ABSTRACT
This study was undertaken to identify and characterize the Ag responsible for the induction of experimental autoimmune anterior uveitis (EAAU). Melanin-associated Ag isolated from bovine iris and ciliary body was digested with the proteolytic enzyme V8 protease to solubilize the proteins and the pathogenic protein was purified to homogeneity. Lewis rats were sensitized to various fractions and investigated for the development of anterior uveitis and an immune response to the purified Ag. The uveitogenic Ag had a mass of 22 kDa (SDS-PAGE) and an isoelectric point of 6.75. The N-terminal amino acid sequence of this protein demonstrated 100% homology with the bovine type I collagen alpha-2 chain starting from amino acid 385 and will be referred to as CI-alpha2 (22 kDa). Animals immunized with bovine CI-alpha2 (22 kDa) developed both cellular and humoral immunity to the Ag. They developed anterior uveitis only if the CI-alpha2 chain underwent proteolysis and if the bound carbohydrates were intact. EAAU induced by CI-alpha2 (22 kDa) can be adoptively transferred to naive syngenic rats by primed CD4(+) T cells. EAAU could not be induced by the adoptive transfer of sera obtained from animals immunized with CI-alpha2 (22 kDa). The alpha-1 and alpha-2 chains (intact or proteolytically cleaved) of type I collagen from calfskin were not pathogenic. Although human anterior uveitis has been historically characterized as a collagen disease, this is first time collagen has been directly identified as the target autoantigen in uveitis.
Subject(s)
Autoantigens/immunology , Autoimmune Diseases/etiology , Collagen Type I/immunology , Uveitis, Anterior/etiology , Adoptive Transfer , Amino Acid Sequence , Animals , Autoantigens/isolation & purification , Autoimmune Diseases/immunology , Immune Sera/immunology , Male , Molecular Sequence Data , Organ Specificity , Protein Processing, Post-Translational , Rats , Rats, Inbred Lew , Uveitis, Anterior/immunologyABSTRACT
Age-related macular degeneration (AMD) is the leading cause of blindness after age 55 in the industrialized world. Severe loss of central vision frequently occurs with the exudative (wet) form of AMD, as a result of the formation of a pathological choroidal neovasculature (CNV) that damages the macular region of the retina. We tested the effect of an immunotherapy procedure, which had been shown to destroy the pathological neovasculature in solid tumors, on the formation of laser-induced CNV in a mouse model simulating exudative AMD in humans. The procedure involves administering an Icon molecule that binds with high affinity and specificity to tissue factor (TF), resulting in the activation of a potent cytolytic immune response against cells expressing TF. The Icon binds selectively to TF on the vascular endothelium of a CNV in the mouse and pig models and also on the CNV of patients with exudative AMD. Here we show that the Icon dramatically reduces the frequency of CNV formation in the mouse model. After laser treatment to induce CNV formation, the mice were injected either with an adenoviral vector encoding the Icon, resulting in synthesis of the Icon by vector-infected mouse cells, or with the Icon protein. The route of injection was i.v. or intraocular. The efficacy of the Icon in preventing formation of laser-induced CNV depends on binding selectively to the CNV. Because the Icon binds selectively to the CNV in exudative AMD as well as to laser-induced CNV, the Icon might also be efficacious for treating patients with exudative AMD.
Subject(s)
Choroid/blood supply , Choroid/drug effects , Immunotherapy/methods , Macular Degeneration/therapy , Neovascularization, Pathologic , Adenoviridae/genetics , Animals , Cells, Cultured , DNA, Complementary/metabolism , Disease Models, Animal , Gene Library , Genetic Vectors , Humans , Lasers , Liver/metabolism , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Protein Binding , Retina/drug effects , Swine , Thromboplastin/metabolismABSTRACT
PURPOSE: To investigate whether 2'-benzoyl-oxycinnamaldehyde (BCA) induces apoptosis in human retinal pigment epithelial (hRPE) cells and has an antiproliferative effect in a proliferative vitreoretinopathy (PVR) model in the rabbit. METHODS: Fifty percent growth inhibition doses of hRPE cells at 50%, 75%, and 100% confluence were determined by MTT assay. Apoptosis in hRPE cells induced by BCA was shown by DAPI staining. Expression of p53, p21, Bcl-2, GADD45, cyclin D, phospho-MAP kinase, cdk2, and Akt1 at various concentrations of BCA in cultured hRPE cells was examined by immunoblot analysis. In the efficacy study, 2.0 x 10(5) rabbit RPE cells were injected into the vitreous cavity after gas compression, and the eyes subsequently received either sham injections or 600 micro M BCA. Fundus examination was performed before and 1, 7, 14, 21, and 28 days after BCA injection. The toxicity studies were conducted by the same protocol as used for the efficacy evaluation but without the RPE cell injection. Simultaneous electroretinograms were recorded on days 1, 7, 14, 21, and 28 after exposure to the drug. RESULTS: BCA treatment induced apoptosis in hRPE cells. Furthermore, an increase in p53 expression, phosphorylation of Bcl-2, and downregulation of Akt1 expression were observed in BCA-induced apoptotic cells. BCA effectively prevented the proliferation of rabbit RPE cells in the experimental PVR model. BCA exhibited a wide safety margin, showing no evidence of causing retinal toxicity, even at the 600- micro M concentration. CONCLUSIONS: The results of this study suggest that BCA effectively inhibits proliferation of RPE cells and has a very wide safety margin, indicating a potential therapeutic usefulness in treating PVR.
