RESUMEN
Zika is a systemic inflammatory disease caused by infection with Zika virus (ZIKV). ZIKV infection in adults is associated with encephalitis marked by elevated expression of pro-inflammatory cytokines and chemokines, as well as increased brain infiltration of immune cells. In this study, we demonstrate that ZIKV encephalitis in a mouse infection model exhibits increased brain TSPO expression. TSPO expression on brain-resident and infiltrating immune cells in ZIKV infection correlates with disease and inflammation status in the brain. Brain TSPO expression can also be sensitively detected ex vivo and in vitro using radioactive small molecule probes that specifically bind to TSPO, such as [3H]PK11195. TSPO expression on brain-resident and infiltrating immune cells is a biomarker of ZIKV neuroinflammation, which can also be a general biomarker of acute viral neuroinflammatory disease.
Asunto(s)
Biomarcadores , Encéfalo , Enfermedades Neuroinflamatorias , Receptores de GABA , Infección por el Virus Zika , Virus Zika , Animales , Infección por el Virus Zika/virología , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/metabolismo , Ratones , Receptores de GABA/metabolismo , Receptores de GABA/genética , Virus Zika/inmunología , Encéfalo/virología , Encéfalo/metabolismo , Encéfalo/patología , Enfermedades Neuroinflamatorias/virología , Enfermedades Neuroinflamatorias/inmunología , Enfermedades Neuroinflamatorias/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones Endogámicos C57BL , Femenino , Citocinas/metabolismoRESUMEN
Glioblastoma multiforme (GBM) is the most common malignant primary brain cancer affecting the adult population. Median overall survival for GBM patients is poor (15 months), primarily due to high rates of tumour recurrence and the paucity of treatment options. Oncolytic virotherapy is a promising treatment alternative for GBM patients, where engineered viruses selectively infect and eradicate cancer cells by inducing cell lysis and eliciting robust anti-tumour immune response. In this study, we evaluated the oncolytic potency of live-attenuated vaccine strains of Zika virus (ZIKV-LAV) against human GBM cells in vitro. Our findings revealed that Axl and integrin αvß5 function as cellular receptors mediating ZIKV-LAV infection in GBM cells. ZIKV-LAV strains productively infected and lysed human GBM cells but not primary endothelia and terminally differentiated neurons. Upon infection, ZIKV-LAV mediated GBM cell death via apoptosis and pyroptosis. This is the first in-depth molecular dissection of how oncolytic ZIKV infects and induces death in tumour cells.
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Glioblastoma , Viroterapia Oncolítica , Virus Oncolíticos , Infección por el Virus Zika , Virus Zika , Humanos , Virus Zika/fisiología , Infección por el Virus Zika/prevención & control , Glioblastoma/terapia , Vacunas Atenuadas , Recurrencia Local de Neoplasia/terapiaRESUMEN
INTRODUCTION: Zika virus (ZIKV) is a neurotropic human pathogen that causes neuroinflammation, whose hallmark is elevated translocator protein (TSPO) expression in the brain. This study investigates ZIKV-associated changes in adult brain TSPO expression, evaluates the effectiveness of TSPO radioligands in detecting TSPO expression, and identifies cells that drive brain TSPO expression in a mouse infection model. METHODS: The interferon-deficient AG129 mouse infected with ZIKV was used as neuroinflammation model. TSPO expression was evaluated by tissue immunostaining. TSPO radioligands, [3H]PK11195 and [18F]FEPPA, were used for in vitro and ex vivo detection of TSPO in infected brains. [18F]FEPPA-PET was used for in vivo detection of TSPO expression. Cell subsets that contribute to TSPO expression were identified by flow cytometry. RESULTS: Brain TSPO expression increased with ZIKV disease severity. This increase was contributed by TSPO-positive microglia and infiltrating monocytes; and by influx of TSPO-expressing immune cells into the brain. [3H]PK11195 and [18F]FEPPA distinguish ZIKV-infected brains from normal controls in vitro and ex vivo. [18F]FEPPA brain uptake by PET imaging correlated with disease severity and neuroinflammation. However, TSPO expression by immune cells contributed to significant blood pool [18F]FEPPA activity which could confound [18F]FEPPA-PET imaging results. CONCLUSIONS: TSPO is a biologically relevant imaging target for ZIKV neuroinflammation. Brain [18F]FEPPA uptake can be a surrogate marker for ZIKV disease and may be a potential PET imaging marker for ZIKV-induced neuroinflammation. Future TSPO-PET/SPECT studies on viral neuroinflammation and related encephalitis should assess the contribution of immune cells on TSPO expression and employ appropriate image correction methods to subtract blood pool activity.
Asunto(s)
Encefalitis , Infección por el Virus Zika , Virus Zika , Adulto , Humanos , Ratones , Animales , Virus Zika/metabolismo , Infección por el Virus Zika/diagnóstico por imagen , Infección por el Virus Zika/metabolismo , Enfermedades Neuroinflamatorias , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Tomografía de Emisión de Positrones/métodos , Modelos Animales de Enfermedad , Receptores de GABA/metabolismoRESUMEN
PURPOSE: Zika (ZIKV) is a viral inflammatory disease affecting adults, children, and developing fetuses. It is endemic to tropical and sub-tropical countries, resulting in half the global population at risk of infection. Despite this, there are no approved therapies or vaccines against ZIKV disease. Non-invasive imaging biomarkers are potentially valuable tools for studying viral pathogenesis, prognosticating host response to disease, and evaluating in vivo efficacy of experimental therapeutic interventions. In this study, we evaluated [18F]fluorodeoxyglucose ([18F]FDG)-positron emission tomography (PET) as an imaging biomarker of ZIKV disease in a mouse model and correlated metabolic tracer tissue uptake with real-time biochemical, virological, and inflammatory features of tissue infection. METHODS: [18F]FDG-PET/CT imaging was performed in an acute, lethal ZIKV mouse infection model, at increasing stages of disease severity. [18F]FDG-PET findings were corroborated with ex vivo wholemount-tissue autoradiography and tracer biodistribution studies. Tracer uptake was also correlated with in situ tissue disease status, including viral burden and inflammatory response. Immune profiling of the spleen by flow cytometry was performed to identify the immune cell subsets driving tissue pathology and enhancing tracer uptake in ZIKV disease. RESULTS: Foci of increased [18F]FDG uptake were consistently detected in lymphoid tissues-particularly the spleen-of ZIKV-infected animals. Splenic uptake increased with disease severity, and corroborated findings in tissue pathology. Increased splenic uptake also correlated with increased viral replication and elevated expression of pro-inflammatory cytokines within these tissues. ZIKV-infected spleens were characterized by increased infiltration of myeloid cells, as well as increased proliferation of both myeloid and lymphoid cells. The increased cell proliferation correlated with increased tracer uptake in the spleen. Our findings support the use of [18F]FDG as an imaging biomarker to detect and track ZIKV disease in real time and highlight the dependency of affected tissue on the nature of the viral infection. CONCLUSION: [18F]FDG uptake in the spleen is a useful surrogate for interrogating in situ tissue viral burden and inflammation status in this ZIKV murine model.
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Infección por el Virus Zika , Virus Zika , Animales , Ratones , Infección por el Virus Zika/diagnóstico por imagen , Infección por el Virus Zika/metabolismo , Infección por el Virus Zika/patología , Virus Zika/metabolismo , Fluorodesoxiglucosa F18/metabolismo , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Distribución Tisular , Tomografía Computarizada por Rayos X , Tomografía de Emisión de Positrones , Tejido Linfoide/metabolismo , Tejido Linfoide/patología , Inflamación/diagnóstico por imagen , Inflamación/metabolismo , Modelos Animales de Enfermedad , Biomarcadores/metabolismo , CitocinasRESUMEN
Dengue virus (DENV) non-structural protein 5 (NS5) is critical for viral RNA synthesis within endoplasmic reticulum (ER)-derived replication complexes in the cytoplasm; however a proportion of NS5 is known to be localized to the nucleus of infected cells. The importance of nuclear DENV NS5 on viral replication and pathogenesis is still unclear. We recently discovered a nuclear localization signal (NLS) residing in the C-terminal 18 amino acid (Cter18) region of DENV NS5 and that a single NS5 P884T amino acid substitution adjacent to the NLS is sufficient to relocalize a significant proportion of DENV2 NS5 from the nucleus to the cytoplasm of infected cells. Here, in vitro studies show that the DENV2 NS5 P884T mutant replicates similarly to the parental wild-type infectious clone-derived virus while inducing a greater type I interferon and inflammatory cytokine response, in a manner independent of NS5's ability to degrade STAT2 or regulate SAT1 splicing. In both AG129 mouse and Aedes aegypti mosquito infection models, the P884T virus exhibits lower levels of viral replication only at early timepoints. Intriguingly, there appears to be a tendency for selection pressure to revert to the wild-type proline in P884T-infected Ae. aegypti, in agreement with the high conservation of the proline at this position of NS5 in DENV2, 3, and 4. These results suggest that the predominant nuclear localization of DENV NS5, while not required for viral RNA replication, may play a role in pathogenesis and modulation of the host immune response and contribute to viral fitness in the mosquito host.
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Virus del Dengue , Animales , Línea Celular , Virus del Dengue/genética , Ratones , Mutación , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
BACKGROUND: Newer antiviral agents for chronic hepatitis B (CHB) are highly effective, with minimal risks of complications and development of resistance. AIM: To identify the proportion of patients with CHB on treatment who will not require alteration of management and the clinical factors of those who will require closer monitoring. METHODS: Patients with CHB who were on entecavir and/or tenofovir between January 2011 and December 2016 were retrospectively studied. According to the initial treatment plan provided by the managing physician, any deviation in the interval of follow up, choice of investigations and alteration of medical therapy were considered a change in CHB management. We also evaluated the predictability of these changes, factors associated with higher frequency of change and the additional cost of managing stable patients with CHB in a tertiary setting. RESULTS: Of the patients, 75.7% (n = 87/115) did not have a change in CHB management; 85.6% of the changes in management were predictable based on liver function tests, hepatitis B virus DNA polymerase chain reaction levels and liver ultrasound. Interpreter use (OR (95% CI) = 2.41 (1.01-5.76)), liver cirrhosis (OR (95% CI) = 4.11 (1.44-11.75)) and immunosuppression (OR (95% CI) = 3.81 (1.2-12.06)) were associated risk factors. Overall, there was an incremental annual cost of AU$60 166 to manage patients who did not require alteration of their CHB management in our institution. CONCLUSION: The majority of stable CHB patients on highly potent antiviral treatment do not require alteration of management. While additional investigations are required, this study highlights the potential for a shared primary care approach in highly selected CHB patients.
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Antivirales/uso terapéutico , Hepatitis B Crónica/tratamiento farmacológico , Adulto , Costos y Análisis de Costo , ADN Viral/sangre , Manejo de la Enfermedad , Femenino , Guanina/análogos & derivados , Guanina/uso terapéutico , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/complicaciones , Hepatitis B Crónica/economía , Humanos , Cirrosis Hepática/virología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Tenofovir/uso terapéuticoRESUMEN
Objective: JIA is an autoimmune, inflammatory disease with involvement of innate and adaptive immune responses. However, the role of neutrophils in JIA pathogenesis remains unclear. This study aimed to identify and validate neutrophil gene expression signatures in JIA using public microarray datasets and new clinical samples. Methods: Three suitable datasets were analysed by significance analysis of microarray and Ingenuity. Neutrophils and peripheral blood mononuclear cells (PBMCs) were isolated from a new cohort of JIA patients and healthy paediatric controls (HCs). Gene expression was validated using quantitative PCR. Serum concentrations of proteins were measured using ELISA. Low-density granulocytes (LDGs) in JIA and HC PBMCs were quantified by flow cytometry using forward/side-scatter properties. Results: Ingenuity identified transcriptional regulation (false discovery rate < 0.05) by G-CSF, GM-CSF and IL-8 along with expression of neutrophil granule protein genes including ELANE, MPO, MMP8 and MMP9 in datasets from JIA PBMCs. LDG counts were elevated in JIA compared with HCs (2.5% vs 1.4%; P = 0.007). Transcripts for MMP8 (P = 0.005), MPO (P = 0.0124) and Fcγ Receptor 1B (FCγR1B) (P = 0.0417) were significantly higher in JIA compared with HC neutrophils. MMP9 protein levels were lower in systemic JIA patient sera [355.95 ng/ml (s.d. 250.03)] compared with HCs [675.41 ng/ml (s.d. 181.17); P = 0.007], but levels of elastase, MPO and MMP8 were not significantly different. Conclusion: LDGs are elevated in JIA and contribute to the transcriptomic profile of JIA PBMCs. JIA neutrophils express higher levels of MMP8 and FCGR1B, which may be implicated in disease pathology through the release of proteases and reactive oxygen metabolites, causing systemic inflammation and damage to joints.
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Artritis Juvenil/inmunología , Granulocitos/inmunología , Activación Neutrófila/genética , Neutrófilos/inmunología , Adolescente , Artritis Juvenil/sangre , Niño , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Interleucina-8/sangre , Recuento de Leucocitos , Leucocitos Mononucleares , Masculino , Metaloproteinasa 8 de la Matriz/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Fc/inmunología , Transcripción Genética , TranscriptomaRESUMEN
Development of antiviral therapy against acute viral diseases, such as dengue virus (DENV), suffers from the narrow window of viral load detection in serum during onset and clearance of infection and fever. We explored a biomarker approach using 18F-fluorodeoxyglucose (18F-FDG) PET in established mouse models for primary and antibody-dependent enhancement infection with DENV. 18F-FDG uptake was most prominent in the intestines and correlated with increased virus load and proinflammatory cytokines. Furthermore, a significant temporal trend in 18F-FDG uptake was seen in intestines and selected tissues over the time course of infection. Notably, 18F-FDG uptake and visualization by PET robustly differentiated treatment-naive groups from drug-treated groups as well as nonlethal from lethal infections with a clinical strain of DENV2. Thus, 18F-FDG may serve as a novel DENV infection-associated inflammation biomarker for assessing treatment response during therapeutic intervention trials.
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The liver is unique in its capacity to regenerate after injury, during which hepatocytes actively divide and establish cell-cell contacts through cell adhesion complexes. Here, we demonstrate that the loss of α-catenin, a well-established adhesion component, dramatically disrupts liver regeneration. Using a partial hepatectomy model, we show that regenerated livers from α-catenin knockdown mice are grossly larger than control regenerated livers, with an increase in cell size and proliferation. This increased proliferation correlated with increased YAP activation, implicating α-catenin in the Hippo/YAP pathway. Additionally, α-catenin knockdown mice exhibited a phenotype reminiscent of clinical cholestasis, with drastically altered bile canaliculi, elevated levels of bile components and signs of jaundice and inflammation. The disrupted regenerative capacity is a result of actin cytoskeletal disorganisation, leading to a loss of apical microvilli, dilated lumens in the bile canaliculi, and leaky tight junctions. This study illuminates a novel, essential role for α-catenin in liver regeneration.
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Colestasis/genética , Regeneración Hepática/fisiología , alfa Catenina/genética , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Canalículos Biliares/patología , Canalículos Biliares/ultraestructura , Proteínas de Ciclo Celular , Proliferación Celular , Colestasis/sangre , Femenino , Hepatocitos/fisiología , Ratones , Ratones Noqueados , Microvellosidades/ultraestructura , Modelos Animales , Fosfoproteínas/metabolismo , Proteínas Señalizadoras YAP , alfa Catenina/deficienciaRESUMEN
BACKGROUND: T cells are important to systemic lupus erythematosus (SLE) disease progression. This study determined the pro-inflammatory potential of T cells within the rare condition juvenile-onset SLE (JSLE). METHOD: IL-17A and Th1/Th2-related cytokine concentrations were measured in plasma/serum from JSLE patients (n = 19, n = 11) and HC (n = 18, n = 7). IL17A, RORC, IL23 and IL23R mRNA were measured in peripheral blood mononuclear cells (PBMCs) from JSLE and healthy controls (HC) (n = 12). Th17-associated cytokine expression was analysed in the supernatant of CD3/CD28 activated JSLE (n = 7) and HC (n = 6) PBMCs. RESULTS: JSLE plasma IL-17A level (21.5 ± 5.2 pg/ml) was higher compared to HC (7.2 ± 2.5 pg/ml, p = 0.028). No differences were found in Th1/Th2 cytokines levels. IL = 17A (p = 0.022), IL-6 (p = 0.028) and IL-21 (p = 0.003) concentrations were increased in supernatants from activated JSLE PBMCs. IL-17 F (p = 0.50) and IL-22 (p = 0.43) were also increased but were not statistically significant. IL17A and IL23 mRNA was significantly higher in JSLE PBMCs (p = 0.018 and p = 0.01). CONCLUSION: JSLE T cells have an increased ability to secrete Th17 associated cytokines once activated, which could contribute to the pro-inflammatory disease phenotype seen in these patients.