RESUMO
Alphavirus vectors are being developed for possible human vaccine and gene therapy applications. We have sought to advance this field by devising DNA-based vectors and approaches for the production of recombinant vector particles. In this work, we generated a panel of alphavirus vector packaging cell lines (PCLs). These cell lines were stably transformed with expression cassettes that constitutively produced RNA transcripts encoding the Sindbis virus structural proteins under the regulation of their native subgenomic RNA promoter. As such, translation of the structural proteins was highly inducible and was detected only after synthesis of an authentic subgenomic mRNA by the vector-encoded replicase proteins. Efficient production of biologically active vector particles occurred after introduction of Sindbis virus vectors into the PCLs. In one configuration, the capsid and envelope glycoproteins were separated into distinct cassettes, resulting in vector packaging levels of 10(7) infectious units/ml, but reducing the generation of contaminating replication-competent virus below the limit of detection. Vector particle seed stocks could be amplified after low multiplicity of infection of PCLs, again without generating replication-competent virus, suggesting utility for production of large-scale vector preparations. Furthermore, both Sindbis virus-based and Semliki Forest virus-based vectors could be packaged with similar efficiency, indicating the possibility of developing a single PCL for use with multiple alphavirus-derived vectors.
Assuntos
Alphavirus/genética , Vetores Genéticos , Vírus da Floresta de Semliki/genética , Sindbis virus/genética , Vacinas Sintéticas , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologia , Vacinas Virais , Animais , Formação de Anticorpos , Linhagem Celular , Transformação Celular Viral , Cricetinae , Feminino , Humanos , Rim , Camundongos , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Viral/genética , Linfócitos T Citotóxicos/imunologia , Transcrição Gênica , Proteínas Estruturais Virais/biossínteseRESUMO
A new method of producing vesicular stomatitis virus (VSV) G protein pseudotyped retroviral vectors is described. In this method, stocks of VSV-G pseudotyped vector were reproducibly obtained by infecting an env-, human, retroviral vector producer cell line with a recombinant murine cytomegalovirus (CMV) which expresses VSV-G protein. The recombinant murine CMV, RMCMVG, expressed VSV-G protein under transcriptional control of the human CMV immediate-early promoter. RMCMVG, like murine CMV, can infect human cells, but the infection is limited to the expression of the viral immediate-early genes; no productive replication of murine CMV occurs. Recombinant murine CMV vector infection of non-permissive cells may be useful in situations where high levels of gene expression are desired without concomitant viral vector replication.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos/genética , Glicoproteínas de Membrana , Muromegalovirus/genética , Retroviridae/genética , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Células 3T3 , Animais , Linhagem Celular , Genes Virais/genética , Terapia Genética/métodos , Células Gigantes , Glicoproteínas/biossíntese , Glicoproteínas/genética , Humanos , Camundongos , Muromegalovirus/crescimento & desenvolvimento , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Recombinação Genética , Retroviridae/crescimento & desenvolvimento , Transfecção , Vírus da Estomatite Vesicular Indiana/genética , Ensaio de Placa ViralRESUMO
Cytotoxic T lymphocyte (CTL) activity and CD4+ helper T cell responses to the hepatitis B virus (HBV) core antigen (HBcAg) have been implicated in clearance of acute and chronic HBV infections. We showed that intramuscular injections of a novel recombinant retroviral vector expressing an HBcAg-neomycin phosphotransferase II (HBc-NEO) fusion protein induces HBc/eAg-specific antibodies and CD4+ and CD8+ T cell responses in mice and rhesus monkeys. We have now immunized three chronically infected chimpanzees, each with 10(10) CFU of nonreplicating retroviral vector particles expressing the HBc-NEO fusion protein. Of two immunized chimpanzees examined for CTL responses, one developed HBcAg-specific CTLs and showed marginal, transient elevations of alanine aminotransferase (ALT) levels following injection. However, both chimpanzees remained positive for serum HBeAg, negative for anti-HBe antibody by conventional assays, and displayed no change in HBV viral load throughout the study. In contrast, the third chimpanzee exhibited a traditional seroconversion evidenced by a loss of serum HBeAg and the subsequent emergence of anti-HBe antibodies within 24 weeks after the first injection. Simultaneously, two transient ALT flares and a significant decrease in the serum HBV DNA levels were noted. Despite its limitations, the present study demonstrates (1) the safety of treatment with high titers of retroviral vector in chimpanzees, (2) the capability of a retroviral vector expressing HBcAg to stimulate immune responses in HBV chronic carrier chimpanzees, and (3) that retroviral vector immunization may be therapeutically beneficial in the treatment of chronic HBV infection.
Assuntos
Terapia Genética , Vetores Genéticos , Antígenos do Núcleo do Vírus da Hepatite B/genética , Hepatite B Crônica/terapia , Retroviridae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Portador Sadio , Primers do DNA , DNA Viral/sangue , Modelos Animais de Doenças , Anticorpos Anti-Hepatite B/biossíntese , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/imunologia , Humanos , Dados de Sequência Molecular , Pan troglodytes , Recombinação Genética , Homologia de Sequência de Aminoácidos , Linfócitos T Citotóxicos/imunologia , Carga ViralRESUMO
Gene delivery via murine-based recombinant retroviral vectors is currently widely used in gene therapy clinical trials. The vectors are engineered to be replication defective by replacing the structural and nonstructural genes of a cloned infectious retrovirus with a therapeutic gene of interest. The retroviral particles are currently generated in packaging cell lines, which supply all retroviral proteins in trans. Recombination between short homologous regions of the retroviral vector and packaging cell line elements can theoretically generate replication-competent retrovirus (RCR) and hence the Food and Drug Administration (FDA) requires the monitoring of clinical trial subjects for the presence of RCR. Sensitive polymerase chain reaction (PCR) assays have been used for the detection of murine leukemia virus (MLV) nucleotide sequences in peripheral blood mononuclear cells (PBMCs). A novel serological enzyme-linked immunosorbent assay (ELISA) for the detection of anti-MLV specific immunoglobulin (Ig) has been developed to be used as an alternative to the PCR assay. Both assays were used to monitor human immunodeficiency virus (HIV)-positive clinical trial subjects who had received multiple injections of HIV-IT (V), a retroviral vector encoding HIV-1 IIIBenv/rev. Western blot analysis and an in vitro vector neutralization assay were used to characterize further a subset of serum samples tested by ELISA. Results show no evidence of RCR infection in clinical trial subjects. PCR and ELISA assays are discussed in terms of their advantages and limitations as routine screening assays for RCR. The PCR assay is our current choice for monitoring clinical trial subjects receiving direct administration of vector, and the ELISA is our choice for those receiving ex vivo treatment regimens.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Vetores Genéticos/uso terapêutico , Reação em Cadeia da Polimerase/métodos , Retroviridae/genética , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Ensaios Clínicos Fase I como Assunto , Estudos de Avaliação como Assunto , Vetores Genéticos/genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Infecções por HIV/virologia , Humanos , Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/imunologia , Replicação ViralRESUMO
Cytotoxic T-lymphocyte (CTL) activity appears to play an important role in resolving hepatitis B virus (HBV) infection, and the ability to induce such responses remains an important goal for developing effective immunotherapeutics. A panel of recombinant retrovirus vectors expressing different forms of the HBV core antigen (HBcAg) or e antigen (eAg) were found to induce antigen-specific major histocompatibility complex-restricted CTL responses in both mice and macaques. In addition, a novel retrovirus vector expressing an HBcAg-neomycin phosphotransferase II (HBc-Neo) fusion protein [LHBc-NEO(6A3)], which allows the measurement of the anti-Neo antibody response as a means of directly tracking biological activity of the vector, was generated. Doses greater than 10(7) CFU were necessary to induce CTL responses in H-2(k) mice. Intramuscular injections with 10(8) CFU of the LHBc-NEO(6A3) retrovirus vector into rhesus monkeys induced HBc/eAg-specific antibody production and CD8+ CTLs. The CTL response from one of the two responder rhesus monkeys was directed against a 9-residue peptide, GELMTLATW, at positions 63 to 71 of the HBc/eAg sequence. The CTL response is long lived, being detectable as late as 16 weeks after immunization, and can be boosted upon reimmunization. The potent ability of recombinant retrovirus vectors to induce HBcAg- and eAg-specific CTL responses may prove beneficial as a therapeutic treatment for chronic hepatitis B infection.
Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos E da Hepatite B/imunologia , Retroviridae/genética , Linfócitos T Citotóxicos/imunologia , Vacinas Sintéticas/imunologia , Animais , Relação Dose-Resposta Imunológica , Feminino , Vetores Genéticos , Antígenos H-2/imunologia , Hepatite B/terapia , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos E da Hepatite B/genética , Imunização , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3HRESUMO
Several DNA-based Sindbis virus vectors were constructed to investigate the feasibility and potential applications for initiating the virus life cycle in cells transfected directly with plasmid DNA. These vectors, when transfected into mammalian cells, have been used to produce virus, to express heterologous genes, and to produce infectious vector particles. This approach involved the conversion of a self-replicating vector RNA (replicon) into a layered DNA-based expression system. The first layer includes a eukaryotic RNA polymerase II expression cassette that initiates nuclear transcription of an RNA which corresponds to the Sindbis virus vector replicon. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the vector, proceeds according to the Sindbis virus replication cycle and results in expression of the heterologous gene. The Sindbis virus DNA vectors expressed reporter genes in transfected cells at levels that were comparable to those of in vitro-transcribed RNA replicons and were approximately 10-fold higher than the levels produced by conventional RNA polymerase II-dependent plasmids in which the promoter and reporter gene were linked directly. Reporter gene expression was also observed in rodent muscle following injection with Sindbis virus DNA vectors. In a second application, packaged vector particles were produced in cells cotransfected with complementing replicon and defective helper DNAs. The Sindbis virus-derived DNA vectors described here increase the utility of alphavirus-based vector systems in general and also provide a vector with broad potential applications for genetic immunization.
Assuntos
DNA Viral , Vetores Genéticos , Sindbis virus/genética , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Estudos de Viabilidade , Técnicas de Transferência de Genes , Genes Reporter , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Músculos/metabolismo , Músculos/virologia , Plasmídeos , RNA/biossíntese , RNA Polimerase II/metabolismo , RNA Viral/metabolismo , Ratos , Ratos Sprague-DawleyAssuntos
DNA Recombinante/genética , Regulação Viral da Expressão Gênica , Vetores Genéticos/genética , RNA Viral/genética , Sindbis virus/genética , Animais , Bovinos , Células Eucarióticas , Genes Reporter , Hormônio do Crescimento/biossíntese , Hormônio do Crescimento/genética , Anticorpos Anti-Hepatite B/biossíntese , Antígenos da Hepatite B/biossíntese , Antígenos da Hepatite B/genética , Antígenos da Hepatite B/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Vírus da Leucemia Murina de Moloney/genética , RNA Polimerase II/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Viral/biossíntese , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/biossíntese , Sequências Repetitivas de Ácido Nucleico , Replicon , Sindbis virus/fisiologia , Replicação ViralRESUMO
Many protocols for gene therapy employ recombinant retroviral vectors, which are replication-defective retroviruses engineered to serve as gene delivery vehicles. The use of retroviral vectors for human gene therapy requires careful screening of vector-producing cell lines and culture supernatants to ensure the absence of replication competent retrovirus (RCR) in clinical products. In this study we have examined several different culture assays routinely used to test for the presence of RCR. Results indicate that cocultivation of a vector-producing cell line with a permissive cell line can reproducibly detect a low level of contaminating RCR. RCR was detected less frequently in direct tests of cell-free culture supernatants from a contaminated vector-producing line. Further studies revealed that recombinant retroviral vector can interfere, to varying degrees, with the detection of low-level RCR in culture supernatants when a marker rescue assay, an extended mink S+L- assay or a PG-4 S+L- assay is used. Interference can be partially overcome by culturing the vector preparation with a permissive cell line for several days before testing on the indicator cell line. The interference phenomenon we have observed may also occur in other culture assays routinely used for the detection of RCR.
Assuntos
Artefatos , Bioensaio , Linhagem Celular/virologia , Meios de Cultivo Condicionados/análise , Vírus Defeituosos/isolamento & purificação , Terapia Genética/normas , Vetores Genéticos/isolamento & purificação , Retroviridae/isolamento & purificação , Retroviridae/fisiologia , Ensaio de Placa Viral , Replicação Viral , Animais , Técnicas de Cultura/métodos , Cães , Reações Falso-Negativas , Teste de Complementação Genética , Marcadores Genéticos , Vetores Genéticos/normas , Vison , Muridae , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Recombinação Genética , Reprodutibilidade dos Testes , Segurança , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: To demonstrate the safety and enhancement of HIV-1-specific immune responses in HIV-infected asymptomatic patients following treatment with retroviral vector (Retrovector)-transduced autologous fibroblasts (VTAF) expressing HIV-1IIIB Env/Rev proteins. DESIGN: A non-placebo-controlled, single arm Phase I study. PARTICIPANTS: Four HIV-1-seropositive asymptomatic volunteers were selected based on age (18-50 years), CD4/CD3 lymphocyte counts (> 600 x 10(6)/l or > 40%), and positive delayed-type hypersensitivity test to at least one recall antigen. INTERVENTIONS: Patients were treated at 2-week intervals with a total of three intramuscular injections of irradiated autologous fibroblasts transduced with a molecularly engineered, non-replicating amphotropic murine retrovector encoding the HIV-1IIIB Env/Rev proteins. MAIN OUTCOME MEASURES: The clinical status of patients was assessed by history, physical examination, serum chemistry and hematology, CD4/CD3 lymphocyte counts, HIV viral burden, and monitored throughout the study to detect potentially treatment-induced toxic or unwanted side-effects. In addition, HIV-1-specific cytotoxic T-lymphocyte (CTL) activity was measured to determine the biological activity of VTAF. RESULTS: No acute local or systemic adverse events occurred following three injections with VTAF. Furthermore, a statistically significant increase of CD8+ CTL activity against HIV-1IIIB Env/Rev-expressing targets was observed in peripheral blood mononuclear cells from two out of four patients. CONCLUSIONS: This is the first report of the administration of a gene transfer treatment to HIV-1-infected patients and provides initial support for the safety and activity of retrovector-transduced fibroblasts administered to asymptomatic patients. This treatment resulted in the detection of increased HIV-1IIIB Env/Rev-specific CTL activity in two HIV-seropositive patients and could provide a better understanding of the role of CTL activity in HIV disease progression.
Assuntos
Vacinas contra a AIDS/imunologia , Produtos do Gene env/imunologia , Produtos do Gene rev/imunologia , Soropositividade para HIV/imunologia , HIV-1/imunologia , Ativação Linfocitária , Linfócitos T Citotóxicos , Vacinas Sintéticas/imunologia , Adolescente , Adulto , Animais , Transplante de Células , Técnicas de Transferência de Genes , Vetores Genéticos , Soropositividade para HIV/terapia , Soropositividade para HIV/virologia , HIV-1/genética , Humanos , Camundongos , Pessoa de Meia-Idade , Transplante Autólogo/imunologia , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
Sequence analysis of poliovirus vaccine RNA has resulted in the identification of a new point mutation at position 2493 in Sabin 3. cDNA-derived Sabin 3 virus whose genome includes the new mutation has phenotypic properties expected for a vaccine strain. Virus engineered to contain a single base substitution at 2493 has lost the small plaque phenotype, and low neurovirulence characteristics normally associated with Sabin poliovirus strains. The significance of these observations and a potential relationship to the frequency of vaccine-associated poliomyelitis are described.
Assuntos
Mutação , Vacina Antipólio Oral , Poliovirus/genética , RNA Viral/genética , Animais , Composição de Bases , DNA/genética , Humanos , Macaca mulatta , Camundongos , Camundongos Transgênicos , Poliomielite/epidemiologia , Poliomielite/etiologia , Poliomielite/patologia , Poliovirus/patogenicidade , Vacina Antipólio Oral/efeitos adversos , Vacina Antipólio Oral/toxicidade , Estados Unidos/epidemiologia , Células Vero , Virulência/genéticaRESUMO
The attenuated phenotype of Sabin 3 poliovirus compared with its neurovirulent progenitor strain has been largely accounted for by mutations in the genome at positions 472 and 2034 (G. D. Westrop, K. A. Wareham, D. M. A. Evans, G. Dunn, P. D. Minor, D. I. Magrath, F. Taffs, S. Marsden, M. A. Skinner, G. C. Schild, and J. W. Almond, J. Virol. 63:1338-1344, 1989). By sequencing vaccine virus RNA, we recently identified another Sabin 3-specific mutation at position 2493 (U----C), which predicts an Ile----Thr change at the sixth residue of VP1 (C. Weeks-Levy, J. M. Tatem, S. J. DiMichele, W. Waterfield, A. F. Georgiu, and S. J. Mento, Virology 185:934-937, 1991). Viruses generated by using cDNAs which represent the vaccine sequence (LED3) and a derivative (VR318) possessing a single base change to the wild-type nucleotide (U) at 2493 were used to determine the impact of the 2493 mutation on virus phenotype. The VP1 proteins of LED3 and VR318 viruses were distinguishable by denaturing electrophoretic analysis. LED3 produced smaller plaques in Vero cells than VR318 virus did. Neurovirulence testing of these cDNA-derived viruses in monkeys demonstrated that the 2493 mutation in LED3 virus is attenuating.
Assuntos
Capsídeo/genética , Poliomielite/genética , Poliovirus/genética , Animais , Proteínas do Capsídeo , Mutação/genética , Fenótipo , Poliovirus/patogenicidade , Vacinas Atenuadas , Células VeroRESUMO
The complete RNA sequence of Sabin 3 (LED3) used in vaccine in the United States has been determined. The LED3 Sabin 3 sequence contains the attenuating mutations at bases 472 and 2034 but differs from that published by Stanway et al. (Nucleic Acids Res., 11, 5629-5643, 1983) at two other base positions, 2493 and 6061. The change at base 6061 is silent and does not affect amino acid composition. The other base, a C at position 2493, is contained in the viral capsid protein VP1 and predicts a new Sabin 3 specific amino acid change of a threonine instead of an isoleucine at amino acid 6 of the protein [corrected]. Reversion of this base to that present in the pathogenic progenitor strain, Leon, is observed to occur after replication of vaccine virus in the gut of primary vaccines and in nervous tissue of neurovirulence test monkeys. Passage conditions have been identified that lead to the reversion of base 2493 as well as the reversion of the attenuated base to the parental base (Leon) at position 472 in the 5' noncoding region. The observation that these two bases delta position are found to revert during passage suggests that there is a selective advantage for virus containing the parental bases at these positions.
Assuntos
Capsídeo/genética , Vacina Antipólio Oral , Poliovirus/genética , Animais , Proteínas do Capsídeo , Variação Genética/genética , Haplorrinos/microbiologia , Mutação/genética , Poliovirus/patogenicidade , Seleção Genética , Virulência/genéticaRESUMO
Derivatives of Sabin 3 shed from recipients of oral poliovirus vaccine in the United States (U.S.) were examined for genetic changes identified in strains excreted by vaccinees in the United Kingdom [U.K.; Evans et al., 1985; Cammack et al., 1988, Macadam et al., 1989]. Among the eight primary vaccinees studied, the duration of excretion and molecular evolution of type 3 strains varied greatly. The period of virus excretion after vaccination ranged from as few as 2 days to as many as 36 days. Nucleotide sequence analysis of viral RNAs extracted from shed virus indicated that only fifty percent of the vaccinees exclusively excreted strains in which the attenuating mutation at nucleotide 472 in the 5' noncoding region of the genome had reverted from uracil (U) to cytosine (C), the nucleotide found in neurovirulent strains. Compared to the wild-type Leon strain, the low activity of stool isolate KW4 in a complete monkey neurovirulence test demonstrated that presence of C at 472 does not render a type 3 strain pathogenic. Conversely, an isolate was identified which efficiently replicated in monkey nervous tissue and maintained the attenuated U at 472. Oligonucleotide fingerprinting and sequence analysis of viral RNAs from stool isolates indicated that one vaccinee (KW) eventually excreted intertypic recombinant strains consistent with those reported in the U.K. studies. Unique to this study, one vaccinee (KS) excreted nonrecombinant virus possessing U at 472 for up to 21 days. The significance of the KS strain profile in relation to differences in the U.S. vaccine compared to the vaccine distributed in the U.K. and other countries is discussed.
Assuntos
Poliomielite/prevenção & controle , Vacina Antipólio Oral/genética , Poliovirus/crescimento & desenvolvimento , Replicação Viral , Animais , Eletroforese em Gel de Poliacrilamida , Fezes/microbiologia , Humanos , Lactente , Intestinos/microbiologia , Macaca mulatta , Mapeamento de Nucleotídeos , Poliomielite/microbiologia , Poliovirus/genética , Poliovirus/isolamento & purificação , Vacina Antipólio Oral/administração & dosagem , RNA Viral/análise , Estados UnidosRESUMO
Mice, guinea pigs, and rhesus monkeys were immunized with immunoaffinity-purified native glycoprotein D (gD) derived from herpes simplex virus type 1 (HSV1). The native glycoprotein has evoked significant in vivo responses even at low doses. Thus, mice immunized with doses as low as 1 microgram were significantly protected from the morbidity and mortality of lethal HSV2 challenge and from establishment of latent HSV2 infection. Protection was dose-related and correlated with prechallenge serum neutralizing antibody titres to HSV. Similarly, immunized guinea-pigs demonstrated significant reductions in the frequency, severity and duration of genital lesions induced by HSV2 vaginal challenge. In long term immunogenicity studies, immunized rhesus monkeys exhibited significant serum neutralizing antibody responses to both HSV1 and HSV2. In vitro stimulation of monkey peripheral blood leucocytes with purified gD resulted in a significant cellular proliferative response. The results obtained in these animal models with a gD subunit vaccine provide an appropriate foundation for the initiation of human studies.
Assuntos
Anticorpos Antivirais/sangue , Herpes Simples/prevenção & controle , Vacinas contra Herpesvirus , Simplexvirus/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Feminino , Cobaias , Herpes Genital/prevenção & controle , Imunização , Ativação Linfocitária , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Testes de Neutralização , Organismos Livres de Patógenos Específicos , Proteínas do Envelope Viral/química , Vacinas Virais/químicaRESUMO
A cell line used in the production of biologicals should be free of infectious agents, and 'described with respect to cytogenetic characteristics and tumorigenicity'. Vero, a continuous cell line derived from a normal African green monkey kidney, was examined for the presence of retroviruses and for tumorigenic potential. We were unable to detect the presence of retroviruses by reverse transcriptase assay, electron microscopy or hybridization of cellular genomic DNA with Mason-Pfizer monkey virus DNA probes. In addition, passage 156 Vero cells did not form progressively growing tumors in nude mice or grow with high efficiency in soft agarose.
Assuntos
Células Vero/microbiologia , Animais , Adesão Celular , DNA Viral/isolamento & purificação , Feminino , Camundongos , Camundongos Nus , Microscopia Eletrônica , Neoplasias Experimentais/etiologia , Retroviridae/isolamento & purificação , Vírus da Imunodeficiência Símia/isolamento & purificaçãoRESUMO
Although diabetes mellitus is generally subclassified into either of two categories, insulin-dependent (type I) and non-insulin-dependent (type II), the heterogeneity of its clinical expressions, genetics, and etiologies may make a clear distinction in classification difficult. Evidence to date for the involvement of viruses in the etiology of the disease has involved only type I diabetes. However, clinical data derived from the subtle chronicity of type II diabetes, the lack of pathological alterations in the pancreas in some patients with type II diabetes, and animal and human studies with Venezuelan encephalitis virus and rubella virus suggest a possible role for viruses in the pathogenesis of non-insulin-dependent diabetes.
Assuntos
Diabetes Mellitus/etiologia , Viroses/complicações , Animais , Cricetinae , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/terapia , Suscetibilidade a Doenças , Humanos , Mesocricetus , Camundongos , Pâncreas/microbiologia , Viroses/diagnóstico , Viroses/imunologia , Replicação ViralAssuntos
Fibroblastos/citologia , Sindbis virus/fisiologia , Animais , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Cricetinae , Cricetulus/genética , Feminino , Fibroblastos/efeitos dos fármacos , Genes Recessivos , Ovário , Biossíntese de Proteínas , RNA Viral/metabolismo , Receptores Virais/genética , Proteínas Virais/biossínteseRESUMO
Spontaneous and EMS-induced alpha-amanitin-resistant Aedes albopictus cells have been isolated and characterized. Two mutant sublines, one of intermediate resistance (alpha A2) and the other highly resistant (Ama18) contained RNA polymerase II activity, the resistance of which in vitro to alpha-amanitin correlated well with the resistance of these cells in vivo. The resistance of these cells to alpha-amanitin can likely be attributed to the presence of an altered RNA polymerase II.
Assuntos
Aedes/enzimologia , Amanitinas/farmacologia , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerase II/metabolismo , Aedes/genética , Animais , Linhagem Celular , Células Clonais , Resistência a Medicamentos , Metanossulfonato de Etila/farmacologia , Mutação , Fenótipo , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/genéticaRESUMO
The phenotype of a ouabain-resistant Aedes albopictus cell line has been partially characterized. Treatment of ouabain-sensitive cells with 0.005-1.0 mM ouabain resulted in an 80% reduction in the uptake of 86rubidium (86Rb+), an ion with an affinity for the K+ pump binding site; ouabain-resistant cells showed only a 40% reduction with 1.0 mM ouabain. When ouabain-sensitive cells were incubated in the presence of ouabain (0.1 mM) for one and one-half to three hours, the molar ratio of intracellular Na+/K+ rose from 0.2 to 4.2. In ouabain-resistant cells, a similar treatment had very little effect. Based on [3H] ouabain-binding studies, ouabain-resistant cells were estimated to have 60% fewer binding sites per cell than ouabain-sensitive cells. The spontaneous mutation rate from ouabain sensitivity to ouabain resistance was calculated to be 1-6 x 10(-8) mutations/cell/generation, a value similar to that reported for mammalian cells at the analogous locus.