Richard D. Dix

Treatment of the Acute Retinal Necrosis Syndrome with Intravenous Acyclovir

We treated 13 eyes of 12 patients with the acute retinal necrosis syndrome (ARN) with intravenous acyclovir (1500 mg/M2/day) for an average of 10.9 days. All patients were also treated with oral aspirin or Coumadin. in an attempt to prevent thrombotic complications and nine of twelve patients were treated with oral prednisone after intravenous acyclovir had been initiated. Regression of retinal lesions was first seen on average 3.9 days after initiation of therapy and required 32.5 days on average for completion. No eye developed new retinal lesions or progressive optic nerve involvement 48 hours or more after initiation of therapy, although progression within the first 48 hours was occasionally seen. Treatment did not ameliorate vitritis or prevent retinal detachment, which occurred in 11 of 13 eyes, an average of 59 days after the initiation of therapy. There were no evident ocular or systemic complications of therapy. Our data suggest the need for a prospective randomized clinical trial to evaluate the efficacy of intravenous or oral acyclovir in the treatment of the acute retinal necrosis syndrome.

Loss of the Perforin Cytotoxic Pathway Predisposes Mice to Experimental Cytomegalovirus Retinitis

ABSTRACT AIDS-related human cytomegalovirus (HCMV) retinitis continues to be a chronic ophthalmologic problem among human immunodeficiency virus type 1 (HIV-1)-infected patients who do not respond to highly active antiretroviral therapy. Although HCMV retinitis occurs during HIV-1-induced immunosuppression, the precise effector mechanism(s) that fails during the immunopathogenesis of AIDS to allow onset and progression of HCMV retinal disease remains unclear. We therefore performed a series of experiments to explore the relative roles of distinct pathways of lymphocyte-mediated cytotoxicity in either resistance or susceptibility to experimental murine cytomegalovirus (MCMV) retinitis in mice. Whereas mutant C57BL/6 mice deficient in the Fas/FasL cytotoxic pathway (gld mice) were identical to normal C57BL/6 mice and exhibited absolute resistance to retinal necrosis following subretinal MCMV inoculation, knockout C57BL/6 mice deficient in the perforin cytotoxic pathway (PKO mice) were susceptible to MCMV retinitis. Susceptibility of PKO mice to MCMV retinitis correlated with increased ocular MCMV titers when compared with ocular MCMV titers of gld and normal mice. Since mice with retrovirus-induced immunodeficiency syndrome (MAIDS) exhibited a frequency and severity of MCMV retinitis that were equivalent to those observed in PKO mice, we hypothesized that susceptibility to MCMV retinitis during MAIDS correlates with a decrease in the perforin cytotoxic pathway. To test this hypothesis, we developed a quantitative competitive reverse transcription-PCR assay to measure mouse perforin mRNA levels in the splenic T lymphocytes and MCMV-inoculated eyes of normal mice or mice with MAIDS. Perforin mRNA levels in splenic T lymphocytes were significantly decreased during MAIDS, by ∼100-fold, from perforin mRNA levels in normal mice. Moreover, MCMV-inoculated eyes destined to develop retinitis during MAIDS also showed a significant decrease in perforin mRNA levels from the perforin mRNA levels of MCMV-inoculated eyes of normal mice destined to be resistant to retinitis. As expected, perforin mRNA could not be detected in unmanipulated and uninfected eyes of normal mice. These results provide the first evidence that the perforin cytotoxic pathway is more important than the Fas/FasL cytotoxic pathway in providing resistance to experimental MCMV retinitis and that loss of the perforin cytotoxic pathway predisposes to MCMV retinitis.

Viability of herpes simplex virus type 1 on the applanation tonometer.

We performed several experiments to define possible factors that influence the viability of herpes simplex virus type 1 on the applanation tonometer. Infectious virus could be detected on experimentally inoculated tonometer heads for up to two hours during natural drying. If tonometers were kept moist the virus could be detected more than eight hours later. Several ophthalmic solutions, including topical anesthetics, dilating agents, and a fluorescein solution, showed only minimal antiviral activities. Wiping of virus-infected tonometer heads with a dry tissue was ineffective and allowed residual infectious virus to remain. However, no infectious virus could be detected on infected tonometer heads that had been swabbed with 70% isopropyl alcohol. Our results raised concerns regarding effective disinfection of tonometers after eye examinations. We concluded that the applanation tonometer should be swabbed routinely with alcohol after each patient examination to ensure complete virus inactivation and to prevent accidental transmission of virus in the clinical setting.

Infection of Human Neural Cell Aggregate Cultures with a Clinical Isolate of Cytomegalovirus

Human neural cell aggregate cultures were prepared from dissociated fetal brain tissue and maintained in rotation culture. After 35 days in culture, aggregates had the histologic appearance of dense, immature, neural cells in a tightly packed neuropil. Electron microscopy revealed ultrastructural features suggestive of immature neurons and neuroglia. In addition, neuron-specific enolase and glial fibrillary acidic protein associated with radial glial cells were detected within the aggregates by immunoperoxidase staining. When infected with a laboratory-adapted strain of cytomegalovirus (CMV), [AD169], cells containing large, bizarre, nuclei and CMV-induced intranuclear inclusion bodies were dispersed throughout the aggregates at 16 days postinfection. In situ hybridization using a CMV-specific DNA probe and electron microscopy confirmed the presence of virus sequences as well as virus particles at histologic sites of cytopathology. In sharp contrast, aggregate cultures infected with a CMV strain recovered from the retina of an acquired immune deficiency syndrome (AIDS) patient with CMV retinitis and encephalitis displayed distinct foci of cytopathology at 23 days postinfection, a pattern not observed in CMV [AD169]-infected aggregates. Our findings suggest that human neural cell aggregates represent a promising multicellular non-neoplastic culture system in which to study the replication of human neurotropic viruses within neural tissue.

Mice immunosuppressed by murine retrovirus infection (MAIDS) are susceptible to cytomegalovirus retinitis

Although various methods of immunosuppression have been used to enhance susceptibility of mice to murine cytomegalovirus (MCMV) retinitis, none reproduce the unique complexity of immune deficiency experienced by patients during the progression of AIDS. C57BL/6 mice are susceptible to a retrovirus-induced murine acquired immune deficiency syndrome (MAIDS), characterized by progressive immune dysfunction which shares many features with AIDS. We therefore evaluated the frequency and severity of MCMV retinitis in C57BL/6 mice with MAIDS. Following subretinal inoculation of MCMV, nearly 90% of mice with MAIDS developed a necrotizing retinitis 8 to 10 days postinfection, whereas retinitis was observed in only 8% of age-matched immunocompetent control mice. Histopathologic analysis of the retinitis that developed in mice with MAIDS revealed features similar to those found in AIDS-related CMV retinitis. Eyes from MAIDS animals also contained on average higher amounts of infectious virus when compared with eyes fr...

Histopathologic characteristics of two forms of experimental herpes simplex virus retinitis

Herpes simplex virus type 1 (HSV-1) inoculated intracamerally into one anterior chamber of a BALB/c mouse produces retinitis in the uninoculated contralateral eye within 7 to 10 days while the retina of the inoculated eye is spared. In sharp contrast, animals receiving HSV type 2 (HSV-2) by the anterior chamber route develop a dramatic retinitis in the inoculated eye by day 7 postinoculation while the retina of the contralateral eye remains uninvolved. Histopathologic examination of retinal destruction in the HSV-2-infected ipsilateral eye revealed features which were distinct from those observed in the contralateral eye of HSV-1-infected animals. Whereas HSV-1 produced a rapid, explosive, retinitis which led to destruction of all cell layers of the contralateral retina, HSV-2 induced a retinitis in the ipsilateral eye that was more gradual in onset. Ipsilateral HSV-2 retinitis was characterized initially by disruption of the ganglion and inner nuclear layers which progressed by day 10 to 14 to complete replacement of the retina by a fibrocellular scar. These changes were dominated by a vigorous mononuclear cell infiltrate, a feature not observed in the HSV-1-infected contralateral retinitis. These results suggest that experimental retinitides produced by HSV-1 and HSV-2 are of diverse pathogenesis.

Macrophage Activation Associated with Chronic Murine Cytomegalovirus Infection Results in More Severe Experimental Choroidal Neovascularization

The neovascular (wet) form of age-related macular degeneration (AMD) leads to vision loss due to choroidal neovascularization (CNV). Since macrophages are important in CNV development, and cytomegalovirus (CMV)-specific IgG serum titers in patients with wet AMD are elevated, we hypothesized that chronic CMV infection contributes to wet AMD, possibly by pro-angiogenic macrophage activation. This hypothesis was tested using an established mouse model of experimental CNV. At 6 days, 6 weeks, or 12 weeks after infection with murine CMV (MCMV), laser-induced CNV was performed, and CNV severity was determined 4 weeks later by analysis of choroidal flatmounts. Although all MCMV-infected mice exhibited more severe CNV when compared with control mice, the most severe CNV developed in mice with chronic infection, a time when MCMV-specific gene sequences could not be detected within choroidal tissues. Splenic macrophages collected from mice with chronic MCMV infection, however, expressed significantly greater levels of TNF-α, COX-2, MMP-9, and, most significantly, VEGF transcripts by quantitative RT-PCR assay when compared to splenic macrophages from control mice. Direct MCMV infection of monolayers of IC-21 mouse macrophages confirmed significant stimulation of VEGF mRNA and VEGF protein as determined by quantitative RT-PCR assay, ELISA, and immunostaining. Stimulation of VEGF production in vivo and in vitro was sensitive to the antiviral ganciclovir. These studies suggest that chronic CMV infection may serve as a heretofore unrecognized risk factor in the pathogenesis of wet AMD. One mechanism by which chronic CMV infection might promote increased CNV severity is via stimulation of macrophages to make pro-angiogenic factors (VEGF), an outcome that requires active virus replication.

Diagnostic value for culture of cerebrospinal fluid from HIV-1-infected individuals for opportunistic viruses : a prospective study

Objective.To investigate the diagnostic value of cerebrospinal fluid (CSF) culture for opportunistic viruses from HIV-1-infected individuals. Methods.A 4-year prospective study was conducted using a participant population consisting of 186 HIV-1-infected individuals without neurologic disease, 73 HIV-1-infected individuals with encephalopathy, myelopathy, and/or peripheral neuropathy, and 10 controls. CSF samples recovered at 1-year intervals were subjected to virus culture using techniques commonly used in the clinical laboratory setting. Results.CSF samples obtained from only 15 of the 269 (5.6%) participants yielded an opportunistic virus upon culture. Cytomegalovirus, herpes simplex virus types 1 and 2, adenovirus, and presumptive enteroviruses were identified. No consistent correlation was observed between the detection of an opportunistic virus within a CSF sample and the presence or future development of neurologic disease. However, a significant correlation was observed between culture of virus from CSF and the future development of abnormal CD4+ (X2, P= 0.0286) and CD8+ (X2, P= 0.0018) lymphocyte counts in HIV-1-infected participants without neurologic disease. Conclusion.These results show that culture of CSF to screen for opportunistic viruses is neither diagnostic nor predictive of neurologic disease in HIV-1-infected individuals. Nevertheless, the presence of virus within CSF may be an indicator of HIV-1-mediated immune dysfunction and a predictor for future development of abnormal CD4+ and/or CD8+ lymphocyte counts.

Susceptibility to murine cytomegalovirus retinitis during progression of MAIDS: Correlation with intraocular levels of tumor necrosis factor-α and interferon-γ

Purpose. To correlate tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) synthesis with histopathologic disease and virus replication within murine cytomegalovirus (MCMV)-infected eyes during progression of murine acquired immunodeficiency syndrome (MAIDS). Materials and methods. Groups of normal mice and mice with MAIDS of 2-weeks (MAIDS-2), 4-weeks (MAIDS-4), and 12-weeks (MAIDS-12) duration were infected uniocularly with MCMV by subretinal MCMV injection. MCMV-inoculated eyes from all mice were subjected to histopathologic analysis, quantitative plaque assay, or cytometric bead array analysis for quantification of TNF-α and IFN-γ. Results. Whereas MCMV-inoculated eyes of normal, MAIDS-2, and MAIDS-4 mice were resistant to MCMV retinitis, all MCMV-inoculated eyes of MAIDS-12 mice developed retinitis. Surprisingly, MCMV-inoculated eyes of MAIDS-4 mice without retinitis harbored high amounts of infectious virus at a level equivalent to that of MCMV-inoculated eyes of MAIDS-12 mice that developed retinitis. Intraocular TNF-α levels were consistently ∼50% greater in MCMV-inoculated eyes of MAIDS-12 mice when compared with TNF-α levels of normal, MAIDS-2, and MAIDS-4 mice. In contrast, intraocular INF-γ levels within MCMV-inoculated eyes progressively declined as animals became susceptible to retinitis. Conclusions. An inverse relationship exists between TNF-α and INF-γ production within MCMV-inoculated eyes during MAIDS evolution that is characterized by an increase in intraocular TNF-α levels and a concomitant decrease in intraocular INF-γ levels. Susceptibility of MCMV-inoculated eyes to virus replication and development of necrotizing retinitis are independent events with susceptibility to MCMV replication preceding susceptibility to MCMV retinitis by several weeks. Time of Th1/Th2 shift in cytokine profile appears to be a crucial event in the pathogenesis of MAIDS-related MCMV retinitis.

Murine cytomegalovirus downregulates interleukin-17 in mice with retrovirus-induced immunosuppression that are susceptible to experimental cytomegalovirus retinitis.

Interleukin-17 (IL-17), a pro-inflammatory cytokine produced by CD4+ Th17 cells, has been associated with the pathogenesis of several autoimmune diseases including uveitis. The fate of IL-17 during HIV/AIDS, however, remains unclear, and a possible role for IL-17 in the pathogenesis of AIDS-related diseases has not been investigated. Toward these ends, we performed studies using a well-established animal model of experimental murine cytomegalovirus (MCMV) retinitis that develops in C57/BL6 mice with retrovirus-induced immunosuppression (MAIDS). After establishing baseline levels for IL-17 production in whole splenic cells of healthy mice, we observed a significant increase in IL-17 mRNA levels in whole splenic cells of mice with MAIDS of 4-weeks (MAIDS-4), 8-weeks (MAIDS-8), and 10-weeks (MAIDS-10) duration. In contrast, enriched populations of splenic CD4+ T cells, splenic macrophages, and splenic neutrophils exhibited a reproducible decrease in levels of IL-17 mRNA during MAIDS progression. To explore a possible role for IL-17 during the pathogenesis of MAIDS-related MCMV retinitis, we first demonstrated constitutive IL-17 expression in retinal photoreceptor cells of uninfected eyes of healthy mice. Subsequent studies, however, revealed a significant decrease in intraocular levels of IL-17 mRNA and protein in MCMV-infected eyes of MAIDS-10 mice during retinitis development. That MCMV infection might cause a remarkable downregulation of IL-17 production was supported further by the finding that systemic MCMV infection of healthy, MAIDS-4, or MAIDS-10 mice also significantly decreased IL-17 mRNA production by splenic CD4+ T cells. Based on additional studies using IL-10 -/- mice infected systemically with MCMV and IL-10 -/- mice with MAIDS infected intraocularly with MCMV, we propose that MCMV infection downregulates IL-17 production via stimulation of suppressor of cytokine signaling (SOCS)-3 and interleukin-10.