) and the progesterone receptor (PR/NR3C3). Here we review recent advances in our understanding of the structure-and-function relationship of steroid nuclear receptors and discuss their implications for the etiology of human diseases. We focus in particular on the role played by AR dysregulation in both prostate cancer (PCa) and androgen insensitivity syndromes (AIS), but also discuss conditions linked to mutations of the GR gene as well as those in a non-steroidal receptor, the thyroid hormone receptor (TR). Finally, we explore how these recent results might be exploited for the development of novel and selective therapeutic strategies.

Preclinical findings on the potential of intranasal neuropeptide Y for treating hyperarousal features of PTSD.

Acoustic startle response (ASR) assesses hyperarousal, a core symptom of posttraumatic stress disorder (PTSD). Intranasal neuropeptide Y (NPY) administration was shown to prevent hyperarousal in single prolonged stress (SPS) rodent PTSD model. However, it is unclear how ASR itself alters responses to stress. Rats (A-S-A) were exposed to acoustic startle (AS) 1 day before SPS (ASR1) and 2 weeks afterward (ASR2). Other groups were exposed in parallel to either AS (A-A) or SPS or neither. SPS enhanced ASR2. In relevant brain areas, mRNA levels were determined by qRT-PCR. In mediobasal hypothalamus, AS or SPS each increased CRH mRNA levels without an additive effect. Exposure to AS appeared to dampen some responses to SPS. The SPS-triggered reduction of GR and FKBP5 gene expression was not observed in A-S-A group. In locus coeruleus, SPS increased CRHR1 and reduced Y2R mRNAs, but not in A-S-A group. In both regions, AS altered NPY receptor gene expression, which may mediate dampening responses to SPS. In second experiment, intranasal NPY administered 2 weeks after SPS reversed hyperarousal symptoms for at least 7 days. This study reveals important effects of AS on the NPY system and demonstrates that intranasal NPY elicits long-lasting reversal of traumatic stress-triggered hyperarousal.

Dietary trans‐cinnamic acid application for rainbow trout (Oncorhynchus mykiss): II. Effect on antioxidant status, digestive enzyme, blood biochemistry and liver antioxidant gene expression responses

The present study investigated the effects of dietary trans‐cinnamic acid (TrCin) on growth performance, biochemical composition, fatty acid composition, blood biochemistry, antioxidant status, digestive enzyme and liver antioxidant gene (EF1α, SOD1, SOD2, CAT, GPX1, GPX4, GR and GST) expression responses of rainbow trout, Oncorhynchus mykiss. Five experimental groups of fish with mean weights of 17.49 ± 0.08 g were used in the study; four of them were fed with TrCin‐supplemented diets (0.25 g/kg TrCin25, 0.50 g/kg TrCin50, 0.75 g/kg TrCin75 and 1.50 g/kg TrCin150), whereas an additive‐free basal diet served as the control (Cntr). At the end of the 60‐day feeding trial, the growth performance, biochemical composition and fatty acid composition were similar for all experimental groups. A decrease was observed in intestinal and stomach pH, serum triglyceride and AST, ALT, LDH and ALP levels in fish fed with especially 0.50 g/kg TrCin‐supplemented diet. Moreover, dietary TrCin especially at 0.50 g/kg incorporation level significantly increased the serum SOD and liver SOD2, CAT, GST, GPX1, GPX4 and GR gene expression responses. As a conclusion, feeding rainbow trout for a period of 60 days with a diet containing 0.50 g/kg TrCin might be sufficiently enough to improve the levels of antioxidant enzymes and health status in fish.

Monosodium Glutamate Triggers Neuroendocrine Stress Axis Leading to Apoptosis and Neural Progenitor Cell Activation

Can monosodium glutamate (MSG) acts as a stressor and stimulate the hypothalamic‐pituitary‐adrenal (HPA) axis? If so, can its effects be compared to Corticosterone (CORT)? In order to test this hypothesis, we put forth a Glutamate model and CORT induced model system in rats to study the orchestration of molecular signaling pathways. Young adult (2 month old) male Wistar rats weighing about 50–100 g body weight were divided into 4 groups. Group I control rats were injected with saline (NaCl 0.9%) and Group II rats with MSG at a dose of 4 mg/kg body weight. Group III control was ethanol‐saline injected and Group IV was CORT injected at 3 mg/kg body weight. Rats were injected subcutaneously with test materials or vehicles for 7 days and maintained for 8, 15 and 30 days and were sacrificed by decapitation. The MSG model showed significant enhanced Crh gene expression in the hypothalamus as an immediate response to stress. On day 15, the expression level was reduced with a significant increase on day 30. Similarly pituitary Pomc mRNA expression was significantly upregulated on day 8 and 30 with an insignificant downregulation on day 15. GR (Nr3c1) gene expression in the cerebral cortex was significantly upregulated on day 8, a normalization on day 15 and a very significant down regulation on day 30. There was no change in the plasma corticosterone. Caspase‐3 showed an upregulated expression in the hypothalamus on day 8, indicating the activation of Caspase‐3‐dependent apoptotic pathways at the mRNA level. Nestin, a neural stem/progenitor cell marker showed a significant up regulation on day 15 followed by normalized expression on day 30. In the CORT model, Crh gene expression in the hypothalamus showed a significant downregulation on day 8, transient normalization on day 15 and a very significant downregulation on day 30. Pomc mRNA showed a significant downregulation on day 8. GR (Nr3c1) displayed an insignificant downregulation across the three time points. In the CORT group, plasma CORT concentration was not changed significantly across the time points when compared to the control. Nestin gene showed a significant downregulation on day 8. Our results reaffirm that the negative feedback is activated in the CORT model, thereby no cell death and repair. Furthermore, our results raise the question whether there is a shift in the negative feedback mechanism (shut off) activated in MSG‐induced stress? The elevated HPA axis sensitization during the MSG exposure is probably due to impaired negative feedback mechanism or an increased intrinsic excitability of the CRH neurons. This feedback escape may, in turn, attenuate presynaptic glutamate input to the CRH neurons resulting in glutamate neurotoxicity. MSG triggers the apoptotic signaling cascade, thereby causing the neural stem cell to onset repair machinery, to suggest that MSG is a stressor and a neurotoxin.Support or Funding InformationCUSAT Postdoctoral Fellowship‐Dr. Smita Mathew. U.O. No.Ac.B3/PDF/2016‐17(b) dt. 10.5.2017This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.