title: Cell polarity, phagocytosis and viral gene transfer in cultured human retinal pigment epithelial cells.
abstract: OBJECTIVE
  To investigate whether there is a difference in the expression of adenovirus transgenes in human retinal pigment epithelial cells when the vector was exposed to the apical or basal surface, the effect of transgene expression on rod outer segment (ROS) phagocytosis and finally, the role of phagocytosis in gene transfer to RPE cells, using the Royal College of Surgeons (RCS) rat.
  
  
  METHODS
  Monolayers of human retinal pigment epithelium (HRPE) or an RPE cell line (A407) had the apical or basal surfaces exposed to 10(7) pfu/ml of replication deficient adenovirus (Ad.RSV.betagal) carrying the beta-galactosidase marker gene, and the numbers of expressing cells were compared. Parallel cultures were infected and challenged with fluorescein-labelled bovine rod outer segments (FBROS). The fluorescence of infected versus uninfected cells was recorded for both challenged and unchallenged states, using fluorophotometric flow cytometry. Primary cultures of RCS rat RPE were established and the transgene uptake dynamics compared to control Long Evans rat RPE cells.
  
  
  RESULTS
  The expression of transgene in HRPE and A407 cell cultures was an order of magnitude greater when the vector was exposed apically (analysis of variance p < 0.05). There was no difference in the phagocytic capacity of Ad.RSV.betagal-infected and -noninfected cells when challenged with FBROS. There was also no difference in the number of cells expressing transgene, when compared to the RCS or Long Evans control rat RPE.
  
  
  CONCLUSIONS
  The surface of exposure in polarized retinal pigment epithelial cells affects the rate of uptake and expression of adenovirus. The defective ROS phagocytosis in RCS rat RPE cells did not lead to a decrease in transgene expression relative to the Long Evans control cells. Finally we have found that phagocytosis is not significantly altered with adenoviral transgene expression in this in vitro model.
authors.affiliation: Centre for Ophthalmology and Visual Sciences, Lions Eye Institute, Nedlands, Perth, Australia.
authors.forename: L
authors.initials: L
authors.lastname: da Cruz
authors.forename: T
authors.initials: T
authors.lastname: Robertson
authors.forename: M O
authors.initials: MO
authors.lastname: Hall
authors.forename: I J
authors.initials: IJ
authors.lastname: Constable
authors.forename: P E
authors.initials: PE
authors.lastname: Rakoczy
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000293:Adolescent; D000818:Animals; D002461:Cell Line, Transformed; D016764:Cell Polarity / Q000502:physiology*; D002478:Cells, Cultured; D003673:Defective Viruses; D005434:Flow Cytometry; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005814:Genes, Viral / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D010587:Phagocytosis / Q000502:physiology*; D010857:Pigment Epithelium of Eye / Q000502:physiology* / Q000648:ultrastructure / Q000821:virology; D051381:Rats; D011922:Rats, Mutant Strains; D012162:Retinal Degeneration / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D001616:beta-Galactosidase