Fulminant Endogenous Anterior Uveitis due to Listeria monocytogenes.

PURPOSE: To report an unusual case of fulminant anterior uveitis, confirmed as endogenous Listeria monocytogenes infection. SUBJECT: A 67-year-old man with multiple comorbidities acutely developed a severe endogenous anterior uveitis. RESULTS: L. monocytogenes, a ubiquitous Gram-positive bacillus, was directly indicated by culture and PCR. Early and aggressive treatment with intravenous antibiotics likely prevented the endophthalmitis which most cases on record experienced. Our patient regained satisfactory visual acuity. CONCLUSIONS: Prompt antimicrobial therapy is paramount when severe endogenous uveitis develops in a patient with comorbidities, especially with systemic immunosuppression. Treatment solely with corticosteroids should be avoided.

Mitomycin C induces multidrug resistance in glaucoma surgery.

BACKGROUND: Despite the adjuvant use of mitomycin C during trabeculectomy, failures still occur. We investigated whether cultured human Tenon fibroblasts exposed to low-dose mitomycin C developed a multidrug resistance phenotype in vitro, and whether mitomycin C treatment during previous filtration surgery induces P-glycoprotein expression in vivo. METHODS: Cultured human Tenon fibroblasts treated with low-dose 0.01 nM mitomycin C for 2 weeks were subsequently treated with 0.1 to 100 microM mitomycin C in the absence or presence of 4 microM verapamil, and allowed to recover for 24 hours. Low-dose mitomycin C-treated fibroblasts were analysed for P-glycoprotein expression using flow cytometry, immunoblotting, and RT-PCR for mdr-1 mRNA. In addition, fibroblasts were treated with low dose 0.1 nM 5-fluorouracil for 2 weeks and analysed for P-glycoprotein expression using flow cytometry. Expression of P-glycoprotein was analysed in surgically removed Tenon tissue (n = 30) using immunohistochemistry. Of the 30 patients, 20 had a previous trabeculectomy, of which nine had previous adjuvant therapy with mitomycin C during trabeculectomy. RESULTS: Partial resistance to mitomycin C after low-dose mitomycin C pre-treatment was significantly neutralised by the addition of verapamil. Low-dose mitomycin C up-regulated P-glycoprotein expression, but not mdr-1 mRNA expression. 5-Fluorouracil did not induce P-glycoprotein expression. P-glycoprotein expression was detected in all nine patients exposed to mitomycin C during previous trabeculectomies. Only six of 21 specimens from patients not previously exposed to mitomycin C showed faint P-glycoprotein expression. CONCLUSION: The induction of P-glycoprotein by mitomycin C could explain some failures that occur after repeated use of mitomycin C during trabeculectomy. The concomitant use of verapamil or the use of 5-fluorouracil alone could increase the success rate of repeat trabeculectomies.

Synergistic effects of combined cytotoxic and apoptosis-inducing drugs on Tenon's capsule fibroblasts in vitro and in vivo.

BACKGROUND: Highly toxic antimetabolites have gained access to routine clinical use to modulate and reduce the amount of postoperative scarring following glaucomatous filtering procedures. It could be speculated that by combining two different antiproliferative substances with different mechanisms of action total amounts of the substances could be decreased and side effects reduced. METHODS: Twenty-two substances were tested that had antiproliferative effects by acting cytotoxically, inhibiting growth factors, or inducing apoptosis. With combinations of each two substances, cell culture experiments using 3T3 and human Tenon's capsule fibroblasts were performed evaluating cell toxicity, proliferation and migration, the extent of free radicals, and the amount of apoptosis (TUNEL, electron microscopy). The five most potent combinations were used in an animal experiment with rabbits performing filtering procedures. The extent of episcleral scarring was evaluated by histopathology. RESULTS: The results of the various assays revealed consistently strong effects in 5 of the 462 combinations. Of these five combinations, two were highly effective in the rabbit model. Substances with strong effects when applied in combination included staurosporine, mitomycin, and CD95L. CONCLUSIONS: We found synergistic effects in assays that evaluated different aspects of cell function. The amount of scarring in an animal experiment was inhibited to a level comparable with a high single dose of mitomycin. Combination therapy of two antiproliferative acting substances may be a promising concept.

Latanoprost stimulates secretion of matrix metalloproteinases in tenon fibroblasts both in vitro and in vivo.

PURPOSE: To investigate the presence and the possible role of different matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in Tenon capsule fibroblasts. These enzymes are essential for the control of tissue remodeling in the context of wound repair. This aspect is important to further the understanding of and possibly to influence the scarring process of filtering blebs after glaucoma surgery. METHODS: Untreated and latanoprost-treated human Tenon fibroblasts were examined for the presence of MMPs and TIMPs on the mRNA and protein levels. Assays performed included RT-PCR, real-time RT-PCR, immunocytochemistry, Western blot analysis, flow cytometry, and zymography. To investigate the changes in vivo, conjunctival specimens of rabbits treated with latanoprost eye drops were examined by immunohistochemistry. RESULTS: In all assays, both MMP-3 and TIMP-2 were detected. With the real-time RT-PCR technique, MMP-1, -2, -3, -7, -9, and -14 and TIMP-1 and -2 were detected. An upregulation of MMP-3 and TIMP-2 after latanoprost treatment of the fibroblasts was shown and found to occur on the mRNA and the protein levels. The upregulation of MMP-3 and TIMP-2 was confirmed in vivo. CONCLUSIONS: Tenon fibroblasts contain the ability on the mRNA level to synthesize all enzymes of the MMP and TIMP family that are related to remodeling of the extracellular matrix. The levels of MMP-3 and TIMP-2 increase after treatment with latanoprost. Tenon fibroblasts may be the target cells for attempts to influence the tissue levels of MMPs and TIMPs in the context of conjunctival wound healing after glaucoma surgery.

Investigation of laser-induced choroidal neovascularization in the rat.

PURPOSE: Choroidal neovascularization plays an important role in pathogenesis of age-related macular degeneration. Induction of neovascularization by laser photocoagulation in the rat fundus is an established animal model in which the effects of new therapeutic approaches are assessed. The purpose of this study was to compare different detection methods of laser-induced neovascularization in the rat. METHODS: Laser spots were applied to the fundus of Long-Evans rats. Ten days after, four different methods were used to detect laser-induced neovascularization: (1) high-resolution angiography with fluorescein isothiocyanate-dextran, (2) immunohistochemical visualization of platelet endothelial cell adhesion molecule (PECAM)-1, (3) visualization of intravascular lumens by peroxidase perfusion in the living rat with subsequent histologic analysis, and (4) histochemical representation of alkaline phosphatase in endothelial cells. RESULTS: At the rim of the laser scars vessel-forming endothelial cells with intravasal dextran and peroxidase were present. Cross-sections demonstrated that these vessels originated from the retina. The center of the scars contained homogenous endothelial cells of choroidal origin, which was confirmed by immunohistochemistry and electron microscopy. In laser-treated eyes without FITC-dextran perfusion, scars showed unspecific fluorescence, making differentiation from specific FITC-dextran-associated fluorescence difficult. CONCLUSIONS: In the rat model of laser-induced neovascularization, newly developed endothelial cells originate from the retina and the choroid. Whereas ring-like surrounding vessels come from the retina, flat endothelial cells in deeper layers are of choroidal origin or may originate from circulating endothelial precursor cells. Dextran angiography has to be regarded critically for visualizing the choriocapillaris and CNV in laser scars. PECAM-1 immunohistochemistry is best for detection and quantification of neovascularization in laser scars.

Characterization of daunorubicin-induced apoptosis in retinal pigment epithelial cells: modulation by CD95L.

PURPOSE: To characterize daunorubicin-induced cell death in cultured human retinal pigment epithelial (RPE) cells and its modulation by CD95 ligand (CD95L). METHODS: In situ DNA end labeling and an ELISA for histone-associated DNA fragments were used to assess apoptosis. CD95 and CD95L expression were examined by immunohistochemistry, flow cytometry, immunoblot, and RT-PCR. Cell death was measured by crystal violet staining. YVAD- and DEVD-amc cleavage was used to measure caspase-1 and -3-like activity. Total RNA and protein synthesis was measured by incorporation level of [(3)H]-leucine and [(3)H]-uridine. RESULTS: RPE cells expressed both CD95 and CD95L, but only CD95 was expressed at the cell surface. Daunorubicin-induced RPE cell apoptosis was associated with enhanced CD95 and CD95L expression. Enhanced CD95L expression was epiphenomenal to the death process, evidenced by the fact that neutralizing CD95L antibodies failed to modulate daunorubicin cytotoxicity. In contrast, the cytotoxic effects of daunorubicin were synergistically enhanced by exogenous CD95L. Synergy appeared to involve enhanced caspase-3-like activity as well as daunorubicin-mediated inhibition of RNA synthesis. CONCLUSIONS: Apoptosis has been shown to be an important factor in the control of specific cell populations. The synergistic activity of an antiproliferative agent, daunorubicin, and a cytokine, CD95L, induces apoptosis in RPE cells. Such approaches provide a means to reduce the concentration of chemotherapeutic agents with a small therapeutic window owing to retinal toxicity, such as daunorubicin, in the adjuvant therapy of proliferative vitreoretinopathy.

Contribution of TNF-alpha to leukocyte adhesion, vascular leakage, and apoptotic cell death in endotoxin-induced uveitis in vivo.

PURPOSE: To investigate the effect of TNF-alpha on leukocyte adhesion, vascular leakage, and apoptotic cell death in endotoxin-induced uveitis (EIU) in the rat. METHODS: EIU was induced in Long-Evans rats by a single footpad injection of lipopolysaccharide (LPS; 350 microg/kg) from Salmonella typhimurium. A single injection of recombinant TNF receptor P75 (etanercept) was given subcutaneously 24 hours before the administration of LPS. Twenty-four hours after administration of LPS, leukocyte adhesion was evaluated in vivo with SLO-acridine orange angiography and ex vivo with concanavalin A lectin staining of retinal flatmounts. Neutrophil activation was quantified by a myeloperoxidase activity assay. Vascular leakage was assessed by Evans blue extravasation. Retinal cell death was assessed with TUNEL staining and quantified with a modified ELISA protocol. Involvement of caspase-3 and -8 was determined by M30 antibody staining, Western blot analysis, and a test for enzymatic activity. RESULTS: Twenty-four hours after the LPS injection, significant increases in leukocyte rolling, adhesion, and activation were observed. In addition, increased levels of apoptosis in the vascular endothelium and the ganglion cell and inner nuclear layers and activation of caspase-8 and -3 were observed. After administration of the TNF-alpha inhibitor, significant reduction in the leukocyte rolling, adhesion, activation, and apoptosis in all the affected layers was observed. The quantitative analysis of vascular leakage revealed a significant decrease after treatment with etanercept. Retinal cell death quantification showed a significant decrease after treatment with the TNF-alpha inhibitor. CONCLUSIONS: Anti-TNF-alpha treatment reduces the LPS-induced increases in leukocyte rolling, adhesion, and vascular leakage in this rat model of inflammatory uveitis. These results suggest the involvement of TNF-alpha in inflammatory uveitis and its potential use as a therapeutic agent in the reduction of ocular inflammation.

Autologous transplantation of genetically modified iris pigment epithelial cells: a promising concept for the treatment of age-related macular degeneration and other disorders of the eye.

Age-related macular degeneration (ARMD) is the leading cause for visual impairment and blindness in the elder population. Laser photocoagulation, photodynamic therapy and excision of neovascular membranes have met with limited success. Submacular transplantation of autologous iris pigment epithelial (IPE) cells has been proposed to replace the damaged retinal pigment epithelium following surgical removal of the membranes. We tested our hypothesis that the subretinal transplantation of genetically modified autologous IPE cells expressing biological therapeutics might be a promising strategy for the treatment of ARMD and other retinal disorders. Pigment epithelium-derived factor (PEDF) has strong antiangiogenic and neuroprotective activities in the eye. Subretinal transplantation of PEDF expressing IPE cells inhibited pathological choroidal neovascularization in rat models of laser-induced rupture of Bruch's membrane and of oxygen induced ischemic retinopathy. PEDF expressing IPE transplants also increased the survival and preserved rhodopsin expression of photoreceptor cells in the RCS rat, a model of retinal degeneration. These findings suggest a promising concept for the treatment of ARMD and other retinal disorders.

Apoptosis-mediating receptor-ligand systems in human retinal pigment epithelial cells.

BACKGROUND: To establish new strategies for the treatment of proliferative vitreoretinopathy (PVR), we investigated new members of a recently discovered apoptosis-inducing receptor-ligand system in human retinal pigment epithelial (RPE) cells. TRAIL (Apo2-L) and Apo3-L are capable of inducing cell death via their receptors Trail-R1 to Trail-R4 and TRAMP. The goal of this study was to prove the existence of these new apoptosis-inducing receptors and ligands in RPE cells. METHODS: Human RPE cells, cultured or prepared directly from the eye, were examined by RT-PCR. Immunohistochemistry of epiretinal membranes of traumatic PVR was performed for the detection of TRAIL and Trail-R1. Protein expression of Trail-R1 was examined in cultured human RPE cells by western blot. Cell death after TRAIL treatment of human RPE cells was measured by crystal violet staining. RESULTS: For RPE cells derived directly from the eye, we detected mRNAs of Trail-R2, Trail-R3, TRAIL, and APO3-L, but not Trail-R1, Trail-R4, and TRAMP. All the examined transcripts were detected in human P0 RPE cells in vitro. Immunohistochemical studies on PVR membranes identified TRAIL and Trail-R1. Western blot confirmed the presence of Trail-R1 in cultured human RPE cells. TRAIL failed to kill RPE cells in vitro, but showed a strong synergistic killing effect when coincubated with protein (cycloheximide) or RNA (actinomycin D) synthesis inhibitor. CONCLUSIONS: We detected a novel apoptosis-inducing receptor-ligand system in RPE cells. An induction of apoptosis as a treatment of PVR seems to be possible. Further investigations are needed including an animal model of PVR.

Characterization of CD95 ligand (CD95L)-induced apoptosis in human tenon fibroblasts.

Toxic side effects of cytotoxic agents such as 5-fluorouracil or mitomycin-C in glaucomatous filtering procedures call for alternative approaches to control fibroblast proliferation. CD95L is a death ligand that triggers apoptosis in susceptible target cells. Apoptosis allows for the safe disposal of cells without damaging the surrounding tissue. The goal of this study was to characterize and to evaluate the CD95L induced cell death in cultured Tenon fibroblasts. Human Tenon fibroblasts were treated with different concentrations of CD95L. For comparison, murine NIH 3T3 fibroblasts were used. Immunohistochemistry and Western blot were used to investigate the CD95 and CD95L expression. Cytotoxicity was measured by crystal violet assay. Apoptosis was investigated using in situ DNA end labelling (TUNEL). DEVD-AMC caspase 3 like activity was measured and caspase 3 processing was studied by immunoblot and the use of the caspase inhibitor DEVD-CHO in cell culture assays. Tenon and NIH 3T3 fibroblasts express CD95 and CD95L. The authors found concentration dependent inhibition of proliferation after CD95L treatment. Tenon fibroblasts, but not NIH 3T3 fibroblasts, show synergy when combined with actinomycin D or cyclohexamide. CD95L treatment did not alter total protein or RNA synthesis. Cell death induced by CD95L was apoptotic and activated caspase 3, as TUNEL positive cells and the active fragment of caspase 3 were found. CD95L induced cell death could be inhibited by the caspase-inhibitor.Here, it is demonstrated that the CD95L induced cell death in cultured human Tenon fibroblasts is apoptotic and possibly mediated by the caspase 3 pathway. These results suggest that it may be possible to use CD95L in glaucomatous filtering procedures. In vivo studies are necessary for further evaluation.

Vitreous treatment of cultured human RPE cells results in differential expression of 10 new genes.

PURPOSE: To analyze the differential gene expression in cultured human retinal pigment epithelial (RPE) cells after treatment with vitreous. METHODS: Cultured human RPE cells were incubated for 48 hours with 25% human vitreous from donor eyes. Total RNA from treated and untreated cells was extracted. The gene expression was analyzed by differential expression analysis (DEmRNA-PCR). The differentially expressed genes were identified by gene bank searches. Differential expression was verified by a quantitative real-time RT-PCR fluorescent nucleic acid staining system. The in vivo mRNA expression of these genes in RPE cells was shown by gene-specific RT-PCR. RESULTS: Vitreous treatment of human RPE cells resulted in the reduced expression of NFIB2, KE03 (NY-REN-25ag), PIG-B, DKFZp564BC462, LKHA, G3BP, PAM, UEV-1, and MAP1B calibrated to the expression of GAPDH when compared with their expression in untreated cells. The reduced expression after vitreous treatment was quantified by gene-specific quantitative real-time RT-PCR and varied from 0.69 to 0.17 compared with untreated cells. The mRNA expression of UDP-GalNac mRNA remained constant. The mRNA expression of eight of these genes was demonstrated in this study for the first time in human RPE cells. CONCLUSIONS: Vitreous treatment of cultured RPE cells induces the differential expression of a variety of genes with functions in transcription, mediation of signal transduction and inflammation, glycosylation, ubiquitination and protein-protein interaction. Further examination of these genes may locate additional targets for treatment of diseases caused by contact of RPE cells with vitreous, typical in proliferative vitreoretinopathy.

Iris pigment epithelial cells transplanted into the vitreous accumulate at the optic nerve head.

BACKGROUND: Iris pigment epithelial (IPE) cells have mainly been investigated in the past for their proposed potential to rescue or even replace degenerated retinal pigment epithelial (RPE) cells after subretinal transplantation in patients with age-related macular degeneration (AMD). More recent reports have characterised the IPE cell as a potent source of trophic factors and cytokines. In our study we investigated the spatial distribution of IPE cells that were injected into the vitreous instead of being injected subretinally. METHODS: IPE cells from Long Evans rats were isolated and injected into the vitreous cavity of Wistar rats without preculturing. Free melanin granules were injected into the vitreous in the same manner. After a period of 2 months, eyes were prepared for histological analysis. Localisation of the injected IPE cells was defined by topographical mapping of the analysed sections. RESULTS: PVR was not observed in any eye. In 8 of 10 injected eyes, IPE cells had accumulated in the prepapillary region. In 2 of 10 eyes, no IPE cells could be detected. The injected melanin granules also accumulated at the optic nerve head, indicating that this is most likely a passive process. In sections of the papillary region containing retinal vessels, the IPE cells seemed to have migrated into the superficial tissue of the optic nerve head. CONCLUSION: Our results demonstrate a way to access the optic nerve head easily and securely without the danger of damaging its fragile structure. This could have important implications for new therapeutic strategies in ocular neurodegenerative diseases like glaucoma. New prospects in gene therapy will require further characterisation of the potential of the IPE cell to produce neuroprotective trophic factors at the optic nerve head.

Trabeculectomies in fellow eyes have an increased risk of tenon's capsule cysts.

OBJECTIVE: To assess outcomes and complications of primary trabeculectomies in fellow eyes in a large group of patients. The assumption was that first and fellow eyes undergoing fistulizing surgical procedures behave similarly in the postoperative period. DESIGN: Retrospective nonrandomized comparative trial (paired eye study). PARTICIPANTS: Over a 4-year period, 566 consecutive patients underwent primary trabeculectomy, all without antifibrotic agents. One hundred thirty-eight of these patients underwent bilateral surgery. INTERVENTION: A primary trabeculectomy was performed. MAIN OUTCOME MEASURES: Preoperative data, postoperative intraocular pressure, and visual acuity were monitored. In addition, complications and the need for consecutive surgical procedures were noted. RESULTS: The mean follow-up period of these trabeculectomies was 27.4 months (range, 7-62 months). Of the 124 bilateral adult cases, no statistical difference was found in intraocular pressures, number of antiglaucomatous medications, and success or failure rates between the two eyes. The need for enhancement procedures, such as needling or surgical excision of Tenon's capsule cysts, was significantly higher in fellow eyes than in first eyes (16 vs. 6 cases; P = 0.03; Mann-Whitney U test). Hypotony as a complication occurred more frequently in fellow eyes. CONCLUSIONS: Primary trabeculectomies performed in both eyes of patients have a remarkably similar clinical course. Failures of first eyes may be a reason to use antimetabolites in primary trabeculectomy of the fellow eye. The present data suggest that fellow eyes have a greater risk of Tenon's capsule cyst formation. This may be important for patient counseling before surgery. These results may additionally be important for clinical studies. Given that first and fellow eyes do not behave in an absolutely similar manner, study designs making intraindividual comparisons may not be feasible.