41 research outputs found
Real architecture For 3D Tissue (RAFT™) culture system improves viability and maintains insulin and glucagon production of mouse pancreatic islet cells
Modelling the neuropathology of lysosomal storage disorders through disease-specific human induced pluripotent stem cells
Mucopolysaccharidosis II (MPS II) is a lysosomal storage disorder (LSD), caused by iduronate 2-sulphatase (IDS)
enzyme dysfunction. The neuropathology of the disease is not well understood, although the neural symptoms
are currently incurable. MPS II-patient derived iPSC lines were established and differentiated to neuronal
lineage. The disease phenotype was confirmed by IDS enzyme and glycosaminoglycan assay. MPS II neuronal
precursor cells (NPCs) showed significantly decreased self-renewal capacity, while their cortical neuronal differentiation potential was not affected. Major structural alterations in the ER and Golgi complex, accumulation
of storage vacuoles, and increased apoptosis were observed both at protein expression and ultrastructural level
in the MPS II neuronal cells, which was more pronounced in GFAP + astrocytes, with increased LAMP2 expression but unchanged in their RAB7 compartment. Based on these finding we hypothesize that lysosomal
membrane protein (LMP) carrier vesicles have an initiating role in the formation of storage vacuoles leading to impaired lysosomal function. In conclusion, a novel human MPS II disease model was established for the first
time which recapitulates the in vitro neuropathology of the disorder, providing novel information on the disease
mechanism which allows better understanding of further lysosomal storage disorders and facilitates drug testing
and gene therapy approaches
TYK2 Kinase Activity Is Required for Functional Type I Interferon Responses In Vivo
Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2K923E) mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN) induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein’s stability. An inhibitory function was only observed upon over-expression of TYK2K923E
in vitro. Tyk2K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors
Enhanced cardiac differentiation of mouse embryonic stem cells by use of the slow-turning, lateral vessel (STLV) bioreactor
The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods
Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.Toxicolog
A new class of glycomimetic drugs to prevent free fatty acid-induced endothelial dysfunction
Background: Carbohydrates play a major role in cell signaling in many biological processes. We have developed a set of glycomimetic drugs that mimic the structure of carbohydrates and represent a novel source of therapeutics for endothelial dysfunction, a key initiating factor in cardiovascular complications. Purpose: Our objective was to determine the protective effects of small molecule glycomimetics against free fatty acidÂinduced endothelial dysfunction, focusing on nitric oxide (NO) and oxidative stress pathways. Methods: Four glycomimetics were synthesized by the stepwise transformation of 2,5Âdihydroxybenzoic acid to a range of 2,5Âsubstituted benzoic acid derivatives, incorporating the key sulfate groups to mimic the interactions of heparan sulfate. Endothelial function was assessed using acetylcholineÂinduced, endotheliumdependent relaxation in mouse thoracic aortic rings using wire myography. Human umbilical vein endothelial cell (HUVEC) behavior was evaluated in the presence or absence of the free fatty acid, palmitate, with or without glycomimetics (1µM). DAFÂ2 and H2DCFÂDA assays were used to determine nitric oxide (NO) and reactive oxygen species (ROS) production, respectively. Lipid peroxidation colorimetric and antioxidant enzyme activity assays were also carried out. RTÂPCR and western blotting were utilized to measure Akt, eNOS, NrfÂ2, NQOÂ1 and HOÂ1 expression. Results: Ex vivo endotheliumÂdependent relaxation was significantly improved by the glycomimetics under palmitateÂinduced oxidative stress. In vitro studies showed that the glycomimetics protected HUVECs against the palmitateÂinduced oxidative stress and enhanced NO production. We demonstrate that the protective effects of preÂincubation with glycomimetics occurred via upregulation of Akt/eNOS signaling, activation of the Nrf2/ARE pathway, and suppression of ROSÂinduced lipid peroxidation. Conclusion: We have developed a novel set of small molecule glycomimetics that protect against free fatty acidinduced endothelial dysfunction and thus, represent a new category of therapeutic drugs to target endothelial damage, the first line of defense against cardiovascular disease