Treatment of retinal arterial occlusion with local fibrinolysis using recombinant tissue plasminogen activator.

OBJECTIVE: Retinal arterial occlusion is one of the most dramatic problems faced by ophthalmologists because of its sudden onset and the severe consequences it may have on the visual system. In this study, local intra-arterial fibrinolysis (LIF) using recombinant tissue plasminogen activator (rTPA) as a new technique for the treatment of retinal arterial occlusion was investigated. DESIGN: Retrospective, noncomparative case series. PARTICIPANTS: Strict inclusion and exclusion criteria were used to select patients for treatment. Fifty-three patients with central retinal artery occlusion (n = 46) or branch retinal arterial occlusion (n = 7) were enrolled. INTERVENTION: For a maximum of 3 hours, 10- to 20-mg rTPA per hour in 50-ml sodium chloride was infused transfemorally by catheterization of the ophthalmic artery with a variable stiffness microcatheter. MAIN OUTCOME MEASURES: The best-corrected visual acuity for distance by an 18-line logarithmic table was measured on admission, at 24 hours, and at 3 months after intervention. RESULTS: At 3 months, visual acuity had improved in 35 (66%) of 53 patients. Twenty-five (47.2%) patients showed an improvement of more than 2 lines, and in 10 (18.8%) patients, improvements of 1 to 2 lines were observed. No change in visual acuity occurred in 12 (22.6%) patients, and in 6 (11.3%) patients, the visual acuity deteriorated. The mean occlusion time was 14 hours (range, 3-50 hours). No statistically significant correlation was found between occlusion time and visual outcome (P > 0.22). In two patients, a temporary slight hemiplegia was observed during catheterization, and in one patient, a hypertensive crisis after LIF treatment was observed. CONCLUSIONS: The high success rate of LIF using rTPA in patients suffering from retinal arterial occlusion is supposedly due to a causal effect of rTPA on primary platelet-fibrin emboli and secondary thrombi. The local fibrinolytic therapy with rTPA involves little risk for patients selected by strict inclusion and exclusion criteria. It may be used for the treatment of retinal arterial occlusion even later than 8 hours after the acute visual loss. However, a successful outcome of the therapy depends on the prompt referral by well-informed ophthalmologists; a speedy execution of all internal, neurologic, and ophthalmologic diagnostic measures; and a prompt therapy.

Coincidence of cytomegalovirus retinitis and central retinal vein occlusion in a patient with hemophilia B and AIDS.

A 37-year-old man with hemophilia B, acquired immunodeficiency syndrome, and a unilateral cytomegalovirus retinitis developed a central retinal vein occlusion. This vascular complication occurred despite effective antiviral drug treatment with improvement of the fundus and despite decreased blood coagulability due to hemophilia B. Additional analyses of thrombophilic parameters did not reveal hints of systemic thrombophilia, suggesting that toxic and inflammatory effects of cytomegalovirus itself were responsible for the ophthalmologic aggravation.

[Intraocular pseudotumor in an AIDS patient. Block excision, differential diagnosis, histology]. / Intraokularer Pseudotumor bei einem Aids-Patienten. Blockexzision, Differentialdiagnose, Histologie.

BACKGROUND: Intraocular pseudotumors are a rare event in Aids patients and often pose diagnostic problems. CASE REPORT: A 37-year-old patient who had had HIV seroconversion for 7 years was seen to developed progressively growing, multiple, disseminated, subretinal lesions OD > OS, accompanied by exudative retinal detachment and iritis. Since all etiological laboratory diagnostic efforts to detect an infectious, noninfectious and neoplastic systemic lesion failed, a diagnostic and curative therapeutic chorioretinal excisional biopsy specimen of the largest of the tumors (3 x 3 x 2 mm) was taken. The histological work-up demonstrated granulation tissue similar to an intraocular pseudotumor without signs of infection, malignancy or reactive lymphoid hyperplasia. This finding resulted in systemic corticosteroid treatment with complete resolution of the lesions in both eyes and no recurrences. CONCLUSIONS: An invasive diagnostic procedure in patients suffering from lesions of unknown cause resulting in the institution of an appropriate medical treatment may be beneficial for the integrity and vision of the respective eye.

[Secondary prevention of cytomegalovirus retinitis with ganciclovir]. / Zur Sekundärprophylaxe der Zytomegalieretinitis mit Ganciclovir.

The prophylaxis of cytomegalovirus retinitis reactivation is effective in reducing the risk of blindness and in prolonging the remission interval and time of survival if given daily throughout life. In this study, a newly developed therapeutic regimen with 3 infusions a week was compared to the conventional maintenance therapy of 5 infusions a week using the same total weekly dose. For this purpose, ten patients were given 10 mg ganciclovir/kg 3 times a week (group A), and 18 received 6 mg ganciclovir/kg once daily for 5 days a week (group B). Only patients with newly diagnosed retinitis were included in this study. Both groups were comparable regarding their general health and ocular state at the beginning of the study. Induction therapy for stabilization of retinitis had to be given for 17.1 and 16.7 days (P = 0.785). Visual acuity was 0.5 and 0.7, respectively, at the beginning (P = 0.128) and 0.5 each at the end of the study (P = 0.875). Fifty-six percent of both groups presented with central retinal involvement at the beginning, whereas it was 56 and 78%, respectively, at the end (P = 0.250). The retinitis was found to have progressed more than 0.5 papilla diameters (pd) after 63.8 and 64.0 days (P = 0.996) and more than 1 pd after 117.6 and 77.8 days (P = 0.350). New induction therapy had to be performed after 147.9 and 131.5 days, respectively (P = 0.598). The maintenance therapy had to be interrupted due to side effects for 1.4 and 8.3 days, respectively (P = 0.185). According to these results, the prophylaxis of retinitis reactivation with 3 x 10 mg ganciclovir/kg per week is as effective as the established one with 5 x 6 mg/kg per week and can thus be recommended for an improvement in the quality of life for the patients concerned. No problems with this therapy were noted.

[Humoral immune response to retinal S-antigen in patients with uveitis]. / Humorale Immunantwort gegen retinales S-Antigen bei Patienten mit Uveitis.

Experimental autoimmune uveoretinitis (EAU) is a predominantly T-cell-mediated autoimmune inflammatory disease of the retina and uveal tract of the eye. Retinal S-antigen, a protein found in retinal photoreceptor cells, is a potent agent for the induction of EAU in susceptible species and strains. Elevated titers of antibody to S-antigen have been reported in patients with different forms of uveitis. Serum samples from 166 patients and 87 healthy blood donors were tested by immunoblotting against human retinal abstract for IgG, IgM and IgA antibodies to S-antigen. Compared to the controls the patient sera showed a higher incidence of S-specific antibodies (17.5% vs 9.2%). No specific correlation between the presence of any type of uveitis and anti-S antibodies has been found (anterior uveitis 15.1%, posterior 19.6%, panuveitis 18.9%). There was a higher incidence especially with IgG antibodies during active disease (19.7% vs 9.2% in controls). The results suggest that since EAU is T-cell mediated, antibodies in humans may be most important as indicators of autoimmunity rather than mediators of the inflammation. As these anti-S antibodies might be induced by disruption and nonspecific inflammation of the retina and uvea alone, an important and difficult question in patients is whether or not these secondary autoimmune response can contribute to the induction of uveitis.

S-antigen: preparation and characterization of site-specific monoclonal antibodies.

Previous attempts to prepare monoclonal antibodies (MAbs) against S-antigen, a photoreceptor cell protein involved in the visual process and a potent autoantigen for the induction of experimental autoimmune uveitis (EAU), have yielded MAbs which define only carboxyl terminal epitopes. In this study we devised alternate strategies to prepare five MAbs directed to other regions of the molecule. MAbC10C10 and MAbH11-A2 were prepared against synthetic peptides known to be uveitopathogenic and they were selected for more detailed studies. MAbC10C10 was generated against synthetic peptide BSA281-302 which contains a predictive consensus sequence for defined T cell epitopes (GIALD) as well as a consensus sequence for GTP-binding proteins. One human adenosine deaminase synthetic peptide containing an extensive amino acid sequence homology to BSA281-302 was a potent inhibitor of MAbC10C10 binding in a competitive inhibition radioimmunoassay. MAbH11-A2 was generated against peptide BSA303-332 which also contains a uveitopathogenic site. The binding site of MAbH11-A2 was determined to be within amino acid positions 305 to 314 (NLASSTIIKE) in S-antigen. This binding site corresponded closely to the binding site of an affinity-purified rat polyclonal antibody raised to human S-antigen. MAb5C6.47 was isolated from a mouse hyperimmunized with bovine S-antigen and was specific for a highly conserved sequence near the amino terminus, amino acid residues 42 to 48 (DGVVLVD). Both MAbC10C10 and MAb5C.47 were useful in screening gt11 cDNA libraries expressing S-antigen polypeptides as fusion proteins. Our results demonstrate the feasibility of producing site-specific MAbs potentially useful in the study of T cell-mediated immune mechanisms in EAU as well as in the phototransduction of vision.

Selection of antibody epitopes in an immunopathogenic neural autoantigen.

Results with two well-characterized self-antigens, cytochrome c and myelin basic protein, have led to differing opinions regarding the predominant specificities of autoantibodies, whether regions of sequence diversity or 'structurally inherent features' of a protein determine favored antigenic sites. To further examine this question, 16 antibody epitopes have been mapped on a highly immunopathogenic autoantigen, retinal S-antigen (S-Ag). The epitopes were characterized for: (1) sequence diversity and cross-reactivity on S-antigens from several species; (2) conformational dependency; and (3) probability of their occurrence on the surface of S-antigen. A single C-terminal region containing sequence diversity was most frequently recognized, but no evidence for recognition of any other regions of sequence diversity was found. Thirteen of 16 monoclonal antibodies raised to native S-Ag bound epitopes strongly predicted to be on the surface of S-antigen. Conversely, only one of six antibody preparations raised to peptides or affinity-purified on peptides was found to recognize an epitope predicted to be on the surface, suggesting a good correlation between specificity for conformation-dependent sites and surface probability based on the surface prediction algorithm. Three of these six antibodies which preferred denatured epitopes bound sites which overlapped or coincided with T cell sites; two of these T cell sites are immunopathogenic. The epitopes recognized on denatured antigen and peptides were similar whether the antibodies were elicited with intact human or bovine S-antigen or with cyanogen bromide-cleaved peptides. Our data suggests that in the case of S-antigen, structural features are more significant factors in epitope selection than sequence diversity.

Identification of the main immunogenic region of retinal S-antigen: subordinate influence of MHC, IGH, species or strain differences on the specificity of the antibody response.

The factors which lead to selection of dominant antigenic sites concentrated in discreet regions of proteins and polypeptides are important to the development of antigen-specific immunotherapies for autoimmune diseases and for vaccine design. In this study, the main immunogenic regions of the immunopathogenic autoantigen, retinal S-antigen, have been identified by examination of the specificity of antibody responses of different species. Using cyanogen bromide and synthetic peptides in western blots and the ELISA, the specificities of antisera from rabbits, guinea pigs, rats and 19 inbred strains of mice were tested. All animals produced high titers of antibody to S-antigen with the exception of PL/J mice. Antibodies which bound epitopes contained in peptide CB46, a 46 amino acid-containing peptide located at the C-terminus of S-antigen, were dominant in all species and strains tested. The epitopes in CB46 were multiple, overlapping, and concentrated in a stretch of approximately 30 residues. Two overlapping synthetic peptides from that region substantially competed the anti-CB46 response of all animals. Antibodies which recognized peptide CB47, a 47 residue peptide from the N-terminus, comprised the next most common group. This epitope was similar in all mice and overlapped the epitope defined by rat antibodies. All anti-CB47 antibodies mapped to an 11 residue region of CB47. Eleven strains of mice did not respond to CB47 after one immunization with S-antigen; however, multiple immunizations readily converted all animals so tested to CB47 responders. Rabbits and guinea pigs exhibited very weak responses to CB47 following one immunization; multiple immunizations increased the response minimally. Rats produced a strong antibody response to peptide CB123, which contains the known uveitogenic sites, while very little activity to CB123 was raised in rabbits and guinea pigs. Only 3 murine strains, LP, LP.R3, and B10.R3-71, responded with antibodies to CB123 and the epitope was mapped to a 30 residue region which in rats also contains two distinct pathogenic sites and an antibody epitope. Only rats and rabbits made antibody to the CB35 peptide; the epitopes were contained within an 18 residue sequence. The results show that a main immunogenic region is located in S-antigen near the C-terminus and is independent of species or MHC. Less dominant, species and strain-dependent immunogenic regions were found in three other areas, i.e. peptides CB47, CB123 and CB35.

Epitope mapping of bovine retinal S-antigen with monoclonal antibodies.

We examined the binding of seven murine monoclonal antibodies raised to S-antigen, an immunopathogenic, 404 residue photoreceptor cell autoantigen which induces experimental autoimmune uveoretinitis. S-antigen has also been identified as arrestin, a protein involved in the regulation of phototransduction. One additional monoclonal antibody (C10C10), raised to a synthetic peptide (peptide N) corresponding to residues 281 to 302 in bovine S-antigen, was also studied. In preliminary studies we examined the specificity of the antibody response to bovine S-antigen in sera from Balb/c mice. Western blots of mouse sera on the cyanogen bromide digest of bovine S-antigen demonstrated that all animals produced antibody which recognized epitopes within the C-terminal cyanogen bromide peptide designated CB46. Mice of the H-2d haplotype, including the Balb/c strain often used to produce monoclonal antibodies, showed little activity to cyanogen bromide peptides other than CB46. Also, all seven of the monoclonals raised to S-antigen are specific for epitopes in the CB46 peptide. The epitopes recognized by the monoclonal antibodies could be grouped into four distinct sites defined by peptides AE-1 (A2G5), peptide AA (PDS-1), peptide 19-OV (A9C6), and peptide 199 (BDS-1,2,3 and 4). The mono-clonal antibody, C10C10, raised to peptide N recognizes an epitope in the N peptide and binds to a larger cyanogen bromide peptide designated CB123 as well as intact S-antigen. Fine mapping of these epitopes was done with various subpeptides. None of the antibodies bound the known immunopathogenic peptide, peptide M, which resides in CB123 although the C10C10 antibody binds a peptide adjacent to peptide M.

Preparation of overlapping peptides of bovine retinal S-antigen and their localization by immunoblotting with peptide-specific antibodies.

Bovine retinal S-antigen was cleaved by three chemical cleavage procedures including o-iodosobenzoic acid (IBA), mile acid and cyanogen bromide. The resultant peptides were used to study antibody-defined epitopes. Treatment with IBA, which cleaves primarily at tryptophanyl peptide bonds, produced at least 4 major fragments and several minor fragments. The peptides have been identified by their migration on SDS-PAGE and tested for their immunoreactivity to several affinity-purified anti-CNBr-peptide antibodies and to affinity-purified anti-IBA peptide antibodies. The presence of a single tryptophan residue 194 residues from the amino-terminus should result in 2 fragments of approximately 23,000 and 26,000 molecular weight based on the known size of intact S-antigen. The additional fragmentation is due to the presence of acid labile bonds and cleavage at IBA-sensitive tyrosyl residues associated with a side reaction. Western immunoblots using affinity-purified antibodies against the various IBA and CNBr peptides have allowed location of these peptides within the intact molecule. Specifically, IBA23K and IBA21K are overlapping fragments on the carboxy end, mutally exclusive of all other peptides. IBA15K and IBA5.6K overlap, and IBA18K and IBA10K overlap within IBA26K which comprises the N-terminal half of S-Ag. Additionally, IBA10K contains an antibody epitope destroyed by CNBr cleavage of the methionyl residue between CB53 and CB56. Further characterization of these IBA peptides will expedite the location of possible additional uveitogenic epitopes in the amino-terminal half of S-Ag as well as epitopes lost by other peptide generating techniques.

Assignment of several epitopes to cyanogen bromide peptides of bovine retinal S-antigen by immunoblotting with peptide-specific antibodies.

Peptide fragments of bovine S-antigen, an immunopathogenic retinal autoantigen which mediates experimental autoimmune uveoretinitis, were produced by cyanogen bromide cleavage and used to study antibody-defined epitopes, primarily those defined by antibodies from Lewis rats immunized with the intact antigen or various peptide fragments purified from the digests by HPLC. Antibodies from the sera have been affinity-purified on several of the peptides and examined by western blot analysis and enzyme-linked immunosorbent assay on S-antigen, digests and purified fragments. By immunoblotting it could be shown that five of the purified peptides, CB46, CB47, CB67, CB74 and CB123 were immunogenic, eliciting antibodies which recognized the peptides to which they were prepared; all, except for CB67, elicited antibodies which also bound intact S-antigen. Two more peptides, CB14 and CB27 were not immunogenic and did not contain epitopes. An epitope was also found in CB35, a previously uncharacterized peptide. Using these procedures together with amino acid sequence and composition data, we have been able to determine the origins of the peptides which contain antibody epitopes, including those which we have previously determined to possess epitopes recognized by class II MHC-restricted T cell lines raised to S-antigen and several of the peptides. A T cell line specific for the non-uveitogenic CB47 peptide was unable to transfer the disease.