RESUMEN
The virus-specific cytotoxic T lymphocyte (CTL) response is a major obstacle to effective delivery of adenovirus gene therapy. However, its relative role in viral clearance, transgene elimination and hepatotoxicity remains unclear. In this paper, we present an analysis of viral clearance and liver toxicity in relation to the induction of the virus-specific CD8 T-cell response revealed by an MHC class I tetramer. A surprisingly high number of tetramer+ CD8 T cells were found in the liver and lung and reached peak values at days 8 and 10, respectively, post-infection. Nearly 100% of these tetramer+ CD8 T cells expressed high levels of granzyme B and IFNgamma. Remarkably, liver viral load and liver enzyme elevation peaked early, at days 2 and 4, respectively, post-infection, before the specific CTL response was detectable. After generation of CTLs, there was only minimal liver damage or further decrease in virus titer. These results indicated that the primary peak response of tetramer+ CTLs does not correlate with the elimination of adenovirus or liver cytotoxic response.
Asunto(s)
Infecciones por Adenoviridae/inmunología , Adenoviridae , Hígado/virología , Linfocitos T Citotóxicos/inmunología , Alanina Transaminasa/sangre , Animales , Granzimas , Antígenos de Histocompatibilidad Clase I/administración & dosificación , Inyecciones Intravenosas , Interferón gamma/análisis , Hígado/enzimología , Pulmón/virología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/análisis , Factores de TiempoRESUMEN
CD8 T cells drive the protective immune response to lymphocytic choriomeningitis virus (LCMV) infection and are thus a determining force in the selection of viral variants. To examine how escape mutations affect the presentation and recognition of overlapping T-cell epitopes, we isolated an LCMV variant that is not recognized by T-cell receptor (TCR)-transgenic H-2Db-restricted LCMV GP33-41-specific cytotoxic T lymphocytes (CTL). The variant virus carried a single-amino-acid substitution (valine to alanine) at position 35 of the viral glycoprotein. This region of the LCMV glycoprotein encodes both the Db-restricted GP33-43 epitope and a second epitope (GP34-42) presented by the Kb molecule. We determined that the V-to-A CTL escape mutant failed to induce a Db GP33-43-specific CTL response and that Db-restricted GP33-43-specific CTL induced by the wild-type LCMV strain were unable to kill target cells infected with the variant LCMV strain. In contrast, the Kb-restricted response was much less affected. We found that the V-to-A substitution severely impaired peptide binding to Db but not to Kb molecules. Strikingly, the V-to-A mutation did not change any of the anchor residues, and the dramatic effect on binding was therefore unexpected. The strong decrease in Db binding explains why the variant virus escapes the Db GP33-43-specific response but still elicits the Kb-restricted response. These findings also illustrate that mutations within regions encoding overlapping T-cell epitopes can differentially affect the presentation and recognition of individual epitopes.
Asunto(s)
Antígenos Virales , Antígenos H-2/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas Virales , Sustitución de Aminoácidos , Animales , Linfocitos T CD8-positivos/inmunología , Citometría de Flujo , Glicoproteínas/inmunología , Antígenos H-2/genética , Antígeno de Histocompatibilidad H-2D , Interferón gamma/análisis , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Bazo/inmunologíaRESUMEN
Bcl-2 plays a critical role in regulating cell survival and apoptosis. We examined Bcl-2 expression in virus-specific CD8 T cells during the expansion, death, and memory phases of the T cell response following infection of mice with lymphocytic choriomeningitis virus (LCMV). Naive CD8 T cells expressed a basal level of Bcl-2 that was down-regulated in effector CD8 T cells just before the death phase. Bcl-2 levels remained low during the death phase but surviving memory CD8 T cells expressed higher levels of Bcl-2 than naive cells. These changes were shown to occur in LCMV TCR transgenic cells as well as virus-specific CD8 T cells in C57BL/6 and BALB/c mice identified by MHC class I tetramers. In all instances, memory CD8 T cells expressed higher levels of Bcl-2, suggesting that increased Bcl-2 expression plays a role in the long-term maintenance of memory CD8 T cells in vivo.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Epítopos de Linfocito T/inmunología , Memoria Inmunológica , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Femenino , Citometría de Flujo , Receptores de Hialuranos/biosíntesis , Epítopos Inmunodominantes/inmunología , Inmunofenotipificación , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Nucleoproteínas/inmunología , Fragmentos de Péptidos/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
Inhibitory receptors specific for alleles of MHC class I proteins play an important role in determining the reactivity and specificity of NK cells. To determine whether these receptors are also able to regulate T cell functions, we have studied anti-viral immune responses in mice transgenic for a class I-specific inhibitory receptor, Ly49A. Although nontransgenic mice express Ly49A primarily on NK cells and some T cells, the Ly49A transgenic mice express Ly49A on all lymphocytes, including T cells. We have assessed the activation, expansion, cytokine production, and cytotoxic activity of CD8 T cells in both transgenic and nontransgenic mice following infection with lymphocytic choriomeningitis virus. As expected, nontransgenic mice made a potent virus-specific CD8 T cell response following virus infection. However, as measured in cytolysis assays and by cytokine production, virus-specific CD8 T cell activity was reduced in Ly49A transgenic mice. This inhibition was largely, but not always exclusively, dependent upon the presence, either in vivo or in vitro, of the Ly49A ligand, H-2Dd. Strikingly Ly49A transgenic mice have reduced capacity to control infection with the virulent lymphocytic choriomeningitis virus variant clone 13. Overall, these studies demonstrate that expression of killer inhibitory receptors can modulate anti-viral T cell responses in vivo and in vitro.
Asunto(s)
Antígenos Ly , Proteínas Portadoras/biosíntesis , Virus de la Coriomeningitis Linfocítica/inmunología , Proteínas de la Membrana/biosíntesis , Receptores Inmunológicos/biosíntesis , Linfocitos T/inmunología , Linfocitos T/virología , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , División Celular/inmunología , Citocinas/biosíntesis , Antígenos H-2/genética , Antígeno de Histocompatibilidad H-2D , Lectinas Tipo C , Activación de Linfocitos , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Subfamilia A de Receptores Similares a Lectina de Células NK , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores Similares a Lectina de Células NK , Linfocitos T/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/virologíaRESUMEN
We examined the regulation of virus-specific CD8 T cell responses during chronic lymphocytic choriomeningitis virus (LCMV) infection of mice. Our study shows that within the same persistently infected host, different mechanisms can operate to silence antiviral T cell responses; CD8 T cells specific to one dominant viral epitope were deleted, whereas CD8 T cells responding to another dominant epitope persisted indefinitely. These virus-specific CD8 T cells expressed activation markers (CD69(hi), CD44(hi), CD62Llo) and proliferated in vivo but were unable to elaborate any antiviral effector functions. This unresponsive phenotype was more pronounced under conditions of CD4 T cell deficiency, highlighting the importance of CD8- CD4 T cell collaboration in controlling persistent infections. Importantly, in the presence of CD4 T cell help, adequate CD8 effector activity was maintained and the chronic viral infection eventually resolved. The persistence of activated virus-specific CD8 T cells without effector function reveals a novel mechanism for silencing antiviral immune responses and also offers new possibilities for enhancing CD8 T cell immunity in chronically infected hosts.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Anergia Clonal/inmunología , Coriomeningitis Linfocítica/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Anticuerpos Monoclonales , Antígenos CD/análisis , Linfocitos T CD4-Positivos/metabolismo , Enfermedad Crónica , Supresión Clonal , Antígenos de Histocompatibilidad Clase I/metabolismo , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/metabolismo , Interferón gamma/análisis , Activación de Linfocitos/inmunología , Cooperación Linfocítica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Citotóxicos/química , Proteínas Estructurales Virales/inmunologíaRESUMEN
The goal of therapeutic vaccination is to elicit an antiviral immune response within persistently infected individuals and consequently eradicate the infection. CD8+ T cells are potent mediators of viral clearance; however, during chronic infections CD4+ T cell help is required to sustain antiviral CD8+ T cell activity. Therapeutic vaccination should be targeted towards enhancing both CD4+ and CD8+ T cell virus-specific responses.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunación , Vacunas Virales/uso terapéutico , Virosis/inmunología , Animales , Enfermedad Crónica , Humanos , Activación de Linfocitos , Virosis/tratamiento farmacológicoRESUMEN
Viral infections often induce potent CD8 T cell responses that play a key role in antiviral immunity. After viral clearance, the vast majority of the expanded CD8 T cells undergo apoptosis, leaving behind a stable number of memory cells. The relationship between the CD8 T cells that clear the acute viral infection and the long-lived CD8 memory pool remaining in the individual is not fully understood. To address this issue, we examined the T cell receptor (TCR) repertoire of virus-specific CD8 T cells in the mouse model of infection with lymphocytic choriomeningitis virus (LCMV) using three approaches: (a) in vivo quantitative TCR beta chain V segment and complementarity determining region 3 (CDR3) length repertoire analysis by spectratyping (immunoscope); (b) identification of LCMV-specific CD8 T cells with MHC class I tetramers containing viral peptide and costaining with TCR Vbeta-specific antibodies; and (c) functional TCR fingerprinting based on recognition of variant peptides. We compared the repertoire of CD8 T cells responding to acute primary and secondary LCMV infections, together with that of virus-specific memory T cells in immune mice. Our analysis showed that CD8 T cells from several Vbeta families participated in the anti-LCMV response directed to the dominant cytotoxic T lymphocyte (CTL) epitope (NP118-126). However, the bulk (approximately 70%) of this CTL response was due to three privileged T cell populations systematically expanding during LCMV infection. Approximately 30% of the response consisted of Vbeta10+ CD8 T cells with a beta chain CDR3 length of nine amino acids, and 40% consisted of Vbeta8.1+ (beta CDR3 = eight amino acids) and Vbeta8.2+ cells (beta CDR3 = six amino acids). Finally, we showed that the TCR repertoire of the primary antiviral CD8 T cell response was similar both structurally and functionally to that of the memory pool and the secondary CD8 T cell effectors. These results suggest a stochastic selection of memory cells from the pool of CD8 T cells activated during primary infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Regiones Determinantes de Complementariedad , Infecciones/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Modelos Animales de Enfermedad , Cadenas alfa de Inmunoglobulina/inmunología , Memoria Inmunológica/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Ratones , Ratones Endogámicos BALB C , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Viral infections induce extensive T cell proliferation in vivo, but the specificity of the majority of the responding T cells has not been defined. To address this issue we used tetramers of MHC class I molecules containing viral peptides to directly visualize antigen-specific CD8 T cells during acute LCMV infection of mice. Based on tetramer binding and two sensitive assays measuring interferon-gamma production at the single-cell level, we found that 50%-70% of the activated CD8 T cells were LCMV specific [2 x 10(7) virus-specific cells/spleen]. Following viral clearance, antigen-specific CD8 T cell numbers dropped to 10(6) per spleen and were maintained at this level for the life of the mouse. Upon rechallenge with LCMV, there was rapid expansion of memory T cells, but after infection with the heterologous vaccinia virus there was no detectable change in the numbers of LCMV-specific memory CTL. Therefore, much of the CD8 T cell expansion seen during viral infection represents antigen-specific cells and warrants a revision of our current thinking on the size of the antiviral response.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos , Recuento de Linfocitos/métodos , Coriomeningitis Linfocítica/inmunología , Animales , Presentación de Antígeno , Linfocitos T CD8-positivos/citología , Femenino , Citometría de Flujo , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunidad Activa , Memoria Inmunológica , Interferón gamma/biosíntesis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fragmentos de Péptidos/inmunología , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Following infection with lymphocytic choriomeningitis virus (LCMV), normal adult mice generate virus-specific, major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL) which clear the virus after intraperitoneal infection or cause death following intracranial (i.c.) infection. We have investigated the response of beta2-microglobulin-deficient (beta2m-) mice of the H-2d haplotype (KOD mice) to LCMV infection. Unlike H-2b beta2m- mice, which generate CD4+ MHC class II-restricted CTL in response to LCMV, KOD mice generate high levels of CD8+ MHC class I-restricted, virus-specific CTL. These CTL are specific for the LCMV nucleoprotein epitope (residues 118 to 126) in association with the Ld class I molecule, analogous to the CTL response in wild-type mice. KOD mice are also susceptible to lethal LCM disease, with 75 to 80% of the mice dying 7 to 9 days following i.c. infection with virus. Similar to results with normal mice, lethal LCM disease in KOD mice is prevented by in vivo depletion of CD8+ T cells prior to i.c. infection. In contrast to wild-type mice, however, KOD mice cannot control LCMV and become persistently infected. Overall, these results demonstrate that beta2m is not an absolute requirement for presentation of endogenous antigen on Ld or for induction of virus-specific Ld-restricted CTL in vivo.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Nucleoproteínas/inmunología , Fragmentos de Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Microglobulina beta-2/fisiología , Animales , Femenino , Antígenos H-2/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones NoqueadosRESUMEN
beta 2-Microglobulin-deficient (beta 2m-) mice generate a CD4+ major histocompatibility complex class II-restricted cytotoxic T-lymphocyte (CTL) response following infection with lymphocytic choriomeningitis (LCM) virus (LCMV). We have determined the cytotoxic mechanism used by these CD4+ CTLs and have examined the role of this cytotoxic activity in pathogenesis of LCM disease in beta 2m- mice. Lysis of LCMV-infected target cells by CTLs from beta 2m- mice is inhibited by addition of soluble Fas-Ig fusion proteins or by pretreatment of the CTLs with the protein synthesis inhibitor emetine. In addition, LCMV-infected cell lines that are resistant to anti-Fas-induced apoptosis are refractory to lysis by these virus-specific CD4+ CTLs. These data indicate that LCMV-specific CD4+ CTLs from beta 2m- mice use a Fas-dependent lytic mechanism. Intracranial (i.c.) infection of beta 2m- mice with LCMV results in loss of body weight. Fas-deficient beta 2m- Jpr mice develop a similar wasting disease following i.c. infection. This suggests that Fas-dependent cytotoxicity is not required for LCMV-induced weight loss. A potential mediator of this chronic wasting disease is tumor necrosis factor (TNF)-alpha, which is produced by LCMV-specific CD4+ CTLs. In contrast to LCMV-induced weight loss, lethal LCM disease in beta 2m- mice is dependent on Fas-mediated cytotoxicity. Transfer of immune splenocytes from LCMV-infected beta 2m- mice into irradiated infected beta 2m- mice results in death of recipient animals. In contrast, transfer of these splenocytes into irradiated infected beta 2m- Jpr mice does not cause death. Thus a role for CD4+ T-cell-mediated cytotoxicity in virus-induced immunopathology has now been demonstrated.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Citotoxicidad Inmunológica , Coriomeningitis Linfocítica/inmunología , Receptor fas/inmunología , Traslado Adoptivo , Animales , Humanos , Coriomeningitis Linfocítica/metabolismo , Ratones , Microglobulina beta-2/deficienciaRESUMEN
We have investigated the induction and role of natural killer (NK) activity in lymphocytic choriomeningitis virus (LCMV)-infected beta 2-microglobulin-deficient (beta 2m-) mice. We demonstrate that LCMV infection is more effective than polyinosinic:polycytidylic acid (poly I:C) at stimulating NK activity in beta 2m- mice. In addition, beta 2m- NK cells respond poorly to in vitro treatment with IL-12. The target specificity of the virally induced NK cells is similar to that previously reported for chemically induced beta 2m- NK cells. In both cases they can lyse YAC-1 tumor cells but are unable to kill beta 2m- or beta 2m+ T cell blasts. We have also found that the time course of induction of NK and cytotoxic T lymphocyte (CTL) activity by LCMV in beta 2m- mice is delayed compared with normal mice. Maximal NK and CTL activity is attained at day 8 and 10 post-infection respectively in beta 2m- mice compared with day 4 and 6-8 in B6 mice. Whereas normal mice die approximately 7 days following intracranial infection with LCMV, the course of disease in beta 2m- mice is protracted and characterized by a marked loss of body weight. We show that although the CD4+ CTL response in these mice is intimately involved in mediating weight loss, the virus-induced NK cells do not appear to play a role in the disease.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Células Asesinas Naturales/inmunología , Coriomeningitis Linfocítica/inmunología , Linfocitos T Citotóxicos/inmunología , Microglobulina beta-2/deficiencia , Animales , Citotoxicidad Inmunológica , Progresión de la Enfermedad , Femenino , Antígenos H-2/inmunología , Humanos , Interleucina-12/farmacología , Células Asesinas Naturales/efectos de los fármacos , Depleción Linfocítica , Coriomeningitis Linfocítica/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Poli I-C/farmacología , Pérdida de PesoRESUMEN
The PE2 cleavage signal in a full-length cDNA clone of the alphavirus Venezuelan equine encephalitis virus (VEE) was ablated by site-directed mutagenesis. RNA transcripts derived from the resulting plasmids programmed the production of nonviable particles upon transfection of baby hamster kidney (BHK) cells. However, the mutant RNAs also gave rise to a small proportion of viable revertants. Analysis of these biological revertants and their molecularly cloned homologs demonstrated that second-site suppressor mutations at either E2 position 243 or E1 position 253 were able to restore viability to PE2 cleavage signal mutants. The viable revertants incorporated unprocessed PE2 into particles which showed normal infectivity for BHK cells, but reduced ability to grow in C6/36 mosquito cells. A mutant carrying a lethal PE2 cleavage signal mutation in combination with a suppressor at E1 253 was either avirulent or highly attenuated in adult mice when inoculated by the subcutaneous, intracerebral, or intranasal route and conferred complete protection against both intraperitoneal and intranasal challenge with virulent VEE. These results indicate the close functional association of the E2 and E1 proteins in the alphavirus spike. They also have implications for the design of recombinant live virus vaccines for VEE, for other alphaviruses, and for other viruses that use a similar mechanism for glycoprotein maturation.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/genética , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Animales , Virus de la Encefalitis Equina Venezolana/inmunología , Femenino , Genes Letales , Genes Supresores , Glicoproteínas/genética , Glicoproteínas/metabolismo , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Procesamiento Proteico-Postraduccional , Vacunas Atenuadas/genética , Proteínas del Envoltorio Viral/metabolismo , Vacunas Virales/genética , Replicación ViralRESUMEN
The lengthy 5' nontranslated region (5'NTR) of hepatitis A virus (HAV) forms a highly ordered secondary structure, which has been suggested to play an important role in controlling viral translation by allowing for translation initiation by internal ribosome entry. To test this hypothesis, synthetic bicistronic RNAs, with all or part of the HAV 5'NTR in the intercistronic space, were translated in rabbit reticulocyte lysates. In the presence of an upstream cistron designed to block ribosomal scanning, the HAV 5'NTR was capable of directing the internal initiation of translation, confirming the presence of an internal ribosome entry site (IRES). Analysis of various deletion mutants demonstrated that the 5' border of the IRES is located between nucleotides 151 and 257, while the 3' border extends to the 3' end of the 5'NTR, between nucleotide 695 and the first initiation codon at 735. Except for a segment between bases 638 and 694, deletion of stem-loop structures between bases 151 and the 3' end of the 5'NTR inhibited or abolished translation. The addition of a 5' cap structure (m7GpppN) to monocistronic or bicistronic transcripts decreased the translation of a reporter gene downstream of the HAV 5'NTR but enhanced translation of the upstream cistron in bicistronic transcripts. This finding indicates that a 5' cap structure is inhibitory to HAV IRES-directed translation initiation and that the cap structure and the HAV IRES probably compete for the same limiting translation factors. The efficiency with which monocistronic constructs containing the HAV 5'NTR directed translation in reticulocyte lysates was compared with results for monocistronic constructs containing the IRES of the more rapidly growing encephalomyocarditis virus (EMCV). These results indicated that the HAV 5'NTR was more than 25-fold less active than the EMCV IRES in producing translation product. HAV 5'NTR-directed translation was inhibited by the presence of a one-fifth molar quantity of RNA containing the EMCV IRES, while a fivefold molar excess of the HAV 5'NTR did not inhibit EMCV IRES-directed translation. The relatively weak activity of the HAV IRES may thus be due to a reduced affinity for cellular translation factors which are present in limiting quantities in rabbit reticulocyte lysate.
Asunto(s)
Virus de la Encefalomiocarditis/genética , Hepatovirus/genética , Biosíntesis de Proteínas , ARN Viral/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Ribosomas/metabolismo , Animales , Sistema Libre de Células , Análisis Mutacional de ADN , Virus de la Encefalomiocarditis/crecimiento & desarrollo , Hepatovirus/crecimiento & desarrollo , ARN Mensajero/genética , Conejos , Secuencias Repetitivas de Ácidos Nucleicos/genética , Reticulocitos/metabolismo , Eliminación de Secuencia , Replicación Viral/genéticaRESUMEN
In this study we have investigated the role of CD4+, MHC class II-restricted cytotoxic T lymphocytes (CTLs) in the disease caused by lymphocytic choriomeningitis virus (LCMV) in beta 2-microglobulin deficient (beta 2m-) mice. Intracranial (i.c.) infection with LCMV resulted in death of six out of 11 beta 2m- mice. Mice that survived showed a marked loss in body weight. Death and loss of body weight could be prevented by immunosuppressing the mice with irradiation or cyclosporine prior to i.c. injection of LCMV. This treatment also prevented induction of virus-specific, MHC class II-restricted CTL following peripheral inoculation with LCMV. In vivo depletion of CD4+ cells with antibody also prevented death following i.c. injection whereas in vivo depletion of CD8+ cells had no effect. Disease could be transferred to recipient beta 2m- mice by adoptive transfer of beta 2m- derived immune spleen cells. Transfer of non-immune spleen cells did not result in illness. In vitro treatment of immune spleen cells with anti-CD4 antibody and complement eliminated class II-restricted CTL activity and also prevented mortality of recipients after adoptive transfer. Treatment with anti-CD8 antibody had no effect. We were unable to transfer LCM disease to beta 2m- recipients by adoptive transfer of immune spleen cells from C57BL/6 mice. These results suggest that, unlike normal mice, the pathology of LCM disease in beta 2m- mice is dependent upon virus-specific, CD4+CD8-, MHC class II-restricted T cells.
Asunto(s)
Antígenos CD4/análisis , Linfocitos T CD4-Positivos/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos CD8/análisis , Células Cultivadas , Femenino , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunoterapia Adoptiva , Coriomeningitis Linfocítica/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Microglobulina beta-2/deficienciaRESUMEN
Remote afterloading high-dose-rate brachytherapy (RAHDRB) was used endobronchially for the management of malignant airway obstruction in 82 patients, 72 of whom had primary disease in the lung. Treatment was palliative (n = 58) or definitive (n = 24). The extent of airway compromise was determined at bronchoscopy and with symptoms of hemoptysis, dyspnea, or cough or with radiographic evidence of atelectasis. RAHDRB doses were 1,000-4,700 cGy in one to five fractions. External beam radiation was used in previously unirradiated patients. A substantial reduction ,N airway disease and an improvement in symptoms were seen in 82% of patients. Obstruction scores showed an overall 74% improvement. Complications occurred in only 10 patients (two of whom died). Median survival was short (palliative group, 5 months; definitive group, 12 months); however, symptoms remained palliated in 62 patients (76%) until death or the last follow-up examination. RAHDRB is effective and can be applied with equal success in all patients with malignant airway obstruction, even those whose disease has recurred after external beam irradiation.
Asunto(s)
Braquiterapia , Neoplasias Pulmonares/radioterapia , Anciano , Anciano de 80 o más Años , Braquiterapia/efectos adversos , Braquiterapia/métodos , Bronquios/patología , Constricción Patológica , Femenino , Humanos , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Cuidados Paliativos , Dosificación Radioterapéutica , Tasa de SupervivenciaRESUMEN
The mutation responsible for the temperature-sensitive (ts) phenotype of mutant tsN19 (complementation group E) of respiratory syncytial virus has been located to the P protein gene. Viral protein synthesis was completely restricted at 39 degrees C, and the tsN19 P protein did not react with an anti-P monoclonal antibody (MAb) (3-5) at 33 degrees C. Reversion of temperature sensitivity restored reactivity with MAb 3-5. Nucleotide sequence determination and in vitro expression of cDNA clones of P mRNA derived from wild-type, tsN19 and non-ts revertant-infected cells, revealed that temperature sensitivity and loss of reactivity with MAb 3-5 were consequences of a Gly----Ser amino acid change at position 172. A low M(r) polypeptide, which represented the C-terminal 93 amino acids of the P protein, was produced by internal initiation in the P open reading frame during in vitro translation, and a similar product was detected transiently in vivo.
Asunto(s)
Antígenos Virales/genética , Genes Virales/genética , Proteína HN , Virus Sincitiales Respiratorios/genética , Proteínas Virales , Animales , Anticuerpos Monoclonales , Antígenos Virales/biosíntesis , Secuencia de Bases , Clonación Molecular , ADN de Cadena Simple/genética , Epítopos/genética , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteínas del Envoltorio ViralRESUMEN
The calibration of a Nucletron MicroSelectron High Dose Rate Remote Afterloader unit and the radiation survey around the facility is discussed. The radiation survey of the facility indicates that the use of an existing linear accelerator vault will provide adequate shielding. The methodologies for performing source calibration are presented.
Asunto(s)
Braquiterapia/instrumentación , Neoplasias/radioterapia , Calibración , Humanos , Radioisótopos de Iridio , Exposición Profesional , Protección RadiológicaRESUMEN
We have investigated the interactions of purified radiolabelled hepatitis A virus (HAV) with a variety of continuous cell lines. Virus labelled either in vitro with radiolabelled iodine or in vivo with radiolabelled uridine bound to cells with similar efficiency. Attachment to BS-C-1 cells was calcium ion-dependent and this correlated with infectivity assay results. The cell tropism of HAV attachment was examined using cell suspensions and confluent cell monolayers at both 4 degrees C and 37 degrees C. The maximum level of attachment was observed at 4 degrees C with cells in suspension, but was severely inhibited by 2% foetal calf serum; these results again correlated with infectivity assays. The components of serum which inhibit attachment have been characterized by gel filtration chromatography, sucrose density gradient analysis, immunoprecipitation and Western blotting. The data show that such components are of high Mr and that the serum glycoprotein, alpha 2-macroglobulin, can partly mimic the inhibitory effect of whole serum.
Asunto(s)
Hepatovirus/fisiología , Adsorción/efectos de los fármacos , Animales , Antígenos Virales/análisis , Proteínas Sanguíneas/metabolismo , Western Blotting , Calcio/farmacología , Cápside/inmunología , Adhesión Celular/efectos de los fármacos , Línea Celular , Centrifugación por Gradiente de Densidad , Cromatografía en Gel , Hepatovirus/crecimiento & desarrollo , Hepatovirus/inmunología , Humanos , Magnesio/farmacología , Pruebas de Precipitina , Radioinmunoensayo , Temperatura , Virión/inmunologíaRESUMEN
Paget's disease of bone is often discovered incidentally, but can have extensive metabolic and local mechanical complications. Treatment is not required for all patients and should only be undertaken for certain indications, and with a clear understanding of the three types of drugs available. Bone pain unmanageable with analgesics and pathologic fractures are the most common indications, while neurologic symptoms, hypercalcemia and congestive heart failure are less frequent ones. Calcitonin or mithramycin is used for the more urgent indications, and calcitonin or the diphosphonate, etidronate sodium (EHDP), for the more chronic ones. The drugs are generally efficacious and well tolerated.