No abstract

Antigenic peptide loading onto class I MHC molecules in the endoplasmic reticulum requires the generation of peptides by the cytosolic proteasome and subsequent peptide transport into the endoplasmic reticulum. Dendritic cells express several endocytic receptors, including membrane lectins such as the mannose receptor and DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin) that may be used to increase antigen presentation (2).The mannose receptor, first characterized in macrophages (3), binds and takes up glycoproteins and glycoconjugates containing terminal mannose, GlcNAc, or fucose (4). Calcium-dependent recognition of these sugars is mediated by C-type carbohydrate recognition domains (CRDs) in the extracellular region of the receptor (5). Glycoproteins internalized by the mannose receptor follow a well characterized endocytic pathway leading to lysosomes (6). The cytoplasmic tail of the mannose receptor contains a tyrosine-based motif involved in the internalization process in association with clathrin-coated vesicles, and the mannose receptor traffics through early endosomes. Our experiments with fluorescein-labeled neoglycoproteins suggested that the mannose receptor is also expressed on monocyte-derived dendritic cells (7), and this finding was subsequently confirmed (8).Recently, DC-SIGN, a new dendritic cell-specific membrane lectin was characterized (9). DC-SIGN binds the gp120 envelope glycoprotein on the surface of human immunodeficiency virus-1 (10). Its extracellular domain is a tetramer stabilized by an ␣-helical stalk, whereas its C-type CRDs bind high mannose oligosaccharides (11). The presence of an internalization motif in its cytoplasmic tail suggests that DC-SIGN acts as an endocytic receptor, and it has been shown that complexes of DC-SIGN with an anti DC-SIGN antibody are targeted to late endosomes/lysosomes (12). Therefore, in dendritic cells that express both the mannose receptor and DC-SIGN, the traffick-

Polyethylenimine (PEI) is one of the most potent non-viral vectors. We have developed a lactosylated PEI (Lac-PEI) to enhance cell-specific transfection and have shown that Lac-PEI is more efficient than unsubstituted PEI for gene transfer into immortalized cystic fibrosis airway epithelial SigmaCFTE29o-cells. As both intact PEI/plasmid and Lac-PEI/plasmid complexes are found in the cell nucleus, we have investigated the transcription efficiency of the plasmid complexed with PEI or Lac-PEI, according to the polymer nitrogen/DNA phosphate (N/P) ratio (from 0 to 20). The initiation of transgene transcription was analyzed in an acellular nuclease S1 transcription assay. For both PEI and Lac-PEI complexes, transcription efficiency varied with the N/P ratio of the complexes. Transcription inhibition was observed when plasmid DNA was either loosely (N/P<5) or tightly condensed (N/P>15). For an N/P ratio of 5 and up to 15, transcription of the complexed plasmid was as efficient as that of the free plasmid. Similar results were observed when gene expression was studied after nuclear microinjection of the complexes into SigmaCFTE29o-cells. Our study shows that condensation of DNA influences the accessibility of the plasmid to the transcription machinery. Interestingly, the charge ratios that allow the most efficient transcription are those usually known to be the most efficient for gene transfer in vitro and in vivo.