RESUMO
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
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 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
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
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Although various viruses have been proposed to contribute to MS pathology, the etiology of MS remains unknown. Since intrathecal antibody synthesis is well documented in chronic viral infection and neuroinflammatory diseases, we hypothesized whether the patterns of antigen-specific antibody responses associated with various viral exposures may define patients with CNS chronic immune dysregulation. The pan-viral antibody profiling in cerebrospinal fluid (CSF) and serum of patients with MS showed significant differences from those in healthy volunteers and a pattern of antibody responses against multiple viruses, including the previously identified Epstein-Barr virus. These findings demonstrate that virus-specific antibody signatures might be able to reflect disease-associated inflammatory milieu in CSF of subjects with neuroinflammatory diseases.
Assuntos
Infecções por Vírus Epstein-Barr , Esclerose Múltipla , Humanos , Herpesvirus Humano 4 , Doenças Neuroinflamatórias , AntiviraisRESUMO
BACKGROUND: The "central vein sign" (CVS), a linear hypointensity on T2*-weighted imaging corresponding to a central vein/venule, is associated with multiple sclerosis (MS) lesions. The effect of lesion-size exclusion criteria on MS diagnostic accuracy has not been extensively studied. OBJECTIVE: Investigate the optimal lesion-size exclusion criteria for CVS use in MS diagnosis. METHODS: Cross-sectional study of 163 MS and 51 non-MS, and radiological/histopathological correlation of 5 MS and 1 control autopsy cases. The effects of lesion-size exclusion on MS diagnosis using the CVS, and intralesional vein detection on histopathology were evaluated. RESULTS: CVS+ lesions were larger compared to CVS- lesions, with effect modification by MS diagnosis (mean difference +7.7 mm3, p = 0.004). CVS percentage-based criteria with no lesion-size exclusion showed the highest diagnostic accuracy in differentiating MS cases. However, a simple count of three or more CVS+ lesions greater than 3.5 mm is highly accurate and can be rapidly implemented (sensitivity 93%; specificity 88%). On magnetic resonance imaging (MRI)-histopathological correlation, the CVS had high specificity for identifying intralesional veins (0/7 false positives). CONCLUSION: Lesion-size measures add important information when using CVS+ lesion counts for MS diagnosis. The CVS is a specific biomarker corresponding to intralesional veins on histopathology.
Assuntos
Esclerose Múltipla , Encéfalo/patologia , Estudos Transversais , Humanos , Imageamento por Ressonância Magnética/métodos , Esclerose Múltipla/patologia , Veias/diagnóstico por imagemRESUMO
BACKGROUND: Dramatic improvements in visualization of cortical (especially subpial) multiple sclerosis (MS) lesions allow assessment of impact on clinical course. OBJECTIVE: Characterize cortical lesions by 7 tesla (T) T2*-/T1-weighted magnetic resonance imaging (MRI); determine relationship with other MS pathology and contribution to disability. METHODS: Sixty-four adults with MS (45 relapsing-remitting/19 progressive) underwent 3 T brain/spine MRI, 7 T brain MRI, and clinical testing. RESULTS: Cortical lesions were found in 94% (progressive: median 56/range 2-203; relapsing-remitting: 15/0-168; p = 0.004). Lesion distribution across 50 cortical regions was nonuniform (p = 0.006), with highest lesion burden in supplementary motor cortex and highest prevalence in superior frontal gyrus. Leukocortical and white matter lesion volumes were strongly correlated (r = 0.58, p < 0.0001), while subpial and white matter lesion volumes were moderately correlated (r = 0.30, p = 0.002). Leukocortical (p = 0.02) but not subpial lesions (p = 0.40) were correlated with paramagnetic rim lesions; both were correlated with spinal cord lesions (p = 0.01). Cortical lesion volumes (total and subtypes) were correlated with expanded disability status scale, 25-foot timed walk, nine-hole peg test, and symbol digit modality test scores. CONCLUSION: Cortical lesions are highly prevalent and are associated with disability and progressive disease. Subpial lesion burden is not strongly correlated with white matter lesions, suggesting differences in inflammation and repair mechanisms.
Assuntos
Pessoas com Deficiência , Esclerose Múltipla , Substância Branca , Adulto , Encéfalo/patologia , Humanos , Imageamento por Ressonância Magnética/métodos , Esclerose Múltipla/patologia , Substância Branca/patologiaRESUMO
BACKGROUND AND OBJECTIVES: The central vein sign (CVS), a central linear hypointensity within lesions on T2*-weighted imaging, has been established as a sensitive and specific biomarker for the diagnosis of multiple sclerosis (MS). However, the CVS has not yet been comprehensively studied in newly developing MS lesions. We aimed to identify the CVS profiles of new white matter lesions in patients with MS followed over time and investigate demographic and clinical risk factors associated with new CVS+ or CVS- lesion development. METHODS: In this retrospective longitudinal cohort study, adults from the NIH MS Natural History Study were considered for inclusion. Participants with new T2 or enhancing lesions were identified through review of the radiology report and/or longitudinal subtraction imaging. Each new lesion was evaluated for the CVS. Clinical characteristics were identified through chart review. RESULTS: A total of 153 adults (95 relapsing-remitting MS, 27 secondary progressive MS, 16 primary progressive MS, 5 clinically isolated syndrome, and 10 healthy; 67% female) were included. Of this cohort, 96 had at least 1 new T2 or contrast-enhancing lesion during median 3.1 years (Q1-Q3: 0.7-6.3) of follow-up; lesions eligible for CVS evaluation were found in 62 (65%). Of 233 new CVS-eligible lesions, 159 (68%) were CVS+, with 30 (48%) individuals having only CVS+, 12 (19%) only CVS-, and 20 (32%) both CVS+ and CVS- lesions. In gadolinium-enhancing (Gd+) lesions, the CVS+ percentage increased from 102/152 (67%) at the first time point where the lesion was observed, to 92/114 (82%) after a median follow-up of 2.8 years. Younger age (OR = 0.5 per 10-year increase, 95% CI = 0.3-0.8) and higher CVS+ percentage at baseline (OR = 1.4 per 10% increase, 95% CI = 1.1-1.9) were associated with increased likelihood of new CVS+ lesion development. DISCUSSION: In a cohort of adults with MS followed over a median duration of 3 years, most newly developing T2 or enhancing lesions were CVS+ (68%), and nearly half (48%) developed new CVS+ lesions only. Importantly, the effects of edema and T2 signal changes can obscure small veins in Gd+ lesions; therefore, caution and follow-up is necessary when determining their CVS status. TRIAL REGISTRATION INFORMATION: Clinical trial registration number NCT00001248. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that younger age and higher CVS+ percentage at baseline are associated with new CVS+ lesion development.
Assuntos
Progressão da Doença , Esclerose Múltipla/diagnóstico por imagem , Esclerose Múltipla/patologia , Substância Branca/diagnóstico por imagem , Substância Branca/patologia , Adulto , Veias Cerebrais/diagnóstico por imagem , Veias Cerebrais/patologia , Feminino , Humanos , Estudos Longitudinais , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Substância Branca/irrigação sanguíneaRESUMO
OBJECTIVE: Human T-cell lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive myelopathy. A high proviral load (PVL) is one of the main risk factors for HAM/TSP. Recently, it was shown that raltegravir could inhibit cell-free and cell-to-cell transmission of HTLV-1 in vitro. Given the substantial clinical experience in human immunodeficiency virus infection and its excellent safety profile, this agent may be an attractive therapeutic option for HAM/TSP patients. METHODS: Sixteen subjects with HAM/TSP received raltegravir 400 mg orally twice daily in an initial 6-month treatment phase, followed by a 9-month post-treatment phase. HTLV-1 PVLs were assessed using droplet digital PCR from the PBMCs every 3 months, and from the CSF at baseline, month 6, and month 15. We also evaluated the ability of raltegravir to regulate abnormal immune responses in HAM/TSP patients. RESULTS: While a downward trend was observed in PBMC and/or CSF PVLs of some patients, raltegravir overall did not have any impact on the PVL in this HAM/TSP patient cohort. Clinically, all patients' neurological scores and objective measurements remained relatively stable, with some expected variability. Immunologic studies showed alterations in the immune profiles of a subset of patients including decreased CD4+ CD25+ T cells and spontaneous lymphoproliferation. INTERPRETATION: Raltegravir was generally well tolerated in this HAM/TSP patient cohort. A subset of patients exhibited a mild decrease in PVL as well as variations in their immune profiles after taking raltegravir. These findings suggest that raltegravir may be a therapeutic option in select HAM/TSP patients. CLINICAL TRIAL REGISTRATION NUMBER: NCT01867320.
Assuntos
Inibidores de Integrase/farmacologia , Paraparesia Espástica Tropical/tratamento farmacológico , Raltegravir Potássico/farmacologia , Adulto , Idoso , Feminino , Humanos , Inibidores de Integrase/administração & dosagem , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Raltegravir Potássico/administração & dosagem , Resultado do TratamentoRESUMO
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) characterized by varying degrees of secondary neurodegeneration. Retinal ganglion cells (RGC) are lost in MS in association with optic neuritis but the mechanisms of neuronal injury remain unclear. Complement component C3 has been implicated in retinal and cerebral synaptic pathology that may precede neurodegeneration. Herein, we examined post-mortem MS retinas, and then used a mouse model, experimental autoimmune encephalomyelitis (EAE), to examine the role of C3 in the pathogenesis of RGC loss associated with optic neuritis. First, we show extensive C3 expression in astrocytes (C3+/GFAP+ cells) and significant RGC loss (RBPMS+ cells) in post-mortem retinas from people with MS compared to retinas from non-MS individuals. A patient with progressive MS with a remote history of optic neuritis showed marked reactive astrogliosis with C3 expression in the inner retina extending into deeper layers in the affected eye more than the unaffected eye. To study whether C3 mediates retinal degeneration, we utilized global C3-/- EAE mice and found that they had less RGC loss and partially preserved neurites in the retina compared with C3+/+ EAE mice. C3-/- EAE mice had fewer axonal swellings in the optic nerve, reflecting reduced axonal injury, but had no changes in demyelination or T cell infiltration into the CNS. Using a C3-tdTomato reporter mouse line, we show definitive evidence of C3 expression in astrocytes in the retina and optic nerves of EAE mice. Conditional deletion of C3 in astrocytes showed RGC protection replicating the effects seen in the global knockouts. These data implicate astrocyte C3 expression as a critical mediator of retinal neuronal pathology in EAE and MS, and are consistent with recent studies showing C3 gene variants are associated with faster rates of retinal neurodegeneration in human disease.
Assuntos
Complemento C3/metabolismo , Esclerose Múltipla/patologia , Doenças Neuroinflamatórias/patologia , Células Ganglionares da Retina/patologia , Animais , Astrócitos/imunologia , Astrócitos/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Humanos , Camundongos , Esclerose Múltipla/imunologia , Degeneração Neural/imunologia , Degeneração Neural/patologia , Doenças Neuroinflamatórias/imunologia , Nervo Óptico/patologia , Neurite Óptica/imunologia , Neurite Óptica/patologiaRESUMO
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
OBJECTIVE: We sought to characterize spinal cord atrophy along the entire spinal cord in the major multiple sclerosis (MS) phenotypes, and evaluate its correlation with clinical disability. METHODS: Axial T1-weighted images were automatically reformatted at each point along the cord. Spinal cord cross-sectional area (SCCSA) were calculated from C1-T10 vertebral body levels and profile plots were compared across phenotypes. Average values from C2-3, C4-5, and T4-9 regions were compared across phenotypes and correlated with clinical scores, and then categorized as atrophic/normal based on z-scores derived from controls, to compare clinical scores between subgroups. In a subset of relapsing-remitting cases with longitudinal scans these regions were compared to change in clinical scores. RESULTS: The cross-sectional study consisted of 149 adults diagnosed with relapsing-remitting MS (RRMS), 49 with secondary-progressive MS (SPMS), 58 with primary-progressive MS (PPMS) and 48 controls. The longitudinal study included 78 RRMS cases. Compared to controls, all MS groups had smaller average regions except RRMS in T4-9 region. In all MS groups, SCCSA from all regions, particularly the cervical cord, correlated with most clinical measures. In the RRMS cohort, 22% of cases had at least one atrophic region, whereas in progressive MS the rate was almost 70%. Longitudinal analysis showed correlation between clinical disability and cervical cord thinning. CONCLUSIONS: Spinal cord atrophy was prevalent across MS phenotypes, with regional measures from the RRMS cohort and the progressive cohort, including SPMS and PPMS, being correlated with disability. Longitudinal changes in the spinal cord were documented in RRMS cases, making it a potential marker for disease progression. While cervical SCCSA correlated with most disability and progression measures, inclusion of thoracic measurements improved this correlation and allowed for better subgrouping of spinal cord phenotypes. Cord atrophy is an important and easily obtainable imaging marker of clinical and sub-clinical progression in all MS phenotypes, and such measures can play a key role in patient selection for clinical trials.
Assuntos
Medula Cervical , Esclerose Múltipla Recidivante-Remitente , Esclerose Múltipla , Adulto , Atrofia/patologia , Medula Cervical/diagnóstico por imagem , Medula Cervical/patologia , Estudos Transversais , Avaliação da Deficiência , Progressão da Doença , Humanos , Estudos Longitudinais , Imageamento por Ressonância Magnética , Esclerose Múltipla/diagnóstico por imagem , Esclerose Múltipla/patologia , Esclerose Múltipla Recidivante-Remitente/diagnóstico por imagem , Esclerose Múltipla Recidivante-Remitente/patologia , Fenótipo , Medula Espinal/patologiaRESUMO
OBJECTIVE: To test the hypothesis that teriflunomide can reduce ex vivo spontaneous proliferation of peripheral blood mononuclear cells (PBMCs) from patients with human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). METHODS: PBMCs from patients with HAM/TSP were cultured in the presence and absence of teriflunomide and assessed for cell viability, lymphocyte proliferation, activation markers, HTLV-1 tax and HTLV-1 hbz messenger ribonucleic acid (mRNA) expression, and HTLV-1 Tax protein expression. RESULTS: In culture, teriflunomide did not affect cell viability. A concentration-dependent reduction in spontaneous proliferation of PBMCs was observed with 25 µM (38.3% inhibition), 50 µM (65.8% inhibition), and 100 µM (90.7% inhibition) teriflunomide. The inhibitory effects of teriflunomide were detected in both CD8+ and CD4+ T-cell subsets, which are involved in the immune response to HTLV-1 infection and the pathogenesis of HAM/TSP. There was no significant change in HTLV-1 proviral load (PVL) or tax mRNA/Tax protein expression in these short-term cultures, but there was a significant reduction of HTLV-1 PVL due to inhibition of proliferation of CD4+ T cells obtained from a subset of patients with HAM/TSP. CONCLUSIONS: These results suggest that teriflunomide inhibits abnormal T-cell proliferation associated with HTLV-1 infection and may have potential as a therapeutic option in patients with HAM/TSP.
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Crotonatos/farmacologia , Hidroxibutiratos/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Nitrilas/farmacologia , Paraparesia Espástica Tropical/tratamento farmacológico , Toluidinas/farmacologia , Adulto , Idoso , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Produtos do Gene tax/metabolismo , Infecções por HTLV-I/complicações , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , RNA Mensageiro/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacosRESUMO
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
In multiple sclerosis (MS), a subset of chronic active white matter lesions are identifiable on magnetic resonance imaging by their paramagnetic rims, and increasing evidence supports their association with severity of clinical disease. We studied their potential role in differential diagnosis, screening an international multicenter clinical research-based sample of 438 individuals affected by different neurological conditions (MS, other inflammatory, infectious, and non-inflammatory conditions). Paramagnetic rim lesions, rare in other neurological conditions (52% of MS vs 7% of non-MS cases), yielded high specificity (93%) in differentiating MS from non-MS. Future prospective multicenter studies should validate their role as a diagnostic biomarker. ANN NEUROL 2020;88:1034-1042.
Assuntos
Esclerose Múltipla/diagnóstico por imagem , Adulto , Idoso , Diagnóstico Diferencial , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/epidemiologia , Doenças do Sistema Nervoso/diagnóstico por imagem , Neuroimagem , Prevalência , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto JovemRESUMO
Progressive multifocal leukoencephalopathy (PML) is a rare opportunistic brain infection caused by the JC virus and associated with substantial morbidity and mortality. Accurate MRI assessment of PML lesion burden and brain parenchymal atrophy is of decisive value in monitoring the disease course and response to therapy. However, there are currently no validated automatic methods for quantification of PML lesion burden or associated parenchymal volume loss. Furthermore, manual brain or lesion delineations can be tedious, require the use of valuable time resources by radiologists or trained experts, and are often subjective. In this work, we introduce JCnet (named after the causative viral agent), an end-to-end, fully automated method for brain parenchymal and lesion segmentation in PML using consecutive 3D patch-based convolutional neural networks. The network architecture consists of multi-view feature pyramid networks with hierarchical residual learning blocks containing embedded batch normalization and nonlinear activation functions. The feature maps across the bottom-up and top-down pathways of the feature pyramids are merged, and an output probability membership generated through convolutional pathways, thus rendering the method fully convolutional. Our results show that this approach outperforms and improves longitudinal consistency compared to conventional, state-of-the-art methods of healthy brain and multiple sclerosis lesion segmentation, utilized here as comparators given the lack of available methods validated for use in PML. The ability to produce robust and accurate automated measures of brain atrophy and lesion segmentation in PML is not only valuable clinically but holds promise toward including standardized quantitative MRI measures in clinical trials of targeted therapies. Code is available at: https://github.com/omarallouz/JCnet.
Assuntos
Aprendizado Profundo , Leucoencefalopatia Multifocal Progressiva , Encéfalo/diagnóstico por imagem , Humanos , Processamento de Imagem Assistida por Computador , Leucoencefalopatia Multifocal Progressiva/diagnóstico por imagem , Imageamento por Ressonância Magnética , Redes Neurais de ComputaçãoRESUMO
OBJECTIVE: Human T cell lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive, neurological disease. Chronic activation of CD8+ T cells, as evidenced by increased spontaneous lymphoproliferation and HTLV-1-specific cytotoxic T cells, has been demonstrated in HAM/TSP patients. Since IL-2 and IL-15 stimulate memory CD8+ T cell activity, these cytokines have been implicated in the immunopathogenesis of HAM/TSP. In this phase I trial, we evaluated the safety, pharmacokinetics, and ability of Hu-Mikß1, a humanized monoclonal antibody directed toward the IL-2/IL-15 receptor ß-chain (IL-2/IL-15Rß: CD122), to saturate CD122 and regulate abnormal immune responses in patients with HAM/TSP by inhibition of IL-15 action. METHODS: Hu-Mikß1 was administered intravenously at doses of 0.5 mg/kg, 1.0 mg/kg, or 1.5 mg/kg in a total of nine HAM/TSP patients. Five doses of Hu-Mikß1 were administered at 3-week intervals. The clinical response was evaluated using standardized scales. Viral and immunologic outcome measures were examined including HTLV-1 proviral load, T cell phenotypic analysis and spontaneous lymphoproliferation in HAM/TSP patients. RESULTS: There was no significant toxicity associated with Hu-Mikß1 administration in HAM/TSP patients. Saturation of CD122 by Hu-Mikß1 was achieved in five out of nine HAM/TSP patients. Administration of Hu-Mikß1 was associated with inhibition of aberrant CD8+ T cell function including spontaneous lymphoproliferation and degranulation and IFN-γ expression, especially in HAM/TSP patients that achieved CD122 saturation. INTERPRETATION: The treatment with Hu-Mikß1 had a number of immunological effects on HAM/TSP patients although no clinical efficacy was observed. We also did not see any dose-related toxicity.
Assuntos
Anticorpos Monoclonais/farmacologia , Subunidade beta de Receptor de Interleucina-2/antagonistas & inibidores , Paraparesia Espástica Tropical/imunologia , Paraparesia Espástica Tropical/terapia , Administração Intravenosa , Adulto , Idoso , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Citocinas/biossíntese , Feminino , Infecções por HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Humanos , Interferon gama/metabolismo , Interleucina-15 , Interleucina-2 , Subunidade beta de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Doenças do Sistema Nervoso , Linfócitos T Citotóxicos/imunologiaRESUMO
Importance: In multiple sclerosis (MS), chronic active lesions, which previously could only be detected at autopsy, can now be identified on susceptibility-based magnetic resonance imaging (MRI) in vivo as non-gadolinium-enhancing lesions with paramagnetic rims. Pathologically, they feature smoldering inflammatory demyelination at the edge, remyelination failure, and axonal degeneration. To our knowledge, the prospect of long-term in vivo monitoring makes it possible for the first time to determine their contribution to disability and value as a treatment target. Objective: To assess whether rim lesions are associated with patient disability and long-term lesion outcomes. Design, Setting, Participants: We performed 3 studies at the National Institutes of Health Clinical Center: (1) a prospective clinical/radiological cohort of 209 patients with MS (diagnosis according to the 2010 McDonald revised MS criteria, age ≥18 years, with 7-T or 3-T susceptibility-based brain MRI results) who were enrolled from January 2012 to March 2018 (of 209, 17 patients [8%] were excluded because of uninterpretable MRI scans); (2) a radiological/pathological analysis of expanding lesions featuring rims; and (3) a retrospective longitudinal radiological study assessing long-term lesion evolution in 23 patients with MS with yearly MRI scans for 10 years or more (earliest scan, 1992). Main Outcomes and Measures: (1) Identification of chronic rim lesions on 7-T or 3-T susceptibility-based brain MRI in 192 patients with MS and the association of rim counts with clinical disability (primary analysis) and brain volume changes (exploratory analysis). (2) Pathological characterization of 10 expanding lesions from an adult with progressive MS who came to autopsy after 7 years of receiving serial in vivo MRI scans. (3) Evaluation of annual lesion volume change (primary analysis) and T1 times (exploratory analysis) in 27 rim lesions vs 27 rimless lesions. Results: Of 209 participants, 104 (50%) were women and 32 (15%) were African American. One hundred seventeen patients (56%) had at least 1 rim lesion regardless of prior or ongoing treatment. Further, 84 patients (40%) had no rims (mean [SD] age, 47 [14] years), 66 (32%) had 1 to 3 rims (mean [SD] age, 47 [11] years), and 42 (20%) had 4 rims or more (mean [SD] age, 44 [11] years). Individuals with 4 rim lesions or more reached motor and cognitive disability at an earlier age. Normalized volumes of brain, white matter, and basal ganglia were lower in those with rim lesions. Whereas rimless lesions shrank over time (-3.6%/year), rim lesions were stable in size or expanded (2.2%/year; P < .001). Rim lesions had longer T1 times, suggesting more tissue destruction, than rimless lesions. On histopathological analysis, all 10 rim lesions that expanded in vivo had chronic active inflammation. Conclusions and Relevance: Chronic active lesions are common, are associated with more aggressive disease, exert ongoing tissue damage, and occur even in individuals treated with effective disease-modifying therapies. These results prompt the planning of MRI-based clinical trials aimed at treating perilesional chronic inflammation in MS.
Assuntos
Encéfalo/diagnóstico por imagem , Esclerose Múltipla/diagnóstico por imagem , Adulto , Encéfalo/patologia , Estudos de Coortes , Progressão da Doença , Imagem Ecoplanar , Feminino , Humanos , Imageamento Tridimensional , Inflamação , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/fisiopatologia , Tamanho do Órgão , PrognósticoRESUMO
BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is an opportunistic brain infection that is caused by the JC virus and is typically fatal unless immune function can be restored. Programmed cell death protein 1 (PD-1) is a negative regulator of the immune response that may contribute to impaired viral clearance. Whether PD-1 blockade with pembrolizumab could reinvigorate anti-JC virus immune activity in patients with PML was unknown. METHODS: We administered pembrolizumab at a dose of 2 mg per kilogram of body weight every 4 to 6 weeks to eight adults with PML, each with a different underlying predisposing condition. Each patient received at least one dose but no more than three doses. RESULTS: Pembrolizumab induced down-regulation of PD-1 expression on lymphocytes in peripheral blood and in cerebrospinal fluid (CSF) in all eight patients. Five patients had clinical improvement or stabilization of PML accompanied by a reduction in the JC viral load in the CSF and an increase in in vitro CD4+ and CD8+ anti-JC virus activity. In the other three patients, no meaningful change was observed in the viral load or in the magnitude of antiviral cellular immune response, and there was no clinical improvement. CONCLUSIONS: Our findings are consistent with the hypothesis that in some patients with PML, pembrolizumab reduces JC viral load and increases CD4+ and CD8+ activity against the JC virus; clinical improvement or stabilization occurred in five of the eight patients who received pembrolizumab. Further study of immune checkpoint inhibitors in the treatment of PML is warranted. (Funded by the National Institutes of Health.).
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Fatores Imunológicos/uso terapêutico , Vírus JC/isolamento & purificação , Leucoencefalopatia Multifocal Progressiva/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Adulto , Idoso , Encéfalo/diagnóstico por imagem , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Líquido Cefalorraquidiano/virologia , Regulação para Baixo , Feminino , Humanos , Síndrome Inflamatória da Reconstituição Imune/etiologia , Leucoencefalopatia Multifocal Progressiva/diagnóstico por imagem , Leucoencefalopatia Multifocal Progressiva/imunologia , Contagem de Linfócitos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/metabolismo , Carga Viral , Substância Branca/diagnóstico por imagem , Substância Branca/patologiaRESUMO
BACKGROUND: Brain- and lesion-volumes derived from magnetic resonance images (MRI) serve as important imaging markers of disease progression in neurodegenerative diseases and aging. While manual segmentation of these volumes is both tedious and impractical in large cohorts of subjects, automated segmentation methods often fail in accurate segmentation of brains with severe atrophy or high lesion loads. The purpose of this study was to develop an atlas-free brain Classification using DErivative-based Features (C-DEF), which utilizes all scans that may be acquired during the course of a routine MRI study at any center. METHODS: Proton-density, T2-weighted, T1-weighted, brain-free water, 3D FLAIR, 3D T2-weighted, and 3D T2*-weighted images, collected routinely on patients with neuroinflammatory diseases at the NIH, were used to optimize the C-DEF algorithm on healthy volunteers and HIV + subjects (cohort 1). First, manually marked lesions and eroded FreeSurfer brain segmentation masks (compiled into gray and white matter, globus pallidus, CSF labels) were used in training. Next, the optimized C-DEF was applied on a separate cohort of HIV + subjects (cohort two), and the results were compared with that of FreeSurfer and Lesion-TOADS. Finally, C-DEF segmentation was evaluated on subjects clinically diagnosed with various other neurological diseases (cohort three). RESULTS: C-DEF algorithm was optimized using leave-one-out cross validation on five healthy subjects (age 36 ± 11 years), and five subjects infected with HIV (age 57 ± 2.6 years) in cohort one. The optimized C-DEF algorithm outperformed FreeSurfer and Lesion-TOADS segmentation in 49 other subjects infected with HIV (cohort two, age 54 ± 6 years) in qualitative and quantitative comparisons. Although trained only on HIV brains, sensitivity to detect lesions using C-DEF increased by 45% in HTLV-I-associated myelopathy/tropical spastic paraparesis (n = 5; age 58 ± 7 years), 33% in multiple sclerosis (n = 5; 42 ± 9 years old), and 4% in subjects with polymorphism of the cytotoxic T-lymphocyte-associated protein 4 gene (n = 5; age 24 ± 12 years) compared to Lesion-TOADS. CONCLUSION: C-DEF outperformed other segmentation algorithms in the various neurological diseases explored herein, especially in lesion segmentation. While the results reported are from routine images acquired at the NIH, the algorithm can be easily trained and optimized for any set of contrasts and protocols for wider application. We are currently exploring various technical aspects of optimal implementation of CDEF in a clinical setting and evaluating a larger cohort of patients with other neurological diseases. Improving the accuracy of brain segmentation methodology will help better understand the relationship of imaging abnormalities to clinical and neuropsychological markers in disease.