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Transcriptional targeting of dendritic cells for gene therapy using the promoter of the cytoskeletal protein fascin In immunotherapy, dendritic cells (DC) are of central importance, since they represent the principal inducers of immune responses. Here we describe isolation and use of the promoter of the murine actin bundling protein fascin to target transcriptionally gene expression to cutaneous DC. Using the reporter gene enhanced green fluorescent protein (EGFP), we demonstrate that the fascin promoter mediates a strong antigen expression that is restricted to mature DC. DNA vaccination with antigen encoding expression vectors under control of the fascin promoter using a gene gun resulted, consistently, in limited antigen expression by few directly transfected DC. Nevertheless, nearly as many antigen specific CD8+ T cells directed against the encoded antigens EGFP and galactosidase, respectively, were induced as with expression constructs under control of the ubiquitously expressed CMV promoter. This result impressively underlines the pivotal role of directly transfected DC in DNA vaccination. Immunization using the fascin promoter induced markedly lower levels of antigen specific antibodies following single or repeated immunization. Thus, our DC targeted DNA vaccination approach induces qualitatively distinct, predominantly cellular immune responses and provides new opportunities for immunotherapy. Keywords: Langerhans cell, dendritic cell, DNA vaccination, fascin, gene therapy Immature dendritic cells (DC) like epidermal Langerhans cells represent sentinel cells of the immune system residing in nearly all peripheral organs. They take up antigen and, once activated by inflammatory stimuli, leave peripheral tissues via lymph vessels to enter the T cell areas of draining lymph nodes. There they interact with T cells by presenting processed antigen peptides via major histocompatibility (MHC) molecules to T cells and provide, if full maturation was induced, all necessary costimulatory signals required for efficient activation of naive T cells (for a review see Banchereau and Steinman1). Owing to their primary stimulatory capacity in the mature state and their unique migratory behavior, DC are crucial for elicitation of T helper cell dependent humoral as well as cytotoxic T cell immune responses. Accordingly, transfer experiments show that in vitro transfected DC elicit immune reactions in vivo,2,3 while other cells fail to do so3 unless they are injected directly into lymphoid tissues.4 Antigen presentation by keratinocytes alone leads to immunological unresponsiveness.5,6 The aim of the present study was to isolate a strong cell type specific promoter to target gene expression to mature DC for use in gene therapy. In the preceeding work, we analyzed differential gene expression in maturing DC and identified several genes of functional importance, which are selectively expressed by mature DC and only very few other cell types.7,8 For a detailed analysis, we isolated the promoter of one of these genes, encoding the actin bundling protein fascin. This structural protein crosslinks actin filaments and was shown by us to be pivotal for dendrite formation in DC.7,9 The following characteristics of fascin expression suggested that the promoter might be useful to transcriptionally target production of transgenes to mature DC: (1) As a structural protein fascin is abundantly produced, promising high expression levels of genes under the control of the fascin promoter. (2) Fascin synthesis is switched on during maturation of DC in mouse and man,7,9 directing expression of transgenes to fully mature, primary stimulatory DC. (3) Expression of fascin in nontransformed cell types other than DC is restricted to neuronal tissues, capillary endothelial cells and, possibly, to follicular DC.7,10 It should, therefore, be possible to restrict expression of transgenes to DC using fascin promoter driven expression constructs, for example, by DNA vaccination via the skin. DNA vaccination using a gene gun is an efficient method to elicit cellular as well as humoral immune responses against plasmid encoded antigens.11 Microscopic gold particles with noncovalently attached antigen encoding plasmid DNA are accelerated by helium pressure, penetrate the surface of the skin and transfect skin cells. The transfected cells, because of high abundance mainly keratinocytes and only few DC, express the encoded antigen, and immune responses against this antigen are elicited. Transfection of the DC was, however, demonstrated to be pivotal for the induction of immune responses following DNA vaccination.12,13 To test the fascin promoter in this system, we first isolated the fascin gene from a genomic library derived from C57BL/6 mice using fascin cDNA as a probe. Partial sequencing and restriction mapping ensured that the clone isolated encompasses the complete fascin gene and several kb of flanking sequences. A 3.5 kb subclone was sequenced completely. By comparison with the 5 ends of several fascin cDNA sequences obtained from our differential screenings7 and from the EMBL database, we identified the putative transcriptional start site. As hallmarks of a typical promoter, we identified stretches of GC rich sequences and consensus binding sites for several transcription factors, including a consensus TATA box in appropriate distance from the putative transcriptional start site. Based on these data, the coding sequence of the reporter genes enhanced green fluorescent protein (EGFP) and galactosidase were set under control of a 2.6 kb fascin promoter fragment and inserted into pCI (Promega, Mannheim, Germany), thereby replacing the CMV promoter of pCI (pFascin EGFP and pFascin Gal, Figure 1a). (a) An arrayed genomic library derived from C57BL/6 mice obtained from the Resource Centre of the German Human Genome Project (RZPD, Berlin) was screened as described24 with a mouse fascin cDNA probe. A 2.6 kb promoter fragment including part of the transcribed but nontranslated sequence of the fascin gene was subcloned into vector pCI (Promega, Mannheim, Germany). The coding sequence of either EGFP (derived from plasmid pEGFP N1, BD Biosciences CLONTECH, Heidelberg, Germany) or galactosidase (derived from pCMV, BD Biosciences CLONTECH) was inserted to drive the expression of EGFP (pFascin EGFP) or galactosidase (pFascin Gal) under control of the fascin promoter. The polyadenylation site (pA) of pCI, derived from SV40, is marked. (b) DC2.4 cells were transfected with pCI based expression vectors, driving synthesis of EGFP under control of the fascin promoter (pFascin EGFP) or the cytomegalovirus immediate early promoter (pCMV EGFP). Cells were analyzed for EGFP expression 24 h following transfection by fluorescence microscopy using a BX50WI microscope (Olympus, Hamburg, Germany). (c) The DC lines DC2.4 and XS52 were transfected with p EGFP (pCI based expression vector harboring the EGFP coding region but without any promoter driving its expression), with pCMV EGFP or with pFascin EGFP. EGFP expression of transfected cells was analyzed by cytofluorometry using a FACSscan (Becton Dickinson, Heidelberg, Germany). Chanel FL1 (EGFP+ cells, EGFP) is plotted against sidewards scatter (SSC). EGFP+ cells are located in the gate on the right hand side of the plots. Corresponding numbers of EGFP+ cells are indicated within the gate of each plot. For transfection, GenePORTER reagent (Gene Therapy Systems, San Diego, CA, USA) was used following the recommendations of the manufacturer. Based on optimization experiments 1 g DNA and 5 l (DC2.4) or 8 l (XS52) GenePORTER, respectively, were used. Full figure and legend (251K) To analyze the activity of the fascin promoter, pFascin EGFP was transfected into cells in vitro and in vivo. In parallel, an EGFP construct under the control of the cytomegalovirus immediate early promoter (pCMV EGFP) was used for transfections. EGFP expression was monitored by fluorescence microscopy and cytofluorometry. By fluorescence microscopy, strong EGFP expression was detected with pFascin EGFP in the murine cell line DC2.4 (Figure 1b), which had been characterized to be of mature DC phenotype.14 In contrast, no EGFP expression was observed with pFascin EGFP in XS52 cells, a DC line of immature phenotype15 (data not shown). Using cytofluorometry, numerous EGFP+ cells were detected following transfection with pCMV EGFP in XS52 cells and DC2.4 cells, as well as with pFascin EGFP in DC2.4 cells, but not in XS52 cells. Given the comparable number of EGFP+ XS52 and DC2.4 cells following pCMV EGFP transfection, we can rule out that lack of EGFP+ XS52 cells transfected with pFascin EGFP is by chance, especially as no positive cells were detected in repeated experiments. Thus, the fascin promoter is active in the mature DC line DC2.4 but not in the immature DC line XS52. Notably, XS52 cells do not express fascin.7 Following biolistic in vivo transfection of murine skin with pCMV EGFP, numerous patches with EGFP expressing cells were detected after 24 h in the epidermis of most skin sections (Figure 2a). The EGFP positive cells analyzed were negative for the LC marker langerin/CD20716 (Figure 2b), as was expected, since biolistic transfection is a stochastic event and LC represent only 1 of the epidermal cells. Following biolistic transfection with pFascin EGFP, rare but clearly EGFP positive cells were detected in a few sections (Figure 2c, arrow). These cells were exclusively located in the dermis, partly in deeper layers not penetrated by gold particles. Part of the EGFP+ cells stained positive for langerin (Figure 2c, arrow), identifying them as emigrating LC. Rare langerin negative, EGFP positive cells in the dermis may represent transfected dermal DC, neuronal cells or LC not accessible to the anti langerin antibody within the section. All EGFP positive cells identified in cell suspensions of draining lymph nodes were langerin positive (Figure 2e g), discouraging the view that langerin negative dermal DC had been transfected. Taken together, the fascin promoter is suited to transcriptionally target epidermal LC, which, as shown before,7 upregulate fascin expression following activation. Ear skin of BALB/c mice was transfected by biolistic transfer of 4 g pFascin EGFP and pCMV EGFP, respectively, using a gene gun (Helios BioRad, Hercules, CA, USA) according to the recommendations of the manufacturer. Mice were killed 24 h following biolistic particle bombardment and transfected ears as well as draining lymph nodes were taken. Cross sections of transfected ears (a were analyzed for langerin expression, using the monoclonal anti langerin antibody 929F3, and for EGFP expression (a: pCMV EGFP, EGFP; b: pCMV EGFP, langerin; c: pFascin EGFP, EGFP; d: pFascin EGFP, langerin). Arrows in (c) and (d) mark a cell that is positive for both, EGFP and langerin. Note that langerin+ LC in (b) and (d) emigrate preferentially from the transfected epidermis (upper site) but not from the opposite nontransfected epidermal layer (lower site). Cytospins of lymph node cells from pFascin EGFP transfected mice (e A transfected cell harboring mens ugg boots uk a gold particle (e, arrow) is positive for langerin (f) and EGFP (g). Langerin+ EGFP cells but no langerin EGFP+ cells were detected (not shown). As a control of specificity of immunofluorescence analysis, a CD86 EGFP+ cell line was mixed with a CD86+ EGFP cell line and cytospins were stained for CD86 (h). Such EGFP peptide specific CD8+ T cells, induced by DNA vaccination, had been shown to represent cytotoxic T lymphocytes.17 Notably, comparable numbers of antigen reactive CD8+ effector T cells were induced with both constructs following one single vaccination (P=0.3), despite the drastically lower number of EGFP expressing cells in the skin of pFascin EGFP treated versus pCMV EGFP treated mice. Even after three rounds of vaccination, the number of IFN producing CD8+ T cells was only slightly lower using pFascin EGFP. Similar results were obtained from analogous DNA vaccinations using pFascin Gal with galactosidase as an antigen (Figure 3a). In contrast to the cellular immune response, major differences between mice transfected with pCMV EGFP and pFascin EGFP were observed, when analyzing levels of EGFP specific IgG (Figure 3b). Low, but significant, EGFP specific IgG titers were detected in the sera of mice following one single immunization with pCMV EGFP, while production of EGFP specific IgG following transfection of pFascin EGFP was barely above the detection limit. Repeated immunization with pCMV EGFP tremendously boosted the production of EGFP specific IgG, whereas the increase in IgG antibody titers after three consecutive immunizations with pFascin EGFP was only weak. Again, comparable results were obtained with pFascin Gal (Figure 3b). BALB/c mice were immunized at the shaved abdomen by a single (1 ) or three weekly (3 ) applications of 4 g plasmid DNA using a gene gun (BioRad) according to the recommendations of the manufacturer. Either CMV promoter or fascin promoter cizme ugg online driven EGFP or galactosidase expression constructs were used. (a) Enumeration of antigen peptide specific IFN producing CD8+ effector T cells. ELISPOT assay was performed as described17 with the modification that 3 amino 9 ethylcarbazole (AEC; Sigma Aldrich, Deisenhofen, Germany) was used as substrate. The frequency of CD8+ effector T cells was analyzed in triplicates for each mouse separately. Left: On day 74 of immunization of mice (n=3) with pCMV EGFP or pFascin EGFP, the number of IFN producing CD8+ effector T cells in spleen cell cultures, stimulated with (open bars) or without (solid bars) 1 g/ml EGFP200 peptide, was determined. Right: On day 49 of a single immunization of mice (n=4) and on day 28 after three immunizations (n=3) with pCMV Gal or pFascin Gal, the number of IFN producing CD8+ effector T cells in spleen cell cultures, stimulated with (open bars) or without (solid bars) 1 g/ml galactosidase876 peptide, was determined. Data are representative of at least three independent experiments. (b) Detection of antigen specific IgG antibodies. Sera were recovered from the retro orbital plexus of immunized mice and EGFP specific or galactosidase specific IgG was measured by ELISA as described25 with the modification that recombinant EGFP (2 g/ml; BD Biosciences CLONTECH) or galactosidase (5 g/ml; Sigma Aldrich) was used as antigen. The antibody titer was defined as the reciprocal serum dilution, yielding an absorbance value of OD=0.2 after linear regression analysis. is presented. Left: EGFP specific IgG in the sera of mice (n=3) transfected with pCMV EGFP and pFascin EGFP, respectively, was measured on day 56. Right: galactosidase specific IgG in the sera of mice (n=4 transfected with pCMV Gal and pFasin Gal, respectively, was measured on day 46. Data are representative of at least three independent experiments. Statistically significant differences (PP Full figure and legend (49K) Taken together, the murine fascin promoter was successfully used to target DC in DNA vaccination and offers new opportunities for gene therapy. Our data confirm and extend previous findings indicating that DC targeted DNA vaccination preferentially induces cellular immune responses. Some of us have reported that mice immunized with DC, which had been transduced in vitro with a recombinant adenovirus encoding EGFP, also developed a predominantly cellular immune response against EGFP.18 Furthermore, Morita et al19 recently used the murine dectin 2 promoter to target EGFP expression to DC applying DNA vaccination. Splenocytes from transfected mice showed enhanced cell proliferation and augmented IFN production in response to recombinant EGFP. We add new information to this picture showing that transgene specific IFN producing CD8+ T cells are induced by DC targeted DNA vaccination and that the number of these cells is comparable to the number of transgene specific CD8+ T cells induced in mice transfected with CMV promoter driven constructs. In addition, we detected low but significant amounts of transgene specific antibodies; this finding may be because of a higher sensitivity of the assay used or because of the fact that the fascin promoter is stronger than the dectin 2 promoter. Morita et al19 compared the activity of the dectin 2 promoter with the SV40 promoter and observed a relative expression level similar to the relative expression level we observed using the much stronger CMV promoter for comparison. Cho et al20 used the antigen presenting cell specific CD11b promoter for DNA immunization and identified low but significant amounts of antigen specific antibodies as well. Brocker et al.21 employed the CD11c promoter to target expression of MHC class II I E molecules to DC. All these various promoters have individual characteristics. Special features of the fascin promoter are the high gene expression level in DC and restriction of expression to mature DC. The latter is of importance for the induction of immune responses as mature DC induce novel immune responses, while immature DC were shown to induce anergy.22 Considering that DNA vaccination is currently being broadly tested in clinical trials, we believe that our fascin promoter based vaccination approach offers significant opportunities, especially when cellular immune responses are curative. Importantly, we isolated the human fascin promoter, which shows very similar characteristics (manuscript in preparation) and allows for the first time transcriptional targeting of DC in human gene therapy. Furthermore, our system is ideally suited to study the mechanisms underlying induction of cellular and humoral immune responses following DNA vaccination. DC targeted DNA vaccination differs in some aspects from conventional DNA vaccination, which might explain the reduced humoral response observed: (1) The amount of antigen synthesized is drastically reduced. Most transfected cells are non DC (mainly keratinocytes) and do not express the antigen after vaccination transcriptionally targeting DC. Following conventional DNA vaccination, nonsecreted proteins like EGFP and galactosidase expressed by keratinocytes will be retained intracellularly until the cells die. The subsequent leakage of the antigen may favor the induction of antibody responses as, presumably, more free native antigen is available to supply B cell epitopes. (2) The sites of dominant antigen exposure are different. This might be relevant, as the tissue distribution of antigen may be of importance for the outcome of an immune response.4,23 (3) Owing to the low number of antigen expressing cells, cross presentation of antigen to DC appears to play a minor role. The contribution of these factors to the outcome of the immune response will be addressed in future experiments. Furthermore, the fascin promoter will prove to be very useful to investigate the biological functions of DC in vivo. This may lead to the development of novel applications for DC based immunotherapy. Top of pageReferencesTop of pageAcknowledgementsThis black ugg boots work was supported by the Deutsche Forschungsgemeinschaft, SFB 548 and SFB 432. We thank Dr Albert DeLeo (University of Pittsburgh School of Medicine, Pittsburgh, PA, USA) for providing the EGFP peptide, Dr Kenneth Rock (UMass Medical School, Worcester, MA, USA) for providing the cell line DC2.4, Drs Sem Saeland and Giorgio Trinchieri (both Schering Plough Laboratory for Immunological Research, Dardilly, France) for providing the anti langerin antibody 929F3 and Dr Akira Takashima (University of Texas Southwestern Medical Center, Dallas, TX, USA) for providing the cell line XS52. The expert technical assistance of Ms Katrin Krause Ms Nadine Wiechmann is gratefully acknowledged.

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