Increased T cell breadth and antibody response elicited in prime-boost regimen by viral vector encoded homologous SIV Gag/Env in outbred CD1 mice

BACKGROUND: A major obstacle for the development of HIV vaccines is the virus’ worldwide sequence diversity. Nevertheless, the presence of T cell epitopes within conserved regions of the virus’ structural Gag protein and conserved structures in the envelope (env) sequence raises the possibility that cross-reactive responses may be induced by vaccination. In this study, the aim was to investigate the importance of antigenic match on immunodominance and breadth of obtainable T cell responses. METHODS: Outbred CD1 mice were immunized with either heterologous (SIVmac239 and HIV-1 clade B consensus) or homologous (SIVmac239) gag sequences using adenovirus (Ad5) and MVA vectors. Env (SIVmac239) was co-encoded in the vectors to study the induction of antibodies, which is a primary target of current HIV vaccine designs. All three vaccines were designed as virus-encoded virus-like particle vaccines. Antibody responses were analysed by ELISA, avidity ELISA, and neutralization assay. T cell responses were determined by intracellular cytokine staining of splenocytes. RESULTS: The homologous Env/Gag prime-boost regimen induced higher Env binding antibodies, and induced stronger and broader Gag specific CD8+ T cell responses than the homologous Env/heterologous Gag prime-boost regimen. Homologous Env/heterologous Gag immunization resulted in selective boosting of Env specific CD8+ T cell responses and consequently a paradoxical decreased recognition of variant sequences including conserved elements of p24 Gag. CONCLUSIONS: These results contrast with related studies using Env or Gag as the sole antigen and suggest that prime-boost immunizations based on homologous SIVmac239 Gag inserts is an efficient component of genetic VLP vaccines—both for induction of potent antibody responses and cross-reactive CD8+ T cell responses

Virus-like vaccines against HIV/SIV synergize with a subdominant antigen T cell vaccine

Additional file 1. Gating strategy for flow cytometry analysis of intracellular cytokine staining. First, the cells were gated for single cells (a) in a side scatter (SSC)-A/SSC-W plot, which were further gated for the lymphocyte population (b) in a plot of forward scatter (FCS)-A and SSC-A. The lymphocyte population was gated for CD8+ B220- cells (c) and CD4+ B220- cells (d). Next, these cells were gated for IFNγ+ CD44+ cells (e, upper rectangle), here shown representatively for the CD8+ population. From these populations the absolute number of IFNγ+ CD44+ B220- CD8+ and CD4+ T-cells was calculated by multiplying the percentage of IFNγ+ CD44+ B220- CD8+/CD4+ cells of the lymphocytes with the number of counted lymphocytes per spleen. To obtain the percentage of double positive (IFNγ+ TNFα+) cells of IFNγ+ CD8+ and CD4+ T-cells, CD8+/CD4+ B220- cells were also gated for CD44+ cells (e, both rectangles) in homologous prime-boost regimen and subsequently for IFNγ- TNFα+ (f, left rectangle) and IFNγ+ TNFα+ (f, right rectangle) cells

Increased immunogenicity and protective efficacy of influenza M2e fused to a tetramerizing protein

The ectodomain of the matrix 2 protein (M2e) of influenza A virus represents an attractive target for developing a universal influenza A vaccine, with its sequence being highly conserved amongst human variants of this virus. With the aim of targeting conformational epitopes presumably shared by diverse influenza A viruses, a vaccine (M2e-NSP4) was constructed linking M2e (in its consensus sequence) to the rotavirus fragment NSP4(98-135); due to its coiled-coil region this fragment is known to form tetramers in aqueous solution and in this manner we hoped to mimick the natural configuration of M2e as presented in membranes. M2e-NSP4 was then evaluated side-by-side with synthetic M2e peptide for its immunogenicity and protective efficacy in a murine influenza challenge model. Here we demonstrate that M2e fused to the tetramerizing protein induces an accelerated, augmented and more broadly reactive antibody response than does M2e peptide as measured in two different assays. Most importantly, vaccination with M2e-NSP4 caused a significant decrease in lung virus load early after challenge with influenza A virus and maintained its efficacy against a lethal challenge even at very low vaccine doses. Based on the results presented in this study M2e-NSP4 merits further investigation as a candidate for or as a component of a universal influenza A vaccine

Scaling Rule for Nonperturbative Radiation in a Class of Event Shapes

We discuss nonperturbative radiation for a recently introduced class of infrared safe event shape weights, which describe the narrow-jet limit. Starting from next-to-leading logarithmic (NLL) resummation, we derive an approximate scaling rule that relates the nonperturbative shape functions for these weights to the shape function for the thrust. We argue that the scaling reflects the boost invariance implicit in NLL resummation, and discuss its limitations. In the absence of data analysis for the new event shapes, we compare these predictions to the output of the event generator PYTHIA.Comment: 23 pages, 3 figures, uses JHEP3.cls (included); v2 - version to appear in JHE

Nuclear expression of FLT1 and its ligand PGF in FUS-DDIT3 carrying myxoid liposarcomas suggests the existence of an intracrine signaling loop

<p>Abstract</p> <p>Background</p> <p>The FUS-DDIT3 fusion oncogene encodes an abnormal transcription factor that has a causative role in the development of myxoid/round-cell liposarcomas (MLS/RCLS). We have previously identified <it>FLT1 </it>(<it>VEGFR1</it>) as a candidate downstream target gene of FUS-DDIT3. The aim of this study was to investigate expression of FLT1 and its ligands in MLS cells.</p> <p>Methods</p> <p>HT1080 human fibrosarcoma cells were transiently transfected with <it>FUS-DDIT3</it>-GFP variant constructs and FLT1 expression was measured by quantitative real-time PCR. In addition, <it>FLT1</it>, <it>PGF, VEGFA and VEGFB </it>expression was measured in MLS/RCLS cell lines, MLS/RCLS tumors and in normal adiopocytes. We analyzed nine cases of MLS/RCLS and one cell line xenografted in mice for FLT1 protein expression using immunohistochemistry. MLS/RCLS cell lines were also analyzed for FLT1 by immunofluorescence and western blot. MLS/RCLS cell lines were additionally treated with FLT1 tyrosine kinase inhibitors and assayed for alterations in proliferation rate.</p> <p>Results</p> <p><it>FLT1 </it>expression was dramatically increased in transfected cells stably expressing FUS-DDIT3 and present at high levels in cell lines derived from MLS. The FLT1 protein showed a strong nuclear expression in cells of MLS tissue as well as in cultured MLS cells, which was confirmed by cellular fractionation. Tissue array analysis showed a nuclear expression of the FLT1 protein also in several other tumor and normal cell types including normal adipocytes. The FLT1 ligand coding gene <it>PGF </it>was highly expressed in cultured MLS cells compared to normal adipocytes while the other ligand genes <it>VEGFA </it>and <it>VEGFB </it>were expressed to lower levels. A more heterogeneous expression pattern of these genes were observed in tumor samples. No changes in proliferation rate of MLS cells were detected at concentrations for which the kinase inhibitors have shown specific inhibition of FLT1.</p> <p>Conclusions</p> <p>Our results imply that <it>FLT1 </it>is induced as an indirect downstream effect of FUS-DDIT3 expression in MLS. This could be a consequence of the ability of FUS-DDIT3 to hijack parts of normal adipose tissue development and reprogram primary cells to a liposarcoma-like phenotype. The findings of nuclear FLT1 protein and expression of corresponding ligands in MLS and normal tissues may have implications for tissue homeostasis and tumor development through auto- or intracrine signaling.</p

EMBL Nucleotide Sequence Database: developments in 2005

The EMBL Nucleotide Sequence Database () at the EMBL European Bioinformatics Institute, UK, offers a comprehensive set of publicly available nucleotide sequence and annotation, freely accessible to all. Maintained in collaboration with partners DDBJ and GenBank, coverage includes whole genome sequencing project data, directly submitted sequence, sequence recorded in support of patent applications and much more. The database continues to offer submission tools, data retrieval facilities and user support. In 2005, the volume of data offered has continued to grow exponentially. In addition to the newly presented data, the database encompasses a range of new data types generated by novel technologies, offers enhanced presentation and searchability of the data and has greater integration with other data resources offered at the EBI and elsewhere. In stride with these developing data types, the database has continued to develop submission and retrieval tools to maximise the information content of submitted data and to offer the simplest possible submission routes for data producers. New developments, the submission process, data retrieval and access to support are presented in this paper, along with links to sources of further information

Distinct Cytoplasmic and Nuclear Functions of the Stress Induced Protein DDIT3/CHOP/GADD153

DDIT3, also known as GADD153 or CHOP, encodes a basic leucine zipper transcription factor of the dimer forming C/EBP family. DDIT3 is known as a key regulator of cellular stress response, but its target genes and functions are not well characterized. Here, we applied a genome wide microarray based expression analysis to identify DDIT3 target genes and functions. By analyzing cells carrying tamoxifen inducible DDIT3 expression constructs we show distinct gene expression profiles for cells with cytoplasmic and nuclear localized DDIT3. Of 175 target genes identified only 3 were regulated by DDIT3 in both cellular localizations. More than two thirds of the genes were downregulated, supporting a role for DDIT3 as a dominant negative factor that could act by either cytoplasmic or nuclear sequestration of dimer forming transcription factor partners. Functional annotation of target genes showed cell migration, proliferation and apoptosis/survival as the most affected categories. Cytoplasmic DDIT3 affected more migration associated genes, while nuclear DDIT3 regulated more cell cycle controlling genes. Cell culture experiments confirmed that cytoplasmic DDIT3 inhibited migration, while nuclear DDIT3 caused a G1 cell cycle arrest. Promoters of target genes showed no common sequence motifs, reflecting that DDIT3 forms heterodimers with several alternative transcription factors that bind to different motifs. We conclude that expression of cytoplasmic DDIT3 regulated 94 genes. Nuclear translocation of DDIT3 regulated 81 additional genes linked to functions already affected by cytoplasmic DDIT3. Characterization of DDIT3 regulated functions helps understanding its role in stress response and involvement in cancer and degenerative disorders

Designing a memorial place

The design and selection of a memorial stone and the site of the grave, both of which represent the deceased, can be a central issue for people bereaved by traffic accidents. This was revealed in an interview survey of recent Swedish roadside memorials and other memorial places. In this article we consider the design and selection of the memorial stone and gravesite as expressions of continuing care for the deceased and as a way to offer comfort to the bereaved. Materiality, representation and presence will be discussed as crucial parts of the link between the living and the dead. Communicative, spatial and physical values are important also in the professional's design of common public memorial places. Of specific interest for this text are two design practice-based terms, memory object and passage landscape, which may be used by landscape architects when designing memorial places, such as cemeteries and public monuments. Throughout this text, we argue that memorial places like these are capable of bridging the gap between the space of life and the space of death, as well as supporting the regeneration of present memories and the construction of future ones

Motivationshöjande arbetssätt och stödinsatser i matematikundervisning : En småskalig intervjustudie med lärare i grundskolan

Lärares arbetssätt och stödinsatser undersöktes, som kan gynna elevers motivation och matematikutveckling med inriktning på att kunskapskraven ska kunna uppnås. Det är en kvalitativ intervjustudie  med  ett urval av lärare i matematik, speciallärare och specialpedagoger som arbetar i grundskolan. Data analyserades tematiskt och resulterade i tre teman: relationsskapande och trygghet; struktur, hjälpmedel och lustyllt lärande samt undervisningsnivå och tempo.

Organiserat samarbete mellan lärare och kollegor inom IKT-baserad distansundervisning

Abstrakt Detta är en hermeneutisk studie där vi använt oss av intervjuer som datainsamlingsmetod. Vi har studerat samarbete ur ett lärarperspektiv i distansundervisning på Blekinge Tekniska högskola (BTH). Vad vi fann var att lärarna och deras kollegor efterfrågade ett mer organiserat samarbete dem emellan. Två former för ett ökat samarbete är, samarbete i team och i distansnätverk, vilka kan existera parallellt med varandra. I nätverket ingår alla personer som arbetar med distanskurser, allt ifrån lärare till tekniker. Team är en mindre enhet för samarbete och är uppbyggd runt en specifik distanskurs. Deltagarna i teamet är experter på olika yrkesområden och kan komplettera och hjälpa varandra. Lärarna är experter på sitt område, vilket är undervisning, och vill inte behöva känna sig tvingade att bli experter på nya områden, för att kunna undervisa på distans. Undersökningen visar att de teammedlemmar som kan vara till hjälp för lärarna är: Andra lärare, Administratörer, Teknikpedagoger, Bibliotekarier, Inlånade kompetenser, Tekniker och Studentrepresentanter. Kursen och temadeltagarna, i huvudsak lärarna, avgör när och hur mycket de olika experterna behövs under kursen. Men det är viktigt att vara medveten om att alla inte vill arbeta i team eller anser att samarbete alltid är positivt. Samarbete och teamarbete ska ses som ett stöd för lärarnas i deras arbete