Kathleen A. Yates

Human Cathelicidin (LL-37), a Multifunctional Peptide, is Expressed by Ocular Surface Epithelia and has Potent Antibacterial and Antiviral Activity

Purpose: This study determined whether LL-37 (cathelicidin) is expressed by conjunctival and corneal epithelia as part of ocular host defense. The antimicrobial activity of LL-37 was also assessed in vitro against Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Staphylococcus epidermidis (SE), herpes simplex virus type 1 (HSV-1), and adenovirus (Ad). Methods: Expression of LL-37/hCAP 18 mRNA and LL-37 protein was determined by reverse transcription–polymerase chain reaction (RT-PCR) and immunoblotting, respectively, in scraped human corneal epithelium and primary cultured human corneal and conjunctival epithelial cells. The EC50 values for three strains of PA and one each of SA and SE were determined for LL-37. LL-37 antiviral inhibition of HSV-1 and adenovirus was assessed by direct inactivation assays. Toxicity of LL-37 to A549 cells was evaluated by a MTT assay. Results: LL-37/hCAP18 mRNA and LL-37 peptide were expressed by human corneal and conjunctival epithelial cells. Antibacterial activity for LL-37 was demonstrated (EC50 values for the three PA strains were 2.8 ± 1.3, 1.9 ± 0.3, and 3.6 ± 2.1; for SA: 1.6 ± 1.5; for SE: 1.3 ± 1.9 μ g/ml). LL-37 produced a significant reduction (p < 0.001 ANOVA) in HSV-1 and Ad19 viral titers with distinctly different time-kill curves (p < 0.001). LL-37 (up to 111 μ M) produced no toxicity in A549 cells. Conclusions: Corneal and conjunctival epithelia express LL-37 as part of mucosal innate immunity to protect against bacterial and viral ocular infections.

Topical corticosteroids of limited potency promote adenovirus replication in the Ad5/NZW rabbit ocular model

Purpose. To determine the effect of topical therapy with several corticosteroids with limited potency on viral clearance in the adenovirus type 5 (Ad5) rabbit ocular model. Methods. Sixty rabbits were inoculated in both eyes with Ad5. On the first day, the rabbits were equally divided into four topical treatment groups: 0.12% prednisolone acetate (PA), 0.1% fluorometholone (FM), 1% rimexolone (RMX), and control. Treatment was administered four times daily, in both eyes, for 3 days. All eyes were cultured for virus on days 0, 1, 3, 4, 5, 7, 9, 11, 14, 16, 18, and 21. Results. Compared with the control group, treatment with PA, FM, and RMX significantly increased the number of Ad5 positive eye cultures from days 7–21. Fluorometholone and RMX prolonged the duration of Ad5 shedding, and FM increased the mean combined Ad5 titer from days 1–5 and 7–21. Conclusions. Treatment of an experimental ocular adenovirus infection with PA, FM, and RMX for 3 days significantly enhanced adenovirus replication compared with the control group. Short-term treatment of EKC with several commercially available topical corticosteroids with limited potency may offer symptomatic relief, but may also delay viral clearance and promote office and community epidemics.

Experimental laser-assisted in situ keratomileusis induces the reactivation of latent herpes simplex virus

PURPOSE We determined whether laser-assisted in situ keratomileusis acts as a trigger for the reactivation and ocular shedding of herpes simplex virus type-1 in a rabbit latency model. METHODS Herpes simplex virus type-1 latently infected rabbits were divided into three treatment groups: Group I received surface excimer laser ablation in both eyes (positive control), Group II received laser-assisted in situ keratomileusis in both eyes, and Group III received no treatment (negative control). Eyes were cultured daily for 10 days to determine herpes simplex virus type-1 reactivation. RESULTS The number of herpes simplex virus type-1 positive eye cultures and total herpes simplex virus type-1 shedding days were significantly greater after surface excimer laser ablation and laser-assisted in situ keratomileusis compared with the untreated control group (P < 0.002 and P < 0.000001, respectively). CONCLUSION Laser-assisted in situ keratomileusis as well as surface excimer laser ablation act as a trigger for the reactivation of herpes simplex virus type-1 in the rabbit latency model.

Sequence changes in the human adenovirus type 5 DNA polymerase associated with resistance to the broad spectrum antiviral cidofovir

Although there is currently no FDA approved antiviral treatment for adenovirus (Ad) infections, the broad spectrum antiviral cidofovir (CDV) has demonstrated potent inhibitory activity against many Ad serotypes in vitro and in an in vivo ocular replication model. The clinical potential of CDV prompted the assessment for the emergence of CDV resistance in Ad5. Serial passage of Ad5 in increasing concentrations of CDV resulted in derivation of four different Ad5 variants with increased resistance to CDV. CDV resistance was demonstrated by ability to replicate viral DNA in infected cells at CDV concentrations that inhibit the parental virus, by ability to form plaques in CDV concentrations of >20 microg/ml and by increased progeny release following infection and growth in media containing CDV. Using marker rescue, the loci for CDV resistance in variant R1 was shown to be mediated by one residue change L741S, one of two mutations within the R1 encoded DNA polymerase. The CDV-resistant variants R4, R5 and R6 also contained mutations in their respective DNA polymerase sequences, but these were different from R1; variant R4 contained two changes (F740I and V180I), whereas both R5 and R6 variants contained the non-conserved mutation A359E. R6 contained additional alterations L554F and V817L. The location of the R1 change is close to a region of the DNA polymerase which is conserved with other polymerases that is predicted to involve nucleotide binding.

Antiviral prophylaxis with twice daily topical cidofovir protects against challenge in the adenovirus type 5/New Zealand rabbit ocular model

Adenoviral ocular infections are the most common external ocular infections world wide and there is no approved treatment. Topical cidofovir has been shown to be effective in vitro, in animal models and in case studies for the treatment of adenoviral ocular infections. Prophylaxis to prevent transmission within households and to reduce community epidemics remains an important public health goal. The current study examined whether antiviral prophylaxis with cidofovir, twice daily dosing, would restrict viral replication following a large challenge inoculum of adenovirus type 5 (Ad5) in the New Zealand white rabbit ocular model. The results showed that antiviral prophylaxis with 1 and 0.5% cidofovir significantly reduced mean daily Ad5 ocular titers (days 0-5), the number of Ad5 positive cultures/total (days 1-14), serial Ad5 positive cultures/total (days 1, 2, 3, 4, 5, 7), and the number of eyes with Ad5 replication beyond day 0 (1% cidofovir only). Antiviral prophylaxis appears to be an effective strategy to reduce and restrict adenovirus replication experimentally.

Valacyclovir inhibition of recovery of ocular herpes simplex virus type 1 after experimental reactivation by laser in situ keratomileusis

Purpose: To determine whether the systemic administration of valacyclovir (Valtrex®) reduces ocular shedding of herpes simplex virus type 1 (HSV‐1) after laser in situ keratomileusis (LASIK) in the New Zealand White (NZW) rabbit latency model. Setting: Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. Methods: New Zealand White rabbits latently infected with HSV‐1 W strain were divided into 3 groups. The first received 100 mg/kg/day of valacyclovir; the second, 200 mg/kg/day of valacyclovir; and the third (control), saline. One half the total dose of valacyclovir was delivered via intraperitoneal injections twice daily for 7 days beginning with 1 dose before LASIK. The HSV‐1 ocular shedding was determined from eye cultures for 7 days after LASIK. Results: The administration of both 100 mg/kg/day and 200 mg/kg/day of valacyclovir significantly reduced the number of eyes (1/16 in both groups) and the total number of HSV‐1 shedding days (1/122 and 2/122, respectively) from which HSV‐1 was recovered compared to the control group (7/16 [P = .0396] and 14/129 [P < .007], respectively). Conclusions: Systemic administration of valacyclovir significantly reduced HSV‐1 ocular shedding after LASIK in the NZW rabbit latency model. The clinical implications of this study suggest that patients with a history of recurrent ocular herpes may be able to safely have LASIK with less risk of a recurrent herpetic episode while on valacyclovir antiviral prophylaxis.

Short-term Treatment With a Potent Topical Corticosteroid of an Acute Ocular Adenoviral Infection in the New Zealand White Rabbit

Purpose. To determine the effect of short-term topical therapy with 1% prednisolone acetate (PA) on normal immune adenoviral clearance in the rabbit ocular model. Methods. Thirty rabbits were topically inoculated in both eyes with adenovirus type 5 (Ad5). On day 1, the rabbits were divided into three topical treatment groups: 1% PA four times daily for 3 days, 1% PA four times daily for 5 days, control (artificial tears) four times daily for 5 days. Eyes were cultured for virus on days 0, 1, 3, 4, 5, 7, 9, 11, and 14. Results. Compared with the control group, treatment with 1% PA for 3 or 5 days significantly increased the total and daily number of Ad5-positive cultures from days 7 to 14, prolonged the duration of Ad5 shedding, and increased the mean combined Ad5 titer from days 1 to 5. In addition, treatment with 1% PA for 5 days increased the mean combined Ad5 titer from days 7 to 14. Conclusion. Treatment of an ocular adenoviral infection with 1% PA for as little as four times daily for 3 days significantly enhanced adenoviral replication compared with the control group. Short-term corticosteroid treatment of acute adenoviral ocular infections with 1% PA should be used judiciously.

Benzalkonium Chloride Enhances the Antibacterial Efficacy of Gatifloxacin in an Experimental Rabbit Model of Intrastromal Keratitis

PURPOSE The aim of this study was to determine whether a preservative (0.005% benzalkonium chloride [BAK]) enhances the antibacterial efficacy of an antibiotic (0.3% gatifloxacin, [GAT]) in vivo. METHODS Rabbits were inoculated intrastromally with GAT-resistant, methicillin-resistant Staphylococcus aureus or Staphylococcus epidermidis and then divided into four treatment groups: 0.3% GAT + 0.005% BAK; 0.3% GAT without BAK; vehicle including 0.005% BAK; and saline control. At 4 h postinoculation, topical treatment was initiated in both eyes every 15 min for 5 h. One (1) h after therapy, corneal colony counts were determined. RESULTS For S. aureus, duplicate experiments demonstrated that GAT + BAK and GAT without BAK significantly reduced colony counts, compared with BAK or saline (P < 0.05). Further, GAT + BAK significantly reduced colony counts, compared with GAT without BAK. BAK alone was equivalent to the saline control. For S. epidermidis, duplicate experiments demonstrated that GAT + BAK and GAT without BAK significantly reduced colony counts, compared with BAK or saline (P < 0.05). There were no differences between GAT + BAK and GAT without BAK for S. epidermidis. CONCLUSIONS For the first time, we demonstrated that a preservative (0.005% BAK) significantly enhanced the antibacterial efficacy of an antibiotic (0.3% GAT) in an experimental rabbit model of intrastromal keratitis.

The Effects of Xalatan® On the Recovery of Ocular Herpes Simplex Virus Type 1 (HSV-1) in the Induced Reactivation and Spontaneous Shedding Rabbit Models

PURPOSE Xalatan treatment has been reported both clinically and experimentally to promote recurrences of herpetic keratitis. Our goal was to determine the effects of topical Xalatan and its components on the recovery of ocular herpes simplex virus type 1 (HSV-1) in the Induced Reactivation (IR) and Spontaneous Shedding (SS) HSV-1/NZW rabbit latency models using virological outcome measures. METHODS HSV-1 latently-infected rabbits in both the IR and SS studies were divided into different topical treatment groups to evaluate commercial Xalatan, its preservatives, and vehicle against appropriate negative and positive controls. In the IR Studies, 91 rabbits received intra-stromal injections of water in both eyes to promote ocular shedding of latent HSV-1. All eyes were then treated and cultured for 10 days. In the SS Studies, 65 rabbits were treated and cultured in both eyes for 30 days. RESULTS Dexamethasone, a positive control, promoted extensive ocular shedding of HSV-1 in both the IR and SS Models. In general, neither Xalatan nor its components demonstrated any adverse effects, but some experimental variation was noted. All groups demonstrated comparable recovery of latent HSV-1 from respective trigeminal ganglia. CONCLUSIONS Our experimental studies support the world wide clinical epidemiological experience that commercial Xalatan does not appear to promote HSV-1 ocular shedding.

Lomefloxacin is an effective treatment of experimental bacterial keratitis.

Purpose. Lomefloxacin was evaluated as a potential topical therapy for bacterial keratitis. Methods. Lomefloxacin was compared with ciprofloxacin in different rabbit keratitis models. A total of 216 corneas were infected with Staphylococcus aureus (ciprofloxacin-susceptible and -resistant), Streptococcus viridans, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Serratia marcescens and were treated with lomefloxacin (0.3%), ciprofloxacin (0.3% Ciloxan), and the control phosphate-buffered saline (PBS), respectively. The data were analyzed statistically comparing the decrease in the number of recovered viable bacteria. Results. Compared with PBS-treated control corneas, the colony counts for all bacterial isolates were significantly reduced (p < 0.05) after topical treatment with either lomefloxacin or ciprofloxacin. For Gram-positive bacteria, lomefloxacin and ciprofloxacin were equally effective. For Gram-negative bacteria, lomefloxacin, while effective, was less so than ciprofloxacin under experimental conditions (p < 0.05). Conclusion. Our data, using multiple bacterial keratitis models, suggest that lomefloxacin is promising for therapy of bacterial keratitis. Further clinical studies are needed to expand its use for keratitis therapy.