RESUMO
Due to late diagnosis and a pronounced chemoresistance, most patients with hepatocellular carcinoma (HCC) have an overall poor prognosis. Measles vaccine viruses (MeV) have been shown to possess anti-tumor properties and their efficacy has been enhanced by arming with suicide genes. To test armed MeV for the treatment of HCC, we equipped it with the suicide gene Super-cytosine deaminase (SCD) and tested the efficacy in cell culture and in a mouse xenograft model of human HCC. Prodrug conversion was investigated in cell culture and quantified by high-performance liquid chromatography. We observed a strong oncolytic activity of MeV-SCD against human HCC in vitro and in vivo. The prodrug was efficiently converted in infected cells leading to a significant enhancement of the cytotoxic effect. Treatment of HCC xenografts with MeV caused long-term virus replication in tumor tissue. We show that the suicide gene therapy induces an apoptosis-like cell death but is not dependent on intact apoptosis pathways. These results demonstrate that MeV-based suicide gene therapy is a promising novel therapy regimen for HCC overcoming resistance towards conventional therapy. The independence from apoptosis raises hopes for the treatment of patients whose tumor cells exert defects in this cell death mechanism.
Assuntos
Apoptose , Carcinoma Hepatocelular/terapia , Citosina Desaminase/genética , Vírus do Sarampo , Terapia Viral Oncolítica , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Cromatografia Líquida , Terapia Combinada , Citosina Desaminase/metabolismo , Resistencia a Medicamentos Antineoplásicos , Genes Transgênicos Suicidas , Terapia Genética , Células Hep G2 , Humanos , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/terapia , Vacina contra Sarampo , Vírus do Sarampo/genética , Camundongos , Camundongos Nus , Vírus Oncolíticos/genética , Células Tumorais Cultivadas , Células Vero , Replicação Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Four protein fragments which span the entire hemagglutinin-neuraminidase protein (HN) of mumps virus were expressed in HeLa cells and cell extracts were tested for their capability to induce neutralizing antibodies in mice. Fragment HN3 (aa 213-372) was able to induce the production of hemagglutination-inhibiting and neutralizing antibodies. When a subfragment of HN3, the synthetic peptide NSTLGVKSAREF (aa 329-340 of HN) was used for immunization, hemagglutination-inhibiting and neutralizing antibodies against mumps wild type virus but not against the Urabe Am9 vaccine virus were raised. The peptide could, therefore, contain a new epitope, which may be critical for protective host humoral immune response.
Assuntos
Mapeamento de Epitopos , Epitopos de Linfócito B/análise , Proteína HN/imunologia , Vírus da Caxumba/imunologia , Neuraminidase/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais , Linhagem Celular , Chlorocebus aethiops , Epitopos de Linfócito B/imunologia , Feminino , Proteína HN/química , Proteína HN/isolamento & purificação , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Vacina contra Caxumba , Vírus da Caxumba/genética , Vírus da Caxumba/patogenicidade , Neuraminidase/química , Neuraminidase/isolamento & purificação , Testes de Neutralização , Peptídeos/análise , Peptídeos/síntese química , Células VeroRESUMO
The promoter of the interferon regulatory factor-1 (IRF-1) gene contains at position -47 to -38 an evolutionary conserved binding sequence for the inducible transcription factor NF-kappa B. This site is highly homologous to a transcriptionally active site from the MHC class I enhancer. In this study, we show by in vitro assays using purified NF-kappa B that the kappa B motif in the IRF-1 promoter binds the factor specifically and with high affinity, comparable to various other cis-acting kappa B elements. Two copies of the IRF-1 kappa B site fused to the heterologous c-fos promoter conferred induction of a chloramphenicol acetyl transferase (CAT) reported gene in response to stimulation of L929 fibroblasts with various NF-kappa B inducers, such as tumor necrosis factor alpha (TNF alpha) or phorbol 12-myristate 13-acetate (PMA). Mutation of the binding site completely abolished transcriptional inducibility of the heterologous promoter. Surprisingly, the same IRF-1 kappa B motif in context of the homologous IRF-1 promoter was transcriptionally inactive in CAT assays. The very weak induction of the IRF-1 promoter in response to TNF treatment or infection of fibroblasts with Newcastle disease virus (NDV) was barely affected by point mutation of the kappa B site or loss of the site by truncation of the promoter. Analysis of the occupational state of the chromosomal IRF-1 kappa B site by in vivo foot-printing revealed that no footprint was induced over the kappa B motif in the IRF-1 promoter after PMA treatment of L929 fibroblast cells, despite the simultaneous induction of IRF-1 mRNA and NF-kappa B binding activity. Constitutive footprints were detected at a CCAAT and GC-rich region in the promoter. This is the first example of a high-affinity NF-kappa B binding site within a promoter which may not participate in transcriptional regulation under conditions activating NF-kappa B DNA binding and gene expression.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , NF-kappa B/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas , Ativação Transcricional , Animais , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Fibroblastos/metabolismo , Fator Regulador 1 de Interferon , Camundongos , Sondas Moleculares/genética , Dados de Sequência Molecular , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/genéticaRESUMO
A technique is described for the small-scale determination of antibodies in blood and liquor. Small sample volumes of about 3 microliter blood and 15 microliter liquor are sufficient to detect even low antibody titers against different proteins. Thus, for studying the timecourse of antibody titers, repeated examinations of small laboratory animals are possible. The method includes the highly reproducible separation of the antigens in a 45 X 35 X 0.5 mm polyacrylamide gel (6.5%) using the Phast-system (Pharmacia), followed by electrotransfer and immunostaining. The whole procedure takes less than five hours.
Assuntos
Anticorpos Antivirais/análise , Western Blotting/métodos , Animais , Anticorpos Antivirais/líquido cefalorraquidiano , Antígenos Virais/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Técnicas Imunoenzimáticas , Camundongos , Vírus da Parainfluenza 1 Humana/imunologia , Infecções por Paramyxoviridae/imunologia , Ratos , Proteínas Virais/imunologia , Proteínas Virais/isolamento & purificaçãoRESUMO
In a comparative study the factors influencing the recovery of recombinant Sendai viruses (SeV) from plasmid based cDNA were analysed systematically in order to establish an efficient and robust method for virus rescue. The amounts and ratios of transfected helper plasmids encoding the viral N, P and L proteins proved to be crucial for virus rescue, and they were optimised step-by-step for enhanced virus release. When the C open reading frame from the P gene was expressed at low level, virus rescue was generally possible but virus release could be improved when C gene expression was abolished completely. SeV particle formation could be increased greatly when the transcription initiation site for T7 polymerase in the cDNA was modified or when the genomic ribozyme instead of the antigenomic ribozyme of hepatitis delta virus was used for processing the 3'end of the viral RNA transcript. Heterologous helper viruses vTF7-3 and MVA-T7, which are necessary for T7 polymerase production in transfected cells, were compared for their use in SeV recovery and subsequent elimination of the helper virus from recombinant SeV. Interference with SeV replication was less severe with MVA-T7, and MVA-T7 was eliminated efficiently without the need for any inhibitors by serial passages in Vero cells. Optimal combination of all parameters led to a highly efficient generation of recombinant SeV from cDNA. Titres of the released virus particles are high enough to enable analysis of the recombinant SeV directly on test cells or propagation in cell cultures without the need for amplification in embryonated chicken eggs. The system is very robust and allows rapid generation of defined SeV mutants that require specialised host cells for propagation.
Assuntos
DNA Viral , Recombinação Genética , Respirovirus/isolamento & purificação , Animais , Embrião de Galinha , Chlorocebus aethiops , DNA Complementar , Células HeLa , Humanos , Respirovirus/genética , Respirovirus/fisiologia , Fatores de Tempo , Células Vero , Interferência ViralRESUMO
The determination of the complete DNA sequence of the large (L) polymerase gene of Sendai virus strain Fushimi was used to explore the potential and feasibility of primer walking with fluorescent dye-labelled dideoxynucleotide terminators on an automated ABI DNA sequencer. The rapid identification of the complete sequence demonstrated that this approach is a time- and cost-saving alternative to classical sequencing techniques. Analysis of the data revealed that the L gene of Sendai virus strain Fushimi consists of exactly 6800 nucleotides and that the deduced amino acid sequence identifies a single open reading frame encoding a protein of 252.876 kDa. In contrast to Sendai virus strain Enders, the L mRNA of strain Fushimi is monocistronic. The comparison of the deduced amino acid sequences of the L genes of three different Sendai virus strains confirmed the existence of conserved as well as variable regions in the L protein and revealed a high grade of conservation in the carboxyterminal third. Furthermore, functional amino acid sequence motifs, like elements of RNA-dependent RNA polymerases and ATP-binding sites as postulated previously, were identified.
Assuntos
Passeio de Cromossomo/métodos , RNA Polimerases Dirigidas por DNA , Genes Virais/genética , Vírus da Parainfluenza 1 Humana/genética , Proteínas Virais/química , Sequência de Bases , DNA de Cadeia Simples , DNA Viral , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Proteínas Virais/genéticaRESUMO
Numerous ubiquitous ribonucleic acid and deoxyribonucleic acid viruses, inducing acute, monophasic infections (mostly childhood diseases) have been considered as potential causes of multiple sclerosis. The present hypothesis reconsiders the role of the viral agent: not the virus, but the reaction of the defense system to the viral persistence, appearing after the acute phase, is postulated as a key factor. A prerequisite of multiple sclerosis is polygenetically determined or acquired immunodeficiency; the defense system is not able to stop repeated viral reactivations induced by a set of exogenous and/or endogenous factors. Thus, an aberrant virus production can appear repeatedly. If the virus spreads from primary target--the lymphoreticular system--into the central nervous system, the multiple sclerosis process can be initiated. Activated T cells and endothelial cells serve as first-host cells. Their infection triggers a set of reactive events: multiple microthrombosis and inflammation play a key role, both of which can result in nonspecific degradation of the myelin. An increased release of myelin antigens induces a homeostatic autoimmunity. Long-term repetition of the shifts and the infection of inflammatory cells can lead to disturbances in self-tolerance. A dysregulated pathological autoimmunity can develop, which acts as a main effector of the specific demyelination.
Assuntos
Esclerose Múltipla/imunologia , Esclerose Múltipla/virologia , Síndrome da Imunodeficiência Adquirida , Criança , Vírus de DNA/patogenicidade , Doenças Desmielinizantes/imunologia , Doenças Desmielinizantes/virologia , Endotélio/fisiologia , Humanos , Inflamação , Ativação Linfocitária , Modelos Biológicos , Linfócitos T/imunologia , TromboseRESUMO
The cause of otosclerosis is still unknown. Recently, measles virus involvement has been implicated. The aim of this study was to analyze the presence of measles virus RNA within the otosclerotic focus and to evaluate the perilymphatic antibody pattern. Bone and perilymph specimens from 40 patients with the spontaneous form of otosclerosis and from control patients were investigated by reverse transcription polymerase chain reaction (RT-PCR), Western blot techniques, and cell culture. By the use of RT-PCR, measles virus RNA could be detected in 32 patients, but not in controls. Analysis of perilymph revealed the presence of antibodies to N, F1, and M measles virus proteins in all cases, and antibodies against H protein in 2 additional cases. In preosteoblasts cultured from otosclerotic bone chips, no measles virus RNA could be amplified. We conclude that the spontaneous form of otosclerosis is, in the vast majority of cases, a measles virus-associated disease of the otic capsule.
Assuntos
Vírus do Sarampo/isolamento & purificação , Otosclerose/virologia , Adulto , Idoso , Anticorpos Antivirais/análise , Western Blotting , Feminino , Humanos , Masculino , Sarampo/complicações , Vírus do Sarampo/genética , Vírus do Sarampo/imunologia , Pessoa de Meia-Idade , Perilinfa/imunologia , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Histologic and immunohistochemical studies of otosclerotic lesions have shown that there is a chronic inflammatory reaction of the otic capsule with bone resorption resulting from vascular invasion accompanied by inflammatory cells. During the active lytic stage of otosclerosis, paramyxoviral structures have been identified by electron microscopy and measles virus antigen expression by immunohistochemistry. Recently, measles virus related sequences have been detected in tissue of otosclerotic lesions. Because the otosclerotic focus has a close relation to the perilymphatic space, the expression of measles virus antigens within it should represent an immunologic challenge to the immune system of the endolymphatic sac. In this study, measles virus specific antibodies were detected in all of the perilymph samples from 19 patients suffering from otosclerosis, and the relative amount of these IgG antibodies was much higher than in serum samples of the same patients or in perilymph of control patients. These findings support the hypothesis that measles viruses play an crucial role in the pathogenesis of otosclerosis.
Assuntos
Anticorpos Antivirais/análise , Antígenos Virais/análise , Orelha Interna/imunologia , Vírus do Sarampo/imunologia , Otosclerose/virologia , Orelha Interna/virologia , Feminino , Humanos , Masculino , Vírus do Sarampo/isolamento & purificação , Otosclerose/imunologia , Perilinfa/virologia , Reação em Cadeia da PolimeraseRESUMO
The etiopathogenesis of otosclerosis is still largely unexplained and remains controversial. Morphologic examinations have shown the presence of a chronic inflammation in otosclerotic tissue. Among the proposed explanations for this inflammation are an immunologic reaction against collagen, mutations of collagen gene 1A1, and a viral infection. In this paper, we focus on the role of measles virus in otosclerosis, and we review the current literature, devoting particular attention to a suspected paramyxoviral etiopathogenesis in Paget's disease. Our examination of footplate fragments by reverse transcription polymerase chain reaction testing in 95 patients with otosclerosis revealed the presence of measles virus RNA in 83% of cases. Quantification of measles virus immunoglobulin G (IgG) in otosclerosis patients indicated that the ratio of antimeasles virus IgG in total IgG was higher in perilymph than in serum. Furthermore, an almost identical incidence of otosclerosis and measles virus-caused mortality in women suggests that women are more susceptible to measles virus infection. Finally, since the introduction of the measles virus vaccination program in Europe, there has been a decline in the incidence of otosclerosis. Moreover, the average age of patients at diagnosis and surgery at our hospital has increased to 54 years. Our findings, when they are considered along with findings regarding the presence of paramyxoviral RNA in Paget's disease, support the hypothesis that measles virus is involved in the etiopathogenesis of otosclerosis.
Assuntos
Vírus do Sarampo/isolamento & purificação , Sarampo/complicações , Osteíte Deformante/virologia , Otosclerose/virologia , Adolescente , Adulto , Idoso , Anticorpos Antivirais/análise , Southern Blotting , Estudos de Casos e Controles , Doença Crônica , Feminino , Predisposição Genética para Doença , Alemanha/epidemiologia , Humanos , Incidência , Masculino , Sarampo/epidemiologia , Sarampo/imunologia , Sarampo/virologia , Vírus do Sarampo/genética , Vírus do Sarampo/imunologia , Pessoa de Meia-Idade , Otosclerose/epidemiologia , Otosclerose/etiologia , Otosclerose/imunologia , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores SexuaisRESUMO
Measles virus (MeV) might play an important role as an environmental stimulus in the etiopathogenesis of otosclerosis. Chronic inflammation was shown in morphologic investigations of otosclerotic foci and MeV N, P, and F proteins were detected within cells of the otosclerotic focus by immunohistochemical investigations. MeV RNA was extracted from fresh-frozen otosclerotic tissue by the use of in vitro RT-PCR. This result was validated through amplification of MeV genome sequences by RT-PCR from celloidin-embedded sections with morphologically ascertained otosclerotic foci. In searching for an immune response of the inner ear immune system against MeV proteins, elevated anti-MeV IgG levels were detected in the perilymph of patients with otosclerosis in comparison with the serum levels. In situ RT-PCR allowed the localization of MeV sequences in osteoclasts, osteoblasts, chondrocytes, macrophages, and epithelial cells in middle ear mucosa of otosclerotic tissue. Further evidence for MeV persistence has recently been given. Genotyping of MeV in otosclerotic foci demonstrated the presence of MeV genotype A, which circulated in Europe around 1960. All the above results confirm a strong association between MeV and otosclerosis.
Assuntos
Vírus do Sarampo/genética , Otosclerose/virologia , RNA Viral/genética , Anticorpos Antivirais/metabolismo , Orelha Média/imunologia , Orelha Média/patologia , Orelha Média/virologia , Genoma Viral/genética , Genótipo , Humanos , Imunoglobulina G/metabolismo , Inflamação , Vírus do Sarampo/imunologia , Otosclerose/imunologia , Otosclerose/patologia , Perilinfa/imunologia , Perilinfa/virologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
An essential prerequisite for generating a stable helper cell line, which constitutively expresses functional Sendai virus RNA-dependent RNA polymerase, is the expression of all three Sendai virus nucleocapsid (NC) proteins, NP, P, and L, simulataneously. Generating a stable helper cell line was accomplished by cotransfecting cell line 293 with all three corresponding viral genes under the control of cytomegalovirus promoter-enhancer elements. Cotransfection with a dominant selectable marker enabled selection for stably transfected cells. The levels of the expressed P and NP proteins reached up to 1/10th and 1/20th of the protein levels in Sendai virus-infected cells, respectively. The Sendai virus polymerase activity of the coexpressed proteins was demonstrated by an in vivo polymerase assay. The cell clone H29 gave the strongest signal and produced DI genomes continuously for at least 3 months. This result demonstrates that it is possible to stably express adequate levels of all three viral NC proteins to form Sendai virus polymerase activity, thereby performing the replication and encapsidation of viral RNA, essential prerequisites for a helper cell line to be competent in producing recombinant viruses.
Assuntos
Capsídeo/biossíntese , Vírus Defeituosos/metabolismo , Vírus da Parainfluenza 1 Humana/metabolismo , Proteínas do Core Viral/biossíntese , Sequência de Bases , Capsídeo/análise , Capsídeo/isolamento & purificação , Linhagem Celular , Células Clonais , Citomegalovirus/genética , Primers do DNA , Elementos Facilitadores Genéticos , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Polimerase Dependente de RNA/biossíntese , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Linfócitos T Auxiliares-Indutores , Transfecção , Proteínas do Core Viral/análise , Proteínas do Core Viral/isolamento & purificaçãoRESUMO
The cell line Cl-E-8 showing expression of Sendai-6/94 viral antigens after the original isolation was reexamined after approximately 160 subcultures. In the virus fraction of cell supernatants 6/94 virus particles (termed 6/94 cl) could be demonstrated; no infectivity, however, was monitored. We were also unable to activate the viral infectivity of 6/94 cl virus by trypsin treatment. The analysis of viral RNA revealed that the virus contains a high-molecular-weight (50 S) single-stranded RNA. After cocultivation of Cl-E-8 cells with several standard cell lines the production of an infectious 6/94 virus, termed 6/94 co, was detected. The infectivity titer in the supernatants was very low, about three orders of magnitude lower than in cultures infected with the egg-grown 6/94 virus (6/94 ST). Surprisingly, the production of infectious 6/94 cl virus invariably ceased several subcultures after cocultivation even in the presence of foreign cells. However, the infectivity could be repeatedly reinduced by adding fresh foreign cells.
Assuntos
Vírus da Parainfluenza 1 Humana/isolamento & purificação , Antígenos Virais/análise , Linhagem Celular , Peso Molecular , Vírus da Parainfluenza 1 Humana/crescimento & desenvolvimento , RNA Viral/análise , Tripsina/metabolismo , Ativação ViralRESUMO
Several catalytic antisense RNAs directed against different regions of the genomic or antigenomic RNA of Sendai virus were constructed. All RNAs contained the same catalytic domain based on hammerhead ribozymes but some had deletions or mutations resulting in imperfect helices I and III. Pre-annealed substrate/ribozyme complexes were used to determine the rates of the cleavage process for the different ribozymes under single-turnover conditions. It was found that the sequence context surrounding the cleavable motif influenced the cleavage efficiencies. Deletions or mutations of nucleotides 2.1 or 15.1 and 15.2 according to the numbering system for hammerhead ribozymes of Hertel et al. destroyed catalytic activity. Deletions of nucleotide 2.2 or additional nucleotides in the helix I-forming region of the ribozyme did not destruct, but only reduced the cleavage efficiencies. Similar results were observed for a deletion of nucleotide 15.3. Simultaneous deletions within helices I and III resulted in alternative cleavage sites. The potential consequences for the specificity of the ribozyme reaction are discussed.
Assuntos
Conformação de Ácido Nucleico , RNA Antissenso/química , RNA Catalítico/química , Sequência de Bases , Catálise , Cinética , Dados de Sequência Molecular , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/genética , Vírus da Parainfluenza 1 Humana/química , Mutação Puntual/fisiologia , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA Viral/metabolismo , Deleção de Sequência/fisiologiaRESUMO
Sendai virus RNA species were quantitated in lytically and persistently infected cultured cells by Northern blot hybridization to region- and strand-specific cloned cDNA probes. Levels of NP, P and M mRNA in lytically infected cells were equally high, but F and HN mRNA were present in about 3-fold, and L mRNA in 30-fold, lower amounts, reflecting transcriptional attenuation especially at the M-F and HN-L gene junction. Two persistently infected cell lines, which release only 1% of the virus particles of lytically infected cells, were shown to contain only 4- to 8-fold-less amounts of each viral mRNA and 2- to 3-fold-less genomic RNA than lytically infected cells. Additionally, transcription was neither defective nor more attenuated as compared to the lytical infection. Taken together the results suggest the existence of an additional regulatory mechanism for the virus release. A cell-associated state of infection therefore seems to be achievable by a relatively weak general reduction of the copy numbers of viral mRNA and genomic RNA.
Assuntos
Expressão Gênica , Vírus da Parainfluenza 1 Humana/genética , Animais , Sequência de Bases , Northern Blotting , Linhagem Celular , Embrião de Galinha , Sondas de DNA , DNA Viral/genética , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , RNA Viral/genética , Mapeamento por Restrição , Transcrição Gênica , Células VeroRESUMO
The complete nucleoprotein (NP) gene sequences of the Sendai virus Fushimi and 6/94 strains were determined. For both viruses an open reading frame of 524 amino acids can be predicted for the NP proteins. By comparing the sequences with others reported in the literature, the 5' noncoding region and the middle third of the coding region were found to be highly conserved. The carboxyl terminal part carries nine amino acid changes and a completely different sequence of the carboxyl terminus with a seven amino acid extension. This carboxyl terminus of the Sendai virus NP protein was confirmed using tryptic peptide sequence analysis.