Richard Ornberg

Complement Deposition and Microglial Activation in the Outer Retina in Light-Induced Retinopathy: Inhibition by a 5-HT1A Agonist

PURPOSE Increasing evidence supports a role for complement in the pathogenesis of age-related macular degeneration (AMD). This study evaluated retinal microglia, T-lymphocytes, and complement deposition in a light-induced retinopathy model. The effect of a serotonin (5-hydroxytryptamine, 5-HT(1A)) agonist on these processes was investigated. METHODS Rats were dark adapted for 24 hours before a 6-hour blue light exposure. Some animals were predosed subcutaneously with AL-8309A. Retinas were evaluated at different times after light exposure. Paraffin sections were stained with antibody for a microglial marker (Iba1), a T-lymphocyte marker (CD3), and complement components C1q, C3, factor B, factor H, and membrane attack complex (MAC). RESULTS Light exposure resulted in substantial photoreceptor and RPE loss. Robust microglia activation and migration to the outer retina occurred rapidly. Substantial T-lymphocyte recruitment did not occur. Complement alternative pathway was strongly activated, resulting in the deposition of C3, factor B, factor H, and MAC in the area of photic lesions. Dosing with AL-8309A prevented retinal lesions and decreased microglia activation/recruitment and complement deposition in the outer retina. CONCLUSIONS In blue light exposed retinas, microglia were activated and migrated toward the outer retina, whereas a T-lymphocyte response was minimal. The innate immune system was markedly activated, with substantial complement deposition in the outer retina after light exposure. This complement deposition was prevented by AL-8309A. This model may be useful in the evaluation of complement inhibitors and other neuroprotectants intended for ocular use. AL-8309 is under evaluation in the clinic and may be useful in the treatment of AMD.

AL-78898A Inhibits Complement Deposition in a Primate Light Damage Model

PURPOSE To determine the association between ocular dominance and spherical/astigmatic anisometropia, age, and sex in hyperopic subjects. METHODS The medical records of 1274 hyperopic refractive surgery candidates were filtered. Ocular dominance was assessed with the hole-in-the-card test. Refractive error (manifest and cycloplegic) was measured in each subject and correlated to ocular dominance. Only subjects with corrected distance visual acuity of >20/22 in each eye were enrolled, to exclude amblyopia. Associations between ocular dominance and refractive state were analyzed by means of t-test, χ(2) test, Spearman correlation, and multivariate logistic regression analysis. RESULTS Right and left eye ocular dominance was noted in 57.4 and 40.5% of the individuals. Nondominant eyes were more hyperopic (2.6 ± 1.27 diopters [D] vs. 2.35 ± 1.16 D; P < 0.001) and more astigmatic (-1.3 ± 1.3 D vs. -1.2 ± 1.2 D; P = 0.003) compared to dominant eyes. For spherical equivalent (SE) anisometropia of >2.5 D (n = 21), the nondominant eye was more hyperopic in 95.2% (SE 4.7 ± 1.4 D) compared to 4.8% (1.8 ± 0.94 D; P < 0.001) for the dominant eye being more hyperopic. For astigmatic anisometropia of >2.5 D (n = 27), the nondominant eye was more astigmatic in 89% (mean astigmatism -3.8 ± 1.1 D) compared to 11.1% (-1.4 ± 1.4 D; P < 0.001) for the dominant eye being more astigmatic. CONCLUSIONS The present study is the first to show that the nondominant eye has a greater degree of hyperopia and astigmatism than the dominant eye in hyperopic subjects. The prevalence of the nondominant eye being more hyperopic and more astigmatic increases with increasing anisometropia.

AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice.

Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year.

Long-acting protein drugs for the treatment of ocular diseases

Protein drugs that neutralize vascular endothelial growth factor (VEGF), such as aflibercept or ranibizumab, rescue vision in patients with retinal vascular diseases. Nonetheless, optimal visual outcomes require intraocular injections as frequently as every month. Here we report a method to extend the intravitreal half-life of protein drugs as an alternative to either encapsulation or chemical modifications with polymers. We combine a 97-amino-acid peptide of human origin that binds hyaluronan, a major macromolecular component of the eyes vitreous, with therapeutic antibodies and proteins. When administered to rabbit and monkey eyes, the half-life of the modified proteins is increased ∼3–4-fold relative to unmodified proteins. We further show that prototype long-acting anti-VEGF drugs (LAVAs) that include this peptide attenuate VEGF-induced retinal changes in animal models of neovascular retinal disease ∼3–4-fold longer than unmodified drugs. This approach has the potential to reduce the dosing frequency associated with retinal disease treatments.

Conjunctivitis: 55 Ocular Signs and Symptoms Elicited by a Naturalistic Allergen Challenge in an Environmental Exposure Chamber Model Versus a Direct Allergen Instillation Model

Background Direct-instillation ocular models are well established for eliciting allergic responses in research and clinical testing. This study compared direct ocular instillation of allergen to a more naturalistic airborne allergen exposure. Methods Thirteen subjects with histories of ragweed allergy and positive skin prick responses attended screening, dose-finding, dose, confirmation, and analysis study visits. For conjunctival allergen provocation testing (CAPT), 1 drop of ragweed allergen was administered to each eye, at the lowest possible subject-specific concentration between 1.6 and 100 protein nitrogen units per 25 &mgr;l drop. For environmental exposure chamber (EEC) testing, subjects were exposed to continual airborne ragweed pollen at 3500 ± 500 particles/m3. Symptoms of itching and tearing were self-assessed on diary cards by subjects. Signs of hyperemia, swelling, and mucous discharge were assessed by clinicians. Assessment time points started at 30 minutes before exposure and continued through 180 minutes after exposure. Results At baseline, there were minimal signs and symptoms. Maximum mean hyperemia with CAPT was 2.3 ± 0.6 units (between moderate and severe) and with EEC was 1.9 ± 0.5 units (approximately moderate); these maxima occurred after 30 minutes with CAPT (rapid spike) and after 180 minutes with EEC (gradual increase). Mean swelling was <1 unit out of 4 units at all times (CAPT and EEC), and mucous discharge was observed in only 1 subject during the study (with CAPT). Maximum mean itching with both CAPT and EEC was 2.8 ± 1.0 units (approximately severe), but this maximum occurred after 20 minutes with CAPT (rapid spike) and after 180 minutes with EEC (gradual increase). Maximum mean tearing with CAPT was 1.2 ± 0.7 units (approximately mild) and with EEC was 1.6 ± 0.6 units (between mild and moderate); these maxima occurred after 15 minutes with EEC (rapid spike) and after 120 minutes with EEC (gradual increase). Conclusions The time courses of allergic signs and symptoms differed between CAPT and EEC models; however, both models evoked similar maximum response levels. This demonstrates that the EEC model is a useful challenge model for mimicking natural airborne ocular allergen exposure.