Cytokine expression in murine corneas during recurrent herpetic stromal keratitis.

PURPOSE: Recurrent herpetic stromal keratitis (HSK) is a potentially blinding, immune-mediated disease. To better understand the immunopathology of recurrent HSK, we examined the cytokine profile of mouse corneas with the condition. METHODS: The eyes of latently infected mice were examined for corneal pathology and cytokine content following UV-B-stimulated herpes simplex virus (HSV) reactivation. RESULTS: Peak HSV-induced corneal disease, manifested by stromal opacification, occurred 7-14 days after viral reactivation in latently infected mice. In qualitative RT-PCR analyses, IFNgamma, IL-10, IL-4, and IL-12 p40 mRNA were simultaneously expressed before and during recurrent HSK. Competitive, semi-quantitative RT-PCR evaluation of cytokine mRNA revealed highest IFNgamma expression before and during clinical disease with a decline thereafter. IL-4 levels peaked and declined before day 14, while IL-10 peaked on days 7 or 14 and paralleled IFNgamma at lower levels. Small amounts of IL-12 p40 mRNA were detected late in the disease course. ELISA evaluation of corneal extracts demonstrated similar results, featuring early expression of Th2 cytokines relative to disease. CONCLUSIONS: The presence of Th2 cytokines during early stages of recurrent herpetic corneal lesions indicate the presence of a mixed Th1 and Th2 cell infiltrate, which is likely associated with a memory response to viral antigens. These data suggest that disease resolution in corneas with recurrent HSK may depend upon the balance between destructive and protective cytokines at individual sites of viral recurrence.

Three-dimensional localization: from image-guided surgery to information-guided therapy.

Image-guided surgery has become the standard of care for intracranial procedures. However, significant development is required before the benefits of this technology are brought to the majority of patients undergoing surgery. This article categorizes the areas wherein progress is needed, and indicates recent advances that may form the basis for the broad acceptance of this exciting technology. Emphasis is placed on a technique whereby preoperative imaging can be updated using low-resolution intraoperative imaging to reflect changes in anatomy caused by surgery, and on transforming image-guided surgery to information-guided therapy, in which diverse sources can be brought to bear at the time of greatest possible benefit, when the patient's anatomy is exposed for therapeutic intervention.

IL-1 and TNF-alpha are important factors in the pathogenesis of murine recurrent herpetic stromal keratitis.

PURPOSE: To better understand the role of interleukin (IL)-1 and tumor necrosis factor (NF)alpha in recurrent herpetic stromal keratitis (HSK), the cytokine content and the effects of anti-cytokine antibodies on mouse corneas with the disease were examined. METHODS: Competitive reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent analyses of IL-1alpha and TNF-alpha content were performed on corneas removed 3, 5, 7, 10, 14, and 21 days after latently infected NIH mice were irradiated with UV-B light to reactivate herpes simplex virus (HSV). In separate experiments, mice were injected with anti-IL-1 or anti-TNF-a antibodies 1 day before and 7 days after reactivation. RESULTS: UV-B irradiation stimulated an increase in corneal IL-la mRNA in reactivated (virus shedding) mice. This increase persisted longer and was higher than in UV-B irradiated uninfected control animals. IL-1alpha and TNF-alpha protein in corneas of reactivated mice was significantly elevated on days 3 to 10 compared with day 0 levels, and exceeded levels in control corneas on the same days. Anti-IL-1 and anti-TNF-alpha antibody administration both resulted in significantly decreased virus-induced corneal opacity between 7 and 21 days after UV-B exposure. CONCLUSIONS: IL-1alpha and TNF-alpha are upregulated in corneas in mice experiencing recurrent HSK. Abrogation of virus-induced corneal disease by anti-cytokine antibodies suggests that these cytokines play important roles in the pathogenesis of recurrent disease. Therefore, neutralization of specific proinflammatory cytokines may have potential therapeutic value.

Human cytomegalovirus infection in a retinoblastoma cell line in vitro.

BACKGROUND: The focus of these studies was to determine whether the Y79 human retinoblastoma cell line could function as a good in vitro model system for studying human cytomegalovirus (HCMV) infection. METHODS: Y79 cells were exposed to an HCMV mutant carrying a LacZ gene, and the resulting beta-galactosidase expression in infected cells was assessed by flow cytometry. The extent to which the three classes of viral gene products immediate early, early, and late proteins - were expressed was analyzed by immunohistochemical staining and Western blotting. Infected Y79 cells were also co-cultivated on human foreskin fibroblast (SF cell) cultures to recover virus. RESULTS: Infection of Y79 cells with the virus resulted in beta-galactosidase expression as detected by flow-cytometric analysis. Immunohistochemical staining revealed that a portion of Y79 cells expressed antigens reactive to monoclonal antibodies against immediate early, early, and late HCMV proteins. The 43-kDa early gene product was also detected by Western blotting. Infected Y79 cells co-cultivated on SF cell cultures yielded infectious foci, which turned blue following X-gal staining, demonstrating productive HCMV infection in the Y79 cells. CONCLUSION: These results demonstrate that while HCMV can productively infect Y79 cultures, it does so in a highly inefficient manner, leading these authors to conclude that this cell line does not provide a particularly good model system to study HCMV infection.

Reproduction of antiviral effect in an in vivo model of human cytomegalovirus retinal infection.

BACKGROUND: Cytomegalovirus retinitis remains a serious problem in AIDS patients, and the species specificity of human cytomegalovirus (HCMV) has hindered the development of animal models suitable for testing new therapeutic agents. Having previously described an in vivo model of HCMV retinal infection, we investigated its ability to reproduce the antiviral effects of the established anti-HCMV agent ganciclovir in order to determine the model's potential for evaluating novel agents. METHODS: Athymic rats had human fetal retinal tissue implanted in both anterior chambers. At 14 or 28 days post implantation, a suspension of a beta-galactosidase (lacZ+) mutant of HCMV was injected into each anterior chamber. Commencing 3 days prior to the injection of virus, rats in the treatment group received twice-daily intraperitoneal injections of ganciclovir (identical to a total of 100 mg/kg per day) for the duration of the study. The control rats received no drug. Twenty days after virus injection, the eyes of all rats were removed, sectioned and developed with X-gal substrate to detect any beta-galactosidase expression in the human tissue implants. RESULTS: Blue-staining foci of infection were detected in the implanted retinal tissue in 8 of 10 eyes from untreated control rats, but no beta-galactosidase expression was found in any of 12 eyes from animals which had received ganciclovir treatment. CONCLUSION: Intraperitoneal administration of ganciclovir successfully prevented HCMV replication in the intraocular retinal implants. This model of HCMV retinal infection is therefore suitable for preliminary evaluation of systemically administered antiviral agents.

Postexposure vaccination with a virion host shutoff defective mutant reduces UV-B radiation-induced ocular herpes simplex virus shedding in mice.

A herpes simplex virus type 1 (HSV-1) recombinant (UL41NHB) deficient in the virion host shutoff (vhs) function was tested as a therapeutic vaccine in an ultraviolet (UV) light-induced mouse ocular reactivation model. Mice were infected with HSV-1 via the cornea. Following the establishment of latency by HSV-1 the mice were subsequently vaccinated intraperitoneally with one dose of UL41NHB or with uninfected cell extract. Mice were subsequently UV-irradiated to induce viral reactivation and during the 7 days post-UV irradiation, numbers of mice shedding virus were reduced from 13/23 (57%) to 3/25 (12%), and numbers of virus-positive eye swabs were reduced from 40/161 (25%) to 6/175 (3%) by the vaccine (P < 0.001). These data suggest that deletion of vhs may be a useful strategy in the development of attenuated therapeutic HSV vaccines.

In vitro and in vivo effects of polyhexamethylene biguanide against herpes simplex virus infection.

PURPOSE: The differential diagnosis of Acanthamoeba keratitis frequently includes herpes simplex viral keratitis. Previous in vitro studies with chlorhexidine, a drug with antiacanthamoebic action, have suggested concomitant antiviral activity against herpes simplex virus. We tested another related antiacanthamoebic compound, polyhexamethylene biguanide (PHMB), to determine its activity against herpes simplex virus (HSV) in vitro and herpes simplex viral keratitis in vivo. METHODS: Equal aliquots of HSV-1 (McKrae) strain were incubated in a medium with no PHMB or with PHMB at 0.01, 0.02, or 0.05 for 5 min at 35 degrees C and the inoculum was then titered on a monolayer of E-2 cells (human corneal fibroblasts). Monolayers were examined on consecutive days and the percentage of plaque reduction was calculated. Eighteen rabbits (36 eyes) were inoculated with HSV-1 McKrae strain (10(5) pfu [plaque-forming units]/per eye). Rabbits were divided into three groups and treatment was initiated on day 3 postinfection. Group I received trifluorothymidine, group II received PHMB, and group III received artificial tears, each given five times daily in both eyes until day 10. Daily corneal swabbing to detect viral shedding and slit-lamp examination every 3 days were performed during this period. RESULTS: In vitro studies showed 62.5, 100, and 100% plaque reduction with 0.01, 0.02, and 0.05% PHMB, respectively. Slit-lamp examination of the rabbit corneas revealed faster resolution of dendrites in animals in group I treated with trifluorothymidine. Virus was not recoverable from corneal swabs in nine of 10 rabbits in group I by day 5, but all animals in groups II and III were still shedding HSV through day 8. CONCLUSION: Although PHMB has potent in vitro activity against HSV, it was not an effective treatment in the in vivo rabbit model of primary HSV keratitis at the concentration commonly used for treatment of Acanthamoeba infection. This suggests that 0.02% PHMB will not provide adequate antiherpetic coverage with treatment of keratitis of undetermined etiology in which the clinical differential diagnosis includes both herpes simplex and Acanthamoeba.

Efficacy of a recombinant glycoprotein D subunit vaccine on the development of primary and recurrent ocular infection with herpes simplex virus type 1 in mice.

The protective efficacy of a glycoprotein D subunit vaccine (gD2 SB AS4) was evaluated in a mouse model of human recurrent herpetic stromal keratitis (HSK). When administered before primary infection, gD2 SB AS4 protected mice against corneal pathology, mortality, and latency resulting from ocular viral challenge with herpes simplex virus type 1 (HSV-1) McKrae strain. In addition, gD2 SB AS4 significantly decreased postreactivation corneal disease. A control vaccine, gD2 alum, protected against acute ocular infection only. When administered after primary infection, gD2 SB AS4 vaccination decreased postreactivation ocular shedding but had no other significant effects. Vaccination with gD2 SB AS4 was associated with high anti-gD antibody responses and low delayed-type hypersensitivity responses. These results have identified a prophylactic vaccine, gD2 SB AS4, with activity against acute and recurrent HSK in mice and emphasize the need for vaccine evaluation in both primary and recurrent ocular herpetic disease models.

An in vivo model of human cytomegalovirus retinal infection.

PURPOSE: To develop an animal model system in which human retina implanted in the anterior chamber of the eyes of rats would support human cytomegalovirus replication. Cytomegalovirus retinitis currently represents the most common cause of posterior uveitis in many urban areas in North America. Despite the tremendous interest in cytomegalovirus retinitis as a result of the acquired immunodeficiency syndrome (AIDS) epidemic, human cytomegalovirus infection has been difficult to model in vivo because of its extreme species-specificity. METHODS: Human retina was introduced into the anterior chamber of athymic rats and allowed to attach to the rat iris. A human cytomegalovirus mutant carrying a beta-galactosidase indicator gene was then injected into the anterior chamber to infect the implanted tissue. After 4 weeks, the eyes were removed, sectioned, and developed with a chromogenic substrate to demonstrate the presence and location of beta-galactosidase expression. RESULTS: Multiple spreading foci of beta-galactosidase expression were found in the retinal implants, indicating that human cytomegalovirus replication had occurred within the human tissue. There was no infection of rat tissue. CONCLUSIONS: This model allows human cytomegalovirus infection of human retina to be established in vivo and sustained long enough to permit multiple cycles of viral replication to occur. The model thus has potential for evaluating antiviral therapies directed against human cytomegalovirus retinal disease.

Differential effects of HSV-1 and HCMV infection on adhesion molecule expression on human corneal keratocytes.

PURPOSE: Previous studies have shown that keratoplasty buttons obtained at surgery from patients with herpes simplex virus-1 (HSV-1) keratitis have elevated localized expression of the adhesion molecule ICAM-1, which plays a critical role in the initiation and amplification of an immune response. We performed studies to determine whether changes in expression of ICAM-1 and HLA class I are direct effects of productive infection of human corneal fibroblasts with HSV-1. METHODS: Immunocytologic and flow cytometric analyses were performed to analyze the ability of HSV-1 to induce ICAM-1 and HLA class I expression in a primary cornea-derived keratocyte cell line, E-2. Positive controls for these experiments were E-2 cells infected with human cytomegalovirus (HCMV), which has been shown to increase ICAM-1 expression in selected cells, and E-2 cells treated with IFN-gamma, which upregulates both ICAM-1 and HLA class I expression in most cell types. RESULTS: Kinetic cytometric analysis indicated decreased ICAM-1 expression 3 hours following HSV-1 infection of E-2 cells. In contrast, HCMV led to detectable increases in ICAM-1 expression starting 6 hours after infection. Infections with either HSV-1 or HCMV resulted in reduced HLA class I expression on E-2 and SF cells. CONCLUSIONS: These studies suggest that increased ICAM-1 expression seen on corneal stromal cells during clinical HSV-1 infection is not a direct result of productive viral infection, but of other mechanisms such as cytokine release by infiltrating mononuclear cells.

A critical evaluation of hepatitis C testing of cadaveric corneal donors.

PURPOSE: Enzyme immunoassays (EIAs) for hepatitis C virus (HCV) antibodies used to screen corneal donors are optimized for testing premortem sera. This study evaluated their efficiency when screening cadaveric sera. METHODS: Abbott HCV EIA 2.0 was used to rescreen 101 cadaveric sera, 70 of which had tested positive and 31 negative by EIA 1.0. Matrix-HCV recombinant immunoblot assay was used as a reference standard. Antibody titers and reactivities were compared in premortem and cadaveric sera. Selected sera from confirmed seropositive donors were screened for virus by polymerase chain reaction (PCR). RESULTS: HCV EIA 2.0 was 100% sensitive and 92.7% specific. EIA and Matrix-HCV gave similar end-point titers with pre- and postmortem sera. Viral RNA was detected in only three of 15 sera from seropositive donors. CONCLUSIONS: EIA 2.0 and Matrix-HCV efficiently screen cadaveric sera. However, HCV seropositivity does not necessarily indicate the presence of viral genomes in sera and tissues.

Screening potential corneal donors for HIV-1 by polymerase chain reaction and a colorimetric microwell hybridization assay.

PURPOSE: Current screening of potential corneal donors for human immunodeficiency virus type 1 (HIV-1) involves serologic detection of antibodies to the virus. However, this approach cannot detect infection during the seronegative window period of the disease. We therefore evaluated the polymerase chain reaction (PCR) assay for viral nucleic acid as a possible alternative to screening cadaveric blood for HIV-1. METHODS: Blood specimens from cadavers diagnosed at autopsy with acquired immunodeficiency syndrome (AIDS) (n = 21), at high risk for HIV-1 infection (n = 47), and at no known risk (n = 350) were screened by PCR for HIV-1 proviral DNA and human leukocyte antigen (HLA)-DQ alpha sequences, and for HIV antibodies. RESULTS: All AIDS group samples were seropositive; of these, 18 (86%) and 20 (95%) of 21 were positive for HIV by PCR of proteinase K- and Chelex-extracted pellets, respectively. The seropositive samples negative by PCR testing were shown to inhibit PCR amplification. Nine (19%) of 47 high-risk specimens were HIV-positive. The no-known-risk group yielded negative results. The overall sensitivities for PCR in the proteinase K- and Chelex-treated groups were 90% and 97%, respectively, compared with Western blot reactivity. If PCR-inhibitory samples and HLA-DQ alpha-negative samples had been eliminated, sensitivity would have been 100%. Specificity was 100% for each group. CONCLUSIONS: Screening cadaveric blood by PCR may be feasible, but further refinement of the assay and blood specimen collection practices will be necessary for it to become routine. Future studies should focus on optimizing specimen procurement and preparation to reduce or eliminate specimens that inhibit PCR.

A comparison of recurrent and primary herpes simplex keratitis in NIH inbred mice.

Herpes simplex virus (HSV) infection is one of the leading causes of corneal blindness. This study compared the clinical, virologic, and immunopathologic features of primary and recurrent murine models of herpes simplex keratitis (HSK) in the National Institutes of Health (NIH) inbred mouse strain. Primary infection resulted in multiple epithelial dendrites, followed by diffuse stromal opacification, symptoms that do not mimic what is seen in human HSK. In contrast, recurrent infection presented clinical features that included microdendrites, focal stromal opacities, disciform endotheliitis, and corneal neovascularization, which were similar to those observed in human disease. Immunohistochemical characterizations indicated that the number and duration of T cells and macrophages in recurrent HSK resemble those observed in primary disease. Results also indicated that the amount of infectious virus detected in the cornea during primary and recurrent disease was similar. However, when corneas were stained for HSV-1 antigens, mice with primary HSK displayed diffuse HSV antigen expression throughout the cornea, whereas HSV antigens were more focally distributed in recurrent disease. These data suggest that the clinical differences between the recurrent and primary herpetic keratitis may, in part, reflect the different distribution of HSV-1 antigens within the cornea.

AIDS-related optic neuropathy: a histological, virological and ultrastructural study.

BACKGROUND: Clinical and histopathological evidence of optic nerve axonal loss has been reported in AIDS patients without retinitis. The study was carried out to investigate the possible involvement of HIV-infected cells in the development of optic nerve degeneration. METHODS: Optic nerves were obtained from eight AIDS patients and four normal controls. These nerves were morphologically and immunohistochemically analyzed. Additionally, using PCR amplification techniques, the retina and optic nerve samples obtained from three HIV-seropositive patients and one control were examined for the presence of HIV and cytomegalovirus antigens. RESULTS: We noted various stages of axonal degeneration in the optic nerves obtained from AIDS patients in whom there was an absence of retinal findings. Characteristic glial changes involving hypertrophic astrocytes, vacuolated oligodendrocytes, and mononuclear phagocyte series cells were also seen in the AIDS optic nerves. HIV DNA was present in at least four of five optic nerves but in only one of five retinas. Control specimens were each negative for all cytomegalovirus and HIV antigens. CONCLUSIONS: Degeneration in the optic nerve may be mediated by HIV-infected macrophages rather than by direct viral infection of neurons. Axonal degeneration due to AIDS at the level of the optic nerve can occur independently of retinal infection.

Poor correlation between reactive syphilis serology and human immunodeficiency virus testing among potential cornea donors.

PURPOSE: The current practice in which eye banks screen cornea donors for syphilis is based mainly on the potential utility of positive syphilis serology as a surrogate marker for human immunodeficiency virus-1 (HIV-1) infection. We examined the correlation between positive syphilis and HIV-1 serologies within the potential cornea donor population. METHODS: We distributed a questionnaire to 94 eye banks in the United States regarding their rates of positive serology for syphilis and HIV-1 between Feb. 1 and July 30, 1992. We subsequently used the polymerase chain reaction for HIV-1 to further evaluate the whole blood of 21 rapid plasma reagin and fluorescent treponemal antibody-positive, HIV-1 enzyme-linked immunosorbent assay (ELISA)-negative cornea donors to determine whether these donors were infected with HIV-1 but were within a seronegative window for HIV-1 antibodies at their time of death. RESULTS: Of 8,932 donors screened, 103 (1.15%) had reactive screening for syphilis serology and 35 (0.39%) were HIV-1 seropositive. No donor with positive syphilis serology was also HIV-1 seropositive. Twelve of 31 donors who originally tested seropositive for syphilis by nontreponemal screening tests (Venereal Disease Research Laboratory or rapid plasma reagin tests) proved seronegative for syphilis when further tested with a treponemal test (FTA-ABS or microhemagglutination-Treponema pallidum), suggesting a high (38.7%) false-positive rate for the syphilis screening tests. Additionally, all 21 rapid plasma reagin and fluorescent-treponemal antibody-positive, HIV-1 ELISA-negative donors further tested were also negative for HIV-1 by the polymerase chain reaction. CONCLUSIONS: Among potential cornea donors, a population prescreened for identifiable HIV-1 risk factors, positive syphilis serology appears to be a poor marker for HIV-1 infection. The role of syphilis screening of potential cornea donors may need to be reevaluated.

Screening corneas for human immunodeficiency virus type 1 proviral DNA by polymerase chain reaction.

PURPOSE: We evaluated the sensitivity of the polymerase chain reaction as a technique to directly screen potential donor corneas for human immunodeficiency virus type 1 (HIV-1) proviral DNA. METHODS: DNA from the central 8.0-mm cornea, limbal cornea, aqueous humor, and retina from 22 eyes of 11 cadavers seropositive for HIV was extracted and amplified by polymerase chain reaction using primers specific for the gag and env regions of the HIV-1 genome. The identity of amplification products was confirmed by Southern blot hybridization. RESULTS: Viral DNA was detected in four (18.2%) of 22 central corneas, one (4.5%) of 22 limbal corneas, one (6.3%) of 16 aqueous humor samples, and seven (31.8%) of 22 retinas. No correlation was noted between the presence of HIV-1 proviral DNA in samples from the central cornea and from the other tissues tested from the same eye. CONCLUSIONS: Within the limits of our assay, processing and analysis of limbal cornea, aqueous humor, and retina by polymerase chain reaction may not reliably ascertain the presence of HIV-1 in the central, transplantable cornea.

A structural and functional comparison of the latency-associated transcript promoters of herpes simplex virus type 1 strains KOS and McKrae.

The promoters of the latency-associated transcripts (LATs) of herpes simplex virus type 1 (HSV-1) strains KOS and McKrae were compared to examine their influence upon the reactivation phenotypes of these two strains. Unlike strain KOS, McKrae is readily reactivable using in vivo reactivation models. We found greater than 96% sequence conservation between KOS and McKrae in the LATs promoter region, and both promoters showed equivalent basal and inducible activities. An inter-strain recombinant (termed MK13) was constructed in which the LATs promoter of HSV-1 McKrae was recombined into the background of HSV-1 strain KOS. In a murine u.v. light-induced reactivation model, virus shedding was detected by eye swabbing in two of 44 (5%) mice infected with KOS, 20 of 42 (48%) mice infected with McKrae and none of 45 (0%) mice infected with MK13. These data show that the LATs promoters of these viruses are structurally and functionally similar and that transfer of the LATs promoter from McKrae into KOS is insufficient to confer a reactivatable phenotype.

The role of nerve growth factor in modulating herpes simplex virus reactivation in vivo.

The role of nerve growth factor (NGF) in the modulation of herpes simplex virus (HSV) latency and reactivation was investigated in a mouse model. To determine whether NGF depletion would reactivate latent virus, concentrated anti-NGF serum antibodies were administered intraperitoneally to latently infected mice for 9 consecutive days. To determine whether NGF given prophylactically could suppress UV-B-induced viral reactivation, mice were irradiated with UV-B while being treated with NGF using diverse regimes over a 4-day period. Following intraperitoneal administration of anti-NGF antibodies, viral shedding was detected in a small number (10%) of mice, but it was not possible to pharmacologically suppress UV-B-induced viral reactivation with NGF. It would appear, therefore, that HSV latency in neurons innervating the cornea can be sustained and disrupted by physiological factors independent of NGF levels.

Comparative diagnosis of neonatal chlamydial conjunctivitis by polymerase chain reaction and McCoy cell culture.

Cervical and ocular swabs from 100 mother/newborn pairs delivering on the clinic service were assayed for Chlamydia trachomatis with standard McCoy cell culture and with standard and biotinylated polymerase chain reaction techniques, using primers directed against the major outer membrane protein gene and C. trachomatis-specific cryptic plasmid, respectively. Using the polymerase chain reaction, 20 (20%) mothers and seven (7%) neonates were positive for Chlamydia. All neonates positive by polymerase chain reaction were from mothers positive by polymerase chain reaction, yielding a 35% transmission rate. Only five of 20 (25%) mothers and two of seven (28%) neonates positive by polymerase chain reaction were positive by cell culture. All cell culture samples were positive by polymerase chain reaction testing. Culture and polymerase chain reaction analysis two weeks after treatment with oral erythromycin were negative. The polymerase chain reaction assay appears to be equally specific and more sensitive than McCoy cell culture for the detection of C. trachomatis from ocular specimens.