Immunogenicity of targeted lentivectors

To increase the safety and possibly efficacy of HIV-1 derived lentivectors (LVs) as an anti-cancer vaccine, we recently developed the Nanobody (Nb) display technology to target LVs to antigen presenting cells (APCs). In this study, we extend these data with exclusive targeting of LVs to conventional dendritic cells (DCs), which are believed to be the main cross-presenting APCs for the induction of a TH1-conducted antitumor immune response. The immunogenicity of these DC-subtype targeted LVs was compared to that of broad tropism, general APC-targeted and non-infectious LVs. Intranodal immunization with ovalbumin encoding LVs induced proliferation of antigen specific CD4(+) T cells, irrespective of the LVs' targeting ability. However, the cytokine secretion profile of the restimulated CD4(+) T cells demonstrated that general APC targeting induced a similar TH1-profile as the broad tropism LVs while transduction of conventional DCs alone induced a similar and less potent TH1 profile as the non-infectious LVs. This observation contradicts the hypothesis that conventional DCs are the most important APCs and suggests that the activation of other APCs is also meaningful. Despite these differences, all targeted LVs were able to stimulate cytotoxic T lymphocytes, be it to a lesser extent than broad tropism LVs. Furthermore this induction was shown to be dependent on type I interferon for the targeted and non-infectious LVs, but not for broad tropism LVs. Finally we demonstrated that the APC-targeted LVs were as potent in therapy as broad tropism LVs and as such deliver on their promise as safer and efficacious LV-based vaccines

Enhancement of the antigen-specific cytotoxic T lymphocyte-inducing ability in the PMDC11 leukemic plasmacytoid dendritic cell line via lentiviral vector-mediated transduction of the caTLR4 gene.

The aim of the present study was to enhance the efficiency of leukemia immunotherapy by increasing the antigen-specific cytotoxic T lymphocyte-inducing ability of leukemia cells. The leukemic plasmacytoid dendritic cell line PMDC05 containing the HLA-A02/24 antigen, which was previously established in our laboratory (Laboratory of Hematology and Oncology, Graduate School of Health Sciences, Niigata University, Niigata, Japan), was used in the present study. It exhibited higher expression levels of CD80 following transduction with lentiviruses encoding the CD80 gene. This CD80-expressing PMDC05 was named PMDC11. In order to establish a more potent antigen-presenting cell for cellular immunotherapy of tumors or severe infections, PMDC11 cells were transduced with a constitutively active (ca) toll-like receptor 4 (TLR4) gene using the Tet-On system (caTLR4-PMDC11). CD8(+) T cells from healthy donors with HLA-A02 were co-cultured with mutant WT1 peptide-pulsed PMDC11, lipopolysaccharide (LPS)-stimulated PMDC11 or caTLR4-PMDC11 cells. Interleukin (IL)-2 (50 IU/ml) and IL-7 (10 ng/ml) were added on day three of culture. Priming with mutant WT1 peptide-pulsed PMDC11, LPS-stimulated PMDC11 or caTLR4-PMDC11 cells was conducted once per week and two thirds of the IL-2/IL-7 containing medium was replenished every 3-4 days. Immediately prior to the priming with these various PMDC11 cells, the cultured cells were analyzed for the secretion of interferon (IFN)-γ in addition to the percentage and number of CD8(+)/WT1 tetramer(+) T cells using flow cytometry. caTLR4-PMDC11 cells were observed to possess greater antigen-presenting abilities compared with those of PMDC11 or LPS-stimulated PMDC11 cells in a mixed leukocyte culture. CD8 T cells positive for the WT1 tetramer were generated following 3-4 weeks of culture and CD8(+)/WT1 tetramer+ T cells were markedly increased in caTLR4-PMDC11-primed CD8(+) T cell culture compared with PMDC11 or LPS-stimulated PMDC11-primed CD8(+) T cell culture. These CD8(+) T cells co-cultured with caTLR4-PMDC11 cells were demonstrated to secrete IFN-γ and to be cytotoxic to WT1-expressing target cells. These data suggested that the antigen-specific cytotoxic T lymphocyte (CTL)-inducing ability of PMDC11 was potentiated via transduction of the caTLR4 gene. The present study also suggested that caTLR4-PMDC11 cells may be applied as potent antigen-presenting cells for generating antigen-specific CTLs in adoptive cellular immunotherapy against tumors and severe viral infections

"The centre of pleasure and magnificence": Paul and Thomas Sandby's London

In this essay John Bonehill examines Six London Views, a set of prints published between late 1766 and early 1768 by Edward Rooker, mainly after designs by Paul and Thomas Sandby. These prints are considered in relation to rival pictorial visions of the city as well as to architectural debates regarding the capital's preservation and modernization—its pasts, presents, and futures. These London views advanced the argument for a more “magnificent” and scenographic cityscape, one indebted to the grand visions of court architects of the past, preeminently Inigo Jones, and more befitting the imperial age ushered in by the victories of the Seven Years' War

Designs on the landscape: Paul and Thomas Sandby in North Britain

In 1747, a young Paul Sandby took up the role of chief draughtsman on the Military Survey of North Britain undertaken by the Board of Ordnance. This ambitious mapping project was part of a series of measures directed at ‘pacifying’ the Scottish highlands in the wake of the Jacobite rebellion of 1745, linking with a large-scale road building programme and the planning of new settlements. In his work for the Board, Sandby was employed in projecting as well as recording the progress of these improvements. Taking forward issues the authors first addressed in the catalogue to a 2009 exhibition of Sandby’s art, this article reviews the artist’s work in North Britain and its role in the remaking of the nation state, locating it in relation to various designs on the landscape. It focuses on drawings made in connection with ‘Utopian’ improvement schemes for planned settlements: firstly, in prospects of the west-coast town of Inveraray, made in collaboration with Sandby’s elder brother Thomas, and secondly, in a view of a surveying party at work near Loch Rannoch, in the southern highlands. They are works which open up wider perspectives on the roles of drawing and design in the period, raising questions too about the significance and scope of the ‘topographical’ as a genre

Design of an optimized Wilms' tumor 1 (WT1) mRNA construct for enhanced WT1 expression and improved immunogenicity in vitro and in vivo

Tumor antigen-encoding mRNA for dendritic cell (DC)-based vaccination has gained increasing popularity in recent years. Within this context, two main strategies have entered the clinical trial stage: the use of mRNA for ex vivo antigen loading of DCs and the direct application of mRNA as a source of antigen for DCs in vivo. DCs transfected with mRNA-encoding Wilms' tumor 1 (WT1) protein have shown promising clinical results. Using a stepwise approach, we re-engineered a WT1 cDNA-carrying transcription vector to improve the translational characteristics and immunogenicity of the transcribed mRNA. Different modifications were performed: (i) the WT1 sequence was flanked by the lysosomal targeting sequence of dendritic cell lysosomal-associated membrane protein to enhance cytoplasmic expression; (ii) the nuclear localization sequence (NLS) of WT1 was deleted to promote shuttling from the nucleus to the cytoplasm; (iii) the WT1 DNA sequence was optimized in silico to improve translational efficiency; and (iv) this WT1 sequence was cloned into an optimized RNA transcription vector. DCs electroporated with this optimized mRNA showed an improved ability to stimulate WT1-specific T-cell immunity. Furthermore, in a murine model, we were able to show the safety, immunogenicity, and therapeutic activity of this optimized mRNA. This work is relevant for the future development of improved mRNA-based vaccine strategies K

HR consultant wellness in the South African working environment

This dissertation focuses on the positive and negative influences on wellness in the South African working environment, as well as three of the aspects that constitute today’s modern definition of wellness, namely: psychological, social, and physical wellness, as perceived by HR consultants in the target organisation. The results of the study determine what the main positive and negative influences on the wellness of HR Consultants were as perceived by the study’s participants. Nature of the Job, State of Mind, and Relationships are some of the most significant identified categories because they best reflect what is perceived to affect each aspect of HR Consultant wellness in the working environment.Dissertation (MCom)--University of Pretoria, 2012.Human Resource Managementunrestricte

Staging grounds: Loutherbourg and Warley

In 1778, Philippe-Jacques de Loutherbourg began work on a pair of companion pictures marking George III's attendance at a spectacular military review on the broad expanse of Essex wasteland that was Warley Common. Scholars of the painter's art have largely overlooked these ambitious, large-scale landscapes, but their commission and subsequent display at the Royal Academy played a key role in advancing Loutherbourg's career. Strikingly novel, the paintings attracted a good deal of critical attention for their curious mix of the patriotic and the satirical, the topographic and the scenographic. Taking its cue from this latter stage set-like quality, this essay situates Loutherbourg’s Warley scenes in relation to a series of dramatic spaces, moving from the ‘battlefield’ depicted to the London stage to the Academy exhibition room, highlighting a series of connections and interactions that shed new light on the performativity of Georgian art, culture and spaces of display