Sally S. Atherton

Amplification of epstein-barr virus genomic sequences in blood cells, lacrimal glands, and tears from primary Sjögren's syndrome patients

Based on observations of primary Sjögrens syndrome (SS) following acute Epstein-Barr virus (EBV) infection, the authors hypothesized that EBV may play a role in the pathogenesis of SS. This hypothesis was tested by evaluating ten peripheral blood mononuclear (PBMN) cell specimens, ten lacrimal gland biopsies, and five tear specimens from 15 EBV-seropositive primary SS patients for EBV genomic sequences using polymerase chain reaction (PCR). Epstein-Barr virus DNA sequences were detected in 50% of SS PBMN cell specimens and 80% of SS lacrimal gland and tear specimens. In six SS patients, specimens were obtained from two or more sites (i.e., PBMN cell and lacrimal gland and/or tears), and EBV genomic sequences were amplified in the PBMN cells and the lacrimal gland or tears in three of these subjects. The authors previously detected EBV genomes in 32% (11/34) of normal human lacrimal glands from EBV-seropositive donors using PCR and concluded that the normal human lacrimal gland may be a site of EBV persistence; however, they were unable to amplify EBV sequences in DNA from PBMN cells or tear specimens from normal donors. Amplification of EBV DNA in PBMN cells, lacrimal glands, and tears of primary SS patients at a greater frequency (P less than 0.01) than normal controls suggests that EBV may be a risk factor in the pathogenesis of SS.

Presence of Langerhans cells in the central cornea linked to the development of ocular herpes in mice

Recurrences of herpetic stromal keratitis are believed to be initiated by reactivation of herpes simplex virus infection, probably in the trigeminal ganglion. Genetic features of the virus and the host as well as the immune status of the host influence the outcome of infection. Following infection on the snout with HSV-1, mice with normal corneas usually develop mild anterior segment disease. We studied the induction of herpetic infection in mice that had abnormal corneas, containing center due to trauma or a spontaneous dystrophy. The corneal abnormality led to more frequent herpetic stromal keratitis and more severe anterior chamber reaction. In addition, we found that snout-infected mice with dystrophic corneas had an increased risk of dying from viral infection. Our data suggest that not only the strain of virus and the genetic background of the mouse, but also the state of the cornea itself, can contribute to susceptibility to ocular herpes infection.

Retinitis in euthymic mice following inoculation of murine cytomegalovirus (MCMV) via the supraciliary route.

Cytomegalovirus (CMV) retinitis is the most frequent infectious ocular complication of the acquired immune deficiency syndrome (AIDS). Currently, there are few animal models to study the virologic and immunologic factors which contribute to the pathogenesis of CMV retinitis. In these experiments, 1-2 X 10(4) PFU of murine cytomegalovirus (MCMV) was inoculated into the supraciliary space of BALB/c mice. Within three days of inoculation, moderate iridocyclitis was observed which progressed to necrosis of the ciliary body by day 14. Approximately 60% of the mice developed typical retinitis characterized by virus-infected cytomegalic cells in the retina and retinal pigment epithelium, focal retinal infiltrates, transition zones between uninvolved and involved retina, and optic neuritis. The remaining animals exhibited atypical retinitis characterized by non-specific retinal inflammation in the absence of obvious viral infection. This murine model of CMV retinitis shares some features with retinitis observed in AIDS patients with CMV retinitis and may be useful to evaluate the efficacy of immunologic and/or pharmacologic treatment strategies for CMV retinitis.

Herpetic stromal keratitis in mice: less reversibility in the presence of Langerhans cells in the central cornea.

Infection on the snout with HSV-1 in mice with normal corneas produced a mild ocular disease, characterized by a zosteriform skin lesion around the eye, enlargement of the pupil, hyperemia of the iris and, sporadically, transient keratitis. By contrast, snout infection after prior cauterization of the cornea induced significantly more frequent and more severe corneal disease, in which keratitis was usually permanent. Corneal cauterization also produced increased numbers of Langerhans cells in the central cornea. We speculate that the combination of virus and increased numbers of Langerhans cells within the cornea may lead to an exaggerated ocular immune response that is destructive to the cornea.

A critical role for ACAID in the distinctive pattern of retinitis that follows anterior chamber inoculation of HSV-1.

To test the hypothesis that ACAID induction is instrumental in producing the distinctive pattern of retinal pathology that follows anterior chamber inoculation of HSV-1 in BALB/c mice, panels of mice received uniocular anterior chamber, uniocular intravitreal, and bilateral anterior chamber inoculations of HSV-1. It was found that contralateral retinitis developed after the first two routes, and ACAID was induced by all three. Enucleation of eyes inoculated with HSV-1 before 3 days post-inoculation (but not thereafter) prevented both ACAID and contralateral retinitis. Intracameral inoculations of HSV-2 induced vigorous delayed hypersensitivity and failed to incite contralateral retinitis. It is concluded that ACAID induction plays a crucial role in the pathogenesis of contralateral retinitis following anterior chamber inoculation of HSV-1.

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.

Virus-specific DTH prevents contralateral retinitis following intracameral inoculation of HSV-2

Following inoculation of HSV-1 (KOS strain) into the anterior chamber of one eye of a BALB/c mouse, the virus travels to the uninoculated contralateral eye and contributes to the devastating retinal necrosis which occurs in this eye 10-12 days p.i. In parallel experiments, HSV-2 (MS strain) was inoculated uniocularly via the anterior chamber route; animals were sacrificed at intervals and their eyes removed for histopathology and virus culture. Histopathological examination revealed that, in contrast to the retinal destruction observed in HSV-1 inoculated animals, the retina of the contralateral eye of HSV-2 injected animals was unaffected. Culture studies demonstrated that, similar to the spread of HSV-1 after anterior chamber inoculation, HSV-2 reached the contralateral eye in two temporally separate waves and also revealed that the amount of virus recovered from the uninoculated eye of HSV-2 infected animals was significantly less than that recovered from the uninoculated contralateral eye of HSV-1 injected mice. Further examination demonstrated that animals inoculated with HSV-2 via the anterior chamber route did not develop the suppression of virus-specific delayed hypersensitivity (DH) which is characteristic of HSV-1 induced anterior chamber associated immune deviation (ACAID). Taken together, the sparing of the contralateral retina and the ability to generate a virus-specific DH response suggest that DH assists in the preservation of the contralateral retina of animals inoculated with HSV-2 via the anterior chamber route by limiting virus replication and the amount of virus which reaches the uninoculated eye.Following inoculation of HSV-1 (KOS strain) into the anterior chamber of one eye of a BALB/c mouse, the virus travels to the uninoculated contralateral eye and contributes to the devastating retinal necrosis which occurs in this eye 10-12 days p. i. In parallel experiments, HSV-2 (MS strain) was inoculated uniocularly via the anterior chamber route; animals were sacrificed at intervals and their eyes removed for histopathology and virus culture. Histopathological examination revealed that, in contrast to the retinal destruction observed in HSV-1 inoculated animals, the retina of the contralateral eye of HSV-2 injected animals was unaffected. Culture studies demonstrated that, similar to the spread of HSV-1 after anterior chamber inoculation, HSV-2 reached the contralateral eye in two temporally separate waves and also revealed that the amount of virus recovered from the uninoculated eye of HSV-2 infected animals was significantly less than that recovered from the uninoculated contralateral eye of HSV-1 in...

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.

ACAID requires early replication of HSV-1 in the injected eye.

After uniocular anterior chamber inoculation of the KOS strain of HSV-1 in BALB/c mice, acute retinal necrosis develops in the uninoculated eye. These mice exhibit suppression of virus-specific delayed hypersensitivity which is the hallmark of anterior chamber associated immune deviation (ACAID). In contrast, inoculation of C57BL/6 mice with KOS induces vigorous virus-specific DTH, and there is no retinal necrosis in the uninoculated eye. We performed experiments using RH116, a mutant of KOS and SC16, a neurovirulent strain of HSV-1, in BALB/c and C57BL/6 mice to examine whether virus or host factors determine the outcome of anterior chamber inoculation of HSV-1 (ACAID or DTH, retinal necrosis or retinal preservation). We found that high titers of virus (approximately 10(5)) within the first 24 hours in the inoculated eye were linked to the induction of ACAID and that the induction of ACAID following anterior chamber inoculation of HSV-1 is neither an exclusive feature of a strain of virus nor is the ability to develop ACAID a distinguishing characteristic of an individual strain of mice. Taken together, our findings suggest that the interplay of both virus and host factors results ultimately in acute retinal necrosis following anterior chamber inoculation of HSV-1 in the mouse.

Apoptosis and increased expression of Fas ligand after uniocular anterior chamber (AC) inoculation of HSV-1

Purpose. To determine if apoptosis occurs in the eyes and brain following uniocular anterior chamber (AC) inoculation of HSV-1 and if expression of Fas ligand (FasL) or Fas is altered by virus infection. Methods. After inoculation of HSV-1 via the AC route, apoptotic cells in the eyes and brain were identified by TUNEL assay, and TUNEL results indicating DNA fragmentation in the brain were confirmed by DNA laddering assays. Expression of FasL and Fas in the eyes and brain was determined by immunohistochemistry and Western blotting and confirmed by RT-PCR, Southern blotting of RT-PCR products, and sequencing. Results. After AC inoculation of HSV-1, HSV-1 positive cells and apoptotic cells were observed in the eyes and brain. Immunofluorescent staining revealed that although both FasL and Fas were expressed in the cornea, retinal pigment epithelium cells (RPE), and photoreceptor cells of normal and virus infected mice, Fas and FasL were expressed at different locations in these cells. On day 8 p.i., more FasL DNA expression was observed in HSV-infected mice compared with uninfected mice. Conclusions. HSV-1 infection following AC inoculation of virus induced apoptosis in the eyes and brain. Although FasL expression was upregulated by HSV-1 infection, these results suggest there are likely to be factors in addition to FasL and Fas, such as Bcl-2 and caspases, that are involved in apoptosis observed in the eyes and brain of HSV-1- infected mice.