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.     

Bromodeoxyuridine incorporation and Ki 67 expression in oral hairy leukoplakia

OBJECTIVES: Oral hairy leukoplakia (HL) is an acan-thotic, hyperparakeratotic lesion characterised by the presence of a replicative Epstein-Barr virus (EBV) infection in the superficial and adjoining layers of the epithelium. EBV or its gene products are capable of modifying epithelial differentiation. The aim of this study was to establish whether the presence of EBV was associated with an alteration in cell turnover by assessing bromodeoxyuridine (BrdU) incorporation and Ki 67 expression in lesional tissue and control mucosa.
METHODS: Biopsies of HL together with age, site and sex matched controls ( n = 7 and 8 respectively) were incubated in 200 μM BrdU in vitro , fixed in methacarn and processed to paraffin wax. Following acid hydrolysis, incorporated BrdU and Ki 67 were identified in serial 5 fim sections using a three-stage immunoperoxidase technique and cell density expressed as the number of positive cells per mm basement membrane length.
RESULTS: Overall, there was no difference in the number of BrdU positive cells per mm basement membrane length between control and HL tissue. However, within HL alone, the presence'of focal EBV replication was associated with a significant reduction in the number of basal cells incorporating BrdU compared to adjacent EBV free areas (P < 0.05). There was no significant difference between Ki 67 positive cells in control and HL tissue and no evidence of a reduction of Ki 67 positive cells in areas associated with EBV replication.
CONCLUSIONS: These results suggest that there is no evidence of a generalised alteration of the proliferative capacity of basal cells in HL, although the focal reduction in BrdU incorporation may reflect subtle changes on cell turnover by EBV infection.     

Fine structure of EBV-infected keratinocytes in oral hairy leukoplakia

We evaluated biopsy specimens of 42 cases of clinically suspected oral hairy leukoplakia for the pattern and frequency of ultrastructural alterations specific to epithelial cells infected with Epstein-Barr virus. Some structures could clearly be identified as Epstein-Barr virus at different stages of assembly, but other intranuclear and cytoplasmic alterations were not conclusively identifiable as any known structure. Keratinocytes producing Epstein-Barr virus contained intranuclear particles of different size and shape; some of them were arranged in a monodispersed pattern and others formed arrays. In contrast, both lesional keratinocytes not producing virus and keratinocytes in uninvolved mucosa contained intranuclear particles reminiscent of perichromatin granules. The nuclei of productive cells also contained marginated chromatin, tubular structures, and, occasionally, crystalline and fibrillar formations as well as enveloped virus. Formations of electron-dense bilayers were seen on both sides of the nuclear membrane. In the cytoplasm of productive cells we observed aggregates of parallel tubules and enveloped electron-dense bodies. Although many of these observations are of diagnostic and pathobiological significance, the morphogenesis, composition, and function of alterations with uncertain morphological identification remain unclear.     

Application of molecular techniques in the rapid diagnosis of EBV-associated oral hairy leukoplakia

A rapid method for the detection of EBV-DNA in paraffin sections of lesions of oral hairy leukoplakia (OHL) is described. The method makes use of advances in molecular technology, including the use of synthetic oligonucleotides with digoxigenin labelling in an in situ hybridisation (ISH) reaction, which can be completed in 24 h. Using this method, sections from 15 of 17 patients clinically diagnosed as having OHL contained readily detectable EBV-DNA in small foci along the upper layers of the stratum spinosum. The sections examined from the two remaining patients appeared to be EBV-DNA negative but both patients were on AZT therapy and one was in addition receiving acyclovir.     

Oral hairy leukoplakia: observations in 95 cases and review of the literature

Oral hairy leukoplakia (HL) was observed in 25.4% of 373 HIV-seropositive patients (n = 95). 87 were men of an average age of 37.1 yr, 8 were women (30.3 yr). 71.6% of the patients were male homosexuals. At initial diagnosis of HL the majority of cases was classified as CDC IVc1 (45.3%) and CDC II (22.1%). Average CD4/CD8 ratio (n = 19) was 0.24 with a range of 0.04-1.0. Thirty biopsies of HL revealed some of the histologic features thought to be characteristic. In only 20 of 30 biopsies EB-virus-specific-capsid antigen was detected. The problems of clinical and histological diagnosis of HL are discussed. Further strict criteria are necessary in order to define HL more distinctly.     

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.     

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.     

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.     

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.     

A rapid microwave-in situ hybridization method for the definitive diagnosis of oral hairy leukoplakia: comparison with immunohistochemistry

As a diagnostic technique, in situ hybridization requires a long processing time, a degree of expertise and may he difficult to handle routinely in some laboratories. To simplify the in situ hybridization method, we have modified a microwave in situ hybridization technique and applied it to oral hairy leukoplakia (OHL) biopsies obtained from 10 HIV-seropositive patients (definitively diagnosed by a conventional in situ hybridization technique) with appropriate controls. It was neccessary to design a novel chamber to avoid drying of sections during the hybridization step. This modified microwave in situ hybridization technique was equispecific and equisensitive to the conventional technique and it shortens the hybridization time from overnight incubation to 14 minutes. To determine the sensitivity of our microwave in situ hybridization method we applied it to previously documented tongue tissue obtained from an AIDS autopsy without clinical evidence of OHL. but found to contain Epstein-Barr virus (EBV) by conventional in situ hybridization. This tissue specimen acted as a low EBV copy number, positive control. The sensitivity of immunohistochemistry using three different commercial detection kits was compared to that of in situ hybridization on the same tissues, following optimisation steps. This included the use of 2 cycles of primary and biotinylated secondary antibodies (antibody double cycling). Clearly positive signals for EBV were detected in all OHL biopsies with the Vectastain Elite ABC and the Histostain-SP kits. The sensitivity of the three commercial detection kits was evaluated at immunohistochemistry level by their application to the low-EBV copy number positive control specimen. Signals for EBV antigen in the low copy number positive control specimen were obtained only with the Vectastain Elite ABC kit. This indicates that, in this application, use of the Vectastain Elite ABC kit gives comparable sensitivity for immunohistochemistry to that found by in situ hybridiation.     

Apoptosis-associated proteins in oral hairy leukoplakia

OBJECTIVE To test the hypothesis that the anti-apop totic ability of Epstein-Barr virus (EBV) may result in altered expression of apoptosis-associated proteins in oral hairy leukoplakia (HL), we evaluated HL tissue and normal epithelium for these proteins by immunohistochemistry.
MATERIALS AND METHODS Twenty formalin-fixed, paraffin-embedded specimens of HL lesions and six spedmens of normal control mucosa were selected from archived tissue specimens. Bcl-2. Bcl-x, Bax and p53 apoptoris-associated proteins were evaluated in immu-nohistochemically stained tissue sections according to staining intensity and pattern. The percentage of p53-positive burl cells was estimated in sequential fields.
RESULTS: Generalty, there were only slight differences in the expression of Bcl-2 and Bcl-x proteins in the epithelium of HL and control tissue. The staining for Bcl-2 was weaker in keratinocytes than in putative melano-cytes and Langerhans cells. Equivocal diffuse cytoplasmic staining of prickle cells was also noted. Keratinocytes throughout the epithelium stained positively for Bcl-x protein, although upper layers were more weakly stained. The 'balloon' keratinocytes in HL were infrequently positive for Bcl-x. Bax staining in HL differed from that in control tissue in being more heterogeneous. The staining reaction in HL was weak to negative in upper epithelial levels where 'balloon' keratinocytes were located. Weak to moderate nuclear p53 protein staining was detected in a mean of 25.3% of basal keratinocytes in all but one of the HL specimens; weak staining was seen in only two control specimens.
CONCLUSIONS: We found only slight immunohistochemid evidence that expression of the apoptosis-associated proteins is altered in HL. p53 appears to be over-expressed in HL; we speculate that this may be related to upregulation or stabilization of wild-type p53 protein related to EBV infedon.     

Further ultrastructural findings in epithelial cells of hairy leukoplakia

Epstein-Barr virus and multivesicular structures appearing in lesions of hairy leukoplakia have been examined ultrastructurally in serial sections. It was found that both structures probably represent various levels of sectioning of transversely cut cell processes. The so-called viral nucleocapsid having a single membrane results when the plane of section is at right angles to one of the membranes but tangential to the other. While the so-called viral particle having two membranes would result if the plane of section is perpendicular to both the membranes of adjacent cells. The internal electron-dense core probably represents filaments enclosed within these processes and therefore does not represent DNA nucleotides. In addition, these nuclei contained bundles of filaments which probably represented tonofilaments. This was found to be due to tangential sectioning of the nuclear membrane surrounding deep cytoplasmic invaginations which led to the appearance of these filaments as if free within the nucleus.     

Oral hairy leukoplakia in an HIV-seronegative heart transplant patient

While oral hairy leukoplakia has been observed predominantly in patients with HIV-infection at various stages, recent reports have shown that HL may also occur in patients immunosuppressed for other reasons. This report describes oral hairy leukoplakia in a heart transplant recipient with negative HIV serology. The histopathologic diagnosis of HL was confirmed by immunohistochemical detection of EBV-VCA in the surface epithelium of the lesion and by negative staining electron microscopy.     

Oral hairy leukoplakia in an HIV-negative renal transplant recipient

Oral hairy leukoplakia (HL) has been seen exclusively in those infected with HIV or at risk for AIDS. This case report describes an example of HL seen in a renal transplant recipient who was negative for HIV on serology and culture. The diagnosis of HL was confirmed using in situ hybridization for EBV DNA.     

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.     

A retrospective analysis of oral hairy leukoplakia in South Australia

BACKGROUND: The features of oral hairy leukoplakia (OHL) have been widely reported in the literature. However, no studies have described this lesion in the Australian setting. This study retrospectively examines, with respect to specific clinical factors, the prevalence of OHL in a South Australian HIV-infected population. METHODS: Clinical data were collected from the records of 197 HIV-infected patients who had attended the Adelaide Dental Hospital between January 1986 and February 1995. Data were analysed using the chi-square test. RESULTS: The prevalence of OHL in South Australian HIV-infected patients was 45.2 per cent. The study found the presence of OHL was not related to CD4+ T-lymphocyte count or AIDS-defining illness nor did the length of time a patient had been infected with HIV relate to the presence of OHL. An association was observed between a reduced prevalence of OHL in patients who were taking antiviral medication. CONCLUSION: The prevalence of OHL in South Australia is comparable with results of other studies. This study supports the notion that OHL is not an indicator of immunosuppression in South Australian HIV-infected patients. Further longitudinal studies are required to ascertain the relationship of OHL to HIV disease progression.     

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.     

Altered patterns of keratin expression in oral hairy leukoplakia: prognostic implications

To establish why the lateral border of tongue is the site of predilection for the development of hairy leukoplakia (HL) and to understand its likely behavior, the pattern of keratin expression was compared in 8HL lesions with matched controls in an immunocytochemical study. Keratins 7, 8, 18 were absent in HL and normals; uniform basal keratin 19 was present in normals but much reduced in HL. Loss of conformationally sensitive epitopes of keratin 14 in lower epithelial layers was seen in HL. Overall expression of non-cornifying keratins 4/13 was reduced in HL and completely lost in the parakeratin zone. Expression of the high-turnover keratins 6/16 was reduced in HL. The HL keratin phenotype suggests that no dysplastic change is likely, but in contrast there is enhanced differentiation, which suggests a benign course for the condition.     

云南地区成年人类免疫缺陷病毒感染患者口腔毛状白斑发病情况分析

目的探讨云南地区成年人类免疫缺陷病毒（HIV）感染患者口腔毛状白斑（OHL）的发病情况、临床特点及其与免疫状态的关系。方法以2008年1月-2010年6月收治的1 060例成年HIV感染患者为研究对象,收集信息包括每位患者的年龄、性别、教育程度、HIV阳性确诊时间、传播途径、口干症、口腔念珠菌病、临床高效抗逆转录病毒药物的使用及CD4细胞计数等,并通过口腔检查记录OHL的发病情况,分析OHL发病与CD4细胞计数的关系。结果1 060例HIV感染者中,检出OHL患者94例（8.9%）,其平均年龄为（39.33±10.45）岁。90%的OHL发生在舌的两侧缘,70.2%的患者其CD4细胞计数低于200 mm-3。结论OHL经常发生在严重的免疫抑制的患者中,与CD4细胞计数下降有关。     

In situ hybridization and the polymerase chain reaction (PCR) in the analysis of biopsies and exfoliative cytology specimens for definitve diagnosis of oral hairy leukoplakia (DHL)

The definitive diagnosis of oral hairy leukoplakia (OHL) demands that Epstein-Barr virus (EBV) is demonstrated in the lesional tissue, since the histopathological features on conventional light microscopy are not pathognomonic. We have investigated the possible use of polymerase chain reaction (PCR) technology in reaching a definitive diagnosis of this lesion by its application to ten biopsy specimens with definitive diagnoses of OHL determined by in situ hybridization. EBV DNA was demonstrated by PCR in all ten OHL biopsy specimens analysed, and none of ten control specimens. Furthermore, we have investigated the role of PCR in analysis of exfoliative cytology samples collected from the lateral border of the tongue by a minimally-invasive scraping technique. EBV DNA was not only detected in all OHL lesional scrapings but also in more than one-third of healthy controls, due to viral presence in saliva at the time of sampling. In this application, the highly sensitive PCR technique has low specificity and cannot be recommended.