RESUMO
In this study, we employ antisense RNA technology to block Hepatitis B Virus (HBV) gene expression in cell culture by gene transfer as an approach to block immune recognition and pathogenic sequelae. Retroviral vectors encoding antisense and sense copies of the HBV surface antigen gene (HBsAg) were constructed, respectively. To assay the inhibition of HBV gene expression by antisense RNA, the antisense retroviral construct was co-transfected with HBV expression vector (pTHBV) in hepatoma cell line, HepG2 cells. Expression of surface antigen was assessed by a standard HBsAg assay. The results indicated that HBsAg expression was reduced (40-50%) in antisense co-transfected cells as compared to the control vector co-transfected cells. Furthermore, HepG2 was transduced with antisense retroviral vector and transfected with pTHBV. HBsAg expression was reduced 75% in the antisense retrovirus transduced HepG2 cells as compared to control vector transduced cells. The retroviral vectors developed in this study can be used to identify the target antigen of cytotoxic T lymphocytes, which contribute to the immune mediated damage in chronic HBV patients. The retroviral mediated antisense gene transfer combined with liver (or hepatocyte) transplant could also provide a molecular targeting approach for treating chronic hepatitis patients.
Assuntos
Regulação Viral da Expressão Gênica/efeitos dos fármacos , Terapia Genética/métodos , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , RNA Antissenso/farmacologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Vetores Genéticos/genética , Antígenos de Superfície da Hepatite B/biossíntese , Antígenos de Superfície da Hepatite B/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , RNA Antissenso/uso terapêutico , RNA Viral/biossíntese , RNA Viral/farmacologia , RNA Viral/uso terapêutico , Transfecção/métodos , Replicação Viral/efeitos dos fármacosRESUMO
A critically ill, HBV seronegative girl who received a liver from a HBsAg+ donor is described. Despite HBV Ig prophylaxis, she was seropositive for HBsAg shortly after transplantation. Although the postoperative period was complicated, HBV-related problems were not encountered. Liver dysfunction was noted 7 months after transplantation. At that time, she became anti-HBc IgM-positive, with liver histologic findings suggestive of chronic active hepatitis B. The liver function normalized after a reduction of immunosuppressive therapy and introduction of ciprofloxacin. The patient had low level HBV replication during the entire follow-up period (HBV DNA-positive by PCR only) and sequencing of the virus on 4 occasions revealed only wild-type HBV. She subsequently lost serum HBsAg and HBV DNA (even by PCR) and has remained well 2 years after transplantation.
Assuntos
Antígenos de Superfície da Hepatite B/análise , Hepatite B/transmissão , Transplante de Fígado/imunologia , Doadores de Tecidos , Sequência de Bases , Criança , Doença Crônica , DNA Viral/análise , Feminino , Vírus da Hepatite B/fisiologia , Humanos , Imunossupressores/uso terapêutico , Dados de Sequência MolecularRESUMO
Live attenuated vaccines prepared from simian immunodeficiency virus (SIV) have provided the best protective immunity in challenge experiments. In animals vaccinated with attenuated SIV, immune responses may be elicited owing to endogenous expression of native SIV proteins and/or antigen presentation in the native replication site of virus. However, replication-competent viral vaccines raise safety concerns for clinical trials in humans. To ensure the safety and maintain the immunogenicity of a live, attenuated vaccine, we have developed a replication-defective HIV pseudotyped with vesicular stomatitis virus G protein (VSV-G). The polymerase gene of HIV was truncated to construct the replication-defective HIV. This pseudotyped HIV can infect many cell types, including human and simian cells, and undergoes only one round of replication. Furthermore, antibody immune response can be detected in mice immunized with VSV-G-pseudotyped replication-defective HIV.
Assuntos
Vacinas contra a AIDS/imunologia , Infecções por HIV/prevenção & controle , HIV-1/fisiologia , Glicoproteínas de Membrana , Vacinas Atenuadas/imunologia , Proteínas do Envelope Viral/genética , Replicação Viral , Vacinas contra a AIDS/genética , Animais , Linfócitos B , Linhagem Celular , Produtos do Gene pol/genética , Anticorpos Anti-HIV/biossíntese , Infecções por HIV/imunologia , HIV-1/genética , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Linfócitos T Citotóxicos , Transdução Genética , Vacinas Atenuadas/genéticaRESUMO
Direct in situ introduction of retroviral producer cells might provide a form of treatment for localized tumors. A possible undesirable consequence of this treatment could be uncontrolled proliferation of the injected producer cells. To test this possibility, severe combined immunodeficiencies (SCID) mice were reconstituted with human peripheral blood lymphocytes which were marked with a retroviral vector using a coculture method. Although specific measures were taken to remove the possible contaminating producer cells, a high percentage of mice developed fibrosarcoma 2-6 weeks after reconstitution. We hypothesized that tumors arose from a small number of contaminating producer cells in the inoculum. Tumor cells were consistently DNA tetraploid, a characteristic of the producer cell line. DNA extracted from tumor tissue was found to contain the gene (neomycin phosphotransferase) used to mark the producer cell line. Furthermore, SCID mice injected with 1 x 10(4) producer cells developed tumors with analogous characteristics. This report indicates that the retroviral producer cell line is tumorigenic in immune-deficient animals. Copyright 1995 S. Karger AG, Basel
RESUMO
cDNA clones mapping within the first 2601 bases of the 3' end of the TGEV genome were sequenced completely or in part by the method of Maxam and Gilbert and open reading frames were examined. One reading frame yielding a protein having properties of the matrix (M) protein was identified. It is positioned at the immediate 5' side of the nucleocapsid (N) gene but is separated by an intergenic region of 12 bases. The deduced M protein is comprised of 262 amino acids, has a molecular weight of 29,544, is moderately hydrophobic, and has an amino acid sequence homology of approximately 36% with the mouse hepatitis coronavirus, 37% with the bovine enteric coronavirus, and 28% with the avian infectious bronchitis virus. Judging from an alignment with MHV and IBV proteins, the amino terminus of the TGEV M protein extends 54 amino acids from the virion envelope which compares with 26 for MHV and 21 for IBV.
Assuntos
Coronaviridae/genética , Genes Virais , Vírus da Gastroenterite Transmissível/genética , Proteínas da Matriz Viral/genética , Sequência de Aminoácidos , Sequência de Bases , DNA/genética , Dados de Sequência Molecular , Peso MolecularRESUMO
It has been demonstrated that human immunodeficiency virus (HIV) replication can be effectively blocked by an antisense sequence that was introduced into the lymphoid cell line through retroviral-mediated gene transfer. In this study, it is demonstrated that antisense RNA can also inhibit simian immunodeficiency virus (SIV) replication in the peripheral blood mononuclear cells (MNCs) of healthy donors. MNCs were transduced with amphotropic recombinant virus encoding either sense or antisense constructs of SIV DNA fragments. After challenge with SIV, the viral replication was suppressed in the antisense-recombinant virus-transduced MNCs compared to sense-recombinant virus-transduced and untransduced MNCs. These data indicate that amphotropic retroviral vectors can be used to introduce antiviral factors (antisense sequence) into human primary MNCs and render them resistant to viral replication.
Assuntos
Leucócitos Mononucleares/microbiologia , RNA Antissenso/farmacologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Sequência de Bases , Depressão Química , Vetores Genéticos , Humanos , Dados de Sequência Molecular , Vírus da Imunodeficiência Símia/fisiologia , TransfecçãoRESUMO
To test the feasibility of gene therapy for AIDS patients, an animal model is needed to evaluate the efficacy and safety of this approach. Antiviral genes (encoding antisense RNA or viral protein) derived from Simian immunodeficiency virus (SIV) were efficiently targeted into CD4+ lymphocytes through retroviral-mediated gene transfer. After challenging with infectious viruses, the transduced lymphocytes that received antiviral genes were not only protected from SIV infection, but also from infection with HIV, for at least 25 days. Furthermore, little or no cytolytic effect (syncytium formation) was observed in the protected cells. These data demonstrated that SIV or HIV replication could be effectively blocked by antisense sequence(s) or negative dominant factors which were introduced into targeted cells through retroviral-mediated gene transfer.
Assuntos
Linfócitos T CD4-Positivos/microbiologia , Técnicas de Transferência de Genes , HIV-1/fisiologia , Vírus da Imunodeficiência Símia/fisiologia , Replicação Viral/genética , Animais , Sequência de Bases , Northern Blotting , Linhagem Celular , Efeito Citopatogênico Viral , Primers do DNA/química , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos , Células Gigantes/microbiologia , Infecções por HIV/terapia , HIV-1/genética , Macaca , Dados de Sequência Molecular , RNA Antissenso/análise , RNA Antissenso/genética , RNA Viral/análise , DNA Polimerase Dirigida por RNA/análise , Mapeamento por Restrição , Retroviridae , Vírus da Imunodeficiência Símia/genética , Transdução GenéticaRESUMO
Antisense RNA-mediated inhibition of HIV showed mixed success in previous experiments. In order to elucidate the parameters influencing the efficacy of an antisense RNA approach, retroviral vectors encoding 4.5 kb, 3.5 kb, or 2.5 kb antisense RNA of the gag-pol region of SIV (simian immunodeficiency virus) were constructed and used to transduce a CD4-positive CEM174 cell line. The growth rate of transduced cells was measured, and results showed that antisense RNAs have no detrimental effect on cell growth. Similar levels of antisense RNA expression were observed in all transduced cells by Northern analysis. The transduced cells were challenged with uncloned SIVmac239 at a m.o.i. (multiplicity of infection) of 1.0, 0.1, or 0.01. At a m.o.i. of 1.0 or 0.1, no significant inhibition of viral replication was observed in any antisense construct transduced cells up to 9 days postinfection. At a lower m.o.i. (0.01), viral replication was effectively inhibited in 3.5 kb antisense transduced cells as compared to 4.5 kb and 2.5 kb antisense transduced cells at 15 days postinfection. These data suggest that the size of antisense RNA and the challenge dose play a significant role in achieving effective inhibition.
Assuntos
Antivirais/farmacologia , RNA Antissenso/farmacologia , RNA Viral , Vírus da Imunodeficiência Símia/fisiologia , Replicação Viral/efeitos dos fármacos , Sequência de Bases , Northern Blotting , Linhagem Celular , Transformação Celular Viral/genética , DNA Viral/análise , Vetores Genéticos/genética , Dados de Sequência Molecular , Provírus/genética , Retroviridae/genética , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/genética , Proteínas Virais/análise , Replicação Viral/genéticaRESUMO
The open reading frame potentially encoding a 78 amino acid, 9101 Da hydrophobic protein (HP) and, mapping at the 3' end of the porcine transmissible gastroenteritis coronavirus (TGEV) genome, was shown to be expressed during virus replication. The cloned HP gene was placed in a plasmid under control of the T7 RNA polymerase promoter and in vitro translation of transcripts generated in vitro yielded a 9.1-kDa protein that was immunoprecipitable with porcine hyperimmune anti-TGEV serum. Antiserum raised in rabbits against a 31 amino acid synthetic polypeptide that represented the central hydrophilic region of HP specifically immunoprecipitated HP from TGEV-infected cells. HP was further shown to become associated with microsomal membranes during synthesis in vitro and was found to be closely associated with the endoplasmic reticulum and cell surface membranes in infected cells. The intracellular location of HP suggests that it may play a role in the membrane association of replication complexes or in virion assembly.
Assuntos
Membrana Celular/microbiologia , Retículo Endoplasmático/microbiologia , Vírus da Gastroenterite Transmissível/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA Viral , Imunofluorescência , Dados de Sequência Molecular , Vírus da Gastroenterite Transmissível/metabolismo , Proteínas Virais/metabolismo , Replicação ViralRESUMO
cDNA clones mapping within the first 2601 bases of the 3' end of the porcine transmissible gastroenteritis corona-virus (TGEV) genome were sequenced by the method of Maxam and Gilbert and an open reading frame yielding a protein having properties of the matrix (M or E1) protein was identified. It is positioned at the 5' side of the nucleocapsid (N) gene from which it is separated by an intergenic stretch of 12 bases. The deduced M protein comprises 262 amino acids, has a molecular weight of 29,544, is moderately hydrophobic, and has a net charge of +7 at neutral pH. Thirty-four percent of its amino acid sequence is homologous with the M protein of the bovine coronavirus (BCV), 32% with that of the mouse hepatitis coronavirus (MHV), and 19% with that of the avian infectious bronchitis coronavirus (IBV). Judging from alignment with the BCV, MHV, and IBV M proteins, the amino terminus of the TGEV M protein extends 54 amino acids from the virion envelope which compares with only 28 for BCV, 26 for MHV, and 21 for IBV. Eleven of the sixteen amino-terminal amino acids are hydrophobic and the positions of charged amino acids around this sequence suggest that the first 16 amino acids comprise a potentially cleavable signal peptide for membrane insertion. A similar sequence is not found in the M proteins of BCV, MHV, or IBV. When mRNA from infected cells, or RNA prepared by in vitro transcription of the reconstructed M gene, was translated in vitro in the presence of microsomes, the M protein became translocated and glycosylated. When a protein without the amino-terminal signal peptide was made by translating a truncated version of the M gene transcript, some translocation and glycosylation also occurred suggesting that the amino-terminal signal peptide on the TGEV M protein is not an absolute requirement for membrane translocation. Interestingly, the amino-terminal peptide did not appear to be cleaved during in vitro translation in the presence of microsomes suggesting that a step in virion assembly may be required for proper exposure of the cleavage site to the signal peptidase.
Assuntos
Coronaviridae/fisiologia , Sinais Direcionadores de Proteínas/fisiologia , Vírus da Gastroenterite Transmissível/fisiologia , Proteínas da Matriz Viral/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Membrana Celular/metabolismo , Clonagem Molecular , Glicosilação , Dados de Sequência Molecular , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Relação Estrutura-Atividade , Proteínas da Matriz Viral/genéticaRESUMO
This was a two-step study performed to assess the validity of a computer system in the quantification of coronary arterial angiography (CAG). First, the in vitro study involved 10 aluminum tubes in different internal diameters but the same thickness which were pre-filled with 76% urograffin followed by cinefilm taking. Acquisition and digitization of the stop film was undertaken by a videocamera linking to a computer system. The internal diameters and mean gray levels of all tubes were measured to compare with the real diameters and areas. Second, the in vivo study calculated the percentage of stenosis by visual estimation, diameter measurement and gray level measurement by the computer system from 22 vessels in 13 coronary angiograms. The percentage of patency is the ratio of the diameter or gray level from stenotic region to that of normal region in the CAG. The calculated interobserver variability by visual estimation, diameter measurement and Mean gray level measurement were studied. Good correlation was found between measured diameters and true tube diameters (r = 0.99), and between gray level and tube areas (r = 0.94). The diameter measured by computer and the gray level had lower variability (4.0% and 5.7% respectively) than visual estimation (9.8%) did. Thus, the diameter and densitometric gray level measurements might have clinical implications to assess the coronary arterial stenosis from the coronary angiograms.
Assuntos
Angiografia Coronária , Idoso , Constrição Patológica/diagnóstico por imagem , Doença das Coronárias/diagnóstico por imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Interpretação de Imagem Radiográfica Assistida por ComputadorRESUMO
A patient with hepatitis B virus (HBV) precore mutant (seropositive for hepatitis B surface antigen [HBsAg], anti-hepatitis B e antigen [HBeAg], and HBV DNA) who underwent orthotopic liver transplantation for end-stage liver disease is described. Sequencing of the HBV precore region of the pretransplant serum sample confirmed the presence of the precore stop-codon mutant (G-->A mutation in codon 1896) only. The patient received HBV immunoglobulin prophylaxis for 6 months but HBV recurred thereafter with a mild hepatitic flare, and he remained seropositive for HBsAg, anti-HBe, and HBV DNA. The initial hepatitic illness resolved in 3 months. The patient remained well for another 16 months before presenting with fibrosing cholestatic hepatitis (FCH). During his entire initial hepatitic flare, quiescent period, and final FCH phase, he remained seropositive for HBsAg, anti-HBe, and HBV DNA. Moreover, sequencing of the serum HBV DNA in final FCH phase showed the presence of the identical HBV precore mutant. Immunohistochemical staining showed extensive expression of HBsAg/pre-S1, pre-S2, and hepatitis B core antigen, but HBeAg was scarcely detectable. This case illustrates that (1) recurrence of HBV precore mutant infection can occur in liver; (2) it can give rise to FCH; and (3) hepatic accumulation of HBeAg is not essential for the development of FCH.