Cell-mediated immunity against human retinal extract, S-antigen, and interphotoreceptor retinoid binding protein in onchocercal chorioretinopathy

Autoimmune mechanisms are thought to be involved in the pathogenesis of onchocercal chorioretinopathy. Cell-mediated immune responses to human retinal S-antigen, interphotoreceptor retinoid binding protein (IRBP), and crude retinal extract were investigated in patients with onchocerciasis from Sierra Leone, West Africa using a two-step migration-inhibition factor assay. Patients were subdivided into three groups: (1) without ocular involvement (n = 10), (2) with ocular onchocerciasis limited to the anterior segment (n = 19), and (3) with onchocercal chorioretinopathy (n = 21). A group of endemic controls (n = 25) from Sierra Leone were also studied. The cellular immune response to concanavalin A (Con A) was measured to assess the general capacity of lymphocytes to respond to a mitogen. Four of 50 (8%) patients with onchocerciasis and four of 25 (16%) endemic controls reacted with at least one retinal antigen. From the patients with onchocercal chorioretinopathy two of 21 (10%) showed a positive cellular response. The general mitogen response tested with Con A was positive in all these individuals. A role for an antiretinal autoimmune mechanism in the pathogenesis of onchocercal chorioretinopathy, as studied with human S-antigen, IRBP, or crude retinal extract, could not be shown because the cellular response to these antigens did not differ in patients with or without onchocercal chorioretinopathy or in endemic controls.     

Analysis of aqueous humour in ocular onchocerciasis

The development of an onchocercal chorioretinopathy from the first detectable signs to a full blown oncho fundus is not fully understood. We investigated the intraocular humoral immune response against Onchocerca volvulus, human S-antigen, IRBP and crude retinal extract (using an ELISA) by examining paired aqueous humour and serum samples obtained from onchocerciasis patients (without [n = 10] and with ocular symptoms [n = 8]) and endemic controls [n = 14] from Sierra Leone (West Africa). A local intraocular anti-retinal IgG antibody production could not be demonstrated in onchocerciasis patients, whether they had ocular symptoms or not. A significantly higher level of O. volvulus antibodies and IgG was measured in the aqueous of onchocerciasis patients with ocular involvement, as compared to patients without ocular symptoms (Mann-Whitney ranksum test; p less than 0.001 and p less than 0.02 respectively). Since interleukin-6 (IL-6) plays an essential role in the differentiation of B cells into immunoglobulin producing plasma cells, we therefore measured this cytokine in paired aqueous and serum samples. Elevated IL-6 levels were found in the aqueous of two out of eight onchocerciasis patients tested. In view of these findings it seems improbable that retinal autoimmunity is a major factor in the pathogenesis of onchocercal chorioretinopathy. The high intraocular levels of antibodies against the parasite suggest a direct involvement of the parasite in the pathogenesis of onchocercal chorioretinopathy.     

Antibody affinity to retinal S-antigen in patients with retinal vasculitis.

Using a modified enzyme-linked immunosorbent antibody method that included dissociation of antigen antibody complexes with sodium thiocyanate, we examined the functional affinity of antibody to retinal S-antigen in 48 patients with retinal vasculitis and 46 age-matched healthy control subjects. Antibody affinity was markedly lower in patients with retinal vasculitis than in healthy subjects. Low-affinity antibody was more prevalent in acute retinal vasculitis and in patients with normal levels of circulating immune complexes. We found distinct differences between the antiretinal antibodies found in patients with retinal vasculitis and those in control subjects. The association of low-affinity antibody with normal levels of circulating immune complexes may suggest defective regulation of antiretinal autoimmunity and have important pathogenic implications.     

Human antiretinal antibodies in toxoplasma retinochoroiditis

BACKGROUND/AIMS—Toxoplasma retinochoroiditis (TR) is an important cause of blindness and visual morbidity, affecting young adults. It has been postulated that some of the retinal damage observed in TR is due to antiretinal autoimmune mechanisms.
METHODS—Humoral antiretinal autoimmunity in TR was investigated by indirect immunofluorescence (IIF) on normal human cadaveric retina and by a human retinal S-antigen ELISA. 36 patients with TR were separated on clinical grounds into those with first recurrence of disease (n=18) or those with multiple recurrences (n=18). Patients were also segregated into those with active (n=28) or quiescent disease (n=8). Serum from 16 normal controls (six with positive toxoplasma serology and 10 without) with no evidence of eye disease and 12 patients with idiopathic retinal vasculitis (IRV) were also tested.
RESULTS—Sera from 34 of the 36 patients (94%) with TR demonstrated photoreceptor layer reactivity by IIF contrasting with six of 16 normal controls (p= <0.001) and three of 12 IRV patients (p= <0.001). Titres of antiphotoreceptor antibody were also higher among TR patients than controls. Sera from 27 of the 36 TR patients, 10 of 16 normals, and nine of 12 retinal vasculitis patients possessed anti-human retinal S-antigen antibodies at a titre of 1:400 or more as assessed by ELISA (p= >0.05). Antiretinal autoantibody as detected by IIF did not run in parallel with S-antigen reactivity.
CONCLUSIONS—The data indicate that the extent of antiretinal reactivity within TR is not accounted for by anti-S-antigen antibodies alone. This remarkably high prevalence of antiphotoreceptor antibody in TR as opposed to that found in either healthy or disease controls suggest that these antibodies may be co-pathogenic in toxoplasma retinochoroiditis.

Keywords: retinochoroiditis; toxoplasma; immunofluorescence; autoimmunity     

Does autoimmunity to S-antigen play a role in Fuchs' heterochromic cyclitis?

Autoimmunity directed against retinal or choroidal antigens has been suggested to play a role in the chorioretinal lesions observed in patients with Fuchs'' heterochromic cyclitis. This hypothesis was addressed and patients with Fuchs'' heterochromic cyclitis were tested for cellular immunity (migration inhibitory factor assay) against human retinal S-antigen. A significantly higher percentage of patients with Fuchs'' heterochromic cyclitis had a positive cellular autoimmune response to S-antigen than healthy controls and other patients with anterior uveitis. This finding is remarkable since Fuchs'' heterochromic cyclitis is generally classified as an anterior uveitis and patients with Fuchs'' heterochromic cyclitis without chorioretinal lesions also had a positive test. In view of these results and a sensitisation against a corneal antigen reported earlier in Fuchs'' heterochromic cyclitis, it is suggested that a chronic low grade grade anterior uveitis or chorioretinitis of unknown origin may cause the release of potent autoantigens in these patients.     

An Immunologic Study on Age-related Macular Degeneration

Forty-one patients with age-related macular degeneration(AMD) were detected for serum autoantibodies against normal humanretinal protein by means of Western immunoblot analysis.Twenty-sevenout of the 41 patients showed positive response,with a rate of 66 percent.The positive rate of antiretinal antibody in the AMD patients wassignificantly higher than that in normal controls (18%) and in patients withother retinal diseases (24%) (p<0.0005).These antiretinal antibodies fromthe AMD patients partly reacted with the retinal protein of molecular weightbetween 28 and 32 Kd,partly with Mr of 38 to 42 Kd,48 to 52 Kd,62 to65 Kd or 110 to 130 Kd.Of them,the antiretinal antibody against theprotein with Mr of 28 to 32 Kd in the AMD patients was higher than innormal controls (p<0.05).Two or more antibodies were found in theserum from AMD patients,showing a significant difference between thepatients and the controls (p<0.005).The results indicated that in theoccurrence of AMD and/or during its developing process there wereinflammation and immunological response,involving antibodies againstretinal proteins of various molecular weight.Eye Science 1993;9:113-120.     

Cellular immune responses to retinal antigens in retinitis pigmentosa

Patients with retinitis pigmentosa and a group of controls were tested for their cellular immune response toward two retinal proteins, S-antigen and inter-photoreceptor retinoid-binding protein (IRBP), as well as their reaction against two synthetic peptides (M and N) derived from the sequence of S-antigen and peptide R14, derived from IRBP. Positive responses to the retinal antigens were found in larger proportions and with higher levels in the patient group than in the controls. The difference between the two groups was statistically significant in their response to S-antigen, but the patients reacted better than the controls against the other antigens as well. Of particular interest was the finding that several patients responded to both retinal proteins and/or to their peptides. These patients suffered from severe retinal changes and the data are thus interpreted as suggesting that the responses to the retinal antigens are secondary to these changes and to non-physiological release of retinal antigens. Offprint requests to: M. Mochizuki     

IRBP免疫鼠血清特异性抗体的测定及动态变化

我们首次在国内提纯了光感受器间维生素 A类结合蛋白(interphotoreceptor retinoid-binding protein,IRBP),将其免疫 Lewis 大鼠后动态测定了鼠血清抗IRBP 抗体和抗视网膜 S 抗原抗体.发现抗 IRBP 抗体于免疫后第7天出现,以后逐渐上升,于第26天达高峰,未测出抗 S 抗原抗体.根据特异性抗体与实验性自身免疫性葡萄膜视网膜炎(experimental autoimmune uveoretinitis,EAU)之间的关系,讨论了特异性体液免疫反应在 EAU发生中的作用.     

A point prevalence study of 150 patients with idiopathic retinal vasculitis: 2. Clinical relevance of antiretinal autoimmunity and circulating immune complexes.

This study describes the occurrence of antiretinal antibodies and circulating immune complexes in the sera of a large series of patients with idiopathic retinal vasculitis whose ophthalmological and clinical features are presented in Part 1. Antiretinal antibodies were measured by indirect immunofluorescence and passive haemagglutination, and circulating immune complexes were measured by polyethylene glycol precipitation and Clq binding. The occurrence of antiretinal antibodies and that of circulating immune complexes were analysed in relation to each other, to severity of retinal disease, to the type of associated systemic inflammatory disease, and to the presence of individual features of retinal inflammation. In patients with retinal vasculitis together with systemic inflammatory disease circulating immune complexes were usually accompanied by antiretinal antibodies. However, those patients with antiretinal antibodies in the absence of circulating immune complexes tended to have more severe retinal vasculitis, a feature particularly evident in Behçet''s disease (p = 0.028). In patients with isolated retinal vasculitis, severity of disease was associated with antiretinal antibody (p = 0.013), as well as with the occurrence of both antiretinal antibody and circulating immune complexes together (p = 0.010). In the series as a whole there was a tendency for individual features of retinal vasculitis to be associated with antiretinal antibodies unaccompanied by circulating immune complexes; especially in macular oedema (p = 0.028). In isolated retinal vasculitis there was also an additive effect of antiretinal antibodies and circulating immune complexes in relation to disease severity; in contrast, in patients with systemic inflammatory disease, the coexistence of antiretinal antibodies and concluded that both antiretinal autoimmunity and circulating immune complexes may act as immunopathogenetic factors in idiopathic retinal vasculitis but that, in certain patients, circulating immune complex formation seems to protect against the more severe forms of autoimmune retinal inflammatory disease.     

Immune response to retinal antigens in patients with gyrate atrophy and other hereditary retinal dystrophies

PURPOSE: Gyrate atrophy (GA) is a rare hereditary disease that causes retinal destruction. Retinal damage in GA and other heredodegenerative diseases such as retinitis pigmentosa (RP) releases sequestered antigens and may trigger immune response to these molecules. Here, we studied the immune response to retinal antigens in patients with GA and RP and compared it with that of patients with inactive posterior uveitis and normal volunteers. Patients and methods : Peripheral blood was collected from 24 patients with RP, 10 patients with GA, 10 patients with inactive posterior uveitis, and 16 normal volunteers. Cell-mediated immune responses to human S-antigen (HS-Ag), bovine S-antigen (BS-Ag), and interphotoreceptor retinoid-binding protein (IRBP) were investigated by lymphocyte proliferation assay. In addition, serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were studied by ELISA. Immunologic data were correlated with clinical and electrophysiological findings. RESULTS: Patients with GA or RP responded to HS-Ag and BS-Ag more vigorously than patients with uveitis or healthy controls, as shown by higher mean stimulation indices and larger proportions of responders. Unlike S-Ag, IRBP stimulated low lymphocyte responses in only a small proportion of RP patients. The mean sVCAM-1 levels were significantly higher in the sera from patients with GA than in that from normal controls. CONCLUSION: An elevated cellular immune response to S-Ag is common in patients with GA and RP. This elevated cellular immune response to S-Ag may exacerbate retinal destruction in patients with GA and RP.     

Experimental ocular onchocerciasis in cynomolgus monkeys. IV. Chorioretinitis elicited by Onchocerca volvulus microfilariae

Onchocerciasis is a major cause of blindness worldwide, and much of the blindness is caused by onchocercal chorioretinitis. In an experimental animal model for ocular onchocerciasis, intravitreal injections of 10,000 live Onchocerca volvulus microfilariae isolated from infected humans into the eyes of cynomolgus monkeys (Macaca fascicularis) resulted in patchy, progressive loss of retinal pigment with pigment clumping. Areas of pigment loss were less extensive in animals that had been sensitized with microfilariae. Intravitreal injections of dead O. volvulus microfilariae resulted in mild vitritis with relatively less clinical change noted in the retina and choroid. Histopathologic examination revealed thinning and loss of outer retinal layers with pigment migration into the retina, and inflammation was more pronounced in eyes that received live microfilariae. Clinical changes appeared in eyes receiving live microfilariae before the development of significant antibody or cell-mediated immune responses. O. volvulus microfilariae appear to be more suitable than O. lienalis microfilariae in producing lesions which resemble human onchocerciasis in the primate model.     

Autoimmunity in hereditary retinal degeneration. I. Basic studies.

One hundred and sixteen patients with retinitis pigmentosa (RP), 64 patients with other eye diseases, and 36 control subjects with no known eye disease were examined for antiretinal autoimmune activity. Sera were screened by indirect immunofluorescence on normal donor human eye sections to detect antibodies to human retinal antigens. Forty three of 116 RP patients (37%), 21 out of 64 non-RP patients with other eye diseases (33%), and 1 out of 42 controls (2%) had antibodies reacting with donor eye retinal antigens. Lymphocytes were tested by an in vitro transformation assay to detect cell mediated immunity to retinal antigens. Sixteen RP patients (19%), 11 non-RP patients (18%), and four controls (10%) showed lymphocyte sensitisation. Autoimmune responses were detected in many degenerative ocular disorders, but it is not known if they play a contributory pathogenic role.     

Autoimmunity in hereditary retinal degenerations. II. Clinical studies: antiretinal antibodies and fluorescein angiogram findings.

Testing by indirect immunofluorescence for the detection of antiretinal antibodies and lymphocyte stimulation for cell-mediated immunity to retinal antigens was performed on blood obtained from 59 patients with retinitis pigmentosa (RP) and 29 without RP who had other types of retinal disease. The results from the patients' immunological studies were correlated in a masked fashion with six parameters of the fluorescein angiogram: disc staining, peripapillary oedema, vascular arcade oedema, macular oedema, and focal vascular staining (late phases), and disc telangiectasia (early phases). Significant correlations for both groups together were found for IgG antiretinal antibody reactivity and macular oedema (p less than 0.038) and disc staining (p less than 0.033). The non-RP retinal disease group had more significant correlations, including IgG antiretinal antibody reactivity with vascular arcade oedema (p less than 0.018), disc staining (p less than 0.018), and peripapillary oedema (p less than 0.023); the RP patients had significant correlation with IgG reactivity and arcade oedema (p less than 0.045). With combinations of IgG, IgM, and lymphocyte reactivity various significant correlations were found with the fluorescein angiogram.     

Differential expression of nitric oxide synthase in experimental uveoretinitis.

PURPOSE: To investigate the site and the cellular source of inducible nitric oxide synthase (iNOS) expression in human S-antigen peptide-induced experimental autoimmune uveoretinitis (EAU). METHODS: Twenty-one Lewis rats were sensitized with human S-antigen peptides. Three rats were killed each consecutive day from day 6 through day 12 after sensitization. Frozen sections of the enucleated eyes were analyzed for iNOS by the dual immunohistochemical method. Primary antibodies included rabbit anti-mouse iNOS combined with anti-human endothelium NOS, anti-rat lysosomal protein (ED1), or anti-rat major histocompatibility complex class II molecule (OX6) monoclonal antibodies. Secondary antibodies were fluorescein-conjugated anti-mouse IgG and streptavidin rhodamine-labeled anti-rabbit IgG. The adjacent sections were separately stained with ED1, iNOS, and glial fibrillary acidic protein (GFAP). The mouse macrophage cell line RAW 264.7 was exposed to either interferon (IFN)gamma/lipopolysaccharide (LPS) or S-antigen and to interphotoreceptor retinoid-binding protein (IRBP), myelin basic protein, and bovine serum albumin for 12 hours. Cells were harvested for detection of iNOS expression by northern blot analysis hybridization and detection of protein by immunohistochemistry. RESULTS: In the retina of eyes with EAU, ED1+/iNOS+ and OX6+/iNOS+ cells were first detected on day 9 after sensitization. These iNOS+ cells increased in number on subsequent days in parallel with the increasing severity of retinal damage. Most of the cells localized around the outer retina. In contrast, a large number of ED1+ and OX6+ cells that were localized in the uvea and conjunctiva were negative for iNOS. Retinal pigment epithelial cells did not stain for iNOS. Macrophages exposed to IFNgamma/LPS, S-antigen, and IRBP showed expression of iNOS mRNA and the protein. CONCLUSIONS: Macrophages are an important source of NO production in eyes with EAU. These macrophages preferentially express iNOS in the retina. Such a differential expression of iNOS by the macrophages appears to be related to retinal soluble proteins.     

Progressive subretinal fibrosis and blindness associated with multifocal granulomatous chorioretinitis: A variant of sympathetic ophthalmia

OBJECTIVE: To report a case of bilateral progressive subretinal fibrosis and blindness with multifocal granulomatous chorioretinitis occurring after intraocular surgery. We propose that this is a variant of sympathetic ophthalmia. DESIGN: Clinicopathologic case report. METHODS: The left enucleated globe was examined by histopathologic methods. The patient's sera were subjected to immunohistochemical studies against retinal antigens, and collagen 2 types in areas of fibrosis were identified. Polymerase chain reaction was used to test for herpes virus DNA in microdissected, formalin-fixed, paraffin-embedded tissue. RESULTS: The enucleated globe demonstrated histopathologic features similar to an entity previously described as progressive subretinal fibrosis with multifocal granulomatous chorioretinitis. The patient's sera demonstrated antibodies directed against retinal photoreceptors and pigment epithelium. Polymerase chain reaction for herpes virus was negative. Immunohistochemical studies demonstrated types III, IV, V, and VI collagen in areas of fibrosis. CONCLUSIONS: The clinical history along with the histopathologic and immunohistochemical findings suggest that progressive subretinal fibrosis with multifocal granulomatous chorioretinitis may represent a variant of sympathetic ophthalmia and that retinal autoimmunity may play a role in its pathogenesis.     

Electroretinographic abnormalities in multiple sclerosis: possible role for retinal autoantibodies

Background Multiple sclerosis (MS) has been associated with inflammation of the uveal tract, suggesting an immunological link between the uvea and central nervous system (CNS) in this disease. The retina is embryologically derived from the CNS, and it is conceivable that retinal antigens may also be recognized by the immune system in MS. Electroretinographic abnormalities, as well as retinal autoantibodies, have previously been described in MS. We performed this study to further explore the possibility of retinal autoimmunity in MS.Methods Thirty-four patients with clinically definite MS and thirty-seven healthy controls were recruited. All patients and controls had standard electroretinographic (ERG) testing done, as well as a brightflash ERG protocol to isolate rod photoreceptor function. Patient and control sera were analyzed for the presence of antiretinal antibodies using Western blot techniques.Results We found statistically significant differences between MS patients and controls in four ERG parameters. In the MS group, implicit times of the rod-cone b-wave response, cone b-wave response, and rod photoreceptor response were increased. The amplitudes of the photopic oscillatory potentials were reduced in the MS group. Patients with the highest titres of retinal autoantibodies had delayed rod-cone b-wave implicit times and diminished photopic oscillatory potential amplitudes.Conclusions We report ERG evidence of retinal dysfunction in patients with MS. We also report the first use of the brightflash ERG protocol in MS, which demonstrated rod photoreceptor dysfunction. Patients with the highest antiretinal antibody titres had abnormal ERG recordings. Retinal autoimmunity is a possible explanation for these observed ERG abnormalities in MS patients.     

Interphotoreceptor retinoid binding protein is a potent tolerogen in Lewis rat: suppression of experimental autoimmune uveoretinitis is retinal antigen specific

AIMS—Administration of unfractionated retinal antigen(s) (retinal extract, RE) suppresses RE induced experimental autoimmune uveoretinitis (EAU) and offers a potential therapeutic alternative to non-specific immunosuppressive therapies for posterior uveitis and autoimmune diseases. S-Ag and interphotoreceptor retinoid binding protein (IRBP) are two major autoantigens within soluble RE. It was aimed to assess, firstly, as has previously been shown with S-Ag, if IRBP can induce intranasal tolerance and, secondly, the contribution of both these major autoantigens to tolerance induction by whole RE.
METHODS—Animals were tolerised by intranasal administration with S-Ag or IRBP, either alone or in combination, or RE before immunisation with either IRBP or RE. Control animals were administered nasally either PBS or MBP. Daily clinical responses were recorded biomicroscopically and histological grades were obtained using a semiquantitative scoring system. Weekly serum antibody levels to retinal antigens were measured by ELISA and delayed hypersensitivity responses (DTH) were assessed by skin reactivity to intradermal inoculation with retinal or non-specific antigens.
RESULTS—Microgram doses of IRBP successfully suppressed both clinically and histologically IRBP induced EAU. This suppression was accompanied by reduced antigen specific DTH reactivity but maintained T cell dependent (IgG2a) antibody responses. Furthermore, combined S-Ag and IRBP administration afforded equal suppression of RE induced EAU when compared with RE therapy alone. Suppression of RE induced EAU was not achieved with administration of a non-retinal specific autoantigen, MBP. Although individually, both S-Ag and IRBP suppressed RE induced EAU, whole RE was unable to protect against IRBP induced disease.
CONCLUSIONS—Intranasal administration of IRBP suppressed IRBP induced EAU in the Lewis rat. S-Ag and IRBP are the major contributors to the tolerogenicity within RE, despite the known uveogenicity of other retinal antigens within RE and induction of tolerance was retinal antigen specific. Furthermore, suppression induced by single antigen administration is antigen specific although concomitant bystander suppression may also play a role. RE was unable to protect against IRBP induced disease despite tolerogenic levels of antigen within RE. Although this may be due in part to a dose effect of either tolerising or immunising antigen, further investigation into the possible antigen dominance of IRBP or mucosal processing of combinations of antigens is necessary so that the full efficacy of mucosal tolerance therapy can be assessed.

     

Serum antibody level to S-antigen in children with chronic uveitis.

Bovine S-antigen was purified by gel filtration and ion exchange chromatography according to previously described techniques. An enzyme linked immunosorbent assay (ELISA) using antiserum to bovine S-antigen raised in guinea-pigs was employed to detect S-antigen in the chromatographic fractions. The purity of the S-antigen was determined by SDS-PAGE electrophoresis, where a single band was found. The purified S-antigen in microgram quantities together with Freund's complete adjuvant induced uveitis in rats two weeks after injection into the foot pad. Serum samples from children suffering from chronic uveitis and healthy children were tested for antibodies to S-antigen by the ELISA. A statistically significant difference in the level of specific antibodies between patients and controls was found. There was no clear-cut correlation between the severity of uveitis and antibody titre, but cases with retinal involvement and aggressive uveitis all showed definite elevations of antibodies to S-antigen.     

A longitudinal study of clinical and immunological findings in 52 patients with relapsing retinal vasculitis.

Fifty-two patients with retinal vasculitis--26 with idiopathic disease and 26 with associated systemic inflammatory disease--were followed up for periods ranging from six months to 12 years. The aim of the study was to determine the relationship between relapse of uveitis, visual outcome, and the occurrence of circulating immune complexes (CIC) and antiretinal antibodies. In a total of 69 relapses, CIC were increased in one-third of patients and antiretinal antibodies in one-half. In those 34 patients who expressed antiretinal antibodies 27 (79%) of the relapses were characterised by antiretinal antibodies in the absence of raised CIC levels (p less than 0.01). These findings support our previous hypothesis that CIC may have a protective role in autoimmune retinal vasculitis and that antiretinal autoimmunity is of pathogenetic importance in relapse. In individual patients the visual outcome was not related to the number of relapses or to the CIC-autoantibody pattern, suggesting the operation of additional features which merit identification.     

Analysis of autoantibodies against human retinal antigens in sera of patients with glaucoma and ocular hypertension

PURPOSE: The aim of this study was to show that complex antibody patterns against retinal antigens in sera of patients with glaucoma, found in previous studies, are autoantibodies against human antigens. METHODS: Sera of 179 patients were collected at the Department of Ophthalmology (University of Mainz, Germany): non-glaucomatous control patients (n=45), primary open-angle glaucoma (n=45), ocular hypertension (n=44), and normal tension glaucoma patients (n=45). The sera were tested against Western blots of human retinal antigens. IgG antibody patterns were analyzed by multivariate statistical techniques, and some significant antigens were identified by mass spectrometry. RESULTS: All subjects, even healthy ones, showed different and complex banding patterns. Glaucoma groups showed up- and down-regulations of antibody reactivities compared to the control group. The multivariate analysis of discriminance found significant differences (p<0.05) in IgG antibody profiles between glaucoma groups, ocular hypertension, and healthy subjects against human retinal antigens. The antigen band at 12 kDa was identified as Histone H4 via mass spectrometry, the 29 kDa band as cellular retinaldehyde-binding protein, and one at 49 kDa as retinal S-antigen. CONCLUSIONS: Using human retinal antigen, we demonstrated that complex autoantibody patterns exist in sera of patients with glaucoma. Large correlations with previous studies using bovine retinal antigens could be seen.