The expression of the Epstein-Barr virus nuclear antigen (EBNA-I) in oral hairy leukoplakia

OBJECTIVE: Oral hairy leukoplakia (OHL) is characterised by the presence of a replicative Epstein-Barr virus (EBV) in the superficial layers of the epithelium. There is some doubt, however, whether this reflects activation of a latent infection of the basal epithelial cells. EBV latency is associated with the expression of the viral gene product EBNA-I and the aim of this study was to investigate EBNA-I expression in OHL. METHODS: 22 biopsies of clinically suspicious OHL and three cases of normal mucosa were available as fresh frozen or paraffin embedded material. EBNA-I was detected immunocytochemically using a rat monoclonal antibody (IH4-I) following microwave irradiation. Lytic EBV infection was confirmed by the identification of the BZLF-I protein. RESULTS: 16 of the 22 cases displayed focal replicative EBV meeting the criteria for OHL, and in 13 of these, EBNA-I expression was restricted to the nuclei of epithelial cells in the upper layers of the epithelium. EBNA-I expression was absent from the basal cells in all cases and in the nine BZLF-I negative mucosal biopsies. CONCLUSION: These findings suggest that lytic EBV infection in OHL is not the result of activation of a latent infection of basal cells and suggests a role for EBNA-I, not only in latent EBV infection, but also in virus replication.     

Demonstration of Epstein-Barr virus in scrape material of lateral border of tongue in heart transplant patients by negative staining electron microscopy

Scrape material from the lateral border of the tongue of 50 heart transplant patients and 20 controls was studied for the presence of EBV by negative staining electron microscopy. Mild oral hairy leukoplakia was observed in two cases. Particles of the herpes virus were found in 20% of the specimens. Controls were negative for EBV. The study has shown that EBV may be expressed at the lateral border of the tongue during immunosuppression, occasionally resulting in the clinical appearance of hairy leukoplakia.     

A simple and rapid technique for the detection of Epstein-Barr virus DNA in HIV-associated oral hairy leukoplakia biopsies

A method of generating nucleic acid probes by polymerase chain reaction (PCR) for the detection of Epstein-Barr virus (EBV)-DNA by in situ hybridization in oral hairy leukoplakia (OHL) lesions is described. This method has the advantage over older methods of being cheaper, quicker and retaining sensitivity and specificity. Purified PCR products of Epstein-Barr virus DNA of 110 bp and 328 bp were labelled with biotin by nick translation or random primer labelling and were compared in in situ hybridization experiments with probes prepared by incorporation of biotin-labelled nucleotides in the PCR reaction mixture, with EBV viral DNA as a template. These probes were applied to 18 OHL tongue biopsies known to be positive for EBV-DNA, using a commercially available biotin-labelled BamHI "V" fragment EBV-DNA probe. To determine the specificity of the probes, we applied them to 20 normal tongue tissue samples and to 12 biopsies taken from keratotic tongue lesions from patients without risk factors for HIV infection and known to be negative for EBV-DNA. Clear positive signals for EBV-DNA were detected in all 18 cases of OHL biopsies using the amplimer of 328 bp labelled by PCR and random primer labelling. However, nick translation labelling was less efficient and sensitive. All control specimens were negative for EBV-DNA.     

Epstein-Barr virus: Biology and disease

Epstein—Barr virus is a human herpes virus which, whilst found as a widespread asymptomatic infection, is also associated with certain tumours of lymphoid and epithelial origin including Burkitt's lymphoma (BL), immunoblastic lymphoma (IBL), Hodgkin's Disease (HD) and nasopharyngeal carcinoma (NPC). A unique characteristic of EBV is its ability to infect and transform primary resting B lymphocytes in vitro into permanently growing lymphoblastoid cell lines (LCLs); this effect is associated with constitutive expression of a limited set of viral genes. Interestingly, the pattern of EBV gene expression observed in LCLs in vitro is also a feature of IBLs, a tumour associated with immunosuppression. The other EBV associated tumours display a more restricted pattern of EBV latent protein expression. B cell lines can be activated in vitro into the virus replicative cycle, where a large number of viral genes associated with EBV DNA replication and virus assembly are synthesised. Whilst EBV can be detected in throat washings from seropositive individuals, the only in vivo situation where full virus replication can be reliably observed is hairy leukoplakia (HL), a benign lesion of lingual epithelium frequently found in AIDS patients. Thus, the relative contribution of lymphoid cells and epithelial cells to latent EBV infection/persistence vs replication in vivo remains controversial. Recent studies suggest that HL represents a focus of EBV replication in the absence of a truly latent infection and this supports the contention that EBV persistence resides in the lymphoid compartment. These aspects together with the role of EBV in oral diseases and the effect of certain EBV genes on the control of epithelial cell growth and differentiation will be discussed.     

Epstein-Barr virus in oral diseases

Epstein-Barr virus (EBV), a B-lymphotropic gamma-herpesvirus, causes infectious mononucleosis and oral hairy leukoplakia, and is associated with various types of lymphoid and epithelial malignancies. Saliva is the main vehicle for EBV transmission from individual to individual. Recent studies have also implicated EBV in the pathogenesis of advanced types of periodontal disease. EBV DNA is detected in 60-80% of aggressive periodontitis lesions and in 15-20% of gingivitis lesions or normal periodontal sites. The periodontal presence of EBV is associated with an elevated occurrence of periodontopathic anaerobic bacteria. Moreover, EBV active infection occurs in approximately 70% of symptomatic and large-size periapical lesions. EBV and cytomegalovirus often co-exist in marginal and apical periodontitis. Periodontal therapy can markedly suppress the EBV load in periodontal pockets as well as in saliva, which has the potential to reduce the risk of viral transmission between close individuals. EBV proteins up-regulate cytokines and growth factors, which seem to play a central role in the proliferative response of tongue epithelial cells in oral hairy leukoplakia and in the cell-transformation process of EBV-associated malignancies. Further research is needed to identify the full range of EBV-related diseases in the human oral cavity.     

Detection of Epstein-Barr virus DNA in tongue tissues from AIDS autopsies without clinical evidence of oral hairy leukoplakia

Epstein-Barr virus (EBV) DNA was detected by in situ hybridization at 3 sites of 30 samples taken from clinically normal lateral border of tongue mucosa from 15 AIDS autopsies and in none of 20 samples from 10 controls. The first positive case showed a thin layer of parakeratosis correlated with positive signals for EBV in one area and an adjacent area without obvious parakeratosis was also positive for EBV. These findings were present on both sides of the tongue. The second case was unilaterally positive for EBV and parakeratosis was absent. The hybridization signals were localised to koilocyte-like cells in the stratum spinosum, as in oral hairy leukoplakia (OHL). These observations suggest that the in situ hybridization technique can detect very early or subclinical OHL, and supports the role of EBV in the pathogenesis of this lesion.     

Epstein-Barr virus infection and expression in oral lesions

OBJECTIVE: The prevalence of Epstein-Barr virus (EBV) and the recently discovered Kaposi's sarcoma associated herpes virus, human herpesvirus 8 (KSHV or HHV8), was determined within oral lesions common to HIV infection including OHL, pseudoOHL (PHL), oral lymphoma, oral aphthous ulcers, and an oral Kaposi's sarcoma. METHODS: DNA and RNA were extracted from oral lesions. EBV and HHV8 genomes were detected by Southern blot and polymerase chain reaction (PCR), and viral expression was analyzed using PCR amplification of cDNA. RESULTS: Multiple EBV strains were detected within OHL with recombination across repeat sequences generating new viral variants. EBV expression in OHL included expression of some viral genes, usually expressed in latent infections, that induce the EBV receptor. EBV replication was detected only within OHL lesions but not within adjacent Kaposi's tissue or oral aphthous ulcers while HHV8 was only detected within the Kaposi's lesions. CONCLUSIONS: These findings indicate that the OHL lesion is unique with viral replication and superinfection with additional EBV strains. Expression of the EBV receptor within the OHL lesion may promote superinfection which then activates EBV replication. The consistent detection of EBV replication only within OHL lesions and the detection of HHV8 only within Kaposi's sarcoma, strengthens the etiologic link between EBV and HHV8 infection to these specific pathologies.     

Epstein-Barr virus BMRF-2 and BDLF-3 expression in hairy leukoplakia

Hairy leukoplakia (HL) is a lesion found on the side of the tongue of immunocompromised individuals, including those with human immunodeficiency virus (HIV) infection. The lesion has unique histopathologic features and is characterised by high-level Epstein-Barr virus (EBV) replication, multiple EBV strains, and extensive inter-and intra-strain recombination. Expression of EBV genes spanning the entire viral life cycle from latency-associated genes to late, replicative genes has been detected in the lesion. HL thus provides a unique opportunity to study EBV expression in oral epithelium, and to study expression of novel EBV genes. We therefore constructed a cDNA library from an HL biopsy and detected expression of two genes not previously described in vivo: BMRF-2 and BDLF-3. Sequence analysis of the cDNAs revealed few amino acid changes from the B95-8 sequence. Expression of both genes was localized to the lower prickle cell layer of the tongue epithelium. BMRF-2 protein expression was primarily detected in the cell nuclei of the upper prickle cell layer. BDLF-3 protein expression was observed in the perinuclear space and Golgi compartment. The function of these proteins is currently under investigation.     

Integrin expression in oral hairy leukoplakia and normal tongue epithelium

OBJECTIVE: To describe the expression of integrins in the epithelium of oral hairy leukoplakia (HL) and compare to that of normal lateral tongue epithelium. MATERIALS AND METHODS: Immunohistochemistry to identify integrins (alpha 2, alpha 3, alpha 5, alpha 6, alpha v, beta 1) was performed, using a standard biotin-streptavidin-peroxidase technique on five clinically and histologically confirmed frozen biopsy specimens of HL and five normal lateral tongue control tissues. RESULTS: Expression of integrins alpha 2, alpha 3, alpha 6, alpha v, beta 1 was seen both in HL epithelium and in normal control tissue. alpha 5 expression was not seen in HL or in control tissue epithelium. alpha 2 and alpha 3 were expressed mainly in the basal and suprabasal layers; alpha 6 expression was most intense on the basal surface of the basal cells, alpha v was expressed in the basal and suprabasal layers with more expression seen in the higher differentiated cell layers than the other integrins. beta 1 expression was seen in the basal and suprabasal layers only. No apparent difference between HL and normal oral mucosa was noted in the staining pattern of the various integrins. CONCLUSION: Integrins alpha 2, alpha 3, alpha 6, alpha v, beta 1 are expressed in HL and the expression pattern is not different from that of normal oral mucosa. alpha 5 is not expressed in HL or in normal oral epithelium.     

Detection of Epstein-Barr virus in oral scrapes in HIV infection, in hairy leukoplakia, and in Healthy non-HIV-infected people

Epstein-barr Virus (EBV) has been implicated in the pathogenesis of oral hairy leukoplakia (OHL). EBV is normally detected by lesional biopsy. The objectives of this study were to examine oral scrapes containing squamous epithelial cells from healthy non-HIV-infected people with and without clincial lesions of OHL, and from healthy non-HIV-infected controls, for EBV-DNA by the polymerase chain reaction (PCR). EBV-DNA was detected in 65% of HIV-infected individuals it was found as frequently in those without OHL as in those with. Moreover, EBV-DNA was not detected in all HIV-infected individuals, nor in all OHL as in those with. Moreover, EBV-DNA was not detected in all Hiv-infected individuals, nor in all OHL> The results suggest that the presence or absenc e of detectable EBV-DNA in oral scrapes, though a guide, cannot be regarded as absolutely reliable in the diagnosis or exclusion of OHL.     

Human immunodeficiency virus-positive individuals with oral hairy leukoplakia are able to mount cytotoxic T lymphocyte responses to Epstein-Barr virus

OBJECTIVE: Oral hairy leukoplakia (OHL) is a white lesion of the tongue that is caused by Epstein-Barr virus (EBV) and occurs mainly in people infected with human immunodeficiency virus (HIV). The aim of this study was to determine whether the presence of OHL reflects the absence of EBV-specific cytotoxic T lymphocyte (CTL) activity. SUBJECTS AND METHODS: EBV-specific CTL responses were measured in HIV-positive homosexual men with OHL, HIV-positive homosexual men without OHL, and HIV-negative homosexual men. Also, the phenotypes of cells responsible for EBV-specific responses were studied. RESULTS: Eighty percent (8/10) of HIV-positive subjects with OHL, 52% (12/23) of HIV-positive subjects without OHL, and 83% (15/18) HIV-negative subjects had a positive anti-EBV CTL response (P = 0.004, Kruskal-Wallis test). Two HIV-positive subjects showed a greater anti-EBV CTL response after developing OHL than before the appearance of OHL Additional experiments showed that CD8-positive T cells and CD4-positive T cells were responsible for the EBV-specific CTL responses. CONCLUSION: Our data show more EBV-specific CTL activities in HIV-positive individuals with OHL than in HIV-positive individuals without OHL. Whether the presence of EBV-specific CTL contributes to resolution of OHL remains to be clarified.     

Negative staining EM for the detection of Epstein-Barr virus in oral hairy leukoplakia

The performance of two different EM techniques applied for the detection of Epstein-Barr Virus (EBV) in oral hairy leukoplakia (HL) was assessed, i.e. the conventional two-step method of negative staining (CNS) and negative staining after Airfuge enrichment (ANS). Scrape specimens from the lateral borders of tongue of 66 HIV-positive patients with or without HL, of 3 patients with infectious mononucleosis and of 10 HIV-negative patients were evaluated. While CNS resulted in virus detection only in 25% of clinically diagnosed HL cases, EBV was detected by ANS in 85% of clinically suspected cases of HL. Scrape specimens of individuals negative for HIV were negative in EM while 2 of 3 mononucleosis patients were positive without clinical evidence for HL. Due to this high sensitivity the method of negative staining after Airfuge enrichment appears to be useful in the diagnosis of HL. The finding of EBV in clinically normal oral mucosa in HIV-seropositive individuals is interesting and indicates that EBV expression may precede the clinical appearance of HL.     

Detection of Epstein-Barr virus and human papillomavirus type 16 DNA in hairy leukoplakia by in situ hybridisation and the polymerase chain reaction

Demonstration of Epstein-Barr virus (EBV) is considered desirable for the accurate diagnosis of hairy leukoplakia (HL). Previous studies have reported possible associations with human papillomavirus (HPV) infection although this is not a universal finding. Presence of EBV and HPV 16 was examined in biopsy specimens from 18 cases of HL and ten control specimens by in situ hybridisation using digoxigenin-labellcd synthetic oligonucleotide probes and by the polymerase chain reaction (PCR). The presence of EBV was demonstrated in 12 cases by both techniques. Of the remaining six cases EBV could be detected in three by in situ hybridisation but not by PCR; EBV was not detected by either method in a further three cases. All samples were negative for HPV 16 by both techniques under conditions of high stringency, although when stringency of in situ hybridisation was reduced, four samples appeared to harbour HPV DNA sequences. This study provides further evidence to support the role of EBV in the pathogenesis of HL and suggests that HPV 16 is not regularly encountered.     

Comparison of cytopathic changes in oral hairy leukoplakia with in situ hybridization for EBV DNA

OBJECTIVE: It has been observed that the cytopathic changes in hairy leukoplakia (HL) correlate with ultra-structural evidence of intra-keratinocyte herpes-type viral particles. In situ hybridization is considered to be the definitive confirmation of Epstein-Barr virus (EBV)-induced HL. This study evaluated the consistency of histopathological findings, which many believe to be diagnostic, with in situ hybridization for EBV-DNA in 60 patients with lesions clinically suggestive of HL.
MATERIALS AND METHODS: Hematoxylin and eosin (H&E)-stained sections were reviewed independently by three oral pathologists who did not know the hybridization results. The presence in keratinocytes of nuclear inclusions and/or homogenization, believed to be specific for EBV in these lesions, was used as an indicator for infection. Cytoplasmic changes were evaluated separately.
RESULTS: With in situ hybridization, 48 cases were positive and 12 were negative. When the two methods were compared, pathologist concurrence ranged from 83% to 92%. False negatives ranged from 6% to 19%, and false positives ranged from 8% to 25%. Cytoplasmic ballooning, homogenization, and perinuclear clearing were evident in all cases of hybridization-confirmed HL; however, these changes were also noted in 75% (9/12) of the cases with negative hybridization results. Most confirmed HL cases exhibited both nuclear homogenization and inclusions, although the former was more consistently seen.
CONCLUSION: Cytoplasmic changes did not agree well with EBV-DNA hybridization results, whereas nuclear changes demonstrated good, but not complete, agreement. In appropriate clinical settings, the finding of nuclear inclusions and/or homogenization may be of diagnostic value. However, because the potential for false positives and negatives is high, H&E cytopathology should not be used as a substitute for in situ hybridization in the definitive diagnosis of oral hairy leukoplakia.     

Epstein-Barr virus in tobacco-induced oral cancers and oral lesions in patients from India

D'Costa J, Saranath D, Sanghvi V, Mehta AR: Epstein-Barr virus in tobacco-induced oral cancers and oral lesions in patients from India. J Oral Pathol Med 1998; 27: 78–82. © Munksgaard, 1998.
We examined 103 oral squamous cell carcinomas (OSCC), 100 oral lesions consisting primarily of leukoplakia (82 cases), and 76 clinically normal mucosa specimens from the contralateral site in the oral cavity of individuals with oral lesions, for the presence of Epstein-Barr virus (EBV). Polymerase chain reaction (PCR) was used to amplify a 239 bp fragment of the BamHIL region of the EBV genome, followed by Southern blot hybridization with EBV oligonucleotide probe to increase further the specificity and sensitivity of the assay system. Since EBV seropositivity is frequent in populations, we also examined the peripheral blood cells (PBC) from 141 patients (50 oral cancer patients, 91 patients with oral lesions) for the presence of EBV We detected EBV in 25 of 103 (25%) OSCC, 13 of 100 (13%) oral lesions, 3 of 76 (4%) clinically normal mucosa samples and 10 of 141 (7%) PBC. Our results indicate that EBV may contribute as one of the multiple factors in oral cancers, in a certain proportion of Indian patients.     

Oral lesions in organ transplant patients

Patients who have undergone organ transplantation can present with a variety of oral lesions that appear to be related either directly to their medication or arise as a consequence of drug-induced immunosuppression. Such lesions include hairy leukoplakia, an increased propensity to both fungal and viral infections and a high incidence of malignant change, especially lip cancer. Cyciosporin remains the immunosuppressant of choice in most transplant patients. Gingival overgrowth is the main unwanted oral effect associated with cyclosporin. Some 30% of dentate transplant patients experience this problem, which is further compounded by concomitant medication with a calcium channel blocker. This review appraises the various oral problems that can arise in this group of patients and emphasises the importance of regular oral screening and the establishment of links with the various transplant teams.     

Epstein-Barr virus receptors but not viral DMA are present in normal and malignant oral epithelium

The presence and distribution of Epstein-Barr Virus receptors (EBVR's) on a range of normal (n = 18), dysplastic (n = 10) and malignant (n = 20) oral mucosa were studied by immunocytochemical methods using the monoclonal antibodies (MAb's) HB5 and B2. EBVR's were demonstrated as membrane staining of the spinous layers of normal non- and parakeratinized epithelium, indicating that EBVR's are differentiation-linked. This distribution was retained in dysplastic epithelium. Tissue from oral squamous cell carcinomas (SCC's) showed variable reactivity of only a few cells scattered randomly within the samples. Furthermore, a sensitive in situ hybridization (ISH) technique was used to determine if Epstein-Barr virus (EBV) was present in normal (n = 15) and oral squamous cell carcinoma tissue (n = 20). No EBV DNA was demonstrated within either normal or malignant epithelium, suggesting that the virus does not persist in normal oral stratified squamous epithelium nor is there any evidence for a role in oral carcinogenesis.     

Determinants of gingival overgrowth severity in organ transplant patients

Abstract The role of HLA phenotype as a risk factor for drug-induced gingival overgrowth was investigated in a cohort of 172 transplant recipients. Clinically significant overgrowth warranting surgical correction was observed in 72 patients (42%). Using stepwise regression modelling. 6 clinical parameters were identified as significant risk factors for the severity of gingival overgrowth. These were; age. sex. creatinine plasma level, duration of therapy, papilla bleeding index and concomitant medication with a calcium channel blocking drug. 3 HLA alleles were also identified as risk factors when adjusted for other clinically significant risk factors (HLA -DR2, A24, B37). However, when the p-values for the HLA variables were corrected to compensate for the use of multiple significance testing, only HLA-B37 remained statistically significant at the 5% level. Organ transplant patients are at risk of developing gingival overgrowth, with approximately 25% medicated with cyclosporin alone requiring corrective gingival surgery. This figure more than doubles in patients concomitantly medicated with a calcium blocking drug. The data at present available would suggest that the severity of gingival overgrowth is also significantly associated with the HLA-B37 phenotype.     

Bioluminescence assay of enzymes obtained from buccal epithelium by superficial scraping

abstract — A method is presented for the simultaneous assay of buccal enzymes by measuring reduced nicotinamide adenine dinucleotide and adenosine triphosphate with the aid of the bioluminescence of luciferase extracts. The activity of glucose-6-phosphate dehydrogenase (G6PDH) was shown earlier to be increased in homogeneous leukoplakias of the oral mucosa. Since smoking has been implicated as an etiologic factor of leukoplakia, G6PDH was measured in the normal buccal epithelium of cigarette smokers. No difference was found in the activity of G6PDH between smokers and nonsmokers when related to the activity of pyruvate kinase, which is known to be invariable in healthy and leukoplakic oral mucosa. A new compact kinetic luminescence analyzer is briefly described.     

Detection of Epstein-Barr virus (EBV) DNA and antigens in oral mucosa of renal transplant patients without clinical evidence of oral hairy leukoplakia (OHL)

The use of the polymerase chain reaction (PCR) to detect the presence of Epstein-Barr virus (EBV) DNA in oral mucosa in the absence of specific lesions gives rise to the problem of identifying the real viral replication sites. To verify whether the detection of EBV is due to salivary contamination or its true replicative capacity in oral mucosa, saliva samples and exfoliated cells from four different oral mucosa sites were taken from 40 renal transplant patients and 20 normal subjects for examination by PCR using two pairs of primers specific for the BamHI-L and BamHI-K genomic regions. EBV-specific sequences were detected in one or more of the oral mucosa samples from 29 transplant patients (72.5%) and six healthy controls (30%), and in the sliva samples of 16 transplant patients (40%) and three healty controls (15%). A total of 89 oral mucosa smears from 29 transplant patients, and 13 from healthy subjects, were EBV-positive. The positive samples were also investigated by means of in situ hybridization in order to confirm the intracellular presence of the viral genome, and by means of immunofluorescence testing with monoclonal antibodies to assess the possible expression of viral antigens. Hybridization with the EBV-specific probe was observed in 40/89 and 2/13 samples, respectively. Latent antigens (with or without lytic antigens) were detected in only 23 of the 40 samples (collected from eight different transplant patients) that were positive by in situ hybridization. Our data show that EBV is more frequently present in the oral mucosa of immunodeficient patients (where it can efficiently replicate) than in normal subjects.