Inhibition of rabbit keratocyte and human fetal lens epithelial cell proliferation by retrovirus-mediated transfer of antisense cyclin G1 and antisense MAT1 constructs.

The aim of this study is to evaluate the potential of gene transfer of cell cycle control genes as treatment of corneal haze or secondary cataract formation. The guiding hypothesis is that strategic modulation of the cyclin G1 or MAT1 gene by retrovirus-mediated gene transfer will inhibit proliferation of rabbit keratocytes (RabK) and fetal human lens epithelial (FHLEpi) cells in vitro. RabK and FHLEpi cell cultures were transduced in triplicate with retroviral vectors bearing either a nuclear-targeted beta-galactosidase, an antisense cyclin G1 (aG1), an antisense MAT1 (aMAT1) construct, or the neo(r) gene. The presence of beta-galactosidase activity in the transduced cultures was detected by immunohistochemical X-Gal staining, while cyclin G1 and MAT1 protein expression levels were evaluated by Western analysis. Proliferation of RabKs and FHLEpi cells was analyzed by counting the number of cells in the aG1 and aMAT1 vector-transduced cultures over 5 days. The mean transduction efficiency was 34.4% (SD 1.41) for RabKs and 19.7% (SD 1.83) for FHLEpi cells. Downregulation of cyclin G1 and MAT1 protein expression was noted 24 hr after transduction of RabK cultures with the respective vectors. Cytostatic effects of the aG1 and aMAT1 vectors in both RabKs and FHLEpi cells were most pronounced on the fifth day (RabKs, p < 0.0007; FHEpi cells, p < 0.001). An increased incidence of apoptosis was identified in both aG1 and MAT1-transduced FHLEpi cells. Taken together, these data suggest the potential utility of developing aG1 and aMAT1 retroviral vectors in gene therapy protocols for corneal haze and secondary cataract formation.

Explanation for good visual acuity in uncorrected residual hyperopia and presbyopia after radial keratotomy.

It has been observed that, after radial keratotomy (RK), uncorrected residual hyperopes may have better visual acuity than expected and that some presbyopes, corrected only for distance, have adequate near-vision. We offer a possible explanation for these observations by showing that the increased spherical aberration after RK may produce a second focus 1.5 diopters or more in front of the principal focus of the eye.

Human papillomavirus DNA in tissues and ocular surface swabs of patients with conjunctival epithelial neoplasia.

DNA from human papillomavirus (HPV) type 16 has been recently identified in conjunctival epithelial dysplasia and carcinoma. In other body sites, HPV 16 is thought to play a role in the development of dysplastic lesions. To further explore the relationship between HPV and conjunctival neoplasia, we examined paraffin-embedded tissue samples from 42 biopsies or excisions from 38 patients whose lesions ranged from mild dysplasia to infiltrating squamous carcinoma of the conjunctiva. We also examined limbal swabs from six patients with dysplasia or carcinoma, five of whom also had tissue samples available for study. HPV 16 DNA was present in 37 (88.1%) tissue samples, including duplicate samples from four patients. Five (83.3%) of six patients who had conjunctival swabs had HPV 16 DNA present in the swabs, including two patients whose lesions had been excised one and eight years before swabs were done. We conclude there is a high prevalence of HPV 16 DNA in conjunctival epithelial neoplasia, suggesting that the development of neoplasia is related somehow to the presence of this virus. However, based on its presence in clinically uninvolved eyes and on the persistence of infection many years after successful eradication of the lesions, HPV apparently does not act alone in the development of conjunctival epithelial neoplasia.

Corneal tensile strength in fully healed radial keratotomy wounds.

PURPOSE: To investigate the susceptibility to rupture of a human donor cornea with fully healed radial keratotomy (RK) incisions using biomechanical measurement methods and finite element analysis. METHODS: A human cornea 8 years after RK was cut into four strips and subjected to tensile testing until rupture occurred. The fellow cornea was pressurized on an artificial anterior chamber beyond the initiation of wound gape. The tensile strengths of nine strips from unincised corneas were measured as controls. To investigate the effect of epithelial plugs on the RK strip test results, a finite element model of a strip containing an epithelial plug was analyzed. RESULTS: Rupture occurred along the wound site with wide variability in the measured tensile strengths. There was no significant difference in tensile strength between the RK corneal strips (16.6 +/- 4.43 N/mm2) and the control strips (19.1 +/- 3.50 N/mm2). Four of the eight incisions of the fellow RK cornea gaped, but the cornea did not rupture up to a pressure of 2740 mm Hg. An epithelial plug of 10% of the corneal thickness was found in one of the incisions. The finite element results demonstrated higher stresses at the wound site that increased with the size of the epithelial plug. CONCLUSIONS: The presence of an epithelial plug in a fully healed radial keratotomy incision will create a stress concentration at the incision site that may predispose the cornea to rupture. The variability in the strength measurements indicates that the increase in rupture susceptibility due to RK may be hard to predict and may depend on factors such as the size of the plug and the strength of the wound collagen.

In vitro effects of aminoglycosides and fluoroquinolones on keratocytes.

PURPOSE: Commonly used fluoroquinolones are reported to have less of an effect than aminoglycosides on corneal epithelial cells. The purpose of this study was to assess the effects of these antibiotics on stromal keratocytes in vitro. METHODS: Cultured rabbit keratocytes were incubated with various concentrations of gentamicin, tobramycin, ofloxacin, norfloxacin, and ciprofloxacin. Evaluations were performed by means of phase-contrast microscopy and [3H]thymidine uptake assay after 24 hours and 48 hours of incubation with the drug (concentrations from 3 to 0.003 mg/ml). RESULTS: At a concentration of 3 mg/ml, all three fluoroquinolones inhibited keratocyte proliferation significantly more than either aminoglycoside after 24 hours (P<0.001) and after 48 hours (P<0.001). In contrast to the aminoglycosides, all three fluoroquinolones induced a dose- dependent inhibition of proliferation after 24 hours. Even at the lowest concentration (0.003 mg/ml), ofloxacin and norfloxacin inhibited keratocyte proliferation significantly (P=0.001) compared to control after 24 hours. Concentrations of fluoroquinolones ranging from 0.09 to 0.24 mg/ml produced a 50% inhibition of proliferation, a level of inhibition not observed with any tested concentration of aminoglycosides. After 24 hours, all three fluoroquinolones, but neither of the aminoglycosides, showed moderate to severe signs of cytotoxicity at a concentration of 3 mg/ml. CONCLUSIONS: Relative effects of fluoroquinolones and aminoglycosides on epithelial cells and stromal keratocytes appear to be different. This might have an impact on choosing the optimal antibiotic drug to be applied prophylactically in clinical situations in which the epithelium is absent, such as after photorefractive keratectomy or chemical burn.

Ablation rates and surface ultrastructure of 193 nm excimer laser keratectomies.

PURPOSE: To determine whether photorefractive keratectomy can be performed at lower energies than are currently employed in clinical trials. METHODS: Fresh pig corneas were ablated using a clinical excimer laser to study the effects of various energy densities (100-200 mJ/cm2) and beam diameters on ablation rates and on the surface ultrastructure of the ablated cornea. RESULTS: A 20-mJ increase in energy density was associated with a 0.03 micron per pulse increase in the ablation rate. A nearly linear increase in the pseudomembrane thickness occurred with increasing energy densities (r2 = 0.83) or decreasing ablation area diameter (r2 = 0.86). CONCLUSIONS: Our findings suggest that fluences less than those currently used in clinical trials (160-180 mJ) are capable of ablating tissue while producing thinner electron-dense pseudomembranes on the corneal surface. The relationship between pseudomembrane thickness and clinical factors such as reepithelialization and postoperative haze remains to be determined. Operating at lower fluences does have the advantages of allowing larger diameter ablations, reducing possible shockwave damage, and reducing the maintenance requirements for the laser.

Studies of adenovirus-induced eye disease in the rabbit model.

PURPOSE: To achieve a better understanding of the pathogenic processes associated with human adenovirus (Ad)-induced ocular disease. METHODS: Growth curves of Ad5 and Ad14 were performed in cell cultures derived from rabbit and human corneal epithelium (CE) and corneal keratocytes (CK). For in vivo studies, rabbit eyes were inoculated intrastromally and topically with 10(6) plaque-forming units per eye of Ad5 and ultraviolet light-inactivated (UV-1) Ad5 or Ad14, and the clinical features of the eyes were evaluated by biomicroscopic slit lamp examinations. Duration and quantitation of virus in tear samples were monitored. Humoral response was evaluated by enzyme-linked immunosorbent assay and serum neutralization titrations. Histopathologic and immunocytochemical staining of frozen corneal tissues was performed to determine the expression of major histocompatibility complex (MHC) class I and II and the presence of CD4+ and CD8+ T lymphocytes and CD18+ cells after the immunopathologic response elicited by virus inoculation. RESULTS: Both Ad5 and Ad14 replicated in all human cell cultures studied. In cells of rabbit origin, Ad5 replicated in cultured CE and CK cells, whereas Ad14 replication appeared restricted. Virus titers in ocular samples from Ad5-inoculated eyes peaked on postinoculation days 3 through 4, with approximately a 100-fold increase in infectious virus in comparison to initial titers. The duration of Ad5 shedding was 8.9 +/- 2.4 days. Ad5, Ad5 UV-I, and Ad14 induced seroconversion and subepithelial opacities. CD4+ and CD8+ T lymphocytes and CD18+ cells were present in these intrastromal immune cell infiltrates. Expression of MHC class I and II was observed in keratocytes and immune cells; MHC class I also was expressed on CE cells in inflamed areas. CONCLUSIONS: Ad5 is capable of replicating in both CE and CK cells of the rabbit eye. The presence of Ad antigens within the corneal stroma originating from infectious virus (Ad5), UV-inactivated virus (Ad5), or nonreplicating infectious virus (Ad14) can elicit indistinguishable immunopathologic responses in the stroma composed of CD4+, CD8+, and CD18+ cells.

Detection of human papillomavirus type 6/11 DNA in conjunctival papillomas by in situ hybridization with radioactive probes.

Twenty-three conjunctival papillomas and 28 conjunctival dysplasias were examined for human papillomavirus (HPV)-DNA sequences by in situ hybridization with nick-translated 35S-labeled HPV probes. Adjacent paraffin sections were hybridized with HPV type 2, 6, 16, and 18 probes at Tm - 17 degrees C. Fifteen tissues, all papillomas, displayed positive hybridization with the HPV-6 probe. Infection with HPV-6 (or the closely related HPV-11) appeared to be responsible for most of the conjunctival papillomas of children and young adults. The presence of genital tract HPV-6 in these lesions suggests that some of the infections were acquired during passage through an infected birth canal. The lack of hybridization in adult conjunctival dysplasias indicates either that HPVs are not associated with this condition or that the probes and the technique utilized were not adequate for demonstration of this association.

Evaluation of Cidofovir (HPMPC, GS-504) against adenovirus type 5 infection in vitro and in a New Zealand rabbit ocular model.

The antiviral inhibitory activity of Cidofovir [1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine dihydrate, HPMPC, GS-504] against adenovirus type 5 (Ad5) in the New Zealand rabbit ocular replication model was evaluated. The 50% inhibitory dose (ID50) of Cidofovir was determined to be 4.7-9.5 micrograms/ml against four adenoviruses (two Ad5, Ad8 and Ad14) by plaque reduction assay in A549 cells. Twenty-four New Zealand rabbits received intrastromal inoculation and topical application of 2 x 10(6) plaque-forming units (PFU) per eye of Ad5 McEwen, a clinical isolate. Cidofovir was administered topically at three different concentrations twice per day, beginning 16 h postinoculation and continuing for 20 consecutive days. The inhibitory effects were determined by measuring suppression of virus replication and by observation of the clinical effects. Compared to the placebo group, the 1% and 0.5% Cidofovir-treated groups showed significantly reduced Ad5 ocular titers, fewer days of viral shedding and less severe subepithelial opacities (P = 0.0001). The 1% Cidofovir group had the lowest humoral antibody titer against adenovirus antigens, but the difference was not significant (P = 0.24). Cidofovir proved to have potent antiviral activity against adenovirus replication and may have great promise for the treatment of adenovirus infection. Further investigation is recommended.

Empirical or culture-guided therapy for microbial keratitis? A plea for data.

In the United States, about 30,000 bacterial corneal ulcers are treated annually. Compared with 100, or even 20, years ago ophthalmologists today have available to them many diagnostic tools (including special media and bacterial identification techniques), and in impressive assortment of antibiotics. Many reviews and book chapters describe the uses of microbiologic studies--from Gram and Giemsa staining to media inoculation to immunofluorescence and even molecular genetics--to identify causative organisms. This literature also describes the formulas for preparation of highly concentrated, "fortified" antibiotics for initial treatment of bacterial keratitis, until culture and sensitivity results are available to guide modifications in therapy. It would seem, therefore, that "experts" in the field of corneal and external diseases have reached consensus on an appropriate initial microbiologic evaluation and treatment of suspected microbial keratitis, and the large body of literature on this topic might be considered to represent practice guidelines. It comes as a surprise to many that these published "guidelines" apparently are routinely ignored in current clinical practice.

Early changes in refractive error following radial keratotomy.

One hundred twenty-six eyes that had undergone radial keratotomy were analyzed to assess the early changes in refractive error after surgery. Between two weeks and three months, 38% of eyes became more myopic by less than 1 diopter (D), 34% by 1.00 to 1.87 D, and 25% by 2.00 to 3.50 D. Four eyes (3%) had a decrease of 0.25 to 1.25 D. The average increase between two weeks and three months was 1.31 D (SD, 0.9 D). To decrease this change, 15 eyes were semi-pressure patched at night and treated with topical steroids for four weeks following surgery. The average change between two weeks and three months in these eyes was 0.3 D (SD, 1.0 D). This retrospective study suggests that semi-pressure patching after surgery may be useful for eyes that are undercorrected or overcorrected by less than 1 D.

Computer-assisted corneal topography in parents of patients with keratoconus.

Recently developed computer-assisted devices allow detection of early topographic abnormalities of the cornea, including mild or abortive forms of keratoconus. To address the possibility that keratoconus is an inherited condition, we examined both parents of 12 randomly selected patients with keratoconus whom we were following up. Of the 12 sets of parents, at least one parent in each of seven sets had abnormal corneal characteristics. Using previously determined quantitative criteria (eg, central corneal steepening, greater steepening inferiorly than superiorly, and asymmetry between the two eyes) to distinguish normal from keratoconic corneas we found evidence of keratoconus in at least one of the 14 parents. The remaining five patients had parents with normal corneal characteristics. These data support the hypothesis that keratoconus is sometimes an inherited condition, exhibiting, at least in some families, autosomal dominant inheritance with incomplete penetrance.

Computer-assisted corneal topography in family members of patients with keratoconus.

In about 6% of patients with keratoconus, there is a history of familial disease. High keratometric astigmatism and mildly irregular mires observed by placidois disc have been suggested to represent variable forms of expression of a gene in family members of patients with keratoconus. We used a computer-assisted digital videophotokeratoscope to map the corneas of 28 family members of 5 patients with keratoconus. Abnormalities observed in family members included central steepening, greater steepening of the cornea inferior to the apex, and substantial asymmetry in the central dioptric power between the two eyes of the same individual. These findings were similar to, but less severe than, those found in patients with keratoconus and may represent the variable expression of a gene contributing to the development of keratoconus. Pedigree analysis in these families suggests an autosomal dominant mode of inheritance.

Surface ultrastructure after excimer laser ablation. Expanding vs contracting apertures.

Excimer laser corneal ablation (photorefractive keratectomy) for myopia using a diaphragm delivery system was performed on eye bank and animal eyes by either progressively expanding or contracting the iris aperture. Use of an expanding aperture, in which the final ablations cover the entire treatment zone, produced a pseudomembrane over the zone that had few discontinuities. Ultrastructural examination of the corneas ablated using a progressively contracting aperture, however, revealed numerous discontinuities in the surface pseudomembrane. Use of an expanding iris aperture to perform ablations to correct myopia may be preferable as it produces a pseudomembrane with few discontinuities. Clinical studies of surface healing in humans are necessary to determine the clinical relevance of improved continuity of the surface pseudomembrane.

Prospective corneal topographic analysis in surgery for postkeratoplasty astigmatism.

Computer-assisted photokeratoscopy was used to evaluate the topographic characteristics of corneas preoperatively and postoperatively in seven patients who underwent surgery for correction of postkeratoplasty astigmatism. The steep hemimeridians were typically separated by an angle other than 180 degrees (mean, 162.5 degrees) and the flat hemimeridians were often not orthogonal to the steep hemimeridians. Asymmetry of power (1.5 or more diopters) between these two major hemimeridians was also observed in three patients. Relaxing incisions were placed in the two steep hemimeridians and compression sutures were placed in the flat hemimeridians. The mean percent of reduction of astigmatism (vector-corrected) was 81.1%. The amount of keratometric astigmatism, and the degree of asymmetry of the hemimeridians were not correlated with the percent of reduction of astigmatism after placement of the relaxing and compression sutures. Computer-assisted topographic analysis may prove useful in planning transverse keratotomies centered on the steep hemimeridians and in placement of compression sutures in flat hemimeridians.

Topical ultraviolet light-absorbing chromophore protects against experimental photokeratitis.

Ultraviolet radiation may play a causative role in a number of human ocular and dermatologic conditions; reduction of environmental UV light exposure may decrease their incidence. We investigated the effects of two different UV light-absorbing chromophores, octyl methoxycinnamate (Parsol-MCX) and butyl methoxydibenzoylmethane (Parsol-1798) on corneal UV light transmittance, and evaluated topically applied Parsol-MCX for its protective effect against experimental UV light-induced keratitis in New Zealand white rabbits. Both Parsol-1789 (UV-A light absorber) and Parsol-MCX (UV-B light absorber) are well tolerated by New Zealand white rabbit eyes. Pretreatment with a single drop of Parsol-MCX reproducibly protected against UV light-induced keratitis, apparently by absorbing greater than 75% of UV-B (peak wavelength, 308 nm) irradiation.

Immunoglobulin deposition in the cornea after application of autologous serum.

A 47-year-old man with a history of multiple corneal allografts for recurrent herpes simplex keratitis developed a subtotal nonhealing corneal epithelial defect. The patient was treated with hourly drops of autologous serum. A ringlike infiltrate was subsequently observed, followed by reepithelialization of the graft. The patient later suffered allograft rejection of the cornea and recurrence of the epithelial defect, and a repeated penetrating keratoplasty was performed. Examination of the excised button demonstrated a total epithelial defect, changes compatible with allograft rejection, and, in addition, eosinophilic granular deposits within the superficial corneal stroma that corresponded to the "immune ring" observed clinically. Immunoperoxidase staining was positive for IgG, IgM, IgA, and kappa and lambda light chains. These pathologic changes lend credence to the hypothesis that the precorneal tear film may be a source of immunoglobulin that becomes deposited within the stroma.

Posterior capsule opacification. An in vitro model.

Posterior capsule opacification results from migration of lens epithelial cells onto the posterior capsule, where they produce collagen and contract to cause wrinkling of the capsule. We have designed an in vitro model that reproduces the process of opacification. Lens capsules removed from enucleated bovine eyes are placed within specially designed holders, creating a culture dish with the capsule as its bottom surface. Albino rabbit lens epithelial cells are then plated on the capsule. These capsules develop clinical and ultrastructural features of opacification very similar to those observed in humans. Membranes composed of confluent epithelial cells closely resemble the cellular membranes observed with specular microscopy in human patients. Transmission electron microscopy demonstrates that a membrane several cells thick forms on the capsules. This model allows careful morphologic, metabolic, and biochemical studies of the process of opacification and the investigation of therapies designed to prevent opacification of the posterior capsule.

Corneal thickness changes after high-risk penetrating keratoplasty. Collaborative Corneal Transplantation Study Group.

OBJECTIVE: To determine whether ultrasonic measurements of corneal thickness are of prognostic value after high-risk penetrating keratoplasty. DESIGN: A prospective, multicenter, randomized trial. PATIENTS: Four hundred fifty patients at high risk for graft failure because of previous immunologic graft failure or because of two or more quadrants of stromal vascularization. Patients underwent surgery and were treated according to a specific protocol and observed at frequent intervals. INTERVENTION: At each postoperative visit, ultrasonic measurement of central corneal thickness was performed and the corneal status was determined by biomicroscopic examination. MAIN OUTCOME MEASURE: Graft failure owing to immunologic or nonimmunologic causes. RESULTS: Corneal thickness stabilized by 3 months at a median thickness of 0.54 mm. The range of corneal thicknesses in patients with corneal grafts judged to be clear was large. In 49% of eyes, development of an allograft reaction episode was accompanied by an increase in corneal thickness of at least 10%; the greater the increase in thickness, the more likely the graft would fail. Clear grafts with central thicknesses of 0.59 mm or greater at 1, 3, or 6 months had a much greater risk of failure than those with thicknesses of less than 0.59 mm. CONCLUSION: Corneal thickness measurements after high-risk penetrating keratoplasty are of prognostic value.

Topical anti-inflammatory agents in an animal model of microbial keratitis.

Topical steroids potentiate Pseudomonas keratitis in the absence of concomitant antibiotic therapy. The effects of other anti-inflammatory agents on microbial keratitis are unknown, but if these agents do not intensify the infection, they may be useful in the initial management of microbial keratitis by limiting ulceration and thus the size of the resultant corneal scar. In rabbit models of untreated Pseudomonas keratitis and pneumococcal keratitis, topical 1% prednisolone phosphate, 0.03% flurbiprofen sodium, and vehicle were applied hourly. In cases of Pseudomonas keratitis, prednisolone worsened the clinical disease, and flurbiprofen further worsened the disease. When combined with effective antibiotic therapy, neither anti-inflammatory agent worsened the disease. In pneumococcal keratitis, neither anti-inflammatory agent worsened the disease. Topical anti-inflammatory agents should be used with caution in cases of microbial keratitis at least until effective antibiotic therapy has been instituted.