Samir C. Patel

Cellular response in rabbit eyes after human fetal RPE cell transplantation

Abstract · Background: This study was carried out to study inflammatory and proliferative cellular responses in the rabbit eye after subretinal transplantation of human fetal retinal pigment epithelial (HFRPE) cells. · Methods: 5-Bromo-2-deoxyuridine (BrdU)-labeled HFRPE cells were injected subretinally into rabbit eyes at three different concentrations. Macrophage, glial, and proliferative responses of the eye tissues were studied by immunohistochemistry and light microscopy at different times after the surgery. · Results: In transplanted eyes, the HFRPE cells were distributed irregularly either as multilayers or monolayers. In eyes receiving high-density cell suspensions, retinal breaks were seen. No retinal breaks were noted in the eyes receiving low-density HFRPE cell suspensions. The highest intensity of inflammatory response was seen at 4–14 days after surgery, with greater expression in transplanted eyes receiving high-density cell suspensions. The host cellular response was characterized initially by local infiltration of the retina and subretinal space by macrophages and glial cells. After day 14, a decline in the number of donor cells was noted in all eyes. At later stages the host cellular response was characterized mainly by local choroidal thickening and infiltration by inflammatory cells. Proliferative response was expressed mainly by retinal cells. · Conclusion: Initial inflammatory and proliferative responses after the xenogenic human to rabbit HFRPE cell transplantation were expressed by retinal cells with later involvement of the choroid. Our results showed a decline in the number of donor cells starting from day 14 after the transplantation. This may suggest a possibility of rejection. The initial quantity of injected cells may be critical for the intensity of the immune and inflammatory responses.

Human fetal retinal pigment epithelium suppresses the activation of CD4(+) and CD8(+) T-cells.

Abstract · Background: The suppressive effect of human fetal retinal pigment epithelium (HFRPE) on the activation of human CD4+ and CD8+ T-cells was evaluated in vitro. · Methods: Pure populations of CD4+ and CD8+ T-cells were isolated from human peripheral blood-derived buffy coats by negative immunomagnetic selection. The purity of the cells was examined by flow cytometry using anti-CD3-FITC, anti-CD4-FITC, anti-CD8-PE, and anti-CD20-PE mAbs. HFRPE cells were isolated from fetal eyes and pure cultures were obtained. The effect of normal or IFN-γ-activated HFRPE cells at early (P3) or late (P6) passages on the activation of CD4+ and CD8+ T-cells was assessed in two different T-cell activation assays. In both activation models anti-CD3 mAb (OKT3) provided the antigen-specific signal. The secondary signal for the activation of CD4+ and CD8+ T-cells was provided with anti-CD18 mAb (TS1/18) and anti-CD28 mAb (9.3) in the first and the second assay respectively. Cross-linking of these soluble mAbs was performed with sheep-anti-mouse IgG-coated latex beads. The T-cell activation was determined by cell proliferation measured by [3H]thymidine incorporation. In each activation assay T-cells were incubated with HFRPE cells in a ratio of T-cells to HFRPE of 1:1 or 1:4. · Results: CD4+ and CD8+ T-cells were activated by cross-linking CD3 and CD18 in the first assay (CD3/CD18) and CD3 and CD28 in the second assay (CD3/CD28). In both assays HFRPE inhibited the activation of CD4+ and CD8+ T-cells. IFN-γ-activated HFRPE cells totally suppressed the T-cell activation at a 1:1 ratio. This suppressive effect was weaker at lower cell ratios. Some donor variation was observed in the inhibition at the lower cell ratios, especially for the inhibition of CD8+ T-cell activation with anti-CD3/CD18. The passaging of HFRPE cells did not alter their suppressive effect on CD4+ and CD8+ T-cells. · Conclusions: HFRPE cells suppressed the activation of both CD4+ and CD8+ T-cells in vitro. These findings suggest that RPE-induced immune suppression may play a significant role in maintaining immune privilege in the subretinal space.

Rheumatoid hyperviscosity syndrome: reversibility of microvascular abnormalities after treatment

PURPOSE To report a case of rheumatoid hyperviscosity syndrome involving both retinal and choroidal circulation that resolved after treatment. DESIGN Interventional case report. METHODS A 58-year-old woman with clinical and serologic evidence of an inflammatory connective tissue disease without any visual complaints was referred for a funduscopic evaluation. RESULTS Funduscopic examination revealed marked dilation and beading of the venous system, microaneurysms, and telangiectatic capillary beds in the posterior pole. Fluorescein angiography disclosed delayed choroidal filling, prolonged arteriovenous transit time, and areas of capillary nonperfusion. These findings were accompanied by a severe polyclonal hypergammaglobulinemia and a 10-fold increase in serum viscosity. The ocular findings were reversible after plasmapheresis and steroid treatment. CONCLUSION Rheumatoid hyperviscosity syndrome can involve both retinal and choroidal circulation. The prominent microvasculopathy is reversible after appropriate treatment.

Growth of human fetal retinal pigment epithelium as microspheres.

Abstract · Background: The aim was to develop a three-dimensional cell culture system for human fetal retinal pigment epithelial (HFRPE) cells for in vitro cellular studies and for possible application in subretinal transplantation. · Methods: Pieces of freshly isolated HFRPE monolayer tissue were grown on crosslinked fibrinogen (CLF) films. The growth pattern and morphologic characteristics of the implanted tissue were studied using phase-contrast microscopy, photography, and light and electron microscopy. The cells were screened immunohistochemically for HLA-ABC, HLA-DR, ICAM-1, B7, and Cytokeratin. Cell proliferation was studied using 5-bromo-2-deoxyuridine incorporation. · Results: After attachment to CLF, HFRPE monolayer tissue formed small tumor-like formations, i.e. microspheres. HFRPE microspheres survived and proliferated in a floating state for at least 4 months. After attachment of the microspheres to the culture dish floor, formation of a confluent HFRPE cell monolayer with high proliferative activity was noted around the microspheres. HFRPE cells stained positive for HLA-ABC, ICAM-1, and cytokeratin and negative for B7 and HLA-DR. The microspheres could be easily detached from the dish and they were able to initiate similar growth after reattachment. · Conclusion: HFRPE grown on CLF resemble a three-dimensional culture system with high yield of pure cells that can be useful for a wide variety of in vitro studies. Because of their adjustable size, spherical shape, and ability to initiate growth of cells with a high proliferative potential, HFRPE microspheres may be successfully utilized as a source of donor cells for subretinal transplantation.

Cytokine modulation of costimulatory molecules on human fetal retinal pigment epithelial cells.

Purpose. Experiments were performed to evaluate the effect of various pro- and anti-inflammatory cytokines on the human fetal retinal pigment epitheliums (HFRPE) expression of major histocompatibility complex (MHC) and costimulatory molecules. Methods. Pure cultures of HFRPE cells were isolated. HFRPE cells were incubated in the presence of Interferon-? (IFN-?), IFN-ß, Tumor Necrosis Factor-a (TNF-a), Interleukin-1ß (IL-1ß), Tumor Growth Factor-ß (TGF-ß), and a combination of IFN-? and TGF-ß (pre-incubation and simultaneously incubated). The expression of MHC class I and class II, Intercellular cell adhesion molecule (ICAM-1), B7-1 (CD80), and B7-2 (CD86) molecules was quantitatively analyzed by flow cytometry. Results. The cultured HFRPE cells expressed high levels of MHC class I and low levels of MHC class II and ICAM-1 molecules. After culture with the above mentioned cytokines, IFN-? up-regulated the HFRPE’s expression of MHC class II and ICAM-1. IFN-ß and IL-1ß only up-regulated the expression of ICAM-1. TGF-ß was unable to suppress the up-regulatory effect of IFN-? in HFRPE cells (pre-incubated and simultaneously incubated). The other cytokines did not have any significant effect on HFRPE’s expression of MHC I and II or the selected costimulatory molecules. Conclusions. Our findings indicate that TGF-ß cannot suppress up-regulating effects of IFN-? on HFRPE’s expression of MHC and costimulatory molecules. Overall, the weak or lack of expression of costimulatory molecules after stimulation with various cytokines further confirms that HFRPE cells are weak antigen presenting cells.

A model for xenogenic immune response

Abstract Purpose: To develop a model for analyzing the immune response after xenogenic human fetal retinal pigment epithelium (HFRPE) transplantation. Materials and methods: Pure sheets of HFRPE cells were isolated and attached to poly (dl-lactide-co-glycolide) polymer films and HFRPE spheroids were formed. The spheroids were transplanted into the subretinal space of New Zealand albino rabbits and were observed for 5 months. Bare polymer films were transplanted into the subretinal space of Dutch Belted pigmented rabbits, serving as control. Results: The polymer film was bioegraded within 3 weeks in the subretinal space. No signs of inflammation in the retina or choroid were observed. The HFRPE spheroids were easily transplanted into the subretinal space. The immune response was followed with ophthalmoscopy. Light microscopy indicated a localized immune response in the transplanted area in which the retina and the choroid were infiltrated with immune cells. This infiltration was denser in the choroid. Conclusions: HFRPE spheroid transplantation may be utilized as a model for studying the xenogenic immune response after HFRPE transplantation. This model may also have applications in evaluating the role of immune suppressive agents in preventing rejection after HFRPE transplantation.

Human fetal retinal pigment epithelium induces apoptosis in human T-cell line Jurkat which is independent from its expression of TRAIL

Purpose. To evaluate whether human fetal retinal pigment epithelial (HFRPE) cells express TRAIL (tumor necrosis factor related apoptosis inducing ligand). The role of TRAIL in HFRPE induced apoptosis was evaluated. Methods. Pure cultures of HFRPE cells were isolated. The expression of TRAIL protein and mRNA in non-activated and IFN-? activated HFRPE cells was evaluated with RT-PCR. The role of TRAIL in HFRPE induced apoptosis was assessed by incubating HFRPE cells with human T-cell leukemia line Jurkat (Jkt) in the presence or absence of neutralizing TRAIL antibodies. Cultures were pulsed with [ 3 H]-thymidine to measure Jkt cell proliferation. The role of TRAIL was further examined by western blott evaluating the cleavage of caspases 8 and 10 in Jkt cells after their incubation with HFRPE cells. Results. HFRPE cells expressed TRAIL mRNA. The expression of TRAIL mRNA and protein was up-regulated by IFN-? activation. However, anti-TRAIL antibodies were not able to prevent the HFRPE induced suppression of Jkt cell proliferation. The caspases 8 and 10 were also not cleaved in Jkt cells after their incubation with IFN-? activated HFRPE cells. Conclusions. Although HFRPE cells express TRAIL and its expression is upregulated by IFN-? activation, TRAIL is not involved in HFRPE induced apoptosis in Jkt cells. Currently the role of TRAIL in HFRPE cells is under investigation.