Identification of Rare High-Avidity, Tumor-Reactive CD8+ T Cells by Monomeric TCR-Ligand Off-Rates Measurements on Living Cells.

The avidity of the T-cell receptor (TCR) for antigenic peptides presented by the peptide-MHC (pMHC) on cells is a key parameter for cell-mediated immunity. Yet a fundamental feature of most tumor antigen-specific CD8(+) T cells is that this avidity is low. In this study, we addressed the need to identify and select tumor-specific CD8(+) T cells of highest avidity, which are of the greatest interest for adoptive cell therapy in patients with cancer. To identify these rare cells, we developed a peptide-MHC multimer technology, which uses reversible Ni(2+)-nitrilotriacetic acid histidine tags (NTAmers). NTAmers are highly stable but upon imidazole addition, they decay rapidly to pMHC monomers, allowing flow-cytometric-based measurements of monomeric TCR-pMHC dissociation rates of living CD8(+) T cells on a wide avidity spectrum. We documented strong correlations between NTAmer kinetic results and those obtained by surface plasmon resonance. Using NTAmers that were deficient for CD8 binding to pMHC, we found that CD8 itself stabilized the TCR-pMHC complex, prolonging the dissociation half-life several fold. Notably, our NTAmer technology accurately predicted the function of large panels of tumor-specific T cells that were isolated prospectively from patients with cancer. Overall, our results demonstrated that NTAmers are effective tools to isolate rare high-avidity cytotoxic T cells from patients for use in adoptive therapies for cancer treatment.

Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.

BACKGROUND: Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades. RESULTS: We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage. CONCLUSIONS: These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements.

Editorial: Serial Fecal Calprotectin and Lactoferrin Measurements for Early Diagnosis of Pouchitis After Proctocolectomy for Ulcerative Colitis: Is Pouchoscopy No Longer Needed?

This editorial discusses the role of serial measurements of fecal calprotectin or fecal lactoferrin for the early detection of pouchitis in patients with ulcerative colitis having undergone procto-colectomy with ileo-pouch-anal anastomosis. Furthermore, the role of fecal calprotectin and fecal lactoferrin for the monitoring of pouchitis is highlighted.

Reliability of maximal grip strength measurements and grip strength recovery following a stroke.

STUDY DESIGN: Clinical measurement. PURPOSE: The test-retest reliability of maximal grip strength measurements (MGSM) is examined in subjects for 12 weeks post-stroke together with maximal grip strength recovery and the maximal-grip and upper-extremity strength measurements' relationship with capacity and performance test scores. METHODS: A Jamar dynamometer and the Motricity Index (MI) were used for strength measurements. The Chedoke Arm and Hand Activity Inventory and ABILHAND questionnaire for evaluating capacities and performances. RESULTS: MGSM were reliable (Intraclass Correlation Coefficients = 0.97-0.99, Minimal Detectable Differences = 2.73-4.68 kg). Among the 34 participants, 47% did not have a measurable grip strength one week post-stroke but 50% of these recovered some strength within the first eight weeks. The MGSM and MI scores were correlated with scores of tests of capacity and performance (Spearman's Rank Correlation Coefficients = 0.69-0.94). CONCLUSIONS: MGSM are reliable in the first weeks after a stroke. LEVEL OF EVIDENCE: N/A.

Image quality in CT: From physical measurements to model observers.

Evaluation of image quality (IQ) in Computed Tomography (CT) is important to ensure that diagnostic questions are correctly answered, whilst keeping radiation dose to the patient as low as is reasonably possible. The assessment of individual aspects of IQ is already a key component of routine quality control of medical x-ray devices. These values together with standard dose indicators can be used to give rise to 'figures of merit' (FOM) to characterise the dose efficiency of the CT scanners operating in certain modes. The demand for clinically relevant IQ characterisation has naturally increased with the development of CT technology (detectors efficiency, image reconstruction and processing), resulting in the adaptation and evolution of assessment methods. The purpose of this review is to present the spectrum of various methods that have been used to characterise image quality in CT: from objective measurements of physical parameters to clinically task-based approaches (i.e. model observer (MO) approach) including pure human observer approach. When combined together with a dose indicator, a generalised dose efficiency index can be explored in a framework of system and patient dose optimisation. We will focus on the IQ methodologies that are required for dealing with standard reconstruction, but also for iterative reconstruction algorithms. With this concept the previously used FOM will be presented with a proposal to update them in order to make them relevant and up to date with technological progress. The MO that objectively assesses IQ for clinically relevant tasks represents the most promising method in terms of radiologist sensitivity performance and therefore of most relevance in the clinical environment.

Speciation and Bioavailability Measurements of Environmental Plutonium Using Diffusion in Thin Films.

The biological uptake of plutonium (Pu) in aquatic ecosystems is of particular concern since it is an alpha-particle emitter with long half-life which can potentially contribute to the exposure of biota and humans. The diffusive gradients in thin films technique is introduced here for in-situ measurements of Pu bioavailability and speciation. A diffusion cell constructed for laboratory experiments with Pu and the newly developed protocol make it possible to simulate the environmental behavior of Pu in model solutions of various chemical compositions. Adjustment of the oxidation states to Pu(IV) and Pu(V) described in this protocol is essential in order to investigate the complex redox chemistry of plutonium in the environment. The calibration of this technique and the results obtained in the laboratory experiments enable to develop a specific DGT device for in-situ Pu measurements in freshwaters. Accelerator-based mass-spectrometry measurements of Pu accumulated by DGTs in a karst spring allowed determining the bioavailability of Pu in a mineral freshwater environment. Application of this protocol for Pu measurements using DGT devices has a large potential to improve our understanding of the speciation and the biological transfer of Pu in aquatic ecosystems.

Comparison of the prognostic value of pretreatment measurements of systemic inflammatory response in patients undergoing curative resection of clear cell renal cell carcinoma.

PURPOSE: Pretreatment measurements of systemic inflammatory response, including the Glasgow prognostic score (GPS), the neutrophil-to-lymphocyte ratio (NLR), the monocyte-to-lymphocyte ratio (MLR), the platelet-to-lymphocyte ratio (PLR) and the prognostic nutritional index (PNI) have been recognized as prognostic factors in clear cell renal cell carcinoma (CCRCC), but there is at present no study that compared these markers. METHODS: We evaluated the pretreatment GPS, NLR, MLR, PLR and PNI in 430 patients, who underwent surgery for clinically localized CCRCC (pT1-3N0M0). Associations with disease-free survival were assessed with Cox models. Discrimination was measured with the C-index, and a decision curve analysis was used to evaluate the clinical net benefit. RESULTS: On multivariable analyses, all measures of systemic inflammatory response were significant prognostic factors. The increase in discrimination compared with the stage, size, grade and necrosis (SSIGN) score alone was 5.8 % for the GPS, 1.1-1.4 % for the NLR, 2.9-3.4 % for the MLR, 2.0-3.3 % for the PLR and 1.4-3.0 % for the PNI. On the simultaneous multivariable analysis of all candidate measures, the final multivariable model contained the SSIGN score (HR 1.40, P < 0.001), the GPS (HR 2.32, P < 0.001) and the MLR (HR 5.78, P = 0.003) as significant variables. Adding both the GPS and the MLR increased the discrimination of the SSIGN score by 6.2 % and improved the clinical net benefit. CONCLUSIONS: In patients with clinically localized CCRCC, the GPS and the MLR appear to be the most relevant prognostic measures of systemic inflammatory response. They may be used as an adjunct for patient counseling, tailoring management and clinical trial design.

Uncertainty evaluation in activity measurements using ionization chambers

Pressurized re-entrant (or 4 pi) ionization chambers (ICs) connected to current-measuring electronics are used for activity measurements of photon emitting radionuclides and some beta emitters in the fields of metrology and nuclear medicine. As a secondary method, these instruments need to be calibrated with appropriate activity standards from primary or direct standardization. The use of these instruments over 50 years has been well described in numerous publications, such as the Monographie BIPM-4 and the special issue of Metrologia on radionuclide metrology (Ratel 2007 Metrologia 44 S7-16, Schrader1997 Activity Measurements With Ionization Chambers (Monographie BIPM-4) Schrader 2007 Metrologia 44 S53-66, Cox et al 2007 Measurement Modelling of the International Reference System (SIR) for Gamma-Emitting Radionuclides (Monographie BIPM-7)). The present work describes the principles of activity measurements, calibrations, and impurity corrections using pressurized ionization chambers in the first part and the uncertainty analysis illustrated with example uncertainty budgets from routine source-calibration as well as from an international reference system (SIR) measurement in the second part.

Evaluation of drug-induced neurotoxicity based on metabolomics, proteomics and electrical activity measurements in complementary CNS in vitro models.

The present study was performed in an attempt to develop an in vitro integrated testing strategy (ITS) to evaluate drug-induced neurotoxicity. A number of endpoints were analyzed using two complementary brain cell culture models and an in vitro blood-brain barrier (BBB) model after single and repeated exposure treatments with selected drugs that covered the major biological, pharmacological and neuro-toxicological responses. Furthermore, four drugs (diazepam, cyclosporine A, chlorpromazine and amiodarone) were tested more in depth as representatives of different classes of neurotoxicants, inducing toxicity through different pathways of toxicity. The developed in vitro BBB model allowed detection of toxic effects at the level of BBB and evaluation of drug transport through the barrier for predicting free brain concentrations of the studied drugs. The measurement of neuronal electrical activity was found to be a sensitive tool to predict the neuroactivity and neurotoxicity of drugs after acute exposure. The histotypic 3D re-aggregating brain cell cultures, containing all brain cell types, were found to be well suited for OMICs analyses after both acute and long term treatment. The obtained data suggest that an in vitro ITS based on the information obtained from BBB studies and combined with metabolomics, proteomics and neuronal electrical activity measurements performed in stable in vitro neuronal cell culture systems, has high potential to improve current in vitro drug-induced neurotoxicity evaluation.