Receptor-Mediated Gene Delivery

Receptor-mediated gene delivery capitalises on the presence of specific cell surface molecules for DNA uptake into cells and represents a particularly appealing approach for targeting vector DNA to specific cell types in vivo and in vitro. Various ligand/DNA and antibody/DNA transfer complexes were generated that, following binding to cells, are internalised and reach the endosomal compartment. Vector complexes contain endosomolytic components that ensure vector release from the endosome and translocation of vector DNA into the nucleus where transcription occurs. Thus, receptor-mediated gene delivery encompasses several critical steps that must be considered when designing and applying such vector systems

RNA-containing adenovirus/polyethylenimine transfer complexes effectively transduce dendritic cells and induce antigen-specific T cell responses

Background: Dendritic cells (DCs) are the most potent antigen‐presenting cells in initiating primary immune responses. Given the unique properties of DCs, gene‐modified DCs represent a particularly attractive approach for immunotherapy of diseases such as cancer. Methods: Gene‐modified DCs were obtained by a receptor‐mediated gene delivery system using adenovirus (Ad) particles as ligand and RNA or DNA condensed by polyethylenimine (PEI). In vitro transcribed polyadenylated or non‐polyadenylated RNA was used. RNA‐transduced DCs were generated expressing chicken ovalbumin (OVA) or chimeric constructs thereof, and compared with DNA‐transduced DCs. Results: Ad/PEI transfection complexes efficiently delivered RNA into DCs. Such RNA‐transduced DCs induced OVA‐specific T cell responses more effectively than DNA‐transduced DCs. Furthermore, DCs transduced with polyadenylated RNA were more potent in stimulating CD4+ and CD8+ T cell responses than DCs transduced with non‐polyadenylated RNA and this was particularly important for CD4+ T cell responses. Conclusions: Ad/PEI/RNA transfection is an efficient means for generating RNA‐transduced DCs and for stimulating antigen‐specific T cell responses. Polyadenylation of RNA enhances CD8+ T cell responses and is essential for CD4+ T cell responses

RNA interference-mediated gene silencing in murine T cells: in vitro and in vivo validation of proinflammatory target genes

BACKGROUND: T cells play a central role in many inflammatory diseases, hence the identification and validation of T cell-specific target genes will increase the understanding of T cell function in pathologic inflammatory situations. RNA interference (RNAi), with its ability to induce specific gene silencing in mammalian cells, represents a powerful technology to investigate and validate the function of pharmaceutical target genes in vitro and in vivo. The aim of the present study was to systematically explore RNAi-mediated gene-silencing of known T cell-specific model signaling molecules in primary murine T cells in vitro and in vivo. RESULTS: We demonstrate that siRNA delivery and subsequent silencing of T cell specific genes is substantially increased, if murine T cells were activated prior siRNA transfection. Silencing of ZAP70, p56Lck as well as PLC-γ1 protein expression resulted in impaired function of T cells in vitro. Furthermore, delayed type hypersensitivity (DTH) was ameliorated in vivo after adoptive transfer of ZAP70-silenced T cells. COCLUSION: The combination of RNAi-mediated gene silencing and adoptive transfer of gene-silenced T cells, thus, may allow the identification and analysis of T cell-specific targets for therapeutic intervention. Additionally, this model system may represent an alternative to conventional time consuming and cost intensive gene targeting approaches

The impact of c-met/scatter factor receptor on dendritic cell migration

Dendritic cells (DC) are professional antigen-presenting cells that possess both migratory properties and potent T cell stimulatory activity, and that allow the uptake of antigenic material in peripheral tissues and its subsequent presentation in the T cell areas of lymphoid organs. Thus motility represents a central property that is required for DC function. Here we report on the expression of the receptor tyrosine kinase c-met in DC. c-Met is the high affinity receptor for scatter factor (SF)/hepatocyte growth factor, and ligand-activated c-met exhibits mitogenic, morphogenic and motogenic activity in vivo and in vitro. c-Met is signaling competent in DC since it is effectively tyrosine phosphorylated in response to SF ligand. It is demonstrated here that ligand-activated c-met regulates DC adhesion to the extracellular matrix component laminin but leaves antigen presenting function unaffected. Importantly, in ear sheet explant experiments activation of c-met by ligand induces emigration of cutaneous DC (Langerhans cell, LC) from skin, but SF is not a chemoattractant factor for DC. Our results suggest an important role of the c-met/SF system in DC/LC migration

RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes-1

stealth™ siRNAs (S#1, S#2, and S#3) by using nucleofection. Cell lysates were prepared 24 and 48 h after transfection and ZAP70 expression was analyzed by RT-PCR (A) and Western blot analysis (B). As control cells were mock transfected ("-", no siRNA added), or transfected with a control siRNA pool (Ctrl). (A) RT-PCR of ZAP70 mRNA expression in transfected T cells determined 24 h after nucleofection as fold expression of the house keeping gene β-actin. (B) Western blot analysis of transfected T cells 48 h after nucleofection with siRNAs indicated in (A). Protein knockdown of ZAP70 was detected by ZAP70-specific antibody. β-actin was used as loading control. Shown are the mean values of triplicates of a representative experiments of two independent experiments.<p><b>Copyright information:</b></p><p>Taken from "RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes"</p><p>http://www.biosignaling.com/content/6/1/3</p><p>Cell communication and signaling : CCS 2008;6():3-3.</p><p>Published online 6 Aug 2008</p><p>PMCID:PMC2517589.</p><p></p

RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes-2

nucleofection. Cell lysates were prepared 24 and 48 h after transfection and mRNA and protein expression were analyzed by RT-PCR (A) and Western blot analysis (B). As control, cells were transfected with a control siRNA pool (Ctrl). (A) RT-PCR results of transfected T cells determined 24 h after nucleofection with Control ("siCtrl"), ZAP70 ("siZAP70"), PLC-γ ("siPLC-γ1") and Lck ("siLck") specific siRNAs. (B) Western blot analysis of transfected T cells 48 h after nucleofection. Protein knockdown of ZAP70, PLC-γ and Lck was detected by specific antibodies. β-actin was used as loading control. Shown are the mean values of triplicates of a representative experiments of two independent experiments.<p><b>Copyright information:</b></p><p>Taken from "RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes"</p><p>http://www.biosignaling.com/content/6/1/3</p><p>Cell communication and signaling : CCS 2008;6():3-3.</p><p>Published online 6 Aug 2008</p><p>PMCID:PMC2517589.</p><p></p

RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes-5

stealth™ siRNAs (S#1, S#2, and S#3) by using nucleofection. Cell lysates were prepared 24 and 48 h after transfection and ZAP70 expression was analyzed by RT-PCR (A) and Western blot analysis (B). As control cells were mock transfected ("-", no siRNA added), or transfected with a control siRNA pool (Ctrl). (A) RT-PCR of ZAP70 mRNA expression in transfected T cells determined 24 h after nucleofection as fold expression of the house keeping gene β-actin. (B) Western blot analysis of transfected T cells 48 h after nucleofection with siRNAs indicated in (A). Protein knockdown of ZAP70 was detected by ZAP70-specific antibody. β-actin was used as loading control. Shown are the mean values of triplicates of a representative experiments of two independent experiments.<p><b>Copyright information:</b></p><p>Taken from "RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes"</p><p>http://www.biosignaling.com/content/6/1/3</p><p>Cell communication and signaling : CCS 2008;6():3-3.</p><p>Published online 6 Aug 2008</p><p>PMCID:PMC2517589.</p><p></p

RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes-0

Sfected with 0.9 μM ZAP70-specific siRNA pool by nucleofection. Cell lysates were prepared 24, 48 and 72 h after transfection and ZAP70 expression was analyzed by RT-PCR (A) and Western blot analysis (B). As control, cells were mock ("-", no siRNA added) transfected () or cells were transfected with a control siRNA pool (Ctrl). (A) RT-PCR of ZAP70 mRNA expression determined as fold expression of the house keeping gene β-actin. (B) Western blot analysis of ZAP70 was detected by ZAP70-specific antibody. β-actin was used as loading control. Shown are the mean values of triplicates of a representative experiments of four independent experiments.<p><b>Copyright information:</b></p><p>Taken from "RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes"</p><p>http://www.biosignaling.com/content/6/1/3</p><p>Cell communication and signaling : CCS 2008;6():3-3.</p><p>Published online 6 Aug 2008</p><p>PMCID:PMC2517589.</p><p></p

Je suis habille de plumes pour gagne ma vie a chanter

A young man who dons a costume of feathers to earn his living as a minstrel recounts his adventures on meeting a hunter, some birds, and a young girl.Laforte CCFF II Chansons __ques, p. 229; Young: "Vielles Chansons de Nouvelle France", Archives de Folklore #7, pp. 88-89; Lacourciere: "Archives de Folklore" IV (1949), 88