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L da Cruz

Publications and source records attributed to L da Cruz.

7 recordsLinked to original sources
PubMed1999

Dynamics of phosphorothioate oligonucleotides in normal and laser photocoagulated retina.

AIMS: To investigate the distribution, persistence, and stability of fluorescently labelled phosphorothioate oligonucleotides (PS-ODNs) in normal and laser photocoagulated retina following intravitreal injection in the rat. METHODS: Fluorescently labelled PS-ODNs were injected intravitreally into pigmented eyes at doses of 0.5-10.0 nmol in 2.0 microl solution. The dynamics of PS-ODNs was evaluated by fluorescent microscopy of cryosections and flat mounted retinal pigment epithelium (RPE)-choroid-sclera. Genescan analysis was used to assess the integrity of PS-ODNs in the retina after injection. The dynamics of PS-ODNs was also evaluated in the retina following krypton laser photocoagulation with a protocol producing choroidal neovascularisation (CNV). RESULTS: Following intravitreal injection the PS-ODNs demonstrated dose and time dependent distribution and persistence in the retina, where they accessed all neural layers. However, they preferentially accumulated in the RPE layer, demonstrated as bright granules in the cytoplasm of the cells. Injections of 5.0 and 7.5 nmol of PS-ODNs exhibited strong fluorescence in the retina for 6 weeks after injection. Genescan analysis demonstrated that the PS-ODNs remained almost completely intact for at least 12 weeks. Following laser treatment, the PS-ODNs were concentrated in the regions of laser photocoagulation and retained high intensity for at least 8 weeks after injection, particularly localised to macrophages, RPE, and the local choroidal tissue. CONCLUSIONS: These results indicate that PS-ODNs are stable and accessible to most neural layers of the retina, and they preferentially accumulate in the RPE layer following intravitreal injection. The successful delivery of PS-ODNs into normal and laser photocoagulated retina suggests that PS-ODNs may have potential in the development of therapy for attenuating retinal degenerations and CNV.

Animals↗
PubMed1998

Cell polarity, phagocytosis and viral gene transfer in cultured human retinal pigment epithelial cells.

PURPOSE: 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.

Adenoviridae↗
PubMed1997

Ocular gene therapy: the basic science and current state of research.

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.

Adenoviruses, Human↗
PubMed1996

Dynamics of gene transfer to retinal pigment epithelium.

PURPOSE: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Adenoviruses, Human↗