title: Analysis of reporter gene expression in ovine dermis and afferent lymph dendritic cells in vitro and in vivo.
abstract: Plasmid DNA administration has revolutionised approaches to vaccination, and many studies have demonstrated the generation of both humoral and cytotoxic T cell responses which confer protection against live pathogen challenge. However, the mechanisms underlying DNA vaccination are poorly understood. Several studies have suggested the involvement of professional antigen presenting cells such as dendritic cells (DC), but direct evidence for this is lacking. We have used the pseudoafferent lymphatic cannulation model in sheep to study the expression of a plasmid encoding enhanced green fluorescent protein (EGFP) by afferent lymph DC following administration to skin. The cells were analysed by flow cytometry. Preliminary studies were carried out to determine if the pEGFP would function in sheep cells in vitro. The results showed that electroporation of sheep skin fibroblasts, primary macrophages, and afferent lymph DC with 30 microg pEGFP resulted in varying degrees of fluorescence in these cells e.g. 35% of skin cells examined at 48 h, and 7% of afferent lymph DC examined after 4 h. Following intradermal injection of 120 microg of pEGFP, small numbers of fluorescent DC (1-5%) were evident by flow cytometry after 1-4 h. The fluorescent DC continued to drain into the lymphatics over a period of 24 h. Analysis by PCR showed that free pEGFP appeared in the afferent lymph plasma within 1 h of injection, peaking at 2 h and becoming undetectable after 6 h. The results suggest that primary immune responses may be initiated by uptake of soluble protein antigen by afferent lymph DC and by free plasmid rapidly draining to the lymphatics where it may be taken up by DC in the lymph plasma and the local lymph node.
authors.affiliation: Department of Veterinary Pathology, University of Edinburgh, Summerhall, UK.
authors.forename: C
authors.initials: C
authors.lastname: Watkins
authors.forename: S
authors.initials: S
authors.lastname: Lau
authors.forename: R
authors.initials: R
authors.lastname: Thistlethwaite
authors.forename: J
authors.initials: J
authors.lastname: Hopkins
authors.forename: G D
authors.initials: GD
authors.lastname: Harkiss
mesh_terms: D000818:Animals; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D003713:Dendritic Cells / Q000276:immunology*; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D018274:Electroporation / Q000662:veterinary; D005434:Flow Cytometry / Q000662:veterinary; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000737:chemistry; D007271:Injections, Intradermal / Q000662:veterinary; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012756:Sheep / Q000235:genetics / Q000276:immunology*; D012867:Skin / Q000276:immunology*; D014611:Vaccination / Q000662:veterinary*; D019444:Vaccines, DNA / Q000276:immunology
chemical_list: D017931:DNA Primers; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D019444:Vaccines, DNA; D049452:Green Fluorescent Proteins; D004247:DNA