Evaluation of an Epstein-Barr virus (EBV) immunoglobulin M enzyme-linked immunosorbent assay using a synthetic convergent peptide library, or mixotope, for diagnosis of primary EBV infection.
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An immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) was developed by using a 24-amino-acid peptide of the 18-kDa Epstein-Barr virus (EBV) viral capsid antigen (VCAp18). IgM detection was increased by 23% by using this antigen. Detection of IgM antibodies to the EBV proteins in the new ELISA was 100% specific and 95% sensitive. (+info)
Nitric oxide (NO), a mediator of biological functions, has an antimicrobial activity against a variety of pathogens including viruses. In this study, we found that a constitutive, low level of inducible NO synthase (iNOS) mRNA was expressed in the EBV-infected gastric tissue-derived GT38 and GT39 cell lines, by analysis with the reverse transcription-polymerase chain reaction (RT-PCR) and Southern blotting. Treatment of these cells with a specific NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), induced the immediate-early, EBV transactivator gene BZLF1 protein ZEBRA, suggesting a significant increase in EBV reactivation by L-NMMA. Northern blotting demonstrated that BZLF1 and BRLF1 transcripts were also induced by 12-O-tetradecanoylphorbol-13 acetate (TPA). Meanwhile, constitutive expression of iNOS mRNA was inhibited by TPA. L-NMMA also enhanced TPA-induced expression of the BZLF1 gene. On the other hand, a NO donor, S-nitroso-N-acetylpenicillamine (SNAP), which releases NO in an aqueous solution, inhibited the TPA-induced BZLF1 gene expression in a dose-dependent manner at both mRNA and protein levels. These results demonstrated that NO is a regulatory factor in maintaining virus latency via inhibiting EBV reactivation in the infected epithelial cells. (+info)
The oncogenic 30 and 69 bp deletion variants of the EBV LMP-1 gene are common in HIV-negative lymphoproliferations, both malignant and benign.
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BACKGROUND: In vitro studies have shown that the 30 and 69 base pair (bp) deletion variants of the latent membrane protein (LMP)-1 gene of the Epstein-Barr virus (EBV) have a higher transforming capacity than the wild-type variant. In recent years these studies have triggered an in vivo search for such potentially oncogenic variants in lymphoid tissues. PATIENTS AND METHODS: We used polymerase chain reaction (PCR) to investigate the prevalence of LMP-1 gene variants in EBV-positive lymph nodes from 60 HIV-negative Italian patients with benign and malignant lymphoid disorders. RESULTS: The 30 bp variant was detected in 10 of 39 (25.6%) malignant lymphomas but also in 4 of 13 (30%) reactive lymphadenitis with follicular hyperplasia. Of note is the fact that the 69 bp variant was detected in three cases of malignant lymphoproliferation but also in two cases of localized Castleman's disease of hyalin vascular type. CONCLUSIONS: The molecular detection of the oncogenic variants of the LMP-1 gene in a lymph node biopsy as an indicator of the aggressiveness of the EBV-associated lymphoproliferative disease must be considered with caution. The relatively high frequency of the 69 bp variant in our series compared with that reported in the literature probably reflects a different incidence of LMP-1 variants in healthy populations from different geographical areas. (+info)
Definition of the transcription factors which bind the differentiation responsive element of the Epstein-Barr virus BZLF1 Z promoter in human epithelial cells.
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Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus and an important human pathogen. Initiation of the EBV lytic cycle is dependent upon transcription of the EBV BZLF1 gene. Our previous studies of transcriptional regulation of the BZLF1 Z promoter (Zp) in human SCC12F epithelial cells identified a region within Zp that is responsive to epithelial cell differentiation. In the present study, we localize this differentiation responsive element to the CREB/AP-1-like binding site (TGACATCA) between -67 to -60 bp within Zp, previously designated ZII, and furthermore show that homodimers and heterodimers of CREB and ATF-1 specifically bind ZII. Consistent with a regulatory role for CREB and ATF-1 in differentiation dependent BZLF1 expression, ZII was able to bind approximately 3-fold more CREB and ATF-1 when incubated with nuclear extract obtained from populations of SCC12F cells enriched for the differentiated phenotype than when incubated with extract obtained from populations enriched for the undifferentiated phenotype. In addition, CREB and ATF-1 were found to increase in abundance during SCC12F differentiation. These results indicate a regulatory role for CREB and ATF-1 in differentiation-dependent expression of BZLF1 in human epithelial cells. (+info)
Interleukin 13 is secreted by and stimulates the growth of Hodgkin and Reed-Sternberg cells.
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Gene expression patterns can provide vital clues to the pathogenesis of neoplastic diseases. We investigated the expression of 950 genes in Hodgkin's disease (HD) by analyzing differential mRNA expression using microarrays. In two independent microarray experiments, the HD-derived cell lines L428 and KMH2 were compared with an Epstein-Barr virus (EBV)-immortalized lymphoblastoid B cell line, LCL-GK. Interleukin (IL)-13 and IL-5 were found to be highly expressed in the HD-derived cell lines. Examination of IL-13 and IL-5 expression by Northern blot analysis and enzyme-linked immunosorbent assay confirmed these results and revealed the expression of IL-13 in a third HD-derived cell line, HDLM2. Control LCL and EBV-negative non-Hodgkin lymphoma-derived cell lines did not express IL-13. In situ hybridization of lymph node tissue from HD patients showed that elevated levels of IL-13 were specifically expressed by Hodgkin/Reed-Sternberg (H/RS) tumor cells. Treatment of a HD-derived cell line with a neutralizing antibody to IL-13 resulted in a dose-dependent inhibition of H/RS cell proliferation. These data suggest that H/RS cells produce IL-13 and that IL-13 plays an important role in the stimulation of H/RS cell growth, possibly by an autocrine mechanism. Modulation of the IL-13 signaling pathway may be a logical objective for future therapeutic strategies. (+info)
Endogenous CD8+ T cell expansion during regression of monoclonal EBV-associated posttransplant lymphoproliferative disorder.
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There are experimental data which suggest that the primary immune effector cell responsible for maintaining immune surveillance against the outgrowth of EBV-transformed B cells in humans is the CTL, but in vivo proof of this is lacking. In this study we perform a series of cellular and molecular assays to characterize an autologous, endogenous immune response against a transplantation-associated, monoclonal, EBV+ posttransplant lymphoproliferative disorder (PTLD). Following allogeneic bone marrow transplantation, a patient developed a monoclonal PTLD of donor B cell origin. With a decrease in immune suppression, we document the emergence of endogenous, donor-derived CD3+CD8+ CTLs, followed by regression of the PTLD. The TCR Vbeta repertoire went from a polyclonal pattern prior to the development of PTLD to a restricted TCR Vbeta pattern during the outgrowth and regression of PTLD. Donor-derived CD3+CD8+ T lymphocytes displayed MHC class I-restricted cytolytic activity against the autologous EBV+ B cells ex vivo without additional in vitro sensitization. The striking temporal relationship between the endogenous expansion of a TCR Vbeta-restricted, CD3+CD8+ population of MHC class I-restricted CTL, and the regression of an autologous monoclonal PTLD, provides direct evidence in humans that endogenous CD3+CD8+ CTLs can be responsible for effective immune surveillance against malignant transformation of EBV+ B cells. (+info)
Phenylbutyrate induces cell differentiation and modulates Epstein-Barr virus gene expression in Burkitt's lymphoma cells.
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Although Burkitt's lymphoma (BL) is a readily treated malignancy, recurrences, as well as disease arising in immunosuppressed patients, are notoriously resistant to conventional therapeutic approaches. The EBV is associated with a significant proportion of these lymphomas that evade immune surveillance through decreased expression of both viral and cellular antigens. Increasing the immunogenicity of BL cells may, therefore, represent a potentially beneficial therapeutic maneuver. Using in vitro models of EBV-transformed lymphoblastoid as well as BL cell lines, we demonstrate increased expression of genes coding for HLA class I and EBV latent proteins by the differentiation inducer phenylbutyrate (PB). The aromatic fatty acid also caused cytostasis associated with sustained declines in c-myc expression, a direct antitumor effect that was independent of the EBV status. We conclude, therefore, that differentiation therapy of BL with PB may lead to growth arrest with increased tumor immunogenicity in vivo. The findings may have clinical relevance because the in vitro activity has been observed with PB concentrations that are well tolerated and nonimmunosuppressive in humans, a desirable feature for the different patient populations afflicted with this disease. (+info)
Human monoclonal antibodies isolated from spontaneous Epstein-Barr virus-transformed tumors of Hu-SPL-SCID mice and specific for fusion protein display broad neutralizing activity toward respiratory syncytial virus.
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Two human monoclonal antibodies, RF-1 and RF-2, specifically recognize the fusion protein of the human respiratory syncytial virus (RSV). These were isolated from spontaneous tumors in SCID mice reconstituted with human splenocytes and boosted with fusion protein. The tumors consisted of Epstein-Barr virus-transformed human B cells in animals with antigen-specific antibody titers>105. The binding affinity of RF-1 and RF-2 to the fusion protein is 1010 and 109 M-1, respectively. The antibodies bind specifically to a conformational epitope of the fusion protein on RSV-infected HEp-2 cells. Both antibodies display virus-neutralizing properties in vitro at concentrations varying between 8 and 1000 ng/mL. Virus neutralization applies to a broad variety of wild and laboratory-adapted virus strains belonging to both virus types A and B. These antibodies are potential candidates for passive immunotherapy of severe RSV infections. (+info)