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

3D-printed patient specific instruments for corrective osteotomies of the lower extremity

3D-printing has become a promising adjunct in orthopedic surgery over the past years. A significant drop in costs and increased availability of the required hardware and software needed for using the technique, have resulted in a relatively fast adaptation of 3D-printing techniques for various indications. In this review, the role of 3D-printing for deformity corrections of the lower extremity is described.</p

The size and shape of the Milky Way disc and halo from M-type brown dwarfs in the BoRG survey.

We have identified 274 M-type brown dwarfs in the Hubble Space Telescope\u27s Wide Field Camera 3 pure parallel fields from the Brightest of Reionizing Galaxies (BoRG) survey for high-redshift galaxies. These are near-infrared observations with multiple lines of sight out of our Milky Way. Using these observed M-type brown dwarfs, we fitted a Galactic disc and halo model with a Markov chain Monte Carlo analysis. This model worked best with the scalelength of the disc fixed at h = 2.6 kpc. For the scaleheight of the disc, we found z0=0.29+0.02−0.019 z0=0.29−0.019+0.02 kpc and for the central number density, ρ0=0.29+0.20−0.13 ρ0=0.29−0.13+0.20 # pc−3. For the halo, we derived a flattening parameter κ = 0.45 ± 0.04 and a power-law index p = 2.4 ± 0.07. We found the fraction of M-type brown dwarfs in the local density that belong to the halo to be fh = 0.0075 +0.0025−0.0019 −0.0019+0.0025 . We found no correlation between subtype of M-dwarf and any model parameters. The total number of M-type brown dwarfs in the disc and halo was determined to be 58.2+9.81−6.70×109 58.2−6.70+9.81×109 . We found an upper limit for the fraction of M-type brown dwarfs in the halo of 7 +5−4 −4+5  per cent. The upper limit for the total Galactic disc mass in M-dwarfs is 4.34+0.73−0.5×109 4.34−0.5+0.73×109 M⊙, assuming all M-type brown dwarfs have a mass of 80 MJ

Decidual memory T‐cell subsets and memory T‐cell stimulatory cytokines in early‐ and late‐onset preeclampsia

Problem: Preeclampsia is a major cause of fetal and maternal mortality and morbidity. Disturbed fetal-maternal immune tolerance, and therewith memory T cells, might be involved in its etiology. This study aims to give insight into memory T-cell populations and its associated cytokines in the decidual layers in early-onset preeclampsia (EO-PE) and late-onset preeclampsia (LO-PE). Method of Study: Lymphocytes were isolated from the decidua parietalis and basalis from EO-PE (n = 6), LO-PE (n = 8) and healthy (n = 15) pregnancies. CD4+ and CD8+ central- (CCR7+), effector- (CCR7−), tissue resident- (CD103+), and regulatory- (Foxp3+) memory cell (CD45RO+) populations and their activation status (CD69+) were analyzed using flow cytometry. qRT-PCR analysis was performed on decidua parietalis and basalis biopsies to detect mRNA expression of interferon-gamma, interleukin-1B, IL2, IL6, IL7, IL8, IL10, IL15, and IL23. Results: CD4+ central-memory (CM) cell proportions were lower in the decidua parietalis in LO-PE (P <.0001) and EO-PE (P <.01) compared to healthy pregnancies. CD8+ memory (P <.05) and CD8+ CM (P <.01) cell proportions were also lower in the decidua parietalis in EO-PE compared to healthy pregnancies. This was accompanied by higher IL15 (P <.05) and IL23 (P <.05) and lower IL7 (P <.05) mRNA expression in decidua basalis biopsies from EO-PE compared to healthy pregnancies, analyzed by qPCR. Conclusion: In conclusion, decidual memory T-cell proportions, their activation status, and associated cytokines are altered in preeclampsia and might therefore be involved in fetal-maternal immune tolerance and the pathophysiology of preeclampsia

Wave modelling - the state of the art

This paper is the product of the wave modelling community and it tries to make a picture of the present situation in this branch of science, exploring the previous and the most recent results and looking ahead towards the solution of the problems we presently face. Both theory and applications are considered. The many faces of the subject imply separate discussions. This is reflected into the single sections, seven of them, each dealing with a specific topic, the whole providing a broad and solid overview of the present state of the art. After an introduction framing the problem and the approach we followed, we deal in sequence with the following subjects: (Section) 2, generation by wind; 3, nonlinear interactions in deep water; 4, white-capping dissipation; 5, nonlinear interactions in shallow water; 6, dissipation at the sea bottom; 7, wave propagation; 8, numerics. The two final sections, 9 and 10, summarize the present situation from a general point of view and try to look at the future developments

Analysis of prehospital perimortem caesarean deliveries performed by Helicopter Emergency Medical Services in the Netherlands and recommendations for the future

Background: Prehospital perimortem caesarean delivery (PCD) is a rarely performed procedure. In this study, we aimed to examine all PCDs performed by the four Helicopter Emergency Medical Services in the Netherlands; to describe the procedures, outcomes, complications, and compliance with the reco

Decision making in prehospital traumatic cardiac arrest; A qualitative study

Background: Despite improving survival of patients in prehospital traumatic cardiac arrest (TCA), initiation and/or discontinuation of resuscitation of TCA patients remains a subject of debate among prehospital emergency medical service providers. The aim of this study was to identify factors that influence decision making by prehospital emergency medical service providers during resuscitation of patients with TCA. Methods: Twenty-five semi-structured interviews were conducted with experienced ambulance nurses, HEMS nurses and HEMS physicians individually, followed by a focus group discussion. Participants had to be currently active in prehospital medicine in the Netherlands. Interviews were encoded for analysis using ATLAS.ti. Using qualitative analysis, different themes around decision making in TCA were identified. Results: Eight themes were identified as being important factors for decision making during prehospital TCA. These themes were: (1) factual information (e.g., electrocardiography rhythm or trauma mechanism); (2) fear of providing futile care or major impairment if return of spontaneous circulation was obtained; (3) potential organ donation; (4) patient age; (5) suspicion of attempted suicide; (6) presence of bystanders or family; (7) opinions of other team members; and (8) training and education. Several ambulance nurses reported they do not feel adequately supported by the current official national ambulance guidelines on TCA, nor did they feel sufficiently trained to perform pre-hospital interventions such as endotracheal intubation or needle thoracocentesis on these patients. Conclusion: Eight themes were identified as being important for decision making during prehospital TCA. While guidelines based on prognostic factors are important, it should be recognized that decision making in TCA is impacted by more than factual information alone. This should be reflected in educational programs and future guidelines

Erratum: “Milky Way red dwarfs in the Borg survey; galactic scale-height and the distribution of dwarfs stars in WFC3 imaging .

In the catalog of M-dwarfs presented in Holwerda et al. (2014, H14 hereafter), there is an issue with the conversion from celestial coordinates to Galactic ones, done with PYEPHEM a wrapper around a trusted and vetted library ephermis. Here we present the corrected coordinates (using ASTROPY) and distances based on AB magnitudes. We have amended the tables and figures accordingly. The relation between vertical scale-height (z0) and M- dwarf subtype found in H14 is no longer present. We find a scale-height of 600 pc for all types, in part due to the presence of a second Galactic structural component