[Show abstract][Hide abstract] ABSTRACT: We have designed a low-cost, reusable incubation system that allows cells to be cultured in either plated or suspension culture under complete atmospheric control for radiotracer characterization. We demonstrate its utility here in the first quantification of the hypoxia-dependent accumulation of Cu-diacetyl bis(N4-methylthiosemicarbazone) (Cu-ATSM) in adult rat ventricular myocytes (ARVMs).
ARVMs were allowed to adhere overnight in 9 cm culture plates (2×10 cells/dish) or were used in suspension culture, placed inside the chamber and equilibrated with either oxic (95 or 21% O2/5% CO2) or anoxic gas (95% N2/5% CO2). Cu-ATSM of 100 kBq was administered, and the cells were incubated for 30 or 60 min. Cells were then harvested, counted and fractionated to determine intracellular Cu biodistribution.
After 1 h, the average cellular Cu retention in plated ARVMs under oxygenated conditions was 23.9±2.5 mBq/cell (95% O2), increasing to 27.3±5.1 under 21% O2 (P<0.05) and to 36.1±3.1 under 0% O2 (P<0.05). When ARVMs were cultured in suspension, normoxic-hypoxic contrast was less marked but still significant [63.2±14.1 vs. 53.4±10.9% mBq/cell after 30 min (P<0.05)]. Sixty percent of tracer accumulated in the cytosol, and, although total cellular retention increased during hypoxia, there was no enrichment in any particular cellular compartment.
This apparatus allows the conduction of radiotracer uptake studies in cells under complete atmospheric control, as evidenced by our first demonstration of the hypoxia-dependent uptake of Cu-ATSM in ventricular myocytes. It is ideally suited for screening, validating and characterizing novel hypoxia-selective radiotracers.
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DCs) generated in vitro to present tumour antigens have been injected in cancer patients to boost in vivo anti-tumour immune responses. This approach to cancer immunotherapy has had limited success. For anti-tumour therapy, delivery and subsequent migration of DCs to lymph nodes leading to effective stimulation of effector T cells is thought to be essential. The ability to non-invasively monitor the fate of adoptively transferred DCs in vivo using magnetic resonance imaging (MRI) is an important clinical tool to correlate their in vivo behavior with response to treatment. Previous reports of superparamagnetic iron oxides (SPIOs) labelling of different cell types, including DCs, have indicated varying detrimental effects on cell viability, migration, differentiation and immune function. Here we describe an optimised labelling procedure using a short incubation time and low concentration of clinically used SPIO Endorem to successfully track murine DC migration in vivo using MRI in a mouse tumour model. First, intracellular labelling of bone marrow derived DCs was monitored in vitro using electron microscopy and MRI relaxometry. Second, the in vitro characterisation of SPIO labelled DCs demonstrated that viability, phenotype and functions were comparable to unlabelled DCs. Third, ex vivo SPIO labelled DCs, when injected subcutaneously, allowed for the longitudinal monitoring by MR imaging of their migration in vivo. Fourth, the SPIO DCs induced the proliferation of adoptively transferred CD4(+) T cells but, most importantly, they primed cytotoxic CD8(+) T cell responses to protect against a B16-Ova tumour challenge. Finally, using anatomical information from the MR images, the immigration of DCs was confirmed by the increase in lymph node size post-DC injection. These results demonstrate that the SPIO labelling protocol developed in this study is not detrimental for DC function in vitro and in vivo has potential clinical application in monitoring therapeutic DCs in patients with cancer.
[Show abstract][Hide abstract] ABSTRACT: The hexokinases are fundamental regulators of cardiac glucose uptake; by phosphorylating free intracellular glucose, they maintain the concentration gradient driving myocardial extraction of glucose from the bloodstream. Hexokinases are highly regulated proteins, subject to activation by insulin, hypoxia or ischaemia, and inhibition by their enzymatic product glucose-6-phosphate. In vitro and in many non-cardiac cell types, hexokinases have been shown to bind to the mitochondria, both increasing their phosphorylative capacity, and having a putative role in the anti-apoptotic function of protein kinase B (PKB)/Akt. Whether hexokinase-mitochondrial interaction is a dynamic and responsive process in the heart has been difficult to prove, but there is growing evidence that this association does indeed increase in response to insulin stimulation or ischaemia. In this review I discuss the relevance of hexokinase-mitochondrial interaction to cardiac glycolytic control, our interpretation of (18)FDG cardiac PET scans, and its possible role in protecting the myocardium from ischaemic injury.
Journal of Bioenergetics 06/2009; 41(2):187-93.
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