The effects of Ridostin on the transcription of IFN family genes in human fibroblasts and lymphocytes were studied by quantitative real-time PCR. The degree of gene induction by Ridostin was most pronounced in fibroblasts, and was significantly higher than the induction by Kagocel: transcription of IFN-β, oligoadenylate synthetase, and double-stranded RNA-dependent protein kinase genes increased by about 2000, 100, and 20 times, respectively. In lymphocytes, Ridostin also activated a wide variety of IFN family genes, including genes of IFN-β, IFN-γ, and IFN-dependent enzymes, but this induction was less pronounced than in the fibroblasts. It was shown that gene response in lymphocyte from a child with cancer is reduced in comparison with that of adult healthy participant. Ridostin, and even more so Reaferon up-regulated activities of β-actin, glycerophosphate dehydrogenase, and β2-microglobulin genes, thus making impossible or limiting their use as constitutive stable reference genes (standards) in PCR-assays of IFN and their inductors.
Objective. To study of known interferons (IFN) type I and IFN-inductors drugs different chemical structure on cell differentiation and expression group of genes of TLR/RLRs, referring to group of pattern-recognition receptors. Materials and methods. Cellular lines ТНР-1 (acute monocytic leukemia) и НСТ-116 (colon adenocarcinoma) were treated by interferons drugs Reaferon, Altevir, Genfakson, Infibeta 10 5-10 6 МЕ, IFN-inductors Ridostin 10 2-10 3 μg/ml, Cycloferon 625 μg/ml and Imunomax 2 МЕ during 24 h or 96 h at 37 °С. Gene expression were estimated by method qRT-PCR (CFX-96, Bio-Rad). CD-immunophenotypes of ТНР-1 cells were detected by flow cytometric fluorescence method with FITC and PE monoclonal antibodies (FACSCanto II, Becton Dickinson). Results. It was shown that tumor cells THP-1 and HCT-116 have low and variable constitutive levels gene expression of TLR-receptors 2, 3, 4, 7, 8, 9 during passages. This gene status may be connected with incorrect processing of mature mRNA forms. Genes TLR4, TLR8 and factor NFkB are stimulated by interferons and IFN-inductors more high in THP-cells and genes TLR7, TLR8, TLR9 and NFkB-in HCT-cells. Ridostin (mix ssRNA and dsRNA S. cerevisiae) is the best activator of TLR/RLRs receptors. Ridostin increases
Objective: to study drugs ingavirin and thymogen as activators of signal TLR and RLR reactions in a sensitive cell model of THP-1 monocytes and blood cells of donors.Materials and methods . Investigated drugs ingavirin (imidazolylethanamide pentanedioic acid – 6-[2-(1H-imidazol-4-yl)ethylami- no]-5-oxohexanoic acid; Valenta Pharmaceutics, Russia) and thymogen (alpha-glutamyl-tryptophan; Cytomed, Russia), registered in Russia as medicines. The expression of TLR/RLR receptor genes was determined under the action of ingavirin 50–300 μg/ml and thymogen 0.1–5 μg/ml (24 h, 37 °C) using quantitative RT-PCR. The level of fluid cytokines was determined using ELISA kits (Vec- tor-Best, Russia) in the culture fluid. Transfection of small inhibitory RNA (siRNA) MAVS was performed using the reagent Lipofect- amine 2000 (Invitrogen). The immunophenotype of the THP-1 cell line was determined by flow cytometry with labeled monoclonal antibodies FITC CD14 and PE CD34 (BD Biosciences) on a FACSCanto II instrument (Becton Dickinson).Results . For the first time, it has been shown that ingavirin (imidazolylethanamide) and thymogen (dipeptide Glu-Trp) preparations are activators of the immune TLR/RLR receptors and their signaling factors genes in the cultures of monocytic leukemia THP-1 and blood of healthy donors. In these cellular systems, ingavirin and thymogen preparations elicited similar immune responses and stimulated the expression of genes: endosomal TLR3/7/8/9 receptors, RIG1/MDA5 cytoplasmic sensors and NFκB1 and MAVS signaling factors. Induced cells secrete inflammatory cytokines of TNF-α and IL1-β. Ingavirin in THP-1 cell culture monocytes caused a decrease in CD34+ blast cells. Activation the genes of MAVS and co-receptor B2M of the main histocompatibility complex (MHCII) by ingavirin were interrelated. Transfection of siRNA MAVS reduced the level of homologous mRNA MAVS and heterologous mRNA B2M. Conclusion . The results obtained suggest that the antiviral and immunomodulating properties of the drugs ingavirin and thymogen are associated with the activation of a group of TLR/RLR signaling pathways of the innate and adaptive immunity and the differentiation of hematopoietic cell precursors.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.