Molecular Vision 2004; 10:132-7 <http://www.molvis.org/molvis/v10/a17> Received 21 November 2003 | Accepted 2 February 2004 | Published 25 February 2004 Glaucoma, one of the leading causes of blindness in the world [1], is a group of ocular disorders that are characterized by a loss of retinal ganglion cells and their axons, damage to the optic nerve, and progressive loss of visual field. People of all ages can be affected by this disease. At the age of 70, about 7% of the population suffers from glaucoma. The most com- mon form of glaucoma, primary open-angle glaucoma, is as- sociated with an elevation of the intraocular pressure. How- ever, more than 30% of glaucoma patients do not have an el- evated intraocular pressure (normal tension glaucoma). How- ever, the most common therapy even in those cases is to lower the intraocular pressure towards unphysiological values. Several recent findings support the hypothesis that au- toimmune mechanisms may be involved in the pathogenesis of normal tension glaucoma [2]. Several antibodies against ocular antigens could be found that play a possible neurodegenerative role in glaucoma, including antibodies against heat shock proteins (HSPs) [3], rhodopsin [4], γ-eno- lase [5], glutathione-S-transferase (GST) [6], tumor necrosis factor-α [7], and γ-synuclein [8]. Patients suffering from nor- mal tension glaucoma have an elevated serum titer of autoan- tibodies against small heat shock proteins (25 to 30 kDa) [9,10]. Although some autoantibodies in the sera of glaucoma patients have been identified and correlated with glaucoma, as described hereinbefore, many are still unknown. Western blotting has surfaced as a powerful tool for detecting specific autoantibodies in autoimmune diseases. Complicating the straightforward identification of pathogenically relevant anti- gens, however, is that normal sera contain large amounts of natural antibodies which manifest themselves in complex stain- ing patterns [11-14]. This can complicate the differentiation of disease-associated autoantibodies from the complex back- ground of “auto-immune noise” (naturally occurring autoan- tibodies). The role of these natural autoantibodies is still un- clear. Nonetheless, it is widely accepted that an individual rep- ertoire remains stable for a specific individual [15]. Most studies evaluate one or a few specific disease-re- lated antibodies and screen only for a limited number of puri- fied homologous or heterologous proteins (antigens) by means of ELISA or RIA [4]. However, in most autoimmune diseases, a diagnosis based on a single autoantibody was impossible to establish. On the other hand, the western blotting technique permits simultaneous screening for a wide spectrum of differ- ent autoantigens. Using digital image analysis techniques, these complex banding patterns of antigen-antibody reactions can be quantified and statistically analyzed, allowing the detec- tion of minor changes in the antigenic composition of autoan- tibody repertoires [15]. In this approach, pattern matching al- gorithms that are based on statistical techniques (like discrimi- nant analysis) or artificial neural networks can be used to dif- ferentiate between the mean banding patterns of groups, and to measure the extent to which multiple dependent variables (like the intensity of a antigen-antibody reaction at a specific molecular weight region) contribute to differences between the groups [16-19]. This technique has been successfully used in studies of myasthenia gravis, Graves’ disease, experimen- tal autoimmune uveitis, Guillan-Barré, and Tourette syndrome [20-27]. ©2004 Molecular Vision Complex autoantibody repertoires in patients with glaucoma Franz H. Grus, Stephanie C. Joachim, Esther M. Hoffmann, Norbert Pfeiffer Universitaets-Augenklinik, Department of Ophthalmology, University of Mainz, Mainz, Germany Purpose: Glaucoma is one of the leading causes for blindness in the world. It is characterized by a progressive loss of retinal ganglion cells. An elevated intraocular pressure cannot explain the disease in all subjects. Autoimmune mecha- nisms maybe involvemed in the pathogenesis of the disease. The aim of our study was to analyze the IgG autoantibody repertoires in sera of glaucomatous subjects against optic nerve antigens. Methods: Nintey-four subjects were included in this comparative cross-sectional study of healthy (CTRL), primary- open-angle-glaucoma (POAG), ocular-hypertension (OHT), and normal-tension-glaucoma (NTG) volunteers. Sera of subjects were tested against western blots of optic nerve antigens. For each western blot, a densitograph was built by digital image analysis and subsequently a multivariate analysis of discriminance was performed. Results: Complex IgG autoantibody repertoires could be found in all subjects, even in healthy subjects. The multivariate analysis of discriminance can test for statistical differences between the groups using the whole complex staining pattern for the calculation. A significant difference between all groups against optic nerve antigens was found. The NTG group had the highest variance from controls (p<0.01). Conclusions: This study demonstrates immunological effects in POAG and NTG and may provide further evidence for an involvement of autoantibodies in the pathogenesis of both NTG and POAG. Using the techniques presented in this study, the differences in the complex autoantibody repertoires were assessed by means of statistical analysis. Further studies are needed to determine whether these changes in autoantibodies could be helpful in the diagnosis of glaucoma. Correspondence to: Franz H. Grus, MD, PhD, Universitaets- Augenklinik, Department of Ophthalmology, Langenbeckstrasse 1, 55101 Mainz, Germany; Phone: +496131-173328; email: F@grus.de 132