Corticosteroids induce a sialated glycoprotein (Cort-GP) in trabecular cells in vitro.
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Animals↗
Biomedical subjects
R C Tripathi
Publications and source records attributed to R C Tripathi.
Localization of urokinase-type plasminogen activator in human eyes: an immunocytochemical study.
We investigated the presence of urokinase-type plasminogen activator (u-PA) in various structures of the human eye by using a biotin-strepavidin complex immunoperoxidase technique with a monoclonal antibody specific for human u-PA. A moderate to intense reaction product was seen in the corneal endothelium, episclera, sclera, iris muscles, lens equator and posterior capsule, peripheral vitreous, epithelium of pars plana, retinal pigment epithelium, axons, uveal fibroblasts, optic nerve fibers, and extraocular muscles. A weak reaction product was seen in the conjunctival and corneal epithelium, the trabecular meshwork, melanocytes and stroma of the iris and choroid, and the posterior layers of the retina. u-PA was not localized in the corneal stroma, conjunctival goblet cells, and certain parts of the lens. Comparison of the present results with those of our previous study (Tripathi, Geanon and Tripathi, 1987, Ophthalmology, 94, 1434-8) on the localization of tissue plasminogen activator (t-PA) in the eye indicate that the distribution of u-PA and t-PA overlaps in many tissues, but differs significantly in others, and that this finding may be related to their complementary and specific roles. As in other body systems, u-PA in ocular tissues is probably involved in processes such as fibrinolysis, wound healing, turnover of intra- and extracellular macromolecules, muscles and neuromuscular regeneration, synapse formation, and maintenance of general neuronal function.
Adult↗
Qualitative and quantitative analyses of sialic acid in the human trabecular meshwork.
By using colloidal iron and polycationic ferritin at low pH (1.8 and 1.9, respectively) as electron histochemical stains, we localized sialic acid moieties in the trabecular meshwork of normal human eyes. The markers were distributed continuously over the entire surface of the trabecular cells, but were localized linearly as well as randomly in the basal lamina. Within the beams, the markers were located irregularly in the collagen core, but were absent from the elastic tissue and from the 100 nm banded structures in the cortical zone. Pretreatment of the tissue with neuraminidase abolished this staining which indicates that sialated moieties are present in the stained structures. Biochemical analysis with the lectin Limax flavus agglutinin revealed that the major sialated polypeptides in individual samples of human trabecular meshwork migrated at apparent molecular weights of 56, 75, 95, 128, 140, 180 and 220 kDa under reducing conditions. The fractions at approximate molecular weights of 180 and 220 kDa include the sialoglycoproteins thrombospondin and fibronectin, respectively. The total content of sialic acid in the human trabecular meshwork (patient age range, 27-57 yr) was 3.6 +/- 0.6 mumol g-1 wet weight, as determined by a colorimetric assay. We conclude that significant quantities of sialic acid are present in the normal human trabecular meshwork as neuraminidase-sensitive alpha-ketosidically linked terminal residues of the polypeptides.
Adult↗
Cytotoxicity of hydrogen peroxide to human corneal epithelium in vitro and its clinical implications.
We investigated the cytotoxicity of hydrogen peroxide by exposing primary confluent cultures of human corneal epithelium to a single dose of this agent at concentrations ranging from 30 ppm to 100 ppm in the tissue culture medium. Our criteria for cytotoxicity included alterations in cytokinesis and movement of the cells, changes in cell morphology, and mitotic index, and cell degeneration and death. At a concentration as low as 30 ppm, hydrogen peroxide caused rapid cell retraction as well as cessation of cytokinesis and mitotic activity; formation of membranous vesicles preceded cell death, which occurred by seven to eight hours after exposure. At a concentration of 50 ppm, normal cell activity ceased almost instantaneously; numerous surface vesicles formed by 1.5 hours of exposure, and the cells died within four to five hours. Higher concentrations (70 to 100 ppm) of hydrogen peroxide caused cell death within a few minutes. Because neutralization of hydrogen peroxide and patient compliance with the manufacturer's instructions are critical in the proper use of peroxide-based disinfection systems, users should be aware of possible short- and long-term iatrogenic toxicity of residual peroxide on the cornea.
Cell Division↗
An unusual necrotizing dermatitis after a single application of topical benzoin and pressure bandage for enucleation of an eye.
We report on a patient who developed necrotizing contact dermatitis after a single topical application of tincture of benzoin and a pressure bandage following enucleation of an eye. The toxicity manifested in this case was more severe than any documented previously in the ophthalmic literature.
Administration, Topical↗
Corneal growth factors: a new generation of ophthalmic pharmaceuticals.
Several of the known growth factors either have an effect on corneal tissues or can be isolated from them. Both epidermal growth factor (EGF) and fibroblast growth factor (FGF) stimulate the proliferation of corneal epithelium, keratocytes, and endothelium. Compared to FGF, EGF is more potent in stimulating the growth of endothelial cells; it also increases the tensile strength of corneal stromal wounds. The corneal epithelium produces an angiogenic growth factor as well as a neuronotrophic growth factor. Insulin-like growth factor stimulates the growth of keratocytes and enhances the effect of EGF on the corneal endothelium. Mesodermal growth factor stimulates the proliferation of keratocytes and increases the rate of healing of damaged corneal endothelial cells. It is evident that growth factors have many potential clinical applications especially in accelerating corneal wound repair after surgery, chemical burns, or ulcers, and in increasing the numbers of corneal endothelial cells in aging and diseased corneas, as well as in donor corneas to be used for transplantation. Several parameters require evaluation, e.g. the dose, concentration, combination and formulation, exposure time, receptor affinity, and tissue interdependence of the growth factor(s), as well as the variability in patient response and severity of disease and/or injury. Investigations are also needed for the development of effective delivery systems and for determining whether growth factors are specific and safe in humans. Growth factors are emerging as a new generation of ophthalmic pharmaceuticals, and they will soon be integrated into the advancing practice of ophthalmic surgery and medicine.
Animals↗
Evidence for the neuroectodermal origin of the human lacrimal gland.
Immunohistochemical staining with antineuronal-specific enolase antibodies showed a positive reaction product in the acini and in the nerves of human major and accessory lacrimal glands. The fibrous tissue interstitium, septae, capsule, and blood vessels reacted negatively. Our findings indicate that the acinar portion of the lacrimal gland, but not the fibrous tissue component, is derived from neuroectoderm. We propose that the acini of the human lacrimal glands develop, not as an ingrowth into the mesenchymal stroma from the surface ectoderm that forms the conjunctiva, but as an outgrowth from the embryonic neuroectoderm, and more specifically, from cells that have migrated from the neural crest.
Adult↗
Indomethacin for the treatment of idiopathic orbital myositis.
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Adult↗
Neural crest origin of human trabecular meshwork and its implications for the pathogenesis of glaucoma.
We used an immunohistochemical method to examine the distribution of neuronal-specific enolase in the trabecular meshwork of adult normal human eyes, as well as in primary cultures of human trabecular cells maintained in vitro for up to 44 days with or without the addition of nerve growth factor. In tissue sections of the globes, neuronal-specific enolase was present in the cells of the anterior region of the meshwork and those of the inner uveal beams. The cells of the posterior region of the meshwork stained variably with antiserum against neuronal-specific enolase. Cultured trabecular cells were initially neuronal-specific enolase-positive, but became negative after 18 to 21 days in vitro. The addition of nerve growth factor restored the expression of neuronal-specific enolase in these cultured cells. Because the presence of neuronal-specific enolase in normal cells is believed to indicate their differentiation from neuroectoderm, our results provide evidence that the trabecular cells in human eyes are derived from the embryonic neural crest. Our finding also supports the hypothesis that abnormal migration or a defective terminal induction of the neural crest cells has a role in certain ocular diseases that are characterized by chamber angle anomalies or primary glaucoma.
Adult↗
Hydrogen peroxide damage to human corneal epithelial cells in vitro. Implications for contact lens disinfection systems.
We investigated the cytotoxicity of hydrogen peroxide by exposing primary cell cultures of human corneal epithelium to a single dose of this agent at concentrations ranging from 30 to 100 ppm. Hydrogen peroxide, at a concentration as low as 30 ppm, caused cell retraction as well as cessation of cell movement and mitotic activity. Formation of membranous vesicles preceded cell death that occurred by 7 to 8 hours after exposure to 30 ppm. With a concentration of 50 ppm, normal cell activity ceased almost instantaneously. Numerous surface vesicles formed by 1.5 hours of exposure, and the cells died by 4 to 5 hours. Higher concentrations of hydrogen peroxide (70 to 100 ppm) caused cell death within a few minutes. Because neutralization of hydrogen peroxide and patient compliance are critical in the proper use of hydrogen peroxide-based contact lens disinfection systems, users will be well served if the long-term effects of residual peroxide on the cornea are subjected to continued study.
Cell Survival↗
Cautions on the reanalysis of epidemiologic databases.
The statistical and epidemiologic literatures contain many examples of the reanalyses of medical databases. Some are confirmatory, such as the many reanalyses of the Stanford heart transplant data. Others, however, concern issues that differ from those that the data originally intended to address. We demonstrate in this paper that the second kind of reanalysis, if undertaken uncritically, can produce biased results. Utilizing a log-linear model, we show analytically that the reanalysis of a designed study without adjustment for the design variable can produce biased results. We express the magnitude of this bias as a function of second- and third-order interactions in the data. We demonstrate further that preliminary testing for the existence of second- and third-order interactions can provide substantial bias reduction.
Analysis of Variance↗
Protein composition of human aqueous humor: SDS-PAGE analysis of surgical and post-mortem samples.
We analyzed the protein composition of human aqueous humor. Samples were obtained by paracentesis from 25 human eyes (age range 64-92 years) at elective cataract surgery, and from 20 age-matched post-mortem eyes within 1.5 to 18 hr after death. Individual samples were assayed for total protein, and the polypeptides were separated by qualitative SDS-PAGE into high-, medium- and low-molecular-weight ranges and then silver-stained. The clinical samples showed a remarkable consistency in the total protein values (mean +/- SEM: 12.4 +/- 2.0 mg per 100 ml) and no detectable variations in the profiles of the silver-stained proteins. Twelve major protein fractions, with apparent molecular weights of 140, 80 (doublet), 67, 60 (doublet), 35, 27, 25, 17, 14.6 and 9 kDa, were present. A preliminary analysis showed that the 17 kDa band contained a molecule resembling basic fibroblast growth factor. Two additional samples of aqueous humor from patients whose blood/aqueous barrier was compromised during paracentesis showed a quantitative and qualitative increase in the polypeptides that were present. Compared with the samples of aqueous humor obtained at surgery, the post-mortem samples exhibited a greater variability in total protein content (56.1 +/- 11.6 mg per 100 ml) and an increased number of high- and low-molecular-weight protein fractions. In view of wide differences in the clinical parameters, including ocular and systemic medications, systemic illness, surgical premedications, anesthesia and total serum protein values, the similarity in the protein profiles of the carefully drawn surgical samples is most remarkable. Our results indicate that, in patients who underwent elective cataract surgery, the levels of major proteins in human aqueous humor are not affected by wide individual variations in the clinical parameters. We attribute this finding to the care taken in the collection of aqueous humor samples.
Aged↗
Hydrocortisone-induced DNA endoreplication in human trabecular cells in vitro.
Primary monolayer cultures of trabecular cells and organ cultures of trabecular meshwork, obtained from normal eyes of human subjects (age range, 40 to 70 yr) were exposed to hydrocortisone at concentrations ranging from 10(-4) M to 10(-6) M for periods up to 4 weeks. Phase-contrast microscopy of cultured cells showed an increase in the size of the nuclei (up to three times) and in the extent of the cell cytoplasm compared to those in control cultures, and vesicular structures frequently accumulated in the cytoplasm. Microdensitometry of Feulgen-stained cell nuclei indicated that the cells of the trabecular meshwork in vivo have predominantly diploid levels (2C) of DNA. Many nuclei in the trabecular cell cultures were polyploid and contained DNA values of 4C, 8C, and 16C. Cultures which had been exposed to hydrocortisone showed a significant shift toward the higher DNA classes, in contrast to the untreated control cultures; the average increase in the amount of DNA per nucleus was 36%. We discuss the relevance of these findings to the disease glaucoma in man.
Adult↗
Detection of glycated polypeptides in human aqueous humor by lectin-binding analysis.
The proteins from 30 samples of normal human aqueous humor were separated into major fractions by sodium dodecyl-sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with lectins to characterize the glycated polypeptides. The lectins used were derived from Canavalia ensiformis (concanavalin-A), Phaseolus vulgaris (erythroagglutinin), Arachis hypogaea (peanut agglutinin), and Ulex europaeus agglutinin-I. Using this microanalytical technique, the glycated polypeptides in the aqueous humor were separated into nine major fractions with apparent molecular weights of 17, 41, 50, 55, 61, 77, 80, 120 and 151 kDa. The predominant oligosaccharides detected on these fractions were complex chains usually with sialic acid residues. The possible roles of the glycated polypeptides in the aqueous humor in health and disease are discussed. Our investigation of these molecules in human aqueous humor provides baseline data for a rapid diagnostic analysis of microsamples of this fluid obtained from diseased eyes.
Aged↗
Monoclonal antibodies and lectins as probes for investigation of the cell biology of human trabecular meshwork: a preliminary report.
Two classes of ligand-binding molecules, (1) monoclonal antibodies (MABs) and (2) lectins, were used as probes to investigate the cell biology of human trabecular cells. We raised MABs by using human trabecular meshwork as the immunogen. One MAB (KAa25D7) consistently recognized two polypeptides with molecular weights of approximately 42 and 49 kilodaltons. The former comigrated with actin and the latter was shown by fractionation and extraction studies to be a macromolecular complex. Four lectins (wheat germ agglutinin, concanavalin A, Phaseolus vulgaris leukoagglutinin I, and Ulex europaeus agglutinin I) were used to identify the glycosylated polypeptides in human trabecular meshwork on Western blots. Compared to normal age-matched controls, there was a significant decrease in binding of the lectins to most polypeptides of the trabecular meshwork samples that were obtained from 4 eyes of 2 patients with primary open-angle glaucoma. Our preliminary studies show that lectins applied to Western blots and MABs are useful methods to probe the cell biology of human trabecular meshwork in health and disease.
Adult↗
Cytotoxic effects of benzalkonium chloride and chlorobutanol on human corneal epithelial cells in vitro.
By exposing primary cultures of human corneal epithelial cells to a single dose of either benzalkonium chloride or chlorobutanol, evaluated the cytotoxicity of these two preservatives which are commonly used in artificial tear solutions and other topical ophthalmic medications. Control and experimental cultures were analyzed by continuous time-lapse videomicrographic recordings as well as by sequential phase-contrast microscopy. Both benzalkonium chloride at a concentration of 0.01% and chlorobutanol at 0.5% caused immediate cessation of normal cytokinesis and mitotic activity, and epithelial cells degenerated within two hours and eight hours, respectively.
Benzalkonium Compounds↗
Extracellular release of tissue plasminogen activator is increased with the phagocytic activity of the retinal pigment epithelium.
Primary confluent cultures of retinal pigment epithelium (RPE) cells harvested from porcine eyes were challenged with latex spheres. The extracellular release of tissue plasminogen activator (t-PA) that was associated with phagocytosis of the beads by the RPE was measured by using enzyme-linked immunosorbent and chromogenic substrate assays. In the presence of serum and as compared to the controls, the phagocytosis of latex spheres produced a 5.5-fold increase in the amount of t-PA released into the ambient medium. Cell lysis was not a significant feature in either treated or control groups. Because the RPE in vivo is exposed to serum through the fenestrated choriocapillaris, and because phagocytosis by the RPE outer segments shed by the photoreceptors contributes to the maintenance of the functional integrity of the neural retina, we suggest that the release of t-PA associated with the phagocytic activity of RPE cells is an important physiologic event.
Animals↗
Epinephrine-induced toxicity of human trabecular cells in vitro.
To investigate the possible side effects of epinephrine on the aqueous outflow pathway, we studied the response of human trabecular cells in vitro exposed daily to the drug at various concentrations. Epinephrine at 10(-6) M caused abnormal cytokinesis and cell retraction, inhibited mitosis and phagocytosis, and induced a 4- to 5-fold increase in cAMP. After 7-10 days of exposure, notable degenerative changes were observed in the trabecular cells. Within certain limits, these effects were reversible when the drug was withdrawn. Exposure to epinephrine at 10(-5) M caused cell death within 96 hours. When a 10(-7) M concentration of epinephrine was administered for up to 10 days, no degenerative changes were seen, but mitotic activity was reduced somewhat. Pretreatment with timolol reduced, but did not completely eliminate, the cytotoxic effects of epinephrine. Cells with loose contact to the substrate were affected most. The morphologic changes and the observed loss of intracellular actin filaments indicate that the deleterious effects of epinephrine are probably mediated through damage to cellular contractile proteins. Although the precise cytotoxic action of epinephrine in vivo remains to be established, our results for primary cell cultures indicate a toxic action of this drug at concentrations of 10(-6) M or higher on human trabecular cells and suggest that, for prolonged use, the maximal dose of this drug at target sites should be in the region of 10(-7) M.
Adolescent↗