Proteomics of Human Dendritic Cell Subsets Reveals Subset-Specific Surface Markers and Differential Inflammasome Function.

Dendritic cells (DCs) play a key role in orchestrating adaptive immune responses. In human blood, three distinct subsets exist: plasmacytoid DCs (pDCs) and BDCA3+ and CD1c+ myeloid DCs. In addition, a DC-like CD16+ monocyte has been reported. Although RNA-expression profiles have been previously compared, protein expression data may provide a different picture. Here, we exploited label-free quantitative mass spectrometry to compare and identify differences in primary human DC subset proteins. Moreover, we integrated these proteomic data with existing mRNA data to derive robust cell-specific expression signatures with more than 400 differentially expressed proteins between subsets, forming a solid basis for investigation of subset-specific functions. We illustrated this by extracting subset identification markers and by demonstrating that pDCs lack caspase-1 and only express low levels of other inflammasome-related proteins. In accordance, pDCs were incapable of interleukin (IL)-1β secretion in response to ATP

Anastral spindle assembly and γ-tubulin in Drosophila oocytes

<p>Abstract</p> <p>Background</p> <p>Anastral spindles assemble by a mechanism that involves microtubule nucleation and growth from chromatin. It is still uncertain whether γ-tubulin, a microtubule nucleator essential for mitotic spindle assembly and maintenance, plays a role. Not only is the requirement for γ-tubulin to form anastral <it>Drosophila </it>oocyte meiosis I spindles controversial, but its presence in oocyte meiosis I spindles has not been demonstrated and is uncertain.</p> <p>Results</p> <p>We show, for the first time, using a bright GFP fusion protein and live imaging, that the <it>Drosophila </it>maternally-expressed γTub37C is present at low levels in oocyte meiosis I spindles. Despite this, we find that formation of bipolar meiosis I spindles does not require functional γTub37C, extending previous findings by others. Fluorescence photobleaching assays show rapid recovery of γTub37C in the meiosis I spindle, similar to the cytoplasm, indicating weak binding by γTub37C to spindles, and fits of a new, potentially more accurate model for fluorescence recovery yield kinetic parameters consistent with transient, diffusional binding.</p> <p>Conclusions</p> <p>The FRAP results, together with its mutant effects late in meiosis I, indicate that γTub37C may perform a role subsequent to metaphase I, rather than nucleating microtubules for meiosis I spindle formation. Weak binding to the meiosis I spindle could stabilize pre-existing microtubules or position γ-tubulin for function during meiosis II spindle assembly, which follows rapidly upon oocyte activation and completion of the meiosis I division.</p

Exposure to Apoptotic Activated CD4+ T Cells Induces Maturation and APOBEC3G- Mediated Inhibition of HIV-1 Infection in Dendritic Cells

Dendritic cells (DCs) are activated by signaling via pathogen-specific receptors or exposure to inflammatory mediators. Here we show that co-culturing DCs with apoptotic HIV-infected activated CD4+ T cells (ApoInf) or apoptotic uninfected activated CD4+ T cells (ApoAct) induced expression of co-stimulatory molecules and cytokine release. In addition, we measured a reduced HIV infection rate in DCs after co-culture with ApoAct. A prerequisite for reduced HIV infection in DCs was activation of CD4+ T cells before apoptosis induction. DCs exposed to ApoAct or ApoInf secreted MIP-1α, MIP-1β, MCP-1, and TNF-α; this effect was retained in the presence of exogenous HIV. The ApoAct-mediated induction of co-stimulatory CD86 molecules and reduction of HIV infection in DCs were partially abrogated after blocking TNF-α using monoclonal antibodies. APOBEC3G expression in DCs was increased in co-cultures of DCs and ApoAct but not by apoptotic resting CD4+ T cells (ApoRest). Silencing of APOBEC3G in DC abrogated the HIV inhibitory effect mediated by ApoAct. Sequence analyses of an env region revealed significant induction of G-to-A hypermutations in the context of GG or GA dinucleotides in DNA isolated from DCs exposed to HIV and ApoAct. Thus, ApoAct-mediated DC maturation resulted in induction of APOBEC3G that was important for inhibition of HIV-infection in DCs. These findings underscore the complexity of differential DC responses evoked upon interaction with resting as compared with activated dying cells during HIV infection

Background matching in the brown shrimp Crangon crangon : adaptive camouflage and behavioural-plasticity

A combination of burrowing behaviour and very efficient background matching makes the brown shrimp Crangon crangon almost invisible to potential predators and preys. This raises questions on how shrimp succeed in concealing themselves in the heterogeneous and dynamic estuarine habitats they inhabit and what type of environmental variables and behavioural factors affect their colour change abilities. Using a series of behavioural experiments, we show that the brown shrimp is capable of repeated fast colour adaptations (20% change in dark pigment cover within one hour) and that its background matching ability is mainly influenced by illumination and sediment colour. Novel insights are provided on the occurrence of non-adaptive (possibly stress) responses to background changes after long-time exposure to a constant background colour or during unfavourable conditions for burying. Shrimp showed high levels of intra- and inter-individual variation, demonstrating a complex balance between behavioural-plasticity and environmental adaptation. As such, the study of crustacean colour changes represents a valuable opportunity to investigate colour adaptations in dynamic habitats and can help us to identify the mayor environmental and behavioural factors influencing the evolution of animal background matching

Protamine-stabilized RNA as an ex vivo stimulant of primary human dendritic cell subsets

\u3cp\u3eDendritic cells (DCs) are key in connecting innate and adaptive immunity. Their potential in inducing specific immune responses has made them interesting targets for immunotherapeutic approaches. Our research group was the first to exploit the naturally occurring myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in therapeutic vaccination trials against melanoma. To develop primary DC subsets as an optimal vaccine, the identification of a clinically applicable adjuvant activating both subsets is required. Although the expression of pathogen recognition receptors differs distinctly between the DC subsets, both pDCs and mDCs can respond to single-stranded RNA (ssRNA) via Toll-like receptors 7 and 8, respectively. Since ssRNA is easily degraded by RNases, we stabilized anionic RNA by complexing it with the positively charged protein protamine. This leads to the formation of protamine-RNA complexes with varying features depending on ionic content. We subsequently investigated the immunostimulatory effect of complexes that formed various salt concentrations on purified DC subsets. Both mDCs and pDCs upregulated maturation markers and produced pro-inflammatory cytokines in a dose-dependent way to the protamine-RNA complexes. This was dependent on endosomal acidification and correlated partly with the uptake of protamine-RNA complexes. Furthermore, both DC subsets induced T cell proliferation and IFN gamma secretion in a beneficial ratio to IL-10. These results indicate that protamine-RNA complexes can be used to stimulate human mDC and pDC ex vivo for use in immunotherapeutic settings.\u3c/p\u3

Proteomics of human dendritic cell subsets reveals subset-specific surface markers and differential inflammasome function

\u3cp\u3eDendritic cells (DCs) play a key role in orchestrating adaptive immune responses. In human blood, three distinct subsets exist: plasmacytoid DCs (pDCs) and BDCA3+ and CD1c+ myeloid DCs. In addition, a DC-like CD16+ monocyte has been reported. Although RNA-expression profiles have been previously compared, protein expression data may provide a different picture. Here, we exploited label-free quantitative mass spectrometry to compare and identify differences in primary human DC subset proteins. Moreover, we integrated these proteomic data with existing mRNA data to derive robust cell-specific expression signatures with more than 400 differentially expressed proteins between subsets, forming a solid basis for investigation of subset-specific functions. We illustrated this by extracting subset identification markers and by demonstrating that pDCs lack caspase-1 and only express low levels of other inflammasome-related proteins. In accordance, pDCs were incapable of interleukin (IL)-1β secretion in response to ATP.\u3c/p\u3