Epstein-Barr virus and the lacrimal gland pathology of Sjögren's syndrome.

The lacrimal gland (LG) immunopathology of Sjögren's syndrome (SS) consists of a proliferation of B and CD4 lymphocytes surrounding epithelial structures (Pepose JS, et al: Ophthalmology 1990, 97:1599-1605). Based on the detection of EBV genomes in a greater percentage of SS than normal LG biopsies, we previously postulated that Epstein-Barr virus (EBV) is a risk factor for LG lymphoproliferation in SS (Pflugfelder SC, et al: Ophthalmology 1990, 97:976-984). The purpose of this study was to determine the cellular site(s) of infection, virus type, and antigen expression of EBV infecting normal and SS LGs. EBV DNA was detected by in situ hybridization in intraductal epithelia in 13-33% of lobules in 21% of normal LGs and in cells in areas of B lymphoproliferation as well as the majority of epithelia in 86% of SS LGs. EBV genomic sequences were amplified from 36% of normal and 88% of SS LG biopsies by polymerase chain reaction. Only type 1 EBV sequences were amplified in SS LGs; in contrast EBV nuclear antigen 2-deleted but not type 1 sequences were amplified in normal LGs. Immunohistochemistry with EBV-specific monoclonal antibodies was performed on normal and SS LGs. No EBV antigens were detected in normal LGs. In contrast, latent antigens (latent membrane protein, EBV nuclear antigen 2) were detected in lymphocytes in areas of B lymphoproliferation, and early and late lytic cycle antigens were observed in epithelia in SS LGs. These studies suggest that EBV may play a role in the LG B lymphoproliferation and epithelial pathologic changes observed in SS.

Detection of herpesvirus DNA in vitreous and aqueous specimens by the polymerase chain reaction.

Members of the herpesvirus family, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex virus (HSV), have been recognized as causal agents of chorioretinal inflammatory diseases. We investigated the use of the polymerase chain reaction for the detection of CMV, HSV, and EBV genomes in aqueous, subretinal fluid, and vitreous specimens in patients with clinically diagnosed CMV retinitis. Cytomegalovirus but not HSV or EBV genomic sequences were detected in all of these clinical specimens. We also investigated 18 normal aqueous and eight normal vitreous specimens obtained from patients undergoing cataract or vitrectomy surgery. Cytomegalovirus, HSV, and EBV DNA were not detected in any of the normal aqueous specimens. There was one weakly positive CMV normal vitreous, but none was HSV or EBV positive by the polymerase chain reaction. These results indicate that the polymerase chain reaction may be useful as a rapid and sensitive diagnostic technique to aid in the confirmation of clinical observations.

Detection of herpes viral genomes in normal and diseased corneal epithelium.

Herpetic ocular disease is one of the major causes of corneal blindness. Clinical diagnosis of corneal disease is based principally on corneal appearance. However, abnormal morphology of the corneal epithelium (CE) is not an indicator for the presence of a herpes virus. Further, it has not been established if herpes viruses are present in normal corneal epithelial tissue. In these studies, the polymerase chain reaction was used to evaluate normal and diseased corneal epithelium for the presence of herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) genomic sequences. Thirty-two normal corneal epithelium specimens obtained from cadavers shortly after death were analyzed for HSV-1, EBV and CMV genomic sequences. Three of the 32 normal CE specimens were positive for amplified EBV DNA, 1 was positive for HSV-1 DNA, and none was positive for CMV DNA. We also tested eight herpetic dendritic lesions of which 3 were HSV-1 culture and PCR positive. The remaining five dendritic lesions were HSV-1 culture and PCR negative. Since these lesions were not evaluated for other herpesviruses, the etiology of these dendritic lesions is unknown. Six corneal epithelium samples from HIV-infected donors were negative for EBV, CMV and HSV-1 amplified sequences. Positive EBV, CMV and HSV-1 serology on all normal donors and on donors with clinically apparent disease did not correlate with positive PCR results. The results of these studies suggest that EBV and HSV-1 DNA can be amplified from a small percentage of apparently normal corneal epithelium.

Detection of Epstein-Barr virus genomes in normal human lacrimal glands.

Epstein-Barr virus (EBV) has been implicated in several ocular diseases; however, detection of the EBV genome in ocular tissues has not been documented. We report the detection of amplified EBV genomic sequences in 11 of 26 normal lacrimal gland DNA samples by using the polymerase chain reaction. Serum was available for 19 of the lacrimal gland donors. All 19 were EBV seropositive, although of the 19 lacrimal gland-seropositive patients, EBV sequences were detected in only 10 of the samples. Further, amplified EBV sequences were not detected in circulating lymphocyte DNA from normal seropositive volunteers, most likely because of the low frequency of circulating EBV-infected B cells. Amplification of EBV from cadaver lacrimal gland DNA was possible with minute quantities of DNA, whereas peripheral blood mononuclear cell DNA from normal volunteers did not amplify EBV sequences. Interestingly, the peripheral blood mononuclear cell polymerase chain reactions contained approximately 100 times more DNA than the lacrimal gland polymerase chain reactions. We conclude that the lacrimal gland may be a site for EBV persistence and that positive EBV serology is not an indicator of which individuals may have EBV harbored within their lacrimal glands.

Detection of the complement (CD21)/Epstein-Barr virus receptor in human lacrimal gland and ocular surface epithelia.

CD21, the receptor for the C3d complement fragment, has been reported to be the Epstein-Barr virus (EBV) receptor in B lymphocytes and cultured squamous epithelial cells. This receptor has previously been found to be expressed in the epithelia of nonocular mucosal-associated lymphoid tissues (MALT) which are also sites of persistent EBV infection. We recently found evidence of persistent EBV infection in human ocular MALT and hypothesized that the epithelia in these tissues may also express the complement (C3d)/EBV receptor. To test this hypothesis, histologic sections of human lacrimal gland, ocular surface tissue (conjunctiva, limbus, and cornea), and conjunctival impression cytology specimens were stained by immunohistochemical techniques using three different anti-CD21 monoclonal antibodies (HB-5, B2, OKB7). HB-5 stained lacrimal gland ductal and all ocular surface epithelia, except limbus. B2 stained lacrimal gland ductal and limbal epithelia, and OKB7 stained only the limbal and corneal epithelia. The intensity of limbal and corneal epithelial staining with all anti-CD21 antibodies correlated with the level of epithelial differentiation, with the weakest staining noted in the cells which are thought to be the stem and transient amplifying cells of the cornea. These results suggest that external ocular tissues, similar to other MALT, have CD21-positive epithelia which may be potential targets for EBV infection.

Molecular cloning and sequencing of the MTV-1 LTR: evidence for a LTR sequence alteration.

The vertically transmitted Mtv-1 provirus is the primary causative factor of mammary neoplasia in certain C3Hf strains that lack the horizontally transmitted mouse mammary tumor virus (MMTV). The studies here report the molecular cloning of the germ line 4.5 kb Mtv-1 3' EcoRI fragment and sequencing of the 3' Mtv-1 LTR. The Mtv-1 LTR sequence is closely related to the 5' Mtv-11 LTR sequence also reported here, as well as to known Mtv-8 and MMTV LTR sequences in the portion of MMTV and Mtv-8 LTRs previously demonstrated to contain transcriptional regulatory sequences. A 91 bp unique sequence region, Mtv-1 bp 862 to 952, exists in the Mtv-1 LTR, which is upstream of the sequence homology with the MMTV transcriptional regulatory domain. The Mtv-1 unique sequence region is distinct from a 117 bp sequence, bp 862 to 978, in the Mtv-11 LTR sequence as well as reported Mtv-8 and MMTV LTR sequences, and is present in the germ line Mtv-1 5' and 3' LTR-containing restriction fragments. S1 nuclease mapping experiments of C3Hf/Se mammary tumor poly(A) RNA with the cloned Mtv-1 and Mtv-11 LTRs exhibited a specific set of S1 protected fragments demonstrating that Mtv transcripts which accumulate in C3Hf spontaneous mammary tumors are encoded by the Mtv-1 provirus.