Choroidal γδ T cells in protection against retinal pigment epithelium and retinal injury.

γδ T cells located near the epithelial barrier are integral components of local inflammatory and innate immune responses. We have previously reported the presence of choroidal γδ T cells in a model of chronic degeneration of the retinal pigment epithelium (RPE). The goals of the current study were to further define the functions of choroidal γδ T cells and to explore the underlying mechanisms of their action. Our data demonstrate that choroidal γδ T cells are activated by RPE injury in response to NaIO3 treatment, and that they express genes that encode immunosuppressive cytokines, such as IL-4 and IL-10. γδ-T-cell-deficient mice developed profound RPE and retinal damage at doses that caused minimal effects in wild-type mice, and adoptive transfer of γδ T cells prevented sensitization. Intravitreal injection of IL-4 and IL-10 ameliorated RPE toxicity that was induced by NaIO3Ex vivo coculture of γδ T cells with RPE explants activated the production of anti-inflammatory cytokines via an aryl hydrocarbon receptor (AhR)-dependent mechanism. AhR deficiency abolished the protective effects of γδ T cells after adoptive transfer. Collectively, these findings define important roles for choroid γδ T cells in maintaining tissue homeostasis in the outer retina.-Zhao, Z., Liang, Y., Liu, Y., Xu, P., Flamme-Wiese, M. J., Sun, D., Sun, J., Mullins, R. F., Chen, Y., Cai, J. Choroidal γδ T cells in protection against retinal pigment epithelium and retinal injury.

Mouse genetics and proteomic analyses demonstrate a critical role for complement in a model of DHRD/ML, an inherited macular degeneration.

Macular degenerations, inherited and age related, are important causes of vision loss. Human genetic studies have suggested perturbation of the complement system is important in the pathogenesis of age-related macular degeneration. The mechanisms underlying the involvement of the complement system are not understood, although complement and inflammation have been implicated in drusen formation. Drusen are an early clinical hallmark of inherited and age-related forms of macular degeneration. We studied one of the earliest stages of macular degeneration which precedes and leads to the formation of drusen, i.e. the formation of basal deposits. The studies were done using a mouse model of the inherited macular dystrophy Doyne Honeycomb Retinal Dystrophy/Malattia Leventinese (DHRD/ML) which is caused by a p.Arg345Trp mutation in EFEMP1. The hallmark of DHRD/ML is the formation of drusen at an early age, and gene targeted Efemp1(R345W/R345W) mice develop extensive basal deposits. Proteomic analyses of Bruch's membrane/choroid and Bruch's membrane in the Efemp1(R345W/R345W) mice indicate that the basal deposits comprise normal extracellular matrix (ECM) components present in abnormal amounts. The proteomic analyses also identified significant changes in proteins with immune-related function, including complement components, in the diseased tissue samples. Genetic ablation of the complement response via generation of Efemp1(R345W/R345W):C3(-/-) double-mutant mice inhibited the formation of basal deposits. The results demonstrate a critical role for the complement system in basal deposit formation, and suggest that complement-mediated recognition of abnormal ECM may participate in basal deposit formation in DHRD/ML and perhaps other macular degenerations.

IL1beta, IL6 and IL8 gene polymorphisms involvement in recurrent corneal erosion in patients with hereditary stromal corneal dystrophies.

TGFBI gene mutations cause corneal stromal dystrophies of autosomal dominant inheritance. The most frequent complication of stromal dystrophies is recurrent corneal erosion with varying degree of accompanying inflammation. IL-1beta, IL-6 and IL-8 are main cytokines involved in corneal erosion healing. This study aimed to investigate the association between IL1B gene -511C/T, IL6 gene -174G/C and IL8 gene -781C/T polymorphisms and risk of recurrent erosion development in patients with hereditary corneal stromal dystrophies. A trend to decrease of IL1B gene -511TT genotype frequency in group with erosion (3.7%) comparing to control (6.7%) was observed. IL6 gene -174C allele carriers frequency in control group (65.9%) was significantly (P < 0.05) lower comparing to patients with erosion (80.5%). Frequency of IL8 -781TT genotype was significantly (P < 0.05) lower in the group with erosion (10.7%) comparing to patients without erosion (30.8%) and control (25%). IL6 gene -174C allele may be considered as genetic marker of corneal erosion risk in patients with hereditary stromal corneal dystrophies, whereas IL8 -781TT genotype is associated with negative recurrent erosion prognosis in such patients.

Heavy-chain amyloidosis in TGFBI-negative and gelsolin-negative atypical lattice corneal dystrophy.

PURPOSE: An atypical case of late-onset lattice corneal dystrophy is described in a 61-year-old man without a family history of eye disease. Mutational analysis of the TGFBI gene excluded any pathogenic sequence variants. However, 2 years later, renal impairment and nephrotic syndrome were diagnosed, resulting in a diagnosis of systemic heavy-chain amyloidosis. METHODS: Slit-lamp examination, corneal photography, and in vivo confocal microscopy were performed. General systemic evaluation included blood and urine assessment, bone marrow and renal biopsies, and cardiologic evaluation. A DNA sample underwent initial mutational analysis of TGFBI and, subsequently, gelsolin. The renal biopsy sample was subject to direct protein sequencing by mass spectrometry. RESULTS: A bilateral, atypical, fine, midperipheral lattice corneal dystrophy with minor central subepithelial scarring was clinically characterized. Subsequently, abnormal renal functions with proteinuria, IgG lambda paraproteinemia, extensive deposition of amyloid in renal glomeruli, and increased plasma cells in bone marrow were identified. No pathogenic sequence mutations were identified in TGFBI or the gelsolin genes. Direct protein sequencing by mass spectrometry showed amyloid to be heavy-chain deposition rather than the more usual light-chain deposition. CONCLUSIONS: Atypical midperipheral lattice corneal dystrophy presenting with adult onset and negative family history should arouse suspicion for an association with paraproteinemias or amyloidosis. Exclusion of TGFBI mutations should alert the clinician to the possibility of potentially life-threatening conditions, with referral for careful systemic evaluation.

Immunophenotypes of macular corneal dystrophy in India and correlation with mutations in CHST6.

PURPOSE: To determine the immunophenotypes of macular corneal dystrophy (MCD) in Indian patients and to correlate them with mutations in the carbohydrate 6-sulfotransferase (CHST6) gene. METHODS: Sixty-four patients from 53 families with MCD that were previously screened for mutations in CHST6 were included in an immunophenotype analysis. Antigenic keratan sulfate (AgKS) in serum as well as corneal tissue was evaluated in 31 families. Only cornea was evaluated in 11 families, and only serum was evaluated in 11 families. AgKS was detected in formalin-fixed, paraffin-embedded corneal sections by immunohistochemistry and in serum by ELISA using a monoclonal antibody against sulfated forms of KS in patients with MCD as well as normal controls. RESULTS: Analysis of corneal and/or serum AgKS disclosed MCD type I (27 families), MCD type IA (5 families), and MCD type II (3 families) in the cases studied. An additional 10 families were either MCD type I or MCD type IA since only serum AgKS data were available. Seven families manifested atypical immunophenotypes since the corneal AgKS expression was either of MCD type I or MCD type IA, but serum AgKS levels ranged from 19 ng/ml to 388 ng/ml. More than one immunophenotype was detected amongst siblings in two families. Each immunophenotype was associated with mutational heterogeneity in CHST6. CONCLUSIONS: MCD type I was the predominant immunophenotype in the Indian population studied followed by MCD type IA and then MCD type II. We detected further immunophenotypic heterogeneity by finding atypical patterns of AgKS reactivity in a subset of families. There were no simple correlations between immunophenotypes and specific mutations in CHST6, suggesting that factors other than CHST6 mutations may be contributing to the immunophenotypes in MCD.

Sequencing of the CHST6 gene in Czech macular corneal dystrophy patients supports the evidence of a founder mutation.

AIMS: To characterise the role of the carbohydrate sulfotransferase gene (CHST6) in macular corneal dystrophy (MCD) in Czech patients. METHODS: The coding region of the CHST6 gene was directly sequenced in 10 affected and five unaffected members from eight apparently unrelated MCD families. The type of MCD was determined by enzyme-linked immunosorbent assay of antigenic keratan sulfate (KS) in serum and by immunohistochemical staining of corneas with monoclonal anti-KS antibody. RESULTS: The following changes in the coding sequence of the CHST6 gene were observed; homozygous mutation of c.1A>T (p.M1?); homozygous mutation c.599T>G (p.L200R); compound heterozygosity for c.599T>G and c.614G>A (p.R205Q); compound heterozygosity for c.494G>A (p.C165Y) and c.599T>G; heterozygous c.599T>G mutation and no other change in the coding sequence. One proband exhibited no changes. The pathogenic mutation c.599T>G (p.L200R) was in allelic association with the c.484C>G (p.R162G) polymorphism. Nine patients from seven families were of MCD type I including the subtype IA. CONCLUSION: Four different CHST6 missense mutations, of which p.C165Y is novel, were identified. Allelic association of the c.[484C>G; 599T>G] in six probands out of eight, as well as occurrence of this particular allele in a heterozygous state in one healthy control individual, supports a common founder effect for MCD in the Czech Republic.

Cell proliferation and apoptosis in stromal corneal dystrophies.

The aim of our study was to evaluate corneal cell proliferation and apoptosis in cases of granular, macular and lattice dystrophy, and to provide evidence which may help to clarify whether apoptosis is a pathogenic factor in any of these dystrophies. The study group comprised 39 eyes (from 33 patients) which had undergone penetrating keratoplasty (PK) for stromal dystrophies: these comprised 12 eyes (from 9 patients, 55.5% males) with granular dystrophy, 13 eyes (12 patients, 33.3% males) with macular dystrophy, and 14 eyes (13 patients, 61.5% males) with lattice type I dystrophy. A further 4 corneal buttons from enucleated eyes of 4 patients with choroideal melanoma served as controls. Immunocytochemical analysis of Ki67 (DNAcon Kit, DakoCytomation A/S, Glostrup, Denmark) was used for evaluation of cell proliferation. Apoptosis was detected by use of the TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling) assay method (Apoptag reagent, Q-Biogene, Strasbourg, France). Statistical comparisons were made using the Mann-Whitney test. No Ki67-positive cells were detected in the study-group or control corneas. In control corneas no apoptotic activity was found. In the study group the mean (normalised) apoptotic keratocyte number was 1.1+/-1.7 in granular dystrophy and 0.5+/-1.1 in lattice type I dystrophy (p = 0.36, 0.63 respectively). Compared to the controls, the difference was statistically significant only for macular dystrophy (1.6+/-1.2; p = 0.01). Keratocyte apoptosis seems to be a concomitant or pathogenic factor in macular dystrophy. However, the pathways that are triggered to result in increased apoptotic cell death remain to be clarified.

[Chronic endothelial cell loss of the graft after penetrating keratoplasty: influence of endothelial cell migration from graft to host]. / Chronischer Endothelzellverlust des Transplantats nach perforierender Keratoplastik: Einfluss der Migration von Endothelzellen vom Transplantat zur Wirtshornhaut.

BACKGROUND: Chronic endothelial cell loss of the graft is common after penetrating keratoplasty. Some kind of subclinical immunological reaction that is not visible at the slitlamp has been suspected as main cause for this phenomenon. Furthermore, migration of graft endothelial cells towards the host cornea has been discussed to add to this loss in special cases. In this study, 3 homogenous patient groups with similar risk of immunological reactions were examined. Main difference between these groups was the potential of graft endothelial cells to migrate towards the host cornea. PATIENTS AND METHODS: Patients with keratoconus without cataract surgery (group I with little migration potential, n = 273), patients with Fuchs endothelial dystrophy without/with cataract surgery (groups IIa/IIb with moderate migration potential, n = 89/n = 165) and patients with bullous keratopathy after cataract surgery (group III with potentially large migration tendency, n = 188) were included in the study. All patients had a first keratoplasty. Patients with glaucoma or further intraocular procedures after keratoplasty were excluded from the study. Clear graft survival and ratio of grafts without immune reactions were estimated according to Kaplan and Meier. Endothelial analysis concerned only patients without immune reactions and with at least 3 postoperative endothelial cell density values of the graft center (76 patients in I, 18 patients in IIa, 41 patients in IIb and 23 patients in III). RESULTS: Mean relative loss of endothelial cells per year was 14.0 +/- 19.0 % in group I, 17.0 +/- 19.1 % in group IIa, 20.8 +/- 18.2 % in group IIb and 29.4 +/- 17.6 % in group III (ANOVA, p < 0.01). Five years postoperatively in group I 99 %, in group IIa 98 %, in group IIb 93 % and in group III 69 % of the grafts were centrally clear (log rank test, p < 0.001). In the same period in group I 88 %, in group IIa 86 %, in group IIb 83 % and in group III 81 % of the grafts were free of immune reactions (log rank test, p < 0.05). Reasons for irreversible graft failure were immune reactions (0 in group I; 0 in group IIa; 1 in group IIb; 9 in group III, ), surface disorders (1 in group I; 0 in IIa; 1 in group IIb; 3 in group III) and endothelial failure (0 in group I; 1 in group IIa; 5 in group IIb; 6 in group III) (chi square test, p < 0.01). CONCLUSIONS: In patients with bullous keratopathy endothelial cell loss via migration seems to add significantly to the general chronic loss which is suspected to be immunological. Peripheral migration of endothelial cells, therefore, most probably contributes to limited prognosis of patients with bullous keratopathy in terms of clear graft survival. In consequence, corneal grafts for bullous keratopathy should be as large as immunologically tolerable, and endothelial cell density should be as high as possible in order to counteract this special endothelial loss factor.

Macular corneal dystrophy in Saudi Arabia: a study of 56 cases and recognition of a new immunophenotype.

PURPOSE: To determine the immunophenotype or immunophenotypes of macular corneal dystrophy in Saudi Arabia. METHODS: We studied 56 cases of macular corneal dystrophy. Tissue from 60 corneal transplant buttons was stained by the avidin-biotin complex method using an anti-keratan sulfate monoclonal antibody. The serum antigenic keratan sulfate was measured in 23 of the 56 patients, four unaffected relatives, and 13 individuals with chronic actinic keratopathy. Serum and corneal tissue were studied in 17 of the 50 affected individuals with corneal transplant material. RESULTS: Thirty-five corneas (58.3%) of 29 of 50 patients did not react with anti-keratan sulfate monoclonal antibody. The stroma and abnormal intracellular and extracellular corneal accumulations reacted with anti-keratan sulfate monoclonal antibody in seven corneas (11.7%). The stroma in the other 18 corneas (30.0%) from 15 patients did not react with the anti-keratan sulfate monoclonal antibody, but corneal fibroblasts did. Twenty-one of the 23 patients with macular corneal dystrophy had no detectable serum antigenic keratan sulfate (< 9 ng/ml); two had values of 12 and 51 ng/ml, respectively, and their corneal stroma and abnormal accumulations reacted with anti-keratan sulfate monoclonal antibody. CONCLUSIONS: We detected macular corneal dystrophy type IA, a new immunophenotype characterized by the lack of detectable antigenic keratan sulfate in the serum (< 9 ng/ml), and a corneal stroma that did not react with the keratan sulfate monoclonal antibody but in which corneal fibroblasts did react with keratan sulfate monoclonal antibody (in 15 of 50 patients).

Immunoglobulins in granular corneal dystrophy Groenouw type I.

Three patients with granular corneal dystrophy Groenouw type I underwent corneal grafting, and cryostat sections of the corneal buttons were examined immunohistochemically for immunoglobulins. Positive results were obtained for IgG, Kappa-, and Lambda chains with immunofluorescence technique. The reactions were seen exclusively in the same localizations as the Masson trichrome positive deposits.

Altered antigenicity of keratan sulfate proteoglycan in selected corneal diseases.

Monoclonal antibody against keratan sulfate (KS) was used for immunofluorescent staining of sections of human corneas from 8 normal eyes, 19 with keratoconus, 4 with pellucid marginal degeneration, 5 with primary macular corneal dystrophy, and 1 with recurrent macular corneal dystrophy. The anti-KS monoclonal antibody did not stain the corneas with primary macular corneal dystrophy, but stained all other corneas to varying degrees. Staining intensity was weaker than normal in most keratoconus and pellucid marginal degeneration corneas, and was very weak in a case of macular corneal dystrophy that had recurred in a transplanted normal cornea. In several corneas with keratoconus, normal staining was seen at the periphery, and staining intensity decreased in the thinned central portion of the stroma. The decreased KS staining was not localized in stromal scar tissue found in the keratoconus and pellucid marginal degeneration corneas. Quantitation of relative staining intensity found keratoconus and pellucid marginal degeneration corneas to be 49% and 40% as intensely stained, respectively, as normal corneas, a statistically significant decrease (P less than 0.01). Distribution of staining intensities of the keratoconus corneas demonstrated a single modality. These results are in agreement with findings of previous biochemical studies, which show reduction of highly sulfated keratan sulfate epitopes in corneas from keratoconus and pellucid marginal degeneration, and absence of sulfated keratan sulfate epitopes in macular corneal dystrophy.

Reis-Bücklers' corneal dystrophy. Immunofluorescent and electron microscopic studies.

The anterior stroma, epithelium, and Bowman's layer have been proposed as the site of primary pathology in Reis-Bücklers' corneal dystrophy (RBCD). Immunofluorescent localization of laminin and bullous pemphigoid antigen (BPA) was compared with the ultrastructure of RBCD. As previously reported, patchy deposition of characteristic "peculiar curly" filaments was found in the supra-Bowman's, subepithelial fibrous tissue. We also recognized areas of early involvement with deposition of this "peculiar curly" material between a distorted epithelial basal lamina and a normal undisturbed Bowman's layer. In normal cornea, laminin and BPA localized to the epithelial basal lamina. In RBCD, laminin and BPA were in a piebald mosaic distribution throughout the aberrant subepithelial fibrous tissue between the basal lamina and the buried Bowman's layer. This indicates that RBCD is an epithelial disease, with the "peculiar curly" material paralleling the distribution of the attachment proteins.

[Clinical, secretory and immunologic studies in Terrien disease]. / Klinische, sekretorische und immunologische Untersuchungen beim Morbus Terrien.

The present paper begins with a description of the clinical picture of Terrien's disease. Using radionuclide dacryography (RND) to determine secretion, reduced tear secretion was found unilaterally in four patients and bilaterally in one (as indicated by the half-lives of the conjunctival drainage curves). In four patients RND failed to reveal any reduction in tear secretion on either side. The slightly elevated IgG values found in the tear fluid are in agreement with the clinically detected signs of inflammation. The serum parameters determined, such as iron, magnesium, and copper levels and the electrolyte, uric acid, and creatinine concentrations were normal.

Macular corneal dystrophy: immunochemical characterization using monoclonal antibodies.

Macular corneal dystrophy is an inherited corneal disease characterized by corneal opacities resulting from intra- and extracellular deposits within the corneal stroma. Several monoclonal antibodies developed against antigens of corneal fibroblasts were screened for their reactivity with these abnormal deposits in corneas with macular dystrophy using an indirect peroxidase-conjugated immunostaining technique. One of these monoclonal antibodies (designated 8F1-3) reacted very strongly with these abnormal deposits. Although the antigen recognized by this monoclonal antibody was present in the normal corneal stromal and endothelial cells, its concentration in the cells in the corneas with macular dystrophy appeared to be considerably higher, based on the intensity of the immunostaining reaction. Corneal fibroblasts grown in tissue culture were employed for further characterization of the antigen. After fixing with paraformaldehyde and permeabilizing with Triton X-100, immunofluorescent staining of the corneal fibroblasts using these monoclonal antibodies revealed a filamentous pattern of staining which resembled that seen for vimentin filaments. On treatment of corneal fibroblasts with colchicine, the filaments recognized by this antibody were withdrawn from their cytoplasmic array to form a perinuclear cap as also observed for vimentin-containing intermediate filaments. Immunoelectron microscopic studies using colloidal gold-conjugated antimouse IgG indicated that this monoclonal antibody recognized an antigen associated with intermediate-type filament. However, antivimentin antibody did not react with the abnormal deposits in the corneas with macular dystrophy, indicating that the antigen identified in the present study, although associated with intermediate filaments, was not vimentin. Analyses of cytoskeletal antigens by the immunoblotting technique further revealed that this monoclonal antibody recognized two polypeptides with Mr48,000 and 45,000, while antivimentin antibody reacted with 58,000 Mr polypeptide (vimentin).

Monoclonal antibodies to proteokeratan sulfate of rabbit corneal stroma.

Hybridomas were developed that secreted monoclonal antibodies against two proteokeratan sulfates (PKS) from rabbit corneal stroma. A total of 28 antibodies were isolated, all of the IgG type with kappa light chains. All were found to react with both PKSs. As judged by immunohistochemical staining, none of them reacted with scleral or conjunctival components, nor with sections of skin, but all reacted with nasal cartilage. When tested by enzyme-linked immunosorbent assays, against components of the proteoglycans, all of the antibodies reacted with keratan sulfate-peptide (isolated from papain digests of PKS or of cartilage proteoglycan), and all but two reacted with oligosaccharide-containing protein cores (prepared by keratanase treatment of PKS). Reactivity with cores was probably due to residual portions of the keratan sulfate chains since the endogenous oligosaccharide-peptides (non-sulfated, non-keratan sulfate oligosaccharides isolated after papain digestion of PKS) were not active. None of the antibodies reacted with protein cores made by removal of carbohydrate by hydrolysis with trifluoromethanesulfonic acid. All except one reacted with fragments of keratan sulfate (made by keratanase digestion). These observations are evidence for structural requirements at three different levels of completeness of the antigen for recognition among the various antibodies. In addition, none of the antibodies reacted immunohistochemically with macular dystrophic corneas, confirming the finding of others that the defect lies in the keratan sulfate portion of the proteoglycans.