Donor‐Dependent and Other Nondefined Factors Have Greater Influence on the Hepatic Phenotype Than the Starting Cell Type in Induced Pluripotent Stem Cell Derived Hepatocyte‐Like Cells

Drug‐induced liver injury is the greatest cause of post‐marketing drug withdrawal; therefore, substantial resources are directed toward triaging potentially dangerous new compounds at all stages of drug development. One of the major factors preventing effective screening of new compounds is the lack of a predictive in vitro model of hepatotoxicity. Primary human hepatocytes offer a metabolically relevant model for which the molecular initiating events of hepatotoxicity can be examined; however, these cells vary greatly between donors and dedifferentiate rapidly in culture. Induced pluripotent stem cell (iPSC)‐derived hepatocyte‐like cells (HLCs) offer a reproducible, physiologically relevant and genotypically normal model cell; however, current differentiation protocols produce HLCs with a relatively immature phenotype. During the reprogramming of somatic cells, the epigenome undergoes dramatic changes; however, this “resetting” is a gradual process, resulting in an altered differentiation propensity, skewed toward the lineage of origin, particularly in early passage cultures. We, therefore, performed a comparison of human hepatocyte‐ and dermal fibroblast‐derived iPSCs, assessing the impact of epigenetic memory at all stages of HLC differentiation. These results provide the first isogenic assessment of the starting cell type in human iPSC‐derived HLCs. Despite a trend toward improvement in hepatic phenotype in albumin secretion and gene expression, few significant differences in hepatic differentiation capacity were found between hepatocyte and fibroblast‐derived iPSCs. We conclude that the donor and inter‐clonal differences have a greater influence on the hepatocyte phenotypic maturity than the starting cell type. Therefore, it is not necessary to use human hepatocytes for generating iPSC‐derived HLCs. Stem Cells Translational Medicine 2017;6:1321–133

Polymorphic variants of SCN1A and EPHX1 influence plasma carbamazepine concentration, metabolism and pharmacoresistance in a population of Kosovar Albanian epileptic patients

Aim The present study aimed to evaluate the effects of gene variants in key genes influencing pharmacokinetic and pharmacodynamic of carbamazepine (CBZ) on the response in patients with epilepsy. Materials & Methods Five SNPs in two candidate genes influencing CBZ transport and metabolism, namely ABCB1 or EPHX1, and CBZ response SCN1A (sodium channel) were genotyped in 145 epileptic patients treated with CBZ as monotherapy and 100 age and sex matched healthy controls. Plasma concentrations of CBZ, carbamazepine-10,11-epoxide (CBZE) and carbamazepine-10,11-trans dihydrodiol (CBZD) were determined by HPLC-UV-DAD and adjusted for CBZ dosage/kg of body weight. Results The presence of the SCN1A IVS5-91G>A variant allele is associated with increased epilepsy susceptibility. Furthermore, carriers of the SCN1A IVS5-91G>A variant or of EPHX1 c.337T>C variant presented significantly lower levels of plasma CBZ compared to carriers of the common alleles (0.71±0.28 vs 1.11±0.69 μg/mL per mg/Kg for SCN1A IVS5-91 AA vs GG and 0.76±0.16 vs 0.94±0.49 μg/mL per mg/Kg for EPHX1 c.337 CC vs TT; PG showed a reduced microsomal epoxide hydrolase activity as reflected by a significantly decreased ratio of CBZD to CBZ (0.13±0.08 to 0.26±0.17, pT SNP and SCN1A 3148A>G variants were not associated with significant changes in CBZ pharmacokinetic. Patients resistant to CBZ treatment showed increased dosage of CBZ (657±285 vs 489±231 mg/day; P<0.001) but also increased plasma levels of CBZ (9.84±4.37 vs 7.41±3.43 μg/mL; P<0.001) compared to patients responsive to CBZ treatment. CBZ resistance was not related to any of the SNPs investigated. Conclusions The SCN1A IVS5-91G>A SNP is associated with susceptibility to epilepsy. SNPs in EPHX1 gene are influencing CBZ metabolism and disposition. CBZ plasma levels are not an indicator of resistance to the therapy

Lewis acid mediated cascade reactions of silyl-substituted methylenecyclopropyl ketones

The Lewis acid mediated cyclisation of various silyl-substituted methylenecyclopropyl ketones has been investigated. The presence of the silyl-substituent enhances the reactivity of the methylene cyclopropane in comparison to our earlier study on non-silyl-substituted methylenecyclopropyl ketones, allowing milder Lewis acids (BF3 . Et2O or BF3 . 2AcOH) to be used for the cyclisation reaction. The mild conditions used allow the allyl cation, formed as an intermediate in the cyclisation, to be trapped in further carbon-carbon bond-forming reactions

Nuclear factor-erythroid 2 (NF-E2) p45-related factor-2 (Nrf2) modulates dendritic cell immune function through regulation of p38 MAPK-cAMP-responsive element binding protein/activating transcription factor 1 signaling.

Nrf2 is a redox-responsive transcription factor that has been implicated in the regulation of DC immune function. Loss of Nrf2 results in increased co-stimulatory molecule expression, enhanced T cell stimulatory capacity, and increased reactive oxygen species (ROS) levels in murine immature DCs (iDCs). It is unknown whether altered immune function of Nrf2-deficient DCs (Nrf2(-/-) iDCs) is due to elevated ROS levels. Furthermore, it is unclear which intracellular signaling pathways are involved in Nrf2-mediated regulation of DC function. Using antioxidant vitamins to reset ROS levels in Nrf2(-/-) iDCs, we show that elevated ROS is not responsible for the altered phenotype and function of these DCs. Pharmacological inhibitors were used to explore the role of key MAPKs in mediating the altered phenotype and function in Nrf2(-/-) iDCs. We demonstrate that the increased co-stimulatory molecule expression (MHC II and CD86) and antigen-specific T cell activation capacity observed in Nrf2(-/-) iDCs was reversed by inhibition of p38 MAPK but not JNK. Importantly, we provide evidence for increased phosphorylation of cAMP-responsive element binding protein (CREB) and activating transcription factor 1 (ATF1), transcription factors that are downstream of p38 MAPK. The increased phosphorylation of CREB/ATF1 in Nrf2(-/-) iDCs was sensitive to p38 MAPK inhibition. We also show data to implicate heme oxygenase-1 as a potential molecular link between Nrf2 and CREB/ATF1. These results indicate that dysregulation of p38 MAPK-CREB/ATF1 signaling axis underlies the altered function and phenotype in Nrf2-deficient DCs. Our findings provide new insights into the mechanisms by which Nrf2 mediates regulation of DC function