title: Comparison of three techniques for generation of tolerogenic dendritic cells: siRNA, oligonucleotide antisense, and antibody blocking.
abstract: In recent years, a new view of dendritic cells (DCs) as a main regulator of immunity to induce and maintain tolerance has been established. In vitro manipulation of their development and maturation is a topic of DC therapeutic application, which utilizes their inherent tolerogenicity. In this field, the therapeutic potential of antisense, siRNA, and blocking antibody are an interesting goal. In the present study, the efficiency of these three methods--siRNA, antisense, and blocking antibody--against CD40 molecule and its function in DCs and BCL1 cell line are compared. DCs were separated from mouse spleen and then cultured in vitro using Lipofectamine 2000 to deliver both silencers; the efficacy of transfection was estimated by flow cytometry. mRNA expression and protein synthesis were assessed by real time-PCR and flow cytometry, respectively. By Annexin V and propidium iodine staining, we could evaluate the viability of transfected cells. Knocking down the CD40 gene into separate groups of DCs by siRNA, antisense, and blocking antibody treated DCs can cause an increase in IL-4, decrease in IL-12, IFN-γ production, and allostimulation activity. Our results indicated that, in comparison to antisense and blocking antibody, siRNAs appear to be quantitatively more efficient in CD40 downregulation and their differences are significant.
authors.affiliation: Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. karimimh@sums.ac.ir
authors.forename: Mohammad Hossein
authors.initials: MH
authors.lastname: Karimi
authors.forename: Padideh
authors.initials: P
authors.lastname: Ebadi
authors.forename: Ali Akbar
authors.initials: AA
authors.lastname: Pourfathollah
authors.forename: Mohammad
authors.initials: M
authors.lastname: Moazzeni
authors.forename: Zahra Soheila
authors.initials: ZS
authors.lastname: Soheili
authors.forename: Shahram
authors.initials: S
authors.lastname: Samiee
mesh_terms: D000818:Animals; D017304:Annexin A5; D019138:Antibodies, Blocking / Q000276:immunology* / Q000494:pharmacology; D019013:CD40 Antigens / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology*; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology*; D055785:Gene Knockdown Techniques; D007108:Immune Tolerance / Q000276:immunology*; D066298:In Vitro Techniques; D008055:Lipids; D051379:Mice; D016376:Oligonucleotides, Antisense / Q000276:immunology* / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011419:Propidium; D034741:RNA, Small Interfering / Q000276:immunology* / Q000494:pharmacology
chemical_list: D017304:Annexin A5; D019138:Antibodies, Blocking; D019013:CD40 Antigens; D008055:Lipids; C086724:Lipofectamine; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; D011419:Propidium