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1.
J Virol ; 87(10): 5746-54, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23487452

RESUMO

H7 subtype influenza A viruses, responsible for numerous outbreaks in land-based poultry in Europe and the Americas, have caused over 100 cases of confirmed or presumed human infection over the last decade. The emergence of a highly pathogenic avian influenza H7N3 virus in poultry throughout the state of Jalisco, Mexico, resulting in two cases of human infection, prompted us to examine the virulence of this virus (A/Mexico/InDRE7218/2012 [MX/7218]) and related avian H7 subtype viruses in mouse and ferret models. Several high- and low-pathogenicity H7N3 and H7N9 viruses replicated efficiently in the respiratory tract of mice without prior adaptation following intranasal inoculation, but only MX/7218 virus caused lethal disease in this species. H7N3 and H7N9 viruses were also detected in the mouse eye following ocular inoculation. Virus from both H7N3 and H7N9 subtypes replicated efficiently in the upper and lower respiratory tracts of ferrets; however, only MX/7218 virus infection caused clinical signs and symptoms and was capable of transmission to naive ferrets in a direct-contact model. Similar to other highly pathogenic H7 viruses, MX/7218 replicated to high titers in human bronchial epithelial cells, yet it downregulated numerous genes related to NF-κB-mediated signaling transduction. These findings indicate that the recently isolated North American lineage H7 subtype virus associated with human conjunctivitis is capable of causing severe disease in mice and spreading to naive-contact ferrets, while concurrently retaining the ability to replicate within ocular tissue and allowing the eye to serve as a portal of entry.


Assuntos
Conjuntivite/virologia , Vírus da Influenza A Subtipo H7N3/patogenicidade , Infecções por Orthomyxoviridae/virologia , Tropismo Viral , Animais , Modelos Animais de Doenças , Células Epiteliais/virologia , Feminino , Furões , Humanos , Vírus da Influenza A Subtipo H7N3/isolamento & purificação , Masculino , México , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/transmissão , Sistema Respiratório/virologia
2.
Mol Cell Biol ; 22(11): 3610-20, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11997498

RESUMO

mtCLIC/CLIC4 (referred to here as mtCLIC) is a p53- and tumor necrosis factor alpha-regulated cytoplasmic and mitochondrial protein that belongs to the CLIC family of intracellular chloride channels. mtCLIC associates with the inner mitochondrial membrane. Dual regulation of mtCLIC by two stress response pathways suggested that this chloride channel protein might contribute to the cellular response to cytotoxic stimuli. DNA damage or overexpression of p53 upregulates mtCLIC and induces apoptosis. Overexpression of mtCLIC by transient transfection reduces mitochondrial membrane potential, releases cytochrome c into the cytoplasm, activates caspases, and induces apoptosis. mtCLIC is additive with Bax in inducing apoptosis without a physical association of the two proteins. Antisense mtCLIC prevents the increase in mtCLIC levels and reduces apoptosis induced by p53 but not apoptosis induced by Bax, suggesting that the two proapoptotic proteins function through independent pathways. Our studies indicate that mtCLIC, like Bax, Noxa, p53AIP1, and PUMA, participates in a stress-induced death pathway converging on mitochondria and should be considered a target for cancer therapy through genetic or pharmacologic approaches.


Assuntos
Apoptose/fisiologia , Canais de Cloreto/metabolismo , Dano ao DNA , Proteínas Proto-Oncogênicas c-bcl-2 , Proteína Supressora de Tumor p53/metabolismo , Animais , Sítios de Ligação/genética , Células Cultivadas , Canais de Cloreto/genética , Expressão Gênica , Genes p53 , Queratinócitos/citologia , Queratinócitos/metabolismo , Potenciais da Membrana , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transfecção , Proteína X Associada a bcl-2
3.
Cancer Res ; 65(2): 562-71, 2005 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-15695400

RESUMO

mtCLIC/CLIC4 is a p53 and tumor necrosis factor alpha (TNFalpha) regulated intracellular chloride channel protein that localizes to cytoplasm and organelles and induces apoptosis when overexpressed in several cell types of mouse and human origin. CLIC4 is elevated during TNFalpha-induced apoptosis in human osteosarcoma cell lines. In contrast, inhibition of NFkappaB results in an increase in TNFalpha-mediated apoptosis with a decrease in CLIC4 protein levels. Cell lines expressing an inducible CLIC4-antisense construct that also reduces the expression of several other chloride intracellular channel (CLIC) family proteins were established in the human osteosarcoma lines SaOS and U2OS cells and a malignant derivative of the mouse squamous papilloma line SP1. Reduction of CLIC family proteins by antisense expression caused apoptosis in these cells. Moreover, CLIC4-antisense induction increased TNFalpha-mediated apoptosis in both the SaOS and U2OS derivative cell lines without altering TNFalpha-induced NFkappaB activity. Reducing CLIC proteins in tumor grafts of SP1 cells expressing a tetracycline-regulated CLIC4-antisense substantially inhibited tumor growth and induced tumor apoptosis. Administration of TNFalpha i.p. modestly enhanced the antitumor effect of CLIC reduction in vivo. These results suggest that CLIC proteins could serve as drug targets for cancer therapy, and reduction of CLIC proteins could enhance the activity of other anticancer drugs.


Assuntos
Apoptose/fisiologia , Neoplasias Ósseas/patologia , Canais de Cloreto/antagonistas & inibidores , DNA Antissenso/genética , NF-kappa B/antagonistas & inibidores , Osteossarcoma/patologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/terapia , Bovinos , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Canais de Cloreto/genética , Humanos , Camundongos , Camundongos Nus , NF-kappa B/metabolismo , Osteossarcoma/genética , Osteossarcoma/terapia , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Immunol Res ; 55(1-3): 135-45, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22948808

RESUMO

While the need for CD4 T cells in the generation of CD8 T cell memory has been well documented, the mechanism underlying their requirement remains unknown. Here, we detail an immunization method capable of generating CD8 memory T cells that are indifferent to CD4 T cell help. Using a subunit vaccination that combines polyIC and an agonistic CD40 antibody, we program protective CD4-independent CD8 T cell memory. When cells generated by combined polyIC/CD40 immunization are compared to cells produced following a CD4-dependent vaccination, Listeria monocytogenes, they display dramatic differences, both phenotypically and functionally. The memory cells generated in a CD4-deficient host by polyIC/CD40 immunization provide protection against secondary infectious challenge, whereas cells generated by LM immunization in the same environment do not. Interestingly, combined polyIC/CD40 immunization generates long-term memory cells with low Blimp-1 and elevated Eomes expression despite high expression of Blimp-1 during the primary response. The potency of combined polyIC/CD40 to elicit CD8+ T cell memory in the absence of CD4 T cells suggests that it could be considered as a vaccine adjuvant in clinical situations where CD4 responses/numbers are compromised.


Assuntos
Antígenos CD40/imunologia , Linfócitos T CD8-Positivos/imunologia , Poli I-C/imunologia , Animais , Anticorpos/imunologia , Antígenos CD40/genética , Genes MHC da Classe II/genética , Imunização , Memória Imunológica , Listeria monocytogenes/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Vacínia/imunologia
5.
Vaccine ; 24(44-46): 6588-93, 2006 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-17030078

RESUMO

Because of the time required to identify and produce an antigenically well-matched pandemic vaccine, vaccines that offer broader cross-reactive immunity and protection are desirable. We have compared a live attenuated influenza vaccine (LAIV) and inactivated influenza vaccine (IIV) based on a related H5 hemagglutinin (HA) from a nonpathogenic avian influenza virus, A/Duck/Pottsdam/1042-6/86 (H5N2), for the ability to induce cross-reactive immunity and/or cross-protective efficacy against a contemporary highly pathogenic H5N1 viruses. Both LAIV and IIV provided cross-protection from systemic infection, severe disease, and death following lethal challenges with antigenically distinct A/Vietnam/1203/2004 (VN/1203) virus. Substantial levels of serum anti-VN/1203 HA IgG were detected in mice that received either IIV or LAIV, while nasal wash anti-VN/1203 HA IgA was detected in mice that received LAIV. Formulation of IIV with alum adjuvant augmented neutralizing antibody responses and protective efficacy. These results demonstrated that vaccination of mice with H5 IIV or LAIV induced a high degree of cross-protection from illness and death following lethal challenges with a heterologous H5N1 virus.


Assuntos
Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Vacinas contra Influenza/administração & dosagem , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de Produtos Inativados/administração & dosagem , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Reações Cruzadas , Modelos Animais de Doenças , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Vacinas de Produtos Inativados/imunologia
6.
Genomics ; 82(1): 57-67, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12809676

RESUMO

Protein kinase C-delta (PKC-delta) is a ubiquitously expressed kinase involved in a variety of cellular signaling pathways including cell growth, differentiation, apoptosis, tumor promotion, and carcinogenesis. While signaling pathways downstream of PKC-delta are well studied, the regulation of the gene has not been extensively analyzed. A mouse genomic DNA fragment containing the PKC-delta gene was sequenced by the primer-walking method, and the subsequent DNA sequence data were used as a query to clone Caenorhabditis elegans and human genomic homologs from the publicly available genomic databases. The genomic structures of C. elegans, mouse, rat, and human PKC-delta were analyzed, and the result revealed that PKC-delta genes comprise 12, 18, 19, and 18 exons for C. elegans, mouse, rat, and human, respectively. The translation start methionine resides in the second exon in mouse and human and in the third exon in rat. The first intron between the first exon and the exon with the translation start methionine in mammalian genes represents a very large gap, as long as 17 kb in human, indicating a complexity involved in gene splicing. Overall exon-intron genomic structure is highly conserved among mammals, while significantly diverged in C. elegans. Putative transcription factor binding sites on the 1.7-kb promoter region of the mouse gene suggest that PKC-delta might be involved in spermatogenesis, embryogenesis, development, brain generation, immune response, oxidative environment, and oncogenesis. Studies on the promoter and subsequent biological testing on mouse keratinocytes indicate that tumor necrosis factor (TNF)-alpha increases the expression of PKC-delta, and this correlates with the time of NFkappaB nuclear translocation and activation. This TNF-alpha-mediated upregulation of PKC-delta is repressed in keratinocytes that are preinfected with IkappaB superrepressor adenovirus, suggesting that NFkappaB is involved directly in PKC-delta expression.


Assuntos
Genes/genética , Isoenzimas/genética , Regiões Promotoras Genéticas , Proteína Quinase C/genética , Animais , Animais Recém-Nascidos , Sequência de Bases , Caenorhabditis elegans/genética , Células Cultivadas , Evolução Molecular , Éxons , Genoma , Genoma Humano , Humanos , Íntrons , Isoenzimas/metabolismo , Queratinócitos/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase C/metabolismo , Ratos , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA , Especificidade da Espécie , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/fisiologia
7.
J Biol Chem ; 279(6): 4632-41, 2004 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-14610078

RESUMO

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to the mitochondria and cytoplasm of keratinocytes and participates in the apoptotic response to stress. We now show that multiple stress inducers cause the translocation of cytoplasmic CLIC4 to the nucleus. Immunogold electron microscopy and confocal analyses indicate that nuclear CLIC4 is detected prior to the apoptotic phenotype. CLIC4 associates with the Ran, NTF2, and Importin-alpha nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress-inducing agents. Deletion or mutation of the nuclear localization signal in the C terminus of CLIC4 eliminates nuclear translocation, whereas N terminus deletion enhances nuclear localization. Targeting CLIC4 to the nucleus via adenoviral transduction accelerates apoptosis when compared with cytoplasmic CLIC4, and only nuclear-targeted CLIC4 causes apoptosis in Apaf null mouse fibroblasts or in Bcl-2-overexpressing keratinocytes. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis.


Assuntos
Apoptose/fisiologia , Canais de Cloreto/metabolismo , Proteínas Mitocondriais/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Sequência de Bases , Células Cultivadas , Canais de Cloreto/genética , DNA/genética , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Proteínas Mitocondriais/genética , Mutagênese Sítio-Dirigida , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Organelas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência
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