RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most intractable and devastating malignant tumors. Epigenetic modifications such as DNA methylation and histone modification regulate tumor initiation and progression. However, the contribution of histone variants in PDAC is unknown. Here, we demonstrated that the histone variant H2A.Z is highly expressed in PDAC cell lines and PDAC patients and that its overexpression correlates with poor prognosis. Moreover, all three H2A.Z isoforms (H2A.Z.1, H2A.Z.2.1, and H2A.Z.2.2) are highly expressed in PDAC cell lines and PDAC patients. Knockdown of these H2A.Z isoforms in PDAC cell lines induces a senescent phenotype, cell cycle arrest in phase G2/M, increased expression of cyclin-dependent kinase inhibitor CDKN2A/p16, SA-ß-galactosidase activity and interleukin 8 production. Transcriptome analysis of H2A.Z-depleted PDAC cells showed altered gene expression in fatty acid biosynthesis pathways and those that regulate cell cycle and DNA damage repair. Importantly, depletion of H2A.Z isoforms reduces the tumor size in a mouse xenograft model in vivo and sensitizes PDAC cells to gemcitabine. Overexpression of H2A.Z.1 and H2A.Z.2.1 more than H2A.Z.2.2 partially restores the oncogenic phenotype. Therefore, our data suggest that overexpression of H2A.Z isoforms enables cells to overcome the oncoprotective barrier associated with senescence, favoring PDAC tumor grow and chemoresistance. These results make H2A.Z a potential candidate as a diagnostic biomarker and therapeutic target for PDAC.
Assuntos
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , beta-Galactosidase/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Envelhecimento/genética , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica/genética , Dano ao DNA/efeitos dos fármacos , Metilação de DNA/genética , Reparo do DNA/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética/genética , Xenoenxertos , Histonas/genética , Humanos , Camundongos , GencitabinaRESUMO
Flaviviruses are positive, single-stranded, enveloped cytoplasmic sense RNA viruses that cause a variety of important diseases worldwide. Among them, Zika virus, West Nile virus, Japanese encephalitis virus, and Dengue virus have the potential to cause severe disease. Extensive studies have been performed to elucidate the structure and replication strategies of flaviviruses, and current studies are aiming to unravel the complex molecular interactions between the virus and host during the very early stages of infection. The outcomes of viral infection and rapid establishment of the antiviral state, depends on viral detection by pathogen recognition receptors and rapid initiation of signalling cascades to induce an effective innate immune response. Extracellular and intracellular pathogen recognition receptors play a crucial role in detecting flavivirus infection and inducing a robust antiviral response. One of the main hallmarks of flaviviral nonstructural proteins is their multiple strategies to antagonise the interferon system. In this chapter, we summarize the molecular characteristics of flaviviral proteins and discuss how viral proteins target different components of the interferon signalling pathway by blocking phosphorylation, enhancing degradation, and downregulating the expression of major components of the Janus kinase/signal transducer and activator of transcription pathway. We also discuss how the interactions of viral proteins with host proteins facilitate viral pathogenesis. Due to the lack of antivirals or prophylactic treatments for many flaviviral infections, it is necessary to fully elucidate how these viruses disrupt cellular processes to influence pathogenesis and disease outcomes.
Assuntos
Infecções por Flavivirus/imunologia , Flavivirus/imunologia , Imunidade Inata , Interferons/imunologia , Transdução de Sinais/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Flavivirus/patogenicidade , Infecções por Flavivirus/patologia , Humanos , Janus Quinases/imunologiaRESUMO
This study was undertaken to evaluate the feasibility of using recombinant dengue proteins to discriminate between acute dengue infections versus uninfected dengue samples. Dengue virus proteins E, NS1, NS3, and NS4B were cloned as fusion proteins and expressed in Escherichia coli. Recombinant products were tested in 100 serum samples obtained from acute dengue fever cases collected from 3 states of Mexico where dengue is endemic. Sera from 75 healthy individuals living in nonendemic areas for dengue were used as a control group. In sera from the dengue patients group, antibody responses to E protein were demonstrated in 91% of cases and NS1 protein was recognized to various extents (99%) within the first 7 days of infection. The antibody responses to NS3 and NS4B were frequently of low magnitude. Consistent negative antibody responses to all proteins were found in sera from the control group. These data suggest that the glutathione-S-transferase (GST)-dengue fusion proteins may be feasible antigens for a sensitive and specific serological assay.
Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Vírus da Dengue/imunologia , Dengue/diagnóstico , Proteínas não Estruturais Virais/imunologia , Adolescente , Adulto , Western Blotting , Estudos de Casos e Controles , Criança , Dengue/epidemiologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Estudos de Viabilidade , Feminino , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Masculino , México/epidemiologia , Pessoa de Meia-Idade , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Testes SorológicosRESUMO
Attenuated Salmonella strains are used widely as live carriers of antigens because they elicit both mucosal and systemic immunity against passenger antigens. However, they generally evoke poor cytotoxic T cell (CTL) responses because Salmonella resides within vacuolar compartments and the passenger antigens must travel to the cytosol and be processed through the MHC class I-dependent pathway to simulate CTLs. To address this problem, we designed a fusion protein to destabilize the phagosome membrane and allow a dengue epitope to reach the cytosol. The fusion protein was displayed on the bacterial surface of Salmonella enterica serovar Typhimurium SL3261 through the beta domain of the autotransporter MisL. The passenger alpha domain contained, from the N-terminus, a fusogenic sequence, the NS3 protein 298-306-amino acid CTL epitope from the dengue virus type 2, a molecular tag, and a recognition site for the protease OmpT to release it to the milieu. Display of the fusion protein on the bacterial surface was demonstrated by IFA and flow cytometry using antibodies against the molecular tag. Cleavage of the fusogenic protein-dengue peptide was demonstrated by flow cytometry using OmpT+ Escherichia coli strains. The recombinant Salmonella strains displaying the fusogenic-dengue peptide were able to lyse erythrocytes, induced specific proliferative responses, and elicited CTL responses. These results suggest that the recombinant fusion proteins containing fusogenic sequences provide a promising system to induce CTLs by live vector vaccines.
Assuntos
Vacinas contra Dengue/biossíntese , Vacinas contra Dengue/imunologia , Salmonella enterica/metabolismo , Linfócitos T Citotóxicos/imunologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Cromo/metabolismo , Dengue/imunologia , Vacinas contra Dengue/genética , Vírus da Dengue/imunologia , Epitopos/imunologia , Eritrócitos/efeitos dos fármacos , Escherichia coli/metabolismo , Citometria de Fluxo , Imunofluorescência , Hemólise/efeitos dos fármacos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Oligonucleotídeos , Plasmídeos , Salmonella enterica/genética , Ovinos , Vacinas de Subunidades Antigênicas/biossíntese , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Proteínas Virais de Fusão/biossíntese , Proteínas Virais de Fusão/imunologiaRESUMO
Dengue is considered a reemerging disease of worldwide distribution. The Dengue virus non-structural protein 3 (NS3) is known to possess ATPase, helicase, and protease activities that are a constitutive part of the replication complex of Dengue virus. In this report, we discuss the cloning, expressing, and purifying of the Dengue-2 NS3 protein, to immunize mice and then generate monoclonal antibodies (MAbs). Our results show the production of MAbs specific to NS3 protein of Dengue-2 virus, which by immunofluorescence recognize the native protein in experimentally infected endothelial cells (HMEC). Likewise, C6/36-infected lisates were used in Western blots, and observed the specific characteristic band that defines the NS3 protein. We conclude that these antibodies may be a useful tool, not only to study the replicative process of Dengue virus, but also to generate specific diagnostic tools for Dengue infection.
Assuntos
Adenosina Trifosfatases/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Vírus da Dengue/imunologia , Imunização , Serina Endopeptidases/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Western Blotting , Clonagem Molecular , Vírus da Dengue/química , Vírus da Dengue/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Células Endoteliais/virologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Técnica Indireta de Fluorescência para Anticorpo , Glutationa Transferase/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismoRESUMO
The transfer of spleen cells from (BALB/c x C57Bl/6) F1 mice recovered from a Plasmodium chabaudi chabaudi AS infection into irradiated syngeneic recipients conferred protection. Neither elimination of Thy-1+ cells nor in vitro irradiation of immune cells before transfer affected protection while both anti-Thy-1 treatment and irradiation abolished the appearance of anti-P. c. chabaudi antibodies in the recipients. Superinfection of immune spleen cell donors did not improve their capability to transfer protection which was also unaffected by anti-Thy-1 treatment. The serum of mice after one infection was only marginally protective when transferred into irradiated recipients and a second infection improved the protective activity of serum which was not further improved by six infections. The co-transfer of immune serum and immune cells did not result in any synergistic effect. On the other hand, when P. c. chabaudi AS (BALB/c x C57Bl/6) F1 infected mice were challenged with a high dose of Plasmodium yoelii 17XL at crisis, the mice were unable to control the heterologous parasite. When mice were challenged with P. yoelii 17XL several weeks after infection with P. c. chabaudi AS, a good degree of cross-protection was observed.