The rockefeller university

DCs are critical for initiating immunity. The current paradigm in vaccine biology is that DCs migrating from peripheral tissue and classical lymphoid-resident DCs (cDCs) cooperate in the draining LNs to initiate priming and proliferation of T cells. Here, we observe subcutaneous immunity is Fms-like tyrosine kinase 3 ligand (Flt3L) dependent. Flt3L is rapidly secreted after immunization; Flt3 deletion reduces T cell responses by 50%. Flt3L enhances global T cell and humoral immunity as well as both the numbers and antigen capture capacity of migratory DCs (migDCs) and LN-resident cDCs. Surprisingly, however, we find immunity is controlled by cDCs and actively tempered in vivo by migDCs. Deletion of Langerin(+) DC or blockade of DC migration improves immunity. Consistent with an immune-regulatory role, transcriptomic analyses reveals different skin migDC subsets in both mouse and human cluster together, and share immune-suppressing gene expression and regulatory pathways. These data reveal that protective immunity to protein vaccines is controlled by Flt3L-dependent, LN-resident cDCs.

Novel methods are required for site-specific, quantitative fluorescent labeling of G-protein-coupled receptors (GPCRs) and other difficult-to-express membrane proteins. Ideally, fluorescent probes should perturb the native structure and function as little as possible. We evaluated bioorthogonal reactions to label genetically encoded p-acetyl-l-phenylalanine (AcF) or p-azido-l-phenylalanine (azF) residues in receptors heterologously expressed in mammalian cells. We found that keto-selective reagents were not truly bioorthogonal, possibly owing to post-translational protein oxidation reactions. In contrast, the strainpromoted [3+ 2] azide-alkyne cycloaddition (SpAAC) with dibenzocyclooctyne (DIBO) reagents yielded stoichiometric conjugates with azF-rhodopsin while undergoing negligible background reactions. As one application of this technique, we used Alexa488-rhodopsin to measure the kinetics of ligand uptake and release in membrane-mimetic bicelles using a novel fluorescence-quenching assay.

Environmental Surveyor of Natural Product Diversity (eSNaPD) is a web-based bioinformatics and data aggregation platform that aids in the discovery of gene clusters encoding both novel natural products and new congeners of medicinally relevant natural products using (meta) genomic sequence data. Using PCR-generated sequence tags, the eSNaPD data-analysis pipeline profiles biosynthetic diversity hidden within (meta) genomes by comparing sequence tags to a reference data set of characterized gene clusters. Sample mapping, molecule discovery, library mapping, and new clade visualization modules facilitate the interrogation of large (meta) genomic sequence data sets for diverse downstream analyses, including, but not limited to, the identification of environments rich in untapped biosynthetic diversity, targeted molecule discovery efforts, and chemical ecology studies. eSNaPD is designed to generate a global atlas of biosynthetic diversity that can facilitate a systematic, sequence-based interrogation of nature's biosynthetic potential.

Immunological memory has been regarded as a unique feature of the adaptive immune response mediated in an antigen-specific manner by T and B lymphocytes. However, natural killer (NK) cells and gamma delta T cells, which traditionally are classified as innate immune cells, have been shown in recent studies to have hallmark features of memory cells. Invariant NKT cell (iNKT cell)-mediated antitumor effects indicate that iNKT cells are activated in vivo by vaccination with iNKT cell ligand-loaded CD1d(+) cells, but not by vaccination with unbound NKT cell ligand. In such models, it previously was thought that the numbers of IFN-gamma-producing cells in the spleen returned to the basal level around 1 wk after the vaccination. In the current study, we demonstrate the surprising presence of effector memory-like iNKT cells in the lung. We found long-term antitumor activity in the lungs of mice was enhanced after vaccination with iNKT cell ligand-loaded dendritic cells. Further analyses showed that the KLRG1(+) (Killer cell lectin-like receptor subfamily G, member 1-positive) iNKT cells coexpressing CD49d and granzyme A persisted for several months and displayed a potent secondary response to cognate antigen. Finally, analyses of CDR3 beta by RNA deep sequencing demonstrated that some particular KLRG1(+) iNKT-cell clones accumulated, suggesting the selection of certain T-cell receptor repertoires by an antigen. The current findings identifying effector memory-like KLRG1(+) iNKT cells in the lung could result in a paradigm shift regarding the basis of newly developed extrathymic iNKT cells and could contribute to the future development of antitumor immunotherapy by uniquely energizing iNKT cells.

The skin protects mammals from insults, infection and dehydration and enables thermoregulation and sensory perception. Various skin-resident cells carry out these diverse functions. Constant turnover of cells and healing upon injury necessitate multiple reservoirs of stem cells. Thus, the skin provides a model for studying interactions between stem cells and their microenvironments, or niches. Advances in genetic and imaging tools have brought new findings about the lineage relationships between skin stem cells and their progeny and about the mutual influences between skin stem cells and their niches. Such knowledge may offer novel avenues for therapeutics and regenerative medicine.

Asymmetric cell divisions allow stem cells to balance proliferation and differentiation. During embryogenesis, murine epidermis expands rapidly from a single layer of unspecified basal layer progenitors to a stratified, differentiated epithelium. Morphogenesis involves perpendicular (asymmetric) divisions and the spindle orientation protein LGN, but little is known about how the apical localization of LGN is regulated. Here, we combine conventional genetics and lentiviral-mediated in vivo RNAi to explore the functions of the LGN-interacting proteins Par3, mInsc and G alpha(i3). Whereas loss of each gene alone leads to randomized division angles, combined loss of Gnai3 and mInsc causes a phenotype of mostly planar divisions, akin to loss of LGN. These findings lend experimental support for the hitherto untested model that Par3-mInsc and G alpha(i3) act cooperatively to polarize LGN and promote perpendicular divisions. Finally, we uncover a developmental switch between delamination-driven early stratification and spindle-orientation-dependent differentiation that occurs around E15, revealing a two-step mechanism underlying epidermal maturation.

Background. Only a minority of individuals infected with Mycobacterium tuberculosis develop clinical tuberculosis. Genetic epidemiological evidence suggests that pulmonary tuberculosis has a strong human genetic component. Previous genetic findings in Mendelian predisposition to more severe mycobacterial infections, including by M. tuberculosis, underlined the importance of the interleukin 12 (IL-12)/interferon gamma (IFN-gamma) circuit in antimycobacterial immunity. Methods. We conducted an association study in Morocco between pulmonary tuberculosis and a panel of single-nucleotide polymorphisms (SNPs) covering 14 core IL-12/IFN-gamma circuit genes. The analyses were performed in a discovery family-based sample followed by replication in a case-control population. Results. Out of 228 SNPs tested in the family-based sample, 6 STAT4 SNPs were associated with pulmonary tuberculosis (P=.0013-.01). We replicated the same direction of association for 1 cluster of 3 SNPs encompassing the promoter region of STAT4. In the combined sample, the association was stronger among younger subjects (pulmonary tuberculosis onset <25 years) with an odds ratio of developing pulmonary tuberculosis at rs897200 for GG vs AG/AA subjects of 1.47 (1.06-2.04). Previous functional experiments showed that the G allele of rs897200 was associated with lower STAT4 expression. Conclusions. Our present findings in a Moroccan population support an association of pulmonary tuberculosis with STAT4 promoter-region polymorphisms that may impact STAT4 expression.

Ethnopharmacological relevance: Antrodia cinnamomea - a medicinal fungus that is indigenous to Taiwan - has been used as a health tonic by aboriginal tribes and the Asian population. Recent studies indicate that Antrodia cinnamomea extracts exhibit hepato-protective, anti-hypertensive, anti-oxidative, anti-inflammatory, immuno-modulatory, and anti-cancer effects on cultured cells and laboratory animals. This study aims to explore the anti-inflammatory activity of an Antrodia cinnamomea ethanol extract (ACEE) and elucidate its underlying mechanisms of action using lipopolysaccharide (LPS)-primed, ATP-stimulated human THP-1 macrophages. Materials and methods: The effects of ACEE on cell viability were studied using the MTT assay. The expressions of genes, proteins, and pro-inflammatory cytokines were measured by quantitative real-time RT-PCR, Western blotting and ELISA, respectively. The ACEE was further investigated for its effects on reactive oxygen species (ROS) production using ROS detection kit. Results: Our results showed that ACEE significantly inhibits ATP-induced secretion of interleukin-1 beta (IL-1 beta), interleukin-18 (IL-18) and tumor necrosis factor-alpha (TNF-alpha) by LPS-primed macrophages. ACEE also suppresses the transcription and activation of caspase-1, which is responsible for the cleavage and activation of IL-1 beta and IL-18. Of note, ACEE not only reduces expression of the inflammasome component NLRP3 and the purinergic receptor P2X(7)R but also inhibits ATP-induced ROS production and caspase-1 activation. Furthermore, the anti-inflammatory properties of ACEE correlate with reduced activation of the MAPK and NF-kappa B pathways. Conclusion: The results of the present study indicate that Antrodia cinnamomea suppresses the secretion of IL-1 beta and IL-18 associated with inhibition of the NLRP3 inflammasome in macrophages. These findings suggest that ACEE may have therapeutic potential for the treatment of inflammatory diseases. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

A search for WV gamma triple vector boson production is presented based on events containing a W boson decaying to a muon or an electron and a neutrino, a second V (W or Z) boson, and a photon. The data correspond to an integrated luminosity of 19.3 fb(-1) collected in 2012 with the CMS detector at the LHC in pp collisions at root s = 8 TeV. An upper limit of 311 fb on the cross section for the WV gamma production process is obtained at 95% confidence level for photons with a transverse energy above 30 GeV and with an absolute value of pseudorapidity of less than 1.44. This limit is approximately a factor of 3.4 larger than the standard model predictions that are based on next-to-leading order QCD calculations. Since no evidence of anomalous WW gamma gamma or WWZ gamma quartic gauge boson couplings is found, this paper presents the first experimental limits on the dimension-eight parameter f(T,0) and the CP-conserving WWZ gamma parameters kappa(W)(0) and kappa(W)(C). Limits are also obtained for the WW gamma gamma parameters a(0)(W) and a(C)(W).