RESUMO
Progressive multifocal leukoencephalopathy (PML) is a frequent neurological complication in immunosuppressed patients. PML is caused by the JC virus (JCV), a neurotropic DNA polyomavirus that infects oligodendrocytes and astrocytes, causing inflammation and demyelination which lead to neurological dysfunction. The pathogenesis of PML is poorly understood due to the lack of in vitro or animal models to study mechanisms of disease as the virus most efficiently infects only human cells. We developed a human-derived brain organotypic system (also called brain organoid) to model JCV infection. The model was developed by using human-induced pluripotent stem cells (iPSC) and culturing them in 3D to generate an organotypic model containing neurons, astrocytes, and oligodendrocytes which recapitulates aspects of the environment of the human brain. We infected the brain organoids with the JCV MAD4 strain or cerebrospinal fluid of a patient with PML. The organoids were assessed for evidence of infection by qPCR, immunofluorescence, and electron microscopy at 1, 2, and 3 weeks post-exposure. JCV infection in both JCV MAD4 strain and PML CSF-exposed brain organoids was confirmed by immunocytochemical studies demonstrating viral antigens and electron microscopy showing virion particles in the nuclear compartment of oligodendrocytes and astrocytes. No evidence of neuronal infection was visualized. Infection was also demonstrated by JCV qPCR in the virus-exposed organoids and their media. In conclusion, the brain organoid model of JCV infection establishes a human model suitable for studying the mechanisms of JCV infection and pathogenesis of PML and may facilitate the exploration of therapeutic approaches.
Assuntos
Vírus JC , Leucoencefalopatia Multifocal Progressiva , Infecções por Polyomavirus , Animais , Encéfalo , DNA Viral/genética , Humanos , Vírus JC/genética , Organoides/patologia , Infecções por Polyomavirus/genéticaRESUMO
Multiple sclerosis (MS) is a progressive inflammatory demyelinating disease of the CNS of unknown cause that remains incurable. Inflammasome-associated caspases mediate the maturation and release of the proinflammatory cytokines IL-1ß and IL-18 and activate the pore-forming protein gasdermin D (GSDMD). Inflammatory programmed cell death, pyroptosis, was recently shown to be mediated by GSDMD. Here, we report molecular evidence for GSDMD-mediated inflammasome activation and pyroptosis in both myeloid cells (macrophages/microglia) and, unexpectedly, in myelin-forming oligodendrocytes (ODCs) in the CNS of patients with MS and in the MS animal model, experimental autoimmune encephalomyelitis (EAE). We observed inflammasome activation and pyroptosis in human microglia and ODCs in vitro after exposure to inflammatory stimuli and demonstrate caspase-1 inhibition by the small-molecule inhibitor VX-765 in both cell types. GSDMD inhibition by siRNA transduction suppressed pyroptosis in human microglia. VX-765 treatment of EAE animals reduced the expression of inflammasome- and pyroptosis-associated proteins in the CNS, prevented axonal injury, and improved neurobehavioral performance. Thus, GSDMD-mediated pyroptosis in select glia cells is a previously unrecognized mechanism of inflammatory demyelination and represents a unique therapeutic opportunity for mitigating the disease process in MS and other CNS inflammatory diseases.
Assuntos
Caspase 1/metabolismo , Inibidores de Caspase/farmacologia , Dipeptídeos/farmacologia , Modelos Biológicos , Esclerose Múltipla/enzimologia , Oligodendroglia/enzimologia , Piroptose/efeitos dos fármacos , para-Aminobenzoatos/farmacologia , Células Cultivadas , Humanos , Esclerose Múltipla/patologia , Oligodendroglia/patologiaRESUMO
Accumulating evidence corroborates the role of the "central vein sign" in the radiological diagnosis of multiple sclerosis (MS). Here, we report human magnetic resonance imaging (MRI) and corresponding pathological data that inflammation-dependent intracerebral remodeling of the vessel wall is directly associated with the prominence of intralesional veins on susceptibility-based MRI. In adult marmosets with experimental autoimmune encephalomyelitis, vessel-wall fibrosis was detected early in the demyelinating process, even in lesions <2 weeks old, though fibrosis was more evident after 6 weeks. Vascular remodeling consisted of both luminal enlargement and eccentric thickening of the perivascular space (fibrillar collagen type I deposition) and affected almost exclusively white matter, but not subpial cortical, lesions. The long-term effect of vessel remodeling in MS lesions is currently unknown, but it might potentially affect tissue repair. ANN NEUROL 2019;85:934-942.
Assuntos
Veias Cerebrais/química , Veias Cerebrais/diagnóstico por imagem , Doenças Desmielinizantes/diagnóstico por imagem , Colágenos Fibrilares/análise , Colágenos Fibrilares/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Callithrix , Veias Cerebrais/metabolismo , Doenças Desmielinizantes/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: JC virus (JCV) infection is a lytic infection of oligodendrocytes in progressive multifocal leukoencephalopathy; less common forms of central nervous system manifestations associated with JCV infection include granule cell neuronopathy, encephalopathy, and meningitis. Presented is the first case of fatal JCV encephalopathy after immunosuppressive therapy that included ruxolitinib. METHODS: Postmortem analysis included next generation sequencing, Sanger sequencing, tissue immunohistochemistry, and formalin-fixed hemisphere 7T magnetic resonance imaging. RESULTS: JCV DNA isolated from postmortem tissue samples identified a novel 12bp insertion that altered the transcription site binding pattern in an otherwise "wild-type virus," which has long been thought to be the nonpathogenic form of JCV. Anti-VP1 staining demonstrated infection in cortical neurons, hippocampal neurons, and glial and endothelial cells. INTERPRETATION: This expands the spectrum of identified JCV diseases associated with broad-spectrum immunosuppression, including JAK-STAT inhibitors, and sheds light on an additional neurotropic virus strain of the archetype variety. ANN NEUROL 2019;86:878-884.
Assuntos
Encefalopatias/tratamento farmacológico , Encefalopatias/genética , Vírus JC/genética , Janus Quinases/genética , Pirazóis/uso terapêutico , Adolescente , Sequência de Bases , Encefalopatias/diagnóstico por imagem , Evolução Fatal , Feminino , Humanos , Vírus JC/isolamento & purificação , Nitrilas , PirimidinasRESUMO
Neuroinflammatory diseases such as multiple sclerosis are characterized by infiltration of lymphocytes into the central nervous system followed by demyelination and axonal degeneration. While evidence suggests that activated T lymphocytes induce neurotoxicity and impair function of neural stem cells, the effect of T cells on oligodendrocyte progenitor cells (OPCs) is still uncertain, partly due to the difficulty in obtaining human OPCs. Here we studied the effect of activated T cells on OPCs using OPCs derived from human hematopoietic stem cells or from human fetal brain. OPCs were exposed to supernatants (sups) from activated T cells. Cell proliferation was determined by EdU incorporation and CellQuanti-Blue assays. Surprisingly, we found that sups from activated T cells induced OPC proliferation by regulating cell cycle progression. Vascular endothelial growth factor A (VEGF-A) transcripts were increased in T cells after activation. Immunodepletion of VEGF-A from activated T cell sups significantly attenuated its effect on OPC proliferation. Furthermore, VEGF receptor 2 (VEGFR2) was expressed on OPCs and its inhibition also attenuated activated T cell-induced OPC proliferation. Thus, activated T cells have a trophic role by promoting OPC proliferation via the VEGFR2 pathway.
Assuntos
Proliferação de Células/fisiologia , Citocinas/metabolismo , Células Precursoras de Oligodendrócitos/fisiologia , Regulação para Cima/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Encéfalo/citologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feto/anatomia & histologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Células Precursoras de Oligodendrócitos/efeitos dos fármacos , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transfecção , Regulação para Cima/efeitos dos fármacos , Ureia/análogos & derivados , Ureia/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Viral infections of the central nervous system (CNS) are of increasing concern, especially among immunocompromised populations. Rodent models are often inappropriate for studies of CNS infection, as many viruses, including JC virus (JCV) and HIV, cannot replicate in rodent cells. Consequently, human fetal brain-derived multipotential CNS progenitor cells (NPCs) that can be differentiated into neurons, oligodendrocytes, or astrocytes have served as a model in CNS studies. NPCs can be nonproductively infected by JCV, while infection of progenitor-derived astrocytes (PDAs) is robust. We profiled cellular gene expression at multiple times during differentiation of NPCs to PDAs. Several activated transcription factors show commonality between cells of the brain, in which JCV replicates, and lymphocytes, in which JCV is likely latent. Bioinformatic analysis determined transcription factors that may influence the favorable transcriptional environment for JCV in PDAs. This study attempts to provide a framework for understanding the functional transcriptional profile necessary for productive JCV infection.
Assuntos
Astrócitos/citologia , Diferenciação Celular , Vírus JC/fisiologia , Células-Tronco Multipotentes/citologia , Neurônios/citologia , Fatores de Transcrição/metabolismo , Infecções Tumorais por Vírus/metabolismo , Infecções Tumorais por Vírus/virologia , Astrócitos/metabolismo , Astrócitos/virologia , Encéfalo/citologia , Encéfalo/metabolismo , Linhagem Celular , Células Cultivadas , Humanos , Vírus JC/genética , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/virologia , Neurônios/metabolismo , Neurônios/virologia , Fatores de Transcrição/genética , Infecções Tumorais por Vírus/genéticaRESUMO
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
Epstein-Barr virus (EBV) is an aetiologic risk factor for the development of multiple sclerosis (MS). However, the role of EBV-infected B cells in the immunopathology of MS is not well understood. Here we characterized spontaneous lymphoblastoid cell lines (SLCLs) isolated from MS patients and healthy controls (HC) ex vivo to study EBV and host gene expression in the context of an individual's endogenous EBV. SLCLs derived from MS patient B cells during active disease had higher EBV lytic gene expression than SLCLs from MS patients with stable disease or HCs. Host gene expression analysis revealed activation of pathways associated with hypercytokinemia and interferon signalling in MS SLCLs and upregulation of forkhead box protein 1 (FOXP1), which contributes to EBV lytic gene expression. We demonstrate that antiviral approaches targeting EBV replication decreased cytokine production and autologous CD4+ T cell responses in this ex vivo model. These data suggest that dysregulation of intrinsic B cell control of EBV gene expression drives a pro-inflammatory, pathogenic B cell phenotype that can be attenuated by suppressing EBV lytic gene expression.
Assuntos
Linfócitos B , Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Esclerose Múltipla , Humanos , Herpesvirus Humano 4/genética , Esclerose Múltipla/virologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/virologia , Infecções por Vírus Epstein-Barr/virologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/complicações , Citocinas/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD4-Positivos/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Transcriptoma , Replicação Viral , Regulação Viral da Expressão Gênica , Linhagem Celular , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Perfilação da Expressão Gênica , Adulto , Feminino , MasculinoRESUMO
Wnt/ß-catenin is a neuroprotective pathway regulating cell fate commitment in the CNS and many vital functions of neurons and glia. Its dysregulation is linked to a number of neurodegenerative diseases. Wnt/ß-catenin is also a repressor of HIV transcription in multiple cell types, including astrocytes, which are dysregulated in HIV-associated neurocognitive disorder. Given that HIV proteins can overcome host restriction factors and that perturbations of Wnt/ß-catenin signaling can compromise astrocyte function, we evaluated the impact of HIV transactivator of transcription (Tat) on Wnt/ß-catenin signaling in astrocytes. HIV clade B Tat, in primary progenitor-derived astrocytes and U87MG cells, inhibited Wnt/ß-catenin signaling as demonstrated by its inhibition of active ß-catenin, TOPflash reporter activity, and Axin-2 (a downstream target of Wnt/ß-catenin signaling). Point mutations in either the core region (K41A) or the cysteine-rich region (C30G) of Tat abrogated its ability to inhibit ß-catenin signaling. Clade C Tat, which lacks the dicysteine motif, did not alter ß-catenin signaling, confirming that the dicysteine motif is critical for Tat inhibition of ß-catenin signaling. Tat coprecipitated with TCF-4 (a transcription factor that partners with ß-catenin), suggesting a physical interaction between these two proteins. Furthermore, knockdown of ß-catenin or TCF-4 enhanced docking of Tat at the TAR region of the HIV long terminal repeat. These findings highlight a bidirectional interference between Tat and Wnt/ß-catenin that negatively impacts their cognate target genes. The consequences of this interaction include alleviation of Wnt/ß-catenin-mediated suppression of HIV and possible astrocyte dysregulation contributing to HIV neuropathogenesis.
Assuntos
Complexo AIDS Demência/patologia , Astrócitos/fisiologia , HIV-1/enzimologia , Transdução de Sinais/fisiologia , Proteínas Wnt/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Produtos do Gene rev do Vírus da Imunodeficiência Humana/fisiologia , Western Blotting , Linhagem Celular , Cisteína/fisiologia , Citometria de Fluxo , Produtos do Gene tat/fisiologia , Genes Reporter/genética , Ácido Glutâmico/metabolismo , HIV-1/genética , Humanos , Imunoprecipitação , Luciferases/metabolismo , Plasmídeos/genética , Mutação Puntual/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genéticaRESUMO
Epidemiological studies have demonstrated that Epstein-Barr virus (EBV) is a known etiologic risk factor, and perhaps prerequisite, for the development of MS. EBV establishes life-long latent infection in a subpopulation of memory B cells. Although the role of memory B cells in the pathobiology of MS is well established, studies characterizing EBV-associated mechanisms of B cell inflammation and disease pathogenesis in EBV (+) B cells from MS patients are limited. Accordingly, we analyzed spontaneous lymphoblastoid cell lines (SLCLs) from multiple sclerosis patients and healthy controls to study host-virus interactions in B cells, in the context of an individual's endogenous EBV. We identify differences in EBV gene expression and regulation of both viral and cellular genes in SLCLs. Our data suggest that EBV latency is dysregulated in MS SLCLs with increased lytic gene expression observed in MS patient B cells, especially those generated from samples obtained during "active" disease. Moreover, we show increased inflammatory gene expression and cytokine production in MS patient SLCLs and demonstrate that tenofovir alafenamide, an antiviral that targets EBV replication, decreases EBV viral loads, EBV lytic gene expression, and EBV-mediated inflammation in both SLCLs and in a mixed lymphocyte assay. Collectively, these data suggest that dysregulation of EBV latency in MS drives a pro-inflammatory, pathogenic phenotype in memory B cells and that this response can be attenuated by suppressing EBV lytic activation. This study provides further support for the development of antiviral agents that target EBV-infection for use in MS.
RESUMO
BACKGROUND AND OBJECTIVES: Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that establishes lifelong latency in memory B cells and has been identified as a major risk factor of multiple sclerosis (MS). B cell depletion therapies have disease-modifying benefit in MS. However, it is unclear whether this benefit is partly attributable to the elimination of EBV+ B cells. Currently, there are no EBV-specific antiviral therapies available for targeting EBV latent infection in MS and limited experimental models to study EBV in MS. METHODS: In this study, we describe the establishment of spontaneous lymphoblastoid cell lines (SLCLs) generated ex vivo with the endogenous EBV of patients with MS and controls and treated with either an Epstein-Barr virus nuclear antigen 1 (EBNA1) inhibitor (VK-1727) or cladribine, a nucleoside analog that eliminates B cells. RESULTS: We showed that a small molecule inhibitor of EBNA1, a critical regulator of the EBV life cycle, blocks the proliferation and metabolic activity of these SLCLs. In contrast to cladribine, a highly cytotoxic B cell depleting therapy currently used in MS, the EBNA1 inhibitor VK-1727 was cytostatic rather than cytotoxic and selective for EBV+ cells, while having no discernible effects on EBV- cells. We validate that VK-1727 reduces EBNA1 DNA binding at known viral and cellular sites by ChIP-qPCR. DISCUSSION: This study shows that patient-derived SLCLs provide a useful tool for interrogating the role of EBV+ B cells in MS and suggests that a clinical trial testing the effect of EBNA1 inhibitors in MS may be warranted.
Assuntos
Infecções por Vírus Epstein-Barr , Esclerose Múltipla , Humanos , Linhagem Celular , Proliferação de Células , Cladribina/farmacologia , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Infecções por Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr , Herpesvirus Humano 4 , Estudos de Casos e ControlesRESUMO
Background and objectives: Extracellular vesicles and particles (EVPs) are released from virtually all cell types, and may package many inflammatory factors and, in the case of infection, viral components. As such, EVPs can play not only a direct role in the development and progression of disease but can also be used as biomarkers. Here, we characterized immune signatures of EVPs from the cerebrospinal fluid (CSF) of individuals with HTLV-1-associated myelopathy (HAM), other chronic neurologic diseases, and healthy volunteers (HVs) to determine potential indicators of viral involvement and mechanisms of disease. Methods: We analyzed the EVPs from the CSF of HVs, individuals with HAM, HTLV-1-infected asymptomatic carriers (ACs), and from patients with a variety of chronic neurologic diseases of both known viral and non-viral etiologies to investigate the surface repertoires of CSF EVPs during disease. Results: Significant increases in CD8+ and CD2+ EVPs were found in HAM patient CSF samples compared to other clinical groups (p = 0.0002 and p = 0.0003 compared to HVs, respectively, and p = 0.001 and p = 0.0228 compared to MS, respectively), consistent with the immunopathologically-mediated disease associated with CD8+ T-cells in the central nervous system (CNS) of HAM patients. Furthermore, CD8+ (p < 0.0001), CD2+ (p < 0.0001), CD44+ (p = 0.0176), and CD40+ (p = 0.0413) EVP signals were significantly increased in the CSF from individuals with viral infections compared to those without. Discussion: These data suggest that CD8+ and CD2+ CSF EVPs may be important as: 1) potential biomarkers and indicators of disease pathways for viral-mediated neurological diseases, particularly HAM, and 2) as possible meditators of the disease process in infected individuals.
Assuntos
Vesículas Extracelulares , Doenças do Sistema Nervoso , Paraparesia Espástica Tropical , Humanos , Sistema Nervoso Central , Antígenos CD40 , Doença CrônicaRESUMO
Background: Lytic infection of oligodendrocytes by the human JC polyomavirus (JCPyV) results in the demyelinating disease called progressive multifocal leukoencephalopathy (PML). The detection of viral DNA in the cerebrospinal fluid (CSF) by PCR is an important diagnostic tool and, in conjunction with defined radiological and clinical features, can provide diagnosis of definite PML, avoiding the need for brain biopsy. The main aim of this study is to compare the droplet digital PCR (ddPCR) assay with the gold standard quantitative PCR (qPCR) for the quantification of JC viral loads in clinical samples. Methods: A total of 62 CSF samples from 31 patients with PML were analyzed to compare the qPCR gold standard technique with ddPCR to detect conserved viral DNA sequences in the JCPyV genome. As part of the validation process, ddPCR results were compared to qPCR data obtained in 42 different laboratories around the world. In addition, the characterization of a novel triplex ddPCR to detect viral DNA sequence from both prototype and archetype variants and a cellular housekeeping reference gene is described. Triplex ddPCR was used to analyze the serum from six PML patients and from three additional cohorts, including 20 healthy controls (HC), 20 patients with multiple sclerosis (MS) who had never been treated with natalizumab (no-NTZ-treated), and 14 patients with MS who were being treated with natalizumab (NTZ-treated); three from this last group seroconverted during the course of treatment with natalizumab. Results: JCPyV DNA was detected only by ddPCR for 5 of the 62 CSF samples (8%), while remaining undetected by qPCR. For nine CSF samples (15%), JCPyV DNA was at the lower limit of quantification for qPCR, set at <250 copies/mL, and therefore no relative quantitation could be determined. By contrast, exact copies of JCPyV for each of these samples were quantified by ddPCR. No differences were observed between qPCR and ddPCR when five standardized plasma samples were analyzed for JCPyV in 42 laboratories in the United States and Europe. JCPyV-DNA was undetected in all the sera from HC and MS cohorts tested by triplex ddPCR, while serum samples from six patients with PML tested positive for JCPyV. Conclusion: This study shows strong correlation between ddPCR and qPCR with increased sensitivity of the ddPCR assay. Further work will be needed to determine whether multiplex ddPCR can be useful to determine PML risk in natalizumab-treated MS patients.
Assuntos
Vírus JC , Leucoencefalopatia Multifocal Progressiva , Esclerose Múltipla , DNA Viral/genética , Humanos , Vírus JC/genética , Leucoencefalopatia Multifocal Progressiva/diagnóstico , Leucoencefalopatia Multifocal Progressiva/tratamento farmacológico , Esclerose Múltipla/tratamento farmacológico , Natalizumab/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Carga ViralRESUMO
Background: Progressive multifocal leukoencephalopathy (PML) is a rare and often lethal brain disorder caused by the common, typically benign polyomavirus 2, also known as JC virus (JCV). In a small percentage of immunosuppressed individuals, JCV is reactivated and infects the brain, causing devastating neurological defects. A wide range of immunosuppressed groups can develop PML, such as patients with: HIV/AIDS, hematological malignancies (e.g., leukemias, lymphomas, and multiple myeloma), autoimmune disorders (e.g., psoriasis, rheumatoid arthritis, and systemic lupus erythematosus), and organ transplants. In some patients, iatrogenic (i.e., drug-induced) PML occurs as a serious adverse event from exposure to immunosuppressant therapies used to treat their disease (e.g., hematological malignancies and multiple sclerosis). While JCV infection and immunosuppression are necessary, they are not sufficient to cause PML. Methods: We hypothesized that patients may also have a genetic susceptibility from the presence of rare deleterious genetic variants in immune-relevant genes (e.g., those that cause inborn errors of immunity). In our prior genetic study of 184 PML cases, we discovered 19 candidate PML risk variants. In the current study of another 152 cases, we validated 4 of 19 variants in both population controls (gnomAD 3.1) and matched controls (JCV+ multiple sclerosis patients on a PML-linked drug ≥ 2 years). Results: The four variants, found in immune system genes with strong biological links, are: C8B, 1-57409459-C-A, rs139498867; LY9 (alias SLAMF3), 1-160769595-AG-A, rs763811636; FCN2, 9-137779251-G-A, rs76267164; STXBP2, 19-7712287-G-C, rs35490401. Carriers of any one of these variants are shown to be at high risk of PML when drug-exposed PML cases are compared to drug-exposed matched controls: P value = 3.50E-06, OR = 8.7 [3.7-20.6]. Measures of clinical validity and utility compare favorably to other genetic risk tests, such as BRCA1 and BRCA2 screening for breast cancer risk and HLA-B*15:02 pharmacogenetic screening for pharmacovigilance of carbamazepine to prevent Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis. Conclusion: For the first time, a PML genetic risk test can be implemented for screening patients taking or considering treatment with a PML-linked drug in order to decrease the incidence of PML and enable safer use of highly effective therapies used to treat their underlying disease.
RESUMO
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
BACKGROUND: Xenotropic Murine Leukemia Virus-related (XMRV) virus is a recently identified mouse gammaretrovirus that has the ability to infect certain human cells. In this study, we investigated the susceptibility of primary neuronal cell types to infection with XMRV. FINDINGS: We observed that the human primary progenitors, progenitor-derived neurons, and progenitor-derived astrocytes supported XMRV multiplication. Interestingly, both progenitors and progenitor-derived neurons were more susceptible compared with progenitor-derived astrocytes. In addition, XMRV-infected Jurkat cells were able to transmit infection to neuronal cells. CONCLUSIONS: These data suggest that neuronal cells are susceptible for XMRV infection.
Assuntos
Astrócitos/virologia , Suscetibilidade a Doenças , Células Jurkat/virologia , Células-Tronco Neurais/virologia , Neurônios/virologia , Infecções por Retroviridae/virologia , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/genética , Animais , Astrócitos/citologia , Diferenciação Celular , Humanos , Imuno-Histoquímica , Células Jurkat/citologia , Masculino , Camundongos , Células-Tronco Neurais/citologia , Neurônios/citologia , Cultura Primária de Células , Neoplasias da Próstata/virologia , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Retroviridae/imunologia , Infecções por Retroviridae/metabolismo , Infecções por Retroviridae/transmissão , Células Tumorais Cultivadas , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/metabolismo , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/patogenicidadeRESUMO
Erythro-myeloid progenitors (EMP) are found in a population of cells expressing CD31 and CD45 markers (CD31+CD45+). A recent study indicated that EMPs persist until adulthood and can be a source of endothelial cells. We identified two sub-populations of EMP cells, CD31lowCD45low and CD31highCD45+, from peripheral blood that can differentiate into cells of erythroid lineage. Our novel findings add to the current knowledge of hematopoietic lineage commitment, and our sequential, dual-step, in vitro culture model provides a platform for the study of the molecular and cellular mechanisms underlying human hematopoiesis and erythroid differentiation.
Assuntos
Células Endoteliais , Sistema Hematopoético , Adulto , Diferenciação Celular , Células Eritroides , Hematopoese , HumanosRESUMO
In this study, we examined and characterized disease-specific TCR signatures in cerebrospinal fluid (CSF) of patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). TCR ß libraries using unique molecular identifier-based methodologies were sequenced in paired peripheral blood mononuclear cells (PBMCs) and CSF cells from HAM/TSP patients and normal healthy donors (NDs). The sequence analysis demonstrated that TCR ß repertoires in CSF of HAM/TSP patients were highly expanded and contained both TCR clonotypes shared with PBMCs and uniquely enriched within the CSF. In addition, we analyzed TCR ß repertoires of highly expanded and potentially immunopathologic HTLV-1 Tax11-19-specific CD8+ T cells from PBMCs of HLA-A*0201+ HAM/TSP and identified a conserved motif (PGLAG) in the CDR3 region. Importantly, TCR ß clonotypes of expanded clones in HTLV-1 Tax11-19-specific CD8+ T cells were also expanded and enriched in the CSF of the same patient. These results suggest that exploring TCR repertoires of CSF and antigen-specific T cells may provide a TCR repertoire signature in virus-associated neurologic disorders.
Assuntos
Doenças do Sistema Nervoso/imunologia , Paraparesia Espástica Tropical/líquido cefalorraquidiano , Paraparesia Espástica Tropical/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T CD8-Positivos , Células Clonais , Infecções por HTLV-I , Vírus Linfotrópico T Tipo 1 Humano , Humanos , Leucócitos Mononucleares , Doenças do Sistema Nervoso/virologia , Paraparesia Espástica Tropical/sangue , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/sangueRESUMO
BACKGROUND: Progressive multifocal leukoencephalopathy, a rare disease of the CNS caused by JC virus and occurring in immunosuppressed people, is typically fatal unless adaptive immunity is restored. JC virus is a member of the human polyomavirus family and is closely related to the BK virus. We hypothesised that use of partly HLA-matched donor-derived BK virus-specific T cells for immunotherapy in progressive multifocal leukoencephalopathy would be feasible and safe. METHODS: We did an open-label, single-cohort pilot study in patients (aged 18 years or older) with clinically definite progressive multifocal leukoencephalopathy and disease progression in the previous month at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Overlapping peptide libraries derived from large T antigen and major capsid protein VP1 of BK virus with high sequence homology to JC virus counterparts were used to generate polyomavirus-specific T cells cross-recognising JC virus antigens. Polyomavirus-specific T cells were manufactured from peripheral blood mononuclear cells of first-degree relative donors aged 18 years or older. These cells were administered to patients by intravenous infusion at 1â×â106 polyomavirus-specific T cells per kg, followed by up to two additional infusions at 2â×â106 polyomavirus-specific T cells per kg. The primary endpoints were feasibility (no manufacturing failure based on meeting release criteria, achieving adequate numbers of cell product for clinical use, and showing measurable antiviral activity) and safety in all patients. The safety monitoring period was 28 days after each infusion. Patients were followed up with serial MRI for up to 12 months after the final infusion. This trial is registered at ClinicalTrials.gov, NCT02694783. FINDINGS: Between April 7, 2016, and Oct 19, 2018, 26 patients were screened, of whom 12 were confirmed eligible and received treatment derived from 14 matched donors. All administered polyomavirus-specific T cells met the release criteria and recognised cognate antigens in vitro. 12 patients received at least one infusion, ten received at least two, and seven received a total of three infusions. The median on-study follow-up was 109·5 days (range 23-699). All infusions were tolerated well, and no serious treatment-related adverse events were observed. Seven patients survived progressive multifocal leukoencephalopathy for longer than 1 year after the first infusion, whereas five died of progressive multifocal leukoencephalopathy within 3 months. INTERPRETATION: We showed that generation of polyomavirus-specific T cells from healthy related donors is feasible, and these cells can be safely used as an infusion for adoptive immunotherapy of progressive multifocal leukoencephalopathy. Although not powered to assess efficacy, our data provide additional support for this strategy as a potential life-saving therapy for some patients. FUNDING: Intramural Research Program of the National Institute of Neurological Disorders and Stroke of the NIH.
Assuntos
Vírus BK/imunologia , Imunoterapia/métodos , Leucoencefalopatia Multifocal Progressiva/terapia , Linfócitos T/imunologia , Adulto , Idoso , Doadores de Sangue , Estudos de Coortes , Determinação de Ponto Final , Estudos de Viabilidade , Feminino , Humanos , Imunoterapia/efeitos adversos , Vírus JC/imunologia , Leucoencefalopatia Multifocal Progressiva/diagnóstico por imagem , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Projetos Piloto , Análise de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: HTLV-1 infects over 20 million people worldwide and causes a progressive neuroinflammatory disorder in a subset of infected individuals called HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The detection of HTLV-1 specific T cells in the cerebrospinal fluid (CSF) suggests this disease is immunopathologically mediated and that it may be driven by viral antigens. Exosomes are microvesicles originating from the endosomal compartment that are shed into the extracellular space by various cell types. It is now understood that several viruses take advantage of this mode of intercellular communication for packaging of viral components as well. We sought to understand if this is the case in HTLV-1 infection, and specifically if HTLV-1 proteins can be found in the CSF of HAM/TSP patients where we know free virus is absent, and furthermore, if exosomes containing HTLV-1 Tax have functional consequences. RESULTS: Exosomes that were positive for HTLV-1 Tax by Western blot were isolated from HAM/TSP patient PBMCs (25/36) in ex vivo cultures by trapping exosomes from culture supernatants. HTLV-1 seronegative PBMCs did not have exosomes with Tax (0/12), (Fisher exact test, p = 0.0001). We were able to observe HAM/TSP patient CSF (12/20) containing Tax+ exosomes but not in HTLV-1 seronegative MS donors (0/5), despite the absence of viral detection in the CSF supernatant (Fisher exact test p = 0.0391). Furthermore, exosomes cultivated from HAM/TSP PBMCs were capable of sensitizing target cells for HTLV-1 specific CTL lysis. CONCLUSION: Cumulatively, these results show that there are HTLV-1 proteins present in exosomes found in virus-free CSF. HAM/TSP PBMCs, particularly CD4+CD25+ T cells, can excrete these exosomes containing HTLV-1 Tax and may be a source of the exosomes found in patient CSF. Importantly, these exosomes are capable of sensitizing an HTLV-1 specific immune response, suggesting that they may play a role in the immunopathology observed in HAM/TSP. Given the infiltration of HTLV-1 Tax-specific CTLs into the CNS of HAM/TSP patients, it is likely that exosomes may also contribute to the continuous activation and inflammation observed in HAM/TSP, and may suggest future targeted therapies in this disorder.