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L Kohen

Publications and source records attributed to L Kohen.

At least 19 recordsLinked to original sources
PubMed2001

The influence of pro-inflammatory cytokines on human retinal pigment epithelium cell receptors.

PURPOSE: To investigate the mRNA expression of the receptors for tumour necrosis factor alpha (TNFRp55, TNFRp75), interferon gamma (IFN gamma R alpha, IFN gamma R beta), interleukin 10 (IL-10R, CRFB4) and transforming growth factor beta (TGF beta RII) on human retinal pigment epithelium (RPE) cells and to modulate this expression with the pro-inflammatory cytokines TNF-alpha and IFN-gamma as stimulators. METHODS: The cells were cultured in the presence of TNF-alpha (10 ng/ml), IFN-gamma (1000 U/ml) or a combination of both for 24 h, 48 h and 72 h. The total RNA was prepared, and the receptor mRNA expression was investigated by the reverse-transcription polymerase chain reaction method. The changes in mRNA expression during the modulation were quantified by the ribonuclease protection assay. RESULTS: The mRNA for TNFRp55, TNFRp75, IFN gamma R alpha, IFN gamma R beta, CRFB4 and TGF beta RII was constitutively expressed in vitro. IL-10R mRNA was detected in neither unstimulated nor stimulated RPE cells. Especially the mRNA of the TNF-Rp75 was up-regulated, mainly by IFN-gamma or the combination of both stimulators. CONCLUSION: Our results demonstrate that human RPE cells express the mRNA of different cytokine receptors and the expression may be partially modulated by pro-inflammatory cytokines. This may show that RPE cells act as corresponding cells not only in vitro, but also in inflammation and immunological processes in the eye. In this connection it could be hypothesised that activated RPE cells play a stimulating role in addition to the known suppressive one.

Adult↗
PubMed2001

Molecular and cellular evidence for T-cell stimulation by allogeneic retinal pigment epithelium cells in vitro.

PURPOSE: The rejection of retinal pigment epithelium (RPE) allografts is a major barrier to long-term success after retinal transplantation. The aim of this study was to investigate the action of RPE cells on allogeneic T cells in coculture with or without macrophages. For the detection of T-cell activation the interleukins IL-1beta and IL-2, typical for this process, were investigated. METHODS: Human RPE cells (6 x 10(5) cells/flask) were used as stimulator cells. To investigate the influence of MHC class II molecules the RPE cells were pre-incubated with different concentrations of interferon-gamma (IFN-gamma; 0, 50, 100, or 250 U/ml) for 4 days. This was followed by coculture with either 6 x 10(6) T cells or, in a second trial, the T cells plus 6 x 10(5) macrophages. The mRNAs of the cytokines under study were detected using a reverse-transcriptase polymerase chain reaction and were quantified by colorimetry after 6 h. The cytokine protein content in the supernatants was measured after 20 h using specific enzyme-linked immunoabsorbent assays. RESULTS: Cytokine-specific mRNAs and proteins were found in all samples. After coculture the level of IL-1beta mRNA was higher and that of cytokine-specific protein was significantly increased. Furthermore, the addition of macrophages led to increased cytokine secretion but a general influence of the pre-activation with interferon could not be found. Similar results were detected for IL-2; at the highest dose, IFN-gamma preactivation and, in combination with macrophages, a significant increase in the protein level could be found. CONCLUSION: These results show that RPE cells are able to activate allogeneic T cells in vitro. Professional antigen-presenting cells may promote this process, as may pre-treatment with IFN-gamma. The circumstances modelled here are involved in the rejection process after RPE transplantation in humans and help to explain this immune response.

Adult↗
PubMed2001

Immunosuppression by IL-10-transfected human retinal pigment epithelial cells in vitro.

PURPOSE: Retinal pigment epithelium (RPE) transplantation seems to be a possible therapy for restoring vision in the case of retinal degeneration. As there is a risk of allergic rejection, a gene-transfer of immunosuppressive cytokines into the graft may diminish this reaction. Therefore, we investigated the transfer of interleukin-10 (IL-10) into an immortalised human RPE cell line (hTERT-RPE1) and its effect on the proliferation of allogeneic immune competent cells. METHODS: The hTERT-RPE1 cells were transiently transfected with the cDNA of human IL-10 using a lipid-based transfection reagent. The expression of IL-10 mRNA was ana-lysed by reverse transcription-polymerase chain reaction and an enzyme-linked immunosorbent assay measured the secretion of the cytokine over 7 days. The effect of the secreted IL-10 on the proliferation of allogeneic T cells with and without homologous macrophages was investigated colorimetrically. To enhance this reaction, RPE cells were pre-activated with interferon-gamma (IFN-gamma). Anti-IL-10 antibodies were used in a neutralising assay. RESULTS: A transfection efficiency of 23.3 +/- 9.03% was achieved. IL-10 mRNA could only be shown in IL-10-transfected hTERT-RPE1 cells. The same was found for the level of cytokine, with a maximum on day 3 (10.34 +/- 0.09 ng/ml). A significant suppressive effect of the secreted IL-10 on T-cell proliferation was detectable on days 5 and 6. This effect could be significantly abolished with anti-IL-10 antibodies. CONCLUSIONS: The IL-10-producing hTERT-RPE1 cells had an immunosuppressive action on T-cell proliferation in vitro. A gene-transfer into RPE allografts before transplantation may be able to promote graft survival.

Cell Division↗
PubMed2001

Minor influence of the immunosuppressive cytokines IL-10 and TGF-beta on the proliferation and apoptosis of human retinal pigment epithelial (RPE) cells in vitro.

Undesirable immune reactions such as uveitis or graft rejection after corneal and subretinal transplantations are serious inflammations in the eye. Minimizing this process by means of physiological suppressors, either through systemic or intraocular administration with or without gene therapy, is a future therapeutic possibility. In our study, we used different concentrations of transforming growth factor-beta (TGF-beta; 5, 10, and 50 ng/ml) and interleukin-10 (IL-10; 100, 200, and 500 U/ml), both known as modulators of the suppression process, to treat human retinal pigment epithelium (RPE) cells in vitro. The influence of both cytokines on the viability and proliferation of the RPE cells was measured. Furthermore, the secretion of typical markers of the apoptosis process, such as Fas, soluble Fas ligand, and bcl-2, was investigated. Our results show that the concentrations of TGF-beta and IL-10 used have only a slight influence on RPE cells. Cell proliferation under the influence of TGF-beta was significantly reduced, whereas more Fas protein could be found in the cell lysate of the IL-10 samples. In general, IL-10 seemed to have less effect on the physiology of RPE cells. The discussion of the therapeutic use of an immunosuppressive factor in the eye should therefore be focused more on this cytokine.

Apoptosis↗
PubMed2000

Alterations of sensory retinal explants exposed to choroidal melanoma cells ex vivo.

BACKGROUND: Cultures of retinal explants have been established as a useful tool to investigate effects of pathogenic agents in vitro. We used such cultures as a model to study the effects of choroidal melanoma on retinal organisation and function. METHODS: Rabbit retinal explants were co-cultured with human choroidal melanoma cells, or exposed to supernatants from choroidal melanoma cell cultures, for various periods from 1 day to 10 days. The retinal explants were then studied by histology and immunocytochemistry for glial fibrillary acidic protein (GFAP) and vimentin. The release of the pro-inflammatory interleukins IL-6 and IL-8 into the media was measured by enzyme-linked immunosorbent assay. RESULTS: Both in the co-cultures and after treatment with choroidal melanoma cell supernatants for more than 1 week, the layered structure of the retinae became disorganised. Retinal glial (Müller) cells displayed gliosis as indicated by increased GFAP immunoreactivity and decreased immunoreactivity for vimentin. Additionally, the secretion of cytokines, particularly of IL-8, was significantly modulated. The retinal explants produced much less IL-8 than the melanoma cells in separate cultures but increased their IL-8 release significantly after a few days' exposure to melanoma cell-conditioned medium. CONCLUSION: The results show that in cases of choroidal melanoma, the well-known morphological and inflammatory alterations of the retina are accompanied by glial cell reactivity and up-regulated retinal cytokine secretion, and may be caused by soluble factors secreted and induced by the melanoma.

Animals↗
PubMed2000

Changes in the mRNA expression of cytokines and chemokines by stimulated RPE cells in vitro.

PURPOSE: The transplantation of retinal pigment epithelial (RPE) cells is a possible therapy for degenerative diseases of the retina. However, the immune response and the subsequent rejection of the allografts are major problems in this field. We investigated the effect of pro-inflammatory factors on the cytokine and chemokine mRNA expression of human RPE cells during long-term observations in vitro. METHODS: Human RPE cells were cultured in the presence of tumor necrosis factor-alpha (TNF-alpha, 10 ng/ml), interferon-gamma (IFN-gamma, 1000 U/ml) or with a combination of both up to 96 hours. Cells were harvested and total RNA was isolated. The changes in expression of mRNA coding for RANTES, the interleukines (IL)-6, 8, 10, 15, IFN-gamma, monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-beta1 (TGF-beta1) during the stimulation were investigated using the ribonuclease protection assay. RESULTS: IL-10 and IFN-gamma mRNA were detected in neither unstimulated nor stimulated cells. Human RPE cells constitutively express the mRNA for IL-6, MCP-1, IL-8, IL-15, TGF-beta1 and, at very low levels, for RANTES. The TGF-beta1 mRNA expression was not influenced by either stimulation. The mRNA of the other factors was up-regulated for 24-48 h dependent on the stimulation. CONCLUSIONS: Human RPE cells are able to increase their mRNA expression for the detected cytokines in response to the pro-inflammatory factors which are detectable in the rejection process. These up-regulated cytokines themselves are known to be involved in several inflammatory and immunological processes, suggesting their role in the rejection of transplanted RPE allografts.

Cells, Cultured↗
PubMed2000

The local and systemic secretion of the pro-inflammatory cytokine interleukin-6 after transplantation of retinal pigment epithelium cells in a rabbit model.

The rejection of retinal pigment epithelium (RPE) allografts is one of the major problems for long-term success after retinal transplantation. However, the details of the immunological interactions in the subretinal space after transplantation are still unknown. The aim of our study was to investigate the role of IL-6 in the rejection process in the subretinal space and to use IL-6 monitoring for a possible early sign of rejection after transplantation of allogeneic RPE cells. For this we used a model of transplanting pigmented RPE cells, either activated in vitro with 1000 U/ml interferon-gamma (IFN-gamma) for 8 days or non-activated, into 30 albino rabbits. The IL-6 was investigated 3, 5, 7, 9 and 14 days after transplantation. Additionally, sham operated animals and the untreated eyes served as controls. At these time-points the animals were killed, the liquid in the vitreous cavity and serum was collected and the IL-6 present in these samples was quantified with an enzyme-linked immunosorbent assay. Under these conditions, IL-6 was detected in the liquid of the vitreous cavity and in the serum of all RPE-transplanted rabbits. In the group receiving activated RPE two cytokine peaks were measured, 3 and 7 days after transplantation in the vitreous cavity. In non-activated grafts, a maximum was detected on the 5th day after transplantation. Generally, the detected quantity of IL-6 depended on the host status and on the phase of rejection. No significant changes were seen in the sera from either group. Possibly, the host RPE cells are the main source of this interleukin in the transplantation area. The measuring of IL-6 in the rejection model suggests that it plays a role in the immune cascade in the subretinal space.

Animals↗
PubMed1999

Effective chemokines and cytokines in the rejection of human retinal pigment epithelium (RPE) cell grafts.

OBJECTIVE: In the rejection of transplanted retinal pigment epithelium (RPE) cells, an activation of allografts is probably the pivotal point for long-term success. The detailed immunological interactions involved in the rejection after RPE transplantation are still unknown. The aim of this study is to evaluate the interactions of pro-inflammatory cytokines and chemokines in this activation process in vitro. METHODS: Human RPE cells (2 x 10(5)/ml) were therefore activated through a pre-treatment with different concentrations of interferon (IFN)-gamma (100 or 1000 U/ml), tumour necrosis factor (TNF)-alpha (1 or 10 ng/ml) or combinations of both, or employed in a nonactivated form. Afterwards, the RPE cells were tested by enzyme-linked immunosorbant assay (ELISA) and ribonuclease protection assays (RPA) for the secretion and mRNA content of the different chemokines (RANTES, MCP-1 and IL-8) and cytokines (IL-6) at various time points up to 48 h. MAIN FINDINGS: HRPE cells secrete the investigated cytokines in response to pro-inflammatory activation. This could be demonstrated at both the mRNA (RPA) and the protein levels (ELISA). The secretion was time and dose dependent, and significantly upregulated in comparison to that observed with nonactivated cells. CONCLUSIONS: This study demonstrates that RPE cells efficiently secrete such cytokines as RANTES, MCP-1, IL-6, and IL-8, and have an accountable neutrophil and monocyte chemotactic activity. Thus, it could be indicated that the investigated cytokines play a central role in the activation cascade of RPE and in RPE rejection as well.

Cells, Cultured↗
PubMed1999

Down-regulation of MHC class II expression on bovine retinal pigment epithelial cells by cytokines.

To determine the effect of interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) on the down-regulation of MHC class II antigens, bovine retinal pigment epithelial (RPE) cells were incubated with interferon-gamma (IFN-gamma) in different concentrations. Subsequently, the IFN-gamma pretreated RPE cells were cultured with TGF-beta or IL-10 in distinct concentrations and treatment modi. About 10% of native (totally untreated) RPE cells were positive for MHC class II antigens detected with immunocytostaining. Under the influence of IFN-gamma (1,000 U/ml), the number of MHC class II-bearing cells increased to 49.9 +/- 4.5% positive cells after 8 days' incubation. Expression decreased under the most effective TGF-beta treatment (5 ng/ml) to 2.0 +/- 0.9% after 24 h incubation, and under similar IL-10 treatment (200 U/ml) to 3.8 +/- 1.4% after 72 h. This decreasing number of the MHC class II-positive cells may be useful for various eye research studies and, more especially, to RPE cell transplantation in the future.

Animals↗
PubMed1998

In-vitro methods to decrease MHC class II-positive cells in retinal pigment epithelium cell grafts.

Rejection of RPE transplants may be caused by expression of MHC class II molecules on the graft. We wanted to determine if passage level, hyperoxia, low temperature, or magnetic separation had an influence on this expression or on several other physiological parameters of bovine RPE cells. For this, bovine RPE cells were cultured under normal conditions for several passages (1-5) or were incubated with a high level of oxygen (75%) for 24 h or at low temperature (24 degrees C) for seven days. Magnetic beads coated with monoclonal antibody against MHC class II antigens were used to separate the positive cells from the total cell population. The number of MHC class II-positive RPE cells was not affected by increased passage, oxygen concentration, or low temperature. Using magnetic beads, approximately 7.5% of the cells were separated from the total RPE population as MHC class II-positive cells. Hyperoxia initially increased the number of RPE cells to 178% that of the untreated controls, but the subsequent MTT test showed a decrease in proliferation to 95% of control levels. Similarly, low temperature increased cell number of 110% that of controls, but prolonged proliferation decreased to 76% of the controls. None of the experimental conditions had a significant influence on the viability of the cells. Culture conditions can be modified to increase the yield of RPE cells, and MHC class II-positive RPE cells can be detected and selectively separated from the total cell population, both of which may prove to be useful for RPE cell transplantation.

Animals↗
PubMed1998

Immunological problems of transplantation into the subretinal space.

The objective of retinal transplantation is to substitute destroyed or degenerated retina to improve visual function. Photoreceptors and retinal pigment epithelium cells of embryonic and nonembryonic origin have been transplanted into the subretinal space in different animal models. Recently, retinal cells have also been used for transplantation in untreated or immunosuppressed patients with retinitis pigmentosa and age-related macular degeneration. Transplants performed in animals such as the RCS rat have maintained retinal function at the transplantation site for long periods of time, although such transplantations in humans have not proved conclusively, to date, whether there is a real effect or not. One reason for this phenomenon seems to be an immune response to transplanted retinal cells at the transplantation site. The detectable rejection process shows that the posterior part of the eye is not absolutely immunologically privileged and that rejection is a serious problem in human retinal transplantation. Many questions concerning transplantation technique, graft treatment and postoperative treatment will be answered through more intensive experiments and clinical trials regarding the immunology. However, rejection of transplanted material is one of the main reasons why retinal transplantation has not yet proved successful.

Animals↗
PubMed1998

Secretion of cytokines by human choroidal melanoma cells and skin melanoma cell lines in vitro.

The selective secretion of interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10), basic fibroblast growth factor (b-FGF) and transforming growth factor beta 1 (TGF beta-1) in tissue culture of choroidal melanomas and two established skin melanoma cell lines was investigated with ELISA analysis. Values of choroidal melanoma cells were compared with the melanoma cell lines and human fibroblasts as a physiological control. High secretion of IL-6 was detectable in choroidal melanoma cultures but not in the cell lines. IL-8 secretion was found in all melanoma cultures. However, IL-10 was only secreted by one skin melanoma cell line and in choroidal melanoma cell cultures. B-FGF secretion by choroidal melanomas was higher than by cell lines. No differences were seen in the amounts of TGF beta-1 produced by melanoma cells. Human fibroblasts produce higher amounts of IL-6 and IL-8 but lower of b-FGF in vitro in contrast to the melanoma cells. The secretion of cytokines by choroidal melanoma cells suggests an important role of these soluble factors in the interaction of tumour and healthy tissue.

Choroid Neoplasms↗
PubMed1997

Iris pigment epithelial cells of long evans rats demonstrate phagocytic activity.

The phagocytic activities of iris pigment epithelial (IPE) cells and retinal pigment epithelial (RPE) cells of Long Evans rats towards latex beads and rod outer segments (ROS) were compared in vitro. IPE and RPE cells of Long Evans rats were isolated and pure cultures obtained. The cultures were incubated with latex beads, fixed, and analysed computer morphometrically, IPE and RPE cell cultures were also incubated with isolated ROS and examined using transmission electron microscopy. IPE cells were able to ingest latex beads. There was no significant difference between the number of latex particles phagocytized by IPE and RPE cells. After incubation with isolated ROS, IPE cells also recognized and ingested the ROS particles. However, the specific phagocytic capacity of IPE cells was 76% of that of RPE cells. The autologous IPE cells might have the potential to be used as an alternative to RPE cells for transplantation in the subretinal space.

Animals↗
PubMed1997

Porcine iris pigment epithelial cells can take up retinal outer segments.

This study investigates the ability of iris epithelial cells (IPE) to ingest rod outer segments (ROS) and compares the amount of phagocytosis of porcine RPE and IPE cells by the use of a pH sensitive fluorescent dye (carboxy SNAFL) at the light microscopic level. The dye allowed investigation of ingestion separately from binding of rod outer segments. In a second set of experiments, after exposing ferritin-labeled ROS to the cultured cells, phagosomes were also counted in electron microscopic sections. Additionally immunocytochemical staining was performed with IPE and RPE cells. Both cell types stained positive with polyclonal NaK-ATPase antibodies against the alpha 1 subunit from rat brain and kidney. The epithelial nature of the cultured cells was determined by monoclonal anti-human-cytokeratin antibodies. Moreover, the ultrastructure of the cells revealed high amounts of phagosomes smaller than 1 micron in diameter present in both RPE and IPE cells. The iron label of the phagosomes was determined by EELS spectra taken from individual phagosomes. Electron and light microscopic quantification shows that cultured IPE cells have 64% of the phagocytic capacity of the RPE with respect to phagosomes larger than 1 micron in diameter.

Animals↗
PubMed1997

Iris pigment epithelium transplantation.

BACKGROUND: Iris pigment epithelium (IPE) cells and retinal pigment epithelium (RPE) cells possess the same embryonic origin. It is also known that the pigmented epithelial cells in the eye have a high transdifferentiation potential. In this study we transplanted IPE cells into the subretinal space of albino Royal College of Surgeons (RCS) rats and evaluated their influence on the degeneration of the photoreceptors. METHODS: IPE cells of Long Evans rats were isolated and pure cultures were obtained. The isolated cells were transplanted into the subretinal space of RCS rats. Light microscopic and morphometric analysis were carried out. RESULTS: The IPE transplants survived in the subretinal space and attached themselves to the Bruch's membrane. The transplanted cells were able to delay the degeneration of the photoreceptors for up to 3 months. CONCLUSION: These results suggest that IPE cells could be successfully transplanted and survive in the subretinal space. In the transplanted eyes the photoreceptors were preserved for a period of 3 months. Further studies are needed to explore the capability of IPE cells to assume the main functions of RPE cells in the subretinal space and their potential in the therapy of selective degenerative diseases of the retina.

Animals↗
PubMed1997

Comparison of tight junction permeability for albumin in iris pigment epithelium and retinal pigment epithelium in vitro.

BACKGROUND: The degenerative retinal diseases are one of the major causes of visual loss in the western world. Although heterologous RPE transplants rescue the photoreceptors in the dystrophic rat model, rejection remains a major limiting factor. Given the common embryonic origin, iris pigment epithelial (IPE) cells might be able to take over the functions of retinal pigment epithelial (RPE) cells, serving as an autologous graft for transplantation and thereby preventing rejection. One of the main functions of RPE cells is the generation of tight junctions which form the outer blood-retinal barrier. In this study we compared the tight junction permeabilities of IPE and RPE cells isolated from Long Evans rats by measuring their albumin clearances. METHODS: IPE and RPE cells were cultured on semipermeable filter supports with and without the addition of 0.02% ethylenediaminetetraacetic acid (EDTA). At selected intervals, the albumin clearances of the IPE and RPE cells were measured spectrophotometrically and compared. The morphology of the cells was compared using electron microscopy and fluorescent labeling. RESULTS: IPE and RPE cells both restricted the passage of albumin in vitro. After the modulation of tight junctions with 0.02% EDTA, the clearance increased in both types of cells in a similar fashion. The morphology of tight junctions was visualized with electron microscopy. CONCLUSION: These results indicate that the functional barrier for macromolecules is similar in IPE and RPE cells in vitro. This raises the possibility that IPE cells would form tight junctions in the subretinal space, thereby substituting for the blood-retinal barrier normally formed by RPE cells.

Actins↗
PubMed1997

Mechanisms of graft rejection in the transplantation of retinal pigment epithelial cells.

PURPOSE: The role of activated retinal pigment epithelium (RPE) cells was investigated in the rejection after subretinal transplantation. METHODS: RPE cells from 7 pigmented rabbits were separated and evaluated regarding their MHC class II expression as the sign of activation. The activation of the RPE cells was augmented with a treatment of 1,000 U/ml interferon gamma (IFN-gamma) for 8 days. These cells were then transplanted into 7 albino rabbits. As control, RPE transplantations without a pretreatment were performed in 7 albino rabbits. Six weeks after the transplantation, the transplanted eyes were enucleated and histology was performed. RESULTS: In culture, without IFN-gamma addition, 11.38 +/- 0.94% of the RPE cells presented MHC class II. After IFN-gamma treatment, this quantity increased to 78.26 +/- 1.46% of the RPE cells. These cells transplanted into the rabbits caused an obvious rejection in the transplantation area which was verified histologically. The control group presented a transplantation area without signs of rejection or inflammation. CONCLUSION: In culture, some of the adult RPE cells are activated. These cells may accelerate the rejection cascade after transplantation. An elimination of activated RPE cells from the transplant should be recommended before transplantation.

Animals↗