The microtiter plate technique reported by Baker and colleagues for the glutathione reductase-DTNB recycling assay of total glutathione (GSx) and glutathione disulfide (GSSG) has been modified according to Anderson's recommendations, in order to improve the reliability and accuracy of this miniaturized method for the measurement of glutathione status in cultured/isolated cells. Dilute HCl (10 mmol/L) has been used to lyse cells, before protein removal by centrifugation in the presence of 1.3% sulfosalicylic acid. The final DTNB, GSSG-reductase and NADPH concentrations in the reaction mixture have been increased to 0.7 mmol/L, 1.2 IU/ml and 0.24 mmol/L, respectively. The procedure specificity has been tested by spiking and dilution assays, showing that about 90% of the expected GSx amounts could actually be recovered, while no changes of GSSG concentrations were caused in the cells. Accuracy has been assessed by analysis of within-series precision as well as of intra- and interassay reproducibility, showing coefficient variation of < 10%. Glutathione changes measured either in control rat hepatocytes or in primary cultures treated with paracetamol or menadione were in good agreement with well-known literature data. These data suggest that the experimental conditions reported in this paper are suitable for the analysis of total glutathione and glutathione disulfide concentrations in cultured/isolated cells.

Gene discovery and gene therapy call for advanced technologies to reliably assess gene expression; efficient coupling of gene expression to the expression of reporter genes is critical. Various noninvasive molecular imaging modalities have emerged to track biological processes in animal models. Here, we evaluate various strategies to link transgene expression with that of an (imaging) reporter gene. Using lentiviral vectors containing internal ribosomal entry sites (IRES), 2A-like peptides, or a bidirectional promoter, we compared their ability to ensure efficient coexpression of multiple reporter genes. Although the encephalomyocarditis virus (EMCV) IRES yielded functional bicistronic vectors, the expression level of the reporter downstream of IRES was consistently lower than that of the upstream transgene. Interestingly, peptide 2A constructs performed best in vitro and in vivo, providing effective noninvasive follow-up of transgene expression and having reporter gene expression levels in line with that of the single reporter constructs. The intrinsic "cleavage" property of the peptide 2A sequences allows each protein to be produced at proportional levels, opening ample possibilities for functional genomics and future gene therapeutic applications. Last, using various peptide 2A sequences, we engineered the triple reporter LV-3R (i.e., eGFP, fLuc, HSV1-sr39tk), enabling efficient multimodality readouts in vivo.

BackgroundAccurate and reproducible behavioral tests in animal models are of major importance in the development and evaluation of new therapies for central nervous system disease. In this study we investigated for the first time gait parameters of rat models for Parkinson's disease (PD), Huntington's disease (HD) and stroke using the Catwalk method, a novel automated gait analysis test. Static and dynamic gait parameters were measured in all animal models, and these data were compared to readouts of established behavioral tests, such as the cylinder test in the PD and stroke rats and the rotarod tests for the HD group.ResultsHemiparkinsonian rats were generated by unilateral injection of the neurotoxin 6-hydroxydopamine in the striatum or in the medial forebrain bundle. For Huntington's disease, a transgenic rat model expressing a truncated huntingtin fragment with multiple CAG repeats was used. Thirdly, a stroke model was generated by a photothrombotic induced infarct in the right sensorimotor cortex. We found that multiple gait parameters were significantly altered in all three disease models compared to their respective controls. Behavioural deficits could be efficiently measured using the cylinder test in the PD and stroke animals, and in the case of the PD model, the deficits in gait essentially confirmed results obtained by the cylinder test. However, in the HD model and the stroke model the Catwalk analysis proved more sensitive than the rotarod test and also added new and more detailed information on specific gait parameters.ConclusionThe automated quantitative gait analysis test may be a useful tool to study both motor impairment and recovery associated with various neurological motor disorders.

BackgroundThe prognostic value of body composition in cancer patients has been widely studied during the last decade. The main finding of these studies is that sarcopenia, or skeletal muscle depletion, assessed by CT imaging correlates with a reduced overall survival (OS). By contrast, the prognostic value of fat mass remains ill-defined. This study aims to analyze the influence of body composition including both muscle mass and adipose tissue on OS in a homogeneous population of advanced colorectal cancer (CRC) patients.MethodsAmong 235 patients with chemorefractory advanced CRC included in the SoMore and RegARd-C trials, body composition was assessed in 217 patients on baseline CT images. The relationship between body composition (sarcopenia, muscle density, subcutaneous and visceral fat index and density), body mass index (BMI) and OS were evaluated.ResultsPatients with a higher BMI had a better OS (≥30 versus < 30, HR: 0.50; 0.33–0.76). Those with low muscle index and muscle density had an increased mortality (HR: 2.06; 1.45–2.93 and HR: 1.54; 1.09–2.18, respectively). Likewise, low subcutaneous and visceral fat index were associated with an increased risk of dying (HR: 1.63; 1.23–2.17 and 1.48; 1.09–2.02 respectively), as were a high subcutaneous and visceral adipose tissue density (HR: 1.93; 1.44–2.57 and 2.40; 1.79–3.20 respectively). In multivariate analysis, a high visceral fat density was the main predictor of poor survival.ConclusionsOur results confirm the protective role of obesity in CRC patients at an advanced stage, as well as the negative prognostic impact of muscle depletion on survival. More importantly, our data show for the first time that visceral adipose tissue density is an important prognostic factor in metastatic CRC.Trial registrationNCT01290926, 07/02/2011 and NCT01929616, 28/08/2013.

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The ultimate therapy for ischemic stroke is restoration of blood supply in the ischemic region and regeneration of lost neural cells. This might be achieved by transplanting cells that differentiate into vascular or neuronal cell types, or secrete trophic factors that enhance self-renewal, recruitment, long-term survival, and functional integration of endogenous stem/progenitor cells. Experimental stroke models have been developed to determine potential beneficial effect of stem/progenitor cell-based therapies. To follow the fate of grafted cells in vivo, a number of noninvasive imaging approaches have been developed. Magnetic resonance imaging (MRI) is a high-resolution, clinically relevant method allowing in vivo monitoring of cells labeled with contrast agents. In this study, labeling efficiency of three different stem cell populations [mouse embryonic stem cells (mESC), rat multipotent adult progenitor cells (rMAPC), and mouse mesenchymal stem cells (mMSC)] with three different (ultra)small superparamagnetic iron oxide [(U)SPIO] particles (Resovist, Endorem, Sinerem) was compared. Labeling efficiency with Resovist and Endorem differed significantly between the different stem cells. Labeling with (U)SPIOs in the range that allows detection of cells by in vivo MRI did not affect differentiation of stem cells when labeled with concentrations of particles needed for MRI-based visualization. Finally, we demonstrated that labeled rMAPC could be detected in vivo and that labeling did not interfere with their migration. We conclude that successful use of (U)SPIOs for MRI-based visualization will require assessment of the optimal (U)SPIO for each individual (stem) cell population to ensure the most sensitive detection without associated toxicity.

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PET of gene expression in the brain may greatly facilitate neuroscience research and potential clinical implementation of gene or cell therapy of central nervous system diseases. To date, no adequate PET reporter system is available for the central nervous system because available tracers either do not cross the intact blood-brain barrier or have high background signals. Here we report the first, to our knowledge, PET reporter system for imaging gene expression in the intact brain. Methods: We selected the human type 2 cannabinoid receptor (hCB 2 ) as a reporter because of its low basal expression in the brain. An inactive mutant (D80N) was chosen so as not to interfere with signal transduction. As a reporter probe we used the 11 C-labeled CB 2 ligand, 11 C-GW405833, which readily crosses the blood-brain barrier. Dual-modality imaging lentiviral and adeno-associated viral vectors encoding both hCB 2 (D80N) and firefly luciferase or enhanced green fluorescent protein were engineered and validated in cell culture. Next, hCB 2 (D80N) was locoregionally overexpressed in rat striatum by stereotactic injection of lentiviral and adeno-associated viral vectors. Results: Kinetic PET revealed specific and reversible CB 2 binding of 11 C-GW405833 in the transduced rat striatum. hCB 2 and firefly luciferase expression was followed until 9 mo and showed similar kinetics. Both hCB 2 expression and enhanced green fluorescent protein expression were confirmed by immunohistochemistry. Conclusion: Dual-modality imaging viral vectors encoding hCB 2 (D80N) were engineered, and the reporter system was validated in different animal species. The results support the potential future clinical use of CB 2 as a PET reporter in the intact brain.