Rapid and efficient generation of antigen-specific isogenic T cells from cryopreserved blood samples

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene editing has been leveraged for the modification of human and mouse T cells. However, limited experience is available on the application of CRISPR/Cas9 electroporation in cryopreserved T cells collected during clinical trials. To address this, we aimed to optimize a CRISPR/Cas9-mediated gene editing protocol compatible with peripheral blood mononuclear cells (PBMCs) samples routinely produced during clinical trials. PBMCs from healthy donors were used to generate knockout T-cell models for interferon-gamma, Cbl proto-oncogene B (CBLB), Fas cell surface death receptor (Fas) and T-cell receptor (TCR alpha beta) genes. The effect of CRISPR/Cas9-mediated gene editing on T cells was evaluated using apoptosis assays, cytokine bead arrays and ex vivo and in vitro stimulation assays. Our results demonstrate that CRISPR/Cas9-mediated gene editing of ex vivo T cells is efficient and does not overtly affect T-cell viability. Cytokine release and T-cell proliferation were not affected in gene-edited T cells. Interestingly, memory T cells were more susceptible to CRISPR/Cas9 gene editing than naive T cells. Ex vivo and in vitro stimulation with antigens resulted in equivalent antigen-specific T-cell responses in gene-edited and untouched control cells, making CRISPR/Cas9-mediated gene editing compatible with clinical antigen-specific T-cell activation and expansion assays. Here, we report an optimized protocol for rapid, viable and highly efficient genetic modification in ex vivo human antigen-specific T cells, for subsequent functional evaluation and/or expansion. Our platform extends CRISPR/Cas9-mediated gene editing for use in gold-standard clinically used immune-monitoring pipelines and serves as a starting point for development of analogous approaches, such as those including transcriptional activators and/or epigenetic modifiers

Measuring expectations of inflation: Effects of survey mode, wording, and opportunities to revise

Several national surveys aim to elicit consumers’ inflation expectations. Median expectations tend to track objective inflation estimates over time, although responses display large dispersion. Medians also tend to differ between surveys, possibly reflecting survey design differences. Using a nationally representative Dutch sample, we evaluate the importance of three survey design features in explaining observed differences: mode (face-to-face vs. web), question wording (‘prices in general’ vs. ‘inflation’), and the explicit opportunity to revise responses. We examine effects on item non-responses, revisions, reported inflation expectations and their deviation from the CPI inflation rate. We discuss implications of our findings for survey design

Biochemical basis of 5-aminolaevulinic acid-induced protoporphyrin IX accumulation: a study in patients with (pre)malignant lesions of the oesophagus.

Administration of 5-aminolaevulinic acid (ALA) leads to porphyrin accumulation in malignant and premalignant tissues, and ALA is used as a prodrug in photodynamic therapy (PDT). To understand the mechanism of porphyrin accumulation after the administration of ALA and to investigate whether ALA-induced protoporphyrin IX might be a suitable photosensitizer in Barrett's oesophagus and adenocarcinoma, we determined the activities of porphobilinogen deaminase (PBG-D) and ferrochelatase (FC) in various malignant and premalignant as well as in normal tissues of the human oesophagus. A PDT power index for ALA-induced porphyrin accumulation, the ratio of PBG-D to FC normalized for the normal squamous epithelium of the oesophagus, was calculated to evaluate intertissue variation in the ability to accumulate porphyrins. In malignant and premalignant tissue a twofold increased PBG-D activity and a marginally increased FC activity was seen compared with normal squamous epithelium. A significantly increased PDT power index in Barrett's epithelium and adenocarcinoma was found. Our results suggest that, after the administration of ALA, porphyrins will accumulate in a greater amount in Barrett's epithelium and adenocarcinoma of the oesophagus because of an imbalance between PBG-D and FC activities. The PDT power index here defined might be a useful indicative parameter for predicting the susceptibility of these tissues to ALA-PDT

Thermal Characterization of Polycrystalline CVD Diamond Thin Films

An experimental thermal characterization method is developed for high thermal conductivity thin films. The method utilizes Ta/Pt resistors on microfabricated free-standing thin film structures both for heating and temperature monitoring at different positions on the structures. The steady-state temperature at the heater and the sensor positions are monitored as a function of the power dissipated by the heater under vacuum environment, and the thermal conductivity is estimated by comparing these results to FEA and/or analytical models. The developed method is used to characterize the thermal conductivity of various different CVD diamond films of different grain sizes and films thicknesses. The measured thermal conductivity values range from 15 W/m·K to 300 W/m·K, which are at least one order of magnitude lower than that of natural diamond. It is also shown that the thermal conductivity of such films in the in-plane direction increases with increasing grain size and film