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1.
Sci Rep ; 6: 25806, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-27194388

RESUMO

Human respiratory syncytial virus (hRSV) is a leading cause of acute lower respiratory tract infection in infants, elderly and immunocompromised individuals. To date, no specific antiviral drug is available to treat or prevent this disease. Here, we report that the Smoothened receptor (Smo) antagonist cyclopamine acts as a potent and selective inhibitor of in vitro and in vivo hRSV replication. Cyclopamine inhibits hRSV through a novel, Smo-independent mechanism. It specifically impairs the function of the hRSV RNA-dependent RNA polymerase complex notably by reducing expression levels of the viral anti-termination factor M2-1. The relevance of these findings is corroborated by the demonstration that a single R151K mutation in M2-1 is sufficient to confer virus resistance to cyclopamine in vitro and that cyclopamine is able to reduce virus titers in a mouse model of hRSV infection. The results of our study open a novel avenue for the development of future therapies against hRSV infection.


Assuntos
Vírus Sincicial Respiratório Humano/fisiologia , Transcrição Genética , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Alcaloides/farmacologia , Animais , Linhagem Celular , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Animais de Doenças , Farmacorresistência Viral/efeitos dos fármacos , Feminino , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Receptor Smoothened/metabolismo , Transcrição Genética/efeitos dos fármacos , Veratrum/química , Alcaloides de Veratrum/química , Alcaloides de Veratrum/farmacologia , Alcaloides de Veratrum/uso terapêutico , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
2.
Hong Kong Med J ; 18 Suppl 2: 31-6, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22311359

RESUMO

1. A SARS vaccine was produced based on recombinant native full-length Spike-protein trimers (triSpike) and efficient establishment of a vaccination procedure in rodents. 2. Antibody-mediated enhancement of SARS-CoV infection with anti-SARS-CoV Spike immune-serum was observed in vitro. 3. Antibody-mediated infection of SARS-CoV triggers entry into human haematopoietic cells via an FcγR-dependent and ACE2-, pH-, cysteine-protease-independent pathways. 4. The antibody-mediated enhancement phenomenon is not a mandatory component of the humoral immune response elicited by SARS vaccines, as pure neutralising antibody only could be obtained. 5. Occurrence of immune-mediated enhancement of SARS-CoV infection raises safety concerns regarding the use of SARS-CoV vaccine in humans and enables new ways to investigate SARS pathogenesis (tropism and immune response deregulation).


Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Facilitadores , Glicoproteínas de Membrana/imunologia , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Proteínas do Envelope Viral/imunologia , Internalização do Vírus , Animais , Anticorpos Antivirais/metabolismo , Linhagem Celular Tumoral , Cisteína Proteases/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , Monócitos , Peptidil Dipeptidase A/metabolismo , Receptores Fc/metabolismo , Vírus da SARS/fisiologia , Glicoproteína da Espícula de Coronavírus , Vacinas
3.
J Virol ; 82(22): 11318-30, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18753196

RESUMO

The production of virus-like particles (VLPs) constitutes a relevant and safe model to study molecular determinants of virion egress. The minimal requirement for the assembly of VLPs for the coronavirus responsible for severe acute respiratory syndrome in humans (SARS-CoV) is still controversial. Recent studies have shown that SARS-CoV VLP formation depends on either M and E proteins or M and N proteins. Here we show that both E and N proteins must be coexpressed with M protein for the efficient production and release of VLPs by transfected Vero E6 cells. This suggests that the mechanism of SARS-CoV assembly differs from that of other studied coronaviruses, which only require M and E proteins for VLP formation. When coexpressed, the native envelope trimeric S glycoprotein is incorporated onto VLPs. Interestingly, when a fluorescent protein tag is added to the C-terminal end of N or S protein, but not M protein, the chimeric viral proteins can be assembled within VLPs and allow visualization of VLP production and trafficking in living cells by state-of-the-art imaging technologies. Fluorescent VLPs will be used further to investigate the role of cellular machineries during SARS-CoV egress.


Assuntos
Proteínas do Nucleocapsídeo/metabolismo , Vírus da SARS/fisiologia , Proteínas do Envelope Viral/metabolismo , Proteínas da Matriz Viral/metabolismo , Montagem de Vírus , Animais , Humanos , Glicoproteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas do Nucleocapsídeo/genética , Glicoproteína da Espícula de Coronavírus , Células Vero , Proteínas do Envelope Viral/genética , Proteínas da Matriz Viral/genética , Virossomos/metabolismo , Virossomos/ultraestrutura
4.
J Biol Chem ; 276(28): 26550-8, 2001 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-11352904

RESUMO

The chemokine stromal cell-derived factor 1 (SDF-1) is the natural ligand for CXC chemokine receptor 4 (CXCR4). SDF-1 inhibits infection of CD4+ cells by X4 (CXCR4-dependent) human immunodeficiency virus (HIV) strains. We previously showed that SDF-1 alpha interacts specifically with heparin or heparan sulfates (HSs). Herein, we delimited the boundaries of the HS-binding domain located in the first beta-strand of SDF-1 alpha as the critical residues. We also provide evidence that binding to cell surface heparan sulfate proteoglycans (HSPGs) determines the capacity of SDF-1 alpha to prevent the fusogenic activity of HIV-1 X4 isolates in leukocytes. Indeed, SDF-1 alpha mutants lacking the capacity to interact with HSPGs showed a substantially reduced capacity to prevent cell-to-cell fusion mediated by X4 HIV envelope glycoproteins. Moreover, the enzymatic removal of cell surface HS diminishes the HIV-inhibitory capacity of the chemokine to the levels shown by the HS-binding-disabled mutant counterparts. The mechanisms underlying the optimal HIV-inhibitory activity of SDF-1 alpha when attached to HSPGs were investigated. Combining fluorescence resonance energy transfer and laser confocal microscopy, we demonstrate the concomitant binding of SDF-1 alpha to CXCR4 and HSPGs at the cell membrane. Using FRET between a Texas Red-labeled SDF-1 alpha and an enhanced green fluorescent protein-tagged CXCR4, we show that binding of SDF-1 alpha to cell surface HSPGs modifies neither the kinetics of occupancy nor activation in real time of CXCR4 by the chemokine. Moreover, attachment to HSPGs does not modify the potency of the chemokine to promote internalization of CXCR4. Attachment to cellular HSPGs may co-operate in the optimal anti-HIV activity of SDF-1 alpha by increasing the local concentration of the chemokine in the surrounding environment of CXCR4, thus facilitating sustained occupancy and down-regulation of the HIV coreceptor.


Assuntos
Quimiocinas CXC/farmacologia , Quimiocinas CXC/fisiologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Proteoglicanas de Heparan Sulfato/fisiologia , Receptores CXCR4/fisiologia , Linhagem Celular , Quimiocina CXCL12 , Humanos , Replicação Viral/efeitos dos fármacos
5.
AIDS ; 14(17): 2687-97, 2000 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-11125887

RESUMO

OBJECTIVE: To compare astrocyte toxicity induced by plasma membrane-expressed gp120/41 and soluble gp120. DESIGN: Analysis of morphological alterations and lactate dehydrogenase (LDH) release from astrocytes in culture with monocytes infected with HIV-1, microglia expressing Env of a macrophage-tropic HIV-1 isolate or soluble Env. METHODS: Primary human embryonic astrocytes were cultured with: monocytes infected with two M-tropic HIV-1 isolates (HIV-1(9533), HIV-1(BX08)); human microglia infected with the defective Semliki Forest virus (SFV) vector coding for the env gene of HIV-1(BX08) isolate (SFVenvBX08); and soluble gp140 purified from baby hamster kidney cells transfected with the env gene of HIV-1(BX08) lacking the intracytoplasmic region of gp41 (SFVdelta envBX08). Gp120 mRNA levels were assessed by quantitative reverse transcriptase-polymerase chain reaction and the protein was detected by immunofluorescence in infected monocytes or microglia. RESULTS: Contact of HIV-infected monocytes induced morphological changes in astrocytes and a 137% increase in LDH release at day 2 of co-culture compared with controls (uninfected monocytes). Gp120/41(BX08)-expressing microglia induced a 170% increase in LDH release (relative to SFVLacZ-infected microglia). Pretreatment of co-cultures with an anti-gp120 monoclonal antibody (mAb; NEA-9305) directed against the V3 loop inhibited LDH release. Soluble purified gp140 from BX08 isolate induced only a weak LDH release (104%). Finally, cytotoxicity was not blocked by treatment of the co-culture with Bordetella pertussis toxin, an inhibitor of Gi alpha protein-dependent receptors. CONCLUSION: HIV envelope glycoprotein expressed at the plasma membrane induced astrocyte damage more efficiently than its soluble counterpart. The V3 loop was involved in toxicity induction through a pathway independent of the Gi alpha protein-coupled receptor.


Assuntos
Astrócitos/patologia , Membrana Celular/metabolismo , Efeito Citopatogênico Viral , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/patogenicidade , Macrófagos/virologia , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/isolamento & purificação , Proteína gp120 do Envelope de HIV/toxicidade , Proteína gp41 do Envelope de HIV/toxicidade , HIV-1/genética , HIV-1/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Microglia/citologia , Microglia/metabolismo , Microglia/patologia , Monócitos/citologia , Monócitos/metabolismo , Monócitos/virologia , Toxina Pertussis , RNA Viral/genética , RNA Viral/metabolismo , Vírus da Floresta de Semliki/genética , Solubilidade , Fatores de Virulência de Bordetella/farmacologia
6.
J Biol Chem ; 275(45): 35137-45, 2000 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-10926931

RESUMO

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is thought to exist on the virion surface as a trimer of non-covalently associated gp120/gp41 molecules. We expressed trimeric envelope glycoprotein from three primary, macrophage tropic HIV-1 isolates in baby hamster kidney cells and analyzed the furin-mediated cleavage, stability, and receptor binding properties of the oligomers. The envelope glycoprotein was secreted in a soluble form deleted of its transmembrane anchor and the intracytoplasmic domain (gp140). A mixture of trimers, dimers, and monomers of gp140 as well as monomeric gp120 was detected on polyacrylamide gels. Analysis by sucrose gradient centrifugation revealed that trimers and dimers were essentially composed of uncleaved gp140, whereas most of the gp120 was found in the monomeric fraction. To analyze the effect of the cleavage of gp140 to gp120/Delta41 on trimerization, we co-expressed the furin protease along with gp140. Surprisingly, furin expression changed the subcellular localization of the envelope glycoprotein, which became in majority sequestered in the major furin compartment, the trans-Golgi network, as judged by confocal laser microscopy. The envelope glycoprotein secreted from furin-co-expressing cells was almost completely cleaved to gp120 and Deltagp41, but gp120 was found exclusively in the monomeric fraction, with a few residual oligomers being composed of uncleaved gp140. Secreted uncleaved gp140 trimers were purified to homogeneity and analyzed for their capacity to interact with cellular receptors CD4 and CC chemokine receptor 5 (CCR5). Receptor binding was analyzed on CD4- and CCR5-expressing cells as well as on peripheral blood mononuclear cells. Trimers showed greatly reduced binding to CD4 as compared with monomers. Neither monomers nor trimers bound directly to CCR5. In conclusion, our results show that the cleaved form of the envelope glycoprotein does not form stable trimers, suggesting that gp120/gp41 oligomers on the virion surface might be stabilized by a yet to be identified mechanism and that the virion might attach to CD4 via a monomeric form of gp120. These results are relevant to the development of an envelope-based vaccine against AIDS.


Assuntos
Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Animais , Western Blotting , Antígenos CD4/metabolismo , Linhagem Celular , Centrifugação com Gradiente de Concentração , Cricetinae , Dimerização , Eletroforese em Gel de Poliacrilamida , Endopeptidases/metabolismo , Furina , Produtos do Gene env/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Cinética , Leucócitos Mononucleares/metabolismo , Macrófagos/química , Microscopia Confocal , Plasmídeos/metabolismo , Ligação Proteica , Receptores CCR5/metabolismo , Proteínas Recombinantes/metabolismo , Subtilisinas/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana , Rede trans-Golgi
7.
Virology ; 259(2): 314-23, 1999 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-10388656

RESUMO

HIV-1 envelope proteins gp120 and gp41 are likely to play a role in the pathogenesis of HIV-associated neurocognitive disorders. While detection of gp120 in HIV-infected cell cultures is easy, it has not yet been possible to identify gp120 in human or animal brains in situ. The difficulty in detecting gp120 could be due to low expression levels of the protein, to the shedding of gp120 from infected macrophages/microglia, or to the use of inappropriate gp-specific antibodies. We addressed these questions by analyzing the subcellular localization, oligomeric structure, and shedding behavior of gp120 from a macrophage-tropic, CCR5-dependent primary isolate, BX08, expressed by a Semliki Forest virus replicon (SFVenvBX08) in vitro. We used the same SFV system injected in vivo into the rat brain in an attempt to detect gp120 in situ. Our results show that gp120/41 is expressed as monomers, dimers, and trimers in cell culture. Immunocytochemical analysis revealed that intracytoplasmic gp120 can be recognized by an anti-V3 antibody, whereas gp120 at the plasma membrane is detected exclusively by a conformation-dependent antibody. In the rat brain, the SFV vector allows gene expression in neurons from day 3 to day 9 after injection without any apparent brain damage nor reactive astrogliosis. In SFVenvBX08-infected neurons only conformation-dependent antibodies allowed gp120 labeling. These results suggest that previous difficulties in detecting gp120 in brain tissues may be due to the use of antibodies which were unable to recognize gp120 at the plasma membrane.


Assuntos
Encéfalo/virologia , Proteína gp120 do Envelope de HIV/análise , HIV-1/metabolismo , Macrófagos/virologia , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Linhagem Celular , Proteína Glial Fibrilar Ácida/imunologia , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/análise , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/imunologia , HIV-1/isolamento & purificação , Humanos , Imuno-Histoquímica , Camundongos , Neurônios/imunologia , Neurônios/virologia , Conformação Proteica , Ratos , Replicon , Vírus da Floresta de Semliki/genética
8.
W V Med J ; 94(3): 143-5, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9641072

RESUMO

Idiopathic pulmonary ossification is a rare disease. Most commonly, it affects middle-aged men. Its etiology is unknown. We present a case of nodular type idiopathic pulmonary ossification in a 42-year-old, white male who had one episode of hemoptysis.


Assuntos
Pneumopatias/patologia , Ossificação Heterotópica/patologia , Adulto , Humanos , Pneumopatias/diagnóstico por imagem , Masculino , Ossificação Heterotópica/diagnóstico por imagem , Tomografia Computadorizada por Raios X
9.
Res Virol ; 149(1): 5-20, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9561560

RESUMO

Recombinant Mengo viruses expressing heterologous genes have proven to be safe and immunogenic in both mice and primates, and to be able to induce both humoral and cellular immune responses (Altmeyer et al., 1995, 1996). Several recombinant Mengo viruses expressing either a large region (aa 65-206) of the HIV1 nef gene product, or cytotoxic T lymphocyte (CTL) epitopic regions from the SIV Gag (aa 182-190), Nef (aa 155-178) and Pol (aa 587-601) gene products were engineered. The heterologous antigens were expressed either as fusion proteins with the Mengo virus leader (L) protein, or in cleaved form through autocatalytic cleavage by the foot-and-mouth disease virus 2A protein. Rhesus macaques and BALB/c mice inoculated with the Mengo virus SIV recombinants failed to develop CTL responses against the SIV gene products, while one of the HIV-Nef recombinants induced a weak CTL response in mice directed to an HIV1 Nef peptide spanning positions 182-198. In contrast, BALB/c mice immunized with vaccinia virus recombinants expressing HIV1 Nef developed a strong CTL response to the 182-198 peptide and also responded to a second peptide spanning positions 73-81. These results indicate that Mengo virus recombinants expressing HIV1 Nef and SIV CTL epitopes are weak immunogens. One of the fusion recombinants expressing SIV CTL epitopes failed to infect macaques even when used at high doses, while the recombinant expressing HIV1 Nef as a fusion protein failed to infect BALB/c mice. These results demonstrate that the expression of certain heterologous sequences as fusion proteins with L can result in the loss of the ability of the recombinant to infect normally susceptible animals.


Assuntos
Anticorpos Antivirais/biossíntese , Citotoxicidade Imunológica , HIV-1/imunologia , Mengovirus/genética , Proteínas Recombinantes de Fusão/imunologia , Vírus da Imunodeficiência Símia/imunologia , Sequência de Aminoácidos , Animais , Epitopos de Linfócito T , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Produtos do Gene nef/genética , Produtos do Gene nef/imunologia , Produtos do Gene pol/genética , Produtos do Gene pol/imunologia , Vetores Genéticos , Anticorpos Anti-HIV/biossíntese , HIV-1/genética , Células HeLa , Humanos , Imunização , Macaca mulatta , Mengovirus/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes de Fusão/genética , Vírus da Imunodeficiência Símia/genética , Linfócitos T Citotóxicos/imunologia , Vírus Vaccinia/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana
10.
Proc Natl Acad Sci U S A ; 94(17): 9326-31, 1997 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-9256481

RESUMO

The beta-chemokine receptor CCR-5 is essential for the efficient entry of primary macrophage-tropic HIV-1 isolates into CD4(+) target cells. To study CCR-5-dependent cell-to-cell fusion, we have developed an assay system based on the infection of CD4(+) CCR-5(+) HeLa cells with a Semliki Forest virus recombinant expressing the gp120/gp41 envelope (Env) from a primary clade B HIV-1 isolate (BX08), or from a laboratory T cell line-adapted strain (LAI). In this system, gp120/gp41 of the "nonsyncytium-inducing," primary, macrophage-tropic HIV-1BX08 isolate, was at least as fusogenic as that of the "syncytium-inducing" HIV-1LAI strain. BX08 Env-mediated fusion was inhibited by the beta-chemokines RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory proteins 1beta (MIP-1beta) and by antibodies to CD4, whereas LAI Env-mediated fusion was insensitive to these beta-chemokines. In contrast soluble CD4 significantly reduced LAI, but not BX08 Env-mediated fusion, suggesting that the primary isolate Env glycoprotein has a reduced affinity for CD4. The domains in gp120/gp41 involved in the interaction with the CD4 and CCR-5 molecules were probed using monoclonal antibodies. For the antibodies tested here, the greatest inhibition of fusion was observed with those directed to conformation-dependent, rather than linear epitopes. Efficient inhibition of fusion was not restricted to epitopes in any one domain of gp120/gp41. The assay was sufficiently sensitive to distinguish between antibody- and beta-chemokine-mediated fusion inhibition using serum samples from patient BX08, suggesting that the system may be useful for screening human sera for the presence of biologically significant antibodies.


Assuntos
Anticorpos Antivirais/imunologia , Epitopos/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1 , Macrófagos/virologia , Receptores de Citocinas/imunologia , Receptores de HIV/imunologia , Anticorpos Antivirais/farmacologia , Especificidade de Anticorpos , Fusão Celular/efeitos dos fármacos , Fusão Celular/imunologia , Proteína gp120 do Envelope de HIV/química , Células HeLa , Humanos , Macrófagos/imunologia , Macrófagos/patologia , Conformação Proteica , Receptores CCR5
11.
Curr Opin Neurobiol ; 5(5): 647-55, 1995 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8580717

RESUMO

HIV can invade the CNS, where it replicates principally in macrophages. Yet, neurological disease is more often correlated with levels of neurotoxins or tumor necrosis factor alpha than with viral replication or specific viral determinants in brain. In experimental systems, HIV glycoprotein affects functions of uninfected microglia and astrocytes to eventually cause neuronal death. While the cellular basis of cognitive and neurological dysfunction are unravelled in the simian immunodeficiency virus model, the molecular mechanisms of HIV neurotoxicity are being studied in newly developed mouse models.


Assuntos
Sistema Nervoso Central/virologia , HIV/fisiologia , Microglia/virologia , Replicação Viral/fisiologia , Animais
13.
J Virol ; 69(5): 3193-6, 1995 May.
Artigo em Inglês | MEDLINE | ID: mdl-7707549

RESUMO

Several features make Mengo virus an excellent candidate for use as a vaccine vector. The virus has a wide host range, including rodents, pigs, monkeys, and most likely humans, and expresses its genome exclusively in the cytoplasm of the infected cell. Stable attenuated strains exist which are deleted for part of the 5' noncoding region of the genome. Here we report an attenuated Mengo virus recombinant, vLCMG4, that encodes an immunodominant cytotoxic T-lymphocyte epitope of the lymphocytic choriomeningitis virus (LCMV) nucleo-protein. vLCMG4 induced protective immunity against lethal LCMV infection after a single, low-dose immunization in BALB/c mice and elicited an LCMV-specific CD8+ cytotoxic T lymphocyte response. This demonstrates the potential of recombinant Mengo virus vaccines to confer protection against infectious diseases by the induction of cellular immune responses.


Assuntos
Vetores Genéticos , Mengovirus/genética , Mengovirus/imunologia , Vacinas Sintéticas/isolamento & purificação , Vacinas Virais/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , DNA Viral/genética , Deleção de Genes , Humanos , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/prevenção & controle , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Recombinação Genética , Linfócitos T Citotóxicos/imunologia , Vacinas Atenuadas/isolamento & purificação , Proteínas Virais/genética , Proteínas Virais/imunologia
14.
Proc Natl Acad Sci U S A ; 91(21): 9775-9, 1994 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-7937890

RESUMO

Introduction of a sequence encoding 147 amino acids from human immunodeficiency virus type I (HIV-1) strain MN glycoprotein gp120 into the RNA genome of the stably attenuated Mengo virus strain vM16 yielded an infectious recombinant virus, vMLN450, which expressed the heterologous HIV-1 sequence along with the normal Mengo virus proteins. The HIV-1 gp120 sequence, fused to the amino terminus of the short, nonstructural Mengo virus leader polypeptide was recognized by a gp120 V3 loop-specific monoclonal antibody. When inoculated into mice, recombinant virus vMLN450 elicited a high-titer anti-HIV-1 antibody response as well as an HIV-1MN-specific cytotoxic cellular immune response. An anti-HIV-1 antibody response could also be detected in cynomolgus monkeys after a single immunization. We propose that attenuated Mengo virus can serve as an effective expression vector in cell systems and various animal species and offers another approach to the development of new, live recombinant vaccines.


Assuntos
Vetores Genéticos , Anticorpos Anti-HIV/biossíntese , Proteína gp120 do Envelope de HIV/biossíntese , HIV-1/metabolismo , Mengovirus/genética , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Sequência de Bases , Citotoxicidade Imunológica , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Genoma Viral , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/genética , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Recombinação Genética , Ensaio de Placa Viral
16.
Mol Microbiol ; 7(1): 89-98, 1993 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8382333

RESUMO

Our laboratories have independently identified a gene in Salmonella choleraesuis and Salmonella typhimurium that is necessary for efficient adherence and entry of these organisms into cultured epithelial cells. Introduction of a mutated gene into several Salmonella strains belonging to different serotypes rendered these organisms deficient for adherence and invasion of cultured cells. This effect was most pronounced in the host-adapted serotypes Salmonella gallinarum, S. choleraesuis, and Salmonella typhi. The nucleotide sequence of this gene, which we have termed invH, encodes a predicted 147-amino-acid polypeptide containing a signal sequence. The InvH predicted polypeptide is highly conserved in S. typhimurium and S. choleraesuis, differing at only three residues. The invH gene was expressed in Escherichia coli using a T7 RNA polymerase expression system and a polypeptide of approximately 16,000 molecular weight was observed, in agreement with the predicted size of its gene product. Upon fractionation, the expressed polypeptide was localized in the bacterial membrane fraction. Southern and colony hybridization analyses indicated that the invH gene is present in all Salmonella strains tested (91 strains belonging to 37 serotypes) with the exception of strains of Salmonella arizonae. No homologous sequences were detected in Yersinia, Shigella, Proteus, and several strains of enteroinvasive and enteropathogenic E. coli. Downstream from the S. choleraesuis (but not S. typhimurium) invH gene, a region with extensive homology to the insertion sequence IS3 was detected.


Assuntos
Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Células Epiteliais , Genes Bacterianos , Salmonella/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/metabolismo , Sequência de Bases , Células Cultivadas , Elementos de DNA Transponíveis/genética , Epitélio/microbiologia , Dados de Sequência Molecular , Mutagênese , Fenótipo , Proteínas Recombinantes de Fusão/metabolismo , Salmonella/patogenicidade , Salmonella/fisiologia , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/fisiologia , Homologia de Sequência do Ácido Nucleico , Virulência
17.
Virology ; 184(2): 636-44, 1991 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-1653493

RESUMO

In order to study the properties of foreign antigenic sites expressed on poliovirus a hybrid was constructed in which neutralization antigenic site IA of poliovirus type 1 strain Mahoney [PV1(M)] was replaced by neutralization immunogenic site IA (NImIA) of human rhinovirus 14 (HRV14). The resulting hybrid was viable, but growth was impaired in comparison to PV1(M). The hybrid expressed both PV1(M) and HRV14 antigenic determinants. When inoculated into rabbits it elicited neutralizing antibodies against both PV1(M) and HRV14. Furthermore, the hybrid was efficiently neutralized by polyclonal antisera specific for either PV1(M) or HRV14 and by three out of five monoclonal antibodies directed to NImIA. The monoclonal antibodies also blocked binding of the hybrid to the cellular receptor for poliovirus. One of them is thought to neutralize rhinovirus in this manner, and it appears that NImIA is expressed in a sufficiently favorable context on the hybrid for the same mechanism to be effective. This can be interpreted to mean that the interactions between the parental viruses and their respective cellular receptors are very similar.


Assuntos
Antígenos Virais/genética , Poliovirus/genética , Poliovirus/imunologia , Rhinovirus/genética , Rhinovirus/imunologia , Sequência de Aminoácidos , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Sequência de Bases , Clonagem Molecular , DNA Recombinante , Dados de Sequência Molecular , Testes de Neutralização , Receptores Virais/metabolismo
18.
Chest ; 81(6): 699-700, 1982 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7075305

RESUMO

Infectious mononucleosis (IM) is common among students. These patients often complain of fatigue and dyspnea. To determine whether IM alters respiratory function, we performed spirometric, single-breath diffusing capacity, and maximal static respiratory pressure tests on seven patients with symptoms of IM. These studies were repeated two weeks later and the respiratory pressures were repeated five months later. Each patient served as his own control. Pulmonary function was normal except for respiratory pressures, which were initially low. These pressures, still low after two weeks, improved significantly after five months. We concluded that IM is associated with transient respiratory muscle weakness.


Assuntos
Mononucleose Infecciosa/fisiopatologia , Respiração , Adolescente , Adulto , Feminino , Humanos , Masculino , Capacidade de Difusão Pulmonar , Testes de Função Respiratória
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