RNA and Toll-Like Receptor 7 License the Generation of Superior Secondary Plasma Cells at Multiple Levels in a B Cell Intrinsic Fashion

Secondary plasma cells (PCs) originate from memory B cells and produce increased levels of antibodies with higher affinity compared to PCs generated during primary responses. Here we demonstrate that virus-like particles (VLPs) only induce secondary PCs in the presence of toll-like receptor (TLR) 7 and if they are loaded with RNA. Furthermore, adoptive transfer experiments demonstrate that RNA and TLR7 signaling are required for secondary PC generation, both at the level of memory B cell as well as PC differentiation. TLR7-signaling occurred in a B cell intrinsic manner as TLR7-deficient B cells in an otherwise TLR7-competent environment failed to differentiate into secondary PCs. Therefore, RNA inside VLPs is essential for the generation of memory B cells, which are competent to differentiate to secondary PCs and for the differentiation of secondary PCs themselves. While we have not tested all other TLR or non-TLR adjuvants with our VLPs, these data have obvious implications for vaccine design, as RNA packaged into VLPs is a simple way to enhance induction of memory B cells capable of generating secondary PCs

Semen inhibits Zika virus infection of cells and tissues from the anogenital region

Zika virus (ZIKV) causes severe birth defects and can be transmitted via sexual intercourse. Semen from ZIKV-infected individuals contains high viral loads and may therefore serve as an important vector for virus transmission. Here we analyze the effect of semen on ZIKV infection of cells and tissues derived from the anogenital region. ZIKV replicates in all analyzed cell lines, primary cells, and endometrial or vaginal tissues. However, in the presence of semen, infection by ZIKV and other flaviviruses is potently inhibited. We show that semen prevents ZIKV attachment to target cells, and that an extracellular vesicle preparation from semen is responsible for this anti-ZIKV activity. Our findings suggest that ZIKV transmission is limited by semen. As such, semen appears to serve as a protector against sexual ZIKV transmission, despite the availability of highly susceptible cells in the anogenital tract and high viral loads in this bodily fluid.Peer reviewe

Expression of miRNAs miR-133b and miR-206 in the Il17a/f Locus Is Co-Regulated with IL-17 Production in αβ and γδ T Cells

Differentiation of T helper 17 cells (Th17) is a multistep process that involves the cytokines IL-6, TGF-β, and IL-23 as well as IL-1β, IL-21, and TNF-α. Thereby, robust induction of the capacity to produce IL-17 involves epigenetic modifications of the syntenic Il17a/f locus. Using inbred mouse strains, we identified co-regulation of gene transcription at the Il17a/f locus with the nearby microRNAs miR-133b and miR-206 that are clustered approximately 45 kb upstream of Il17a/f. Expression of these microRNAs was specific for Th17 as compared to other CD4+ T cell subsets and this was equally valid for in vitro polarized and ex vivo derived cells. From all factors analyzed, IL-23 was the most important cytokine for the in vitro induction of miR-133b and miR-206 in naive CD4+ T cells of wild type mice. However, analysis of IL-23R deficient mice revealed that IL-23R signaling was not essential for the induction of miR-133b and miR-206. Importantly, we found a similar co-regulation in CCR6+ and other γδ T cell subsets that are predisposed to production of IL-17. Taken together, we discovered a novel feature of T cell differentiation towards an IL-17-producing phenotype that is shared between αβ and γδ T cells. Notably, the specific co-regulation of miR-133b and miR-206 with the Il17a/f locus also extended to human Th17 cells. This qualifies expression of miR-133b and miR-206 in T cells as novel biomarkers for Th17-type immune reactions

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste

Visual performance in augmented reality systems for mobile use

Users of augmented reality (AR) must direct their attention toward real world as well as artificial information. The authors investigated some aspects of interference between the 2 sources of information that affect performance in completing a visual search task. The search task was carried out under 3 different conditions, 2 of them as found in AR in mobile systems. Participants were asked to detect a target that was superimposed on a background. Target and background were presented on a screen subtending a rectangular area of 55degrees x 43degrees (horizontal x vertical). The target appeared at 6 different locations on the screen. A video recording of a car drive served as the background. In 1 condition, the recording was replayed continuously. Static images of the record were sampled at 5-sec intervals and replayed as background in another condition. A uniform gray background served as a baseline. Detectability (d') of the target was highest in the baseline condition. A reduced detectability was found in the presence of static images. Lowest detectability was found in the condition with continuous playback of the video recording. A deterioration of reaction time was found to increase with the same order of conditions as listed earlier. Participants were more efficient in completing the detection task when the targets were presented in the lower part of the screen than in the upper part. The authors concluded that performance in detecting artificial information depends not only on spatial characteristics but also on temporal variations of the background on which the artificial information is superimposed. Determination of suitability of AR systems used in mobile applications therefore requires the characterization of temporal aspects of the presented visual information. They also concluded that the presentation of artificial information in the upper field of vision is a practicable alternative in the case that the lower field is overloaded. However, this statement is true only in the absence of motion. It can be assumed that a visual task involving real world information may be impeded by the adding of artificial information. Artificial information in AR systems should, therefore, be avoided whenever it is not needed. Because of the particular material used in the experiment, the outcome of the search task might have depended on the participants' driving experience. The results obtained, however, indicate that the total amount of kilometers driven is not correlated with performance on the task

Receptor-Ligand Interactions: Binding Affinities Studied by Single-Molecule and Super-Resolution Microscopy on Intact Cells

Dietz MS, Fricke F, Krueger CL, Niemann H, Heilemann M. Receptor-Ligand Interactions: Binding Affinities Studied by Single-Molecule and Super-Resolution Microscopy on Intact Cells. ChemPhysChem. 2014;15(4):671-676.Protein-ligand interactions play an important role in many biological processes. Notably, membrane receptors are the starting point for a huge variety of cellular signal transduction pathways. Quantifying the binding affinity of a ligand for its transmembrane receptor is of great importance as it provides information on the potency of the ligand. We developed a new experimental procedure to determine binding affinities of ligands for their membrane receptors directly on intact single cells using super-resolution imaging. Dissociation constants were determined by titrating fluorophore-labelled ligand against cells expressing the target protein and applying single-molecule imaging

Dynamic changes in the mouse skeletal muscle proteome during denervation-induced atrophy

Loss of neuronal stimulation enhances protein breakdown and reduces protein synthesis, causing rapid loss of muscle mass. To elucidate the pathophysiological adaptations that occur in atrophying muscles, we used stable isotope labelling and mass spectrometry to quantify protein expression changes accurately during denervation-induced atrophy after sciatic nerve section in the mouse gastrocnemius muscle. Additionally, mice were fed a stable isotope labelling of amino acids in cell culture (SILAC) diet containing C-13(6)-lysine for 4, 7 or 11 days to calculate relative levels of protein synthesis in denervated and control muscles. Ubiquitin remnant peptides (K-epsilon-GG) were profiled by immunoaffinity enrichment to identify potential substrates of the ubiquitin-proteasomal pathway. Of the 4279 skeletal muscle proteins quantified, 850 were differentially expressed significantly within 2 weeks after denervation compared with control muscles. Moreover, pulse labelling identified Lys6 incorporation in 4786 proteins, of which 43 had differential Lys6 incorporation between control and denervated muscle. Enrichment of diglycine remnants identified 2100 endogenous ubiquitination sites and revealed a metabolic and myofibrillar protein diglycine signature, including myosin heavy chains, myomesins and titin, during denervation. Comparative analysis of these proteomic data sets with known atrogenes using a random forest approach identified 92 proteins subject to atrogene-like regulation that have not previously been associated directly with denervation-induced atrophy. Comparison of protein synthesis and proteomic data indicated that upregulation of specific proteins in response to denervation is mainly achieved by protein stabilization. This study provides the first integrated analysis of protein expression, synthesis and ubiquitin signatures during muscular atrophy in a living animal

Acetylation and phosphorylation changes to cardiac proteins in experimental HFpEF due to metabolic risk reveal targets for treatment

Aims: Despite the high prevalence of heart failure with preserved ejection fraction (HFpEF), the pathomechan-isms remain elusive and specific therapy is lacking. Disease-causing factors include metabolic risk, notably obesity. However, proteomic changes in HFpEF are poorly understood, hampering therapeutic strategies. We sought to elucidate how metabolic syndrome affects cardiac protein expression, phosphorylation and acetylation in the Zucker diabetic fatty/Spontaneously hypertensive heart failure F1 (ZSF1) rat HFpEF model, and to evaluate changes regarding their potential for treatment.Main methods: ZSF1 obese and lean rats were fed a Purina diet up to the onset of HFpEF in the obese animals. We quantified the proteome, phosphoproteome and acetylome of ZSF1 obese versus lean heart tissues by mass spectrometry and singled out targets for site-specific evaluation.Key findings: The acetylome of ZSF1 obese versus lean hearts was more severely altered (21 % of proteins changed) than the phosphoproteome (9 %) or proteome (3 %). Proteomic alterations, confirmed by immuno-blotting, indicated low-grade systemic inflammation and endothelial remodeling in obese hearts, but low nitric oxide-dependent oxidative/nitrosative stress. Altered acetylation in ZSF1 obese hearts mainly affected pathways important for metabolism, energy production and mechanical function, including hypo-acetylation of mechan-ical proteins but hyper-acetylation of proteins regulating fatty acid metabolism. Hypo-acetylation and hypo-phosphorylation of elastic titin in ZSF1 obese hearts could explain myocardial stiffening.Significance: Cardiometabolic syndrome alters posttranslational modifications, notably acetylation, in experi-mental HFpEF. Pathway changes implicate a HFpEF signature of low-grade inflammation, endothelial dysfunction, metabolic and mechanical impairment, and suggest titin stiffness and mitochondrial metabolism as promising therapeutic targets