Effects of L-lysine and L-arginine on in vitro replication of feline herpesvirus type-1.

OBJECTIVE: To determine the effects of various concentrations of L-lysine and L-arginine on in vitro replication of feline herpesvirus type-1 (FHV-1). SAMPLE POPULATION: Cultured Crandell-Reese feline kidney (CRFK) cells and FHV-1 strain 727. PROCEDURE: Uninfected CRFK cells or CRFK cells infected with FHV-1 were cultured in Dulbecco's modified Eagle's medium or in 1 of 7 test media containing various concentrations of lysine and arginine. Viral titer and CRFK growth rate were assessed in each medium. RESULTS: Media depleted of arginine almost completely inhibited viral replication, whereas 2.5 or 5.0 microg of arginine/ml of media was associated with a significant increase in FHV-1 replication. In media with 2.5 microg of arginine/ml, supplementation with 200 or 300 microg of lysine/ml reduced viral replication by 34.2 and 53.9%, respectively. This effect was not seen in media containing 5.0 microg of arginine/ml. Growth rates of CRFK cells also were suppressed in media containing these concentrations of amino acids, but they were not significantly different from each other. CONCLUSIONS AND CLINICAL RELEVANCE: Arginine exerts a substantial growth-promoting effect on FHV-1. Supplementation of viral culture medium with lysine attenuates this growth-promoting effect in media containing low concentrations of arginine. Analysis of data from this study indicates that high concentrations of lysine reduce in vitro replication of FHV-1 but only in media containing low concentrations of arginine. Clinical trials will be necessary to determine whether supplemental administration of lysine, with or without arginine restriction, will be useful in the management of cats with FHV-1 infections.

Detection of feline herpesvirus-specific antibodies and DNA in aqueous humor from cats with or without uveitis.

OBJECTIVE: To determine whether uveitis in cats was associated with intraocular production of feline herpesvirus type 1 (FHV-1)-specific antibodies or with detection of FHV-1 DNA in aqueous humor (AH). ANIMALS: 44 cats with idiopathic uveitis, 29 cats with uveitis attributed to Toxoplasma gondii infection, 13 FHV-1 seropositive cats without uveitis, and 9 FHV-1 seronegative cats without uveitis. PROCEDURE: ELISA were used to detect FHV-1-specific antibodies and total IgG antibodies in serum and AH, and the Goldmann-Witmer coefficient (C-value) for intraocular antibody production was calculated. A polymerase chain reaction assay was used to detect FHV-1 DNA in AH. RESULTS: FHV-1 seroprevalence among cats with uveitis was not significantly different from seroprevalence among cats without uveitis. Intraocular FHV-1 antibodies were never detected in cats without uveitis. Significantly more cats with idiopathic uveitis (22/44) or with toxoplasmic uveitis (11/29) had evidence of intraocular antibody production (C-value > 1) than did cats without uveitis. Only cats with idiopathic uveitis had FHV-1 C-values > 8. Among cats with evidence of intraocular antibody production, cats with idiopathic uveitis had a significantly higher median FHV-1 C-value (9.61) than did cats with toxoplasmic uveitis (2.56). Overall, FHV-1 DNA was detected in AH from 12 cats, 11 of which had uveitis. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that FHV-1 can infect intraocular tissues of cats and that intraocular FHV-1 infection may be associated with uveal inflammation in some cats.

Persistence of herpes simplex virus type 1 DNA in chronic conjunctival and eyelid lesions of mice.

Herpes simplex virus type 1 (HSV-1) causes chronic blepharitis and conjunctivitis as well as keratitis in humans. The pathogenesis of these inflammatory ocular and dermal lesions is not well understood. We have examined the persistence of HSV-1 DNA and its relationship to inflammatory lesions in the conjunctiva and eyelid skin of mice which were inoculated with HSV-1 by the corneal route. Viral DNA was detected by in situ PCR in the conjunctiva and eyelid tissue of infected mice at 5, 11, 23, and 37 days postinfection (p.i.). This DNA was localized in the epithelial cells of the conjunctiva and hair follicles and in the epidermal cells of the eyelid skin. Viral proteins were not detected in the conjunctiva or the eyelid skin after 5 days p.i., even though histopathological lesions were found at 23 and 37 days p.i. in both tissues. The DNA-containing cells were adjacent to sites of inflammation in the chronic lesions in both the conjunctiva and the eyelid skin. A similar temporal and spatial relationship between HSV-1 DNA and inflammatory lesions has been previously reported for the cornea. Our data suggest that the lesions in the cornea, conjunctiva, and eyelid skin progress similarly. Further studies are required to determine whether the long-term presence of HSV-1 is involved in the mechanism by which these chronic inflammatory lesions develop. The presence of HSV-1 DNA in these extraocular tissues for extended periods may constitute persistent viral infection of nonneuronal cells.

Detection of feline herpesvirus 1 DNA in corneas of cats with eosinophilic keratitis or corneal sequestration.

OBJECTIVE: To determine whether feline herpesvirus 1 (FHV-1) DNA is in the corneas of clinically normal cats and cats with eosinophilic keratitis or corneal sequestration. SAMPLE POPULATION: Corneal biopsy specimens obtained from cats referred for treatment of corneal sequestration or eosinophilic keratitis. PROCEDURE: Corneal scraping or keratectomy specimens collected from clinically normal cats, cats with eosinophilic keratitis, and cats with corneal sequestration were evaluated for FHV-1 DNA by use of polymerase chain reaction (PCR). DNA was extracted from the tissue, and 1 microgram was assayed for FHV-1 by use of a single-round (40 cycles) PCR assay with primers directed at a 322-bp region of the thymidine kinase gene. Polymerase chain reaction positivity for clinically normal and affected cats of various breeds was compared by chi 2 analysis at alpha = 0.05. RESULTS: The FHV-1 DNA was detected in 5.9% (1/17) of corneas from clinically normal cats, in 55.1% (86/156) of corneal sequestra, and in 76.3% (45/59) of scraping specimens from cats with eosinophilic keratitis. Prevalence was significantly (P < 0.001) greater for cats with corneal sequestration or eosinophilic keratitis than for clinically normal cats. For cats with corneal sequestration, prevalence of FHV-1 DNA was significantly lower in Persian and Himalayan, compared with domestic shorthair and longhair breeds. CONCLUSION: Data strongly imply involvement of FHV-1 in the pathogenesis of eosinophilic keratitis and corneal sequestration. In Persian and Himalayan breeds, however, other nonviral factors also appear to be involved. CLINICAL RELEVANCE: Feline herpesvirus 1 must be considered when treating cats with corneal sequestration or eosinophilic keratitis.

The lens influences aqueous humor levels of transforming growth factor-beta 2.

BACKGROUND: Transforming growth factor-beta 2 (TGF-beta 2) is a pluripotent cytokine which has been suggested to play a number of roles in ocular physiologic and pathologic states. Intraocular fluid (i.o.f.) levels of TGF-beta 2 are quite high. Although the sources of ocular TGF-beta are not completely defined, the retinal pigment epithelium, the epithelium of the ciliary body and trabecular meshwork cells all secrete it. In this study we utilized canine lens and rabbit ciliary pigmented epithelial cell cultures to quantitate the in vitro secretion of TGF-beta 2. In addition, the effects of aphakia or the presence of cataractous lenses on IOF TGF-beta 2 levels were determined. METHODS: Lens and ciliary body epithelial cell culture supernatants and aqueous humors were assayed for total TGF-beta 2 levels by ELISA and bioassay. RESULTS: TGF-beta 2 accumulated in the media bathing lens epithelial cell cultures (0.7 +/- 0.03 ng/ml at day 2) and ciliary pigmented epithelial cell cultures (0.8 +/- 0.06 ng/ml at day 2) in a time-dependent manner. Surprisingly, aqueous humor from aphakic rabbit eyes contained significantly higher levels of TGF-beta 2 than their contralateral phakic controls. Furthermore, aqueous humor from canine eyes with cataracts also contained significantly higher levels of TGF-beta 2 than normal eyes. CONCLUSIONS: These results suggest that the lens secretes TGF-beta 2 and that the presence and status of the lens may influence IOF TGF-beta 2 levels.

Effect of alpha-chymotrypsin on breaking strength and ultrastructural morphology of canine ciliary zonules.

OBJECTIVE: To determine the effect of alpha-chymotrypsin treatment on breaking strength and ultrastructural morphology of canine ciliary zonules. SAMPLE POPULATION: Eyes from young random-source dogs from an animal shelter. PROCEDURE: Eyes were obtained immediately after euthanasia of dogs. The enzyme alpha-chymotrypsin was applied to the ciliary zonules of 1 eye of each dog; the other eye was treated with saline solution as a control. The breaking strength of ciliary zonules was measured, using a linear actuator and force transducer. The lenses and ciliary bodies were then analyzed by scanning and transmission electron microscopy. RESULTS: alpha-Chymotrypsin reduced the breaking strength of ciliary zonules by a mean +/- SD 44 (+/- 20)%, compared with that for saline-treated control eyes. Increasing the volume of enzyme further decreased the breaking strength of the zonules. Differences in the appearance of the ciliary body by electron microscopy were not apparent between enzyme- and saline-treated specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Application of alpha-chymotrypsin to enucleated canine eyes at a concentration used in people significantly reduces the breaking strength of canine ciliary zonules without any apparent damage to the ciliary body. alpha-Chymotrypsin may be useful in the removal of subluxated canine lenses and in removal of cataractous lenses in young dogs, in which phacoemulsification often results in appreciable post operative capsular opacification.

Principles of microsurgery.

The surgical principles and techniques used in ophthalmic microsurgery differ considerably from those used in general surgery. Successful ophthalmic microsurgery requires that the surgeon understand not only the design and complexities of the operating microscope, but how tissues are affected by minute manipulations with microsurgical instruments. Furthermore, ophthalmic microsurgery requires a detailed understanding of how microsurgical techniques need to be adjusted to accommodate the unique features of ocular tissues such as conjunctiva, cornea, and lens. A diligent effort to master the principles of ophthalmic microsurgery is probably the single most important prerequisite to becoming an accomplished ophthalmic surgeon.

Surgery for lens instability.

Lens luxation is a common and potentially blinding disease of dogs. If left untreated, degenerative changes in the pathways for aqueous humor result in glaucoma; however, if the lens is removed by ICLE before significant secondary changes occur, vision can be preserved. In addition, it is now possible to restore excellent vision by replacing the luxated lens with a synthetic IOL.

Effects of valacyclovir in cats infected with feline herpesvirus 1.

OBJECTIVE: To determine whether orally administered valacyclovir can be used safely and effectively to treat cats with primary, feline herpesvirus 1 (FHV-1) infection. ANIMALS: 14 specific-pathogen-free adult cats. PROCEDURE: Cats were infected with FHV-1 strain 87-727 (300 microliters, 10(7) plaque-forming units/ml) by ocular and nasal inoculations, and were treated every 6 hours with dextrose (controls) or valacyclovir (60 mg/kg of body weight, PO). Virus shedding from both eyes and the oropharynx was monitored every 2 days by virus isolation, and subjective clinical scores were assigned daily for ocular and nasal discharge and conjunctival hyperemia. Urinalysis, CBC, and serum biochemical analysis were done prior to inoculation, and on days 2, 5, 7, 9, and 12 of infection. Differences in CBC and serum biochemical indices between groups were compared, as were differences between preinfection values and maximal postinfection values, rectal temperature, and scores for disease severity. RESULTS: All cats developed acute conjunctivitis and rhinitis typical of FHV-1 infection. Beginning between days 6 and 9, valacyclovir-treated cats became noticeably more lethargic and dehydrated than did cats of the control group. Total WBC and neutrophil counts were significantly lower in cats of the valacyclovir group. The experiment was terminated on day 12 for humane reasons. Histologic changes attributable to FHV-1 infection were similar in all cats. Additional histologic abnormalities seen only in the valacyclovir-treated cats were coagulative necrosis of the renal tubular epithelium, centrilobular atrophy and hepatic necrosis, and severe bone marrow depression. CONCLUSIONS: Cats appear to be uniquely sensitive to the toxic effects of valacyclovir, and even high doses appear not to suppress FHV-1 replication in acutely infected cats. CLINICAL RELEVANCE: Use of valacyclovir is of questionable value in cats with acute FHV-1 infection and, at high doses, the drug may be toxic.

High sensitivity polymerase chain reaction assay for active and latent feline herpesvirus-1 infections in domestic cats.

A polymerase chain reaction (PCR) assay was developed and used to detect feline herpesvirus-1 (FHV-1) in conjunctival and oropharyngeal swabs, and in latently infected tissues (trigeminal ganglia, optic nerves, optic chiasma, olfactory bulbs and corneas) collected from 10 experimentally infected cats. There was good agreement between parallel tests of the swab specimens by PCR and virus isolation assay during the phase of acute, latent and recurrent disease episodes (kappa = 0.63, P < 0.001). The PCR reliably detected < or = 240 copies of FHV-1 template DNA, significantly improving upon previously published PCR assays for the agent.

Effect of a live attenuated intranasal vaccine on latency and shedding of feline herpesvirus 1 in domestic cats.

A prospective study was conducted that evaluated duration of virus shedding through acute and experimentally-induced recurrent disease episodes in 12 cats, and tissue distribution of latent infections, following intranasal vaccination with a temperature sensitive (ts) mutant strain of feline herpesvirus 1 (FHV1). Six of these cats were challenged with a virulent field strain of the agent to assess the extent to which vaccination affected subsequent shedding of virus and the establishment of latent infections. Virus isolation (VI) tests were done in parallel with a polymerase chain reaction (PCR) assay to compare the performance of each diagnostic method. The PCR confirmed that all 12 cats shed virus throughout the periods of vaccination, challenge or mock-challenge, and a cyclophosphamide-dexamethasone stress protocol to reactivate latent infections. Shedding to the tsFHV1 was documented by VI for up to 25 days following vaccination and for up to 15 days following challenge, but not after experimental stress. Overall, FHV1 was present in 144 of 300 (48%) cat-days of testing by PCR compared to 32 of 300 (11%) by VI. The frequency and distribution of latent FHV1 detected in neurologic, ophthalmic, and other tissues by PCR were identical for vaccine-only and vaccine-challenge groups, thereby disproving previous hypotheses that tsFHV1 mutants administered by this route protect against latency.

Photocoagulation of limbal melanoma in dogs and cats: 15 cases (1989-1993).

OBJECTIVE: To evaluate immediate clinical effects and long-term results of neodymium:yttrium-aluminum-garnet laser treatment of limbal melanoma in dogs and cats. DESIGN: Retrospective case series. ANIMALS: 13 dogs and 2 cats. RESULTS: At the time of treatment, 9 tumors were progressively enlarging and 4 were static. Recent growth characteristics of 2 tumors were not reported. Total energy applied ranged from 7.5 to 572 J. In all eyes, pigmented tissue shrank after treatment; however, 3 tumors recurred, 1 at 3 months and 2 at 1 year after treatment. CLINICAL IMPLICATIONS: Photocoagulation may be an effective means of treating limbal melanoma in dogs and cats.

Effects of topically applied mitomycin-C on intraocular pressure, facility of outflow, and fibrosis after glaucoma filtration surgery in clinically normal dogs.

The effects of mitomycin-C on intraocular pressure (IOP), facility of outflow (C), and Tenon's capsule fibrosis were studied over 60 days in 10 clinically normal dogs. A-1-piece, silicone glaucoma implant was surgically implanted into both eyes; the filtration site of one eye was treated with a single, 5-minute intraoperative application of mitomycin (0.5 mg/ml), and the fellow eye was treated in a similar manner with balanced salt solution. There were no significant differences in preoperative IOP or C-values between treatment groups. Mean IOP in eyes of both groups initially decreased from the preoperative value, but returned to the baseline value by day 21. Mean facility of aqueous outflow (C-value) increased in all eyes during the first 14 days (mitomycin-C-value = 2.26 +/- 0.72; control C-value = 2.38 +/- 0.81), then reached a plateau that was significantly higher than the baseline value in mitomycin (P = 0.039) and control (P = 0.041) eyes. Histologic evaluation revealed all implants surrounded by a connective tissue capsule composed of regular dense collagen and fibroblasts that was significantly (P = 0.003) thinner in the mitomycin-treated (scleral side = 167 +/- 62 microns; conjunctival side = 122 +/- 41 microns) than the control (scleral side = 261 +/- 92 microns; conjunctival side = 180 +/- 48 microns) group. There were, however, no significant differences in IOP or C-values between groups at any postoperative time interval. Results of this study indicate that intraoperative treatment with mitomycin suppresses, but does not prevent fibrosis around silicone filtering implants.

Iron uptake by cultured lens epithelial cells.

BACKGROUND: Transferrin and Fe concentrations increase in the intraocular fluids in pathological conditions and the lens accumulates Fe during ocular inflammation. Tissues take up Fe from transferrin by two mechanisms, receptor-medicated endocytosis of diferric transferrin and a process occurring at the cell membrane which may be mediated by an oxido-reductase. However, Fe metabolism, transport and storage have not been previously investigated in the lens. This study was designed to characterize the uptake of Fe from transferrin by lens epithelial cells in culture. METHODS: Primary, secondary and tertiary cultures of canine lens epithelial cells and cultures obtained from cataractous lenses were studied. Uptake of 59Fe from transferrin by these cultured cells was measured. Transferrin receptor populations were determined in receptor-binding assays. RESULTS: There was a distinct relationship between the amount of Fe-transferrin added and the amount of Fe taken up, which was linear for the primary cultures but significantly reduced for the secondary, tertiary and cataract cultures (252 +/- 21, 169 +/- 14, 153 +/- 14 and 96 +/- 2 ng Fe/mg protein, respectively). Transferring receptor expression in lens cell cultures was reduced 10-fold within 2 days of addition of serum to cells grown in low-Fe, serum-free medium for 1 week. CONCLUSIONS: The reduction of Fe uptake by the subcultured and cataract cell lines probably reflects a decrease in transferrin receptor expression and in the activity of an alternative pathway for Fe transferrin uptake occurring over time. This reduced Fe uptake may result from long-term exposure to relatively high Fe concentration in the media. A reduction in the expression of the transferrin receptor after incubation with high concentrations of Fe supports this conclusion.

Lens capsule opacification in aphakic and pseudophakic eyes.

BACKGROUND: Posterior capsule opacification (PCO) is the most common complication of lens extraction. Although intraocular lenses (IOLs) are thought to inhibit capsule opacification, the mechanisms by which they do this are poorly understood. This study was done to determine the effects of pseudophakia on secondary cataract and PCO in experimentally lentectomized dogs. METHODS: Twenty-four normal dogs were bilaterally lentectomized by phacoemulsification and unilaterally implanted with a plano-convex polymethylmethacrylate IOL. Secondary cataracts and capsule opacification were evaluated at weeks 1, 2, 4, 10, 14, and 20 after surgery by retrolillumination photography, light microscopy, and scanning and transmission electron microscopy. RESULTS: The pattern of secondary cataract and PCO in dogs was found to be similar to that in other animal species. Production of new lens material was prominent in the equatorial region, and PCO resulted from fibrous metaplasia of lens epithelium and subsequent capsular fibrosis and wrinkling. The presence of an IOL did not prevent the posterior migration of epithelium, nor did it prevent fibrous metaplasia. The presence of an IOL did, however, minimize the capsule-wrinkling effects of fibroplasia and limit the space available for lentoid formation. CONCLUSION: In pseudophakic eyes, IOLs influence secondary cataract formation by limiting the space available for lentoid formation and by maintaining a linear scaffolding for lens epithelial fibrous metaplasia.

The intracapsular extraction of displaced lenses in dogs: a retrospective study of 57 cases (1984-1990).

Records of 48 dogs (57 eyes) that underwent intracapsular lens extraction (ICLE) of displaced lenses were reviewed. Preoperatively, 73% (19/26 eyes) of eyes with anterior luxations had secondary glaucoma compared to 43% (10/23 eyes) with subluxations and 38% (3/8 eyes) with posterior luxations. Forty-one of 57 eyes (72%) had vision and intraocular pressure (IOP) less than 30 mm Hg four to six weeks after surgery. This percentage declined to 61% (22/36 eyes) in three months and 53% (8/15 eyes) in 12 months. Eyes with glaucoma before ICLE had a lower success rate (66%) than eyes without (82%). Since secondary glaucoma was the most common cause of failure, this study suggests that removal of a subluxated lens or a posteriorly luxated lens should be recommended before the lens moves into the anterior chamber.