Abstract
Purpose: :
To test the hypothesis that auto-antibodies (Auto-Abs) are intimately involved with the pathogenesis of AMD and that their binding to ocular tissue autoantigens represents a useful biomarker of the disease state and stage in AMD.
Methods: :
A repository of 270 serum samples from White subjects with AMD (n=97, 73 of whom AREDS category 3 early AMD) and unaffected controls (n=173), all of whom 70 years old or older, is being screened for the presence of auto-Abs against neuroretinal (nRet), retinal pigment epithelium (RPE), and Bruch’s membrane/choroid (BM/Ch) homogenates generated from fresh eye tissues. All participants have been examined and their fundus findings documented photographically and graded in a masked fashion. Additional advanced AMD samples are being collected. Auto-Reactivity is being tested by Western blotting (WB). Auto-antigens in the homogenates are captured by immunoprecipitation (IP) with sera, using agarose A/G beads and eluted by 0.1M glycine pH 3.0. The presence of autoantigens in the eluate is confirmed by silver staining of 1D gels. These eluates are also separated by 2D gels, which allows us to better resolve the individual proteins and identify them by mass spectrometry (MS).
Results: :
We have confirmed our hypothesis that AMD sera display stronger and more frequent autoreactivity than controls against nRet, RPE, and BM/Ch antigens by both WB and IP, and we have preliminary indication that auto-Abs tend to be more common in advanced AMD samples. Complex patterns of autoreactivity have been identified. AMD sera also present with unique autoreactive bands, not observed in WBs and IPs from control sera. More intense and/or unique autoreactivities will be identified by MS.
Conclusions: :
AMD sera present with a complement anti-nRet, anti-RPE and anti-BM/Ch auto-Abs more often and more intensely than control sera. Auto-Abs are seen more frequently in advanced AMD, but are present also in early AMD, likely representing an evolution from progressive inflammatory damage to ocular tissue and desegregation of tissue antigens. Although AMD is certainly not a primary autoimmune disease, the most unique and/or intensely and frequently involved auto-Ab targets have the potential to be, much like carboxyethylpyrrole (Gu et al. JBC 2003; 278: 42027-35), important disease biomarkers and may contribute significantly to AMD pathogenesis and progression.
Keywords: age-related macular degeneration • protein purification and characterization • autoimmune disease