1,721,023 research outputs found
Microbial recognition via toll-like receptor-dependent and -independent pathways determines the cytokine response of murine dendritic cell subsets to CD40 triggering
No full textDendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8α+ and CD8α-CD4- DC to make IL-12 p70. In contrast, exposure of CD8α+, CD4+ and CD8α-CD4- DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets
Relationships among murine CD11chigh dendritic cell subsets as revealed by baseline gene expression patterns
No full textFormulation of a live bacterial vaccine for stable room temperature storage results in loss of acid, bile and bile salt resistance
No full textOptimal protection of stabilised dry live bacteria from bile toxicity in oral dosage forms by bile acid adsorbent resins
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What have dendritic cells ever done for adjuvant design? Cellular and molecular methods for the rational development of vaccine adjuvants
No full textOur new molecular understanding of immune priming states that dendritic cell activation is absolutely pivotal for expansion and differentiation of naïve T lymphocytes, and it follows that understanding DC activation is essential to understand and design vaccine adjuvants. This chapter describes how dendritic cells can be used as a core tool to provide detailed quantitative and predictive immunomics information about how adjuvants function. The role of distinct antigen, costimulation, and differentiation signals from activated DC in priming is explained. Four categories of input signals which control DC activation – direct pathogen detection, sensing of injury or cell death, indirect activation via endogenous proinflammatory mediators, and feedback from activated T cells – are compared and contrasted. Practical methods for studying adjuvants using DC are summarised and the importance of DC subset choice, simulating T cell feedback, and use of knockout cells is highlighted. Finally, five case studies are examined that illustrate the benefit of DC activation analysis for understanding vaccine adjuvant function
Targeted activation of toll-like receptors: conjugation of a toll-like receptor 7 agonist to a monoclonal antibody maintains antigen binding and specificity
No full textTherapeutic activation of Toll-like receptors (TLR) has potential for cancer immunotherapy, for augmenting the activity of antitumor monoclonal antibodies (mAbs), and for improved vaccine adjuvants. A previous attempt to specifically target TLR agonists to dendritic cells (DC) using mAbs failed because conjugation led to nonspecific binding and mAbs lost specificity. We demonstrate here for the first time the successful conjugation of a small molecule TLR7 agonist to an antitumor mAb (the anti-hCD20 rituximab) without compromising antigen specificity. The TLR7 agonist UC-1V150 was conjugated to rituximab using two conjugation methods, and yield, molecular substitution ratio, retention of TLR7 activity, and specificity of antigen binding were compared. Both conjugation methods produced rituximab–UC-1V150 conjugates with UC-1V150: rituximab ratio ranging from 1:1 to 3:1 with drug loading quantified by UV spectroscopy and drug substitution ratio verified by MALDI TOF mass spectroscopy. The yield of purified conjugates varied with conjugation method and dropped as low as 31% using a method previously described for conjugating UC-1V150 to proteins, where a bifunctional cross-linker was first reacted with rituximab and second to the TLR7 agonist. We therefore developed a direct conjugation method by producing an amine-reactive UV active version of UC-1V150, termed NHS:UC-1V150. Direct conjugation with NHS:UC-1V150 was quick and simple and gave improved conjugate yields of 65–78%. Rituximab–UC-1V150 conjugates had the expected pro-inflammatory activity in vitro (EC50 28–53 nM) with a significantly increased activity over unconjugated UC-1V150 (EC50 547 nM). Antigen binding and specificity of the rituxuimab–UC-1V150 conjugates was retained, and after incubation with human peripheral blood leukocytes, all conjugates bound strongly only to CD20-expressing B cells while no nonspecific binding to CD20-negative cells was observed. Selective targeting of Toll-like receptor activation directly within tumors or to DC is now feasible
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Methods for rapid prototyping novel labware: using CAD and desktop 3D printing in the microbiology laboratory
No full textAlthough the microbiology laboratory paradigm has increasingly changed from manual to automated procedures, and from functional to molecular methods, traditional culture methods remain vital. Using inexpensive desktop fused filament fabrication 3D printing, we designed, produced and tested rapid prototypes of customised labware for microbial culture namely frames to make dip slides, inoculation loops, multi-pin replicators, and multi-well culture plates for solid medium. These customised components were used to plate out samples onto solid media in various formats, and we illustrate how they can be suitable for many microbiological methods such as minimum inhibitory concentration tests, or for directly detecting pathogens from mastitis samples, illustrating the flexibility of rapid-prototyped culture consumable parts for streamlining microbiological methods. We describe the methodology needed for microbiologists to develop their own novel and unique tools, or to fabricate and customise existing consumables. A workflow is presented for designing and 3D printing labware and quickly producing easy-to-sterilise and re-useable plastic parts of great utility in the microbiology laboratory
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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