Detection of herpes simplex virus type 1 in human ciliary ganglia

PURPOSE. To determine whether herpes simplex virus type 1 (HSV-1) DNA is present in the ciliary ganglion (CG). METHODS. Fifty CG and 47 trigeminal ganglia (TG) were resected from 63 formalin-fixed cadavers between 56 and 98 years of age that had been embalmed within 12 hours of death. The donors had no known active HSV infection at the time of death. DNA was extracted from all ganglia by proteinase-K digestion (TG) or digestion by a mild lysis buffer (CG). DNA was amplified by polymerase chain reaction for sequences from human chromosome 18, D18S1259 (positive control), and from the HSV-1 DNA polymerase gene, U L 30. The amplified DNA was separated by agarose gel electrophoresis, transferred to nylon membranes, and hybridized with the appropriate digoxigenin-labeled probe that was detected by alkaline phosphatase-conjugated monoclonal antibody. RESULTS. The D18S1259 sequence was amplified from 47 TG and 30 CG samples. Of these samples, 32 (68.0%) of the 47 TG samples and 20 (66.6%) of the 30 CG samples were positive for the UL 30 HSV-1 sequence. CONCLUSIONS. Using amplification of HSV-1 DNA as a surrogate marker of latency, the finding that the frequency of HSV-1 in the CG was approximately the same as that of the TG suggests that the CG may be an additional site of HSV-1 latency in humans. Active infection in or reactivation of HSV-1 from non-TG sites may explain why this virus is able to infect sites, such as the retina, that have no direct connections to the trigeminal nerve

Ocular Reactivation of MCMV after Immunosuppression of Latently Infected BALB/c Mice

PURPOSE. The purpose of this study was to identify the site(s) of MCMV latency and reactivation in the eye. METHODS. Three months after supraciliary inoculation of 5 ϫ 10 2 PFU of MCMV, BALB/c mice underwent immunosuppression with methylprednisolone and antibodies specific for CD4 T cells, CD8 T cells, and NK cells or with methylprednisolone alone. Control mice were infected but did not receive the immunosuppressants. After 2 or 3 weeks of immunosuppression, the mice were killed. Replicating virus and viral antigen were detected in the injected eyes, peripheral blood leukocytes (PBLs), and extraocular tissues by plaque assay and by staining for early antigen (EA) and ␤-galactosidase (␤-gal), respectively. RESULTS. In latently infected, nonimmunosuppressed control mice, replicating-virus-and viral-antigen-positive cells were not detected in the injected eyes or extraocular tissues. After immunosuppression with methylprednisolone and antibodies, EA and ␤-gal were detected, and replicating virus was recovered from the injected eye and from several extraocular sites, including liver, lungs, salivary glands, and kidneys. No virus was recovered from PBLs. ␤-Gal-or EA-positive cells were observed in the RPE of most mice, and a few virus-infected cells were also observed in the nuclear layers and ganglion cells. Microscopic changes, including retinal folding and detachment, photoreceptor atrophy, macrophage infiltration, and a few EA-positive cytomegalic cells, were observed in the injected eye of immunosuppressed mice. CONCLUSIONS. After immunosuppression, MCMV reactivates in the injected eye and extraocular tissues, and RPE cells are the initial site of MCMV ocular reactivation in the eye. The timing of virus recovery from all sites suggests that MCMV observed in the injected eye is from in situ reactivation of virus and not from spread of virus from extraocular sites via infected PBLs. (Invest Ophthalmol Vis Sci. 2005;46:252-258) DOI:10.1167/ iovs.04-0537 H uman cytomegalovirus (HCMV) is a ubiquitous herpesvirus that generally causes asymptomatic infection, but it may also cause pneumonitis, hepatitis, gastroenteritis, myocarditis, and retinitis. 1 Persons who are immunologically immature (neonates) or undergo immunosuppression (patients with AIDS or recipients of organ allografts) are at risk of contracting CMV-related diseases. The host specificity of HCMV has prevented development of HCMV-infected animals as models to study the pathogenic processes whereby HCMV infects human cells, replicates within them, enters latency, and reactivates. 2 Murine cytomegalovirus (MCMV) is a mouse-specific CMV that causes pneumonitis, hepatitis, and retinitis in immunosuppressed mice. 1-4 Like HCMV, MCMV becomes latent after acute infection of immunocompetent mice. 2, 5-10 Because of similarities between HCMV and MCMV, MCMV-infected mice have been used to study the pathogenesis of cytomegalovirus infection and reactivation. The eye is among the organs susceptible to CMV infection. Before the introduction of highly active antiretroviral therapy (HAART), CMV retinitis was the most common ocular opportunistic infection in patients with acquired immunodeficiency syndrome. In the pre-HAART era, although the percentage of patients with CMV retinitis varied depending on the study, up to 46% of patients with AIDS could be expected to have HCMV retinitis at some point during the course of the disease. 11-13 The eye is also a target of congenital or neonatal CMV infection because of an immature blood-retinal barrier. 12,14 A mouse model of acute MCMV retinitis that shares some features with HCMV retinitis has been used to study the pathogenesis of CMV retinitis in immunosuppressed mice. 3,4 Studies of latent and reactivated MCMV infection, in which viral DNA was detected in some inoculated eyes and extraocular tissues several months after clearance of infectious virus from the eye and from all extraocular sites, indicate that the virus becomes latent in the injected eye and in some extraocular sites (such as the salivary gland, kidney, spleen, liver, and lung) after initial ocular infection with MCMV. 16 -20 It has also been shown that immunosuppression induces reactivation of MCMV in the injected eye and in some extraocular sites, as detected by recovery of replicating virus 20 and the presence of mRNA for gH, a late protein. 6 However, the site(s) of latency and reactivation of MCMV in the eye of the mouse have not been defined. It is also not known whether virus detected in the eye of immunosuppressed mice results from virus reactivation in situ in the eye or whether virus detected in the eye results from the spread of replicating virus from nonocular sites. MATERIALS AND METHODS Virus and Cells Two strains of MCMV (RM461 and K181) were used in these experiments. These viruses were kindly provided by Edward S. Mocarski and Cheryl Stoddart (Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA). RM461 was derived by insertion of a modified Escherichia coli LacZ gene (under the control of the HCMV major immediate-early promoter/enhancer) into the wild-type MCMV K181 genome at the HindIII L/J site, just downstream of immediate-early gene 2 (ie2). RM461 expresses the E. coli lacZ gene product ␤-galactosidase (␤-gal) as an immediate-early viral gene product during the viral replication cycle

Recruitment of young women to a trial of chlamydia screening – as easy as it sounds?

BACKGROUND: Recruiting to trials is complex and difficult. The Prevention of Pelvic Infection (POPI) trial aims to see if screening women for chlamydia and treating those found to be infected reduces the incidence of pelvic inflammatory disease in the following twelve months. It focuses on young, sexually active, multiethnic, mainly inner city, female students. The main aim of this paper is to describe our recruitment methods. Secondary aims in two small subgroups, are to compare characteristics of women recruited with those not recruited, and to explore participants' understanding of when their samples would be tested for chlamydia. METHODS: Women students attending lectures or in common rooms at 22 universities and further education colleges were recruited by female research assistants working in pairs. Participants were asked to complete a questionnaire on sexual health and to provide self-taken vaginal swabs. In addition, during 3 recruitment sessions, a female medical student asked non-participants to complete a brief anonymous questionnaire on reasons for not taking part. Finally another female medical student contacted 40 consecutive participants within a month of recruitment and asked if they understood that their samples might not be tested for a year. RESULTS: With enormous effort over 2 years we recruited 2526 women. A survey of 61 non-responders showed only 18 (30%) were eligible to take part (age <28, been sexually active and not been tested for chlamydia in the past 3 months). Eligible non-responders were of similar age to the 35 responders in the same recruitment sessions, but more likely to be from ethnic minority groups (67% 12/18 versus 29% 10/35 p < 0.01). Email and telephone contact with 35/40 (88%) of consecutive participants showed only two (6%) did not understand that their specimen might not be tested for chlamydia for a year. Thirty participants (85%) could name one or more possible consequences of untreated chlamydia infection. CONCLUSION: As in other studies, a key to attaining recruitment targets was the enthusiasm of the research team. Minority ethnic groups were probably under-represented, but understanding of participants was good

Cytokine profiles and inflammatory cells during HSV-1-induced acute retinal necrosis. Invest Ophthalmol Vis Sci.

PURPOSE. To investigate infiltrating cells, cytokines, and kinetics of cytokine expression during acute retinal necrosis (ARN) in the uninoculated eye after inoculation of herpes simplex virus (HSV)-1 into the anterior chamber of one eye of BALB/c mice. METHODS. At different time points after inoculation of 2 ϫ 10 4 plaque-forming units (PFU) HSV-1 (KOS strain) or an equivalent volume of Vero cell extract in cell culture medium, the uninoculated eyes were enucleated. RT-PCRs for TNF␣, IFN␥, and IL-4 and immunohistochemical staining were performed to identify infiltrating cells and cytokines. Cytometric bead array was used to measure the levels of TNF␣, IFN␥, and IL-4 protein. RESULTS. CD4 ϩ T cells, F4/80 ϩ macrophages, Gr-1 ϩ polymorphonuclear cells (PMNs), and CD19 ϩ B cells were detected in the uninoculated eye of virus-infected mice. Furthermore, RPE65 ϩ retinal pigment epithelial (RPE) cells and activated Müller cells were also detected in the ARN lesion. TNF␣, IFN␥, and IL-4 mRNA and protein were upregulated during the evolution of ARN in HSV-1-infected contralateral eyes compared with levels in control subjects. Immunohistochemistry revealed that cytokines were produced by infiltrating cells as well as by resident retinal cells. CONCLUSIONS. The results of these studies support the idea that T cells and cytokines are actively involved in HSV-1 retinitis. They also suggest that PMNs, B cells, and/or macrophages, as well as resident retinal cells, such as RPE and activated Müller cells, also play a role in the pathogenesis of HSV-1 retinitis. (Invest Ophthalmol Vis Sci

Histopathologic study of herpes virus-induced retinitis in athymic BALB/c mice: Evidence for an immunopathogenic process

In order to determine whether antiviral immunity is pathogenic in mouse eyes, HSV-1 was injected into the anterior chamber of one eye of adult athymic BALB/c mice. The eyes of these T cell deficient mice were examined clinically and histopathologically for ocular disease. The anterior segment of injected eyes developed progressive inflammatory reactions that eventually destroyed the ciliary body and then progressed to the posterior compartment where partial necrosis occurred, but only in the inner layers of the retina. A milder form of the same process developed between 7 and 10 days in the contralateral eye. Uninoculated eyes displayed little evidence of choroiditis, hemorrhage, massive necrosis, or disintegration of the architecture of the retina. Since these are features that are found in contralateral retinas of euthymic BALB/c mice infected in one eye via the anterior chamber route, it is concluded that acute retinitis found in contralateral eyes of immunocompetent mice has an immunopathogenic basis. However since euthymic mice develop anterior chamber associated immune deviation (ACAID) (and therefore do not display virus-specific delayed hypersensitivity), the identity of the relevant immune effector remains unknown. Based on these observations and our previous ocular findings following intracameral inoculation of HSV-2, we suggest that in susceptible mice, herpes simplex viruses can induce several pathogenetically distinct forms of retinitis, some of which are mediated by virus-specific immune effector cells