| OCR Text |
Show ORIGINAL CONTRIBUTION Frequency of Anti- Retinal Antibodies in Normal Human Serum Kaori Shimazaki, BS, Guy V Jirawuthiworavong, MD, MA, John R. Heckenlively, MD, and Lynn K. Gordon, MD, PhD Background: Anti- retinal antibodies have been described in the context of autoimmune retinopathies and are often presumed to be pathogenic or disease associated. However, full characterization of patterns of anti- retinal antibody reactivity in normal human serum has been limited. The purpose of this work was to identify the profile of anti- retinal IgG antibodies in serum used as controls in laboratory testing. Methods: Normal human sera used in commercial diagnostic laboratories were tested for the presence of immunoreactivity against soluble human retinal proteins using Western blot analysis of fractionated soluble human retinal proteins. Reactivity was quantified using computerized densitometry, and the level of reactivity was standardized relative to a control positive serum with known reactivity against recoverin. Results: Some anti- retinal reactivity was observed in the majority of all tested normal sera. Reactivity against one to two protein bands was observed in 33%. Reactivity against five or more distinct bands was observed in 22%. There was a tendency for serum from women to react with three or more protein bands compared with serum from men. Conclusions: The presence of anti- retinal antibodies is observed in a majority of normal control human sera, suggesting that identification of new candidate retinal autoantigens should be cautiously interpreted and subject to rigorous testing for disease association. Molecular Biology Institute ( SK) and Jules Stein Eye Institute, Department of Ophthalmology ( GVJ), University of California, Los Angeles David Geffen School of Medicine, Los Angeles, California; Kellogg Eye Center ( JRH), University of Michigan, 1000 Wall Street, Ann Arbor, Michigan; and Ophthalmology Section, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California. This work was presented in part at the North American Neuro-ophthalmology Society Annual Meeting. K. S. and GVJ. contributed equally to this work. This work was supported in part by Grant T32 EY07026- 28 from the National Eye Institute, Bethesda, MD ( GVJ), by a UCLA Academic Senate Faculty Grant ( LKG), and by a James S. Adams Scholars Award from Research to Prevent Blindness, New York ( LKG). Address correspondence to Lynn K. Gordon, MD, PhD, 100 Stein Plaza, Los Angeles, CA 90095; E- mail: lgordon@ ucla. edu Additional studies will aid development of a standardized protocol for validation of potential pathogenic seroreactivity. { JNeuro- Ophthalmol 2008; 28: 5- 11) Autoantibodies against retina are implicated in immune-mediated visual loss ( 1- 4). Anti- retinal reactivity is observed in cancer- associated retinopathy ( CAR), melanoma- associated retinopathy, autoimmune optic neuropathy, and a subset of patients with retinitis pigmentosa ( 5- 8). These disorders may be grouped under the heading of autoimmune- related retinopathies ( AIRs), a variety of clinical entities classified together according to the presence of immune- associated loss of retinal function. Although the exact pathophysiological mechanism of this group of disorders is unknown, it has been postulated that the humoral immune response induces AIRs through antibody internalization and interruption of normal cellular physiologic processes resulting in loss of function ( 1). The best characterized AIR is CAR ( 8) in which anti-retinal activity is generated against a retinal protein that is produced by a primary tumor or against a rumor- associated antigen with shared epitopes with a retinal protein ( 1,9- 11). In CAR, antibodies against recoverin, a 23- kDa protein, have been demonstrated to induce retinal cell death through a pro- apoptotic pathway, a likely cause of the eventual retinal dysfunction ( 11- 15). Other antigenic targets have been implicated in the etiology of CAR and other AIR syndromes ( 3,5,16). Some of the characterized retinal antigenic targets include a- enolase ( 46 kDa) ( 12,17), carbonic anhydrase II ( 30 kDa) ( 6), recoverin ( 23 kDa) ( 18), collapsin response- mediating protein- 5 ( CRMP- 5) ( 62 kDa) ( 19), heat- shock protein 70 ( 65 kDa) ( 20), tubby-like protein 1 ( 78 kDa) ( 21), and lens epithelium- derived growth factor ( LEDGF) ( 22). Other candidate proteins with defined molecular sizes of 22 kDa ( 23), 40 kDa ( 24), and 50 kDa ( 25) have not been formally identified. Despite the interest in this area, anti- retinal reactivity is not universally observed in patients who present with CAR- like signs and J Neuro- Ophthalmol, Vol. 28, No. 1, 2008 5 J Neuro- Ophthalmol, Vol. 28, No. 1, 2008 Shimazaki et al Ocular Specimens Human eyes obtained from deceased donors were procured from the University of California, Los Angeles Eye Bank and the Doheny Eye and Tissue Transplant Bank up to 72 hours after death and processed immediately on arrival. The tenets of the Declaration of Helsinki governed the procurement and management of the tissue. The eyes were stored in a moist chamber at 4° C until dissection. The eyes were bisected in the coronal plane along the pars plana. The posterior segment of the eye was meticulously microdissected to obtain the neurosensory retina and excluded areas immediately adjacent to the ora serrata and optic nerve. The dissected tissues were snap- frozen and stored at 80° C. Proteins Human retinal protein extracts were obtained after tissue homogenization in glass in phosphate- buffered saline ( PBS) containing 2% sodium dodecyl sulfate ( SDS), 100 mM dithiothreitol ( Sigma Chemical Co., St. Louis, MO) and complete protease inhibitor ( Roche Applied Science, Indianapolis, IN), Tissue fragments were removed by centrifugation and the supernatant was frozen at - 80° C until use. Western Blot Analysis Retinal tissue extracts, 10 | Jig per lane, were fractionated by SDS- polyacrylamide gel electrophoresis ( PAGE) on Novex 4%- 20% Tris- glycine gels ( Invitrogen, Carlsbad, CA). Proteins were transferred to nitrocellulose membranes ( Amersham Life Sciences, Buckinghamshire, UK) overnight in Tris- glycine buffer ( National Diagnostics, Atlanta, GA), and adequacy of transfer was verified by ponceau S red staining ( Sigma). The membrane was blocked in 5% nonfat milk in PBS- 0.1% Tween 20 ( PBS- Tween) ( Pierce Biotechnology, Rockford, IL) for 2 TABLE 1. Characteristics Characteristics Sex Female Male Race Asian surname Caucasian of normal Caucasian Hispanic surname African American donors 16- 20 12 12 1 19 3 1 21- 30 14 14 3 21 2 2 31- 40 8 3 0 11 0 0 Age ( years) 41- 50 8 7 0 8 7 0 51- 60 5 4 0 6 2 1 61- 70 0 5 0 5 0 0 Total 47 45 4 70 14 4 symptoms. On the other hand, anti- retinal antibodies have been demonstrated in some patients with cancer in the absence of any documented visual loss or typical symptoms of an AIR ( 16,26,27). This observation suggests that the presence of reactivity of one or multiple autoantibodies is either insufficient to induce the disease phenotype or that reactivity may be a preclinical disease marker. The prevalence of reactivity against these antigens has not been not well characterized in a large control population, and the sensitivity and specificity of the anti-retinal antibodies in relation to AIR disease pathogenesis is still uncertain ( 5,10,16,17,26,28- 31). There is also little standardization regarding the methods of detection of anti-retinal immunoreactivity. Prior studies vary in the species origin of the retinal protein extracts ( ranging from rodents to primates), the methods of protein extraction, and the definition of positive reactivity. The purpose of this study was to use a standardized protocol with internal positive controls to define the pattern and frequency of IgG reactivity against soluble human retinal proteins in a panel of human control serum. METHODS Serum Human serum with high 23- kDa anti- retinal reactivity was used as an internal positive control for Western blot studies ( gift of C. E. Thirkill, University of California- Davis, Davis, CA). A commercially available set of normal human sera from random blood bank donors, available for use in clinical laboratories as normal controls, was obtained ( TheraTest Laboratories, Lombard, IL). Ninety- two samples of normal sera were used in this study and the sex, age, and ethnicity of the subjects are reported in Table 1. 6 © 2008 Lippincott Williams & Wilkins Anti- Retinal Antibodies J Neuro- Ophthalmol, Vol. 28, No. 1, 2008 hours. Several different regimens and times for blocking were initially used to reduce background or nonspecific reactivity, and this protocol was optimal ( data not shown). Blots were incubated for 1 hour with human sera or with positive control serum ( high 23- kDa anti- retinal activity) at a dilution of 1: 1000 in 1% nonfat milk in PBS- Tween. In each experiment one lane was used as a secondary antibody control. After multiple washes with PBS- Tween, bound human IgG was identified by horseradish peroxidase ( HRP)- conjugated goat anti- human IgG ( Pierce) at a dilution of 1: 1000 in 1% nonfat milk in PBS- Tween. The secondary antibody was chosen to specifically detect human IgG antibodies. Reactivity was visualized using the sensitive technique of enhanced chemiluminescence ( ECL) ( Amersham). Semiquantitation of Immunoblots Densitometric quantification of anti- retinal reactivity was performed using the Personal Densitometer SI ( Amersham) followed by ImageQuant ( Molecular Dynamics, Sunnyvale, CA) ( Fig. 1). Intensity of the anti- retinal reactivity of the 23- kDa positive control serum was used as an internal control in each Western blot experiment to control for reactivity. Positive reactivity in the tested serum was denned as greater than or equal to 50% intensity of reactivity of the positive control at 23 kDa. Bands identified as human IgG light chain (- 25 kDa) or heavy chain (~ 50 kDa) are often seen in human tissue extracts when probed with a secondary antibody that recognizes human IgG; these specific bands were excluded from additional analysis or comment. Statistical Analysis Comparison of the groups of normal sera by gender and number of reactive bands was statistically analyzed by the likelihood ratio \ 2 test and Fisher's exact test. A P value of < 0.05 was considered significant. RESULTS Ninety- two human sera samples, commercially used for diagnostic laboratory controls, were screened for anti-retinal antibodies directed against human retinal antigens. Forty- five ( 49%) samples were from men and 47 ( 51%) samples were from women. The ages of the individuals ranged from 16 to 70 years; however, the majority of individuals tested were younger than age 40. Although sera were drawn from individuals of various ethnicities ( African American, Asian, Caucasian, and Hispanic), the majority of the sera were from individuals of Caucasian ethnicity ( Table 1). We initially tested a variety of dilutions of sera for this study ( data not shown), and to optimize testing a 1: 1000 dilution, which both minimized background and disclosed distinguishable reactive bands, was selected for use in this study. ImageQuant Western blot analysis detected the presence of anti- retinal IgG immunoreactivity, denned as being equal to or greater than half of the reactivity of the control anti- recoverin serum, in 57 of 92 ( 62%) normal sera. Thirty ( 33%) had reactivity against only one or two retinal protein bands, whereas 20 ( 22%) were reactive against five or more retinal protein bands ( Table 2). Although 18 tested women had three or more reactive bands compared with only 9 tested men, this gender difference did not reach the level of statistical significance ( P = 0.052 by likelihood ratio x2 analysis). There were no significant gender or age differences in sera exhibiting reactivity compared with sera with no anti- retinal reactivity ( x2 or Fisher's exact test). The numbers of observed immunoreactive bands varied widely among the different sera tested. Half of the sera with positive anti- retinal reactivity demonstrated only one or two bands of reactivity ( Fig. 2). However, five sera exhibited reactivity against at least 10 distinct protein bands, suggesting that a broad range of serum reactivity may be observed in selected control sera. The molecular weights of the observed anti- retinal reactivity also varied widely ( Fig. 3). Reactivity is presented in arbitrary units relative to intensity of the anti- recoverin control where equivalent reactivity is 1.00. The observed calculated molecular weight of detected protein bands ranged from 13 to 148 kDa, and reactivity intensity ranged between 0.59- and 1.71- fold of the anti- recoverin control. There were five distinctive clusters of reactivity observed in the tested sera, which may represent common reactivities against the same protein or against multiple proteins with similar size. Notably, reactivity against a 23 kDa retinal protein, presumed to be recoverin, was not detected in any of the 92 sera tested. DISCUSSION The present work, using a highly sensitive ECL method for detection and formal densitometric quantification, reveals that the majority of " control normal" sera carry IgG antibodies that are able to react against solubilized human retinal proteins and that Western blots against whole tissue extracts may be of limited utility in determining potential pathogenic autoreactivity in human subjects. Although most reports of disease- associated anti-retinal antibodies include a comparison against laboratory normal controls, standardization in these protocols and characterization of normal subjects evaluated in these studies are often unspecified. It has been observed previously that normal individuals may have some antibody reactivity to retinal antigens as detected by Western blot 7 J Neuro- Ophthalmol, Vol. 28, No. 1, 2008 Shimazaki et al oo 0 » ' o » • or • 04 • 05 04 • 0C> I I 19 0 » ' 8: :>.•! 13 o » I'll 01 « 04 nr> 13 o » • r. i . or Ot i 01 • 04 • • CO ^ 50kDa ^ 25kDa 4* T A. « r / v ^ 300 - I I I I I i 4C OO CO TD CO TOO ' . . • J . .^ A to so : UJ 4 « « 30 6CC ? JJ tC XW f > . CO • » -^^ _ ^ \ ceo TX> re ro") r\ , 23kDa DO - r-xo - I - FIG. 1. Densitometric analysis of Western blots using ImageQuant. A. For each gel lane a vertical line is placed manually, and the relative density and band position were generated through the ImageQuant program. Lane 1, the secondary antibody only, reveals the IgG heavy and light chain antibodies present in the tissue extract. Lanes 2 and 3 represent IgG reactivity from two different control samples. Lane 4 demonstrates the 23- kDa reactivity in the control positive serum with known reactivity against recoverin. B. The molecular weight of each measured band is calculated using the position relative to known markers. The bands at 25 and 50 kDa were constantly seen in all lanes and reflect IgG heavy and light chains present in the soluble protein extracts from human tissue. Thus, reactivity at these molecular weights was not used for the determination of the anti- retinal reactivity in the tested sera. B 2008 Lippincott Williams & Wilkins Anti- Retinal Antibodies J Neuro- Ophthalmol, Vol. 28, No. 1, 2008 TABLE 2. Anti- retinal reactivity by sex and age Number of reactive bands Sex Male Female Age ( years) Mean Median Range 0 17 18 33.3 29 16- 60 1- 2 19 11 33.13 25 16- 70 ?>- A 2 5 32.4 33 20- 51 > 5 7 13 32.75 30.5 18- 52 analysis ( 7- 11). Circulating autoantibodies are often observed in human subjects and may result in part from cell degradation and exposure of self- antigens to the immune system ( 7). One report indicates that a large percentage of patients with visual problems ( 43%) have circulating anti- retinal antibodies ( 32). The pathogenetic significance of these antibodies must be questioned in light of our findings of anti- retinal reactivity in the majority of samples in the panel of commercially available normal sera. Detection and identification of autoimmune retinal antigens using sensitive detection methods often lead to complex patterns of immunoreactivity in both patient and normal control sera. To reveal true potential disease-associated reactivity, one has to develop a system to amplify the signal- to- noise ratio. According to the data presented in this paper, definition of new candidate anti- retinal reactivity against whole retinal extract in a patient population would need to meet specific criteria in comparison with the pattern of reactivity observed in controls. For example, our data show that although the majority of normal sera have some reactivity against human retinal proteins, five clusters of activity against proteins of similar molecular weight are present, and there is large variability in the degree of reactivity against retinal proteins. On the basis of these observations, an unknown serum sample could be identified as having potentially abnormal anti- retinal antibodies if the antigen was of a size not recognized by any of the control panels. Alternatively, preliminary identification of a potentially abnormal anti- retinal reactivity of a specific molecular size such as 23 kDa recognized by both the test and the control serum could be made if the intensity of reactivity in the test serum were much higher than the intensity of reactivity in the control serum. Validation of the reactivity as abnormal would then require other confirmatory methodology Subsequent validation could be performed by serial dilutions of the test serum to define a titer of reactivity Other formal characterization of potentially abnormal reactivity should be performed using purified candidate antigens from the retina or by identifying new candidate antigens through Western blots of retinal proteins separated by two- dimensional gel electrophoresis followed by proteomic analysis. The present study has some limitations. Although it is possible that the high level of anti- retinal reactivity observed in this population is secondary to the sensitivity of the detection method, it is also possible that these samples, drawn from a commercial source, came from individuals with some medical histories that predisposed them to development of autoantibodies. We consider this confounder to be unlikely, as most autoimmune retinopathy occurs later in the life, and the sera used in the present study came from relatively young subjects. Despite this potential limitation, analysis of our data did not reveal prominent changes in antibody patterns with aging although additional studies with a larger control cohort population of older individuals might provide additional information. These 18 16 14 12 2 10 03 CO nn rx JZL 8 10 11 12 13 14 15 1S 17 FIG. 2. Anti- retinal reactivity in normal serum. A distribution of anti-retinal reactivity of normal sera was plotted according to the number of highly reactive bands detected by Western blots. Of 92 human sera tested, 57 showed reactivity against human retina. Although most of the sera tended to have a small number of reactive bands, reactivity against multiple retinal proteins was observed in a small group of sera. Reactive Bands 9 J Neuro- Ophthalmol, Vol. 28, No. 1, 2008 Shimazaki et al 1.8 1.7 i. e 1.5 1.4 1.3 1.2 1.1 I 0.3 0.3 0.7 o. e 0.5 t < • • • • 10 20 30 40 50 SO 70 SO SO 100 Molecular Weight ( kDa) - I - 110 - i 1 1 r- 120 130 140 150 - I 160 FIG. 3. Distribution of anti- retinal reactivity in human serum. All reactivities noted in the 57 positive human sera were graphed according to calculated molecular weight on Western blots and relative seroreac-tivity. Despite a wide range of observed reactivity against retinal protein extract in both molecular weight and intensity, reactivity was clustered into multiple regions. No reactivity in control serum was detected at 23 kDa, the location of recoverin. potential limitations suggest that our observations should be validated in a larger control group with detailed medical history and ophthalmic evaluation. Methods for identifying potential disease- associated anti- retinal reactivity should be carefully denned in prospective studies that include a large, well- defined control population. These methods may include preliminary screening evaluation of reactivity against a whole retinal extract, followed by additional testing and analysis. Validation could be performed against denned candidate antigens; alternatively, an initial screening for specific reactivity could be performed against denned candidate antigens using other techniques, such as proteomic analysis ( reviewed in ref 33), enzyme- linked immunosorbent assay ( ELISA) ( 16,34), denned Western ( 16,34) or dot blot ( 34), or antigen arrays ( reviewed in ref. 35), for detection of disease- associated antibodies. Our work suggests that, when tested using sensitive methodology, the prevalence of anti- retinal antibodies is high in a test control population and that determination of disease- associated pathogenic autoantibodies requires rigorous standardization through use of an appropriate and large, clinically defined, and validated evaluation. Acknowledgments The authors thank Fei Yu, PhD, UCLA Department of Ophthalmology, for his assistance with statistical analysis. REFERENCES Adamus G. Autoantibody- induced apoptosis as a possible mechanism of autoimmune retinopathy. Autoimmun Rev 2003; 2: 63- 8. Darnell RB, Posner JB. Paraneoplastic syndromes involving the nervous system. N EnglJ Med 2003; 349: 1543- 54. Hooks JJ, Tso MO, Derrick B. Retinopathies associated with antiretinal antibodies. Clin Diagn Lab Immunol 2001; 8: 853- 8. Jacobson DM, Thirkill CE, Tipping SJ. A clinical triad to diagnose paraneoplastic retinopathy. Ann Neurol 1990; 28: 162- 7. 10. 11. 12. 13. 14. 15. 16. 17. 19. 20. Chan JW. Paraneoplastic retinopathies and optic neuropathies. Surv Ophthalmol 2003; 48: 12- 38. Heckenlively JR, Aptsiauri N, Nusinowitz S, et al. Investigations of antiretinal antibodies in pigmentary retinopathy and other retinal degenerations. Trans Am Ophthalmol Soc 1996; 94: 179- 200. Milam AH, Saari JC, Jacobson SG, et al. Autoantibodies against retinal bipolar cells in cutaneous melanoma- associated retinopathy Invest Ophthalmol Vis Sci 1993; 34: 91- 100. Thirkill CE, FitzGerald P, Sergott RC, et al. Cancer- associated retinopathy ( CAR syndrome) with antibodies reacting with retinal, optic- nerve, and cancer cells. N EnglJ Med 1989; 321: 1589- 94. Maeda T, Maeda A, Maruyama I, et al. Mechanisms of photoreceptor cell death in cancer- associated retinopathy. Invest Ophthalmol Vis Sci 2001; 42: 705- 12. Ohguro H, Maruyama I, Nakazawa M, et al. Antirecoverin antibody in the aqueous humor of a patient with cancer- associated retinopathy Am J Ophthalmol 2002; 134: 605- 7. Shiraga S, Adamus G. Mechanism of CAR syndrome: anti- recoverin antibodies are the inducers of retinal cell apoptotic death via the caspase 9- and caspase 3- dependent pathway. J Neuroimmunol 2002; 132: 72- 8. Adamus G, Machnicki M, Elerding H, et al. Antibodies to recoverin induce apoptosis of photoreceptor and bipolar cells in vivo. J Autoimmun 1998; 11: 523- 33. Adamus G, Machnicki M, Seigel GM. Apoptotic retinal cell death induced by antirecoverin autoantibodies of cancer- associated retinopathy. Invest Ophthalmol Vis Sci 1997; 38: 283- 91. Chen W, Elias RVJ Cao W, et al. Anti- recoverin antibodies cause the apoptotic death of mammalian photoreceptor cells in vitro. J NeurosciRes 1999; 57: 706- 18. Subramanian L, Polans AS. Cancer- related diseases of the eye: the role of calcium and calcium- binding proteins. Biochem Biophys Res Commun 2004; 322: 1153- 65. Adamus G, Ren G, Weleber RG. Autoantibodies against retinal proteins in paraneoplastic and autoimmune retinopathy. BMC Ophthalmol 2004; 4: 5. Adamus G, Aptsiauri N, Guy J, et al. The occurrence of serum autoantibodies against enolase in cancer- associated retinopathy. Clin Immunol Immunopathol 1996; 78: 120- 9. Thirkill CE, Tait RC, Tyler NK, et al. The cancer- associated retinopathy antigen is a recoverin- like protein. Invest Ophthalmol Vis Sci 1992; 33: 2768- 72. Cross SA, Salomao DR, Parisi JE, et al. Paraneoplastic autoimmune optic neuritis with retinitis defined by CRMP- 5- IgG. Ann Neurol 2003; 54: 38- 50. Ohguro H, Ogawa K, Maeda T, et al. Cancer- associated retinopathy induced by both anti- recoverin and anti- hsc70 antibodies in vivo. Invest Ophthalmol Vis Sci 1999; 40: 3160- 7. 10 © 2008 Lippincott Williams & Wilkins Anti- Retinal Antibodies J Neuro- Ophthalmol, Vol. 28, No. 1, 2008 21. Kikuchi T, Arai J, Shibuki H, et al. Tubby- like protein 1 as an autoantigen in cancer- associated retinopathy. JNeuroimmunol 2000; 103: 26- 33. 22. Chin MS, Caruso RC, Derrick B, et al. Autoantibodies to p75/ LEDGF, a cell survival factor, found in patients with atypical retinal degeneration. JAutoimmun 2006; 27: 17- 2. 23. Keltner JL, Thirkill CE. The 22- kDa antigen in optic nerve and retinal diseases. J Neuroophthalmol 1999; 19: 71- 83. 24. Ing EB, Augsburger JJ, Eagle RC. Lung cancer with visual loss. Surv Ophthalmol 1996; 40: 505- 10. 25. Crofts JW, Bachynski BN, Odel JG. Visual paraneoplastic syndrome associated with undifferentiated endometrial carcinoma. Can J Ophthalmol 1988; 23: 128- 32. 26. Bazhin AV, Shifrina ON, Savchenko MS, et al. Low titre autoantibodies against recoverin in sera of patients with small cell lung cancer but without a loss of vision. Lung Cancer 2001; 34: 99- 104. 27. Savchenko MS, Bazhin Ay Shifrina ON, et al. Antirecoverin autoantibodies in the patient with non- small cell lung cancer but without cancer- associated retinopathy. Lung Cancer 2003; 41: 363- 7. 28. Bazhin Ay Savchenko MS, Shifrina ON, et al. Extracts of lung cancer cells reveal antitumour antibodies in sera of patients with lung cancer. Eur Respir J 2003; 21: 342- 6. 29. Bazhin AV, Savchenko MS, Shifrina ON, et al. Recoverin as a paraneoplastic antigen in lung cancer: the occurrence of antirecoverin autoantibodies in sera and recoverin in tumors. Lung Cancer 2004; 44: 193- 8. 30. Heckenlively JR, Jordan BL, Aptsiauri N Association of antiretinal antibodies and cystoid macular edema in patients with retinitis pigmentosa. Am J Ophthalmol 1999; 127: 565- 73. 31. Maruyama I, Ohguro H, Ikeda Y. Retinal ganglion cells recognized by serum autoantibody against gamma- enolase found in glaucoma patients. Lnvest Ophthalmol Vis Set 2000; 41: 1657- 65. 32. Weleber RG, Watzke RC, Shults WT, et al. Clinical and electrophysiologic characterization of paraneoplastic and autoimmune retinopathies associated with antienolase antibodies. Am J Ophthalmol 2005; 139: 780- 94. 33. Fathman CG, Soares L, Chan SM, et al. An array of possibilities for the study of autoimmunity. Nature 2005; 435: 605- 11. 34. Heckenlively JR, Fawzi AA, Oversier J, et al. Autoimmune retinopathy: patients with antirecoverin immunoreactivity and panretinal degeneration. Arch Ophthalmol 2000; 118: 1525- 33. 35. Balboni I, Chan SM, Kattah M, et al. Multiplexed protein array platforms for analysis of autoimmune diseases. Annu Rev Lmmunol 2006; 24: 391^ 18.' 11 |
