RESUMO
A series of novel 1,2,3-triazole-linked isosteviol derivatives were designed and synthesized via Huisgen-click reaction. Their cytotoxicities in vitro against HCT-116 and JEKO-1 cells were screened. The preliminary bioassays indicated that most of the title compounds exhibited noteworthy cytotoxic activities. Particularly, the compound 10b revealed the most potent inhibitory activities against HCT-116 cells with IC50 value of 2.987±0.098µM, which was better than that (3.906±0.261µM) of positive control cisplatin. On the basis of these bioactivity data, hologram quantitative structure-activity relationship (HQSAR) was performed, and a statistically reliable model with good predictive power (r2=0.848, q2=0.544 and R2pred=0.982) was achieved. Additionally, the contribution maps derived from the optimal model explained the individual atomic contributions to the activity for each molecule.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Diterpenos do Tipo Caurano/química , Diterpenos do Tipo Caurano/farmacologia , Triazóis/química , Triazóis/farmacologia , Antineoplásicos/síntese química , Sobrevivência Celular/efeitos dos fármacos , Química Click , Diterpenos do Tipo Caurano/síntese química , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Relação Quantitativa Estrutura-Atividade , Triazóis/síntese químicaRESUMO
Enterovirus 71 (EV71) causes hand, foot, and mouth disease and severe neurological disorders in children. Human scavenger receptor class B member 2 (hSCARB2) and P-selectin glycoprotein ligand-1 (PSGL-1) are identified as receptors for EV71. The underling mechanism of PSGL-1-mediated EV71 entry remains unclear. The endocytosis required for EV71 entry were investigated in Jurkat T and mouse L929 cells constitutively expressing human PSGL-1 (PSGL-1-L929) or human rhabdomyosarcoma (RD) cells displaying high SCARB2 but no PSGL-1 by treatment of specific inhibitors or siRNA. We found that disruption of clathrin-dependent endocytosis prevented EV71 infection in RD cells, while there was no influence in Jurkat T and PSGL-1-L929 cells. Disturbing caveolar endocytosis by specific inhibitor or caveolin-1 siRNA in Jurkat T and PSGL-1-L929 cells significantly blocked EV71 infection, whereas it had no effect on EV71 infection in RD cells. Confocal immunofluorescence demonstrated caveola, and EV71 was directly colocalized. pH-dependent endosomal acidification and intact membrane cholesterol were important for EV71 infection, as judged by the pretreatment of inhibitors that abrogated the infection. A receptor-dominated endocytosis of EV71 infection was observed: PSGL-1 initiates caveola-dependent endocytosis and hSCARB2 activates clathrin-dependent endocytosis.
Assuntos
Endocitose , Enterovirus Humano A/fisiologia , Glicoproteínas de Membrana/metabolismo , Receptores Virais/metabolismo , Internalização do Vírus , Animais , Caveolina 1/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Microscopia ConfocalRESUMO
Coxsackievirus A10 (CVA10) causes hand, foot, and mouth disease (HFMD) and herpangina, which can result in severe neurological symptoms in children. CVA10 does not use the common enterovirus 71 (EV71) receptor, human SCARB2 (hSCARB2, scavenger receptor class B, member 2), for infection but instead uses another receptor, such as KREMEN1. Our research has shown that CVA10 can infect and replicate in mouse cells expressing human SCARB2 (3T3-SCARB2) but not in the parental NIH3T3 cells, which do not express hSCARB2 for CVA10 entry. Knocking down endogenous hSCARB2 and KREMEN1 with specific siRNAs inhibited CVA10 infection in human cells. Co-immunoprecipitation confirmed that VP1, a main capsid protein where virus receptors for attaching to the host cells, could physically interact with hSCARB2 and KREMEN1 during CVA10 infection. It is the efficient virus replication following virus attachment to its cellular receptor. It resulted in severe limb paralysis and a high mortality rate in 12-day-old transgenic mice challenged with CVA10 but not in wild-type mice of the same age. Massive amounts of CVA10 accumulated in the muscles, spinal cords, and brains of the transgenic mice. Formalin inactivated CVA10 vaccine-induced protective immunity against lethal CVA10 challenge and reduced the severity of disease and tissue viral loads. This is the first report to show that hSCARB2 serves as an associate to aid CVA10 infection. hSCARB2-transgenic mice could be useful in evaluating anti-CVA10 medications and studying the pathogenesis induced by CVA10.
Assuntos
Enterovirus Humano A , Infecções por Enterovirus , Enterovirus , Doença de Mão, Pé e Boca , Criança , Humanos , Camundongos , Animais , Células NIH 3T3 , Camundongos Transgênicos , Receptores Depuradores/genética , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismoRESUMO
Nasopharyngeal carcinoma (NPC) is an endemic malignancy prevalent in South East Asia. Epidemiological studies have associated this disease closely with Epstein-Barr virus (EBV) infection. Previous studies also showed that EBV reactivation is implicated in the progression of NPC. Thus, we proposed that recurrent reactivations of EBV may be important for its pathogenic role. In this study, NPC cell lines latently infected with EBV, NA and HA, and the corresponding EBV-negative NPC cell lines, NPC-TW01 (TW01) and HONE-1, were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium n-butyrate (SB) for lytic cycle induction. A single treatment with TPA/SB revealed that DNA double-strand breaks and formation of micronuclei (a marker for genome instability) were associated with EBV reactivation in NA and HA cells. Examination of EBV early genes had identified several lytic proteins, particularly EBV DNase, as potent activators that induced DNA double-strand breaks and contribute to genome instability. Recurrent reactivations of EBV in NA and HA cells resulted in a marked increase of genome instability. In addition, the degree of chromosomal aberrations, as shown by chromosome structural variants and DNA copy-number alterations, is proportional to the frequency of TPA/SB-induced EBV reactivation. Whereas these DNA abnormalities were limited in EBV-negative TW01 cells with mock or TPA/SB treatment, and were few in mock-treated NA cells. The invasiveness and tumorigenesis assays also revealed a profound increase in both characteristics of the repeatedly reactivated NA cells. These results suggest that recurrent EBV reactivations may result in accumulation of genome instability and promote the tumor progression of NPC.
Assuntos
Infecções por Vírus Epstein-Barr/virologia , Instabilidade Genômica , Herpesvirus Humano 4/fisiologia , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/virologia , Ativação Viral/efeitos dos fármacos , Animais , Butiratos/farmacologia , Carcinógenos/farmacologia , Hibridização Genômica Comparativa , Dano ao DNA/efeitos dos fármacos , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/patologia , Imunofluorescência , Dosagem de Genes , Regulação Viral da Expressão Gênica , Genoma Viral/efeitos dos fármacos , Genoma Viral/fisiologia , Humanos , Camundongos , Camundongos SCID , Neoplasias Nasofaríngeas/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/farmacologia , Recidiva , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas , Replicação Viral/efeitos dos fármacosRESUMO
Enterovirus 71 (EV71) has emerged as a neurological virus causing life-threatening diseases in young children and infants. Although EV71 vaccines in development have presented promising results in several clinical trials, the identified key antigen for improving the broad protective efficacy of EV71 vaccines has not been well investigated. In this report, we show that different multiplicities of infection (MOIs) of the B4(E59) virus significantly affect EV71 vaccine production in a serum-free microcarrier bioreactor system. The antigens produced from high MOIs of 10-1 and 10-2 exhibited higher yield and more infectious full particle (FP) contents in the EV71 vaccines than those produced with low MOIs of 10-4 and 10-6, leading to better cross-neutralizing efficacy. The C4(E36) neutralization results showed that only antisera raised from EV71 FPs provided substantial neutralizing titers against C4(E36), whereas empty particles (EPs) of EV71 conferred no efficacy. Competitive ELISA showed that anti-FP mainly binds to FPs and that 20% of antibodies bind to EPs, whereas most anti-EP binds EPs, with only 10% antibodies binding to FPs. VP1-adsorbed anti-FP lost most of the virus neutralization efficiency, suggesting that the VP1 subunit of FP is the major immunogenic antigen determining the ability of the EV71 vaccine to elicit cross-neutralizing antibodies against EV71 virus subtypes. These findings demonstrate that the high-MOI production approach is significantly correlated with FP productivity, thereby improving the cross-neutralization efficacy of an EV71 vaccine and providing the basis for a better vaccine design against widespread EV71 viruses.
Assuntos
Anticorpos Neutralizantes/biossíntese , Enterovirus/genética , Enterovirus/imunologia , Vírion/imunologia , Animais , Especificidade de Anticorpos , Chlorocebus aethiops , Enterovirus/ultraestrutura , Genótipo , Humanos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Células Vero , Vacinas Virais/imunologia , Vírion/ultraestruturaRESUMO
We evaluated the efficacy of a recombinant adenovirus that expresses a membrane-truncated respiratory syncytial virus (RSV) fusion protein (Ad-F0ΔTM) in newborns via maternal immunization (MI) of pregnant cotton rats. Intranasal Ad-F0ΔTM immunization was given to pregnant female rats, and MI-newborn rats were then challenged intranasally with RSV. Anti-RSV IgGs were observed in the serum of MI-newborn rats after birth. The pulmonary viral loads in Ad-F0ΔTM vs. control vector, Ad-LacZ, and MI-newborns on day 3 post-challenge were reduced by 4â¯log10/g lung. The neutralizing antibody remained for up to 3 weeks in the serum of MI-newborns, which is when weaning began. Ad-F0ΔTM protected MI-newborns from RSV challenge for 1 week. Vertical-transferred protective antibodies were examined in the breast milk and placenta as well. Finally, anti-RSV immunity was not boosted but was only primed during the next RSV exposure in Ad-F0ΔTM-MI-newborns. Maternal Ad-F0ΔTM immunization provides acute protection against RSV infection in neonates.
Assuntos
Anticorpos Antivirais/sangue , Portadores de Fármacos/administração & dosagem , Imunidade Materno-Adquirida , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vacinação/métodos , Adenoviridae/genética , Animais , Animais Recém-Nascidos , Anticorpos Neutralizantes/sangue , Feminino , Vetores Genéticos , Pulmão/virologia , Leite Humano/imunologia , Placenta/imunologia , Gravidez , Infecções por Vírus Respiratório Sincicial/virologia , Vacinas contra Vírus Sincicial Respiratório/genética , Vírus Sinciciais Respiratórios/imunologia , Sigmodontinae , Resultado do Tratamento , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Carga ViralRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMO
Enterovirus 71 (EV71) is a major cause of hand, foot and mouth disease (HFMD). The current EV71 propagating in Vero (EV-V) or sub-passaged in RD (EV-R) cells was used as a pathogen. Interestingly, EV-R exhibited differential virulence; challenging human scavenger receptor class B2-expressing (hSCARB2-Tg) mice with EV71 revealed that EV-V was more virulent than EV-R: 100% of mice that received lethal amounts of EV-V died, while all the mice that received EV-R survived. Severe pathogenesis correlated with viral burdens and proinflammatory cytokine levels were observed in EV-V-challenged mice, but controversy in EV-R-challenged mice. Consensus sequence analysis revealed EV-R rapidly acquired complete mutations at E145G and S241L and partial mutations at V146I of VP1, and acquired a T to C substitution at nucleotide 494 of the 5'-UTR. EV-R exhibited higher binding affinity for another EV71 receptor, human P-selectin glycoprotein ligand-1 (hPSGL-1), than EV-V. Both EV71s exhibited no significant difference in binding to hSCARB2. The molecular modelling indicate that these mutations might influence EV71 engagement with PSGL-1 and in vivo virulence.
Assuntos
Regiões 5' não Traduzidas , Enterovirus Humano A/crescimento & desenvolvimento , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/patologia , Glicoproteínas de Membrana/metabolismo , Mutação , Proteínas Estruturais Virais/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Citocinas/sangue , Análise Mutacional de DNA , Modelos Animais de Doenças , Infecções por Enterovirus/virologia , Humanos , Camundongos , Receptores Virais/metabolismo , Análise de Sobrevida , Carga Viral , Proteínas Virais , Proteínas Estruturais Virais/metabolismo , Virulência , Ligação ViralRESUMO
Two series of novel acylthiosemicarbazide and oxadiazole fused-isosteviol derivatives were synthesized based on the 19-carboxyl modification. The target compounds were evaluated for their cytotoxicities against three cancer cell lines (HCT-116, HGC-27, and JEKO-1) using an MTT assay. Lead compounds from the acylthiosemicarbazides (4) showed IC50 values in the lower micromolar range. For example, compounds (4i, 4l, 4m, 4r, and 4s) exhibited significant inhibitory activities against the three cell lines with IC50 values of 0.95-3.36 µm. Furthermore, 2D-HQSAR and 3D-topomer CoMFA analyses were established, which could be used to develop second generation of isosteviol derivatives as anticancer agents.
Assuntos
Simulação por Computador , Diterpenos do Tipo Caurano/síntese química , Diterpenos do Tipo Caurano/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Diterpenos do Tipo Caurano/química , Células HCT116 , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Neoplasias/tratamento farmacológico , Oxidiazóis/síntese química , Oxidiazóis/química , Oxidiazóis/farmacologia , Relação Quantitativa Estrutura-Atividade , Semicarbazidas/síntese química , Semicarbazidas/química , Semicarbazidas/farmacologiaRESUMO
We have developed an efficient cell culture process to scale up the production of a recombinant adenovirus that expresses the membrane-trunked fusion protein of respiratory syncytial virus (RSV; Ad-F0ΔTM). Adherent cells of human embryonic kidney (HEK) 293-derived cell, 293A, which supports the production of E1/E3-deleted Ad-F0ΔTM when cultured in the presence of fetal bovine serum (FBS), were adapted to suspension growth under serum-free medium. In doing so, we studied the immunogenicity of Ad-F0ΔTMsus, which propagated in a bioreactor that was cultured with serum-free suspension of 293A, in comparison with Ad-F0ΔTMadh, which was produced from parental 293A cells that were adherently cultured in medium containing FBS. The size and morphology of Ad-F0ΔTMsus and Ad-F0ΔTMadh virions were identical upon inspection with electron microscopy. The results showed that anti-F IgG and RSV-neutralizing titer were raised in the serum of both mice that were intranasally immunized twice with Ad-F0ΔTMsus or Ad-F0ΔTMadh at two-week injection intervals. Furthermore, the immune responses persisted for six months after vaccination. Activation of F protein-specific CD8(+) T cell's epitope associated IFN-É£ and IL-4 was induced in both Ad-F0ΔTMsus- and Ad-F0ΔTMadh, but not in Ad-LacZsus, -immunized mouse splenocytes. No vaccine-enhanced lung inflammation, airway mucus occlusion or eosinophils infiltration were observed in Ad-immunized mice followed by RSV challenge; however, these symptoms were observed following immunization with formalin-inactivated RSV vaccine. These results indicate that the safety and potency of Ad-F0ΔTM produced from either adherent cells or suspension and serum-free cells are the same.
Assuntos
Adenoviridae/genética , Vetores Genéticos/genética , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/imunologia , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Adenoviridae/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Técnicas de Cultura de Células , Linhagem Celular , Meios de Cultura Livres de Soro , Modelos Animais de Doenças , Expressão Gênica , Vetores Genéticos/imunologia , Células HEK293 , Humanos , Imunização Secundária , Camundongos , Testes de Neutralização , Ratos , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/genética , Vacinas de Produtos Inativados/imunologiaRESUMO
A series of novel isosteviol derivatives bearing amino alcohol and thiourea fragments have been stereo-selectively synthesized and screened for their in vitro cytotoxic activities against three human cancer cell lines (HCT-116, HGC-27 and JEKO-1). The results demonstrated that these compounds exhibited prominent cytotoxicities. Especially, the compound Iw displayed the most potent anticancer activities against HCT-116 cell with IC50 value of 1.450 µM. On the basis of this bioassay results, these derivatives were further investigated by the hologram quantitative structure-activity relationship (HQSAR) technique. The optimal HQSAR model with q(2) = 0.663, r(2) = 0.895, SEE = 0.179 was generated using A/B/H/Ch as fragment distinction parameters and 4-7 as fragment size. This model was employed to predict the cytotoxic activities of test set compounds, and the predicted values were in good agreement with the experimental results. The contribution maps derived from the optimal model explained the individual atomic contribution to the total activity of single molecule.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Diterpenos do Tipo Caurano/química , Diterpenos do Tipo Caurano/farmacologia , Relação Quantitativa Estrutura-Atividade , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Diterpenos do Tipo Caurano/síntese química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura MolecularRESUMO
OBJECTIVES: The regulation of the immunopathology of respiratory syncytial virus (RSV) by regulatory T-cells (CD4(+)CD25(+)Foxp3(+); Tregs) is not understood. METHODS: To deduce the same, Tregs were depleted in BALB/c mice by injecting anti-CD25 antibody followed by RSV infection (anti-CD25-RSV mice). RESULTS: In this model, a decrease in anti-fusion (F) antibody and neutralizing activity, and an increase in anti-nucleocapsid (N) antibody in serum, were seen. Decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activity, increased IgG2a, and an influx of activated CD8(+) T-cells into the lungs were also observed. Co-culture of splenic CD45RA(+) B-cells from RSV-infected normal mice with CD4(+) cells isolated from anti-CD25-RSV mice (B/CD4) increased anti-F antibody secretion. The inclusion of CD25(+) Tregs isolated from isotype Ig-RSV mice into the B/CD4 co-culture substantially enhanced the frequency of anti-F antibody production. However, the same effect was not seen in the co-culture of CD45RA(+) B-cells with dendritic cells (DCs) (B/DCs) or CD8(+) cells (B/CD8) that were obtained from anti-CD25-RSV mice. The transfer of enriched B-cells from anti-CD25-RSV mice into RSV-infected SCID mice increased severe lung inflammation associated with the increased viral load and eosinophil number. CONCLUSIONS: These results indicate that Tregs modulate B-cell activity, particularly in producing F-specific neutralizing antibodies, to regulate RSV-mediated exacerbated diseases.
Assuntos
Linfócitos B/fisiologia , Infecções por Vírus Respiratório Sincicial/imunologia , Linfócitos T Reguladores/fisiologia , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Linfócitos T CD8-Positivos/imunologia , Feminino , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/imunologia , Baço/patologiaRESUMO
Enterovirus 71 (EV71) and coxsackieviruses (CV) are the major causative agents of hand, foot and mouth disease (HFMD). There is not currently a vaccine available against HFMD, even though a newly developed formalin-inactivated EV71 (FI-EV71) vaccine has been tested in clinical trial and has shown efficacy against EV71. We have designed and genetically engineered a recombinant adenovirus Ad-EVVLP with the EV71 P1 and 3CD genes inserted into the E1/E3-deleted adenoviral genome. Ad-EVVLP were produced in HEK-293A cells. In addition to Ad-EVVLP particles, virus-like particles (VLPs) formed from the physical association of EV71 capsid proteins, VP0, VP1, and VP3 expressed from P1 gene products. They were digested by 3CD protease and confirmed to be produced by Ad-EVVLP-producing cells, as determined using transmission electron microscopy and western blotting. Mouse immunogenicity studies showed that Ad-EVVLP-immunized antisera neutralized the EV71 B4 and C2 genotypes. Activation of VLP-specific CD4+ and CD8+/IFN-γ T cells associated with Th1/Th2-balanced IFN-ɣ, IL-17, IL-4, and IL-13 was induced; in contrast, FI-EV71 induced only Th2-mediated neutralizing antibody against EV71 and low VLP-specific CD4+ and CD8+ T cell responses. The antiviral immunity against EV71 was clearly demonstrated in mice vaccinated with Ad-EVVLP in a hSCARB2 transgenic (hSCARB2-Tg) mouse challenge model. Ad-EVVLP-vaccinated mice were 100% protected and demonstrated reduced viral load in both the CNS and muscle tissues. Ad-EVVLP successfully induced anti-CVA16 immunities. Although antisera had no neutralizing activity against CVA16, the 3C-specific CD4+ and CD8+/IFN-γ T cells were identified, which could mediate protection against CVA16 challenge. FI-EV71 did not induce 3C-mediated immunity and had no efficacy against the CVA16 challenge. These results suggest that Ad-EVVLP can enhance neutralizing antibody and protective cellular immune responses to prevent EV71 infection and cellular immune responses against CV infection.
Assuntos
Proteínas do Capsídeo/imunologia , Enterovirus Humano A/imunologia , Doença de Mão, Pé e Boca/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Feminino , Engenharia Genética , Doença de Mão, Pé e Boca/virologia , Soros Imunes/imunologia , Interleucina-13/imunologia , Interleucina-17 , Interleucina-4/imunologia , Camundongos , Camundongos Transgênicos , Vacinas de Produtos Inativados/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Nasopharyngeal carcinoma (NPC) is a head and neck cancer prevalent throughout Southern China and Southeast Asia. Patient death following relapse after primary treatment remains all too common but the cause of NPC relapse is unclear. Clinical and epidemiological studies have revealed the high correlation among NPC development, Epstein-Barr virus (EBV) reactivation and host genomic instability. Previously, recurrent EBV reactivation was shown to cause massive genetic alterations and enhancement of tumor progression in NPC cells and these may be required for NPC relapse. Here, EBV BALF3 has the ability to induce micronuclei and DNA strand breaks. After recurrent expression of BALF3 in NPC cells, genomic copy number aberrations, determined by array-based comparative genomic hybridization, had accumulated to a significant extent and tumorigenic features, such as cell migration, cell invasion and spheroid formation, increased with the rounds of induction. In parallel experiments, cells after highly recurrent induction developed into larger tumor nodules than control cells when inoculated into NOD/SCID mice. Furthermore, RNA microarrays showed that differential expression of multiple cancer capability-related genes and oncogenes increased with recurrent BALF3 expression and these changes correlated with genetic aberrations. Therefore, EBV BALF3 is a potential factor that mediates the impact of EBV on NPC relapse.
Assuntos
Transformação Celular Viral , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Instabilidade Genômica , Herpesvirus Humano 4/metabolismo , Neoplasias Nasofaríngeas/virologia , Proteínas Virais/metabolismo , Animais , Antineoplásicos/farmacologia , Carcinoma , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Variações do Número de Cópias de DNA , Dano ao DNA , Proteínas de Ligação a DNA/genética , Doxiciclina/farmacologia , Endodesoxirribonucleases/genética , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Micronúcleos com Defeito Cromossômico , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Interferência de RNA , Fatores de Tempo , Transfecção , Carga Tumoral , Proteínas Virais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although several factors participating in enterovirus 71 (EV71) entry and replication had been reported, the precise mechanisms associated with these events are far from clear. In the present study, we showed that heat shock protein 90 (HSP90) is a key element associated with EV71 entry and replication in a human rhabdomyosarcoma of RD cells. Inhibition of HSP90 by pretreating host cells with HSP90ß siRNA or blocking HSP90 with a HSP90-specific antibody or geldanamycin (GA), a specific inhibitor of HSP90, as well as recombinant HSP90ß resulted in inhibiting viral entry and subsequent viral replication. Co-immunprecipitation of EV71 with recombinant HSP90ß and colocalization of EV71-HSP90 in the cells demonstrated that HSP90 was physically associated with EV71 particles. HSP90 seems to mediate EV71 replication by preventing proteosomal degradation of the newly synthesized capsid proteins, but does not facilitate viral gene expression at transcriptional level. This was evident by post-treatment of host cells with GA, which did not affect the expression of viral transcripts but accelerated the degradation of viral capsid proteins and interfered with the formation of assembled virions. In vivo studies were carried out using human SCARB2-transgenic mice to evaluate the protection conferred by HSP90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), an analog of geldanamycin, that elicited similar activity but with less toxicity. The results showed that the administration of 17-AAG twice conferred the resistance to hSCARB2 mice challenged with C2, C4, and B4 genotypes of EV71. Our data supports HSP90 plays an important role in EV71 infection. Targeting of HSP90 with clinically available drugs might provide a feasible therapeutic approach to treat EV71 infection.
Assuntos
Benzoquinonas/farmacologia , Enterovirus Humano A/efeitos dos fármacos , Infecções por Enterovirus/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Terapia de Alvo Molecular , Vírion/efeitos dos fármacos , Animais , Anticorpos/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Enterovirus Humano A/genética , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/genética , Infecções por Enterovirus/virologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Transgênicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Células Vero , Vírion/genética , Vírion/patogenicidade , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Enterovirus 71 (EV71) and coxsackievirus (CVA) are the most common causative factors for hand, foot, and mouth disease (HFMD) and neurological disorders in children. Lack of a reliable animal model is an issue in investigating EV71-induced disease manifestation in humans, and the current clinical therapies are symptomatic. We generated a novel EV71-infectious model with hSCARB2-transgenic mice expressing the discovered receptor human SCARB2 (hSCARB2). The challenge of hSCARB2-transgenic mice with clinical isolates of EV71 and CVA16 resulted in HFMD-like and neurological syndromes caused by E59 (B4) and N2838 (B5) strains, and lethal paralysis caused by 5746 (C2), N3340 (C4), and CVA16. EV71 viral loads were evident in the tissues and CNS accompanied the upregulated pro-inflammatory mediators (CXCL10, CCL3, TNF-α, and IL-6), correlating to recruitment of the infiltrated T lymphocytes that result in severe diseases. Transgenic mice pre-immunized with live E59 or the FI-E59 vaccine was able to resist the subsequent lethal challenge with EV71. These results indicate that hSCARB2-transgenic mice are a useful model for assessing anti-EV71 medications and for studying the pathogenesis induced by EV71.
Assuntos
Enterovirus Humano A/genética , Doença de Mão, Pé e Boca/genética , Proteínas de Membrana Lisossomal/genética , Receptores Depuradores/genética , Animais , Células Cultivadas , Chlorocebus aethiops , Infecções por Coxsackievirus/genética , Infecções por Coxsackievirus/imunologia , Modelos Animais de Doenças , Enterovirus/genética , Enterovirus/imunologia , Enterovirus Humano A/imunologia , Infecções por Enterovirus/genética , Infecções por Enterovirus/imunologia , Genótipo , Doença de Mão, Pé e Boca/imunologia , Humanos , Inflamação/imunologia , Proteínas de Membrana Lisossomal/imunologia , Camundongos , Camundongos Transgênicos , Receptores Depuradores/imunologia , Linfócitos T/imunologia , Regulação para Cima/genética , Regulação para Cima/imunologia , Células VeroRESUMO
Identification of HLA-restricted CD8+ T cell epitopes is important to study RSV-induced immunity and illness. We algorithmically analyzed the sequence of the fusion protein (F) of respiratory syncytial virus (RSV) and generated synthetic peptides that can potentially bind to HLA-A*0201. Four out of the twenty-five 9-mer peptides tested: peptides 3 (F33-41), 13 (F214-222), 14 (F273-281), and 23 (F559-567), were found to bind to HLA-A*0201 with moderate to high affinity and were capable of inducing IFN-γ and IL-2 secretion in lymphocytes from HLA-A*0201 transgenic (HLA-Tg) mice pre-immunized with RSV or recombinant adenovirus expressing RSV F. HLA-Tg mice were immunized with these four peptides and were found to induce both Th1 and CD8+ T cell responses in in vitro secondary recall. Effector responses induced by these peptides were observed to confer differential protection against live RSV challenge. These peptides also caused better recovery of body weight loss induced by RSV. A significant reduction of lung viral load was observed in mice immunized with peptide 23, which appeared to enhance the levels of inflammatory chemokines (CCL17, CCL22, and IL-18) but did not increase eosinophil infiltration in the lungs. Whereas, significant reduction of infiltrated eosinophils induced by RSV infection was found in mice pre-immunized with peptide 13. Our results suggest that HLA-A2-restricted epitopes of RSV F protein could be useful for the development of epitope-based RSV vaccine.
Assuntos
Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Fragmentos de Peptídeos/imunologia , Vírus Sinciciais Respiratórios/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas Virais de Fusão/química , Adenoviridae/genética , Sequência de Aminoácidos , Animais , Antígenos CD8/imunologia , Linhagem Celular , Citocinas/biossíntese , DNA Recombinante/genética , Epitopos/química , Epitopos/imunologia , Feminino , Humanos , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Baço/imunologia , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologia , Proteínas Virais de Fusão/genética , Vacinas Virais/química , Vacinas Virais/imunologiaRESUMO
Two recombinant adenoviruses designated rAd-F0DeltaTM and rAd-F0 carrying the transmembrane truncated and full length of the F gene of the RSV-B1 strain, respectively, were engineered. Comparative immunogenicity studies in BALB/c mice showed that each vector was capable of inducing RSV-B1-specific antibodies that cross-reacted with the RSV-long and RSV-A2 viruses. The anti-RSV-B1 antibodies were neutralizing, and exhibited strong cross-neutralizing activity against the RSV-long and RSV-A2 isolates as well. Analysis of the cellular responses revealed that animals immunized with rAd-F0DeltaTM and rAd-F0 elicited CD4(+) T-cell responses of the Th1 and Th2 phenotypes, as well as F protein-specific CTLs. Production of Th2 cytokines (IL-4, IL-5 and IL-13) by splenocytes of the rAd-F0DeltaTM and rAd-F0 immunized mice was markedly lower than those released by animals administered with heat-inactivated RSV-B1 (HIRSV-B1). Comparison of the overall humoral and cellular responses suggests that rAd-F0DeltaTM is significantly more immunogenic than rAd-F0. The anti-viral immunity generated by both recombinant adenovirus vectors has conferred protection against live RSV-B1 challenge as judged by the lower viral load recovered in the lungs, a faster rate of recovery of body weight loss, and a lower count of eosinophils as compared to eosinophilia in mice immunized with HIRSV-B1. Results from these studies suggest that rAd-F0DeltaTM or rAd-F0 possess immunogenic properties that meet the requirements expected from potential RSV vaccine candidates.
Assuntos
Adenoviridae/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/imunologia , Proteínas Virais de Fusão/imunologia , Adenoviridae/genética , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Reações Cruzadas , Citocinas/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Baço/citologia , Baço/imunologia , Proteínas Virais de Fusão/genética , Carga ViralRESUMO
Jaagsiekte sheep retrovirus (JSRV) that causes contagious ovine pulmonary adenocarcinoma (OPA) in sheep carries an oncogenic Envelope gene (Env), which is capable of transforming target cells in vitro and in vivo. We cloned full-length JSRV Env cDNA into an expression vector, SPC/SV40, where the transgene was driven by lung-specific surfactant protein C (SPC) promoter, to obtain SPC-JSRV Env construct. SPC-JSRV Env was microinjected into immunocompetent FVB/N mice embryos to generate Env transgenic mice. We obtained two lines of transgenic mice, both of which were capable of developing spontaneous lung tumors from 1 month onwards and the tumor incidence rate was about 56% at the age of 7 months in Env Transgenic line 1 and about 71% at the age of 6 months in Env Transgenic line 2. We were able to correlate higher tumor incidence rate and tumorigenicity in Env Transgenic line 2 to higher level of expression of Env transgene compared to Env Transgenic line 1. Immunohistochemical analysis showed that the tumor was primarily composed of type II pneumocytes where SPC promoter is known to be active similar to natural infection of JSRV in sheep. Analysis of cellular mitogenic signal transduction pathways revealed significant induction of p44/42 ERK pathway in the transgenic mice lungs with tumors compared to the lungs from non-transgenic FVB/N mice. Tumors in our transgenic mice pose similarities to human lung adenocarcinoma and therefore our mice could serve as a model system for evaluating the mechanisms of lung tumorigenesis in vivo.