Suppression of lymphocyte proliferation and regulation of dendritic cell phenotype by soluble mediators from rat lacrimal epithelial cells.

Lacrimal epithelial cells appear to constitutively secrete autoantigens to their underling stroma. The present experiments address the hypothesis that they also secrete soluble factors that regulate immune responses. Epithelial cells, spleen cells and lymphocytes were obtained from rabbits or rats and cultured in various configurations. Monocytes from rat bone marrow were matured to dendritic cells (DC) ex vivo. Proliferation was measured by [3H]-thymidine incorporation; surface MHC Class II and CD86 using flow cytometry; and mRNA relative abundances using real time RT-PCR. Microporous culture inserts containing rat lacrimal cells inhibited proliferation of rabbit lymphocytes co-cultured with autologous lacrimal cells and of rat lymphocytes co-cultured with TNF-alpha-stimulated DC. They inhibited CD86 and MHC Class II surface expression by maturating DC and reversed surface expression of CD86 but not MHC Class II by partially matured DC. Subsequent exposure of partially matured DC to mediators from rat lacrimal cells reversed the ability to stimulate lymphocyte proliferation. TGF-beta(1) and IL-10 mRNAs increased somewhat when rat lacrimal cells were isolated but decreased markedly in rabbit lacrimal cells. Antibodies to TGF-beta prevented soluble factors from rat lacrimal cells from inhibiting proliferation of rabbit lymphocytes co-cultured with rabbit lacrimal cells, but recombinant TGF-beta alone did not mimic the soluble factors. IL-10 immunopositivity was detected in epithelial cells of interlobular ducts and occasional interstitial cells in rabbit lacrimal gland. Rat lacrimal epithelial cells secrete TGF-beta and other factors that synergize to suppress lymphocyte proliferation and regulate DC maturation. Interlobular duct epithelial cells in rabbit lacrimal glands may express similar functions.

Evaluation of the antiherpetic activity of acyclovir in rabbits.

Acyclovir (3 percent ointment) used topically one to five times a day on acute ocular herpes simplex virus (HSV) infection gave beneficial results as measured by a reduction in corneal involvement, conjunctivitis, iritis, and corneal clouding. Early intensive acyclovir topical treatment every two hours beginning 24 hours after inoculation prevented all but conjunctivitis. Topical treatments did not prevent the establishment of latent HSV infection. Effects of topical acyclovir treatment on rabbit eyes infected with parent McKrae, idoxuridine-resistant, and vidarabine-resistant strains of HSV-1 were studied. Topical acyclovir therapy given four times a day was significantly more effective than idoxuridine and vidarabine in suppressing acute herpetic ocular disease induced by either sensitive or drug-resistant strains of HSV-1. Virus isolation from neural tissues indicated that none of the therapy prevented viral infection in the nervous system. Intravenous acyclovir used twice a day (50 mg/kg) on rabbits with latent HSV infection appeared to suppress HSV in the nervous system but did not eradicate established latent HSV infection.

Human interferon alpha A or alpha D and trifluridine treatment for herpetic keratitis in rabbits.

Recombinant human interferon (IFN) alpha A and alpha D combined with 1% trifluridine ophthalmic solution gave beneficial results when applied topically at a dose of 1 x 10(6) U per eye four times a day commencing 4 hr after eyes were inoculated with herpes simplex virus (HSV-1). Acute herpetic keratitis was suppressed by trifluridine alone and the combined therapies, but the high-titered interferon preparations, alone, had little effect. Duration of HSV-1 shedding into tear film during topical treatment for acute herpetic keratitis was reduced slightly by combined therapy with either IFN alpha A or IFN alpha D with trifluridine.

Reactivation of murine latent HSV infection by epinephrine iontophoresis.

Iontophoresis of epinephrine into the cornea of previously infected mice was used in an attempt to induce reactivation of latent herpes simplex virus (HSV) infection of the trigeminal ganglia. BALB/c mice infected with HSV-1 strain McKrae following corneal scarification developed a latent infection of the trigeminal ganglia within 15 days. At 28 days postinfection, mice were subjected to a 3-day cycle of iontophoresis of epinephrine (0.01%) into the cornea. Ocular shedding of HSV occurred in 16/23 (70%) of stimulated mice; these animals did not shed HSV in the 3-day period prior to iontophoresis. Spontaneous shedding of HSV, however, was noted in 3/97 (3%) mice not subjected to epinephrine iontophoresis. "Infectious" virus was isolated only from the trigeminal ganglia of stimulated mice, whereas "latent" virus was isolated from the trigeminal ganglia of both stimulated and nonstimulated mice. All virus isolates were verified to be HSV by neutralization with a known HSV-1 antiserum. This ocular system thus allows for the study of the full spectrum of latent HSV infections, including latency, ganglionic reactivation, and peripheral virus shedding.

Effect of flurbiprofen on herpes simplex keratitis in rabbits.

The rabbit ocular model was used to determine if flurbiprofen, a new nonsteroidal antiinflammatory agent, caused exacerbation of herpes simplex virus (HSV) infection. Acute ocular HSV infections treated with flurbiprofen (0.1%) or dexamethasone (0.1%) drops four times a day for 10 days were similar in severity and duration as measured by corneal lesions, conjunctivitis, and corneal clouding. Eyes receiving placebo healed more rapidly than eyes treated with either anti-inflammatory drug. This preliminary study suggests that flurbiprofen appears to be comparable with dexamethasone in clinical exacerbation of acute ocular HSV infection.

Effect of acyclovir on acute and latent herpes simplex virus infections in the rabbit.

Acyclovir, a new potent antiviral drug, was used to treat herpes simplex virus (HSV) infection in the rabbit ocular model. Acyclovir (3% ointment) used topically one to five times a day on acute ocular HSV infection gave beneficial results as measured by a reduction in corneal involvement, conjunctivitis, iritis, and corneal clouding. Topical treatment did not prevent the establishment of latent HSV infection. Intravenous acyclovir used two times a day (50 mg/kg) on rabbits with latent HSV infection appeared to suppress HSV in the nervous system but did not eradicate established latent HSV infection.

Assessment of acyclovir on acute ocular infection induced by drug-resistant strains of HSV-1.

Effects of topical acyclovir treatment on rabbit eyes infected with parent McKrae, idoxuridine-resistant, and vidarabine-resistant strains of HSV-1 were studied. Topical acyclovir therapy given four times a day was significantly more effective than idoxuridine and vidarabine at suppressing acute herpetic ocular disease induced by either sensitive or drug-resistant strains of HSV-1. Virus isolation from neural tissues indicated that none of the therapy prevented viral infection of the nervous system.

Human alpha and gamma interferon analogs in rabbits with herpetic keratitis.

Complete gene synthesis methods have been used to construct analogs of human interferons (IFNs): these include a consensus of the known human IFN-alpha S, designated IFN-alpha Con1 and a variant of human IFN-gamma, designated IFN-gamma 4A. These interferons, in purified form, were used topically against herpes simplex virus type 1 (HSV-1) induced ocular keratitis in rabbits. Eyes pretreated with IFN-alpha Con1 had decreased signs of infection and a lower incidence of HSV-1 positive trigeminal ganglia (3 of 14 positive) compared to the placebo treated (10 of 14 positive). IFN-alpha Con1 was as effective as natural IFN-alpha subtypes on a units basis, despite the very high specific activity of this analog. IFN-gamma 4A used under similar conditions do not result in beneficial effects with treatments beginning 24 or 48 hr before or 4 hr after virus inoculation. Rabbits with confirmed latent HSV infection were treated topically with IFN-alpha Con1 (10(6) units per eye each day) either before or before and after attempts to intentionally reactivate the infection by bilateral iontophoresis of 6-hydroxydopamine plus topical epinephrine treatment of the corneas. These IFN-alpha Con1 treatment regimens along with intentional reactivation during latency did not: (1) lessen the frequency of inducible ocular shedding episodes; (2) alter the mean time of 3-5 days between attempts to reactive latent infection and the appearance of HSV in tears; or (3) significantly change the incidence of HSV-positive trigeminal ganglia (83-100% HSV positive).

Evaluation of the antiherpetic activity of 2'-fluoro-5-iodo-ara-C in rabbit eyes and cell cultures.

2-Fluoro-5-iodo-ara-C (FIAC), a new and potent drug, was tested for antiviral activity against several strains of herpes simplex virus (HSV), types 1 and 2. Effective dose-50% (ED-50) determinations for FIAC ranged from 0.023 to 0.51 muM for HSV-2. FIAC-treated cells did not exhibit any toxicity until the drug concentration was increased 2000-fold above the ED-50 level. Ocular herpetic keratitis in New Zealand white rabbits was treated with 1.0%, 0.1%, 0.01% FIAC beginning 3 days after inoculation wit HSV-1 (McKrae strain). Topical chemotherapy was administered five times per day for 7 consecutive days. After 4 days of treatment, corneal epithelial involvement, conjunctivitis, iritis, and clouding were not detectable in eyes receiving 1.0% FIAC. Toxic reactions were not observed in rabbit eyes treated with FIAC drug. HSV was not prevented from spreading into the central nervous system when topical FIAC therapy was initiated on day 3 after inoculation.

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.

Inhibition of human adenoviruses by 1-(2'-hydroxy-5'-methoxybenzylidene)amino-3-hydroxyguanidine tosylate.

Antiviral activities of four Schiff bases of aminohydroxyguanidine, designated ML1, ML4, ATL14 and LK11, were tested against human adenovirus types 5 and 8 (Ad5 and Ad8) in A549 cells by plaque reduction and virus yield reduction methods. Compound (ML1 1-(2'-hydroxy-5'-methoxybenzylidene)amino-3-hydroxyguanidine tosylate gave the best therapeutic indices (TC50/IC50) of 27.2 and 17.8 for Ad5 and Ad8, respectively. Pretreatment of cells with ML1 did not affect the adsorption nor the penetration of virus. Ultrastructure studies showed that only the drug treated infected cells had unidentified irregular shaped electron dense structures that might be drug altered viral macromolecules that were not assembled into complete infectious virus particles. Since these compounds have metal chelating properties, their antiviral activity may involve the early IA (EIA) gene which encodes a viral protein of 289 amino acid which has a zinc finger moiety that is required for its transactivation activity.

Inhibition of herpes simplex virus type 1 and adenovirus type 5 by heterocyclic Schiff bases of aminohydroxyguanidine tosylate.

Eleven heterocyclic Schiff bases of aminohydroxyguanidine tosylate (SB-AHGs), compounds I-XI, were tested for antiviral activity against herpes simplex virus type 1 (HSV-1) and adenovirus type 5 (Ad 5) via plaque reduction and virus yield reduction assays. This work was undertaken to test the hypothesis that low molecular weight SB-AHGs (MW < 235 for the free SB) make better antiviral agents than high MW SB-AHGs (MW > 300). The plaque reduction assay method demonstrated that three compounds, I, VII and IX, had moderate activity against HSV-1, with 50% inhibitory concentration (IC50) values of 38.0, 23.5 and 52.1 microM, respectively. Against Ad 5, compounds I, VIII and XI exhibited moderate activity, with IC50 values of 52.7, 19.3 and 5.1 microM, respectively. Among the compounds screened, compound I (1-[(3'-hydroxy-6'-methyl-2'-pyridyl)methylene]amino-3-hydroxyguanidi ne tosylate) was the most promising antiviral candidate, with selectivity indices (SI) of 10.2 (HSV-1) and 7.6 (Ad 5), respectively. Virus yield reduction assays indicated that compound I had less antiviral potency against HSV-1 than against Ad 5. The antiviral effects of compound I at a high input virus multiplicity of infection (MOI > 5) indicated that compound I had effective anti-adenoviral activity at 24 h post infection. This work demonstrated that some of SB-AHGs only have moderate antiviral activities against Ad 5 and HSV-1 viruses. In general, low MW SB-AHGs have low cytotoxicities to the host cells.

Evaluation of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-ethyluracil in a rabbit model of herpetic keratitis.

The nucleoside analog 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5- ethyluracil (FEAU) was tested in a rabbit model of acute herpetic keratitis and its effectiveness compared with that of acyclovir (ACV). FEAU or ACV was applied topically 3 times daily, beginning 3 days post-HSV-1 inoculation and continued for a period of 7 days. FEAU at a concentration of 1% (w/v) or 3% ACV resulted in significant lessening of the severity of corneal lesions, conjunctivitis, iritis, and corneal clouding at 24 to 48 h after beginning chemotherapy. No toxic reaction was observed in any rabbit eyes treated with either FEAU or ACV. The duration of virus shedding into tear film and colonization of the trigeminal ganglia, however, were not reduced by either FEAU or ACV treatment begun 3 days post-inoculation. Fifty percent effective dose (ED50) of FEAU determinations performed on isolates from tear film and on the virus inoculum in secondary rabbit kidney cultures yielded a range of 4.6-7 microM, with two in vitro resistant isolates having ED50S of greater than or equal to 1500 microM of FEAU. Fifty percent cell growth inhibition for FEAU was 3000 microM at 72 h.

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.

An experimental animal model of adenovirus-induced ocular disease. The cotton rat.

The adenoviruses are a common cause of eye disease in humans and clinically cause three basic syndromes: epidemic keratoconjunctivitis, pharyngo-conjunctival fever, and nonspecific follicular conjunctivitis. Although many serotypes of the adenovirus have been implicated, types 8, 19, and 37 are associated most commonly with ocular disease. There has not been a well-defined and reproducible animal model of this disease. The eyes of cotton rats inoculated with either adenovirus type 5 or type 8 developed clinical features similar to those seen in epidemic keratoconjunctivitis, with subepithelial corneal opacities, seroconversion, and virus shedding. The infectivity of adenovirus type 8 in a control animal illustrated the highly contagious nature of the disease. We conclude that ocular inoculation of at least some adenoviruses (ie, types 5 and 8) in the cotton rat produces an in vivo model for the study of adenovirus-induced ocular disease in humans.

Assessment of diclofenac on herpes keratitis in rabbit eyes.

Topical diclofenac sodium is a non-steroidal anti-inflammatory drug that is being developed for use in the control of postoperative and medical inflammation. A comparison was made of 0.1% diclofenac with 1% prednisolone sodium phosphate, 0.03% flurbiprofen, and a vehicle placebo in rabbit eyes with acute herpetic keratitis in a double-masked study. Maximum corneal epithelial involvement was observed in each group on day 6 postinoculation, and in eyes subsequently treated with prednisolone, the corneal epithelial involvement appeared to be more severe and to resolve more slowly. Conjunctivitis and corneal clouding peaked on days 6 to 7 for all treatment groups and remained most severe in the placebo-treated eyes, followed closely by those treated with prednisolone. The duration of virus shedding was the same for placebo-, flurbiprofen-, and diclofenac-treated groups (50% or more were virus negative by day 10 or 11). Only prednisolone-treated eyes had an extended period of virus shedding, and the rabbit mortality rate in this group was slightly higher. It thus appears that topical diclofenac does not exacerbate acute herpes keratitis; diclofenac-treated eyes displayed less or at least no more severe disease than did the eyes treated with the other anti-inflammatory agents tested, and shedding of virus into tears was not prolonged.

Characterization of infectious crystalline keratitis caused by a human isolate of Streptococcus mitis.

Streptococcus mitis isolated from a human with infectious crystalline keratitis was injected intrastromally into corneas of adult New Zealand white rabbits that were treated with tetracycline hydrochloride, methylprednisolone acetate, or a combination of tetracycline and methylprednisolone. Animals were followed up for up to 44 days; untreated corneas and those treated with tetracycline developed no disease or "fluffy" stromal infiltrates with overlying epithelial defects representing an abscess. Corneas treated with the combination of tetracycline and corticosteroid usually developed crystalline stromal opacities that on histopathologic examination were shown to be intrastromal aggregates of cocci. Transmission electron microscopy of crystalline lesions within 10 days of infection revealed typical cocci intermixed with a fibrillar material having periodicity characteristic of fibrinogen or fibrin, and immunoperoxidase staining for fibrinogen was positive. By 1 month, electron microscopy revealed aggregates of degenerated bacteria that were surrounded by cellular processes of activated keratocytes. Our studies demonstrate a model for crystalline keratitis in which organisms are seen to reside within the stroma for up to 44 days without an inflammatory response. Periocular corticosteroids appear to be necessary to create this model. It is possible that the organisms are isolated from the host response by fibrin or by keratocytes.

Potential of topical norfloxacin therapy. Comparative in vitro activity against clinical ocular bacterial isolates.

Norfloxacin, a new fluoroquinolone antibiotic related to nalidixic acid, was evaluated as a topical agent for clinical efficacy in bacterial eye infections. This study reports on the comparative in vitro activity of norfloxacin and ten topical antibiotics (nalidixic acid, polymyxin B, colistin, bacitracin, chloramphenicol, sulfamethoxazole, tetracycline, erythromycin, gentamicin, and tobramycin) against 203 pathogenic eye isolates of 17 genera (37 species). In general, norfloxacin had the greatest potency and broadest spectrum of activity of the agents tested. It was active against Staphylococcus aureus (minimal inhibitory concentration against 90% [MIC90], less than or equal to 1.0 microgram/mL), coagulase-negative staphylococci (MIC90, less than or equal to 1.0 microgram/mL), Pseudomonas aeruginosa (MIC90, less than or equal to 1.0 microgram/mL), and Haemophilus organisms (MIC90, less than or equal to 1.0 microgram/mL).

Combination antibiotic supplementation of corneal storage medium.

Gram-positive cocci frequently contaminate donor corneal tissue and represent the most common cause of postkeratoplasty endophthalmitis. Although gentamicin is currently added to corneal storage medium in an effort to decrease bacterial contamination of donor tissue, it has poor or variable in vitro activity against many strains of streptococci and staphylococci. To investigate whether the antibiotic supplementation of corneal storage media could be improved, we surveyed 11 antibiotics for antimicrobial efficacy under simulated storage conditions against gentamicin-resistant strains of Staphylococcus aureus, S. epidermidis, Streptococcus pneumoniae, and St. viridans. All antibiotics showed markedly reduced activity at 4 C as compared to their predicted activity at 37 C. Bactericidal activity of streptomycin and tobramycin was enhanced by preceding 4 C storage with a three-hour period at room temperature (23 C). Under these conditions, streptomycin showed the best antimicrobial activity of the 11 antibiotics tested. Addition of gentamicin to streptomycin resulted in further improvement of activity against S. aureus and S. epidermidis, whereas the addition of penicillin G to streptomycin enhanced the activity against St. viridans. Optimal antibiotic activity (99% or more killing) against all four isolates of gentamicin-resistant gram-positive cocci was best achieved with the combination of gentamicin, streptomycin, and penicillin G, coupled with a three-hour period at room temperature before 4 C storage.

Necrotizing retinitis caused by opportunistic virus infection in a patient with Hodgkin's disease.

A 33-year-old woman with Hodgkin's disease developed a painless progressive loss of vision in both eyes. Despite an ophthalmoscopic appearance of white-yellow retinal necrosis and retinal hemorrhage similar to that described with Hodgkin's disease, no sign of the disorder was found at autopsy. Instead, widespread evidence of Herpesviridae family virus infection was present in several organs, including the retinas. Opportunistic infection, including herpes simplex and cytomegalovirus, should be considered when retinitis complicates Hodgkin's disease.