6,012 research outputs found
Relationship Between Non-Hodgkin's Lymphoma and Blood Levels of Epstein-Barr Virus in Children in North-Western Tanzania: A Case Control Study.
No full textNon-Hodgkin's Lymphomas (NHL) are common in African children, with endemic Burkitt's lymphoma (BL) being the most common subtype. While the role of Epstein-Barr Virus (EBV) in endemic BL is known, no data are available about clinical presentations of NHL subtypes and their relationship to Human Immunodeficiency Virus (HIV) infection and Epstein Barr Virus (EBV) load in peripheral blood of children in north-western, Tanzania. A matched case control study of NHL subtypes was performed in children under 15 years of age and their respective controls admitted to Bugando Medical Centre, Sengerema and Shirati district designated hospitals in north-western, Tanzania, between September 2010 and April 2011. Peripheral blood samples were collected on Whatman 903 filter papers and EBV DNA levels were estimated by multiplex real-time PCR. Clinical and laboratory data were collected using a structured data collection tool and analysed using chi-square, Fisher and Wilcoxon rank sum tests where appropriate. The association between NHL and detection of EBV in peripheral blood was assessed using conditional logistic regression model and presented as odds ratios (OR) and 95% confidence intervals (CI). A total of 35 NHL cases and 70 controls matched for age and sex were enrolled. Of NHLs, 32 had BL with equal distribution between jaw and abdominal tumour, 2 had large B cell lymphoma (DLBCL) and 1 had NHL-not otherwise specified (NHL-NOS). Central nervous system (CNS) presentation occurred only in 1 BL patient; 19 NHLs had stage I and II of disease. Only 1 NHL was found to be HIV-seropositive. Twenty-one of 35 (60%) NHL and 21 of 70 (30%) controls had detectable EBV in peripheral blood (OR = 4.77, 95% CI 1.71 - 13.33, p = 0.003). In addition, levels of EBV in blood were significantly higher in NHL cases than in controls (p = 0.024). BL is the most common childhood NHL subtype in north-western Tanzania. NHLs are not associated with HIV infection, but are strongly associated with EBV load in peripheral blood. The findings suggest that high levels of EBV in blood might have diagnostic and prognostic relevance in African children
Immunodominance of lytic cycle antigens in Epstein-Barr virus-specific CD4+ T cell preparations for therapy.
No full textEpstein-Barr virus (EBV) is associated with a number of human malignancies. EBV-positive post-transplant lymphoproliferative disease in solid organ and hematopoietic stem cell transplant recipients has been successfully treated by the adoptive transfer of polyclonal EBV-specific T cell lines containing CD4+ and CD8+ T cell components. Although patients receiving T cell preparations with a higher CD4+ T cell proportion show better clinical responses, the specificity of the infused CD4+ component has remained completely unknown.
We generated LCL-stimulated T cell lines from 21 donors according to clinical protocols, and analyzed the antigen specificity of the CD4+ component in EBV-specific T cell preparations using a genetically engineered EBV mutant that is unable to enter the lytic cycle, and recombinantly expressed and purified EBV proteins. Surprisingly, CD4+ T cell lines from acutely and persistently EBV-infected donors consistently responded against EBV lytic cycle antigens and autoantigens, but barely against latent cycle antigens of EBV hitherto considered principal immunotherapeutic targets. Lytic cycle antigens were predominantly derived from structural proteins of the virus presented on MHC II via receptor-mediated uptake of released viral particles, but also included abundant infected cell proteins whose presentation involved intercellular protein transfer. Importantly, presentation of virion antigens was severely impaired by acyclovir treatment of stimulator cells, as currently performed in most clinical protocols.
These results indicate that structural antigens of EBV are the immunodominant targets of CD4+ T cells in LCL-stimulated T cell preparations. These findings add to our understanding of the immune response against this human tumor-virus and have important implications for the improvement of immunotherapeutic strategies against EBV
Lymphoid hyperplasia and lymphoma in transgenic mice expressing the small non-coding RNA, EBER1 of Epstein-Barr virus
No full textBackground. Non-coding RNAs have critical functions in diverse biological processes, particularly in gene regulation. Viruses, like their host cells, employ such functional RNAs and the human cancer associated Epstein-Barr virus (EBV) is no exception. Nearly all EBV associated tumours express the EBV small, non-coding RNAs (EBERs) 1 and 2, however their role in viral pathogenesis remains largely obscure.Methodology/Principal FindingsTo investigate the action of EBER1 in vivo, we produced ten transgenic mouse lines expressing EBER1 in the lymphoid compartment using the mouse immunoglobulin heavy chain intronic enhancer E++. Mice of several of these E++EBER1 lines developed lymphoid hyperplasia which in some cases proceeded to B cell malignancy. The hallmark of the transgenic phenotype is enlargement of the spleen and mesenteric lymph nodes and in some cases enlargement of the thymus, liver and peripheral lymph nodes. The tumours were found to be of B cell origin and showed clonal IgH rearrangements. In order to explore if EBER1 would cooperate with c-Myc (deregulated in Burkitt's lymphoma) to accelerate lymphomagenesis, a cross-breeding study was undertaken with E++EBER1 and E++Myc mice. While no significant reduction in latency to lymphoma onset was observed in bi-transgenic mice, c-Myc induction was detected in some E++EBER1 single transgenic tumours, indicative of a functional cooperation. Conclusions/SignificanceT his study is the first to describe the in vivo expression of a polymerase III, non-coding viral gene and demonstrate its oncogenic potential. The data suggest that EBER1 plays an oncogenic role in EBV associated malignant disease
Micro RNAs of Epstein-Barr virus promote cell cycle progression and prevent apoptosis of primary human B cells.
No full textCellular and viral microRNAs (miRNAs) are involved in many different processes of key importance and more than 10,000 miRNAs have been identified so far. In general, relatively little is known about their biological functions in mammalian cells because their phenotypic effects are often mild and many of their targets still await identification. The recent discovery that Epstein-Barr virus (EBV) and other herpesviruses produce their own, barely conserved sets of miRNAs suggests that these viruses usurp the host RNA silencing machinery to their advantage in contrast to the antiviral roles of RNA silencing in plants and insects. We have systematically introduced mutations in EBV's precursor miRNA transcripts to prevent their subsequent processing into mature viral miRNAs. Phenotypic analyses of these mutant derivatives of EBV revealed that the viral miRNAs of the BHRF1 locus inhibit apoptosis and favor cell cycle progression and proliferation during the early phase of infected human primary B cells. Our findings also indicate that EBV's miRNAs are not needed to control the exit from latency. The phenotypes of viral miRNAs uncovered by this genetic analysis indicate that they contribute to EBV-associated cellular transformation rather than regulate viral genes of EBV's lytic phase
Binding of the heterogeneous ribonucleoprotein K (hnRNP K) to the Epstein-Barr virus nuclear antigen 2 (EBNA2) enhances viral LMP2A expression.
No full textThe Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties
Messenger RNA Sequence Rather than Protein Sequence Determines the Level of Self-synthesis and Antigen Presentation of the EBV-encoded Antigen, EBNA1
No full textpeer-reviewedViruses establishing persistent latent infections have evolved various mechanisms to avoid immune surveillance. The Epstein-Barr virus-encoded nuclear antigen, EBNA1, expressed in all EBV-associated malignancies, modulates its own protein levels at quantities sufficient to maintain viral infection but low enough so as to minimize an immune response by the infected host cell. This evasion mechanism is regulated through an internal purine-rich mRNA repeat sequence encoding glycine and alanine residues. In this study we assess the impact of the repeat's nucleotide versus peptide sequence on inhibiting EBNA1 self-synthesis and antigen presentation. We demonstrate that altered peptide sequences resulting from frameshift mutations within the repeat do not alleviate the immune-evasive function of EBNA1, suggesting that the repetitive purine-rich mRNA sequence itself is responsible for inhibiting EBNA1 synthesis and subsequent poor immunogenicity. Our comparative analysis of the mRNA sequences of the corresponding repeat regions of different gammaherpesvirus maintenance homologues to EBNA1 highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences. These studies demonstrate the importance of gammaherpesvirus purine-rich mRNA repeat sequences on antigenic epitope generation and evasion from T-cell mediated immune control, suggesting novel approaches to prevention and treatment of latent infection by this class of virus.National Health & Medical Research Council (NH&MRC) Canberra, Australia (#496684 APP1005091); NH&MRC Career Development Award Research Fellowship (#496712
Identification and Functional Analysis of Epstein-Barr Nuclear Antigen 2 (EBNA2) Target Genes
No full textBARF1 AS A NEW THERAPEUTIC TARGET FOR EBV-ASSOCIATED MALIGNANCIES.
No full textWhile Epstein-Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs) have been used successfully for the prophylaxis and treatment of the highly immunogenic post-transplant lymphoproliferative disorders, the clinical experience for other EBV-associated malignancies, such as Hodgkin's lymphoma and undifferentiated nasopharyngeal carcinoma (NPC), is limited and the results obtained so far indicate that EBV-CTLs are less effective in these settings. Decreased CTL efficacy most likely reflects immune evasion strategies by tumor cells, including down-regulation of immunodominant EBV proteins and the weak immunogenicity of the viral proteins expressed. One of the possible approaches to overcome these limitations is the identification of additional immunogenic viral proteins expressed by tumor cells that may serve as tumor-associated antigens to be targeted by improved CTL induction and expansion protocols. We have recently demonstrated that NPC patients show strong spontaneous CD4+ and CD8+ T cell responses specific for the EBV-encoded oncogenic protein BARF1. We also showed that BARF1 provides immunogenic HLA-A*0201-restricted epitopes, suggesting that exploitation of the immunogenic features of this viral antigen may help improve the current immunotherapeutic strategies for EBV-associated malignancies. On these grounds, we characterized more extensively the immunogenic properties of BARF1 with the final goal to develop improved protocols of adoptive immunotherapy based on the use of EBV-CTLs enriched in BARF1-specific effectors. In particular, we identified and validated additional BARF1 CTL epitopes presented in the context of common HLA class I alleles. These results strictly correlate to those deriving from a high-resolution HLA genotyping of a large series of NPC, giving a precise estimate of the immunogenicity of BARF1 in relation to the HLA class I profile of Italian NPC patients. To fully exploit the immunologic properties of BARF1, we are also developing and characterizing BARF1-specific monoclonal antibodies that may be of both diagnostic and therapeutic usefulness in these clinical settings. In future perspective, the proposed research may provide a strong rationale for the clinical application of improved adoptive immunotherapy protocols for the treatment of EBV-associated malignancies, particularly the less immunogenic forms, such as NPC and, possibly, Hodgkin’s lymphoma
Conditional immortalization of human B cells by CD40 ligation
No full textIt is generally assumed that human differentiated cells have a limited life-span and proliferation capacity in vivo, and that genetic modifications are a prerequisite for their immortalization in vitro. Here we readdress this issue, studying the long-term proliferation potential of human B cells. It was shown earlier that human B cells from peripheral blood of healthy donors can be efficiently induced to proliferate for up to ten weeks in vitro by stimulating their receptor CD40 in the presence of interleukin-4. When we applied the same stimuli under conditions of modified cell number and culture size, we were surprised to find that our treatment induced B cells to proliferate throughout an observation period of presently up to 1650 days, representing more than 370 population doublings, which suggested that these B cells were immortalized in vitro. Long-term CD40-stimulated B cell cultures could be established from most healthy adult human donors. These B cells had a constant phenotype, were free from Epstein-Barr virus, and remained dependent on CD40 ligation. They had constitutive telomerase activity and stabilized telomere length. Moreover, they were susceptible to activation by Toll-like receptor 9 ligands, and could be used to expand antigen-specific cytotoxic T cells in vitro. Our results indicate that human somatic cells can evade senescence and be conditionally immortalized by external stimulation only, without a requirement for genetic manipulation or oncoviral infection. Conditionally immortalized human B cells are a new tool for immunotherapy and studies of B cell oncogenesis, activation, and function
The SSC of the Generalised Jahangir’s Graph Jm,k and its Algebraic Characterizations
No full textIn this article, we present important combinatorial and algebraicproperties of spanning simplicial complex (SSC) of the generalised Jahangir’sgraph Jm,k. We describe the relation to find f−vectors associatedto Δs(Jm,k) and determine the Hilbert series for the SR-ring KΔs(Jm,k).In the end, we present the associated primes of the facet ideal IF(Δs(Jm,k))and the Cohen-Macaulay characterization of the SR-ring of Δs(Jm,k).AMS (MOS) Subject Classification Codes: Primary 13-P10, Secondary 13-F20, 13-C14, 13-H10.Corresponding Author: Agha KashifKey Words: Simplicial Complexes, f-vectors, Spanning Trees, Face Ring, Hilbert Series, CohenMacaulay
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