Prolonged survival of corneal allografts incubated in alloantibody fragments.

BACKGROUND: In this study, we determined the binding characteristics of F(ab')2 alloantibody fragments to corneal antigens and assessed the capacity of these antibody fragments to protect corneal allografts from immune attack. METHODS: Goat anti-rabbit alloantibodies were pepsin-digested and labeled with 125I, and the time course of association and dissociation of the F(ab')2 fragments was determined. Corneal allografts were incubated in unlabeled F(ab')2 fragments and transplanted into allogeneic recipients, and the graft survival times were recorded. RESULTS: Binding of radiolabeled F(ab')2 fragments to rabbit cornea cells reached a maximum at 12 hr. At 32 degrees C (rabbit corneal temperature), the radiolabel eluted rapidly from the cornea, reaching baseline at 72 hr. At 4 degrees C (corneal graft storage temperature), significant amounts remained associated with the cornea at 96 hr. Mean survival time for grafts incubated in F(ab')2 anti-rabbit fragments was significantly greater than that of grafts incubated in nonimmune F(ab')2 fragments. Three of the corneal allografts incubated in goat F(ab')2 anti-rabbit fragments survived for 100 days, whereas the longest surviving control allograft incubated in goat F(ab')2 nonimmune fragments was rejected on day 24. Preincubation of corneas in unlabeled, immune F(ab')2 fragments followed by incubation in radiolabeled, immune F(ab')2 fragments suggested that antigen masking was not a factor in the prolongation of graft survival. CONCLUSION: Based on the binding and release kinetics and the graft survival times, it appears that the protective effect of immune F(ab')2 fragments extends well beyond the binding interval of the antibody fragments to corneal cell membranes.

Lack of levamisole effect on experimental herpes keratitis.

Levamisole, which is an anthelminthic, can restore depressed cell-mediated immunity (CMI) under some circumstances. In a controlled trial of experimental herpetic keratis in rabbits, levamisole was found to have no significant effect on acute herpetic keratitis or its recurrence rate. This is consistent with previous findings that other nonspecific CMI stimulation had no effect on recurrences of experimental herpes keratitis. Because of the known tendency of levamisole to produce agranulocytosis, we believe it should not be used in man unless proven effective in a carefully controlled double-blind study.

The lack of effect of tunicamycin and 2-deoxy-D-glucose on corneal stromal herpes in rabbits.

A rabbit model for herpes simplex virus (HSV) stromal keratitis, produced by intrastromal injection of live virus, was used to evaluate the effects of tunicamycin and 2-deoxy-D-glucose therapy. In vivo and in vitro evidence suggests that HSV strains that produce stromal disease secrete relatively large amounts of highly antigenic glycoproteins. Also, various studies have shown that tunicamycin and 2-deoxy-D-glucose inhibit the production of complete HSV-specific glycoproteins. Thus, these drugs might be capable of mitigating the clinical manifestations of HSV stromal keratitis by reducing the antigenic load. However, when topical therapy with tunicamycin and/or 2-deoxy-D-glucose was begun in rabbit eyes, the day after intrastromal inoculation of live RE strain HSV and several days before the appearance of stromal disease, no difference in the clinical course of herpetic ocular disease was seen between the experimental (treated) and control (untreated) groups.

Differences in natural and recombinant interferon for herpes keratitis in two animal models.

Natural human leukocyte interferon (natural HuIFN-alpha) and recombinant leukocyte A interferon (recombinant A HuIFN-alpha) were tested for prophylactic and/or therapeutic effects in reducing the severity of keratitis in rabbit and monkey eyes infected with McKrae strain herpesvirus. The results showed that the two interferons acted differently in the rabbit eye; combined prophylactic and therapeutic administration of natural interferon mitigated the disease, while recombinant interferon had no effect. In monkeys, the two interferons acted similarly. Combined prophylactic and therapeutic administration reduced disease findings, while therapeutic administration alone had no effect. Thus, studies in rabbits are not accurate predictors of primate study results; whether or not nonhuman primate results can be extrapolated to humans remains to be seen.

Cold stress-induced recurrences of herpetic keratitis in the squirrel monkey.

PURPOSE: Models of recurrent herpetic keratitis that depend on tissue damage or immunosuppression have been described. The authors report a model that depends only on minimal temperature stress to produce clinical recurrences in a small primate. METHODS: Squirrel monkeys (Saimiri sciureus) infected by the ocular route with the Rodanus strain of herpesvirus type 1 (HSV-1) were exposed to temperatures approximately 5 degrees C lower than the usual ambient temperature for periods as short as 12 hours. RESULTS: The corneas showed more or larger lesions typical of recurrent herpetic keratitis than are usually seen in these animals under normal conditions. Statistical analysis showed that there were significantly higher frequencies of epithelial keratitis at 18 degrees C and 20 degrees C (P < 0.0001). CONCLUSIONS: A minimal temperature change produced significant recurrences in this small animal within a short time. Tissues were not damaged to produce the recurrences. This approach may provide an efficient primate model for rapid testing of drugs to prevent clinical recurrence of ocular herpetic keratitis.

Virus chemotherapy: antiviral drugs and interferon.

Early antiviral drugs, such as idoxuridine and vidarabine, are less effective than newer drugs, such as trifluorothymidine and acyclovir. However, trifluorothymidine is less subject to the development of drug-resistant strains and can be administered topically as a clear drop, which increases patient compliance. Acyclovir has low toxicity and is selective for virus-infected cells because it must be phosphorylated by the viral thymidine kinase to become active. However, drug-resistant strains are produced relatively easily in vitro and may also develop in man with long-term use. To date, no antiviral drug alone has been shown to be effective in the treatment of stromal disease, and no antiviral drug is able to eradicate virus latent in the ganglia and thereby prevent recurrent herpetic infections. Combinations of antiviral drugs and antiviral drugs and interferon are being tested for enhanced efficacy in the treatment of ocular herpetic disease, and for prophylactic effects. The development of recombinant interferons has reduced cost and increased availability, but the effects of the 'manufactured' interferon are not identical to those of natural human leukocyte interferon in all experimental situations.

Suppression of ocular herpes recurrences by a thymidine kinase inhibitor in squirrel monkeys.

5'-Ethynylthymidine, an inhibitor of viral thymidine kinase (TK), was given intraperitoneally to squirrel monkeys previously infected by the ocular route with Rodanus strain herpes simplex virus. Spontaneous ocular recurrences were reduced during therapy, compared to saline-treated controls. This is the first in vivo demonstration that a viral TK inhibitor can reduce recurrences of HSV-1. Similar benefit would be expected for HSV-2 and perhaps VZV (varicella zoster virus).

Effect of 9-(4-hydroxybutyl)-N2-phenylguanine (HBPG), a thymidine kinase inhibitor, on clinical recurrences of ocular herpetic keratitis in squirrel monkeys.

9-(4-Hydroxybutyl)-N2-phenylguanine (HBPG) is a new viral thymidine kinase inhibitor that we tested for the ability to prevent recurrences of herpetic keratitis. Eighteen squirrel monkeys (Saimiri scuireus) were infected in both corneas with the Rodanus strain of herpes simplex virus type 1 (HSV-1). All corneas showed typical dendritic keratitis 3 days after infection, followed by spontaneous healing. On day 21, the monkeys were randomized into two coded groups and ocular examinations were begun. One group received intraperitoneal (i.p.) injections of HBPG, 150 mg/kg, in a corn oil suspension every 8 h, and the other group received i.p. injections of the corn oil vehicle only. On day 22, recurrences were induced by reducing the temperature of the room in the late afternoon so that a low of 18 degrees C was achieved during the night. After the morning treatment, room temperature was raised to the normal ambient temperature (24-27 degrees C), and treatment was discontinued. Treatment was reinstituted on day 27, the room temperature was lowered again on day 28, and treatment was again discontinued as before. Third and fourth cycles of treatment and cold stress were begun on days 34 and 69. Ocular examinations were continued until day 73, at which point the code was broken. We found that the HBPG treatment significantly reduced the number of corneas with recurrences during the treatment periods, compared with recurrences in untreated, cold-stressed animals (P = 0.01).

Trifluridine, cidofovir, and penciclovir in the treatment of experimental herpetic keratitis.

OBJECTIVE: To compare trifluridine eyedrops, cidofovir eyedrops, and penciclovir ophthalmic ointment for the treatment of herpes simplex virus type 1 keratitis. METHODS: New Zealand white rabbits were infected with the McKrae strain of herpes simplex virus type 1. Three days after viral inoculation, the rabbits were randomly assigned to treatment with 1% trifluridine, 0.2% cidofovir, 3% penciclovir ointment, or phosphate-buffered saline (for control) on various schedules. The severity of keratitis was graded in a masked manner. RESULTS: Treatment with any of the antiviral drugs resulted in significantly less severe keratitis than treatment with phosphate-buffered saline. There was no statistically significant difference between eyes given trifluridine 2, 4, or 7 times a day and eyes given cidofovir 2 times a day (P=.06, P=.43, and P=.19, respectively, using the F test of the analysis of variance). Cidofovir given twice a day was significantly more effective than penciclovir given either 2 or 4 times a day (P<.001 and P=.002, respectively). Even with once-a-day dosage, all 3 drugs were significantly more effective than phosphate-buffered saline (P<.001 for all). There was no significant difference between once-a-day trifluridine and cidofovir treatments (P=.17). Trifluridine administered 5 times a day was as effective as 1% cidofovir. A similar degree of punctate keratitis was seen after 4 to 5 days in eyes treated with trifluridine at the highest frequency, 1% cidofovir, or penciclovir ointment. CONCLUSION: Trifluridine treatment was highly effective in this rabbit model, even when given only once a day. Treatment with cidofovir was as effective as that with trifluridine. CLINICAL RELEVANCE: Cidofovir and penciclovir treatments may prove to be effective against epithelial keratitis. Clinical trials of trifluridine, cidofovir, and penciclovir with lower treatment frequencies appear to be warranted.

Cidofovir and experimental herpetic stromal disease.

OBJECTIVE: To compare topical cidofovir with topical trifluridine for the prevention and treatment of herpes simplex type 1 stromal keratitis in rabbits. METHODS: The RE strain of herpes simplex virus 1 was injected into the central stroma of both eyes of New Zealand white rabbits. Two to 3 days after virus inoculation, the rabbits were randomized to treatment groups of 10 each and treated with 1% trifluridine administered 5 or 7 times a day, 1%, 0.5%, or 0.2% cidofovir administered twice a day, fluorometholone administered twice a day, or balanced salt solution (BSS) administered twice a day (control) until day 21 after injection. The treated corneas were examined 3 times a week and the severity of stromal keratitis was graded in a masked fashion. To evaluate the ability of cidofovir to treat established stromal disease, groups of 10 rabbits each were inoculated with herpes simplex virus and treated with 1% cidofovir twice a day, 1% trifluridine 5 times a day, fluorometholone twice a day, or BSS twice a day beginning on day 7 after virus inoculation through day 21. RESULTS: Treatment with 0.2% cidofovir twice a day was not effective in preventing the appearance of stromal disease (P = .89), whereas treatment with 0.5% (P<.001) or 1% (P<.001) cidofovir twice a day or 1% trifluridine 5 times a day (P<.001) or 7 times a day (P = .006) significantly reduced the appearance of stromal keratitis on the 8 evaluation days, compared with BSS treatment (F test analysis of variance). There was no difference between the eyes treated with 0.5% cidofovir twice a day and those treated with 1% trifluridine 5 times a day. Treatment with 1% cidofovir was not effective in treating established stromal disease. CONCLUSIONS: Cidofovir and trifluridine are highly effective in preventing the appearance of herpetic stromal disease. Cidofovir is as effective as, but no more effective than, trifluridine in this model. Neither cidofovir nor trifluridine benefits established stromal disease, however. CLINICAL RELEVANCE: Cidofovir is a new, potent antiviral that seems similar in efficacy to trifluridine and is effective in the prevention of the development of stromal herpes, but is not effective in the treatment of established stromal disease in which hypersensitivity predominates.

Optisol corneal storage medium.

Optisol is an investigational, intermediate-term corneal storage medium containing chondroitin sulfate and dextran to enhance corneal dehydration during storage. We used scanning electron microscopy to grade endothelial cell morphologic characteristics in terms of cell shape, cell borders, cell swelling, and apical holes in pairs of corneas stored in Optisol and Dexsol. Optisolstored corneas showed significantly fewer morphologic changes after 14 days at 4 degrees C than did Dexsol-stored corneas. No significant differences were seen after 1 to 4 days at 26 degrees C. Temperature-reversal analysis showed no significant change in corneal thickness with warming after 2-week storage at 4 degrees C in either medium, although Optisol-stored corneas were significantly thinner than those stored in Dexsol at all times. The results of scanning electron microscopy suggest that preservation at refrigerator temperature for 2 weeks in Optisol is superior to preservation in Dexsol. Both media may be useful in preserving endothelial structure for limited periods at room temperature, which could provide a measure of safety in shipping or storage where refrigeration is unreliable.

Latanoprost increases the severity and recurrence of herpetic keratitis in the rabbit.

PURPOSE: To determine whether topically applied latanoprost increases the severity of acute herpes simplex keratitis, the rate of recurrence of herpes keratitis, or both, in the rabbit. METHODS: To determine the effect on severity of acute herpetic keratitis, the corneas of New Zealand white rabbits were infected with either the less-corticosteroid-sensitive McKrae strain or the corticosteroid-sensitive F(MP)E strain of herpes simplex virus type 1. Rabbits were randomly assigned to twice-a-day treatment with latanoprost 0.005%, dexamethasone sodium phosphate 0.1%, or balanced saline solution within 3 days of infection and evaluated daily for up to 13 days after infection. The severity of keratitis was graded in a masked manner. To determine the effect on recurrences of herpetic keratitis, animals infected with McKrae strain herpes simplex virus type 1 that survived to day 32 after infection were randomized to treatment with latanoprost 0.005% or balanced saline solution and evaluated for the presence of corneal lesions from postinfection day 32 to day 47. RESULTS: In the severity studies, treatment of F(MP)E-infected corneas with latanoprost or dexamethasone significantly worsened herpetic keratitis; by postinfection day 5, F(MP)E-infected eyes treated with dexamethasone or latanoprost demonstrated significantly higher severity scores than the eyes treated with balanced saline solution (P = .0001 and .008, respectively). Scores of McKrae-infected corneas treated with latanoprost or dexamethasone were not significantly different from scores of balanced saline solution-treated corneas. In the recurrence study, treatment with latanoprost significantly increased the appearance of clinical recurrences in McKrae-infected eyes, compared with balanced saline solution treatment (P = .0064). CONCLUSION: Latanoprost may worsen acute herpetic keratitis in the rabbit eye and increase the risk of recurrences in latently infected animals.

Effect of the herpes simplex virus genome on the response of infection to corticosteroids.

The type and severity of ocular herpetic disease, as well as the pattern of recurrence, have been shown to be determined by the virus genome. We infected rabbit eyes with two closely related recombinant strains of herpes simplex virus type 1 and treated one half of the eyes in each group with corticosteroids before or immediately after virus inoculation. The severity of disease in the first week was similar in the treated and untreated eyes infected with the F(MP)F strain; however, with F(MP)E infection, the disease in the treated eyes was significantly worse than the disease in the untreated eyes. Cultures of corneal virus showed similar titers in all of the groups, but cultures of trigeminal ganglia indicated that increased severity of disease did not result in an increased tendency toward ganglionic colonization. The results suggest that the response to corticosteroids is another factor that is determined by the genetics of the infecting virus, but that there is no correlation between worsening of disease with corticosteroid treatment and the establishment of virus latency.

Acyclovir in the treatment of herpetic stromal disease.

Seventeen patients (ten women and seven men, 23 to 72 years old) with stromal herpetic disease were treated with topical and oral acyclovir for 14 days. Of the 12 patients with disciform edema, five showed minimal improvement, four showed no change, and three showed worsening of their disease. Of the five patients with necrotizing stromal keratitis, one improved, one showed no change, and three became worse. The patients who had been treated with corticosteroids previously had a statistically significantly worse outcome than those who had not been so treated. One patient with necrotizing stromal keratitis showed virus particles in tissue specimens obtained by superficial lamellar keratectomy. Thus, acyclovir was not effective in the treatment of disciform edema or necrotizing stromal keratitis. Further studies are needed to ascertain whether the drug is therapeutically ineffective or whether acyclovir did not reach the stroma in amounts sufficient to affect the course of stromal disease in the human eye.

K-Sol corneal preservation.

K-Sol, a new cornea preserving solution which contains no calf serum of foreign protein and is used for refrigerated storage of donor tissue, has storage procedures identical to those currently used for tissue preservation in McCarey-Kaufman medium. K-Sol can keep corneas alive and usable for penetrating keratoplasty for at least two weeks. The clinical results in a series of 17 patients indicated that tissue preserved in K-Sol for as long as two weeks, even when used by inexperienced surgeons in difficult or unfavorable cases requiring extensive anterior segment reconstruction, including reoperations or retained intraocular lenses, gave results virtually identical to those obtained with tissue preserved in McCarey-Kaufman medium for only two or three days.

Effects of topical unoprostone and latanoprost on acute and recurrent herpetic keratitis in the rabbit.

PURPOSE: To determine the effect of the topical ocular hypotensive drug, isopropyl unoprostone, a docosanoid molecule with very weak prostaglandin activity, on herpes keratitis in the rabbit eye. METHODS: For acute disease, rabbit corneas inoculated with the corticosteroid-sensitive F(MP)E strain of herpes simplex virus type 1 were treated with various combinations of 0.12% isopropyl unoprostone, latanoprost, trifluridine, benzalkonium chloride 0.02%, dexamethasone sodium phosphate, ketorolac tromethamine, or saline solution beginning 1 day after infection. Severity of keratitis was evaluated in a masked manner. For recurrent disease, rabbit corneas infected with McKrae strain herpes simplex virus type 1 were treated with unoprostone or saline solution on postinfection days 25 to 42, and the presence or absence of lesions was recorded. RESULTS: Eyes treated with unoprostone showed significantly less severe disease than saline-treated or latanoprost-treated eyes during acute infection. Unoprostone-treated and saline-treated eyes showed no significant difference in the frequency of recurrent lesions. Eyes treated with latanoprost and/or dexamethasone, separately or in combination, showed increased severity of acute herpes simplex virus keratitis, whereas benzalkonium chloride 0.02%--treated eyes showed no significant difference, compared with saline treatment. Trifluridine resulted in rapid healing. CONCLUSIONS: Unoprostone did not increase the severity or recurrence rate of herpes simplex virus keratitis. Unoprostone requires twice-a-day administration, compared with once-a-day for latanoprost, and unoprostone lowers intraocular pressure less than latanoprost. Nevertheless, unoprostone's superior safety profile may make its use advantageous. Benzalkonium chloride alone did not make the keratitis worse.

Vancomycin-enriched corneal storage medium.

Antibiotics in a corneal preservation solution probably have little effect during storage at 4 C, but are effective as the tissue is warmed. The tissue acts as a sponge, soaking up the antibiotic from the solution and releasing it into the eye, where the bactericidal effect is achieved. Currently, high concentrations of gentamicin (relative to the minimal inhibitory concentration) are used in the preserving solution for this purpose. Presumably, proportionately high concentrations of any proposed new antibiotic added to supplement the bactericidal effect of gentamicin, such as the vancomycin used in this study, would be required. However, neither the ability of donor tissue to tolerate high concentrations of vancomycin nor the stability of vancomycin at neutral pH in appropriate storage media has been documented. We evaluated the addition of vancomycin (100 micrograms/ml) to two corneal storage media that contained gentamicin in terms of stability of the antibiotic in solution and the effect on the endothelial cells of donor tissue stored for two weeks at 4 C. Vancomycin was stable in solution at neutral pH (7.2) during the five-month period of the study; the concentration exceeded 90 micrograms/ml for the first five weeks. The endothelial cells from donor tissue stored in the vancomycin-enriched media showed no notable differences from those stored in the same media without vancomycin in terms of cell shape, cell borders, cell swelling, and apical holes. The stability of vancomycin in storage and the absence of endothelial toxicity in vitro support the potential use of this antibiotic as a supplement to gentamicin for the prevention of endophthalmitis in patients receiving corneal transplants.

K-Sol corneal preservation at room temperature.

K-Sol, a recently developed corneal storage medium that contains purified chondroitin sulphate in tissue culture medium (TC 199), is capable of preserving corneal tissue for 14 days at 4 degrees C. To study the effect of tissue storage in K-Sol at room temperature we preserved rabbit corneas in K-Sol and M-K medium for three or seven days at 25 degrees C. All of the corneal endothelial sheets were intact after three days. At seven days the change in pH of the K-Sol medium was less than that of M-K medium. Corneas preserved in M-K medium showed swelling of mitochondria and a decrease in the number of cytoplasmic organelles. Corneas preserved in K-Sol had organised cytoplasmic organelles and nuclei. Scanning electron micrographs revealed well preserved endothelial sheets. Corneas stored in the two media showed no significant difference in thickness. A pair of human corneas preserved in K-Sol at room temperature for six days maintained about 95% of the endothelial sheet in good condition. Small separations were observed between some of the endothelial cells. However, even in these areas, the cytoplasmic organelles were well preserved. It appeared that K-Sol is more stable than M-K medium at room temperature, and that both rabbit and human corneas can be preserved in good condition in K-Sol for at least six days at 25 degrees C.

Histological study of corneas preserved in two new media.

A new corneal preserving medium (K-Sol), developed by Kaufman and others, contains purified chondroitin sulphate, TC 199, HEPES buffer, and gentamicin. Another new medium (JM) containing bicarbonate-free glucose-phosphate Ringer solution and dextran 70 has been developed in Japan. New Zealand white rabbit corneas with scleral rims were stored in each medium at 4 degrees C for one or two weeks. The condition of the endothelium was evaluated histologically. Corneas preserved in both media were in good condition at the end of one week. Corneas preserved in K-Sol for two weeks showed fewer endothelial changes than similar tissue stored in JM for two weeks. Corneal swelling was also less in corneas preserved in K-Sol, than in corneas preserved in JM.

Endothelial cell damage in human and rabbit corneas stored in K-Sol without antioxidants.

Human and rabbit corneas were stored at 4 degrees C in K-Sol with and without antioxidants (ascorbic acid, reduced glutathione, alpha-tocopherol, and retinol acetate) for two to three weeks. All the corneas were then examined visually and by scanning electron microscopy. They appeared clear and slightly oedematous. Scanning electron micrographs were used to grade corneal endothelial cell morphology in a masked manner in terms of cell shape, cell borders, cell swelling, and apical holes. Corneas stored in K-Sol without antioxidants showed changes in cell shape, cell borders, and apical holes. Human corneas showed more morphological changes than rabbit corneas. The results suggest that antioxidants in K-Sol have an important role in the preservation of endothelial cell morphology.