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Targeted therapy of human leukemia xenografts in immunodeficient zebrafish
Personalized medicine holds tremendous promise for improving safety and efficacy of drug therapies by optimizing treatment regimens. Rapidly developed patient-derived xenografts (pdx) could be a helpful tool for analyzing the effect of drugs against an individual’s tumor by growing the tumor in an immunodeficient animal. Severe combined immunodeficiency (SCID) mice enable efficient in vivo expansion of vital tumor cells and generation of personalized xenografts. However, they are not amenable to large-scale rapid screening, which is critical in identifying new compounds from large compound libraries. The development of a zebrafish model suitable for pdx could facilitate large-scale screening of drugs targeted against specific malignancies. Here, we describe a novel strategy for establishing a zebrafish model for drug testing in leukemia xenografts. We used chronic myelogenous leukemia and acute myeloid leukemia for xenotransplantation into SCID zebrafish to evaluate drug screening protocols. We showed the in vivo efficacy of the ABL inhibitor imatinib, MEK inhibitor U0126, cytarabine, azacitidine and arsenic trioxide. We performed corresponding in vitro studies, demonstrating that combination of MEK- and FLT3-inhibitors exhibit an enhanced effect in vitro. We further evaluated the feasibility of zebrafish for transplantation of primary human hematopoietic cells that can survive at 15 day-post-fertilization. Our results provide critical insights to guide development of high-throughput platforms for evaluating leukemia.
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Prescription Medications Alter Neuronal and Glial Cholesterol Synthesis
Mouse brain contains over 100 million neuronal, glial, and other support cells. Developing neurons and astrocytes synthesize their own cholesterol, and disruption of this process can occur by both genetic and chemical mechanisms. In this study we have exposed cultured murine neurons and astrocytes to six different prescription medications that cross the placenta and blood–brain barriers and analyzed the effects of these drugs on cholesterol biosynthesis by an LC–MS/MS protocol that assays 14 sterols and 7 oxysterols in a single run. Three antipsychotics (haloperidol, cariprazine, aripiprazole), two antidepressants (trazodone and sertraline), and an antiarhythmic (amiodarone) inhibited one or more sterol synthesis enzymes. The result of the exposures was a dose-dependent increase in levels of various sterol intermediates and a decreased level of cholesterol in the cultured cells. Four prescription medications (haloperidol, aripiprazole, cariprazine, and trazodone) acted primarily on the DHCR7 enzyme. The result of this exposure was an increase in 7-dehydrocholesterol in neurons and astrocytes to levels that were comparable to those found in cultured neurons and astrocytes from transgenic mice that carried a Dhcr7 pathogenic mutation modeling the neurodevelopmental disorder Smith–Lemli–Opitz syndrome.
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Respiratory Syncytial Virus Infection of Human Lung Fibroblasts Induces a Hyaluronan-Enriched Extracellular Matrix That Binds Mast Cells and Enhances Expression of Mast Cell Proteases
Human lung fibroblasts (HLFs) treated with the viral mimetic polyinosine-polycytidylic acid (poly I:C) form an extracellular matrix (ECM) enriched in hyaluronan (HA) that avidly binds monocytes and lymphocytes. Mast cells are important innate immune cells in both asthma and acute respiratory infections including respiratory syncytial virus (RSV); however, the effect of RSV on HA dependent mast cell adhesion and/or function is unknown. To determine if RSV infection of HLFs leads to the formation of a HA-enriched ECM that binds and enhances mast cell activity primary HLFs were infected with RSV for 48 h prior to leukocyte binding studies using a fluorescently labeled human mast cell line (LUVA). Parallel HLFs were harvested for characterization of HA production by ELISA and size exclusion chromatography. In separate experiments, HLFs were infected as above for 48 h prior to adding LUVA cells to HLF wells. Co-cultures were incubated for 48 h at which point media and cell pellets were collected for analysis. The role of the hyaladherin tumor necrosis factor-stimulated gene 6 (TSG-6) was also assessed using siRNA knockdown. RSV infection of primary HLFs for 48 h enhanced HA-dependent LUVA binding assessed by quantitative fluorescent microscopy. This coincided with increased HLF HA synthase (HAS) 2 and HAS3 expression and decreased hyaluronidase (HYAL) 2 expression leading to increased HA accumulation in the HLF cell layer and the presence of larger HA fragments. Separately, LUVAs co-cultured with RSV-infected HLFs for 48 h displayed enhanced production of the mast cell proteases, chymase, and tryptase. Pre-treatment with the HA inhibitor 4-methylumbelliferone (4-MU) and neutralizing antibodies to CD44 (HA receptor) decreased mast cell protease expression in co-cultured LUVAs implicating a direct role for HA. TSG-6 expression was increased over the 48-h infection. Inhibition of HLF TSG-6 expression by siRNA knockdown led to decreased LUVA binding suggesting an important role for this hyaladherin for LUVA adhesion in the setting of RSV infection. In summary, RSV infection of HLFs contributes to inflammation via HA-dependent mechanisms that enhance mast cell binding as well as mast cell protease expression via direct interactions with the ECM.
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