[Review of the past year--"cornea"].
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Animals↗
Biomedical subjects
M Knorr
Publications and source records attributed to M Knorr.
[Growth characteristics of bovine retinal pericytes in culture].
The loss of retinal pericytes is one of the earliest changes in diabetic retinopathy. In order to study this phenomenon in vitro, an optimal isolation and cultivation system has to be established. Therefore, pericytes from bovine retinae were isolated enzymatically with 0.4% collagenase in phosphate-buffered saline and identical immunologically by positive staining with antibodies against smooth muscle alpha-actin. Routine cultivation of pericytes was performed by using DMEM supplemented with 10% fetal calf serum. Dependent on the in vitro age of cells, the effect of the following reagents on proliferative activity was determined: fetal calf serum, heparin, ECGF, ECGF+heparin, and glucose. Increasing serum concentrations stimulated the proliferation of pericytes, although the degree of stimulation was reduced with increasing in vitro age. Heparin inhibited the growth in a dose-dependent manner; the achieving 50% inhibition was extrapolated to be 25 micrograms/ml. ECGF increased pericyte proliferation significantly, with a maximum at 10 microliters/ml. In addition, ECGF reversed the inhibitory effect of heparin. Furthermore, all tested glucose concentrations (5.5-27.75 mmol/l) did not show any influence on growth rates of pericytes. The results demonstrate that routine cultivation of retinal pericytes is possible. Moreover, they indicate that enhanced blood glucose concentrations, as observed in diabetic patients, are not the only important factor in the loss of retinal pericytes.
Animals↗
[Effect of platelet-derived growth factor PDGF on replication of cultivated bovine lens epithelial cells].
It has previously been reported that PDGF isoforms AB and BB induce an increase of cytosolic free calcium in cultured bovine lens epithelial cells in a dose-dependent manner. To evaluate the biological capacity of PDGF, we investigated the proliferative response of bovine lens epithelial cells to stimulation with PDGF-AA, -AB and -BB. Since various unspecific components of serum-containing media act as mitogenes and mask the effect of PDGF, serum-free culture conditions were a prerequisite for growth-factor-induced effects. Therefore, a basic medium (Waymouth's MB 752/1 with Ham F12 Nutrient Mixture 1:2, v/v; Gibco BRL) was supplemented with only 2 mM CaCl2, 10 micrograms/ml Transferrin, 10 micrograms/ml Thyroglobulin (both Sigma Chemie) and standard amounts of antibiotics. PDGF isoforms were obtained by separate expression of cloned genes in Escherichia coli, which has been previously described. Under these conditions the isoforms PDGF-AB and -BB caused an increase in proliferation in a dose-dependent manner. The maximum increase of the cell number was 21% for PDGF-AB with an EC5 of 5 ng/ml. PDGF-BB revealed a maximum increase of 33% with an EC5 of 1.5 ng/ml. PDGF-AA, when used in similar concentration was ineffective. These data show the involvement of PDGF isoforms AB and BB in the replicative action of BLEC.
Animals↗
Lens epithelial cell response to isoforms of platelet-derived growth factor.
It has been reported previously that platelet-derived growth factor (PDGF) may play an important role in the regulation of lens growth and differentiation. To evaluate PDGF-induced effects at the cellular level, we investigated the response of cultured bovine lens epithelial cells (BLEC) to PDGF-AB, -AA, and -BB isoforms at the cellular level. Stimulation of BLEC with PDGF isoforms showed no increase in cell proliferation under the culture conditions of this study. In contrast, measurement of cytosolic free calcium concentration ([Ca2+]i), which has been shown to be an important second messenger for controlling multiple cellular processes in the lens, revealed a dose-dependent rise in [Ca2+]i upon stimulation with PDGF-AB and -BB isoforms. PDGF-AA used in similar concentrations was not effective. Our data suggest that PDGF-AB and -BB may play a role in the regulation of cellular functions in BLEC via modulation of intracellular calcium homeostasis.
Aminoquinolines↗
Significance, specificity, and ultrastructural localization of HMB-45 antigen in pigmented ocular tumors.
PURPOSE: To evaluate the expression of the monoclonal HMB-45 antibody in melanocytic and nonmelanocytic ocular tumors and seek "activated" cellular subpopulations in an attempt to distinguish between benign and malignant melanocytes, to compare HMB-45 and S100 activity, and to determine the specificity of this tumor marker for melanocytic ocular lesions. METHODS: Immunohistologic investigations were performed with paraffin-embedded tissue of 10 acquired conjunctival melanoses, 19 conjunctival nevi, 34 conjunctival melanomas, 69 uveal melanomas, 20 basal cell carcinomas of the lid, 20 cystic dermoids, 15 hemangiomas of the lid, 20 conjunctival papillomas, 20 squamous cell carcinomas, 20 pterygia, 11 sebaceous gland carcinomas, 10 retinoblastomas, and 5 choroidal metastatic carcinomas. The avidin-biotin peroxidase technique and monoclonal HMB-45 antibody were used. The distribution of S100 protein was studied in the melanocytic tumors for comparison. To localize the HMB-45 antigen, lowicryl-embedded tissue of uveal melanomas was investigated immunoelectron microscopically. RESULTS: More than 95% of the conjunctival and choroidal melanomas expressed the HMB-45 antigen, while S100 was found in all melanomas of the conjunctiva and in 91% of the uveal melanomas. In benign melanocytic lesions of the conjunctiva (nevi and melanocytes), especially the intraepithelial and junctional components stained with HMB-45, and at the site of tumor invasion, infiltrating cells showed increased HMB-45 reactivity. On the whole, HMB-45 antigen was less evenly distributed in the melanocytic tumors investigated than S100 antigen. All nonmelanocytic ocular tumors revealed no HMB-45 expression. Retinal pigment epithelium and tumor-free choroid were negative for HMB-45. The HMB-45 antigen was immunoelectron microscopically found in melanosomes at stages II and III. CONCLUSION: HMB-45 immunohistology helps in distinguishing melanocytic from nonmelanocytic ocular tumors and often clarifies the front of tumor invasion. The stronger HMB-45 reactivity probably reflects melanocytic activation, but a sharp line between benign and malignant melanocytes cannot be drawn.
Adult↗
[Immunohistologic, ultrastructural and morphometric characterization of organ cultures of the human limbus epithelium].
Transplantations of limbus epithelium play a steadily increasing role in the therapy of chemical burns, recurrent erosions, and impaired differentiation of the limbus epithelium (LE). To assess the vitality of LE under different culture conditions, LE was excised from 30 patients and cultivated in media with serum (F12 + 10% FCS) and without it (S4 and F12). AE5 antigen (64K keratin) was expressed by the LE specimens in these 3 media with an intensity similar to that of uncultivated specimens. All specimens strongly expressed EGF and PDGF-beta receptors under serum-free culture conditions, while serum-containing cultures reduced the expression of these receptors. Among the cells which migrated from the conjunctival preparations into the culture medium, connective tissue cells (anti-vimentin), macrophages (mac 1 and mac 2 antibodies), epithelial cells (AE5 antibody) and cells expressing class II antigen (Tü 39 and Tü 22 antibody) were determined. Only in the S4 medium were neither macrophages nor class II antigen-positive cells found. The epithelial thickness was unchanged before and after incubation with S4 medium. The two other media caused a reduction in thickness of the epithelium. The average size of the epithelial cells increased non-significantly in all cultures. Ultrastructurally, the organ cultures incubated in S4 medium showed practically no degenerative cell changes. On the basis of the criteria used here for quality checks of LE organ cultures, S4 appears to be the medium best suited on the basis of functional (PDGF-beta and EGF receptors) and morphological criteria (keratin expression, epithelial thickness and epithelial cell size).
Antigens↗
[Expression of platelet derived growth factor receptors in cultivated lens epithelial cells].
It has been shown that PDGF plays a role in the regulation of lens growth and differentiation. PDGF occurs in vivo as a homodimer or heterodimer of the two polypeptide chains A and B. These isoforms bind with different affinities to two distinct receptor types, termed alpha and beta. In order to identify the different PDGF receptors on cultured bovine lens epithelial cells (BLEC), we performed receptor binding studies, using 125I-labelled PDGF isoforms. Analysis of our data revealed that BLEC expressed approximately 35,000 PDGF-BB binding sites (KD = 21 ng/ml) and 9000 PDGF-AB binding sites (KD = 11 ng/ml), but only 4800 PDGF-AA binding sites (KD = 7 ng/ml). This study represents the first demonstration that lens epithelial cells express PDGF receptors.
Animals↗
Generalized myositis in Behçet disease: treatment with cyclosporine.
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Adult↗
Neuroepithelial adenocarcinoma of the eye.
A 29-year-old male patient with a microphthalmos on the right side since birth was seen in June 1987 in the University Eye Clinic because of a massive exophthalmus of the former microphthalmic eye. Computer tomography revealed a diffuse, infiltrating tumor of the right orbit. The bulbus was not demarcated, and the whole orbita had to be eviscerated. Histologically, we found a solid, undifferentiated tumor with clear signs of malignancy. It consisted of epithelial cells, partly in nest formation, which were keratin positive and vimentin negative, and of more spindle-shaped formations which were keratin negative and vimentin positive. In some areas, the tumor showed a tubular or an adenoid proliferation. CEA or S-100 could not be demonstrated. The histopathological diagnosis was neuroepithelial adenocarcinoma of the eye. The differential diagnosis and the genesis of the tumor are discussed.
Adenocarcinoma↗
[Ascorbic acid. Cytotoxic effect on cultivated bovine lens epithelium cells].
Ascorbic acid is one of the main components (1.16 mM/l) of the aqueous humor. The molarity of this molecule is 25 times higher than in the plasma of the cow, man or horse. Now the question arises as to which function ascorbic acid has in this extremely high concentration referring to the proliferation of the lens epithelial cells. Thus, the effect of ascorbic acid was investigated upon bovine lens epithelial cells (BLEC) in the range of 0-3 mM/l. These cells were cultivated under various culture conditions (serum-free, serum-containing, aqueous-humor-containing medium) and also incubated with such mitogens as retinal extract (RE), crude endothelial cell growth factor (cECGF), basic fibroblast growth factor (bFGF) or with calcium. In each culture condition 1 mM/l ascorbic acid caused remarkable inhibition of the proliferation of BLEC. Higher concentrations (> 1.5 mM/l) revealed cytotoxic effects. These effects were independent of small variations in the pH value caused by ascorbic acid. In addition, the effect of 2 mM/l ascorbic acid in combination with catalase in a concentration of 500 Um/ml and 1000 Um/ml, respectively, was investigated. It could be shown that catalase is capable of preventing the cytotoxic effect of ascorbic acid. These results show the inhibitory effect of ascorbic acid in its physiological concentration in the proliferation of BLEC.
Animals↗
[Effect of heparin on proliferation of cultivated bovine lens epithelial cells].
The treatments proposed to date for the prevention of secondary cataract have shown limited efficacy or have not been satisfactory due to ocular toxicity. Since it has been demonstrated that heparin can inhibit the proliferative activity of smooth muscle cells and fibroblasts in vitro and in vivo, we examined the effect of heparin at concentrations ranging from 20 to 200 micrograms/ml on the proliferation of cultured bovine lens epithelial cells (BLEC) under various culture conditions: (1) serum-free medium (SFM); (2) SFM + aqueous humor 1:1; (3) SFM +1 and 10% fetal calf serum; (4) SFM +1% retinal extract; (5) SFM +50 micrograms/ml endothelial cell growth factor; (6) SFM +10 ng/ml epidermal growth factor; (7) SFM +10 ng/ml basic fibroblast growth factor. Heparin caused no cytotoxic effects in any of the experiments. With medium 2 and 3, heparin caused dose-dependent inhibition of cell proliferation at concentrations ranging from 10 to 50 micrograms/ml. Cells cultivated in medium 4-7 with the addition of 50 micrograms/ml heparin revealed increased proliferative activity when compared with the corresponding controls. The antiproliferative activity on BLEC in medium containing aqueous humor suggests that heparin is a valuable tool for the prevention of secondary cataract in vivo.
Animals↗
A rabbit corneal epithelial cell line expresses functional platelet-derived growth factor beta-type receptors.
Platelet-derived growth factor (PDGF) is a family of three isoforms termed PDGF-AA, PDGF-AB, or PDGF-BB that induce proliferation in various mesenchymal cells. A rabbit corneal epithelial cell line (SIRC) was chosen to study the effects of the three isoforms of PDGF. These cells express approximately 43,000 PDGF beta-type receptors and less than 2800 alpha-type receptors on their surface. Thus, only PDGF-BB and -AB led to a transient dose-dependent rise in cytosolic free calcium with an effective dose for 50% of cells of 5 ng/ml. The PDGF-induced calcium signal is largely independent of the presence of extracellular calcium.
Animals↗
Effect of PDGF-AB heterodimer on a corneal epithelial cell line.
Exposure of cells of a rabbit corneal epithelial cell line (SIRC) to platelet-derived growth factor-AB heterodimer (PDGF-AB) resulted in a rapid and transient elevation of cytosolic free calcium concentration with a maximum at 2 to 3 min after stimulation. The kinetics of the calcium response were dose-dependent, e.g., higher concentrations of PDGF-AB caused a faster rise in cytosolic free calcium concentration. Maximum response was achieved with 10 ng/ml PDGF; higher concentrations up to 100 ng/ml did not further enhance cytosolic free calcium concentration. The ED50 was calculated to be 5 ng/ml PDGF-AB. After complexing extracellular calcium, PDGF-AB still caused a significant rise in cytosolic free calcium concentration indicating a mobilization of calcium from intracellular stores. This rise, however, was less pronounced than in the presence of extracellular calcium. The elevation in cytosolic free calcium concentration was not accompanied by an increased mitotic or proliferative activity of the cells as checked by [3H]thymidine incorporation and counting of cell numbers after 3 days of continuous incubation with various concentrations of PDGF-AB or by alterations in cell size and cell volume. In contrast, alterations in cell shape with a remarkable amount of rounded and partially detached SIRC cells after addition of PDGF-AB were observed within 24 h. Moreover, PDGF-AB caused a reversible distortion of cytoskeletal components such as actin-containing microfilament bundles, microtubules, and vimentin filaments. The results suggest that PDGF-AB may act only as a competence factor for the stimulation of SIRC cells via modification of the intracellular calcium homeostasis.
Animals↗
[EGF in therapy of corneal diseases. Principles and possible uses].
Numerous recent research results stress the importance of the epidermal growth factor (EGF) for the growth and differentiation of corneal tissues. EGF, a physiological ingredient in the tears, plays a predominant role in the regeneration of corneal epithelium. Furthermore, EGF has been shown to increase the proliferation and differentiation of corneal keratocytes and endothelial cells. Topically applied EGF significantly enhances both the re-epithelialization of denuded areas and the tear strength of stromal cicatrices. These results may lead to interesting pharmacological management procedures for numerous corneal disorders. Thus, the clinical feasibility of EGF therapy is currently the subject of intensive evaluation. A better understanding of its mode of action, both on the molecular and cellular level, and increasing knowledge of the pharmacokinetics, together with the development of suitable application media, may all make EGF an integral part of the next generation of ophthalmic drugs. This survey reviews basic molecular-pharmacological and biochemical findings on EGF and evaluates its possible value for therapeutical use.
Animals↗
[The pathologically changed Descemet membrane. Cultivation of human corneal endothelium on transformed membrane].
Corneal endothelial diseases are connected with structural and biochemical changes of Descemet's membrane (DM). Little is known about the possible effects of these changed basement membranes. In the present study we investigated the influence of changed DM on endothelium. Human corneal endothelial cells were cultured on DM of healthy corneas as well as corneas with Fuchs' endothelial dystrophy or bullous keratopathy, and cell growth and morphology were compared. On healthy DM, cells formed a confluent monolayer within 4 days. Cells cultured on Fuchs' DM showed a similar pattern. The polygonal cell pattern was interrupted merely in the area of the guttae, which were covered by cell processes alone. Culturing cells proved to be difficult on DM from corneas with bullous keratopathy. Even though the cells attached to the DM, they did not spread but became spindle shaped and rarely formed intercellular contacts. Even after 7 days no confluent monolayer was established. These results indicate that some though not all pathological DM changes interfere with cell growth.
Adult↗
Paraproteinemic corneal deposits in plasma cell myeloma.
We treated two patients who had IgG-kappa-light chain monoclonal gammopathy with partially crystalline and partially amorphic corneal deposits. Impairment of vision made keratoplasty necessary for each patient. Histologic examination of the corneal specimens showed deposits that stained positively for Masson's trichrome in all corneal cells. Immunohistochemical tests identified these deposits as IgG-kappa-light chain immunoglobulin fragments. Electron microscopy showed intracellular, rhomboid-shaped deposits enveloped by a membrane. The same deposits appeared in the conjunctival epithelium, within subconjunctival fibrocytes, and in the plasma cells of the bone marrow. Immunoelectrophoresis showed IgG-kappa-light chain fragments in the blood serum, the lacrimal film, and the aqueous humor. This suggests that the intracellular immunoglobulin fragments may have entered the corneal and conjunctival epithelium by way of the lacrimal film, the keratocytes by way of the corneo-scleral limbus vasculature, and the endothelial cells from the aqueous humor.
Aged↗
[Distribution of melanoma-associated antigens (HMB 45 and S 100) in benign and malignant melanocytic tumors of the conjunctiva].
The reactivity of the monoclonal antibody HMB 45 was evaluated in melanocytic tumors of the conjunctiva. Among these are 10 acquired melanoses, 19 nevi and 34 melanomas. Results were compared with the presence of the S 100 antigen. Especially the intraepithelial and junctional components of primarily benign lesions were stained with HMB 45. Within malignant melanoma this antibody reacts with melanocytes in the epithelial, junctional and subepithelial areas. The polyclonal antibody S 100 stains all melanocytes in pigmented lesions of the conjunctiva. Intraepithelial or subepithelial malignant infiltrating tumor cells show very intense staining with HMB 45. HMB 45 has therefore high specificity for stimulated melanocytes, but it does not distinguish benign and malignant proliferating melanocytic cells.
Antibodies, Monoclonal↗
[PDGF-induced calcium increase in cultivated epithelial cells in the cattle lens].
Platelet-derived growth factor (PDGF) is one of numerous growth factors regulating development of the ocular lens and its component parts. Although little is known about its site of action on the cell and the mechanisms of this confirmed mitogenic action, a close connection has been assumed between the influence of PDGF on both the lens epithelium and intracellular calcium homeostasis. The present study therefore examined the influence of PDGF on the concentration of intracellular free calcium [( Ca2+]i) in primary cultivated bovine epithelial cells. A dose-dependent [Ca2+]i increase of up to 300% followed stimulation with PDGF (5-50 ng/ml) in a medium containing Ca2+. [Ca2+]i also rose significantly in Ca(2+)-free medium, indicating mobilization from intracellular storage.
Animals↗