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ABSTRACT: The polyomavirus JC (JCV) causes the demyelinating disease progressive multifocal leukoencephalopathy (PML). Infection by JCV is very common in childhood after which the virus enters a latent state, which is poorly understood. Under conditions of severe immunosuppression, especially AIDS, JCV may reactivate to cause PML. Expression of JC viral proteins is regulated by the JCV non-coding control region (NCCR), which contains an NF-kappaB binding site previously shown to activate transcription. We now report that C/EBPbeta inhibits basal and NF-kappaB-stimulated JCV transcription via the same site. Gel shift analysis showed C/EBPbeta bound to this region in vitro and ChIP assays confirmed this binding in vivo. Further, a ternary complex of NF-kappaB/p65, C/EBPbeta-LIP and JCV DNA could be detected in co-immunoprecipitation experiments. Mutagenesis analysis of the JCV NCCR indicated p65 and C/EBPbeta-LIP bound to adjacent but distinct sites and that both sites regulate basal and p65-stimulated transcription. Thus C/EBPbeta negatively regulates JCV, which together with NF-kappaB activation, may control the balance between JCV latency and activation leading to PML. This balance may be regulated by proinflammatory cytokines in the brain.
Virus Research 09/2009; 146(1-2):97-106. · 2.94 Impact Factor
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ABSTRACT: JC virus (JCV) is a human polyomavirus that can emerge from a latent state to cause the cytolytic destruction of oligodendrocytes in the brain resulting in the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Previous studies described a cis-acting transcriptional regulatory element in the JCV non-coding control region (NCCR) that is involved in the response of JCV to cytokines. This consists of a 23 base pair GGA/C rich sequence (GRS) near the replication origin (5112 to +4) that contains potential binding sites for Sp1 and Egr-1. Gel shift analysis showed that Egr-1, but not Sp1, bound to GRS. Evidence is presented that the GRS gel shift seen on cellular stimulation is due to Egr-1. Thus, TPA-induced GRS gel shift could be blocked by antibody to Egr-1. Further, the TPA-induced GRS DNA/protein complex was isolated and found to contain Egr-1 by Western blot. No other Egr-1 sites were found in the JCV NCCR. Functionally, Egr-1 was found to stimulate transcription of JCV late promoter but not early promoter reporter constructs. Mutation of the Egr-1 site abrogated Egr-1 binding and virus with the mutated Egr-1 site showed markedly reduced VP1 expression and DNA replication. Infection of primary astrocytes by wild-type JCV induced Egr-1 nuclear expression that was maximal at 5-10 days post-infection. Finally, upregulation of Egr-1 was detected in PML by immunohistochemistry. These data suggest that Egr-1 induction may be important in the life cycle of JCV and PML pathogenesis.
Virology 07/2008; 375(2):331-41. · 3.35 Impact Factor
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ABSTRACT: The unfavorable clinical evolution in indolent non-Hodgkin lymphomas suggests defective control of neoplastic growth by the immune system. To address this issue, we evaluated phenotype, function, and maturation profile of CD4(+) and CD8(+) T cells from peripheral-blood, lymph nodes, or bone marrow of patients with B-cell non-Hodgkin lymphoma (NHL) at diagnosis. T cells from these patients frequently showed an activated but apoptosis-prone phenotype with low frequency of tumor-reactive T cells showing a TH2/Tc2 functional profile in the response to autologous tumor. In peripheral blood or in lymph nodes and bone marrow, and, in comparison to healthy donors, patients' T cells showed a skewed differentiation toward Tnaive and Tcentral memory stages, with low expression of granzyme B and perforin. T-cell culture with autologous tumor in the presence of IL-2, IL-15, and autologous bone marrow-derived cells led to massive T-cell expansion and to differentiation of cytotoxic factor(+) CD8(+) T cells releasing IFN-gamma and killing autologous B-cell tumor in an HLA-class I-restricted fashion. These results suggest impaired T-cell differentiation to effector stage in patients with B-cell NHL, but indicate that T-cell responsiveness to gammac cytokines is retained, thus allowing to promote generation of antitumor T cells for immune intervention.
Blood 02/2006; 107(2):602-9. · 9.90 Impact Factor
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Massimo Di Nicola,
Sara Napoli,
Andrea Anichini,
Roberta Mortarini, Luca Romagnoli,
Michele Magni,
Paola Matteucci,
Paola Baldassarri,
Ilaria Bersani,
Carmelo Carlo-Stella,
Alessandro M Gianni
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ABSTRACT: Dendritic cells (DC) primed with tumor antigens can effectively mediate the regression of a variety of established solid malignancies in both murine and human models. Several experimental studies indicate that apoptotic bodies are an optimal source of tumor antigens for ex vivo priming of DC. However, the clinical use of killed tumor cells as a source of antigens will require an optimal methodology to induce effective tumor cell apoptosis.
The goal of this study was to compare the efficiency of three agents for inducing neoplastic B lymphocyte apoptosis; staurosporine, infection by modified vaccinia (MVA) viral particles and ultraviolet C (UVC) radiation.
The three methods were finely tuned to induce apoptosis in more than 90% of tumor cells after 24 hours of exposure. However, the viability of monocyte-derived DC, loaded with B-cell tumor apoptotic bodies induced by staurosporine or MVA viral particles, decreased dramatically within 48 hours after phagocytosis of the killed neoplastic cells. The persistence of the apoptosis-inducing agents in the apoptotic bodies and not in the tumor supernatant, was responsible for the observed damage to DC viability. In contrast, DC viability was not affected after uptake of tumor cells killed through UVC-irradiation. Furthermore, B-lymphoblastic cell line (LCL)-specific T cells were reactivated by DC loaded with apoptotic bodies induced by UVC-rays.
Since the method used to induce tumor cell apoptosis might be detrimental to DC viability, these findings should be considered when designing anticancer vaccination programs.
Haematologica 01/2004; 88(12):1396-404. · 6.42 Impact Factor
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ABSTRACT: The polyomavirus JC (JCV) causes the demyelinating disease progressive multifocal leukoencephalopathy (PML). Infection by JCV is very common in childhood after which the virus enters a latent state, which is poorly understood. Under conditions of severe immunosuppression, especially AIDS, JCV may reactivate to cause PML. Expression of JC viral proteins is regulated by the JCV non-coding control region (NCCR), which contains an NF-κB binding site previously shown to activate transcription. We now report that C/EBPβ inhibits basal and NF-κB-stimulated JCV transcription via the same site. Gel shift analysis showed C/EBPβ bound to this region in vitro and ChIP assays confirmed this binding in vivo. Further, a ternary complex of NF-κB/p65, C/EBPβ-LIP and JCV DNA could be detected in co-immunoprecipitation experiments. Mutagenesis analysis of the JCV NCCR indicated p65 and C/EBPβ-LIP bound to adjacent but distinct sites and that both sites regulate basal and p65-stimulated transcription. Thus C/EBPβ negatively regulates JCV, which together with NF-κB activation, may control the balance between JCV latency and activation leading to PML. This balance may be regulated by proinflammatory cytokines in the brain.
Virus Research.
