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Gyrate atrophy is a progressive blindness associated with deficiency of ornithine aminotransferase (OAT). The strategy of using an autologous keratinocyte graft, modified to express high levels of OAT as an ornithine-catabolizing skin-based enzyme sink, is investigated. Two OAT-containing retroviral vectors were constructed with or without a resistance gene. When packaged in a retroviral vector particle generated with the gibbon ape leukemia (GALV) virus envelope (PG13), these vectors could readily transduce50% of target keratinocytes. The transduced keratinocytes in culture expressed up to 75-fold more OAT than normal control keratinocytes and these gene-modified cells extracted [14C]ornithine more efficiently than controls. The vector prepared without neo transduced cells more efficiently and led to higher levels of OAT expression than the neo-containing vector. Ornithine catabolism was maintained at high levels when the transduced patient keratinocytes were differentiated in vitro as a multilayered cutaneous organoid.

P2 nucleotide receptor expression in cultured human retinal pigment epithelial (RPE) cells was investigated using the photoaffinity ATP analog BzATP, polymerase chain reaction of reverse-transcribed RNA (RT-PCR) and fura-2 fluorescence measurement of changes in intracellular free calcium concentration ([Ca2+]i). In experiments carried out in RPE cells at passage 10-15, addition of micromolar concentrations of ATP, UTP, and ATPgammaS to RPE cells resulted in a rapid, transient 3.5-fold increase in [Ca2+]i followed by a prolonged elevation that was twofold above the original baseline. Similar results were obtained from cells at passage 2. Characteristics of nucleotide-stimulated calcium mobilization in RPE cells, including partial inhibition by pertussis toxin, suggest that a G protein-coupled receptor mediates this response. Consistent with the expression of a P2Y2 nucleotide receptor subtype in RPE cells, [alpha-32P]BzATP labeled a 53-kDa protein in plasma membranes, and RT-PCR revealed the presence of P2Y2 receptor RNA. Adenosine had no effect on [Ca2+]i in RPE cells, indicating that the A2 subtype of P1 receptor described previously in human RPE is not involved in the response to nucleotides. Together the results indicate that human RPE cells express functional P2Y2 nucleotide receptors.

Gene therapy may become a powerful therapeutic strategy. However, the application of this method in the treatment of ocular disease presents us with interesting and unique questions. Gene therapy for ocular inflammatory disease has the potential for both therapeutic interventions and a method for studying mechanism of disease. An evolving philosophy on this subject would support the use of somatic gene therapy for ocular inflammatory disease, even if not life threatening. Major technical questions remain, including the use of the appropriate vector, the best methodology for the stable insertion into the genome, and the duration and intensity of expression of the transgene. Various transgenes encoding a wide variety of proteins can be envisaged for the insertion of genes. The study of gyrate atrophy, an hereditary ocular disorder and an excellent candidate for gene therapy, has given us enormous information in the development of practical therapeutic strategies, as have in vitro studies of gene insertion. Future concerns will need to concentrate on the use of better methods for gene insertion and homologous recombination techniques for the development of animal models and later as a strategy for gene therapy. The use of gene therapy as a drug delivery system must also be considered. In addition, the elucidation of the various events controlling transcription for the expression of transgenes in various resident ocular cells is necessary.

Purpose: To study the effect of acetazolamide on cystoid macular edema in patients with uveitis. Methods: Forty patients with chronic intermediate, posterior, or panuveitis associated cystoid macular edema were randomized into a masked, cross-over trial comparing acetazolamide versus placebo. Patients received an initial 4-week course of either acetazolamide or placebo (course A) followed by a 4-week washout period. They then received a 4-week course of the opposite study medication (course B). Primary endpoints included area of cystoid macular edema measured on late-phase views of fluorescein angiography and visual acuity. Results: Thirty-seven patients completed the trial and were available for analysis; 17 (46%) were randomized to receive acetazolamide and 20 (54%) to receive placebo during course A. Acetazolamide resulted in a 0.5-disc area (25%) decrease in cystoid macular edema over that of placebo ( P = 0.01; estimated treatment effect=-0.5 disc areas; 95% confidence interval, -0.9 to -0.1 ). However, there was no statistically significant effect of acetazolamide on visual acuity ( P = 0.61; estimated treatment effect=0.6 letters; 95% confidence interval, -2 to 3). Conclusions: A 4-week course of acetazolamide therapy results in a statistically significant but small decrease in cystoid macular edema in patients with chronic uveitis, and does not improve visual acuity. In contrast to previous studies in the literature, acetazolamide may have a more limited clinical benefit in patients with long-standing cystoid macular edema associated with chronic uveitis.

The participation of growth factors in wound healing and tissue repair has been well established. Previous studies demonstrated that the expression of keratinocyte growth factor (KGF) was greatly elevated shortly after injury and that topical application of KGF accelerated healing. Steroidal antiinflammatory agents, specifically glucocorticoids, markedly impair wound healing. The participation of KGF in wound healing led us to examine the effect of glucocorticoids on KGF production. The addition of dexamethasone significantly reduced the level of constitutively produced KGF messenger RNA, protein, and bioactivity in conditioned medium from dermal fibroblasts. This inhibitory effect was observed with a variety of glucocorticoids, whereas nonsteroidal antiinflammatory compounds had little effect on KGF synthesis. The mechanisms by which dexamethasone decreased KGF production include a combination of a diminished transcriptional rate and destabilization of the KGF messenger RNA. Cytokines such as interleukin-1 alpha, platelet-derived growth factor-BB, and transforming growth factor-alpha, typically up-regulated during wound healing, augment KGF expression by dermal fibroblasts. We determined that dexamethasone also blocked this inductive effect. These results suggest that glucocorticoids could inhibit KGF production in the setting of wound repair, which may contribute to the impairment of healing associated with glucocorticoid use.

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates cell proliferation, motility, and morphogenesis by activation of its receptor, the c-Met tyrosine kinase. HGF/SF is structurally related to plasminogen, including an amino-terminal hairpin loop, four kringle domains, and a serine protease-like region. A truncated HGF/SF isoform, designated HGF/NK2, which extends through the second kringle domain and behaves as a competitive HGF/SF antagonist, was previously shown to be encoded by an alternative HGF/SF transcript. In this study, we describe a second naturally occurring HGF/SF variant, HGF/NK1, consisting of the HGF/SF amino-terminal sequence and first kringle domain. This product is encoded by a 2-kilobase alternative transcript containing intronic sequence that was contiguous with exon K1b. Analysis of baculovirus-expressed HGF/NK1 revealed that this isoform possesses the heparin binding properties of HGF/SF and modest mitogenic and scattering activity relative to HGF/SF. However, at a 40-fold molar excess, HGF/NK1 inhibited HGF/SF-dependent DNA synthesis. HGF/NK1 stimulated tyrosine phosphorylation of Met, and covalent affinity cross-linking demonstrated a direct HGF/NK1-receptor interaction. These findings establish that the HGF/SF gene encodes multiple alternative products, which include not only a mitogenic agonist (HGF/SF) and a pure antagonist (HGF/NK2) but also a molecule with partial agonist/antagonist properties.

We investigated the expression and distribution of keratinocyte growth factor (KGF) (FGF-7) and its receptor (KGFR) during reepithelialization of human skin. KGF mRNA levels increased rapidly by 8-10-fold and remained elevated for several days. In contrast, KGFR transcript levels decreased early but were significantly elevated by 8-9 d. A KGF-immunoglobulin G fusion protein (KGF-HFc), which specifically and sensitively detects the KGFR, localized the receptor to differentiating keratinocytes of control epidermis, but revealed a striking decrease in receptor protein expression during the intermediate period of reepithelization. Suramin, which blocked KGF binding and stripped already bound KGF from its receptor, failed to unmask KGFRs in tissue sections from the intermediate phase of wound repair. The absence of KGFR protein despite increased KGFR transcript levels implies functional receptor downregulation in the presence of increased KGF. This temporal modulation of KGF and KGFRs provides strong evidence for the functional involvement of KGF in human skin reepithelialization.

We examined three affected members of a Chinese-American family with Bietti's crystalline retinopathy. The clinical characteristics of a 24-year-old proband are contrasted to the clinical findings of her grandmother, for whom we have 26 years of follow-up data. Lymphocytes and fibroblasts from a skin biopsy of the grandmother contained crystalline lysosomal material, which supports the diagnosis. Biochemical studies of the crystalline lysosomal material failed to identify the stored compounds but did not show them to be cholesterol or cholesterol ester. Finally, histopathologic studies performed for this condition demonstrated advanced panchorioretinal atrophy, with crystals and complex lipid inclusions seen in choroidal fibroblasts.

Keratinocyte growth factor (KGF) is a stromally derived member of the fibroblast growth factor family (FGF7) with potent mitogenic activity on a variety of epithelial cells. To identify molecules that regulate the expression of this paracrine mediator of epithelial proliferation, we investigated the effects of various cytokines and growth factors on KGF production by fibroblasts. The proinflammatory cytokine interleukin 1 (IL1) strongly induced the expression of KGF RNA in fibroblasts from multiple sources. Platelet-derived growth factor BB, IL6, and transforming growth factor alpha caused a moderate elevation, while tumor necrosis factor alpha and basic FGF did not alter the level of KGF RNA expression. The induction by IL1 of KGF transcript levels was time and dose dependent and specifically blocked by anti-IL1 antibodies. Nuclear run on experiments indicated that IL1 stimulated KGF gene transcription. Western blot analysis and keratinocyte proliferation assays demonstrated a corresponding increase in mitogenically active KGF protein in conditioned medium obtained from IL1-treated fibroblasts. The stimulation of KGF expression by IL1 and other cytokines such as IL6, transforming growth factor alpha, and platelet-derived growth factor may provide a mechanism for KGF induction during inflammation that would support its proposed role as mediator of reepithelialization and wound healing.

In vitro studies have shown that keratinocyte growth factor (KGF, also known as FGF-7) is secreted by fibroblasts and is mitogenic specifically for epithelial cells. Therefore, KGF may be an important paracrine mediator of epithelial cell proliferation in vivo. Because stromal cells are thought to influence glandular proliferation in the primate endometrium, we investigated the hormonal regulation and cellular localization of KGF mRNA expression in the rhesus monkey uterus. Tissues were obtained both from naturally cycling monkeys in the follicular and luteal phases of the cycle, and from spayed monkeys that were either untreated or treated with estradiol (E2) alone, E2 followed by progesterone (P), E2 plus P, or E2 plus P plus an antiprogestin (RU 486). Northern blot analysis of total RNA with 32P-labeled probes revealed that the level of KGF mRNA in the endometrium was 70-100-fold greater in the luteal phase or after P treatment than in untreated, E2-treated, or follicular phase animals. Northern analysis also showed that KGF mRNA was present in the myometrium but was unaffected by hormonal state. RU 486 treatment prevented the P-induced elevation of endometrial KGF mRNA. P-dependent elevation of endometrial KGF expression was confirmed by measurement of KGF protein in tissue extracts using a two-site enzyme-linked immunosorbent assay. In situ hybridization with nonradioactive digoxigenin-labeled cDNA probes revealed that the KGF mRNA signal, which was present only in stromal and smooth muscle cells, was substantially increased by P primarily in the stromal cells located in the basalis region. Smooth muscle cells in the myometrium and the walls of the spiral arteries also expressed KGF mRNA, but the degree of this expression did not differ with hormonal state. P treatment led to increased proliferation in the glandular epithelium of the basalis region and to extensive growth of the spiral arteries. We conclude that the P-dependent increase in endometrial KGF resulted from a dual action of P: (a) a P-dependent induction of KGF expression in stromal cells, especially those in the basalis (zones III and IV), and (b) a P-dependent increase in the number of KGF-positive vascular smooth muscle cells caused by the proliferation of the spiral arteries. KGF is one of the first examples in primates of a P-induced, stromally derived growth factor that might function as a progestomedin.

The development of new drugs and drug delivery devices for the treatment of posterior eye diseases is critically dependent on the potential for that drug to be approved by the United States Food and Drug Administration (FDA). This approval process is predicated on the successful achievement of endpoints in large multicenter clinical trials. This chapter will discuss the history and evolving nature of endpoints for these clinical trials. Updates on recent novel endpoints will be discussed as well as the potential for the use of readouts from various imaging tools of the retina as FDA acceptable endpoints for clinical trials.

Epithelial-mesenchymal interactions mediate aspects of normal lung growth and development and are important in the restoration of normal alveolar architecture after lung injury. To determine if fibroblasts are a source of soluble growth factors for alveolar type II cells, we investigated the effect of fibroblast-conditioned medium (CM) on alveolar type II cell DNA synthesis. Serum-free CM from confluent adult human lung fibroblasts was concentrated fivefold by lyophilization. Type II cells were isolated from adult rats by elastase dissociation and incubated with [3H]thymidine and varying dilutions of concentrated CM and serum from day 1 to 3 of culture. Stimulation of type II cell DNA synthesis by fibroblast-CM was maximal after 48 h of conditioning and required the presence of serum. The activity of the CM was eliminated by boiling and by treatment with trypsin, pepsin, or dithiothreitol and was additive with saturating concentrations of acidic fibroblast growth factor, epidermal growth factor, and insulin. The growth factor activity bound to heparin-Sepharose and was eluted with 0.6 and 1.0 M NaCl. Neutralizing antibody studies demonstrated that the primary mitogens isolated in the 0.6 and 1.0 M NaCl fractions were keratinocyte growth factor (KGF, fibroblast growth factor 7) and hepatocyte growth factor/scatter factor (HGF/SF), respectively. HGF/SF was demonstrated in the crude CM and KGF was detected in the 0.6 M NaCl eluent by immunoblotting. Northern blot analysis confirmed that the lung fibroblasts expressed both KGF and HGF/SF transcripts. Human recombinant KGF and HGF/SF induced a concentration- and serum-dependent increase in rat alveolar type II cell DNA synthesis. We conclude that adult human lung fibroblasts produce at least two soluble heparin-binding growth factors, KGF and HGF/SF, which promote DNA synthesis and proliferation of rat alveolar type II cells in primary culture. KGF and HGF/SF may be important stimuli for alveolar type II cell proliferation during lung growth and after lung injury.

PURPOSE The goal of this study was to examine elimination pathways when delivering subconjunctivally administered hydrophilic agents to the retinas of rat eyes. METHODS The distribution of sodium fluorescein released from an episcleral implant was compared in live and postmortem eyes. Elimination of the subconjunctivally administered hydrophilic agent IgG through blood and lymphatic vessels was investigated by immunohistochemistry. Additionally, lymphatic elimination of subconjunctivally injected sodium fluorescein was quantitatively evaluated. RESULTS NaFl released from an episcleral implant was successfully delivered to the subretinal space in the postmortem eye but failed to do so in the live eye. Immunohistochemical visualization of the conjunctival tissue demonstrated dense distribution of blood and lymphatic vessels while also confirming the elimination of subconjunctivally administered IgG through these same vessels. The lymphatic elimination rate after injection of 75.6 μg of a hydrophilic agent, sodium fluorescein, into the subconjunctival space was determined to be 105 ng/min between 30 and 60 minutes. CONCLUSIONS Conjunctival blood and lymphatic vessel elimination considerably limit transscleral hydrophilic drug delivery to the retina.

Choroidal neovascularization (CNV) is the major cause of severe vision loss in patients with age-related macular degeneration (AMD). Present drug delivery may be limited by poor delivery to the choroid where CNV originates. The goal of this study was to develop a drug delivery system to deliver an integrin-antagonist peptide to the sub-retinal space. We developed polylactic acid/polylactic acid-polyethylene oxide nanoparticles (PLA/PLA-PEO) encapsulating the water-soluble integrin-antagonist peptide, C16Y (C16Y-NP). The PLA/PLA-PEO nanoparticles were 302+/-85.1 nm in size and demonstrated a two-week sustained release, in vitro, of encapsulated C16Y. Injected nanoparticles did not demonstrate retinal toxicity as determined by histopathology. C16Y peptide solution or C16Y-NP was injected 5 or 9 days post laser photocoagulation. A single intravitreal injection of C16Y peptide and C16Y-NP solution at both 5 days and 9 days post laser photocoagulation statistically inhibited CNV (p

Purpose To evaluate potential safety risks associated with nonspecific inhibition of vascular endothelial growth factor (VEGF). Design A perspective, reviewing the current literature. Methods Herein, we discuss the systemic safety of VEGF-targeted therapies, address safety issues for VEGF-targeted therapies in neovascular age-related macular degeneration, and propose the consideration of methods for identifying low rate systemic safety signals from patients treated with these agents. Results Several prospective, randomized clinical trials have demonstrated that intravitreal anti-VEGF therapies generally are well tolerated. However, within these trials, there is some circumstantial evidence that links systemic VEGF inhibition to systemic adverse events, particularly systemic thromboembolic events. Because all of the intravitreal anti-VEGF agents have been associated with detectable levels in the systemic circulation, there is a scientific rationale for the occurrence of potential systemic adverse events. However, if safety issues are present, they occur at very low rates and may go undetected in controlled clinical trials of premarketed drugs. Conclusions We propose that highly sensitive methodologies be put into place for identifying low rate safety signals, including postmarketing clinical trials, chart reviews, electronic medical records, and various national and international registries and databases, to evaluate the systemic safety of antiangiogenic agents in ocular diseases such as neovascular age-related macular degeneration.

The neonatal Fc receptor (FcRn, FcRp, or Fcgrt) is a heterodimer composed of major histocompatibility complex (MHC)-I, which binds to both albumin and the Fc portion of immunoglobulin G (IgG).1,2 Unlike other Fcγ receptors that bind to the lower hinge region and top of the Cγ2 domain, the FcRn receptor binds to the interface between Cγ2 and Cγ3 of IgG.2,3 The binding site of the FcRn receptor to the Fc fragment contains several histidine residues that account for a pH-dependent interaction.2 This enables the FcRn receptor to transport IgG(s) into the blood by transcytosis, allowing intact IgG molecules to pass through cells and into the systemic circulation. This binding of FcRn to the Fc fragment also aids in increasing the circulating half life of IgG by allowing temporary sequestration in vessel endothelium and protection from catabolism and elimination. Several studies have detailed this transcytosis pathway. First, IgG is pinocytosed nonspecifically by the cell and trafficked to the acidic endosome, where (in the low-pH environment) it binds FcRn with high affinity. FcRn then diverts IgG from a degradative lysosomal fate and transports the IgG back to the cell surface, where (at a neutral pH) it releases IgG into the extracellular space.1 The FcRn receptor was first identified in rodents as the receptor that transfers maternal IgGs from mother to young via the neonatal intestine.4 The mammary gland and placental endothelium also express the FcRn receptor which mediates the transfer of protective immunoglobulins to the newborn.5,6 In addition, it is important to note that the FcRn receptor is expressed in the brain microvasculature and the choroid plexus epithelium.7 Transcytosis of IgG from the brain to blood has been shown to use the FcRn receptor located on brain vascular endothelium.8,9 FcRn receptors are also expressed in mammalian adult endothelial cells, hepatocytes, histiocytes, monocytes, intestinal macrophages, dendritic cells, bronchial epithelial cells, kidney epithelial cells, and adipose tissue.5,10-14 However, FcRn receptor distribution in the eye has not yet been determined. Accordingly, the purpose of this study was to determine whether expression of the FcRn receptor occurs in various ocular tissues.

Objective To report the epidemiology and ocular phenotype of retinal capillary hemangioblastomas associated with von Hippel–Lindau (VHL) disease in a large cohort of patients and to correlate patient and ocular characteristics to visual morbidity in this population. Design Cross-sectional study. Participants In 220 unrelated pedigrees, 335 patients affected with VHL disease and retinal capillary hemangioblastomas (RCHs) in at least 1 eye. Methods Demographics of the patient population were recorded and the ocular phenotype of each patient was obtained with a comprehensive ocular examination. Main Outcome Measures The patient population was characterized and the ocular phenotype described in relationship to tumor location, number, and extent of retinal involvement. Correlations between patient demographics, ocular phenotype, and visual function were analyzed. Results We detected RCHs unilaterally in 42.1% and bilaterally in 57.9% of patients. No correlation was detected between the age, gender, or laterality of involvement. Of involved eyes, 86.6% had tumors that could be individually visualized; of these, tumors were commonly found in the peripheral retina (84.7%) only, and less commonly in the juxtapapillary area (15.3%). The tumor count in the periphery averaged 2.5±1.8 per eye, with 25.2% of eyes having >1 quadrant of retinal involvement. Of involved eyes, 13.4% were enucleated or prephthsical; approximately 1 in 5 patients had ≥1 eyes so affected. Severe visual impairment (visual acuity ≤20/160) in affected eyes were more likely to be associated with increasing age, the presence of juxtapapillary lesions, and an increasing number and extent of peripheral lesions. Conclusions This large cohort of VHL patients with RCHs has enabled a systematic and quantitative characterization of the demographics, ocular features, and visual function in VHL disease. Clinical correlations between the visual morbidity and ocular features of the disease were also performed, producing measures that can help clinicians to estimate visual prognoses better based on the ocular phenotype of the disease.

To determine the elimination rates of subconjunctivally injected model drugs using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI).Gadolinium-diethylenetriaminopentaacetic acid (Gd-DTPA) and gadolinium-albumin (Gd-albumin) were injected in rabbits. Experiments were performed in vivo and post mortem and injection volumes of 200 and 600 microl were administered. Signal intensity values from MR images were converted to concentration of contrast agent to determine the mass clearance rates from subconjunctival space.Injection volume did not have a significant effect on clearance rate for both Gd-DTPA and Gd-albumin. The clearance rate of Gd-DTPA in vivo was about nine times faster than that post mortem. The in vivo and post mortem clearance rates of Gd-albumin were not significantly different. The in vivo half-life of Gd-DTPA was about 22 min while that of Gd-albumin was about 5.3 h.DCE-MRI was used to quantitatively compare the subconjunctival clearance rates of Gd-DTPA and Gd-albumin. Dynamic clearance mechanisms present in vivo significantly reduced the subconjunctival concentration of Gd-DTPA but not Gd-albumin. Lymphatic clearance does not seem to be as significant as clearance by blood, as evidenced by data from Gd-albumin injections. Larger injection volumes may allow for longer retention times and prolonged release of drug.

Objectives To characterize the germline mutations found in a large population of persons having von Hippel-Lindau (VHL) disease mutations with the clinical characteristics of associated retinal capillary hemangioblastomas (RCHs),to measure the prevalence of RCHs among patients with VHL disease generally and specifically for each genotype category, to establish genotype-phenotype correlations between genotype category and phenotypic features of ocular VHL disease, and to establish genotype-phenotype correlations between genotype category and visual function. Methods Cross-sectional and molecular genetic study. Of 890 patients with VHL disease, 335 had ocular involvement in the form of RCHs. Statistical analysis was used to correlate the structure of the mutated VHL protein with the ocular phenotype. Results Three genotype categories (amino acid substitutions, protein-truncating mutations, and complete deletions of VHL protein) were defined in all patients.The prevalence of RCHs was lowest (14.5%) among patients with complete deletions;the overall prevalence of retinal angiomatosis was 37.2%. Genotype category had no correlation with the unilaterality or bilaterality of ocular disease or with the number or extent of peripheral RCHs. The prevalence of RCHs at the juxtapapillary location was lower among patients with protein-truncating mutations compared with those with amino acid substitutions. Complete deletions were associated with the highest mean visual acuity compared with the other 2 genotype categories. Conclusion Patients with complete deletions of VHL protein have the lowest prevalence of ocular disease and the most favorable visual outcome. Clinical Relevance The VHL mutation genotype may be used to predict the prevalence and outcome of ocular VHL disease and to guide ophthalmic follow-up.

Purpose Sustained-release intravitreal drug implants for posterior segment diseases are associated with significant complications. As an alternative, subconjunctival infusions of drug to the episclera of the back of the eye have been performed, but results in clinical trials for macular diseases showed mixed Results To improve understanding of transscleral drug delivery to the posterior segment, the distribution and clearance of gadolinium-diethylene-triamino-penta-acetic acid (Gd-DTPA) infused in the subconjunctival or intrascleral space was investigated by means of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Methods In anesthetized rabbits, catheters were placed anteriorly in the subconjunctival or intrascleral space and infused with Gd-DTPA at 1 and 10 muL/min. Distribution and clearance of Gd-DTPA were measured using DCE-MRI. Histologic examination was performed to assess ocular toxicity of the delivery system. results. Subconjunctival infusions failed to produce detectable levels of Gd-DTPA in the back of the eye. In contrast, intrascleral infusions expanded the suprachoroidal layer and delivered Gd-DTPA to the posterior segment. Suprachoroidal clearance of Gd-DTPA followed first-order kinetics with an average half-life of 5.4 and 11.8 minutes after intrascleral infusions at 1 and 10 muL/min, respectively. Histologic examination demonstrated expansion of the tissues in the suprachoroidal space that normalized after infusion termination. Conclusions An intrascleral infusion was successful in transporting Gd-DTPA to the posterior segment from an anterior infusion site with limited anterior segment exposure. The suprachoroidal space appears to be an expandible conduit for drug transport to the posterior segment. Further studies are indicated to explore the feasibility of clinical applications.

Purpose To determine the feasibility, safety, and effectiveness of an episcleral or deep scleral lamellar sustained release cyclosporine (CsA) device in a naturally occurring animal model of uveitis. Methods A two-compartment perfusion chamber was used to assess in vitro human and equine scleral permeability of fluorescein, dexamethasone-fluorescein, or CsA. A biodegradable, matrix-reservoir CsA implant was designed, and release rates of CsA were determined in vitro. Tissue CsA levels were measured in eyes with the implant. Horses with equine recurrent uveitis (ERU) received episcleral or deep scleral lamellar CsA implants and were monitored for up to 3 years. Results Dexamethasone-fluorescein and CsA penetrated the in vitro equine sclera poorly; however, low but detectable levels of CsA were detected intraocularly in vivo. The implant placed episclerally failed to control inflammatory episodes in ERU. CsA implants placed in the deep sclera adjacent to the suprachoroidal space resulted in high levels of CsA in most ocular tissues. In clinical equine patients with ERU, frequency of uveitic flare-ups was significantly decreased after implantation of a deep scleral lamellar CsA implant. Conclusions Diffusion of CsA across the sclera from the episcleral space was not a feasible method of drug delivery to the equine eye. However, placing a deep scleral lamellar CsA implant adjacent to the suprachoroidal space was effective in achieving therapeutic ocular drug concentrations and controlling uveitis in horses with ERU.

In humans, the vascular endothelial growth factor (VEGF) gene resides on chromosome 6p21.3 (1). Northern blot and primer extension analysis have shown that the human VEGF gene has a single major transcription start site, 1038 bp upstream from the ATG initiation codon. This start site is located near a cluster of potential Sp1 factor binding sites, and the promoter contains potential binding sites for the transcription factors AP-1 and AP-2 (2). A hypoxia response element located upstream of the VEGF genes can bind hypoxia-inducible factor (HIF)-1 and may act as an enhancer (3,4). Hypoxia can also increase the half-life of VEGF mRNA, which is intrinsically labile. Binding of HuR, a hypoxia-induced stability factor that is a member of the Elav-like protein family, to an AU-rich element in the 3′-UTR of VEGF mRNA increases the half-life of VEGF mRNA by three- to eightfold (5).

Purpose To evaluate the human transscleral diffusion and intravitreal delivery of carboxyamido-triazole (CAI) and 2-Methoxyestradiol (2ME2). Methods The transscleral diffusion of two retinal antiangiogenic molecules, CAI and 2ME2, was measured in vitro to assess their potential transscleral delivery. Varying concentrations and different solvents of CAI and 2ME2 were placed in the upper compartment of a two-chamber acrylic perfusion apparatus, on the episcleral side of the sclera obtained from human donor eyes. Samples were taken from the lower compartment (uveal side) for up to 24 hours and measured by high performance liquid chromatography. Results All three solutions that contained CAI efficiently diffused through the sclera with permeability constants that ranged from 2.8 to 5.5 x 10 cm/s. The scleral permeability constant derived for 2ME2 was 9.96 x 10 cm/s. The permeability constants obtained for both CAI and 2ME2 are similar to each other as well as to permeability constants measured for other small molecules such as fluorescein and dexamethasone fluorescein. Conclusion Both CAI and 2ME2 traverse the sclera efficiently. These data combined with the reported inhibition of posterior segment neovascularization observed with these two molecules demonstrates that CAI and 2ME2 are good candidate molecules to treat posterior segment neovascularization by local delivery.

Transscleral delivery of triamcinolone acetonide into the vitreous using sub-Tenon's injections may be a safer alternative to reduce the sight-threatening complications of direct intravitreal injections. However, sub-Tenon's injections have demonstrated low and poorly sustained vitreous drug levels in animal studies. To improve our understanding of the clearance mechanisms of corticosteroids, we evaluated vitreous drug levels following sub-Tenon's injection of triamcinolone acetonide in rabbits with selective elimination of conjunctival lymphatic/blood vessels and the choroid. Pigmented rabbits were given a sub-Tenon's injection of a preservative-free triamcinolone acetonide formulation of either a 10- or 20-mg dose in the superotemporal quadrant. The effect eliminating both conjunctival and choroidal clearance was evaluated by injecting the drug, followed by immediate euthanasia, effectively terminating both lymph and blood flow in the conjunctiva and choroid. To inhibit only the clearance from conjunctival lymphatics/blood vessels of a sub-Tenon's injection of triamcinolone acetonide, a group of rabbits had a 'conjunctival window' created by incising an 7 mmx7 mmx7 mm square through the conjunctiva to bare sclera in the superotemporal quadrant. To eliminate only the clearance of drug from the choroidal circulation, cryotherapy was performed in another group of rabbits creating a chorioretinal scar in the superotemporal quadrant. Following the sub-Tenon's drug injection, the eyes were enucleated in all groups after 3 hr and vitreous drug levels were measured with HPLC. In normal animals, a 10-mg sub-Tenon's injection showed no detectable vitreous drug levels; however, a 20-mg injection showed positive vitreous drug levels. This suggested that collectively, the transscleral clearance mechanisms inhibiting delivery into the vitreous may be saturated with a drug depot that has a higher release rate. A 10-mg sub-Tenon's drug depot was able to deliver drug into the vitreous when both the conjunctival and choroidal drug clearance was eliminated by euthanizing the animal immediately following the drug injection. In rabbits that had only a 'conjunctival window', selectively eliminating conjunctival drug clearance, vitreous drug levels were detected. However, in rabbits that had only cryotherapy, selectively eliminating choroidal drug clearance, vitreous drug levels were not detected suggesting that the conjunctival lymphatics/blood vessels may be an important barrier to the transscleral delivery of triamcinolone acetonide. Variability in the vitreous drug levels between rabbits in each group precluded statistical testing. In summary, the rabbit appeared to demonstrate saturable ocular barriers to transscleral delivery of triamcinolone acetonide into the vitreous following a sub-Tenon's injection. The results suggested that the conjunctival lymphatics/blood vessels may be an important barrier to the delivery of triamcinolone acetonide to the vitreous in this rabbit model. The barrier location and clearance abilities of the ocular tissues are important to consider when developing a successful transscleral drug delivery system. Animal models, retaining the dynamics of blood and lymph flow, may improve the basic understanding of the ocular barriers involved with transscleral drug transport and warrants further investigation.

Purpose To develop a local drug delivery system that provides therapeutic cyclosporine levels to treat lacrimal gland graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Methods Episcleral cyclosporine implants were manufactured with a silicone-based matrix design, and in vitro release rates were determined. Preclinical evaluation included toxicology (clinical examination, serial electroretinography, and histopathology) in normal rabbits and dogs, pharmacokinetics in normal rabbits, and pharmacodynamics in a canine model of aqueous tear deficiency and keratoconjunctivitis sicca. Results The cyclosporine implants showed sustained release of drug over time with in vitro assays. Histopathology showed normal ocular tissues in both dogs and rabbits 6 months after implantation. The cyclosporine implant produced lacrimal gland drug levels 1 to 2 log units higher than those reported with a variety of topical cyclosporine formulations and oral administration. The cyclosporine implant was effective in a canine model of keratoconjunctivitis sicca, with all animals able to discontinue topical cyclosporine and maintain normal Schirmer scores over a 6-month follow-up. Conclusions This preclinical evaluation showed that the episcleral cyclosporine implant was safe, delivered potentially therapeutic cyclosporine levels to the lacrimal gland, and showed efficacy in a clinically relevant model of keratoconjunctivitis sicca. The episcleral cyclosporine implant shows promise in reducing the morbidity associated with lacrimal gland graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. In addition, continuous release of cyclosporine in the subconjunctival space with the episcleral implant was an effective means of delivering drug to the ocular surface and may have potential in treating other ocular inflammatory diseases.

This study examined the association between the sequence variation/expression of CX3CR1, a chemokine receptor, and age-related macular degeneration (AMD). Peripheral blood from 85 AMD patients and 105 subjects without AMD (controls), as well as ocular tissue from 40 pathological sections with AMD and two normal eye sections, were screened for V249I and T280M, two single nucleotide polymorphisms (SNPs) in CX3CR1. An increased prevalence, with the highest odds ratio of 3.57, of the I249 and M280 carriers was found among the AMD cases as compared with the controls. When comparing CX3CR1 expression in the archived eye sections, CX3CR1 transcripts were not detectable in the maculae of AMD eyes bearing T/M280; however, transcripts were detected in the maculae of normal eyes bearing T/T280 or T/M280 as well as in the AMD maculae bearing T/T280. Furthermore, lower CX3CR1 protein expression was observed in the maculae of AMD eyes bearing T/M280 compared with the controls bearing T/T280. The I249 and M280 alleles result in a lowered number of receptor binding sites and a decreased ligand affinity. Our data suggest that a decrease, caused by sequence variation and/or lower CX3CR1 expression, in CX3CR1-induced cellular activities could contribute to AMD development.

Purpose. Feeder vessel photocoagulation using both thermal and indocyanine green (ICG) enhanced applications as a treatment for choroidal neovascularization is under investigation. While closure of feeder vessels is achievable, reperfusion of these vessels occurs. The purpose of the following study was to compare, contrast anatomic, and immunohistochemical findings in rat retinal arterioles following attempts at vessel closure using either the diode (810 nm) laser alone or in conjunction with intravascular ICG. Methods. The retinal arterioles of adult Lewis or Brown Norway rats were treated with diode laser alone or immediately following intravenous injection with 75 mg ml−1 ICG. Retinal vessel closure was determined by examination of retinal flatmounts following FITC-dextran or rhodamine-dextran perfusion. Anatomic changes were examined by electron microscopy and quantitative cellular changes were measured by perfusion with Hoechst 33342 nuclear staining. Recruited macrophages were detected by ED1 immunohistochemistry. Results. Treatment with diode laser alone resulted in partial retinal arteriolar closure seen only in pigmented animals. The use of adjuvant ICG achieved complete vessel closure in albino animals with reperfusion seen in all vessels by 7 days. Electron microscopy revealed an intraluminal clot only in ICG-enhanced diode laser treated animals, but with accompanying endothelial and perivascular cellular damage. Immunohistochemistry of the site of retinal arteriolar closure revealed a large increase in perivascular cellularity with an apparent influx of ED1 positive cells. Conclusion. ICG-enhanced diode laser photocoagulation appears to be superior to diode treatment alone in achieving vessel closure, but is limited by clot resolution due to both excessive vascular damage and an accompanying inflammatory response. These results suggest that more durable feeder vessel closure rates may be achievable with either the use of accompanying anti-inflammatory therapies or with a less vascular damaging photoactivating dye.

A retrospective chart review was performed on seven patients treated with topical ocular corticosteroid therapy for progressive cicatricial conjunctivitis associated with chronic graft-versus-host disease (GVHD) following hematopoietic stem cell transplantation. A clinical grading criteria for conjunctival GVHD based on the degree of cicatrization was developed and patients graded prior to therapy. During the treatment course, the dose and frequency of topical corticosteroids and clinical outcomes were recorded. A complete response was defined as a complete resolution of the conjunctival hyperemia with either total resolution of the conjunctival fibrovascularization or presence of inactive conjunctival scarring. Prednisolone acetate 1% eye drops were used in a total of eight courses of therapy in seven patients. A complete response was documented in all seven patients with a total treatment duration of 7 weeks (median, range: 3-16 weeks). Additional studies are required to determine the long-term safety and efficacy of topical corticosteroids for cicatricial conjunctivitis associated with ocular GVHD in the context of a randomized, prospective clinical trial.

Adenoviruses are viruses with genomes of double-stranded DNA approx 36 kb in size. There are more than 100 different serotypes that have been identified, 47 of them of human origin. Human serotypes have been grouped into six subgenera (A-F) and whereas sequence similarity between genera is low, other molecular biologic aspects of the viruses are almost identical. Of all the human adenoviruses, serotypes 2 and 5 (subgenus C) have been the most extensively studied (1).

PURPOSE. The pathogenesis of choroidal neovascularization (CNV) is postulated to be driven by angiogenesis, a process in which the cellular components of the new vessel complex are derived from cells resident within an adjacent preexisting capillary. Recently, an alternative paradigm, termed postnatal vasculogenesis, has been shown to contribute to some forms of neovascularization. In vasculogenesis, the cellular components of the new vessel complex are derived from circulating vascular progenitors from bone marrow. In the current study, transplantation of green fluorescent protein (GFP)‐labeled bone marrow and laser-induced CNV were combined to examine the contribution of vasculogenesis to the formation of CNV. METHODS. Ten adult C57BL/6 female mice were used as recipients for bone marrow transplantation. Bone marrow was obtained from three C57BL/6 female mice transgenic for the -actin promoter GFP. One month after bone marrow transplantation, CNV was induced in recipient mice by making four separate burns in the choroid of each eye with a red diode laser. Four weeks after CNV was induced, eyes of recipient mice were processed for immunohistochemistry to detect GFP and markers for vascular smooth muscle cells (-smooth muscle actin, desmin, and NG2 chondroitin sulfate proteoglycan), endothelial cells (CD31, BS-1 lectin), or macrophages (F4/80). RESULTS. GFP-labeled cells represented 17% of the total cell population in the lesion. Many of the GFP-labeled cells were immunoreactive for -smooth muscle actin (39%), desmin, NG2, CD31 (41%), BS-1 lectin, or F4/80. GFP-labeled cells were morphologically indistinguishable from cells normally present in CNV lesions. CONCLUSIONS. This study is the first to demonstrate that bone marrow‐ derived progenitor cells are a source of endothelial and smooth musclelike cells in CNV. (Invest Ophthalmol Vis Sci. 2003;44:4914 ‐ 4919) DOI:10.1167/iovs.03-0371

To evaluate the long-term natural history and effects of laser photocoagulation treatment in patients with diabetic retinopathy.Follow-up study of the 214 surviving patients enrolled originally at the Johns Hopkins Clinical Center for the Early Treatment Diabetic Retinopathy Study (ETDRS), which was a clinical trial designed to evaluate the role of laser photocoagulation and aspirin treatment in patients with diabetic retinopathy.Early Treatment Diabetic Retinopathy Study patients enrolled in the Johns Hopkins Clinical Center had complete eye examinations, including best-corrected visual acuity measurements, fundus photographs, and medical questionnaires throughout the 7-year study. They had the same examinations at the final long-term follow-up visit at the National Eye Institute, National Institutes of Health, 13 to 19.5 years after the initial laser photocoagulation (median, 16.7 years).The major outcomes were mortality and the rates of moderate and severe vision loss. The secondary outcomes were progression of diabetic retinopathy and need for other eye surgery.Of the 214 patients who were alive at the end of the original ETDRS in 1989, 130 (61%) were deceased at the time of the re-examination. Of the 84 who were alive, 71 (85%) were examined at their long-term follow-up visit at the National Institutes of Health. At the long-term follow-up examination, 42% had visual acuity of 20/20 or better, and 84% had visual acuity of 20/40 or better in the better eye. Compared with baseline, 20% of patients had moderate vision loss (loss of 3 lines or more vision) in the better eye at follow-up. Only one patient had visual acuity of 20/200 bilaterally. He had visual acuity loss secondary to age-related macular degeneration. No patient had severe vision loss (worse than 5/200). All the initially untreated eyes of patients who had severe nonproliferative diabetic retinopathy or worse by the time of the ETDRS closeout visit of the original study received scatter photocoagulation treatment. Focal photocoagulation was performed in 43% bilaterally and 22% unilaterally. Cataract surgery was performed in 31% of the patients, vitrectomy in 17%, and glaucoma surgery in one patient.As previously reported, the mortality rate of patients with diabetic retinopathy is much higher than that of the general population. For those who survived, aggressive follow-up, with treatment when indicated, seems to be associated with maintenance of good long-term visual acuity for most patients. The need for laser scatter photocoagulation with long-term follow-up seems to be high.

Purpose. The question of whether adult animals maintain a reservoir of endothelial progenitor cells (EPCs) in the bone marrow that is involved in neovascularization is under investigation. The following study was undertaken to examine the potential contribution of EPCs to the development of choroidal neovascularization (CNV) in adult mice and to examine the role of local expression of vascular endothelial growth factor (VEGF) in this process. Methods. Lethally irradiated, adult female nude mice were engrafted with whole bone marrow isolated from male transgenic mice expressing LacZ driven by the endothelial specific Tie-2 promoter. Two months, following bone marrow reconstitution, confirmed by quantitative Taqman PCR, an E1-deleted adenoviral vector expressing vascular endothelial growth factor 165 (Ad.VEGF165) was injected subretinally to induce CNV, confirmed by collagen IV immunohistochemistry. Bone marrow-derived endothelial cells were detected using either X-gal staining or Y chromosome in situ hybridization. Y chromosome positive cells within the CNV were confirmed to be endothelial cells by lectin staining. Results. Subretinal Ad.VEGF165 was capable of inducing CNV. Four-week old lesions were found to contain LacZ expressing cells within the CNV in bone marrow transplanted animals but not in negative control animals. Eighteen percent of all Y chromosome positive cells within the CNV were found to be lectin positive while 27% of all endothelial cells within the CNV were Y chromosome positive. Conclusion. Engrafted bone marrow-derived EPCs were shown to differentiate into endothelial cells at the site of subretinal VEGF-induced CNV in mice. These results suggest that EPCs contribute to the formation of neovascularization and that subretinal expression of VEGF might play an important role in recruitment of these cells to the site of CNV.

A patient with human immunodeficiency virus infection and cytomegalovirus (CMV) retinitis developed immune recovery uveitis as a result of receipt of highly active antiretroviral therapy. Fibrovascular changes occurred in the CMV retinitis scar, were misdiagnosed as CMV retinitis reactivation, and were treated with anti-CMV medication. Fibrovascular membranes can be misdiagnosed as reactivated CMV retinitis, and a proper diagnosis is essential to avoid unnecessary therapy with potentially toxic antiviral medications.

Our understanding of the morphogenesis of epithelial phenotypes has been greatly advanced by the use of in vitro cell culture systems. However, cell cultures often do not faithfully reconstitute many of the differentiated properties of the cell from which they are derived and cannot be used to examine complex physiologic interactions between adjacent tissues. This is particularly true of the retinal pigment epithelium (RPE). Many plasma membrane proteins, in vivo, exhibit a reversed polarity with respect to other epithelia, and RPE-derived cell lines seldom exhibit these same polarity properties. Furthermore, the interaction between the RPE cell and the neuorsensory retina, or the underlying blood supply, the choroid, is absent in cell culture. Most epithelia are difficult to isolate and study in vivo. The RPE is an exception to this. We have explored several aspects of RPE protein transport properties, vision-related physiology, and disease-related pathophysiology in the eye using in vivo gene transfer and electrophysiologic techniques. By injecting replication-defective adenoviruses into the subretinal space of rat eyes, we have been able to easily direct the expression of a test protein and follow its sorting and physiologic effects on RPE cells and adjacent tissues. Due to binding and internalization of adenoviral vectors to integrins found on the RPE apical plasma membrane, expression in a healthy eye is essentially confined to the RPE cell, even under control of a cytomegalovirus promotor. The use of varying amounts of adenoviral vector allows for determination of dose-responsive effects and the comparison of multiple mutants of a protein. In addition, there are substantial savings with respect to time and money in comparison to standard transgenic approaches.

We sought to investigate the role of aging as a susceptibility factor for the capacity of dietary fat intake to increase the development of subretinal deposits. Mice of various ages (2, 9 and 16 months) were fed a normal diet or a diet high in saturated and unsaturated fats for a total four and a half months. Some eyes were also exposed to non-phototoxic levels of blue-green light. The outer retina and choroid were examined by light and transmission electron microscopy, and the characteristics, frequency and severity of subRPE deposits was determined. Aged mice fed normal diets developed only very mild subretinal deposits. However, many eyes of mice aged 9 months or older at the time of initiation of diet developed frequent basal laminar deposits of moderate severity, and only 16 month old mice developed more severe deposits after exposure to blue-green light. Some eyes in this older group also developed endothelial invasion into Bruch's membrane. None of the eyes developed classic drusen or linear deposits. These observations demonstrate that age increases the capacity of dietary fat, especially in the presence of environmental light, to induce subRPE deposits.

PURPOSE: To report a patient in whom clear imaging of the retina, impossible with conventional methods, was obtained using indocyanine green (ICG) videoangiography. DESIGN: Interventional case report. METHODS: A 31-year-old patient with nephropathic cystinosis and complaints of decreased vision in the left eye underwent a complete ophthalmologic evaluation, including conventional photography, fluorescein angiography, and ICG videoangiography. RESULTS: With conventional methods (direct and indirect ophthalmoscopy, photography, and fluorescein angiography), the view of the left retina was obscured by densely packed corneal cystine crystals. During ICG angiography, clear imaging of the retina was obtained with near-infrared illumination. CONCLUSION: Indocyanine green videoangiography can be a useful tool in cases with cystinosis and other corneal opacities, where visualization and imaging of the retina are important but impossible with conventional methods.

For many vascular diseases, aging appears to be an independent risk factor for severity of vascular complications, and blood vessels of aged individuals often demonstrate exaggerated repair responses to injury. This study was undertaken to determine the influence of aging on the severity of neovascularization in a mouse model of laser-induced choroidal neovascularization (CNV).CNV was induced in young (2-month-old) and aged (16-month-old) C57BL/6 mice by making four separate choroidal burns in each eye with a diode red laser (650 nm). At 1, 2, and 4 weeks, the left eyes were removed for histopathology, and the right eyes were removed for flatmount analysis of CNV surface area, vascularity, and cellularity.Aged mice demonstrated a much larger area of CNV than did young mice (3.81 +/- 1.28 vs. 1.36 +/- 0.99 disc areas, P0.001) at 2 weeks, when the lesions showed maximum growth. Aged mice also demonstrated higher ratios for vascularity and cellularity of the CNV (1.34 +/- 0.06 vs. 1.03 +/- 0.11, P0.0001 and 4.06 +/- 1.19 vs. 1.91 +/- 0.81, P0.002 at 2 weeks, respectively). Histopathology revealed that CNV in older eyes was larger, thicker, and more cellular than in young eyes.In mice, age is associated with more severe CNV, defined as larger surface area, greater vascularity, and greater cellularity. Age-related systemic susceptibility factors, independent of local changes in the retina, may contribute to the greater severity of CNV in older than in younger individuals.

Choroidal neovascularization (CNV) is the leading cause of severe vision loss associated with age-related macular degeneration. As the pathogenesis of CNV formation is better understood, mechanism-based therapies, including the use of antiangiogenesis inhibitors, have been investigated. 2-methoxyestradiol (2ME2), an endogenous metabolite of estradiol, has been shown in the chick allantoic membrane model and the corneal micropocket assay to have antiangiogenic properties. The authors sought to determine the safety and pharmacokinetics of sustained-release intravitreal 2ME2 implants in normal rabbit and their efficacy in a rat model of CNV. 2ME2 implants were constructed using two designs: implant A, a silicone-based reservoir implant for the rabnbit eye, and implant B, a microimplant matrix design for the rat eye. In vitro release rates of both implants were determined. New Zealand white (NZW) rabbits had implant A placed in the vitreous cavity of one eye and the ocular toxicity was evaluated by clinical examination, serial electroretinography (ERG), and histopathology over a 28 week period. The steady state clearance of 2ME2 in the rabbit eye was calculated from in vivo release rates divided by steady state vitreous concentrations. A CNV model in the Brown-Norway rat was performed by injecting an adenoviral vector encoding human vascular endothelial growth factor in the subretinal space. Following the injection, a 2ME2 or sham (no drug) microimplant was placed in the vitreous cavity. Animals were killed over a 3 week period and the eyes examined for CNV by histopathology. Results showed that following a short burst, the release rate of implant A followed zero-order kinetics, typical of reservoir devices, and the cumulative release of implant B was proportional to the square root of time, as expected for a matrix delivery device. The safety studies in normal rabbit showed no ocular toxicities by clinical examination, ERG, and histopathology. Pharmacokinetic evaluation in the rabbit showed mean 2ME2 vitreous levels within the therapeutic range for the inhibition of endothelial cell proliferation. The experimental rat model showed a significant reduction in CNV in eyes treated with the 2ME2 implant. In conclusion, sustained-release 2ME2 intravitreal implants, which can be designed to deliver potentially therapeutic vitreous levels of 2ME2 for an extended period of time, appeared to be safe in normal rabbit and effective in a rat model of CNV. Sustained-release 2ME2 intravitreal implants may hold promise in the treatment of recurrent CNV refractory to standard therapy.

Primary intraocular lymphoma (PIOL), also known as primary central nervous system lymphoma, is a rare yet blinding and fatal disease. Often presenting with ocular involvement, it can masquerade as posterior or intermediate uveitis, thus delaying diagnosis. A noninvasive ancillary test such as fluorescein angiography could be helpful in raising the level of suspicion in the diagnosis of this disease.Results of fluorescein angiography (FA) and clinical characteristics of 17 patients (31 eyes) who presented to the National Eye Institute with the diagnosis of PIOL (confirmed by histopathologic analysis) were reviewed.The most common angiographic characteristics included disturbances at the level of the retinal pigment epithelium (RPE), such as granularity (19 eyes [61%]), blockage (17 eyes [55%]), and late staining (14 eyes [45%]). These changes are well correlated to histopathologic findings of lymphoma cells located between the RPE and Bruchs membrane. Perivascular staining or leakage and cystoid macular edema were rare. Other less common findings included pigment epithelial detachments and punctate hyperfluorescent lesions. Clinical characteristics found in eyes for which results of FA were available included vitreitis (29 eyes [94%]), subretinal infiltrates (19 eyes [61%]), and anterior chamber cells (10 eyes [32%]). In some cases, clinical examination did not correlate with FA findings.Although PIOL may present with a normal angiographic phenotype, extensive RPE changes demonstrated by FA, combined with the absence of perivascular staining or leakage and macular edema, may be associated with and distinctive of PIOL.

PURPOSE: To determine the association between cystoid macular edema and vascular endothelial growth factor concentration in the aqueous humor and plasma of uveitis patients. METHODS: Cross-sectional study. Vascular endothelial growth factor concentrations were measured by enzyme-linked immunosorbent assay in the aqueous humor of 20 uveitis patients (9 with and 11 without cystoid macular edema), and in the plasma of 40 uveitis patients (20 with and 20 without cystoid macular edema) and 20 healthy volunteers. RESULTS: Mean aqueous humor vascular endothelial growth factor concentrations for uveitis patients with and without cystoid macular edema were 152.3 and 109.5 pg/ml, respectively, P = .044. Mean plasma vascular endothelial growth factor concentrations in uveitis patients with and without cystoid macular edema and in healthy volunteers were 32.2, 29.6, and 55.0 pg/ml, respectively. Uveitis patients had lower plasma vascular endothelial growth factor levels than did healthy volunteers, P = .0002 CONCLUSION: In uveitis patients, vascular endothelial growth factor concentration is increased in the aqueous humor of eyes with cystoid macular edema. It may be useful to investigate vascular endothelial growth factor antagonists as a treatment for uveitis-associated cystoid macular edema.

To investigate the clinical features associated with immune recovery in human immunodeficiency virus (HIV)-infected patients with cytomegalovirus retinitis who are taking highly active antiretroviral therapy.Sixteen patients were evaluated prospectively at the National Eye Institute, Bethesda, Maryland. Evaluation included a medical history and a complete ophthalmologic examination. The examination included best-corrected visual acuity score measured by means of logarithmic charts, slit-lamp biomicroscopy, dilated retinal examination, retinal photography, and fluorescein angiography. Immune-recovery uveitis was defined as the ocular inflammation associated with clinical immune recovery in patients taking potent antiretroviral regimens. The ophthalmic characteristics of immune-recovery uveitis were identified, and their effect on visual acuity was statistically analyzed.The mean CD4+ T-lymphocyte count for the 16 patients taking highly active antiretroviral therapy at the time of evaluation was 393 cells/microl (range, 97-1,338 cells/microl). Immune-recovery uveitis was characterized by vitreitis and optic disk and macular edema. Clinically important complications of immune-recovery uveitis included cataract and epiretinal membrane formation. The visual acuity scores were significantly worse in the 23 eyes with cytomegalovirus retinitis (mean, 67.2 letters, 20/50) than in the nine eyes without cytomegalovirus retinitis (mean, 89.8 letters, 20/16) (P.001). Regression analysis showed that a lower visual acuity score was associated with the presence of moderate to severe macular edema on fluorescein angiography and vitreous haze (Por =. 001).Immune-recovery uveitis is an important cause of visual morbidity in HIV-infected patients with cytomegalovirus retinitis in the era of highly active antiretroviral therapy. Although immune recovery associated with highly active antiretroviral therapy has allowed some patients to discontinue specific anticytomegalovirus therapy, the rejuvenated immune response can be associated with sight-threatening inflammation.