1,721,156 research outputs found
The role of the dendritic cell actin cytoskeleton in the formation and function of the immunological synapse
Full text linkDuring an immune response, dendritic cells (DCs) capture and process antigen, upregulate co-stimulatory molecules and migrate to lymphoid organs to maximise antigen recognition by rare T cell clones. A crucial step for successful T cell activation is cell-cell interaction at the immunological synapse (IS), the organised contact interface which allows optimal communication. IS formation requires the dynamic remodelling of the actin cytoskeleton to spatially distribute membrane areas with distinct protein compositions. While evidence exists for a role of the T cell cytoskeleton in this process, the driving force behind the specific organisation on the DC side is unknown. One important actin regulator is Wiskott-Aldrich Syndrome protein (WASp), expressed exclusively in immune cells. Using a murine model, we investigated the role of WASp in DC-mediated actin-dependent synapse formation. Utilising both confocal and electron microscopy (EM), we observed a disorganised, asymmetric interface between T cells and WASp-deficient DCs. Immunofluorescent staining shows reduced polarisation of certain synapse markers in WAS knock-out conjugates; while high resolution 3D EM reconstructions show severely impaired cell spreading and a reduced contact area. We have used fluorescence recovery after photobleaching (FRAP) to show reduced stability of the actin network in WASp DCs. Our experiments on supported planar bilayers have shown an unexpected interface organisation and highlight a novel role of the DC cytoskeleton in this process. Crucially, we have demonstrated reduced T cell activating capacity of WASp-deficient DCs, as measured by T cell proliferation, cytokine production and transcription factor expression. The results highlight the critical role of cytoskeletal regulation in DC-mediated IS formation. Further, a second murine immunodeficiency model, Dock8 deficiency, and two novel human primary immunodeficiencies were briefly investigated to examine the role of other actin regulators in normal DC function
Lentiviral vector mediated gene therapy for X-linked lymphoproliferative disease
No full textX-linked lymphoproliferative disease (XLP) is a rare primary immunodeficiency characterised by severe immune dysregulation and is caused by mutations in the SH2D1A gene. Clinical manifestations vary and include haemophagocytic lymphohistiocytosis (HLH), lymphoma and dysgammaglobulinaemia, often triggered by Epstein-Barr virus (EBV) infection. SLAM-associated protein (SAP) is a key regulator of immune function in T, NK, and NKT cells and defects in this protein lead to the cellular and humoral immune defects described in patients. Treatment options for XLP are limited and currently haematopoietic stem cell transplant (HSCT) is the only curative option. Results are variable and dependent on a good donor match and absence of active infection at transplant. Somatic gene therapy is now successfully used to correct certain severe immunodeficiencies and offers a potential cure in XLP. The use of self-inactivating (SIN) lentiviral vectors with transgene expression driven by non-viral promoters has improved the biosafety profile of haematopoietic stem cell gene therapy procedures. In this study we have successfully corrected both cellular and humoral defects in a SAP deficient murine model using a SIN lentiviral vector with a codon optimised SAP transgene under the transcriptional control of the elongation factor 1α short form (EFS) promoter. Initial attempts with a non-codon optimised version of SAP led to insufficient protein expression levels to restore immune function. We also assessed the CD2 locus control region (LCR) to evaluate any lymphoid specificity to permit more regulated SAP expression but were unable to demonstrate any benefit with this regulatory element. The results presented here provide proof of concept for the development of gene therapy for XLP and further work is warranted to improve the efficiency of gene transfer, secure engraftment of long term repopulating haematopoietic stem cell progenitors and additional characterisation of immune reconstitution after gene therapy
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
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Development of Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa
Full text linkRecessive dystrophic epidermolysis bullosa (RDEB) is a debilitating genetic cutaneous blistering condition caused by loss-of-function mutations in COL7A1, encoding type VII collagen (C7), central in anchoring fibril (AF) formation at the dermal-epidermal junction (DEJ). Presently no curative treatments exist for RDEB. Reconstitution of COL7A1 expression in autologous primary keratinocytes (KC) and fibroblasts (FB) by ex vivo gene therapy was hypothesised to restore C7 expression and AF construction at the DEJ and ameliorate the RDEB skin phenotype. Feasibility of this approach was demonstrated using a therapeutic grade, self-inactivating- lentiviral vector, encoding codon-optimised COL7A1 (LV-COL7) to transduce primary RDEB KCs and FBs. Expression and secretion of full-length de novo C7 was confirmed, with transduced cells exhibiting supranormal levels of protein expression and functional recovery in in vitro migration assays. A human:murine chimeric preclinical RDEB skin graft model was developed to assess functional correction mediated by the transduced cells in vivo. RDEB grafts lacking C7 expression exhibited severe blistering recapitulating the disease phenotype. Gene- modified grafts, showed C7 deposition at the DEJ, with re-establishment of basement membrane zone integrity. Functional correction was confirmed by an abundance of de novo synthesised AF structures throughout the DEJ securing dermal-epidermal attachment. Gene- corrected FBs were shown to mediate a superior therapeutic benefit. In addition, an alternative strategy was developed using zinc finger nucleases (ZFN) for the targeted editing of COL7A1 and site-specific restoration of endogenous C7 expression. Incorporation of ZFNs into non-integrating lentiviral vectors (NILV) resulted in a marked improvement of their cleavage activity. ZFN-mediated disruption of COL7A1 in KCs confirmed at both genomic and protein level initially enabled the in vitro modelling of RDEB, with observed regression of migration speeds. A dsDNA donor repair template was designed and codon optimised for co-delivery with the ZFNs. Sequencing across the ZFN binding site confirmed site-specific template insertion by targeted homologous recombination. In conclusion, gene correction of primary RDEB cells by LV-COL7 can mediate restoration of protein and structural defects in an RDEB model forming the proposal for therapeutic application in man. Furthermore, development of an alternative site-specific targeting strategy for correction of COL7A1 provides a promising insight into the realm of patient-tailored therapy
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