Recombinant AAV-transduced iris pigment epithelial cell transplantation may transfer vector to native RPE but suppress systemic dissemination.

PURPOSE: To determine whether adenoassociated virus (AAV) vectors transduced into iris pigment epithelial (IPE) cells and transplanted into the subretinal space of rats will transfer the AAV genome to the host cells and whether the vectors are disseminated systemically. METHODS: Recombinant (r)AAV was transduced into rat IPE cells and transplanted into the subretinal space of rats. For the control, rAAVs alone were injected subretinally. The transplanted IPE cells were detected by LacZ staining. Immunohistochemistry, electron microscopy, electroretinography, and fluorescein-dextran angiography were performed. DNA was extracted from various organs and blood and examined for the AAV genome by polymerase chain reaction. RESULTS: No toxicity from rAAV transduction was observed in vitro. LacZ was expressed in the transplanted cells 1 and 2 weeks after transplantation. At 4 and 12 weeks, fewer transplanted cells were detected than at 1 week, and LacZ expression was occasionally detected at the level of host retinal pigment epithelial (RPE) cells. Expression was also detected in ciliary body epithelial cells. The electroretinograms and fluorescein-dextran angiography were only mildly altered. Significantly lower levels of AAV genome were detected in the organs and blood of rats receiving rAAV-IPE cell transplants than with direct intravenous injection of AAV vectors. CONCLUSIONS: AAV-mediated LacZ was expressed in the transplanted cells after subretinal transplantation, and the transplanted IPE cells may transfer the rAAV to host tissues, such as RPE cells, long after the transplantation. This method of gene delivery did not lead to systemic dissemination of the vectors.

Protection of photoreceptor cells from phototoxicity by transplanted retinal pigment epithelial cells expressing different neurotrophic factors.

Transplantation of cells or tissues and the intravitreal injection of neurotrophic factors are two methods that have been used to treat retinal diseases. The purpose of this study was to examine the effects of combining both methods: the transplantation of retinal pigment epithelial (RPE) cells expressing different neurotrophic factors. The neutrophic factors were Axokine, brain derived-neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF). The enhanced green fluorescence protein (eGFP) gene was used as a reporter gene. These genes were transduced into RPE cells by lipofection, selected by antibiotics, and transplanted into the subretinal space of 108 rats. The rats were examined at 1 week and 3 months after the transplantation to determine whether the transduced cells were present, were expressing the protein, and were able to protect photoreceptors against phototoxicity. The survival of the transplanted cells was monitored by the presence of eGFP. The degree of protection was determined by the thickness of the outer nuclear layer. Our results showed that the degree of photoreceptor protection was different for the different types of neurotrophic factors at 1 week. After 3 months, the number of surviving transplanted cell was markedly reduced, and protection was observed only with the BDNF-transduced RPE cells. A significant degree of rescue was also observed by BDNF-transduced RPE cells in the nontransplanted area of the retina at both the early and late times. Lymphocytic infiltration was not detected in the vitreous, retina, and choroid at any time. We conclude that the transplantation of BDNF-transduced RPE cells can reduce the photoreceptor damage induced by phototoxicity in the transplanted area and weakly in the nontransplanted area.

Photoreceptor protection by iris pigment epithelial transplantation transduced with AAV-mediated brain-derived neurotrophic factor gene.

PURPOSE: To determine whether subretinal transplantation of iris pigment epithelial (IPE) cells transduced with the adeno-associated virus (AAV2)-mediated brain-derived neurotrophic factor (BDNF) gene can protect photoreceptors against phototoxicity. METHODS: The BDNF gene was inserted into AAV2 (AAV2-BDNF), and the recombinant AAV2 was transduced into rat IPE (AAV2-BDNF-IPE) cells at various multiplicities of infection (MOI). The concentrations of AAV capsids and BDNF were determined by enzyme-linked immunosorbent assay (ELISA). The AAV2-BDNF-IPE cells were transplanted into the subretinal space of rats, and the rats were placed under constant light on days 1 and 90 after the transplantation. The thickness of the outer nuclear layer was measured in histologic sections and compared to that of control sections. The expression of beta-galactosidase (LacZ) in the subretinal space was confirmed by LacZ staining after AAV2-LacZ-IPE transplantation. BDNF gene expression after transplantation was confirmed by real-time polymerase chain reaction (PCR). RESULTS: Transduction efficiency increased with successive days in culture and increased with higher MOI in vitro. The expression of the BDNF gene in the subretinal space was higher in AAV-BDNF-IPE than with AAV2-LacZ-IPE or with IPE-only transplantation. LacZ expression was observed in the subretinal space 7 and 90 days after transplantation. A statistically significant photoreceptor protection was observed on days 1 and 90 in eyes receiving the AAV2-BDNF-IPE transplant, in both the superior transplant site and the inferior hemispheres which did not receive the transplant. CONCLUSIONS: Transplantation of AAV2-BDNF-IPE cells may be an alternative method of delivering neurotrophic factors to the lesion.

An instrument capable of grading visual function: results from patients with retinitis pigmentosa.

There was no device to grade visual function in patients with retinitis pigmentosa (RP). We have therefore developed an instrument capable of measuring and quantifying the visual capabilities, and here present the results from patients with RP. In total, 118 eyes of 59 patients, 26 men and 33 women, with RP were studied. Seven eyes had hand movement (HM) and eight had light perception (LP) vision, and the others had better visual acuity. The Low Vision Evaluator (LoVE) consists of a pair of goggles with white, light-emitting diodes as the stimulus, a control box, an on-off button to signal the detection of the stimulus, and a printer for permanent records. There are 15 luminance levels of stimuli (combination of 5 intensities and 3 durations). The stimuli are delivered in a random sequence with an audio signal presented 0.3 seconds prior to the light stimulus. Each eye was tested separately, and each stimulus magnitude (intensity x duration) was presented 3 times for a total of 27 stimuli per eye. With 6 catch trials (audio signal without a light stimulus), a total of 60 trials were examined in a full examination. The conventional visual acuity and kinetic visual fields were determined. 59 patients had different visual acuities that ranged from no light perception (NLP) to 1.5 vision, and visual field sizes that ranged from 0.0001 to 3.96 steradians. The visual acuity and visual field size were significantly correlated with the LoVE score (r=0.58 and 0.64, respectively; p<0.01). These results indicate that the LoVE is capable of grading the visual function of RP patients with various visual acuities and visual fields. The testing procedures are simple for the patient and examiner, and this instrument can be used to assess the effectiveness of medical and surgical therapy.

Transplantation of transduced retinal pigment epithelium in rats.

PURPOSE: To examine the effects of transplanting retinal pigment epithelial (RPE) cells transduced with neurotrophic factor genes into the subretinal space of rats. METHODS: RPE cells were transduced with plasmids carrying the cDNAs of Axokine (ciliary neurotrophic factor [CNTF]; Sumitomo Pharmaceuticals Co., Ltd., Tokyo, Japan), brain derived-neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF) genes. These RPE cells were transplanted into the subretinal space of rats, and the localization was examined. The expression of enhanced green fluorescent protein (eGFP)-BDNF-transduced RPE in the subretinal space was examined by real-time polymerase chain reaction (PCR) after the transplanted cells were collected by cell sorting. The expression of major histocompatibility complex (MHC) class I and -II after gene transduction was examined by real-time PCR. The ratio of CD4(+) and CD8(+) T cells and antibody production against transplanted cells were analyzed by flow cytometry. RESULTS: The transplant sites were not significantly different among the neurotrophic factors tested. The RPE cells expressed the BDNF gene in the subretinal region at approximately the same level as that in vitro. RPE cells transduced with Axokine stimulated MHC-I expression, and the cell transplantation changed the ratio of CD4(+) and CD8(+) T cells. A significant production of antibody against the Axokine-transduced RPE cells was also observed after Axokine-transduced RPE transplantation. CONCLUSIONS: RPE cells transduced with neurotrophic factors express the factors after transplantation into the subretinal space. RPE transduced with Axokine or bFGF, in contrast to RPE transduced with BDNF, stimulate an immunologic reaction of the host.

Interferon gamma expression and clinical features in patients with acute retinal necrosis syndrome.

BACKGROUND: Interferon gamma (IFN-gamma) has been reported to play an important role during virus infections. The purpose of this study was to examine the relationship between IFN-gamma expression and the clinical course of patients with acute retinal necrosis syndrome (ARN) associated with the varicella-zoster virus (VZV). METHODS: Six patients with ARN were studied. The aqueous and/or vitreous were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay during the follow-up period. The presence of VZV genome was also determined by PCR. The results were correlated with the clinical data and features and compared with patients with other ocular diseases. RESULTS: A statistically significant higher level of IFN-gamma was detected in the aqueous and/or vitreous in eyes with ARN than in eyes with other ocular diseases. A statistically significant positive correlation was observed between the level of IFN-gamma in the vitreous and the final visual acuity. IFN-gamma was reduced to undetectable levels within 30 days after the initial eye symptoms. Three of five patients had severe inflammation initially, and the visual acuity gradually recovered with the disappearance of VZV and higher levels of IFN-gamma. Conversely, the other 2 patients showed mild inflammation, had a slow decrease of visual acuity with persistent VZV, and lower levels of IFN-gamma expression. CONCLUSION: Our results suggest that IFN-gamma may be one of the factors that plays an important role in the clinical course of VZV-associated ARN.

Interleukin-1beta and barrier function of retinal pigment epithelial cells (ARPE-19): aberrant expression of junctional complex molecules.

PURPOSE: To examine the effects of interleukin (IL)-1beta on the resistance and permeability of the tight junctions of cultured retinal pigment epithelial cells. METHODS: A human RPE cell line (ARPE-19) cultured on microporous filter-supports was used. IL-1beta and monoclonal anti-IL-1beta antibody-treated IL-1beta (mAbIL-1beta) were added to the standard culture medium. Transepithelial resistance (TER) of confluent RPE cells was measured by epithelial voltmeter. The permeability of the RPE cells to sodium fluorescein, horseradish peroxidase, and inulin was measured. The expression of the occludin and claudin was determined by real-time polymerase chain reaction (PCR), immunohistochemistry, and Western blot analysis. RESULTS: A significantly greater decrease of TER occurred in IL-1beta-supplemented medium than in standard medium plus mAbIL-1beta after several days of stimulation. A significantly greater increase of sodium fluorescein, horseradish peroxidase, and inulin permeability occurred in IL-1beta-supplemented medium than in standard medium. The expression of the occludin gene and some types of claudin genes was observed. The expression of occludin was downregulated and that of claudin-1 upregulated more in IL-1beta-supplemented medium than in standard medium by real-time PCR, immunohistochemistry, and Western blot analysis. CONCLUSIONS: The tight junctions of ARPE-19 cells are altered by IL-1beta supplementation either directly or through other factors activated by IL-1beta. The downregulation of occludin and upregulation of claudin-1 may have participated in the dysfunction of the RPE tight junctions in these in vitro experiments.