Teriflunomide Is an Indirect Human Constitutive Androstane Receptor (CAR) Activator Interacting With Epidermal Growth Factor (EGF) Signaling

The constitutive androstane receptor (CAR) is a nuclear receptor involved mainly in xenobiotic and endobiotic metabolism regulation. CAR is activated directly by its ligands via the ligand binding domain (LBD) or indirectly by inhibition of the epidermal growth factor (EGF) signaling. We found that leflunomide (LEF) and its main metabolite teriflunomide (TER), both used for autoimmune diseases treatment, induce the prototype CAR target gene CYP2B6 in primary human hepatocytes. As TER was discovered to be an EGF receptor antagonist, we sought to determine if TER is an indirect activator of CAR. In primary human hepatocytes and in differentiated HepaRG cells, we found that LEF and TER up-regulate CAR target genes CYP2B6 and CYP3A4 mRNAs and enzymatic activities. TER stimulated CAR+A mutant translocation into the nucleus but neither LEF nor TER activated the CAR LBD, CAR3 variant or pregnane X receptor (PXR) in gene reporter assays. Interestingly, TER significantly up-regulated CAR mRNA expression, a result which could be a consequence of both EGF receptor and ELK-1 transcription factor inhibition by TER or by TER-mediated activation of glucocorticoid receptor (GR), an upstream hormonal regulator of CAR. We can conclude that TER is a novel indirect CAR activator which through EGF inhibition and GR activation controls both detoxification and some intermediary metabolism genes

Laminin-511 and laminin-521-based matrices for efficient hepatic specification of human pluripotent stem cells

Human pluripotent stem cells (hPSCs) have gained a solid foothold in basic research and drug industry as they can be used in vitro to study human development and have potential to offer limitless supply of various somatic cell types needed in drug development. Although the hepatic differentiation of hPSCs has been extensively studied, only a little attention has been paid to the role of the extracellular matrix. In this study we used laminin-511, laminin-521, and fibronectin, found in human liver progenitor cells, as culture matrices for hPSC-derived definitive endoderm cells. We observed that laminin-511 and laminin-521 either alone or in combination support the hepatic specification and that fibronectin is not a vital matrix protein for the hPSC-derived definitive endoderm cells. The expression of the laminin-511/521-specific integrins increased during the definitive endoderm induction and hepatic specification. The hepatic cells differentiated on laminin matrices showed the upregulation of liver-specific markers both at mRNA and protein levels, secreted human albumin, stored glycogen, and exhibited cytochrome P450 enzyme activity and inducibility. Altogether, we found that laminin-511 and laminin-521 can be used as stage-specific matrices to guide the hepatic specification of hPSC-derived definitive endoderm cells. 2016 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND licensePeer reviewe

Genetic Predispositions of Glucocorticoid Resistance and Therapeutic Outcomes in Polymyalgia Rheumatica and Giant Cell Arteritis

Polymyalgia rheumatica (PMR) and giant cell arteritis (GCA) are closely related chronic inflammatory diseases. Glucocorticoids (GCs) are first-choice drugs for PMR and GCA, although some patients show poor responsiveness to the initial GC regimen or experience flares after GC tapering. To date, no valid biomarkers have been found to predict which patients are at most risk for developing GC resistance. In this review, we summarize PMR- and GCA-related gene polymorphisms and we associate these gene variants with GC resistance and therapeutic outcomes. A limited number of GC resistance associated-polymorphisms have been published so far, mostly related to HLA-DRB1*04 allele. Other genes such ICAM-1, TLR4 and 9, VEGF, and INFG may play a role, although discrepancies are often found among different populations. We conclude that more studies are required to identify reliable biomarkers of GC resistance. Such biomarkers could help distinguish non-responders from responders to GC treatment, with concomitant consequences for therapeutic strategy

Radiolabeled 15-mer peptide internalization is mediated by megalin (LRP2 receptor) in a CRISPR/Cas9-based LRP2 knockout human kidney cell model

Abstract Background Megalin (LRP2 receptor) mediates the endocytosis of radiolabeled peptides into proximal tubular kidney cells, which may cause nephrotoxicity due to the accumulation of a radioactive tracer. The study aimed to develop a cellular model of human kidney HK2 cells with LRP2 knockout (KO) using CRISPR/Cas9 technique. This model was employed for the determination of the megalin-mediated accumulation of 68Ga- and 99mTc-labeled 15-mer peptide developed to target the vascular endothelial growth factor (VEGF) receptor in oncology radiodiagnostics. Results The gene editing in the LRP2 KO model was verified by testing two well-known megalin ligands when higher viability of KO cells was observed after gentamicin treatment at cytotoxic concentrations and lower FITC-albumin internalization by the KO cells was detected in accumulation studies. Fluorescent-activated cell sorting was used to separate genetically modified LRP2 KO cell subpopulations. Moreover, flow cytometry with a specific antibody against megalin confirmed LRP2 knockout. The verified KO model identified both 68Ga- and 99mTc-radiolabeled 15-mer peptides as megalin ligands in accumulation studies. We found that both radiolabeled 15-mers enter LRP2 KO HK2 cells to a lesser extent compared to parent cells. Differences in megalin-mediated cellular uptake depending on the radiolabeling were not observed. Using biomolecular docking, the interaction site of the 15-mer with megalin was also described. Conclusion The CRISPR/Cas9 knockout of LRP2 in human kidney HK2 cells is an effective approach for the determination of radiopeptide internalization mediated by megalin. This in vitro method provided direct molecular evidence for the cellular uptake of radiolabeled anti-VEGFR 15-mer peptides via megalin. Graphical abstrac

The pregnane X receptor down-regulates organic cation transporter 1 (SLC22A1) in human hepatocytes by competing for (“squelching”) SRC-1 coactivator

International audienc

A Review: Comparison of Approaches to the Approval Process and Methodology for Estimation of Ammonia Emissions from Livestock Farms under IPPC

Ammonia (NH3) emissions have a negative impact on the welfare of breeding animals, human health, and the environment. These influences of modern intensive agriculture have led to numerous protocols, national regulations, and European Directives. Following previous regulatory measures, the Commission Implementing Decision European Union (EU) 2017/302 on 15 February 2017 has established best available technique (BAT) conclusions, under Directive 2010/75/EU of the European Parliament and the Council, for the intensive rearing of poultry and pigs. This applies to intensive poultry and pig producers with a capacity of over 40,000 poultry, 750 sows, or 2000 fattening pigs. Due to the application of this directive, air emissions have been reduced by between 40% and 75% over the last 15 years. The integrated permit monitors the entire environmental burden of the farm on its surroundings (air pollution, water, soil pollution, waste production, energy use). This review aims to provide a critical overview of how member states (including the United Kingdom) are approaching the implementation of IPPC (Integrated Pollution Prevention and Control) and the conclusions of BAT in their legislation and related documents, and how they monitor NH3 emissions from intensive livestock farming. The data for this review were obtained from 2019 to 2020

A Review: Comparison of Approaches to the Approval Process and Methodology for Estimation of Ammonia Emissions from Livestock Farms under IPPC

Ammonia (NH3) emissions have a negative impact on the welfare of breeding animals, human health, and the environment. These influences of modern intensive agriculture have led to numerous protocols, national regulations, and European Directives. Following previous regulatory measures, the Commission Implementing Decision European Union (EU) 2017/302 on 15 February 2017 has established best available technique (BAT) conclusions, under Directive 2010/75/EU of the European Parliament and the Council, for the intensive rearing of poultry and pigs. This applies to intensive poultry and pig producers with a capacity of over 40,000 poultry, 750 sows, or 2000 fattening pigs. Due to the application of this directive, air emissions have been reduced by between 40% and 75% over the last 15 years. The integrated permit monitors the entire environmental burden of the farm on its surroundings (air pollution, water, soil pollution, waste production, energy use). This review aims to provide a critical overview of how member states (including the United Kingdom) are approaching the implementation of IPPC (Integrated Pollution Prevention and Control) and the conclusions of BAT in their legislation and related documents, and how they monitor NH3 emissions from intensive livestock farming. The data for this review were obtained from 2019 to 2020

Additional file 1 of Radiolabeled 15-mer peptide internalization is mediated by megalin (LRP2 receptor) in a CRISPR/Cas9-based LRP2 knockout human kidney cell model

Additional file 1. The additional file contains information pertaining to the detailed methodology of sgRNA design, peptide synthesis, radiolabeling and purity control, additional results of flow cytometry analysis and FACS sorting, full-length Western blot images, and radiochromatograms of both peptides

3β-Isoobeticholic acid efficiently activates the farnesoid X receptor (FXR) due to its epimerization to 3α-epimer by hepatic metabolism

Bile acids (BAs) are important signaling molecules acting via the farnesoid X nuclear receptor (FXR) and the membrane G protein-coupled bile acid receptor 1 (GPBAR1). Besides deconjugation of BAs, the oxidoreductive enzymes of colonic bacteria and hepatocytes enable the conversion of BAs into their epimers or dehydrogenated forms. Obeticholic acid (OCA) is the first-in-class BA-derived FXR agonist approved for the treatment of primary biliary cholangitis. Herein, a library of OCA derivatives, including 7-keto, 6-ethylidene derivatives and 3β-epimers, was synthetized and investigated in terms of interactions with FXR and GPBAR1 in transaction assays and evaluated for FXR target genes expression in human hepatocytes and C57BL/6 mice. The derivatives were further subjected to cell-free analysis employing in silico molecular docking and a TR-FRET assay. The conversion of the 3βhydroxy epimer and its pharmacokinetics in mice were studied using LC-MS. We found that only the 3β-hydroxy epimer of OCA (3β-isoOCA) possesses significant activity to FXR in hepatic cells and mice. However, in a cell-free assay, 3β-isoOCA had about 9-times lower affinity to FXR than did OCA. We observed that 3β-isoOCA readily epimerizes to OCA in hepatocytes and murine liver. This conversion was significantly inhibited by the hydroxy-Δ5-steroid dehydrogenase inhibitor trilostane. In addition, we found that 3,7-dehydroobeticholic acid is a potent GPBAR1 agonist. We conclude that 3β-isoOCA significantly activates FXR due to its epimerization to the more active OCA by hepatic metabolism. Other modifications as well as epimerization on the C3/C7 positions and the introduction of 6-ethylidene in the CDCA scaffold abrogate FXR agonism and alleviate GPBAR1 activation