RESUMEN
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.
Asunto(s)
Apoptosis , Carcinoma Hepatocelular/terapia , Citosina Desaminasa/genética , Virus del Sarampión , Viroterapia Oncolítica , Animales , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Chlorocebus aethiops , Cromatografía Liquida , Terapia Combinada , Citosina Desaminasa/metabolismo , Resistencia a Antineoplásicos , Genes Transgénicos Suicidas , Terapia Genética , Células Hep G2 , Humanos , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas Experimentales/terapia , Vacuna Antisarampión , Virus del Sarampión/genética , Ratones , Ratones Desnudos , Virus Oncolíticos/genética , Células Tumorales Cultivadas , Células Vero , Replicación Viral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Virus del Sarampión/genética , Otosclerosis/virología , ARN Viral/genética , Anticuerpos Antivirales/metabolismo , Oído Medio/inmunología , Oído Medio/patología , Oído Medio/virología , Genoma Viral/genética , Genotipo , Humanos , Inmunoglobulina G/metabolismo , Inflamación , Virus del Sarampión/inmunología , Otosclerosis/inmunología , Otosclerosis/patología , Perilinfa/inmunología , Perilinfa/virología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We present a complete systematics (excitation functions and system-size dependences) of global stopping and side flow for heavy ion reactions in the energy range between 0.09A and 1.93A GeV. For the heaviest system, Au+Au, we observe a plateau of maximal stopping extending from about 0.2A to 0.8A GeV with a fast drop on both sides. The degree of stopping, which is shown to remain significantly below the expectations of a full stopping scenario, is found to be highly correlated to the amount of side flow.
RESUMEN
Detailed studies of the azimuthal dependence of the mean fragment and flow energies in the Au+Au and Xe+CsI systems are reported as a function of incident energy and centrality. Comparisons between data and model calculations show that the flow energy values along different azimuthal directions could be viewed as snapshots of the fireball expansion with different exposure times. For the same number of participating nucleons more transversally elongated participant shapes from the heavier system produce less collective transverse energy. Good agreement with Boltzmann-Uehling-Uhlenbeck calculations is obtained for a soft nuclear equation of state.
RESUMEN
Current concerns over insertional mutagenesis by retroviral vectors mitigate investigations into alternative, potentially persistent gene therapy vector systems not dependent on genomic integration, such as Sendai virus vectors (SeVV). Prenatal gene therapy requires efficient gene delivery to several tissues, which may not be achievable by somatic gene transfer to the adult. Initially, to test the potential and tropism of the SeVV for gene delivery to fetal tissues, first-generation (replication- and propagation-competent) recombinant SeVV, expressing beta-galactosidase was introduced into late gestation immunocompetent mice via the amniotic and peritoneal cavities and the yolk sac vessels. At 2 days, this resulted in very high levels of expression particularly in the airway epithelium, mesothelium and vascular endothelium, respectively. However, as expected, substantial vector toxicity was observed. The efficiency of gene transfer and the level of gene expression were then examined using a second-generation SeVV. The second generation was developed to be still capable of cytoplasmic RNA replication and therefore high-level gene expression, but incapable of vector spread due to lack of the gene for viral F-protein. Vector was introduced into the fetal amniotic and peritoneal cavities, intravascularly, intramuscularly and intraspinally; at 2 days, expression was observed in the airway epithelia, peritoneal mesothelia, unidentified cells in the gut wall, locally at the site of muscle injection and in the dorsal root ganglia, respectively. Mortality was dramatically diminished compared with the first-generation vector.
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Enfermedades Fetales/terapia , Feto/metabolismo , Terapia Genética/métodos , Vectores Genéticos/toxicidad , Enfermedades Pulmonares/terapia , Proteínas Virales de Fusión/genética , Adenoviridae/genética , Animales , Animales Recién Nacidos , Virus Defectuosos , Inyecciones Intravenosas , Pulmón/enzimología , Enfermedades Pulmonares/embriología , Ratones , Ratones Endogámicos , Modelos Animales , Virus Sendai/genética , beta-Galactosidasa/genéticaRESUMEN
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.
Asunto(s)
Virus del Sarampión/aislamiento & purificación , Otosclerosis/virología , Adulto , Anciano , Anticuerpos Antivirales/análisis , Western Blotting , Femenino , Humanos , Masculino , Sarampión/complicaciones , Virus del Sarampión/genética , Virus del Sarampión/inmunología , Persona de Mediana Edad , Perilinfa/inmunología , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
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Mapeo Epitopo , Epítopos de Linfocito B/análisis , Proteína HN/inmunología , Virus de la Parotiditis/inmunología , Neuraminidasa/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales , Línea Celular , Chlorocebus aethiops , Epítopos de Linfocito B/inmunología , Femenino , Proteína HN/química , Proteína HN/aislamiento & purificación , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Vacuna contra la Parotiditis , Virus de la Parotiditis/genética , Virus de la Parotiditis/patogenicidad , Neuraminidasa/química , Neuraminidasa/aislamiento & purificación , Pruebas de Neutralización , Péptidos/análisis , Péptidos/síntesis química , Células VeroRESUMEN
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.
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Virus del Sarampión/aislamiento & purificación , Sarampión/complicaciones , Osteítis Deformante/virología , Otosclerosis/virología , Adolescente , Adulto , Anciano , Anticuerpos Antivirales/análisis , Southern Blotting , Estudios de Casos y Controles , Enfermedad Crónica , Femenino , Predisposición Genética a la Enfermedad , Alemania/epidemiología , Humanos , Incidencia , Masculino , Sarampión/epidemiología , Sarampión/inmunología , Sarampión/virología , Virus del Sarampión/genética , Virus del Sarampión/inmunología , Persona de Mediana Edad , Otosclerosis/epidemiología , Otosclerosis/etiología , Otosclerosis/inmunología , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores SexualesRESUMEN
Sendai virus (SV) infection and replication lead to a strong cytopathic effect with subsequent death of host cells. We now show that SV infection triggers an apoptotic program in target cells. Incubation of infected cells with the peptide inhibitor z-VAD-fmk abrogated SV-induced apoptosis, indicating that proteases of the caspase family were involved. Moreover, proteolytic activation of two distinct caspases, CPP32/caspase-3 and, as shown for the first time in virus-infected cells, FLICE/caspase-8, could be detected. So far, activation of FLICE/caspase-8 has been described in apoptosis triggered by death receptors, including CD95 and tumor necrosis factor (TNF)-R1. In contrast, we could show that SV-induced apoptosis did not require TNF or CD95 ligand. We further found that apoptosis of infected cells did not influence the maturation and budding of SV progeny. In conclusion, SV-induced cell injury is mediated by CD95- and TNF-R1-independent activation of caspases, leading to the death of host cells without impairment of the viral life cycle.
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Apoptosis , Caspasas/fisiología , Respirovirus/fisiología , Animales , Caspasa 3 , Caspasa 8 , Caspasa 9 , Línea Celular , Embrión de Pollo , Activación Enzimática , Haplorrinos , Humanos , Inhibidores de Proteasas/farmacología , Receptores del Factor de Necrosis Tumoral/fisiología , Replicación Viral , Receptor fas/fisiologíaRESUMEN
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.
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ADN Viral , Recombinación Genética , Respirovirus/aislamiento & purificación , Animales , Embrión de Pollo , Chlorocebus aethiops , ADN Complementario , Células HeLa , Humanos , Respirovirus/genética , Respirovirus/fisiología , Factores de Tiempo , Células Vero , Interferencia ViralRESUMEN
Virus-like particles with genetically defined envelope proteins were generated from cDNA in order to examine the requirement of Sendai virus haemagglutinin-neuraminidase (HN) protein for particle formation, and the role of fusion protein (F) in receptor binding and membrane fusion. Characterization of particles devoid of HN protein showed that particle formation was unimpaired by the absence of HN protein, indicating that HN protein is dispensable for virus assembly and budding. Infection studies further demonstrated that virus adsorption and penetration can be mediated solely by the F protein when the human asialoglycoprotein receptor is present at the surface of host cells.
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Hemaglutininas Virales/genética , Neuraminidasa/genética , Receptores de Superficie Celular/fisiología , Respirovirus/genética , Respirovirus/patogenicidad , Proteínas Virales/genética , Células 3T3 , Animales , Receptor de Asialoglicoproteína , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , ADN Complementario/genética , Genes Reporteros , Hemaglutininas Virales/fisiología , Humanos , Fusión de Membrana/fisiología , Ratones , Microscopía Electrónica , Neuraminidasa/fisiología , Respirovirus/fisiología , Proteínas Virales de Fusión/fisiología , Proteínas Virales/fisiología , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
Mixed infection of cells with both Moloney murine leukemia virus (MoMLV) and related or heterologous viruses produces progeny pseudotype virions bearing the MoMLV genome encapsulated by the envelope of the other virus. In this study, pseudotype formation between MoMLV and the prototype parainfluenza virus Sendai virus (SV) was investigated. We report for the first time that SV infection of MoMLV producer cells results in the formation of MoMLV(SV) pseudotypes, which display a largely extended host range compared to that of MoMLV particles. This could be associated with SV hemagglutinin-neuraminidase (SV-HN) glycoprotein incorporation into MoMLV envelopes. In contrast, solitary incorporation of the other SV glycoprotein, SV fusion protein (SV-F), resulted in a distinct and narrow extension of the MoMLV host range to asialoglycoprotein receptor (ASGP-R)-positive cells (e.g., cultured human hepatoma cells). Since stably ASGP-R cDNA-transfected MDCK cells, but not parental ASGP-R-negative MDCK cells, were found to be transduced by MoMLV(SV-F) pseudotypes and transduction of ASGP-R-expressing cells was found to be inhibited by ASGP-R antiserum, a direct proof for the ASGP-R-restricted tropism of MoMLV(SV-F) pseudotypes was provided. Cultivation of ASGP-R-positive HepG2 hepatoma cells on Transwell-COL membranes led to a significant enhancement of MoMLV(SV-F) titers in subsequent flowthrough transduction experiments, thereby suggesting the importance of ASGP-R accessibility at the basolateral domain for MoMLV(SV-F) pseudotype transduction. The availability of such ASGP-R-restricted MoMLV(SV-F)-pseudotyped vectors opens up new perspectives for future liver-restricted therapeutic gene transfer applications.
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Virus de la Leucemia Murina/genética , Precursores de Proteínas/genética , Recombinación Genética , Respirovirus/genética , Proteínas del Envoltorio Viral/genética , Animales , Línea Celular , Vectores Genéticos , Humanos , RatonesRESUMEN
Biochemical evidence suggests that the asialoglycoprotein receptor (ASGP-R) can be used as an alternative receptor for a temperature-sensitive Sendai virus (SV) mutant. We now have investigated this possible alternative route of infection for SV wild-type (SV-wt) strain Fushimi by using a pair of cell lines which differ only with regard to ASGP-R expression. Infection studies after enzymatic destruction of conventional sialic acid-containing SV receptors (SA-R) revealed that only ASGP-R-expressing cells could be infected by SV-wt. This alternative route of cell entry could be completely blocked by incubation of cells with ASGP-R-specific antibodies prior to infection. Furthermore, cleavage of SV-F0 precursor protein into the subunits F1 and F2 was necessary to establish infection via ASGP-R, suggesting a fusion-mediated cell entry after binding of SV-wt to the ASGP-R on host cells. Interestingly, infection via ASGP-R was found to be nearly as efficient as infection via conventional sialic acid-containing SV receptors. A possible physiological role of the ASGP-R-mediated route of SV infection is discussed.
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Precursores de Proteínas/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Virales/metabolismo , Respirovirus/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Células 3T3 , Animales , Receptor de Asialoglicoproteína , Línea Celular , Chlorocebus aethiops , Perros , Glicoproteínas/metabolismo , Ratones , Respirovirus/fisiología , Replicación ViralRESUMEN
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.
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Anticuerpos Antivirales/análisis , Antígenos Virales/análisis , Oído Interno/inmunología , Virus del Sarampión/inmunología , Otosclerosis/virología , Oído Interno/virología , Femenino , Humanos , Masculino , Virus del Sarampión/aislamiento & purificación , Otosclerosis/inmunología , Perilinfa/virología , Reacción en Cadena de la PolimerasaRESUMEN
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.
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Esclerosis Múltiple/inmunología , Esclerosis Múltiple/virología , Síndrome de Inmunodeficiencia Adquirida , Niño , Virus ADN/patogenicidad , Enfermedades Desmielinizantes/inmunología , Enfermedades Desmielinizantes/virología , Endotelio/fisiología , Humanos , Inflamación , Activación de Linfocitos , Modelos Biológicos , Linfocitos T/inmunología , TrombosisRESUMEN
Women suffer from otosclerosis 1.6 times more often than males. Histologically, otosclerotic foci can be found in temporal bones of females 1.9 times more often than in those of males. Characteristic topographic regions are the oval window, round window niche and promontory. Otosclerosis can also occur principally in any area of the enchondral/periosteal layer of the otic capsule. Evidence is presented that otosclerosis is an inflammatory tissue reaction associated with macrophages, T- and B-lymphocytes, HLA-DR positive cells and plasma cells. Dependent on the stage of the osteolytic bone disease present deposits of complement and immunoglobulins (IgG, IgA) can be found. These immunoglobulins have been identified as antibodies to measles virus proteins. Using the polymerase chain reaction we were successful in demonstrating RNA sequences of measles viruses in otosclerotic bone from footplates removed during stapes surgery. Since most of the otosclerotic lesions were in direct contact to the perilymphatic space, it may be expected that the endolymphatic sac--as the immune competent organ of the inner ear--specifically reacts to antigens delivered from the otosclerosis focus into the perilymph. Perilymph samples from patients were collected during stapes surgery and their antibody titers against measles were compared with that in corresponding blood serum. All samples revealed a significantly elevated-specific anti-measles IgG amount which was significantly higher than in the corresponding serum. In contrast, antibody titer in the perilymph against herpes simplex or cytomegalo viruses did not differ from that of the serum. These findings indicate that otosclerosis is a measles virus associated inflammatory osteolytic disease of the temporal bone. Since women suffer from severe measles virus infections more often than males, it can be hypothesized that females have a higher susceptibility of their cochleo-vestibular tissues to these infections (organotropism). In addition, estrogens are well-known stimulators of osteocytic activity and may play a dominant role during ossification of an otospongeotic bone lesion. This may explain the onset of a conductive hearing loss due to otosclerosis during pregnancy.
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Virus del Sarampión/aislamiento & purificación , Sarampión/patología , Otosclerosis/patología , Adulto , Anciano , Anticuerpos Antivirales/análisis , Susceptibilidad a Enfermedades , Estrógenos/fisiología , Femenino , Humanos , Masculino , Sarampión/genética , Sarampión/inmunología , Virus del Sarampión/inmunología , Virus del Sarampión/patogenicidad , Persona de Mediana Edad , Otosclerosis/genética , Otosclerosis/inmunología , Perilinfa/inmunología , Embarazo , Estribo/inmunología , Estribo/patología , Cirugía del Estribo , Hueso Temporal/inmunología , Hueso Temporal/patologíaRESUMEN
BACKGROUND: The self-sustained sequence replication (3SR) reaction is an isothermal method for nucleic acid amplification that has several features that make it an attractive alternative to PCR. We have studied the feasibility of the in situ 3SR reaction in cells using a measles virus-infected cell line as a model. EXPERIMENTAL DESIGN: The study was carried out in four steps. First, using RNA extracted from a measles-infected Vero Green monkey kidney cell line, conditions for the in vitro amplification of a segment of the nucleocapsid portion of the RNA viral genome were optimized for 420- and 119-bp 3SR products, and the results were compared. Second, 3SR was performed on intact infected cells in suspension, and the amount of RNA product was compared with infected cells without 3SR. Then, the 3SR reaction was conducted on cytospin preparation slides, followed by in situ hybridization for detection of the amplification product. Finally, 3SR was carried out on sections of formalin-fixed, paraffin-fixed, paraffin-embedded cells, and the degree of amplification as detected by ISH was quantified and compared between infected cells with and without 3SR reaction. RESULTS: Specific amplification of measles was observed in each of these types of preparations with an 8.5-fold rate of amplification in paraffin sections of formalin-fixed cells (a mean of 272.5 +/- 65.3 grains/cell after 3SR amplification in comparison to 31.97 +/- 4.2 grains/cell without amplification). CONCLUSIONS: A significant amount of amplification of RNA is possible with in situ 3SR (IS-3SR) and, in combination with ISH, offers several advantages compared with in situ PCR (IS-PCR), such as ease of use, lack of conditions that lead to cell damage, and a specificity for RNA amplification. This is the first report of specific amplification of RNA within cells using the IS-3SR procedure, a technique that has a wide range of potential applications in pathology and molecular biology.