The disinfection of contact lenses contaminated with adenovirus.

PURPOSE: We compared the efficacy of different contact lens disinfection systems to eliminate adenovirus. METHODS: Laboratory study evaluating the elimination of adenoviral ocular isolates by contact lens disinfection systems. Hard (gas permeable) and soft contact lenses were contaminated with adenovirus serotypes 8 and 19, and then they were disinfected with chemical, hydrogen peroxide, and heat sterilization systems. The survival of the adenovirus was determined by the shell vial technique. RESULTS: Adenovirus survived chemical and hydrogen peroxide disinfection but not heat sterilization. CONCLUSION: Because heat sterilization is not readily available to sterilize adenovirus contaminated contact lenses, it may be prudent for patients with adenoviral keratoconjunctivitis to dispose of unclean contact lenses.

Antifungal synergism. A proposed dosage for corneal storage medium.

Fungal infections from eye bank-preserved corneas have led us to search for antifungal agents that will eliminate yeast and mold in McCarey-Kaufman (MK) medium and concurrently be nontoxic. Amphotericin B, natamycin, nystatin, and clotrimazole were tested in synergistic combinations in vitro against nine yeast and six mold specimens. For average yeast and mold concentrations of 3.4 X 10(4) and 1.36 X 10(4) colony-forming units (CFUs)/mL at 5 degrees C, and 3.36 X 10(4) and 2.3 X 10(3) CFUs/mL at 37 degrees C, respectively, a synergistic combination of all four drugs at one twelfth the minimal fungicidal concentrations proved fungicidal. This synergistic combination did not alter donor human corneal morphology under specular microscopy, nor did it inhibit rabbit corneal endothelial cell division preserved and propagated in antifungal supplemented MK medium. The synergistic drug mixture did prove to be fungicidal when the endothelial cells were challenged with fungal inoculum.

Selective cytotoxicity of monoclonal antibodies against fibroblasts: application in growth of human corneal epithelial cells in culture.

Several monoclonal antibodies were developed to cell-surface antigens of human corneal fibroblasts grown in culture. Indirect immunohistochemical analysis showed that the antigens recognized by these monoclonal antibodies were also present on the skin fibroblasts. However, these antigens were not detectable on corneal epithelial cells and, therefore, were specific with respect to cell-type. When screened for complement dependent cytotoxicity by dye-exclusion test and 51Cr-release assay, two of the antibodies, designated 5F2-2 (IgM type) and 7E2-1 (IgG type) were found to be cytotoxic to human embryonic lung and skin fibroblasts but not to corneal epithelial cells grown in culture. These two antibodies were utilized to selectively eliminate corneal fibroblasts in the presence of rabbit complement to establish corneal epithelial cells in culture free of corneal fibroblasts.

Cell-surface associated proteins of corneal fibroblasts: dissection with monoclonal antibodies.

It is now generally accepted that the cell surface is involved in the interaction of the cells with the extracellular matrix. To identify and characterize cell-surface-associated components of corneal fibroblasts, several monoclonal antibodies were developed. Hybridomas were developed by fusing mouse myeloma cells SP2/OAg14 with spleen cells from mice immunized with membrane fractions of corneal fibroblasts grown in culture. Twenty-five hybridomas secreting monoclonal antibodies to cell-surface components were selected by an enzyme-linked immunosorbent assay using corneal fibroblasts grown in microtiter plates as the substrate. Immunohistochemical staining demonstrated that the antigenic determinants recognized by these antibodies were not present on corneal epithelial cells, but were present on skin fibroblasts. The antigenic determinants recognized by two of these antibodies, designated 10D2 and 716, were matrix components of the corneal stroma. Immunochemical characterization of the antigens was carried out by indirect precipitation of the radioactively labeled cellular proteins with the monoclonal antibodies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the precipitates. Four antibodies were able to precipitate antigens from cell extract in detectable amounts. Antibodies designated 5E2, 9G2, and 10D2 recognized antigens consisting of polypeptides of approximate molecular weights 105K and 110K, while antibody 716 recognized an antigen of 100K molecular weight. However, based on the tissue distribution and cell-surface distribution, these antibodies reacted with different antigenic determinants. The antigen recognized by 716 was also secreted by cells in culture but consisted of 220K and 200K polypeptide chains. It was tentatively identified as cellular fibronectin, based on the reaction of this antigen with polyclonal antibodies to plasma fibronectin.

Structure and biosynthesis of rabbit lens capsule collagen.

The present study was designed to compare collagen synthesized by rabbit lens epithelial cells in culture with rabbit lens capsule collagen. Confluent monolayers of rabbit lens epithelial cells were established. Incorporation of [3H]-proline into glycoproteins secreted into the medium and cell surface components were analyzed in the presence of protease inhibitors. Gel filtration chromatography on sodium dodecyl sulfate--agarose (Bio-gel A-5m) of [3H]-labeled newly synthesized proteins by lens epithelial cells in culture resolved into a single precursor of approximate molecular weight of 160,000 daltons. Neither the medium nor the cell layer showed any evidence of low-molecular weight hydroxyproline-containing material. Limited pepsin digestion of this material cleaved the higher molecular weight chains into smaller components ranging from 25,000 to 110,000 daltons. Pepsin digestion and direct extraction of the collagenous components of the rabbit lens capsule revealed materials of high--molecular weight proteins similar to that synthesized in culture. Low--molecular weight (55,000 daltons) protein was only detected in lens capsules after prolonged pepsin digestion. S-Carboxylation of the lens capsules collagens did not affect their mobilities, but repepsinization gave rise to 110,000 dalton protein, although no significant changes in the amino acid composition were noticed. The absence of synthesis of low--molecular weight protein by cell culture and the presence of low--molecular weight components only after prolonged pepsin digestion of lens capsule could be the result of unusual susceptibility of the basement membrane collagens to pepsin attack.

Production of first component of complement by corneal fibroblasts in tissue culture.

Corneal fibroblasts were studied to determine if they have the ability to synthesize and secrete complement components in tissue culture. Culture media were assayed for functional complement activity of C1, C4, C2, C3, C5, C6, and C7 with the use of 50% hemolysis of sensitized sheep RBCs. Only C1 showed a progressive increase in hemolytic activity at days 3, 5, 7, 9, and 11 of tissue culture. This increase in hemolytic C1 could be reversibly inhibited by cycloheximide, an inhibitor of protein synthesis. Corneal fibroblasts may be a potential source of C1 in the cornea.

Selective growth of rabbit corneal epithelial cells in culture and basement membrane collagen synthesis.

Selective isolation of rabbit corneal epithelial cells was achieved with a medium containing D-valine. Long-term cultures of the epithelial cells synthesize basement membrane-type pro-collagen doublet chains of 160,000 and 180,000 daltons and a higher-molecular-weight species at 280,000 daltons. Some of these components are disulfide-linked. These peptides gave rise to a single pepsin-resistant collagen chain of approximate molecular size of 115,000 daltons.

Genetics of the connective tissue proteins: assignment of the gene for human type I procollagen to chromosome 17 by analysis of cell hybrids and microcell hybrids.

Somatic cell hybrids between mouse and human cell lines have been used to identify the specific chromosome that governs the synthesis of type I procollagen. Fourteen hybrid clones and subclones were derived independently from crosses between mouse parents [LM (thymidine kinase-negative) or A9 (hypoxanthine phosphoribosyltransferase-negative)] and human cells (human diploid lung fibroblasts WI-38 or diploid skin fibroblasts GM5, GM17, and GM9). The cultures were labeled with [(3)H]proline in modified Eagle's medium without serum. Radioactive procollagens were purified from the medium by the method of Church et al. [(1974) J. Mol. Biol. 86, 785-799]. DEAE-cellulose chromatography was used to separate collagen and type I and type III procollagen. Human type I procollagen was assayed by double immunodiffusion analysis with type I procollagen antibodies prepared by immunizing rabbits with purified human type I procollagen. These analyses combined with karyology and isozyme analyses of each hybrid line have produced evidence for the assignment of the gene for human type I procollagen to chromosome 17. A human microcell-mouse hybrid cell line containing only human chromosome 17 was positive for human type I procollagen, lending further support to the assignment of the human type I procollagen gene to chromosome 17. Finally, by using a hybrid line containing only the long arm of human chromosome 17 translocated onto a mouse chromosome, the type I procollagen gene can be assigned more specifically to the long arm of chromosome 17.

Escherichia coli mutants permissive for T4 bacteriophage with deletion in e gene (phage lysozyme).

Escherichia coli mutants have been isolated that are permissive for the infection by T4 phage with deletion in the cistron for the phage lysozyme, the e gene. Some, but not all, of these mutants are simultaneously permissive for the infection by T4 phage defective in the t gene, the product of which has also been implicated in the release of progeny phages. Most of these mutants shared the following properties: temperature sensitivity in growth and cell division, increased sensitivity towards a number of unrelated antibiotics and colicins, and increased sensitivity towards anionic detergents (sodium dodecyl sulfate and sodium deoxycholate). The possible biochemical basis for these phenotypes is discussed.

Structure of a transducing mycobacteriophage.

Electron micrographs of the transducing phage I3 for Mycobacterium smegmatis strain SN2 revealed a phage with a contractile tail and a head with isometric symmetry and visible capsomeres.