Disturbance in energy metabolism induced by hepatotoxins in a liver spheroid model XU J, JACKSON SK
Centre for Research in Biomedicine, Faculty of Health and Life Sciences, University of the West of England, Bristol, U.K.
The liver plays a central role in the metabolism and transformation of energy-generating substances. Mitochondria and different metabolic pathways in hepatocytes interact and are often vulnerable targets of toxicants. Using animal tests it is difficult to evaluate liver energy metabolic functions accurately because energy-generating substances are also taken up or released by other cells in the body; it is also difficult to achieve high-throughput. We have developed a rat primary liver 3D spheroid cell model which retains metabolic functions found in vivo. Based on this model, a test called the ‘spheroid cell spreading inhibition test’ (SCSIT) was used for determining suitable exposure concentration ranges for test compounds. The effects of the hepatotoxins diclofenac, isoniazid, paracetamol and galactosamine on the metabolism of energy-generating substances (glucose, pyruvate, lactate and galactose) by liver spheroids were evaluated. The results show that all the toxicants tested significantly reduced glucose and lactate release (p < 0.01). Diclofenac, isoniazid and paracetamol significantly reduced pyruvate uptake (p < 0.01), whereas, galactosamine did not affect pyruvate uptake within the concentration range tested. Diclofenac, galactosamine and paracetamol significantly decreased galactose uptake (p < 0.01); by contrast, isoniazid did not show a significant adverse effect. It is concluded that the primary liver spheroid model can mimic in vivo liver function in terms of the metabolism of energy-generating substances. The combined use of several endpoints of energy metabolism can result in reliable toxicity evaluation. High drug concentrations can kill cells rapidly, and may not therefore generate useful functional information on toxicity. As a method for predetermining toxicant concentration ranges, the SCSIT is therefore a reliable, useful and necessary tool for studying the effects of toxicants on liver functions and will have applications for drug safety screening and high throughput testing.
Biomedical Research at The University of The West of England, Bristol JACKSON SK, AVENT ND, XU J Centre for Research in Biomedicine, Faculty of Health and Life Sciences, University of the West of England, Bristol, U.K.
The Centre for Research in biomedicine (CRIB) is a key functional research grouping of international repute that brings together expertise in genomics, cell signaling, inflammation, molecular biology, gene regulation and cell biology to promote research and knowledge exchange in critical areas of biomedicine. Research teams within the Centre support over 20 post-doctoral researchers and more than 30 post-graduate research students.
The primary aim of CRIB is to understand fundamental cellular and molecular biomedical processes and identify potential markers and targets that can be developed into novel diagnostic and therapeutic strategies. The Centre is not only engaged in finding new insights into the understanding of human disease but has a strong focus in the translation of the knowledge into new health policy, diagnostics and therapeutics.
Current projects include: Non-invasive prenatal diagnosis, genetic blood typing, endotoxin and biomarkers for sepsis, insulin vesicle mobilization and diabetes, molecular microbiology and bioluminescent bacteria, 3D cell culture models and in vitro toxicology, volatile organic chemical analyses for clinical diagnoses.
Targeted Immunotherapy of Cancer Through TCR Gene Transfer XUE SA, GAO L, THOMAS S, HART D, XUE JZ, MORRIS E, STAUSS HJ
University College London, London, United Kingdom
Background: Conventional cancer therapies are limited by their toxicity and lack of specificity. To achieve targeted immunotherapy, we have targeted Wilm’s Tumour antigen (WT1) which is overexpressed in most leukaemias and many solid cancers, and isolated WT1-specific T cell receptor (WT1-TCR) genes. Human T cells transduced with WT1-TCR eliminated leukaemia cell lines in vitro and in a NOD/SCID mouse model. To facilitate the clinical application of TCR gene therapy, we have modified this WT1-TCR retroviral construct aimed at improving TCR functional activity and reducing its cytotoxicity.
Methods: WT1-TCR constructs were generated in a retroviral vector in which the woodchuck hepatitis virus-derived post-transcriptional regulatory element was deleted. To enhance desired pairing, a second disulphide bond was introduced between the TCR and chains and the human TCR constant domains were replaced with murine sequences. Following transduction, the functional activity of WT1-TCR engineered T cells was tested by 51Cr release cytotoxic T lymphocyte (CTL) assay and intracellular cytokine staining. After the engraftment of CD34+ leukaemia progenitor cells from a CML patient in NOD/SCID mice, adoptive immunotherapy was performed with WT1-TCR engineered patient’s T cells. Animal survival and leukaemia burden was monitored.
Results: We have generated WT1-TCR constructs with improved safety features and enhanced functional activities as determined by tetramer staining, CTL killing and intracellular cytokine staining. To mimic the clinical settings, we engrafted CD34+ leukaemia progenitor cells into NOD/SCID mice, followed by adoptive immunotherapy with the patients’ T cells transduced with either WT1-TCR or control TCR. We show that mice treated with WT1-TCR engineered CTL had a greater survival than the mice treated with control CTL. Analysis of the bone marrow showed that control mice had engraftment of leukaemia cells, while WT1 CTL treated mice did not. These data provide a solid basis for a phase I clinical trial.
Conclusions: 1). Genetic modification of TCR genes provides a way of generating safe, efficient reagents for clinical applications. 2). WT1-TCR transduction of patient’s T cells offers a simple and efficient way of producing tumour specific T cells for the treatment of leukaemias. This has important implications for treating other WT1-expressing cancers.
