Drainage tube implants in the treatment of glaucoma following penetrating keratoplasty.

A retrospective review was undertaken to compare outcomes in 26 eyes that underwent penetrating keratoplasty (PKP) and drainage tube surgery (Molteno double-plate implant or Schocket procedure). Drainage tube surgery was performed either before PKP (10 eyes), after PKP (7 eyes), or at the same time as PKP (9 eyes). Mean follow up was 22 months. The average preoperative intraocular pressure (IOP) for all of the eyes was 31 mm Hg; 96% of them achieved a final IOP of less than 18 mm Hg (average, 14 mm Hg on a mean of 0.8 medications). Graft failure occurred in 11/26 (42%). Eight of these eyes were regrafted, and six of these eight have remained clear at a mean follow up of 22 months after regrafting. The overall PKP success rate, including the eyes that underwent repeat PKP, was 81%. Visual acuity remained stable or improved in 70% of the eyes.

Anti-rhodopsin monoclonal antibodies of defined specificity: characterization and application.

A panel of anti-bovine rhodopsin monoclonal antibodies (MAbs) of defined site-specificity has been prepared and used for functional and topographic studies of rhodopsins. In order to select these antibodies, hybridoma supernatants that contained anti-rhodopsin antibodies have been screened by enzyme-linked immunosorbent assay (ELISA) in the presence of synthetic peptides from rhodopsin's cytoplasmic regions. We selected for antibodies against predominantly linear determinants (as distinct from complex assembled determinants) and have isolated antibodies that recognize rhodopsin's amino terminus, its carboxyl terminus, as well as the hydrophilic helix-connecting regions 61-75, 96-115, 118-203, 230-252 and 310-321. Detailed specificities have been further determined by using a series of overlapping peptides and chemically modified rhodopsins as competitors. A group of seven antibodies with epitopes clustered within the amino terminal region of rhodopsin and a group of 15 antibodies with epitopes within the carboxyl terminal region are described. These MAbs have high affinities for rhodopsin with Kas in the range of 10(8)-10(10) M-1. Some MAbs specific for the carboxyl and amino terminal regions were used to compare these bovine rhodopsin sequences to those of different vertebrates. The MAbs cross-reacted with the different species tested to different extents indicating that there is some similarity in the sequences of these regions. However, some differences in the sequences were indicated by a reduced or absent cross-reactivity with some MAbs. In membrane topographic studies the MAbs showed both the presence and the accessibility of rhodopsin sequences 330-348, 310-321 and 230-252 on the cytoplasmic surface of the disk membrane. Similarly, sequences 1-20 and 188-203 were shown to reside on the lumenal surface of the disk and to be accessible to a macromolecular (antibody) probe. Antibodies directed against rhodopsin's carboxyl terminal sequence did not bind well to highly phosphorylated rhodopsin. Similarly, these antibodies as well as those against the V-VI loop inhibited phosphorylation of rhodopsin. Antibody A11-82P, specific for phosphorylated rhodopsin, recognized rhodopsin containing two or more phosphates and inhibited its further phosphorylation.

A practical method for rescuing desired hybridomas during monoclonal antibody production.

We present a practical method for the rescue of previously stable hybridoma clones which increases the proportion of desired cells in the population before cloning by limiting dilution. When the antibody activity of a culture supernatant was lower than that previously obtained, a precloning distribution at a density of 10 cells per microtiter well greatly improved the chances of obtaining a single active clone by subsequent limiting dilution. The Poisson distribution model was used to evaluate the method. Probabilities calculated clearly demonstrate the advantage of this precloning distribution step when attempting to isolate a hybridoma cell line that is relatively rare in a population.

Use of peptides to select for anti-rhodopsin antibodies with desired amino acid sequence specificities.

We have produced monoclonal antibodies directed against defined amino acid sequences of rhodopsin by using peptides for hybridoma selection, rather than for immunization. Mice were immunized with rhodopsin, and hybridoma culture supernatants were assayed by ELISA to detect anti-rhodopsin antibodies. These antibodies were further tested by competition ELISA using synthetic peptides from the rhodopsin sequence in order to select antibodies with the desired amino acid sequence specificity. Site-specific monoclonal antibodies selected in this way have affinities for the protein which are typically higher than that of antibodies which have been generated against peptide-carrier conjugates as antigens.

A monoclonal antibody specific for the phosphorylated epitope of rhodopsin: comparison with other anti-phosphoprotein antibodies.

Monoclonal antibody A11-82P has been prepared and shown to recognize the phosphorylated epitope of bovine rhodopsin. Antibody A11-82P is quite specific for phosphorylated rhodopsin; only the 200K neurofilament protein has been found to cross react, and this requires 10(3) more protein for the same extent of binding as evaluated by competition ELISA. An immunocytochemical study of light-adapted retina showed strong staining of rod outer segments. Survey of a variety of rat tissues showed no specific staining with A11-82P, further demonstrating that this antibody is quite specific for phosphorylated rhodopsin. Two other antibodies were found to bind both phosphorylated rhodopsin and the 200K neurofilament protein: RT-97 (1) and MAP 1B3 (2). Both antibodies also recognized other phosphoproteins and appear to be less specific in their structural requirements for a phosphoprotein epitope than is A11-82P.

The effect of enzymatic contact lens cleaning on adherence of Pseudomonas aeruginosa to soft contact lenses.

Previous studies have demonstrated that Pseudomonas aeruginosa adheres more readily to soft contact lenses with a mucin coating than to unworn contact lenses. The mucin coatings that develop on soft contact lenses may, therefore, play a significant role in the pathogenesis of contact lens-associated Pseudomonas corneal ulceration. We tested the ability of a variety of enzymatic contact lens cleaners and other enzyme solutions to decrease the adherence of Pseudomonas to mucin-coated soft contact lenses. Of the commercially available solutions that were tested, cleaning with Optizyme and Extenzyme significantly reduced the adherence of Pseudomonas to the lenses, whereas cleaning with the Softmate Weekly Cleaning System had no effect. Optizyme and Extenzyme were as effective as a 10% solution of acetylcysteine and more effective than a 0.25% trypsin solution. Neuraminidase at pH 5 was the most effective solution at reducing the adherence of Pseudomonas to the lenses, supporting the finding that sialic acid is a specific receptor for Pseudomonas aeruginosa. Soft contact lenses should be cleaned frequently with an effective enzymatic cleaner to reduce the likelihood of Pseudomonas adhering to the lens and thereby reduce the incidence of Pseudomonas corneal ulceration in soft contact lens wearers.

Radioimmunocytochemical localization of retinal S-antigen with monoclonal antibodies.

Two monoclonal antibodies (RSA1/83 and RSA2/83) were developed against a homogeneous preparation of bovine retinal S-antigen. The two hybridomas produced by mouse X mouse hybrid myeloma cells secrete immunoglobulin G. Indirect autoradiography on glutaraldehyde-fixed preparations of bovine explants was used to locate the antigenic site. Antibody RSA1/83 recognizes the antigen primarily in the apical region of the rod outer segment, while antibody RSA2/83 located the antigen both in the outer and inner segments of the rod photoreceptor cells. A distinct band of silver grains also appeared along the inner limiting membrane with both antibodies. Control explants showed no specific labeling pattern over the various retinal compartments.

Cell-mediated immunity in herpetic keratitis measured by leukocyte migration inhibition.

Leukocyte migration inhibition factor (LMIF) assays have been used as a measure of cell-mediated immune responses. Direct assays of this factor were determined in 30 patients with recurrent herpes simplex stromal keratitis during the quiescent stage of the disease and in 10 of these patients during the acute stage as well. The migration of leukocytes incubated in the presence of HSV antigens was compared to that without viral antigens for the migration index (MI). Only 1 out of 30 patients had a positive response during the quiescent stage, while among the 10 patients with the active disease, four had a positive response.

Hybridoma antibodies to insoluble antigens of the corneal epithelium.

Human corneal epithelial water insoluble proteins were used to immunize mice. Immune splenocytes were fused with Sp 2/0-Ag14 mouse myeloma cells by 40% PEG. Hybridoma antibodies obtained by somatic cell fusion were tested by radioimmunoassay. Supernatants from antibody positive hybrid colonies were used in immunofluorescence and crossreaction assays to locate and characterize corneal epithelial antigens. At least three distinct epithelial cell antigens were detected, one of which cross-reacts with rabbit corneal epithelial cells.

Adherence properties of Pseudomonas pili to epithelial cells of the human cornea.

In vitro adherence of Pseudomonas fluorescens organisms to the human cornea is correlated with bacterial pili. The role of pili in the attachment to human corneal epithelial cells was studied in an in vitro adherence assay system. A homogeneous, purified pilin preparation showed a molecular weight of 16,500 on SDS-polyacrylamide gels. Within 5 minutes incubation, 5.5 pg of pilin adhere to 15,000 epithelial cells. When epithelial cells were preincubated with purified pilin, a subsequent decrease in adherence of labeled pilin was noted. It appears that pili mediate adherence of Pseudomonas organism to human cornea.

Primary herpes simplex subepithelial dendritic keratitis.

A 37-year-old man developed an acute follicular conjunctivitis with preauricular lymphadenopathy believed to be epidemic keratoconjunctivitis. On the eighth day of his disease, subepithelial dendritic opacities developed in the cornea which were not typical of either epidemic keratoconjunctivitis or herpetic keratitis. A diagnosis of primary herpes simplex virus infection was established by positive viral culture and a rise in serum antibody titer to herpes simplex virus. Subepithelial dendritic keratitis as a manifestation of herpes simplex infection of the cornea has not been previously described. The lesions seen in this patient were not reproducible in rabbits and we believe they represent an unusual host response to the virus. This form of herpetic keratoconjunctivitis is extremely difficult to differentiate from epidemic keratoconjunctivitis. Corticosteroids should be used with caution in cases that are not completely typical of epidemic keratoconjunctivitis.

Production of hybridomas secreting antibodies to the cornea.

Major controversies exist in the literature on the presence of blood group antigens on the endothelial and stromal layers of the cornea, and the importance of major histocompatibility typing for keratoplasty. Antibodies were raised in BALB/C mice against water soluble proteins of corneal epithelium. Following fusion of spleen cells with myeloma cells (Sp2/0-Ag14) hybrid colonies were maintained in HAT selective medium. The supernates of each colony were measured and screened for antibody production by radioimmunoassay. Gel electrophoresis of the antigen showed nine major bands. The antibodies were partially characterized by cross reaction against other soluble corneal fractions.

Isolation of the plasma membrane from corneal endothelial cells.

The plasma membtanes of normal rabbit endothelail cells were isolated by the use of an aqueous two-phase polymer system. The membrane fraction was identified by electron microscopy, and a fivefold to eightfold increase in the specific activity of two plasma membrane markers. Na+-K+-ATPase and 5'nucleotidase was found. Recovery of the enzyme markers averaged 45% and 22%, respectively. Analysis of the purified membranes for glucose-6-phosphatase, a marker for endoplasmic reticulum, showed no contamination by this structure. This method for cell membrane characterization is promising in determining the enzymatic alterations of diseased corneas.

Leukocyte migration inhibitory factor in HSV infections.

The cell-mediated immune (CMI) response as measured by a direct assay of leukocyte migration inhibition factor (LMIF) was determined in a population of patients with recurrent herpes simplex virus (HSV) infections in the quiescent stage as well as in healthy volunteers. The migration of leukocytes incubated in the presence of HSV antigens was compared to that without viral antigens for the calculation of the migration index (MI). Eleven of 41 control subjects (16.8%) had a MI below 0.8, indicating a positive CMI response. In contrast, all the herpes patients tested had a MI above 0.8, suggesting an impairment in the production of LMIF at this stage of their disease. This difference was statistically significant (t = 4.296; p less than 0.001) and was not dependent on the age of the population. This study indicates that individuals with recurrent HSV infections have impaired CMI response betweeen attacks which may be associated with the stage of the disease.

In vitro studies on the mechanism of herpesvirus plaque growth inhibition by sensitized lymphocytes.

Inhibition of herpesvirus plaque growth was observed when herpes simplex virus (HSV)-sensitized rabbit lymphocytes were placed in contact with an HSV-infected human foreskin monolayer. This inhibition was obtained as early as 3 h when a ratio of 6 viable lymphocytes to target cells was used, and the supernatants of these cultures also demonstrated plaque size reduction when put onto newly infected cell monolayers. Interferon, which is present in this system, had no effect on HSV when tested on human foreskin monolayers, indicating that interferon was not the mechanism for plaque size reduction. Plaque growth inhibition was attributed to the T lymphocyte, because purified T cells reduced plaque growth and anti-rabbit thymocyte serum eliminated the effect of T cells. The specificity of this assay was determined by the facts that nonsensitized lymphocytes did not reduce the size of a plaque and the recognition of an infected cell by the sensitized lymphocyte was necessary for the release of a soluble mediator into the supernatant fluid. This cytotoxic lymphocyte was detected in the peripheral blood of rabbits as early as 4 days after initial corneal infection, with a maximum response at 7 to 10 days.

Failure of systemically administered adenine arabinoside to affect humoral and cell-mediated immunity.

The effect of a high dosage (250 mg/kg of body weight) of adenine arabinoside or ara-A (9-beta-D-arabinofuranosyladenine) on humoral immunity was studied in New Zealand white rabbits infected with the McKrae strain of herpes simplex virus. The rabbits were treated daily for 14 days with subcutaneous injections of ara-A. The primary and secondary humoral responses, as measured by neutralizing antibody titers, developed similarly in control and treated groups. Similar drug treatment was used on guinea pigs before or after sensitization with BCG vaccine. Subsequent challenge of the sensitized animals with Old tuberculin solution indicated that ara-A treatment had no effect on the induction or previously established cell-mediated immunity. The lack of immunosuppressive activity of ara-A at dosage levels higher than those used in primates makes this drug a potentially effective agent in the systemic treatment of herpetic infections.

Anti-herpes activity of adenine arabinoside monophosphate.

Adenine arabinoside monophosphate (Ara-AMP) demonstrated antiviral activity equivalent to adenine arabinoside (Ara-A) against herpesvirus Types 1 and 2 in tissue culture. Against established herpes virus Type 1 epithelial keratitis in rabbits, Ara-AMP 2 per cent was comparable in efficacy to Ara-A 3 per cent, and Ara-AMP 20 per cent was superior to Ara-A 3 per cent. These results are especially significant in that Ara-A's high solubility will permit (1) more concentrated formulations to be presented topically and (2) adequate absorption by parenteral routes with smaller fluid loads than required for Ara-A.

In vitro transformation of hamster cells by herpes simplex virus type 2 from human prostatic cancer cells.

A cell-associated herpes simplex virus type 2 found in a human prostatic carcinoma induced in vitro transformation of hamster embryo cells. The transformed cells (YW-74) have been shown to be hamster cells by karyotype analysis. Their epithelial morphology and growth pattern, which are different from the parental cell, have remained stable through cell passages. The presence of herpesvirus antigens in the transformed cells was determined by specific immunofluorescence and colony inhibition tests. Immunofluorescence staining with specific anti-herpes simplex virus type 2 serum showed an intense and distinctive nuclear and perinuclear fluorescence in about 95% of the transformed cells. In addition, exposure of these transformed cells to herpes simplex virus type 2-sensitized lymphocytes resulted in inhibition of growth and colony formation, while no effect was seen with nonsensitized lymphocytes. Both observations are consistent with the involvement of herpesvirus type 2 in the transformation event. This virus, which does not produce a lytic infection and is not found either in extracellular spaces or supernatant fluid of the transformed cell cultures, is unique in the fact that it is cell associated, noncytopathogenic, and capable of transforming cells in vitro, and its antigens are clearly demonstrated in the transformed cells.