[Immunohistochemical characterization of collagen IV in control corneas and in corneas obtained from patients suffering from posterior polymorphous corneal dystrophy]. / Imunohistochemická charakterizácia kolagénu IV v zdravých rohovkách a rohovkách pacientov so zadnou polymorfnou dystrofiou.

The expression of all six chains of collagen IV was studied using the indirect fluorescent immunohistochemistry in seven control corneas and seven corneas obtained from patients suffering from the posterior polymorphous corneal dystrophy. Heterogeneous staining, especially in the epithelial basement membrane and Descemet's membrane, was observed in the control corneas. An increase of the staining intensity for the alpha1 and alpha2 chains was observed, especially in the Descemet's membrane and the corneal stroma in samples obtained from the patients compared to the control tissues.

Compositional differences between infant and adult human corneal basement membranes.

PURPOSE: Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development. METHODS: Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components. RESULTS: Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from alpha1-alpha2 to alpha3-alpha4 chains after 3 years of life; in the adult, alpha1-alpha2 chains were retained only in the limbal BM. Laminin alpha2 and beta2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, beta2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin gamma3 chain. In the infant DM, type IV collagen alpha1-alpha6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years. CONCLUSIONS: The distribution of laminin gamma3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.

Ocular copper deposition associated with monoclonal gammopathy of undetermined significance: case report.

To report a case of ocular copper deposition in both eyes at the level of Descemet's membrane associated with a monoclonal gammopathy of undetermined significance (MGUS). A 49-year-old white woman had golden-brown metallic dust-like deposits on Descemet's membrane of both eyes. A systemic examination revealed an elevated serum copper, normal serum ceruloplasmin and a normal level of total protein. Serum protein electrophoresis demonstrated a single peak (M-spike) in the gamma region (M-protein in serum = 11 g/l). Flow cytometric analysis of the marrow aspirate identified a monoclonal plasma cell population that represents approximately 2% of the total marrow cells consistent with the diagnosis of monoclonal gammopathy of undetermined significance. Copper deposits at the level of Descemet's membrane may be a finding in a patient with monoclonal gammopathy of undetermined significance.

Ocular copper deposition associated with monoclonal gammopathy of undetermined significance: case report

Relatar um caso de depósito corneano de cobre em ambos os olhos a nível da membrana de Descemet associado a gamopatia monoclonal de significância indeterminada (GMSI). Paciente feminina, 49 anos, leucodérmica, apresentando depósito corneano de aspecto marrom-ouro a nível da membrana de Descemet em ambos os olhos. Exame sistêmico revelou cobre sérico elevado, ceruloplasmina sérica normal, e proteína total normal. Eletroforese de proteínas séricas demonstrou um pico único na região gama (proteína M = 11 g/l). Análise citométrica de aspirado medular evidenciou uma população de células plasmáticas monoclonais de aproximadamente 2% do total de células medulares consistente com o diagnóstico de gamopatia monoclonal de significância indeterminada. Depósitos de cobre a nível da membrana de Descemet podem ser encontrados em pacientes com gamopatia monoclonal de significância indeterminada.

Evaluation of diagnostic parameters of Wilson's disease in childhood.

BACKGROUND: Wilson's disease (WD) is an autosomal recessive disorder with variable clinical presentation. Its diagnosis depends on a combination of clinical and laboratory findings. We evaluated the sensitivity of various diagnostic tests in children with WD and high liver copper concentrations. METHODS: Thirty-three children (6-15 years old, 19 male) with confirmed WD (hepatic copper >250 mcirog/g dry weight) were evaluated retrospectively. Eyes were examined with biomicroscope for Kayser-Fleischer rings and urinary copper content was determined in 30 patients. Serum ceruloplasmin levels were measured and liver tissue samples were stained with orcein in all. RESULTS: All patients presented with hepatic disease. Four patients also had neurological involvement. Hepatic copper concentration was between 250 and 1200 microg/g. Eighteen patients had liver cirrhosis, 9 chronic hepatitis, and 6 had massive hepatic necrosis on liver biopsy or necropsy. The sensitivity of various tests evaluated was: 100% (30/30) for urinary copper excretion, 88% (29/33) for orcein staining on liver tissues, 82% (27/33) for ceruloplasmin levels, and 63% (19/30) for presence of Kayser-Fleischer ring. Kayser-Fleischer ring was present in all patients with neurological manifestations and in 58% of patients with only hepatic presentation. CONCLUSIONS: 24-hour urinary copper excretion seems to be the most sensitive test for diagnosis of WD, particularly when liver biopsy cannot be performed due to coagulation abnormalities.

The alpha1(VIII) and alpha2(VIII) collagen chains form two distinct homotrimeric proteins in vivo.

The short chain collagen variant, type VIII, is considered to be comprised of two distinct gene products, the alpha1 and alpha2 polypeptide chains. However, recent in vitro translation studies suggest that these chains can form homotrimers. We report here data from biochemical, immunohistochemical and molecular biological experiments, which together provide evidence that alpha1 and alpha2 polypeptides of type VIII collagen exist as homotrimers in cells and tissues. High-performance liquid chromatographic separation of type VIII collagen isolated from Descemet's membrane consistently demonstrated equimolar quantities of the two chains (alpha1:alpha2 1. 03+/-0.02 (S.E.M.); n=41). The availability of highly specific antibodies for the two polypeptides has assisted the in vivo characterisation of type VIII collagen. Immunoprecipitation of trimeric type VIII collagen from Descemet's membrane with purified anti-alpha1(VIII) and anti-alpha2(VIII) yielded fractions that contained only the alpha1(VIII) and alpha2(VIII) chains, respectively. Cultured human mesangial cells synthesised both polypeptides, but the alpha1(VIII) chain was found exclusively in the cell pellet, while the media contained only the alpha2(VIII) chain. The RNA from human mesangial cells and cornea showed message for both chains. However, in peritoneal fibroblast and mesothelial cell RNA, only alpha1(VIII) mRNA was detectable, demonstrating that the transcription of these two genes was not always co-ordinated. Immunohistochemistry showed that both polypeptides were present in cornea, optic nerve, aorta and umbilical cord but did not always co-localise. These results indicate the alpha1(VIII) and alpha2(VIII) chains preferentially form pepsin-resistant, homotrimeric molecules and so can exist as two distinct proteins.

Distribution of ascorbate in the anterior bovine eye.

PURPOSE: To analyze the ascorbate distribution in the anterior eye wall to better understand the functional significance of this compound in the eye. METHOD: Ascorbic acid was determined by high-performance liquid chromatography using an LC-10 system (Shimadzu, Kyoto, Japan). Bovine eye samples were used. RESULTS: The highest ascorbate concentration was observed in the corneal epithelium, with significantly higher values in the central (1.56 mg/g) than in the peripheral (1.39 mg/g) area. The ascorbate content was similar in the corneal stroma (0.22 mg/g), the Descemet's membrane (DM)/endothelium (0.22 mg/g), and the aqueous humor (0.21 mg/ml). By comparison, the sclera (0.15 mg/g) and the conjunctiva (0.11 mg/g) showed lower values, as did the lacrimal gland (0.09 mg/g) and the serum (0.0008 mg/ml). CONCLUSIONS: (1) Peak ascorbate concentration was observed in the central corneal epithelium covering the pupillary area. This is compatible with the idea that the ascorbate may act as an UV filter shielding internal eye structures from radiation damage. (2) The ascorbate concentration in the corneal stroma and DM/endothelium was as high as in the aqueous humor, and it is suggested that the aqueous humor plays a key role in the distribution of ascorbate to the anterior eye wall.

The corneal stroma: an inhomogeneous structure.

PURPOSE: This study was conducted to determine the elemental composition of the human cornea. Special attention was paid to corneal stroma inhomogeneity. METHODS: Seventy human corneas were examined by means of energy-dispersive X-ray analysis. Epithelium, subepithelium, middle stroma, sub-Descemet layer, Descemet's membrane and endothelium were subjected to repeated measurements. RESULTS: In the cellular layers the phosphorus concentrations were high [0.35 mol/kg dry weight (dw) in the epithelium and 0.403 mol/kg dw in the endothelium]. Similar concentrations were found for sulphur (0.38 mol/kg dw in the epithelium). Stromal layers showed high contents of sulphur: 0.26 mol/kg dw. The phosphorus concentration was found to be higher in the subepithelium than in the middle stroma. Sulphur concentrations were highest in Descemet's membrane, followed by the subepithelium and the middle stroma. DISCUSSION: Nucleic acids and energy-containing phosphates explain the high levels of phosphorus in the cellular layers. The high sulphur concentrations may be related to the phosphoadenosinphosphosulfate and protein turnover in the epithelium. We interpret the inhomogeneous distribution of phosphorus in the stroma as a function of the density of keratocytes. An evaluation of all known sulphur-containing biochemical components of the stroma (0.217 mol sulphur/kg dw) corresponds to our measurements. In contrast to former results we find the corneal stroma to be an inhomogeneous structure.

Extrusion of abnormal endothelium into the posterior corneal stroma in a patient with posterior polymorphous dystrophy.

PURPOSE: To report the presence of abnormal endothelium that extruded into the posterior corneal stroma in a patient with posterior polymorphous dystrophy. METHODS: The corneal button of a man who underwent penetrating keratoplasty for posterior polymorphous dystrophy was examined by light and electron microscopy. Immunoperoxidase staining for cytokeratins, vimentin, and the endothelial antigen recognized by monoclonal antibody 2B4.14.1 antigen was performed. Two-color immunofluorescence staining for simultaneous detection of cytokeratins and 2B4.14.1 antigen was also done. RESULTS: Much of the endothelium had characteristic features of epithelium-like cells, and abnormalities in Descemet's membrane were present. Curious oval and slit-like spaces in the posterior stroma were lined by epithelium-like endothelial cells and were continuous with the anterior chamber through defects in Descemet's membrane. CONCLUSION: These abnormalities in the posterior stroma have not previously been described in histopathologic reports of posterior polymorphous corneal dystrophy and are likely an unusual variation in the spectrum of this hereditary disorder.

The distribution of fibronectin and P component in Descemet's membrane: an immunoelectron microscopic study.

Descemet's membrane consists of two zones, the 'anterior banded zone' which contains wide-spaced collagen and the amorphous 'posterior non-banded zone'. It is attached anteriorly to the corneal stroma by a narrow transitional zone termed the 'interfacial matrix'. The distribution of fibronectin and P component within the different layers of Descemet's membrane was investigated using an ultrastructural immunogold technique. Seven normal human corneas from an eye bank and one specimen from an orbital exenteration were examined. Fibronectin was predominantly present in the posterior part of the posterior non-banded zone and in the anterior banded zone. The anterior part of the posterior non-banded zone contained less fibronectin. P component was present throughout the anterior banded and posterior non-banded zones. There was a sharp demarcation at the interfacial matrix since neither substance was observed in the corneal stroma. The differences shown in the distribution of fibronectin and P component within Descemet's membrane may have resulted from their binding to other substances or alternatively from differences in the quantities laid down during the evolution of this basement membrane.

Quantitative analysis of immunogold labellings of collagen types I, III, IV and VI in healthy and pathological human corneas.

BACKGROUND: We studied the distribution of collagen types I, III, IV and VI in one healthy human cornea and in seven pathological human corneas, in which the disorders were three cases of pseudophakic bullous keratopathy (two severe, one moderate) and one case each of stage IV keratoconus, chronic ulcer, vascularized cornea and disciform keratitis. METHODS: Transmission electron microscopy examinations were performed on post-embedding immunogold-labelled sections. The staining was evaluated by gold particle count in the different tissues. The presence or absence of a given antigen was determined by statistical analysis, using a d-value test. RESULTS: Our results on healthy corneal tissues corroborate the data available from previous studies, except for collagen type VI, which we found to be absent in Bowman's layer. In pathological corneas with a collagenous layer posterior to Descemet's membrane, collagen types I, III and especially IV were detected in this collagenous layer. Collagen types I, III and VI were detected in the anterior healed stroma of other pathological corneas, except for the keratoconus cornea, in which intense collagen III staining was observed. CONCLUSION: The presence of collagen types I and III in the posterior collagenous layer of our pseudophakic bullous keratopathy corneas suggests that this layer corresponds to scar tissue secreted by stimulated endothelial cells.

Evidence for separate networks of classical and novel basement membrane collagen. Characterization of alpha 3(IV)-alport antigen heterodimer.

The COOH-terminal non-collagenous domains (NC1) of type IV collagen from glomerular basement membranes (GBM), lens capsule basement membranes, and Descemet's membrane varied in the distribution of their NC1 subunits. All of these basement membranes (BMs) contained both classical (alpha 1(IV) and alpha 2(IV)) and novel collagen chains (alpha 3(IV), alpha 4(IV) and the Alport antigen). Whereas GBM had a predominance of disulfide-bonded subunits, the lens capsule and Descemet's membrane were primarily monomeric, differences that are likely related to the functional and structural diversity of collagen in various tissues. A heterodimer formed from monomeric subunits of alpha 3(IV) and the Alport antigen exists in human and bovine GBM. This dimer represents an important cross-link of the NC1 domain of novel collagen. Additionally, immunoaffinity methodology showed that the novel BM collagen hexamers segregate into populations containing only novel BM subunits without the participation of the classical subunits (alpha 1(IV) and alpha 2(IV)). These data provided evidence for the presence of two separate networks of BM collagen: one containing alpha 1(IV) and alpha 2(IV), and the other consisting of the novel collagen chains.

The primary structure of a triple-helical domain of collagen type VIII from bovine Descemet's membrane.

We have isolated and sequenced a fragment of 469 amino acid residues from bovine type VIII collagen. The sequence was composed of a series of Gly-X-Y repeats which was interrupted 8 times by short imperfections. The number and relative location of these interruptions were similar to those of chicken alpha 1(X) and rabbit alpha 1(VIII) chain triple-helical domains. Comparison to published N-terminal sequences to two triple-helical fragments of bovine type VIII collagen and to the cDNA derived sequence of the rabbit alpha 1(VIII) chain showed that this fragment was the triple-helical domain of a second type VIII collagen chain which we designate alpha 2(VIII).