Stephanie C. Joachim

Autoantibodies in patients with glaucoma: a comparison of IgG serum antibodies against retinal, optic nerve, and optic nerve head antigens

PurposeThe aim of this study was to analyze and compare the entire IgG autoantibody patterns against different ocular antigens (retina, optic nerve, and optic nerve head) in sera of glaucoma patients and healthy subjects.MethodsSixty-six patients were included in this study: healthy volunteers without any ocular disorders (CO, n=30), patients with primary open-angle glaucoma (POAG, n=19), and patients with normal-tension glaucoma (NTG, n=17). The sera were tested for antibodies against retinal, optic nerve, and optic nerve head tissues. Immunodetection was performed using 4-chloro-1-naphthol staining. The autoantibody patterns were digitized and subsequently analyzed by multivariate statistical techniques.ResultsAll patients showed a complex repertoire of IgG antibodies against retinal, optic nerve, and optic nerve head antigens. The analysis of discriminance revealed a statistically significant differences between the patterns of all three groups. Our multivariate approach could quantify the differences in immunoreactivities of patient sera against the three antigens. The POAG group had the most significant difference against retinal antigens (P=0.0021) compared with the other antigens. In the NTG group the highest reactivity appeared against optic nerve head (P=0.00053) and optic nerve (P=0.0025).ConclusionsAll groups showed different and complex antibody patterns against the three ocular tissues. These autoantibodies are highly specific for each patient group. The analysis of these patterns could provide further information about possible autoimmune mechanisms in the pathogenesis of glaucoma.

Antibodies to α B-Crystallin, Vimentin, and Heat Shock Protein 70 in Aqueous Humor of Patients with Normal Tension Glaucoma and IgG Antibody Patterns Against Retinal Antigen in Aqueous Humor

Purpose: To show the existence of IgG antibodies against retinal antigens in aqueous humor of normal tension glaucoma patients. Methods: Forty-two patients were included in this study. Aqueous humor was collected from control subjects (CO; n = 21) and patients with normal tension glaucoma (NTG; n = 21). Western blot methods against bovine retinal antigens were used to detect the IgG antibody patterns. The complex antibody repertoires were analyzed by multivariate statistical techniques. Mass spectrometry was used to identify the most important antigens. Results: Very complex IgG antibody patterns against retinal antigens were found in all analyzed aqueous humor samples. Our multivariate approach could quantify differences in immunoreactivities, and including all peaks, the analysis of discriminance revealed a statistical significant difference between the patterns of the NTG and the CO group (p < 0.001). The antigen band at 21 kDa was identified as α B-crystallin, the 57-kDa antigen band as vimentin, and one at 70 kDa as heat shock protein 70. Conclusions: We could demonstrate that complex IgG antibody patterns against retina exist in aqueous humor. The significant differences in the antibody pattern of the glaucoma group compared with the nonglaucoma group in aqueous humor confirm the results of previous studies using sera of glaucoma patients. These differences in antibody patterns might be further evidence for an autoimmune involvement in the pathogenesis of some glaucoma patients.

Sera of glaucoma patients show autoantibodies against myelin basic protein and complex autoantibody profiles against human optic nerve antigens

BackgroundThe aim of this study was to gain more information about the possible immunological mechanisms in glaucoma. We analyzed the complex autoantibody patterns against human optic nerve antigens in sera of patients with glaucoma and tried to identify important antigens.MethodsSera of 133 patients were included: healthy control subjects (n = 44), primary open-angle glaucoma (n = 44), and normal tension glaucoma patients (n = 45). The sera were tested against Western blots of human optic nerve, and antibody bands were visualized with chloronaphthol. IgG antibody patterns were analyzed by multivariate statistical techniques, and the most significant antigens were identified by mass spectrometry (Maldi-TOFTOF).ResultsAll subjects, even healthy ones, showed different and complex antibody patterns. Glaucoma groups showed specific 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 against human optic nerve antigens between both glaucoma groups and healthy subjects. The identified antigens include: myelin basic protein (up-regulated in the POAG group), glial fibrillary acidic protein (down-regulated in the glaucoma groups), and vimentin (down-regulated in the glaucoma groups in comparison to controls).ConclusionsUsing human optic nerve antigen, we were able to demonstrate that complex IgG autoantibody patterns exist in sera of patients with glaucoma. Large correlations between the given and our previous studies using bovine optic nerve antigens could be seen. Furthermore, anti-myelin basic protein antibodies, which can also be detected in patients with multiple sclerosis, were found in sera of glaucoma patients.

Autoimmunity and glaucoma.

Elevated intraocular pressure does not explain glaucoma in all patients, but there is information that autoimmune mechanisms may be involved in this disorder. This review attempts to reveal the findings about specific changes in autoantibody profiles in glaucoma patients and their possible role in glaucoma. Considering that these changes in natural autoimmunity can be found consistently among different study populations, it might be a promising new tool for glaucoma detection.

Changes in Prostaglandin Levels in Patients Undergoing Femtosecond Laser-Assisted Cataract Surgery

PURPOSE To investigate the intraocular prostaglandin concentrations after femtosecond laser treatment and the potential relationship to miosis. METHODS Aqueous humor was collected from patients after femtosecond laser pretreatment and at the beginning of routine cataract surgery. The total prostaglandin and the prostaglandin E2 concentrations were measured in two independent studies using an enzyme-linked immunoassay. RESULTS A significantly higher level of prostaglandins was noted in the aqueous humor of patients immediately after femtosecond laser treatment. This could be confirmed in two studies consisting of independent patient populations (study I: control = 17.3 ± 4.0 pg/mL [n = 22], femtosecond laser [femto] = 182.1 ± 38.1 pg/mL [n = 22], P = .0001, and study II: control = 17.5 ± 1.4 pg/mL [n = 37], femto: 377.1 ± 83.6 pg/ mL [n = 35], P = .00004). A significant increase of prostaglandin E2 was noted in two measurements (study III: control = 4.5 ± 1.9 pg/mL [n = 13], femto: 19.2 ± 2.5 pg/mL [n = 20], P = .0002, and study IV: control = 11.3 ± 1.6 pg/mL [n = 35], femto: 60.3 ± 16.1 pg/mL [n = 36], P = .004). No correlation was noted between age or cataract density and prostaglandin/prostaglandin E2 level or between corneal incision, suction time, or laser time in the femto groups and prostaglandin/prostaglandin E2 level. CONCLUSIONS Prostaglandins rise immediately after femtosecond laser treatment. Future patients should perhaps be treated with non-steroidal anti-inflammatory drugs to maintain mydriasis before undergoing femto-second laser treatment for cataract surgery.

Inflammatory demyelination induces glia alterations and ganglion cell loss in the retina of an experimental autoimmune encephalomyelitis model

BackgroundMultiple sclerosis (MS) is often accompanied by optic nerve inflammation. And some patients experience permanent vision loss. We examined if the grade of optic nerve infiltration and demyelination affects the severity of clinical signs in an experimental autoimmune encephalomyelitis (EAE) model. The loss of retinal ganglion cells (RGC) and alterations in glia activity were also investigated.MethodsC57BL/6 mice were immunized with peptide MOG35-55 in complete Freund’s adjuvant (CFA) and controls received PBS in CFA. Then 23 days post immunization eyes were prepared for flatmounts and stained with Nissl to evaluated neuronal density. Clinical EAE symptoms as well as cell infiltration and demyelination in the optic nerve were examined. Retinal sections were stained with hematoxylin and eosin and silver stain. Immunohistochemistry was used to label RGCs (Brn-3a), apoptotic cells (caspase 3), macroglia (glial fibrillary acidic protein (GFAP)), microglia (Iba1), macrophages (F 4/80) and interleukin-6 (IL-6) secretion.ResultsEAE symptoms started at day 8 and peaked at day 15. Cell infiltrations (P = 0.0047) and demyelination (P = 0.0018) of EAE nerves correlated with the clinical score (r > 0.8). EAE led to a significant loss of RGCs (P< 0.0001). Significantly more caspase 3+ cells were noted in these animals (P = 0.0222). They showed an increased expression of GFAP (P< 0.0002) and a higher number of microglial cells (P< 0.0001). Also more macrophages and IL-6 secretion were observed in EAE mice.ConclusionsMOG immunization leads to optic neuritis and RGC loss. EAE severity is related to the severity of optic nerve inflammation and demyelination. EAE not only affects activation of apoptotic signals, but also causes a glial response in the retina.

Proteomics in ocular fluids.

The focus of this article is to review recent techniques in proteomic analysis of ocular fluids. These fluids include tears, aqueous humor, and vitreous, they will also be compared to serum analysis. Furthermore, we attempt to summarize some disease correlated biomarkers in ocular fluids that were discovered through different proteomic techniques in eye diseases like dry eye, glaucoma, age‐related macular degeneration, uveitis, or diabetic retinopathy. This review is trying to point out the importance of these biomarkers for clinical applications.

Retinal Ganglion Cell Loss Is Accompanied by Antibody Depositions and Increased Levels of Microglia after Immunization with Retinal Antigens

Background Antibodies against retinal and optic nerve antigens are detectable in glaucoma patients. Recent studies using a model of experimental autoimmune glaucoma demonstrated that immunization with certain ocular antigens causes an immun-mediated retinal ganglion cell loss in rats. Methodology/Principal Findings Rats immunized with a retinal ganglion cell layer homogenate (RGA) had a reduced retinal ganglion cell density on retinal flatmounts (p = 0.007) and a lower number of Brn3+retinal ganglion cells (p = 0.0001) after six weeks. The autoreactive antibody development against retina and optic nerve was examined throughout the study. The levels of autoreactive antibodies continuously increased up to 6 weeks (retina: p = 0.004; optic nerve: p = 0.000003). Additionally, antibody deposits were detected in the retina (p = 0.02). After 6 weeks a reactive gliosis (GFAP density: RGA: 174.7±41.9; CO: 137.6±36.8, p = 0.0006; %GFAP+ area: RGA: 8.5±3.4; CO: 5.9±3.6, p = 0.006) as well as elevated level of Iba1+ microglia cells (p = 0.003) was observed in retinas of RGA animals. Conclusions/Significance Our findings suggest that these antibodies play a substantial role in mechanisms leading to retinal ganglion cell death. This seems to lead to glia cell activation as well as the invasion of microglia, which might be associated with debris clearance.

Analysis of IgG antibody patterns against retinal antigens and antibodies to α-crystallin, GFAP, and α-enolase in sera of patients with “wet” age-related macular degeneration

BackgroundThe aim of this study was to compare the IgG antibody patterns against retinal antigens in sera of patients with age-related macular degeneration (AMD) and healthy subjects to learn more about possible immunological aspects of this disease and to identify some of the most important antigens.MethodsSera of 140 patients were analyzed: healthy volunteers (CO, n=101) and patients with “wet” age-related macular degeneration (AMD, n=39). The sera were tested against western blots of bovine retinal antigens. The IgG antibody patterns were analyzed by multivariate statistical techniques and some antigens were identified via LC-MS/MS.ResultsAll patients showed complex patterns of IgG antibodies against retinal antigens. The discriminant analysis revealed a statistical significant difference between the antibody profiles of the AMD and the CO group (P=0.000023). Not only up-regulations of antigen-antibody-reactivities in the AMD group at some molecular weight ranges, e.g. at 46 and 52 kDa, could be seen, but also down-regulations, e.g. at 18 and 36 kDa. The 18 kDa antigen band was identified as αB-crystallin, the band at 46 kDa as α-enolase, and one at 52 kDa as glial fibrillary acidic protein.ConclusionsWe could demonstrate that both groups (wet AMD and CO) show complex IgG antibody patterns against retinal antigens, which are highly specific for each group. This provides further hints for the immunological basis of the disease. These changes in the antibody profiles in “wet” AMD could represent a secondary response to retinal damage or can play a causative role in the disease.

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.