RESUMO
Progressive multifocal leukoencephalopathy (PML)-derived noncoding control region (NCCR) sequences permitted greater early viral gene expression than kidney-associated NCCR sequences. This was driven in part by binding of the transcription factor Spi-B to unique PML-associated Spi-B binding sites. Spi-B is upregulated in developing B cells in response to natalizumab therapy, a known risk factor for PML. Naturally occurring JCV sequence variation, together with drug treatment-induced cellular changes, may synergize to create an environment leading to an increased risk of PML.
Assuntos
Expressão Gênica , Vírus JC/genética , Leucoencefalopatia Multifocal Progressiva/genética , Linfócitos/imunologia , Sequências Reguladoras de Ácido Nucleico , Proteínas de Ligação a DNA/biossíntese , Estudos de Associação Genética , Humanos , Dados de Sequência Molecular , Medição de Risco , Análise de Sequência de DNA , Fatores de Transcrição/biossínteseRESUMO
Progressive multifocal leukoencephalopathy (PML) induced by JC virus (JCV) is a risk for natalizumab-treated multiple sclerosis (MS) patients. Here we characterize the JCV-specific T cell responses in healthy donors and natalizumab-treated MS patients to reveal functional differences that may account for the development of natalizumab-associated PML. CD4 and CD8 T cell responses specific for all JCV proteins were readily identified in MS patients and healthy volunteers. The magnitude and quality of responses to JCV and cytomegalovirus (CMV) did not change from baseline through several months of natalizumab therapy. However, the frequency of T cells producing IL-10 upon mitogenic stimulation transiently increased after the first dose. In addition, MS patients with natalizumab-associated PML were distinguished from all other subjects in that they either had no detectable JCV-specific T cell response or had JCV-specific CD4 T cell responses uniquely dominated by IL-10 production. Additionally, IL-10 levels were higher in the CSF of individuals with recently diagnosed PML. Thus, natalizumab-treated MS patients with PML have absent or aberrant JCV-specific T cell responses compared with non-PML patients, and changes in T cell-mediated control of JCV replication may be a risk factor for developing PML. Our data suggest further approaches to improved monitoring, treatment and prevention of PML in natalizumab-treated patients.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunidade Celular/efeitos dos fármacos , Vírus JC/imunologia , Leucoencefalopatia Multifocal Progressiva/imunologia , Esclerose Múltipla/imunologia , Anticorpos Monoclonais Humanizados/efeitos adversos , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Citomegalovirus/imunologia , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Feminino , Humanos , Interleucina-10/imunologia , Leucoencefalopatia Multifocal Progressiva/tratamento farmacológico , Leucoencefalopatia Multifocal Progressiva/patologia , Leucoencefalopatia Multifocal Progressiva/virologia , Masculino , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/patologia , Esclerose Múltipla/virologia , Natalizumab , Fatores de RiscoRESUMO
JC virus (JCV) DNA in the cerebrospinal fluid (CSF) provides the laboratory confirmatory diagnosis of progressive multifocal leukoencephalopathy (PML) in patients whose clinical symptoms and magnetic resonance imaging findings are consistent with PML.The Laboratory of Molecular Medicine and Neuroscience (LMMN), National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), made the confirmatory laboratory diagnosis in 35 multiple sclerosis (MS) patients treated with natalizumab. Thirteen patients had 3 or more CSF samples taken from weeks to months following PML diagnosis. Seven of the 13 patients demonstrated persistence of JCV DNA in the CSF even though all patients experienced immune reconstitution inflammatory syndrome (IRIS), 11 patients had plasma exchange, and 2 had immunoabsorption. Specific anti-JCV antibody was measured in plasma/sera samples from 25 of the 35 patients. Most of the samples showed moderate to high or rising antibody levels from the time of PML diagnosis. However, plasma from 1 patient at or near the time of PML diagnosis had a titer considered seronegative and 2 other plasma samples from patients had titers considered at baseline for seropositivity. In several PML cases, viral persistence and neurological deficits have continued for several years, indicating that once initiated, JCV infection may not entirely clear, even with IRIS.
Assuntos
Anticorpos Monoclonais/uso terapêutico , DNA Viral/líquido cefalorraquidiano , Vírus JC/imunologia , Leucoencefalopatia Multifocal Progressiva/tratamento farmacológico , Leucoencefalopatia Multifocal Progressiva/etiologia , Esclerose Múltipla/complicações , Anticorpos/sangue , Anticorpos Monoclonais Humanizados , Variações do Número de Cópias de DNA/fisiologia , DNA Viral/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Leucoencefalopatia Multifocal Progressiva/metabolismo , Leucoencefalopatia Multifocal Progressiva/virologia , Estudos Longitudinais , Masculino , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/virologia , NatalizumabRESUMO
We compared effects of interferon-gamma (IFNgamma) on cyclooxygenase-2 (COX-2) expression in malignant human glioma cell lines and cultured primary human astrocytes. While IFNgamma inhibited interleukin-1beta (IL1beta)-induced expression of COX-2 in the glioma cells, it enhanced expression in primary astrocytes. This differential effect correlated with the observed modulation of NFkappaB and AP-1 DNA binding activity; reduced in the glioma cells, increased in primary astrocytes. Furthermore, IFNgamma had a significantly greater anti-proliferative effect on the glioma cells than COX inhibitors. This inhibitory effect of IFNgamma on expression of COX-2 in human glioma cells may have relevance for immunotherapies directed against high-grade gliomas.
Assuntos
Astrócitos/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Glioma/enzimologia , Interferon gama/farmacologia , Isoenzimas/biossíntese , Prostaglandina-Endoperóxido Sintases/biossíntese , Astrócitos/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Ciclo-Oxigenase 2 , Citocinas/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glioma/genética , Humanos , Mediadores da Inflamação/fisiologia , Interferon gama/fisiologia , Isoenzimas/genética , Proteínas de Membrana , Prostaglandina-Endoperóxido Sintases/genéticaRESUMO
IMPORTANCE: Infection with JC virus (JCV) may lead to development of demyelinating progressive multifocal leukoencephalopathy in patients with multiple sclerosis (MS) who are treated with natalizumab. OBJECTIVE: To determine whether mononuclear cells in circulation from MS patients treated with natalizumab harbor JCV DNA. DESIGN, SETTING, AND PARTICIPANTS: In this prospective investigation, we enrolled 49 MS patients from the Clinical Center for Multiple Sclerosis at The University of Texas Southwestern Medical Center and 18 healthy volunteers. We drew 120-mL blood samples from 26 MS patients at baseline and at approximately 3-month intervals to 10 months during the course of natalizumab infusions. One blood sample was drawn from 23 MS patients receiving natalizumab for more than 24 months and from 18 healthy volunteers. INTERVENTIONS: Natalizumab treatment of MS. MAIN OUTCOMES AND MEASURES: The blood samples were separated using flow cytometry into CD34+, CD19+, and CD3+ cell subsets; DNA templates were prepared using quantitative polymerase chain reaction for JCV DNA identification. Plasma samples were tested for anti-JCV antibodies by enzyme-linked immunosorbent assays performed at the Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological and Communicative Diseases and Stroke. RESULTS: Thirteen of the 26 patients (50%) with baseline and follow-up blood samples had detectable viral DNA in at least 1 cell compartment at 1 or more points. Ten of the 23 patients (44%) receiving treatment for more than 24 months and 3 of the 18 healthy volunteers (17%) also had detectable viral DNA in 1 or more cell compartment. Fifteen of the 49 MS patients (31%) were confirmed to harbor JCV in CD34+ cells and 12 of 49 (24%) in CD19+ cells. Only 1 of 18 healthy volunteers were viremic in CD34+ cells and none in CD19+ cells. Nine patients and 1 healthy volunteer were viremic but had seronegative test results for JCV antibodies. CONCLUSIONS AND RELEVANCE: JC virus DNA was detectable within cell compartments of natalizumab-treated MS patients after treatment inception and longer. JC virus DNA may harbor in CD34+ cells in bone marrow that mobilize into the peripheral circulation at high concentrations. Latently infected cells initiate differentiation to CD19+ cells that favors growth of JCV. These data link the mechanism of natalizumab treatment with progressive multifocal leukoencephalopathy.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antígenos CD19/sangue , Antígenos CD34/sangue , Vírus JC/metabolismo , Esclerose Múltipla/tratamento farmacológico , Animais , Antígenos CD19/genética , Antígenos CD34/genética , Aotidae , Linhagem Celular Tumoral , DNA Viral/sangue , Citometria de Fluxo/métodos , Seguimentos , Humanos , Leucócitos Mononucleares/virologia , Esclerose Múltipla/genética , Esclerose Múltipla/virologia , Natalizumab , Estudos ProspectivosRESUMO
BACKGROUND: JC virus (JCV) is the etiologic agent for progressive multifocal leukoencephalopathy (PML), a demyelinating disease occurring in the brain of patients with underlying immune compromised states. All viable JCV genomes contain a conserved region in the T protein coding nucleotide sequence that when detected by PCR in CSF is a confirmatory diagnostic marker for PML along with clinical and neuroradiological evidence. The non-coding regulatory region (NCRR) is hypervariable, as evidenced by nucleotide sequence of the non-virulent variant, which is predominantly excreted in urine, versus that of virulent variants found in brain and CSF of PML patients. All variants can be found in blood. OBJECTIVE: A single assay that quantifies and identifies JCV DNA in clinical samples and discriminates between variants has significant value to physicians and patients at risk for PML. STUDY DESIGN: Separate primer pairs were tested together to quantitatively detect conserved viral DNA nucleotide sequence in patient samples, while simultaneously detecting the NCRR specific for the non-virulent variant. RESULTS: In testing using control plasmids and patients' CSF, blood, and urine, PML patients predictably demonstrated the non-virulent, archetype NCRR in urine, but virulent NCRR variants in CSF and blood. CONCLUSION: The JCV qPCR multiplex assay targets two regions in JCV genomes to simultaneously identify and measure viral DNA, as well as distinguish between variants associated with PML and those that are not. The multiplex results could signal risk for PML if patients are viremic with JCV variants closely associated with PML pathogenesis.
Assuntos
Vírus JC/classificação , Vírus JC/isolamento & purificação , Leucoencefalopatia Multifocal Progressiva/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Virologia/métodos , DNA Viral/genética , DNA Viral/isolamento & purificação , Genótipo , Humanos , Vírus JC/genética , Vírus JC/patogenicidade , Leucoencefalopatia Multifocal Progressiva/virologia , VirulênciaRESUMO
JC virus (JCV) is a ubiquitous human polyomavirus that causes the demyelinating disease Progressive Multifocal Leukoencephalopathy (PML). JCV replicates in limited cell types in culture, predominantly in human glial cells. Following introduction of a replication defective SV40 mutant that expressed large T protein into a heterogeneous culture of human fetal brain cells, multiple phenotypes became immortalized (SVG cells). A subset of SVG cells could support JCV replication. In the current study, clonal cell lines were selected from the original SVG cell culture. The 5F4 clone showed low levels of viral growth. The 10B1 clone was highly permissive for JCV DNA replication and gene expression and supported persistent and stable JCV infection over months in culture. Microarray analysis revealed that viral infection did not significantly change gene expression in these cells. More resistant 5F4 cells expressed high levels of transcription factors known to inhibit JCV transcription. Interestingly, 5F4 cells expressed high levels of RNA of markers of radial glia and 10B1 cells had high expression of markers of immature glial cells and activation of transcription regulators important for stem/progenitor cell self-renewal. These SVG-derived clonal cell lines provide a biologically relevant model to investigate cell type differences in JCV host range and pathogenesis, as well as neural development. Several transcription regulators were identified which may be targets for therapeutic modulation of expression to abrogate JCV replication in PML patients. Additionally, these clonal cell lines can provide a consistent culture platform for testing therapies against JCV infection of the central nervous system.
Assuntos
Vírus JC/fisiologia , Neuroglia/virologia , Infecções por Polyomavirus/genética , Replicação Viral/genética , Linhagem Celular , Células Clonais , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TranscriptomaRESUMO
The multiplication of the human neurotropic polyomavirus JC virus (JCV) is regulated by cell membrane receptors and nuclear transcription factors. Signaling pathways also play a role in determining the extent to which JCV can productively infect cells. These data show that constitutively active MEK1 protein (CA-MEK1), overexpressed in cultures of human glia, supports a substantial increase in late JCV protein (Vp-1) synthesis. The specificity of this pathway was indicated by no significant enhancement of JCV multiplication through activation of other components of mitogen-activated protein kinase pathways such as p38, Jun N-terminal protein kinase, and protein kinase A. Further evidence supporting the importance of signaling in JCV infection came from addition of transforming growth factor beta1 (TGF-beta1), which stimulated a 200% increase of Vp-1 expression. Specific MEK1/2 inhibitors, flavenoid PD98059 and U0126, decreased the basal and TGF-beta1-stimulated Vp-1 expression by 95% or more. TGF-beta1 is known to phosphorylate/activate Smad DNA binding proteins that could subsequently bind or increase binding to JCV promoter sequences, linking the effects of signaling with JCV transcriptional regulation. The effectiveness with which MEK1/2 inhibitors block JCV multiplication provides insight that may contribute to development of compounds directed against JCV.
Assuntos
Vírus JC/metabolismo , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Fator de Crescimento Transformador beta1/metabolismo , Replicação Viral , Linhagem Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Regulação Viral da Expressão Gênica , Humanos , Transdução de Sinais , Células-Tronco/virologia , TransfecçãoRESUMO
The infectious cycle of the human polyomavirus JC (JCV) is ultimately regulated in cellular nuclei at the level of viral protein expression and genomic replication. Such activity is prompted by interactions between variant nucleotide sequences within the JCV regulatory region (promoter) and cellular transcription factors that bind specific DNA consensus sites. In previous work we identified an NF-1 class member, NF-1X, as a critical transcription factor affecting the JCV cellular host range. Within variant JCV promoters, as well as other viral and cellular promoters, adjacently located NF-1 and AP-1 consensus sites are often found. The close proximity of these two binding sites suggests the opportunity for interaction between NF-1 and AP-1 proteins. Here, by electrophoretic mobility shift assays, we show temporal and dose-dependent interference by an AP-1 family member, c-Jun, upon NF-1 proteins binding an NF-1 consensus site derived from JCV promoter sequence. Moreover, as demonstrated by protein-protein interaction assays, we identify specific binding affinity independent of DNA binding between NF-1X and c-Jun. Finally, to compare the binding profiles of NF-1X and c-Jun on JCV promoter sequence in parallel with in vivo detection of viral activity levels, we developed an anchored transcriptional promoter (ATP) assay. With use of extracts from JCV-infected cells transfected to overexpress either NF-1X or c-Jun, ATP assays showed concurrent increases in NF-1X binding and viral protein expression. Conversely, increased c-Jun binding accompanied decreases in both NF-1X binding and viral protein expression. Therefore, inhibition of NF-1X binding by c-Jun appears to play a role in regulating levels of JCV activity.