RESUMO
1. Chicken erythrocytes in blood vessels are the most abundant circulating cells, which participate in the host's immune responses. The transcription factor nuclear factor-kappa B (NF-κB) plays a vital role in the inflammatory response following viral infections. However, the expression of the NF-κB pathway, and other immune-related genes in chicken erythrocytes infected with low pathogenic avian influenza virus (LPAIV H9N2), has not been extensively studied.2. The following study determined the interaction of LPAIV H9N2 with chicken erythrocytes using indirect immunofluorescence microscopy. This was followed by investigating myeloid differentiation primary response 88 (MyD88), C-C motif chemokine ligand 5 (CCL5), melanoma differentiation-associated protein 5 (MDA5), the inhibitor of nuclear factor-kappa B kinase subunit epsilon (IKBKE), NF-κB inhibitor alpha (NFKBIA), NF-κB inhibitor epsilon (NFKBIE), interferon-alpha (IFN-α), colony-stimulating factor 3 (CSF3) and tumour necrosis factor receptor-associated factor 6 (TRAF6) by mRNA expression using quantitative real-time PCR (qRT-PCR) at four different time intervals (0, 2, 6 and 10 h).3. There was a significant interaction between erythrocytes and LPAIV H9N2 virus. Furthermore, the mRNA expression of the NF-κB pathway and other immune-related genes were significantly up-regulated at 2 h post-infection in infected chicken erythrocytes, except for TRAF6, which were significantly downregulated. While at 0 h post-infection, IFN-α and CSF3 were significantly upregulated, whereas NFKBIA was significantly downregulated. Further expression of MDA5, CCL5 and NFKBIA was upregulated, while TRAF6 was downregulated at 6 h post-infection. In infected erythrocytes, expression of MyD88, CCL5 and IKBKE was upregulated. However, IFN-α and TRAF6 were downregulated at 10 h post-infection.4. These results give initial evidence that the NF-κB pathway, and other genes related to immunity, in chicken erythrocytes may contribute to LPAIV subtype H9N2 and induce host immune responses.
Assuntos
Vírus da Influenza A Subtipo H9N2 , Influenza Aviária , Animais , Galinhas/genética , Eritrócitos , Vírus da Influenza A Subtipo H9N2/genética , Influenza Aviária/genética , NF-kappa B/genéticaRESUMO
Disruption of neuronal iron homeostasis and oxidative stress are closely related to the pathogenesis of Parkinson's disease (PD). Ginkgetin, a natural biflavonoid isolated from leaves of Ginkgo biloba L, has many known effects, including anti-inflammatory, anti-influenza virus, and anti-fungal activities, but its underlying mechanism of the neuroprotective effects in PD remains unclear. The present study utilized PD models induced by 1-methyl-4-phenylpyridinium (MPP(+)) and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to explore the neuroprotective ability of ginkgetin in vivo and in vitro. Our results showed that ginkgetin could provide significant protection from MPP(+)-induced cell damage in vitro by decreasing the levels of intracellular reactive oxygen species and maintaining mitochondrial membrane potential. Meanwhile, ginkgetin dramatically inhibited cell apoptosis induced by MPP+ through the caspase-3 and Bcl2/Bax pathway. Moreover, ginkgetin significantly improved sensorimotor coordination in a mouse PD model induced by MPTP by dramatically inhibiting the decrease of tyrosine hydroxylase expression in the substantia nigra and superoxide dismutase activity in the striatum. Interestingly, ginkgetin could strongly chelate ferrous ion and thereby inhibit the increase of the intracellular labile iron pool through downregulating L-ferritin and upregulating transferrin receptor 1. These results indicate that the neuroprotective mechanism of ginkgetin against neurological injury induced by MPTP occurs via regulating iron homeostasis. Therefore, ginkgetin may provide neuroprotective therapy for PD and iron metabolism disorder related diseases.
Assuntos
Biflavonoides/química , Ferro/química , Fármacos Neuroprotetores/química , Doença de Parkinson/tratamento farmacológico , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/efeitos adversos , 1-Metil-4-fenilpiridínio/efeitos adversos , Animais , Antígenos CD/metabolismo , Apoferritinas/metabolismo , Apoptose , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica , Ginkgo biloba , Homeostase , Humanos , Quelantes de Ferro/efeitos adversos , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Receptores da Transferrina/metabolismo , Superóxido Dismutase/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Glycoprotein 96 (gp96), a member of the heat-shock protein family, can elicit priming of antigen-specfic cytotoxic T lymphocytes, when bound to antigenic viral or tumour peptides. We used direct peptide isolation from purified gp96 and microsequencing to show that a virus-specific peptide is bound to gp96 derived from liver tissues of patients with hepatitis B virus (HBV)-induced hepatocellular carcinoma. This virus-specific peptide has potential for engineering tumour vaccines against hepatocellular carcinoma and chronic HBV infection.
Assuntos
Antígenos de Neoplasias/metabolismo , Carcinoma Hepatocelular/metabolismo , Proteínas de Choque Térmico/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Hepatite B/metabolismo , Neoplasias Hepáticas/metabolismo , Vacinas Anticâncer , Carcinoma Hepatocelular/virologia , Hepatite B/complicações , Humanos , Neoplasias Hepáticas/virologia , Fragmentos de Peptídeos/metabolismo , Ligação ProteicaRESUMO
Immature dendritic cells (iDCs) express the CC chemokine receptor (CCR)5, which promotes chemotaxis toward the CC chemokines regulated on activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta. By contrast, mature DCs downregulate CCR5 but upregulate CXC chemokine receptor (CXCR)4, and as a result exhibit enhanced chemotaxis toward stromal cell-derived factor (SDF)-1alpha. CCR5 and CXCR4 also function as coreceptors for macrophage-tropic (M-tropic) and T cell-tropic (T-tropic) human immunodeficiency virus (HIV)-1, respectively. Here, we demonstrate chemotaxis of iDCs toward M-tropic (R5) but not T-tropic (X4) HIV-1. Furthermore, preexposure to M-tropic HIV-1 or its recombinant envelope protein prevents migration toward CCR5 ligands. The migration of iDCs toward M-tropic HIV-1 may enhance formation of DC-T cell syncytia, thus promoting viral production and destruction of both DC and T helper lymphocytes. Therefore, disturbance of DC chemotaxis by HIV-1 is likely to contribute to immunosuppression in primary infection and AIDS. In addition, migration of iDCs toward HIV-1 may aid the capture of R5 HIV-1 virions by the abundant DC cell surface protein DC-specific intercellular adhesion molecule (ICAM)3-grabbing nonintegrin (DC-SIGN). HIV-1 bound to DC cell-specific DC-SIGN retains the ability to infect replication-permissive T cells in trans for several days. Consequently, recruitment of DC by HIV-1 could combine with the ability of DC-SIGN to capture and transmit the virus to T cells, and so facilitate dissemination of virus within an infected individual.
Assuntos
Moléculas de Adesão Celular , Quimiotaxia , Células Dendríticas/fisiologia , HIV-1/fisiologia , Lectinas Tipo C , Macrófagos/virologia , Linfócitos T/virologia , Animais , Antígenos CD4/metabolismo , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CCL5/metabolismo , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Células Dendríticas/metabolismo , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/imunologia , Humanos , Lectinas/metabolismo , Proteínas Inflamatórias de Macrófagos/metabolismo , Testes de Precipitina , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
The cell surface molecules CD4 and CD8 greatly enhance the sensitivity of T-cell antigen recognition, acting as "co-receptors" by binding to the same major histocompatibility complex (MHC) molecules as the T-cell receptor (TCR). Here we use surface plasmon resonance to study the binding of CD8alphaalpha to class I MHC molecules. CD8alphaalpha bound the classical MHC molecules HLA-A*0201, -A*1101, -B*3501, and -C*0702 with dissociation constants (K(d)) of 90-220 microm, a range of affinities distinctly lower than that of TCR/peptide-MHC interaction. We suggest such affinities apply to most CD8alphaalpha/classical class I MHC interactions and may be optimal for T-cell recognition. In contrast, CD8alphaalpha bound both HLA-A*6801 and B*4801 with a significantly lower affinity (>/=1 mm), consistent with the finding that interactions with these alleles are unable to mediate cell-cell adhesion. Interestingly, CD8alphaalpha bound normally to the nonclassical MHC molecule HLA-G (K(d) approximately 150 microm), but only weakly to the natural killer cell receptor ligand HLA-E (K(d) >/= 1 mm). Site-directed mutagenesis experiments revealed that variation in CD8alphaalpha binding affinity can be explained by amino acid differences within the alpha3 domain. Taken together with crystallographic studies, these results indicate that subtle conformational changes in the solvent exposed alpha3 domain loop (residues 223-229) can account for the differential ability of both classical and nonclassical class I MHC molecules to bind CD8.
Assuntos
Antígenos CD8/química , Antígenos CD8/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/química , Linfócitos T/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Antígenos HLA/química , Antígenos HLA/metabolismo , Antígenos HLA-A/química , Antígenos HLA-A/metabolismo , Antígeno HLA-A11 , Antígeno HLA-B35/química , Antígeno HLA-B35/metabolismo , Antígenos HLA-C/química , Antígenos HLA-C/metabolismo , Antígenos HLA-G , Humanos , Células Matadoras Naturais/imunologia , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Antígenos HLA-ERESUMO
A method to produce alphabeta T-cell receptors (TCRs) in a soluble form suitable for biophysical analysis was devised involving in vitro refolding of a TCR fusion protein. Polypeptides corresponding to the variable and constant domains of each chain of a human and a murine receptor, fused to a coiled coil heterodimerization motif from either c-Jun (alpha) or v-Fos (beta), were overexpressed separately in Escherichia coli. Following recovery from inclusion bodies, the two chains of each receptor were denatured, and then refolded together in the presence of denaturants. For the human receptor, which is specific for the immunodominant influenza A HLA-A2-restricted matrix epitope (M58-66), a heterodimeric protein was purified in milligram yields and found to be homogeneous, monomeric, antibody-reactive, and stable at concentrations lower than 1 microM. Using similar procedures, analogous results were obtained with a murine receptor specific for an influenza nucleoprotein epitope (366-374) restricted by H2-Db. Production of these receptors has facilitated a detailed analysis of viral peptide-Major Histocompatibility Complex (peptide-MHC) engagement by the TCR using both surface plasmon resonance (SPR) and, in the case of the human TCR, isothermal titration calorimetry (ITC) (Willcox et al., 1999). The recombinant methods described should enable a wide range of TCR-peptide-MHC interactions to be studied and may also have implications for the production of other heterodimeric receptor molecules.
Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/química , Sequência de Aminoácidos , Sítios de Ligação , Biofísica/métodos , Dimerização , Antígeno HLA-A2/química , Humanos , Zíper de Leucina , Ligantes , Complexo Principal de Histocompatibilidade , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Oncogênicas v-fos/química , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas Proto-Oncogênicas c-jun/química , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Solubilidade , Ressonância de Plasmônio de SuperfícieRESUMO
The CD8 co-receptor is important in the differentiation and selection of class I MHC-restricted T cells during thymic development, and in the activation of mature T lymphocytes in response to antigen. Here we show that soluble CD8alphaalpha receptor, despite an extremely low affinity for MHC, inhibits activation of cytotoxic lymphocytes by obstructing CD3 zeta-chain phosphorylation. We propose a model for this effect that involves interference of productive receptor multimerization at the T-cell surface. These results provide new insights into the mechanism of T-cell activation and evidence that CD8 function is exquisitely sensitive to disruption, an effect that might be exploited by molecular therapeutics.
Assuntos
Antígenos CD8/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Modelos Imunológicos , Linfócitos T Citotóxicos/efeitos dos fármacos , Complexo CD3/metabolismo , Antígenos CD8/imunologia , Dimerização , Antígenos de Histocompatibilidade Classe I/imunologia , Ligantes , Ativação Linfocitária/imunologia , Complexo Principal de Histocompatibilidade , Peptídeos/imunologia , Peptídeos/farmacologia , Fosforilação , Conformação Proteica , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Solubilidade , Linfócitos T Citotóxicos/imunologiaRESUMO
The nucleotide and deduced amino acid sequences of louping ill (LI) virus isolates, collected from representative regions of the British Isles and Norway, were determined for either the entire envelope gene (20 isolates) or for a portion of the envelope gene that spans a hypervariable region and includes an LI virus specific marker sequence (53 isolates). Phylogenetic analysis reveals the presence of three major geographical populations of LI virus in the British Isles, viz. Irish, Welsh and British LI viruses, which all cause encephalomyelitis in animals, predominantly sheep, and co-habit the same tick population. British LI virus occurs throughout Scotland, England, Ireland and Norway. Irish and Welsh LI viruses occur only in Ireland and Wales, respectively. Phylogenetic analysis also predicts that LI virus initially emerged in Ireland and that a descendant was introduced into Great Britain via Wales and was subsequently transported to the borders of Scotland, from where it was dispersed throughout Scotland, northern England and Norway. More recently, the British LI virus was reintroduced into Ireland and also into south-west England. Dates of lineage divergence, calculated from the synonymous substitution rate, indicate that LI virus emerged in the British Isles less than 800 years ago and most LI virus dispersal occurred during the last 300 years. By combining these data with historical records it appears that livestock movement can be implicated in the dispersal of LI virus.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Proteínas do Envelope Viral/genética , Animais , Vírus da Encefalite Transmitidos por Carrapatos/classificação , Variação Genética , Camundongos , Filogenia , Reino UnidoRESUMO
Louping ill virus isolates from Great Britain, Ireland and Norway were compared antigenically by indirect immunofluorescence, haemagglutination-inhibition and neutralization tests using a panel of five envelope-specific and five non-structural protein NS1-specific monoclonal antibodies raised against louping ill virus. The viruses were grouped according to their reactivities with the antibodies. Group 1, members of which were isolated between 1931 and 1987, consisted of 13 viruses that reacted with all antibodies, whereas group 2, members of which were isolated after 1980, consisted of five viruses that were positive with only eight of the 10 monoclonal antibodies. The two monoclonal antibodies that did not react with the group 2 viruses are known to be neutralizing antibodies and the amino acids that they recognize in the viral envelope protein have been identified. We therefore refer to the group 2 viruses as naturally occurring monoclonal antibody escape variants. When compared with group 1 viruses, the escape variants showed reduced virulence for mice in terms of the time taken to kill and/or the proportion that died, following intraperitoneal inoculation. The nucleotide and deduced amino acid sequences of the envelope gene of one escape variant were compared with those of several group 1 viruses. A single amino acid substitution at residue 308 was detected in the envelope protein of the escape variant which corresponds precisely to the position in experimentally selected attenuated monoclonal antibody escape mutants. The importance and potential implications of these naturally occurring variants in louping ill epizootiology and vaccine-based control are discussed.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Sequência de Bases , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Masculino , Camundongos , Dados de Sequência Molecular , Noruega , Reino Unido , Proteínas do Envelope Viral/genética , Virulência/imunologiaRESUMO
The antigenic, pathogenic and molecular characteristics of Turkish sheep encephalitis (TSE) virus, strain TTE80, were compared with other members of the tick-borne encephalitis (TBE) virus complex. Monoclonal antibodies with defined specificity for the flavivirus envelope glycoprotein distinguished TSE virus from louping ill (LI), western or far eastern TBE, Langat and Powassan virus in indirect immunofluorescence, haemagglutination-inhibition and neutralization tests. On the other hand, TSE virus, which produces an LI-like disease in sheep, resembled LI virus in mouse neurovirulence tests. Molecular homology data of all the structural genes of TSE virus compared with other tick-borne flaviviruses demonstrated that TSE virus is a distinct member in the TBE virus subgroup. The data are consistent with the conclusion that TSE virus has evolved by a separate evolutionary pathway as compared with the close antigenic relatives, western European, far eastern TBE viruses and LI virus. By aligning the encoded amino acids in the viral envelope glycoprotein of mosquito- and tick-borne flaviviruses, we have also identified subgroup-specific pentapeptide motifs for the tick-borne encephalitis, Japanese encephalitis and dengue subgroup viruses of the genus Flavivirus. These pentapeptides have important implications for the evolution, classification and diagnosis of flaviviruses.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/classificação , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Antígenos Virais/imunologia , Sequência de Bases , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Encefalite Transmitida por Carrapatos/microbiologia , Encefalite Transmitida por Carrapatos/veterinária , Feminino , Imunofluorescência , Genes Virais , Camundongos , Dados de Sequência Molecular , Oligopeptídeos/imunologia , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Sorotipagem , Ovinos , Doenças dos Ovinos/microbiologia , Turquia , Proteínas do Envelope Viral/classificação , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas Estruturais Virais/classificação , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologia , VirulênciaRESUMO
We have carried out an antigenic analysis and nucleotide sequence comparison of the envelope glycoprotein of recognized louping ill virus strains isolated from Scotland with that of a Norwegian virus known to cause encephalomyelitis in sheep. Monoclonal antibodies with defined specificity for the louping ill virus envelope glycoprotein failed to distinguish between the Norwegian virus and prototype louping ill virus in indirect immunofluorescence, haemagglutination inhibition and neutralization tests. Nucleotide sequencing of the envelope glycoprotein and alignment of the deduced amino acid sequence with other known sequences revealed that the Norwegian virus closely resembles (> 95% identity for nucleotide and > 98% identity for amino acid sequences) louping ill virus. Maximum variation in identities among four strains of louping ill virus were 4.4% and 1.8% respectively for nucleotide and amino acid alignments. We conclude that sheep encephalomyelitis in Norway is caused by louping ill virus. These results imply that other viruses present in Europe and known to cause encephalitis/encephalomyelitis of sheep could be caused by louping ill virus.