To examine the nature and dynamics of gene transfer to human retinal pigment epithelium (RPE) using an adenoviral vector and adjuvants that may enhance the uptake of recombinant adenoviruses.
Human ...RPE cultures (HRPE7) were transfected in vitro with varying concentrations (4, 20, 40, 120, and 200 pfu/microliter) and for varying periods (1, 2, 4, 16, 24, 48, and 72 hours) with a replication-deficient adenovirus (Ad.RSV. beta gal) containing the bacterial beta-galactosidase transgene (beta gal). The expression of beta gal was monitored by counting after X gal staining. The transgene expression profiles were compared to those of human F2000 fibroblasts under the same conditions. The adjuvant effect of sodium hyaluronate (HA) on the expression of beta gal was tested in F2000 and early and late passage human RPE cells for differing concentrations of HA, viral titers, and incubation times. Immunofluorescent cytochemistry was carried on HRPE7 and F2000 cells for the HA receptors, homing receptor CD44 (CD44), intercellular adhesion molecule 1 (ICAM-1), and the receptor for hyaluronan mediated motility (RHAMM).
The number of HRPE7 and F2000 cells expressing the adenoviral transgene increased consistently with increasing incubation time and viral titer. There was a higher uptake of Ad.RSV. beta gal in HRPE7 cells compared to the F2000 fibroblasts under the same conditions. There was an increase of 28.1% and 41.4% in the number of RPE7 cells expressing adenoviral transgene and 16.2% and 15.8% F2000 fibroblast cells expressing the adenoviral transgene in the presence of 0.001% and 0.005% HA, respectively. Significant adjuvant effects on transgene expression also were shown in HRPE51 cells. It appears that the effects of increasing viral titer, length of incubation, and the presence of HA on transgene expression are at least additive. The appearance of CD44 and ICAM receptors on RPE7 and F2000 cells and RHAMM receptors on F2000 cells was similar. The RHAMM receptors in HRPE7 cells, however, were shown preferentially over the nucleus.
On the basis of these results, the authors propose that adenovirus transgene expression increases with increasing incubation time and viral titer in cell culture. The rate of increase of expression differs between human RPE cells and the F2000 fibroblast cells, which may offer a targeting opportunity. The authors propose that the use of HA can offer both an adjuvant effect and a targeting advantage in terms of transferring adenoviral transgenes to human RPE in culture.
Age Related Macular Degeneration Kelly, C. Simon P.; Edwards, Richard; Elton, Peter ...
BMJ (Online),
06/2003, Letnik:
326, Številka:
7404
Journal Article
At present only subjective criteria exist to determine the legibility of health warnings on billboards that display advertisements for cigarettes. We constructed a set of objective standards and used ...them to assess the legibility of warnings on a sample of 37 billboards in Perth, Western Australia. The three features that were analysed were the apparent size of the letters, the colour contrast between the letters and their background, and the obliqueness of the message. We also compared the size and legibility of the health warning with that of the brand name. Health warnings were not legible to passing motorists in 51% of cases and not legible to motorists who were stopped at nearby intersections in 74% of cases, whereas almost all the brand names could be read easily. These results indicate that the present system of voluntary regulation of advertisements for cigarettes has failed to ensure that health warnings on billboards are legible. For health warnings on advertisements to have any useful effect it is essential that they are legible. The only way to ensure legibility is to adopt objective requirements for it.
Recent technical advances have led to the demonstration of the molecular basis of many genetic eye diseases. Methods now in use have been able to both suppress and insert genes in vitro and in vivo ...in ocular cells. The combination of this new knowledge and these techniques offers the potential for a new therapeutic approach to diseases that currently have no treatments. The scientific basis of gene therapy is introduced and then the current state of research is discussed in relation to the eye and ocular tissues.
PDF
