MGSA/GRO transcription is differentially regulated in normal retinal pigment epithelial and melanoma cells.

We have characterized constitutive and cytokine-regulated MGSA/GRO alpha, -beta, and -gamma gene expression in normal retinal pigment epithelial (RPE) cells and a malignant melanoma cell line (Hs294T) to discern the mechanism for MGSA/GRO constitutive expression in melanoma. In RPE cells, constitutive MGSA/GRO alpha, -beta, and -gamma mRNAs are not detected by Northern (RNA) blot analysis although nuclear runoff experiments show that all three genes are transcribed. In Hs294T cells, constitutive MGSA/GRO alpha expression is detectable by Northern blot analysis, and the level of basal MGSA/GRO alpha transcription is 8- to 30-fold higher than in RPE cells. In contrast, in Hs294T cells, basal MGSA/GRO beta and -gamma transcription is only twofold higher than in RPE cells and no beta or gamma mRNA is detected by Northern blot. These data suggest that the constitutive MGSA/GRO alpha mRNA in Hs294T cells is due to increased basal MGSA/GRO alpha gene transcription. The cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) significantly increase the mRNA levels for all three MGSA/GRO isoforms in Hs294T and RPE cells, and both transcriptional and posttranscriptional mechanisms are operational. Nuclear runoff assays indicate that in RPE cells, a 1-h IL-1 treatment induces a 10- to 20-fold increase in transcription of MGSA/GRO alpha, -beta and -gamma but only a 2-fold increase in Hs294T cells. Similarly, chloramphenicol acetyltransferase (CAT) reporter gene analysis using the MGSA/GRO alpha, -beta, and -gamma promoter regions demonstrates that IL-1 treatment induces an 8- to 14-fold increase in CAT activity in RPE cells but only a 2-fold increase in Hs294T cells. The effect of deletion or mutation of the MGSA/GRO alpha NF-kappa B element, combined with data from gel mobility shift analyses, indicates that the NF-kappa B p50/p65 heterodimer in RPE cells plays an important role in IL-1- and TNF alpha-enhanced gene transcription. In Hs294T cells, gel shift analyses indicate that IL-1 and TNF alpha induce NF-kappa B complex formation; however, transactivation does not occur, suggesting that subtle differences in the NF-kappa B complexes may result in the inability of the cytokines IL-1 and TNF alpha to activate transcription of the MGSA/GRO genes. IL-1 and TNF alpha posttranscriptionally regulate MGSA/GRO mRNA levels in both cell types. In Hs294T cells, IL-1 increases the half-life of MGSA/GRO alpha from 15 min to 6 h (a 24-fold increase in half-life). These data indicate that IL-1 and TNF alpha transcriptionally and posttranscriptionally regulate MGSA/GRO alpha, -beta, and -gamma mRNA levels in RPE cells, while in Hs294T cells, the major effect of IL-1 and TNF alpha is on mRNA stability.

Inhibition of cultured human RPE cell proliferation and lysyl hydroxylase activity by hydroxy derivatives of minoxidil.

PURPOSE: To examine the antiproliferative and lysyl hydroxylase suppressing effects of 3'-hydroxyminoxidil and 4'-hydroxyminoxidil, derivatives of minoxidil devoid of the antihypertensive effect, on human retinal pigment epithelial (RPE) cells in culture. METHODS: Subconfluent and confluent cultures of RPE cells, exposed to 0.01 to 5 mM 3' or 4'-hydroxyminoxidil for 15 minutes to 7 days, were examined for proliferation, viability, and morphologic changes. Lysyl hydroxylase activity in confluent cultures exposed to 1 mM 3'- or 4'-hydroxyminoxidil was determined by measuring the amount of 3H2O released from L-(4,5-3H)lysine-labeled unhydroxylated procollagen substrate after vacuum distillation. RESULTS: Both compounds inhibited the proliferation of subconfluent cultures of RPE cells in a dose-dependent fashion. The 50% effect occurred at 0.25 mM for 3'-hydroxyminoxidil and 0.5 mM for 4'-hydroxyminoxidil. The antiproliferative effect was detectable within 24 hours, required a minimum 1-hour exposure, and persisted even after the drug was removed from the culture medium. Cell viability experiments provided no evidence for toxicity. In contrast, the number of cells at confluent density was not affected. Both 3'-hydroxyminoxidil and 4'-hydroxyminoxidil suppressed lysyl hydroxylase activity by 72%. CONCLUSIONS: The structure of minoxidil can be altered to reduce the antihypertensive activity while preserving the antiproliferative and lysyl hydroxylase suppressing effects. The hydroxy derivatives of minoxidil may be useful in the treatment of proliferative vitreoretinopathy, a disease with unwanted proliferation of RPE cells.

Minoxidil inhibits ocular cell proliferation and lysyl hydroxylase activity.

PURPOSE: To examine the antiproliferative and lysyl hydroxylase-suppressing effects of minoxidil on cultured proliferating and density-arrested human retinal pigment epithelial cells (hRPE) and Tenon's capsule fibroblasts (hTCF). METHODS: Proliferating and density-arrested hRPE and hTCF, exposed to minoxidil (0.1-5 mM) for 15 min to 7 days, were examined by proliferation assays, [3H]thymidine incorporation, trypan-blue exclusion, and phase-contrast microscopy. The lysyl hydroxylase-suppressing effects were examined in confluent hRPE exposed to minoxidil (0.01-1 mM) using L-[4,5-3H]-lysine-labeled procollagen substrate and measuring the amount of tritium released as 3H2O after vacuum distillation. RESULTS: Minoxidil (0.1-5 mM) inhibited the proliferation of subconfluent cultures of hRPE and hTCF in a dose-dependent manner with a half-maximal effect at 1.5 and 2.5 mM, respectively. The antiproliferative effect, detectable within 24 hr, occurred with a limited exposure period and persisted even after removal of minoxidil from the culture medium. In contrast, 1-5 mM minoxidil had minimal effect on density-arrested hRPE and hTCF. However, at doses above 3 mM, although minoxidil had no effect on the number of density-arrested hRPE, morphologic and viability experiments indicated signs of cytotoxicity. Minoxidil (0.1-1 mM) caused a maximum of 71% reduction in the activity of lysyl hydroxylase, an enzyme needed for stable cross-links in collagen. CONCLUSIONS: Minoxidil may be a useful drug for the treatment of conditions such as proliferative vitreoretinopathy and bleb scarring after trabeculectomy, disorders with unwanted cell proliferation and collagen production.

The antiproliferative effect of a transferrin-toxin on human retinal pigment epithelial cells and rabbit fibroblasts.

PURPOSE: To determine the effect of a rabbit transferrin conjugated to recombinant ricin A chain (Tfr-rRA) and the carboxylic ionophore monensin on proliferating and density-arrested human retinal pigment epithelial cells and rabbit dermal fibroblasts. METHODS: Cells were seeded on 24-well plates at 20,000 cells/cm2 and exposed to Tfr-rRA (0.1-10,000 ng/ml) with or without monensin (0.01 microM), and with or without human transferrin (65.7 mg/l) for 5 minutes to 7 days. Cells were studied morphologically and counted at 1, 2, 4, and 7 days. RESULTS: Tfr-rRA (10-10,000 ng/ml) killed proliferating human retinal pigment epithelial cells and rabbit dermal fibroblasts in a dose-dependent manner (p < or = 0.01) up to a maximum of 86% and 93%, respectively. In contrast, Tfr-rRA had minimal effect on density-arrested human retinal pigment epithelial cells and rabbit dermal fibroblasts. The cytotoxicity of Tfr-rRA was inhibited by the addition of human transferrin (65.7 mg/l), an effect that was partially overcome by longer treatment with Tfr-rRA. Monensin (0.01 microM) increased the cytotoxicity of Tfr-rRA by 4.8-fold over Tfr-rRA alone, shortened the onset of cell kill with Tfr-rRA from 48 to 24 hours (P = 0.04), and partially reversed the neutralizing effect of human transferrin. CONCLUSIONS: The results indicate that Tfr-rRA effectively inhibited the proliferation of human retinal pigment epithelial cells and rabbit dermal fibroblasts in vitro. The inhibitory effect could be modified by the addition of human transferrin or monensin. Thus, this ricin A chain conjugate may interrupt the proliferation of cells necessary in the pathogenesis of proliferative vitreoretinopathy.

Retinal pigment epithelial cells of the posterior pole have fewer Na/K adenosine triphosphatase pumps than peripheral cells.

The density of Na/K adenosine triphosphatase (ATPase) pumps in retinal pigment epithelial (RPE) cells in different retinal regions was quantified by measuring the binding of 3H-ouabain to RPE in cow and human eyecups. In bovine eyes, pump density was estimated in RPE samples isolated from three retinal regions outlined with a 7-mm trephine: one from the posterior pole in the area centralis and two from the superior, equatorial retina representing unpigmented (in the tapetum) and pigmented zones. In human eyes, RPE samples were isolated from a posterior region centered around the macula and one superior region. Ouabain binding to RPE of the posterior pole of both species was approximately 40-60% lower than binding to RPE of more peripheral regions in the same eyes. For bovine eyes, ouabain binding did not differ between pigmented and unpigmented cells of the superior retina, suggesting that reduced binding in the relatively amelanotic posterior cells was not related to levels of pigmentation. For human RPE, binding to posterior cells was lower in eyes from donors of all ages (range, 17-90 yr). The data suggest that Na/K ATPase pump site density is lower in posterior RPE cells of both bovine and human eyes, perhaps due to a regional difference in requirements for ionic regulation.

Intravitreal sustained-release dexamethasone device in the treatment of experimental uveitis.

PURPOSE: Uveitis often runs a chronic course requiring long-term therapy. Topical treatment results in poor intravitreal penetration, and systemic therapy is associated with significant side effects. The authors investigated whether an intravitreal sustained-release dexamethasone device was effective in the treatment of severe panuveitis in a rabbit model. METHODS: Twenty New Zealand white rabbits were immunized twice subcutaneously with 10 mg of Mycobacterium tuberculosis H37Ra antigen. Twelve days later, sustained-release dexamethasone devices were implanted into the vitreous of the right eye of 10 rabbits. Ten control rabbits received a sham device. One day later, rabbits were challenged with an intravitreal injection of 33 micrograms of antigen. Three animals in each group were sacrificed on post-challenge days 7 and 13 for aqueous white blood cell (WBC) count, protein determination, and histologic examination. To simulate chronic inflammation with exacerbations, the eight remaining eyes were rechallenged with intravitreal antigen on day 15 and were observed for 3 1/2 months. Inflammation was graded clinically by two masked observers. Retinal function was evaluated by electroretinography (ERG). Light microscopy was used to evaluate the eyes histopathologically. The amount of residual drug in the devices was measured on day 13 and at the end of the experiment. RESULTS: By all clinical criteria measured--anterior chamber cells, flare, and vitreous opacity--treated eyes had significantly less inflammation than untreated eyes (P < 0.05). Clinical examination correlated well with objective data. Both protein concentration (P < 0.05) and aqueous WBCs (P < 0.02) were approximately 10-fold higher, and ERGs were significantly depressed (P < 0.05) in untreated eyes compared to treated eyes. Histopathologic examination showed marked inflammation and tissue disorganization in the untreated compared to the treated eyes. After antigen rechallenge, inflammation in experimental eyes was still less than in control eyes. Late complications such as corneal neovascularization, cataract, and hypotony were also less in the treated eyes than in the untreated eyes. At the end of the experiment (99 days after device implantation), approximately 30% of drug remained in the devices. CONCLUSIONS: The intravitreal sustained-release dexamethasone device is highly effective in suppressing inflammation and preventing complications after two episodes of experimental uveitis in a rabbit model for at least 3 1/2 months. This device may be useful in the management of patients with severe chronic posterior uveitis who cannot tolerate systemic or periocular therapy.

Safety and pharmacokinetics of an intraocular fluocinolone acetonide sustained delivery device.

PURPOSE: To determine the safety and pharmacokinetics of an intraocular fluocinolone acetonide sustained drug delivery device. METHODS: Nonbiodegradable drug delivery devices containing 2 or 15 mg of a synthetic corticosteroid, fluocinolone acetonide, were constructed. The long-term in vitro release rates of these devices were determined in protein-free buffer or buffer containing 50% plasma protein. Fifteen-milligram devices were also implanted into the vitreous cavities of rabbit eyes. Intravitreal drug levels, the amount of drug remaining in explanted devices, and the release rate of explanted devices were determined over a 1-year time period. Drug toxicity was assessed over this same time period by slit lamp examination, indirect ophthalmoscopy, electroretinography, and histologic examination. RESULTS: The drug release rates for the 2-mg device, 1.9 +/- 0.25 microg/d, and for the 15-mg device, 2.2 +/- 0.6 microg/d, remained linear over the 6-month and 45-day testing period, respectively. The release rate increased by approximately 20% when devices were transferred from protein-free buffer to buffer that contained protein (P: < 0.0001). Vitreous levels remained fairly constant (0.10-0.21 microg/ml) over a 1-year period. No drug was present in the aqueous humor during this time period. Based on the device release rates, the predicted life span of the 2- and 15-mg devices are 2.7 and 18.6 years, respectively. There was no evidence of drug toxicity by clinical examination, electroretinography, or histologic examination. CONCLUSIONS: It is feasible to construct a nontoxic fluocinolone acetonide drug delivery device that reproducibly releases fluocinolone acetonide in a linear manner over an extended period. These devices show great promise in the treatment of ocular diseases such as uveitis, which are often managed with chronic corticosteroid therapy.

Suppression of NF-kappaB-dependent proinflammatory gene expression in human RPE cells by a proteasome inhibitor.

PURPOSE: To determine whether nuclear transcription factor-kappaB (NF-kappaB) is activated in human retinal pigment epithelial (hRPE) cells in response to interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), or interferon-gamma (IFN-gamma) alone or in combination and if so, whether expression of proinflammatory genes induced by these agents can be blocked by a proteasome inhibitor, MG-132, which inhibits the degradation of I kappaB, an NF-kappaB inhibitor, thereby preventing nuclear translocation of NF-kappaB. METHODS: Cultured hRPE were pretreated for 60 minutes with medium alone or medium containing the proteasome inhibitor MG-132 (20 microM) and then exposed to TNF-alpha (1.1 x 10(3) U/ml), IL-1beta (5 U/ml), or IFN-gamma (7.5 x 10(3) U/ml) alone or in combination (TII). Nuclear translocation of NF-kappaB was determined by fluorescence staining of the NF-kappaB Rel A (p65) subunit. Cytoplasmic I kappaB protein was measured by western blot analysis. Nuclear extract binding to kappaB DNA motifs was measured by electrophoretic mobility shift assay and antibody supershift assay. Steady state mRNA expression of the chemokines melanoma growth stimulating activity (MGSA)/gro-alpha, regulated on activation normal T-cell expression and secreted (RANTES), and monocyte chemoattractant protein (MCP-1), the cytokines IL-1beta and macrophage colony stimulating factor (M-CSF) and intercellular adhesion molecule-1 (ICAM-1) was evaluated by semiquantitative reverse transcription-polymerase chain reaction. Chemokine and cytokine protein secretion was measured by enzyme-linked immunosorbent assay. Cell-surface ICAM-1 expression was determined by flow cytometry. RESULTS: TNF-alpha, IL-1beta, and TII but not IFN-gamma alone caused degradation of I kappaB, Rel A nuclear translocation, and increased NF-kappaB DNA binding activity, effects that were blocked by pretreatment with MG-132. MG-132 suppressed MGSA/gro-alpha, RANTES, MCP-1, IL-1beta, M-CSF, and ICAM-1 mRNA expression and secreted RANTES, MCP-1, and M-CSF protein, and cell-surface ICAM-1 that were induced by IL-1beta, TNF-alpha, and TII. CONCLUSIONS: TNF-alpha, IL-1beta, and TII induce expression of proinflammatory cytokines and ICAM-1 in hRPE cells through an NF-kappaB-dependent signal transduction pathway. This effect is blocked by MG-132, a proteasome inhibitor that prevents I kappaB degradation. Inhibition of NF-kappaB may be a useful strategy to treat proliferative vitreoretinopathy and uveitis, ocular diseases initiated and perpetuated by cytokine activation.

Expression of three forms of melanoma growth stimulating activity (MGSA)/gro in human retinal pigment epithelial cells.

PURPOSE: To characterize mRNA expression and protein production of the cytokine MGSA/gro in human retinal pigment epithelial (RPE) cells and to determine whether expression of MGSA/gro is modulated by serum and the cytokines interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), or transforming growth factor beta (TGF beta) mediators implicated in proliferative vitreoretinopathy (PVR). METHODS: Reverse-transcription polymerase chain reaction was used to determine the steady-state mRNA expression of three forms of MGSA/gro, alpha, beta, and gamma, by cultured human RPE cells in the presence or absence of recombinant IL-1 beta, TNF alpha, or TGF beta, or when serum-starved cells were re-fed with medium containing serum. Immunocytochemistry was used to characterize RPE cell-associated MGSA/gro protein, and immunoprecipitation of MGSA/gro from cell-conditioned medium was used to demonstrate MGSA/gro secretion. RESULTS: MGSA/gro mRNA was expressed minimally under basal conditions. Expression for all three forms of MGSA/gro mRNA was induced in a dose- and time-dependent manner after exposure to IL-1 beta, to a lesser extent after exposure to TNF alpha, but not after exposure to TGF beta. Serum induced MGSA/gro alpha and gamma transcripts, but not beta transcripts. Cell-associated MGSA/gro was identified on RPE cells grown in the absence of cytokines, but MGSA/gro was not secreted under these conditions. Exposure to IL-1 beta did not consistently cause increased cell-associated MGSA/gro; however, IL-1 beta induced secretion of MGSA/gro in a time-dependent manner. CONCLUSION: MGSA/gro is produced by human RPE in response to mediators implicated in PVR. Because MGSA/gro is a pleiotropic modulator of cell proliferation and inflammation, it may contribute to the intraocular wound healing response that characterizes PVR.

Intravitreal sustained release corticosteroid-5-fluoruracil conjugate in the treatment of experimental proliferative vitreoretinopathy.

PURPOSE: Proliferative vitreoretinopathy (PVR) remains the most common cause of failed retinal detachment (RD) surgery. The authors compared the effectiveness of two intraocular sustained-release codrugs in suppressing PVR in a rabbit model a surgically implantable pellet releasing 5-fluorouracil (FU) and dexamethasone (DX) for 1 week and an injectable intravitreal sustained-release suspension releasing 5-FU and triamcinolone acetonide for 1 month. METHODS: Sustained-release devices and suspensions were prepared to deliver equimolar quantities of corticosteroid and 5-FU. In group 1, devices were implanted surgically into the vitreous of the right eye of 10 New Zealand White rabbits. Ten control rabbits received surgical implantation of the suture only. In group 2, drug suspension was injected into the vitreous of the right eye of 10 New Zealand White rabbits. Ten control rabbits received injection of the vehicle only. One day later, each rabbit was injected intravitreally with 250,000 homologous rabbit dermal fibroblasts. Severity of PVR was graded clinically by two masked observers on days 3, 7, 10, 14, 21, and 28. RESULTS: In group 1, clinical severity of PVR was less in the experimental group than in the control group at all time points, this was only statistically significant on day 10 (P = 0.04). Six eyes developed moderate to severe tractional RD or bullous RD in the control group by day 10 compared with none in the experimental group (P = 0.01). In group 2, the median clinical grading of eyes in the experimental group was significantly less than that in the control group at all time points through day 21 (P < or = 0.01). CONCLUSIONS: Both the intravitreal sustained-release dexamethasone-5-FU device and the triamcinolone-5-FU suspension effectively inhibit the progression of PVR in a rabbit model. Simultaneous delivery of 5-FU and corticosteroid may target different components of the wound-healing process in this disease.

Expression of CD44 and variant isoforms in cultured human retinal pigment epithelial cells.

PURPOSE: CD44 is a major hyaluronic acid receptor that exists as a number of isoforms, generated by alternative splicing of 9 "variant" exons in humans (v2 to v10) and 10 exons in rodents. Little is known about the expression and function of CD44 in human retinal pigment epithelium (RPE) cells. Therefore, the authors determined whether human RPE cells express CD44, and whether the expression differs depending on the proliferative status of the cells. METHODS: Human RPE cells were harvested from normal donor eyes and propagated in culture. Total RNA was extracted from cultured cells. mRNA expression of CD44 was determined by reverse transcription-polymerase chain reaction (RT-PCR), followed by cloning and sequencing of the PCR products, and by Southern hybridization. CD44 cell surface expression was measured by flow cytometry. Western hybridization and immunohistochemistry were used to determine the CD44 immunoreactivity of cultured human RPE cells and normal human RPE cells in situ. RESULTS: The standard form of CD44 mRNA and variant isoforms containing exon v6 or v10 were expressed in cultured human RPE cells. CD44 mRNA and protein levels were increased in proliferating human RPE cells compared with density-arrested counterparts. Addition of 1 microM retinoic acid enhanced the cell density-induced downregulation of CD44 mRNA, but did not significantly affect the CD44 cell surface protein expression. As previously reported, CD44 immunoreactivity was not detected in normal human RPE cells in situ. CONCLUSIONS: Cultured human RPE cells express CD44 standard form and variant isoforms containing exon v6 or v10, which are preferentially expressed by proliferating human RPE cells.

Activin expression by cultured human retinal pigment epithelial cells.

PURPOSE: To determine whether human retinal pigment epithelial (hRPE) cells produce activin, a growth factor in the transforming growth factor beta family, and to characterize growth regulatory effects of activin on retinal pigment epithelium. METHODS: mRNA expression was examined using polymerase chain reaction with primers specific for the beta A and beta B chains of activin and by slot blot analysis with a probe specific for the beta A chain. Protein localization was determined immunocytochemically using antibodies specific for the beta A chain of activin and intact activin A. The effect of activin A on DNA synthesis was studied by measuring (3H) thymidine incorporation after cells were exposed to recombinant human activin A (rhA). Growth regulatory effects of rhA on hRPE cells were examined with cell growth assays. RESULTS: beta A mRNA was expressed constitutively in 8/8 cells lines tested. beta B mRNA was not expressed in any of the six cell lines tested but was expressed in human ovarian granulosa cell controls. Positive immunostaining was observed for both the beta A chain and intact activin A. (3H) thymidine incorporation was inhibited 44% (P < 0.025), 45% (P < 0.025), and 44% (P < 0.015) when RPE cells were exposed to 100 ng/ml rhA and grown in serum-free medium, medium with 0.5% serum, and 1% serum, respectively. Cell growth was inhibited 33.2% (P = 0.0001) after RPE cells were exposed to 100 ng/ml rhA for 8 days. CONCLUSIONS: These results suggest that activin A can act as an autocrine-paracrine growth regulator in RPE cells and may help control cellular growth in ocular development and proliferative eye disease.

Ocular toxicity following high dose chemotherapy and autologous transplant.

A 49-year-old woman received an autologous transplant for breast cancer. Six weeks later she noticed visual disturbance of the left eye which correlated with a visual field abnormality. There was a milder degree of visual disturbance in the right eye. Treatment with high-dose steroids partially stabilized the problem, which was felt to be an ischemic optic neuropathy. She ultimately died of respiratory failure. Pathology of the optic nerves revealed demyelination. Visual disturbances following high-dose chemotherapy are uncommon; the pathology to date has not been elucidated. Steroid therapy may be useful.

Progression of nonproliferative diabetic retinopathy following cataract extraction.

Eight patients experienced progression of nonproliferative diabetic retinopathy following cataract extraction. Six patients underwent an uncomplicated extracapsular cataract extraction with placement of a posterior chamber intraocular lens, and in two patients, surgery was complicated by vitreous loss. In each case the retinopathy progressed to a severe exudative form of diabetic macular edema, characterized by diffuse retinal thickening and fluorescein leakage with increased dot and blot hemorrhages and lipid deposition. In all patients, clinically significant macular edema developed in the eye that had been operated on, and six patients received laser photocoagulation for this condition. Final visual acuity was worse than preoperative visual acuity in six of eight patients, and it was unchanged in two of six patients. No patient achieved a visual acuity better than 20/50. The fellow eyes, which were not operated on, remained stable during the follow-up period.

Histopathologic features of adult-onset foveomacular pigment epithelial dystrophy.

We studied the clinical and histopathologic characteristics of the eyes obtained after death from a patient with adult-onset foveomacular pigment epithelial dystrophy. The pigmentation seen in the central fovea corresponded histologically to a hyperplastic clump of retinal pigment epithelium. The pale yellow rim surrounding the central pigmentation corresponded histologically to dense periodic acid-Schiff-positive material underlying thinned, atrophic retinal pigment epithelium. Fluorescence microscopy demonstrated homogeneous autofluorescence in the retinal pigment epithelium that was similar in intensity to that of an age-matched control. The results of this clinicopathologic study suggest that in adult-onset foveomacular pigment epithelial dystrophy, an alteration of macular retinal pigment epithelium causes an accumulation of abnormal subretinal pigment epithelial material, photoreceptor degeneration, and serous retinal detachment.

Age-related changes of the normal visual field.

We prospectively studied the age-related changes of the visual fields obtained from a selected group of 25 normal patients. The OCTOPUS automated perimeter was used to test both eyes of each patient using Program 32. We calculated the mean threshold sensitivity, volume, and surface area of the visual field and measured a linear decline with age for all three characteristics. The age-related decline in threshold sensitivity and the SE of this decline increased with eccentricity from fixation. Sensitivity declined approximately twice as rapidly at 30 degrees eccentricity as it did at fixation. The general decline in sensitivity of the visual field and the increased rate of decline with eccentricity may be related to a functional or anatomic loss of photoreceptors, ganglion cells, and higher structures.

Ferromagnetic hyperthermia and iodine 125 brachytherapy in the treatment of choroidal melanoma in a rabbit model.

Hyperthermia has been combined with conventional radiation methods to achieve enhanced tumor destruction. We combined iodine 125 seeds with ferromagnetic thermoseeds in a single plaque to simultaneously deliver radiation and heat in a rabbit model of choroidal melanoma. Initially, six ferromagnetic thermoseeds were placed in parallel on a 14-mm episcleral plaque. The plaques were placed on normal rabbit eyes and on eyes containing transvitreally implanted choroidal melanoma. The heating response was assessed in both normal and tumor-containing eyes. Rigid copper-constantan and flexible Baily thermocouples were used to monitor temperature responses. The animals were subjected to an electromagnetic field of 100 kHz, with power of 1100 to 1500 W. The thermoseeds autoregulated at 48.2 degrees C. Scleral temperatures stabilized at 45.8 degrees C +/- 0.4 degrees C (SD), while temperatures at the base of the tumor stabilized at 43.6 degrees C +/- 0.1 degrees C. Ferromagnetic thermoseeds were then combined with iodine 125 seeds. Similar temperature responses were recorded, and autoradiographic findings confirmed a uniform radiation distribution. Varying the amount or type of ferromagnetic material in the thermoseeds allows the delivery of heat at virtually any temperature. Ferromagnetic hyperthermia may provide a more simplified approach over currently available methods of heat delivery.

Intravitreal clearance of tissue plasminogen activator in the rabbit.

The clearance of tissue plasminogen activator (t-PA) injected into the midvitreous cavity was studied in the phakic, vitrectomized rabbit eye with and without intravitreal fibrin clots. The quantity and activity of t-PA in the vitreous, serum, and aqueous were determined at ten minutes and at 3, 6, 15, 24, and 48 hours after initial injection by an enzyme-linked immunosorbent assay (ELISA) and a spectrophotometric solid-phase fibrin assay (SOFIA). In eyes without an intravitreal fibrin clot, the estimated half-life for t-PA was 4.3 hours by SOFIA and 5.8 hours by ELISA. In eyes containing a vitreal fibrin clot, the half-life increased to 9.8 hours by SOFIA and 11.9 hours by ELISA. Both of these half-lives were significantly greater than the half-life for eyes without fibrin. Regardless of the presence of fibrin, intravitreal t-PA activity was significantly less than t-PA quantity, suggesting the presence of a t-PA inhibitor. A peak in aqueous t-PA occurred before six hours, indicating that t-PA was cleared in part through the anterior chamber. There was no measurable serum t-PA at any of the sampling times.

Antitransferrin receptor immunotoxin inhibits proliferating human retinal pigment epithelial cells.

Cultured human retinal pigment epithelial cells were exposed to an immunotoxin composed of a monoclonal antibody, 454A12, directed against transferrin receptors conjugated to a toxin, recombinant ricin A chain. Exposure of proliferating human retinal pigment epithelial cells to the immunotoxin (0.1 to 10,000 ng/mL) caused a statistically significant (P less than .0001) decrease in the number of cells. This inhibitory effect was induced after an exposure to the immunotoxin as short as 5 minutes and was maximal after 24 hours of exposure. The diminution in cell number was dose dependent over the range from 0.1 to 100 ng/mL. Monoclonal antibody alone, recombinant ricin A chain alone, or an irrelevant immunotoxin, MOP21C monoclonal antibody-recombinant ricin A, did not diminish the number of cells. There was a marked decrease in DNA synthesis measured by nuclear tritiated thymidine incorporation that accompanied the immunotoxin-mediated decrease in cell number. Viable cells remaining after exposure to the immunotoxin (0.1 to 10,000 ng/mL) were morphologically abnormal; typically the cells had elongated spindle-shaped processes and had lost their normal cuboidal appearance. In contrast, cell number was not decreased in confluent human retinal pigment epithelial cells after treatment with maximal doses of immunotoxin. Morphologic changes similar to those seen in proliferating cells were observed in confluent cells exposed to more than 100 ng/mL of immunotoxin. The effect of the immunotoxin was species specific because large doses of immunotoxin did not reduce the number of viable cells in proliferating or confluent pig retinal pigment epithelial cells or cause observable morphologic changes in this cell type. Our results indicate that the immunotoxin selectively inhibited proliferating retinal pigment epithelial cells by receptor-mediated internalization of the antitransferrin receptor monoclonal antibody-recombinant ricin A chain conjugate.

Photoablation of ocular melanoma with a high-powered argon endolaser.

We studied the use of a 15-W argon blue-green laser in the treatment of choroidal melanoma in a rabbit model. Greene melanoma cells were used to produce 2- to 4-mm thick tumors posteriorly in the suprachoroidal space in pigmented rabbits. Endophotocoagulation delivered through a 600-micron fiberoptic probe was performed to ablate the tumor tissue and a surrounding margin of normal tissue. A vitreous cutter was used simultaneously to remove liberated necrotic debris. The effect of the laser on tumor and normal ocular tissue was evaluated by light microscopy and the extent of the proliferative response by tritiated thymidine radioautography. Application of 100 to 400 pulses of laser energy using treatment parameters of 12 to 14 W of power and 0.1-s pulses resulted in complete ablation of melanoma tissue, overlying retina, and choroid. There was no substantial intraoperative or postoperative hemorrhage. Material liberated during the laser treatment was found to be nonviable. The effect of the laser on tissue appeared localized to within approximately 1.25 mm of the margin of the central lesion. The high-energy argon laser seems to offer a means of effectively ablating melanoma tissue via an internal resection approach.