The Dox-pDC - A murine conditionally immortalized plasmacytoid dendritic cell line with native immune profile

Plasmacytoid dendritic cells (pDC) constitute a very rare blood cell population and play a significant role in immune response and immune-mediated disorders. Investigations on primary pDCs are hindered not only due to their rarity but also because they represent a heterogeneous cell population which is difficult to culture ex vivo. We generated a conditionally immortalized pDC line (Dox-pDC) from mice with Doxycycline-inducible SV40 Large T Antigen with a comparable immune profile to primary pDCs. The Dox-pDC secrete pro- and anti-inflammatory cytokines upon Toll-like receptor 9 stimulation and upregulate their MHCI, MHCII and costimulatory molecules. Further, the Dox-pDC activate and polarize naïve T cells in vivo and in vitro in response to the model antigen Ovalbumin. Due to their long-term culture stability and their robust proliferation Dox-pDC represent a reliable alternative to primary mouse pDC

Induction of T cell response.

<p>(A) <i>In vivo</i> immunization model with OVA-V antigen. (B, C) Proliferation of CD4+ T cells (B) and effector memory T cells (CD4+ TEM; C) activated with OVA-V-pulsed Dox-pDC. (D, E) Proliferation of CD4+CD8lo T cells (D) and effector memory T cells (CD4+CD8lo TEM; E) activated with OVA-V-pulsed Dox-pDC. (F-H) Frequency of Th1 (IFNγ+CD4+; F), Th17 (RORγt+CD4+; G) and CD8+ T cells (IFNγ+CD8+; H) polarized with OVA-LE-pulsed and TLR9-activated Dox-pDC or BM-pDC. Results are expressed as means ± SD from 3–9 mice per group. Statistical significance is indicated, ***(P<0.005) and ****(P<0.001).</p

Structural basis for membrane recruitment of ATG16L1 by WIPI2 in autophagy.

Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double-membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12-5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12-5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven-bladedß -propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 ß-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12-5-16 L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12-5-16 L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand and ATG8 lipidation on the other

Phenotype of immature Dox-pDC.

<p>(A) Scheme of generating Dox-pDC line. (B) Expression of pDC marker by the final 10 single cell clones. (C) IFNα secretion of the final 10 single cell clones. (D) Flow cytometric analysis of BM-pDC and Dox-pDC for different cell-specific markers. (E) Proliferation of Dox-pDC in the presence or absence of Dox and Flt3l and TLR9 activation detected with the viability proliferation dye 450 at day 0, 3, 5, 7 and analysed by flow cytometry. (F) Apoptosis staining of Dox-pDC in the presence or absence of Dox and Flt3l analysed by flow cytometry. (G) Doubling time of Dox-pDC calculated with an exponential growth equation. The figure shows representative results out of 2–4 experiments.</p

Human hand modelling: Kinematics, dynamics, applications

An overview of mathematical modelling of the human hand is given. We consider hand models from a specific background: rather than studying hands for surgical or similar goals, we target at providing a set of tools with which human grasping and manipulation capabilities can be studied, and hand functionality can be described. We do this by investigating the human hand at various levels: (1) at the level of kinematics, focussing on the movement of the bones of the hand, not taking corresponding forces into account; (2) at the musculotendon structure, i.e. by looking at the part of the hand generating the forces and thus inducing the motion; and (3) at the combination of the two, resulting in hand dynamics as well as the underlying neurocontrol. Our purpose is to not only provide the reader with an overview of current human hand modelling approaches but also to fill the gaps with recent results and data, thus allowing for an encompassing picture.Department of Biomechanical EngineeringMechanical, Maritime and Materials Engineerin