RESUMEN
PURPOSE: To examine the in vitro anticancer activity of Melaleuca alternifolia (tea tree) oil (TTO), and its major active terpene component, terpinen-4-ol, against two aggressive murine tumour cell lines, AE17 mesothelioma and B16 melanoma. METHODS: Effects of TTO and terpinen-4-ol on the cellular viability of two tumour cell lines and fibroblast cells were assessed by MTT assay. Induction of apoptotic and necrotic cell death was visualised by fluorescent microscopy and quantified by flow cytometry. Tumour cell ultrastructural changes were examined by transmission electron microscopy and changes in cell cycle distribution were assessed by flow cytometry, with changes in cellular morphology monitored by video time lapse microscopy. RESULTS: TTO and terpinen-4-ol significantly inhibited the growth of two murine tumour cell lines in a dose- and time-dependent manner. Interestingly, cytotoxic doses of TTO and terpinen-4-ol were significantly less efficacious against non-tumour fibroblast cells. TTO and terpinen-4-ol induced necrotic cell death coupled with low level apoptotic cell death in both tumour cell lines. This primary necrosis was clarified by video time lapse microscopy and also by transmission electron microscopy which revealed ultrastructural features including cell and organelle swelling following treatment with TTO. In addition, both TTO and terpinen-4-ol induced their inhibitory effect by eliciting G1 cell cycle arrest. CONCLUSION: TTO and terpinen-4-ol had significant anti-proliferative activity against two tumour cell lines. Moreover, the identification of primary necrotic cell death and cell cycle arrest of the aggressive tumour cells highlights the potential anticancer activity of TTO and terpinen-4-ol.
Asunto(s)
Antineoplásicos/uso terapéutico , Ciclo Celular/efectos de los fármacos , Aceite de Árbol de Té/uso terapéutico , Terpenos/uso terapéutico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Melanoma Experimental/tratamiento farmacológico , Mesotelioma/tratamiento farmacológico , Ratones , Necrosis/inducido químicamenteRESUMEN
The beneficial effects of low-dose orally administered type I interferon (LDOA IFN) have been demonstrated in various animal models of disease and in some human clinical trials. The mechanisms by which LDOA IFN therapy has its effects, however, remain to be established. In the present study, groups of mice were administered 10 IU murine IFN-alpha/beta (MuIFN-alpha/beta) orally for 7 days. Spleens were then collected and analyzed. No differences were detected between the spleen weights of treated mice compared with controls, although reductions in total splenic white blood cell (WBC) number ranging from 15.5% to 35% were observed. Further analysis showed this reduction to be largely restricted to the B cell population, with only minor reductions in CD4(+) or CD8(+) populations being detected. Dose-response studies showed the WBC loss from the spleen to be optimal at 1 IU MuIFN-alpha/beta, whereas both higher and lower doses showed less significant effects. Time course studies show these effects had developed after 2 days of treatment. It is hypothesized that this observed WBC movement from the spleen is part of the mechanism of action of LDOA IFN.
Asunto(s)
Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Interferón Tipo I/administración & dosificación , Leucocitos/efectos de los fármacos , Bazo/citología , Administración Oral , Animales , Muerte Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Recuento de Leucocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Necrosis , Tamaño de los Órganos/efectos de los fármacos , Bazo/inmunologíaRESUMEN
In vivo, low-dose orally administered type I interferon (LDOA IFN) therapy has been shown to provide beneficial effects in a number of diseases. These diseases vary in nature (viral, autoimmune, and neoplastic), yet LDOA IFN therapy is able to provide effective treatment. Despite the growing knowledge of the efficacy of such treatment and ongoing human clinical trials, the mechanism by which LDOA IFN acts remains largely unknown. In this study, we examined the phenomenon known as "priming" as a potential mechanism by which LDOA IFN effects may be mediated. Priming is a phenomenon in which pretreatment of cells or entire organisms with type I IFN causes significantly enhanced IFN production after induction of the endogenous IFN system by virus or polyI:C. This phenomenon of priming has been exploited in commercial industry for the mass production of type I IFN for medical and research use. It was found that LDOA IFN treatment did not cause priming in vivo.
Asunto(s)
Interferón Tipo I/administración & dosificación , Interferón Tipo I/biosíntesis , Administración Oral , Animales , Antivirales/administración & dosificación , Antivirales/sangre , Relación Dosis-Respuesta Inmunológica , Esquema de Medicación , Interferón Tipo I/sangre , Ratones , Ratones Endogámicos BALB CRESUMEN
Overcoming the massive and rapid death of injected donor myoblasts is the primary hurdle for successful myoblast transfer therapy (MTT), designed as a treatment for the lethal childhood myopathy Duchenne muscular dystrophy. The injection of male myoblasts into female host mice and quantification of surviving male DNA using the Y-chromosome-specific (Y1) probe allows the speed and extent of death of donor myoblasts to be determined. Cultured normal C57BL/10Sn male donor myoblasts were injected into untreated normal C57BL/10Sn and dystrophic mdx female host mice and analyzed by slot blots using a 32P-labeled Y1 probe. The amount of male DNA from donor myoblasts showed a remarkable decrease within minutes and by 1 h represented only about 10-18% of the 2.5 x 10(5) cells originally injected (designated 100%). This declined further over 1 week to approximately 1-4%. The host environment (normal or dystrophic) as well as the extent of passaging in tissue culture (early "P3" or late "P15-20" passage) made no difference to this result. Modulation of the host response by CD4+/CD8+ -depleting antibodies administered prior to injection of the cultured myoblasts dramatically enhanced donor myoblast survival in dystrophic mdx hosts (15-fold relative to untreated hosts after 1 week). NK1.1 depletion also dramatically enhanced donor myoblast survival in dystrophic mdx hosts (21-fold after 1 week) compared to untreated hosts. These results provide a strategic approach to enhance donor myoblast survival in clinical trials of MTT.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Supervivencia Celular , Trasplante de Células , Células Asesinas Naturales/inmunología , Músculos/citología , Animales , Antígenos/análisis , Antígenos Ly , Antígenos de Superficie , Células Cultivadas , ADN/análisis , Femenino , Lectinas Tipo C , Depleción Linfocítica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Músculos/metabolismo , Músculos/trasplante , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/terapia , Subfamilia B de Receptores Similares a Lectina de Células NK , Proteínas/análisis , Cromosoma Y/genéticaRESUMEN
Interferon-alpha (IFN-alpha) given orally has biological activity in humans and other animals. The dose providing the most benefit delivers IFN-alpha to the oral mucosa in a concentration (10(2)-10(3) IU), similar to that naturally produced in the nasal secretions during respiratory infections. In contrast, conventional IFN therapy employs parenteral doses of > 10(6) IU and, for this reason, orally administered IFN therapies have been called low-dose treatments. Efficacy in both animal disease models and human studies has been reported, and the mechanisms whereby oral administration has a systemic effect are under active study in a number of laboratories.
Asunto(s)
Antivirales/uso terapéutico , Interferones/uso terapéutico , Administración Oral , Animales , Relación Dosis-Respuesta a Droga , Humanos , Resultado del TratamientoRESUMEN
Cytomegalovirus (CMV) infection has been associated with the development of myocarditis in humans. Our established mouse model for CMV myocarditis allows detailed investigation of the immunopathogenic mechanisms and therapies for cardiovascular disease. The type I interferons (IFN-alpha/beta) are part of the innate immune response to CMV infections. Previously, we have reported that daily treatment with low doses of murine IFN-alpha/beta administered by the oral-mucosal route significantly reduces early virus replication of murine CMV in the spleen and liver of infected mice. The oral-mucosal route provides an alternate delivery system to the current modes of IFN administration and is associated with fewer side effects. Since prophylactic treatment with type 1 IFNs may result in both antiviral and immunomodulatory effects that may lessen the development of disease, we wished to study the effect of IFN-alpha/beta on the development of myocarditis. Low-dose oral use of type I IFN (10 IU/day for 7 days prior to virus infection) did not abrogate myocarditis but suppressed the inflammatory response in both the acute and chronic phase of the disease. Furthermore, low-dose oral use of IFN was as effective at inhibiting myocarditis as a single injection of a high dose of IFN (20,000 IU) on the day of virus infection. These findings indicate the need for evaluation of low-dose use of oral IFN in the development of improved clinical therapies for the treatment of cardiovascular disease.
Asunto(s)
Antivirales/uso terapéutico , Infecciones por Citomegalovirus/tratamiento farmacológico , Interferón Tipo I/uso terapéutico , Miocarditis/tratamiento farmacológico , Adyuvantes Inmunológicos/uso terapéutico , Administración Oral , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Ratones , Ratones Endogámicos BALB C , Miocarditis/virologíaRESUMEN
Oral administration of type I interferons (IFNs; murine IFN-alpha and IFN-beta) reduces early replication of murine cytomegalovirus (MCMV) in both the spleen and liver of MCMV-infected BALB/c mice. Examination of a range of doses of IFN (1 to 1000 IU) showed that 10 IU administered daily for 1 week prior to virus infection was optimal for inhibition of MCMV replication. Furthermore, low-dose orally administered IFN (10 IU/day) was effective in mice challenged with lethal and sublethal virus inocula. The antiviral efficacy of low-dose orally administered IFN was not restricted by either the route of virus inoculation or the mouse genotype. Analysis by immunohistochemistry of IFN-alpha receptor-bearing cells of the gastrointestinal tract revealed predominant staining of perivascular smooth muscle and the lamina propria of the anterior tongue, small intestine and rectum. These tissues, dense in IFN-alpha receptor-bearing cells, are likely to be the sites of interaction of the orally administered IFNs with the mucosal immune system. In conclusion, we propose that low-dose oral use of type I IFN therapy may have broad applications in the treatment of CMV infections.
Asunto(s)
Antivirales/uso terapéutico , Infecciones por Citomegalovirus/tratamiento farmacológico , Interferón Tipo I/uso terapéutico , Administración Oral , Animales , Sistema Digestivo/efectos de los fármacos , Sistema Digestivo/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Genotipo , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Receptores de Interferón/metabolismo , Resultado del TratamientoRESUMEN
The IFN-alpha cytokines belong to a multigene family. However, the in vivo biological functions of each of the IFN-alpha subtypes is unknown. Recently, we developed an experimental model in which the tibialis anterior muscles of mice were transfected in situ with naked DNA plasmids encoding an IFN transgene. Here we use this model to investigate the in vivo effect of the expression of three murine IFN-alpha subtypes (A1, A4, and A9) on murine CMV replication in C57BL/6, BALB/c, and A/J mice. CMV was shown to replicate in the tibialis anterior muscles of mice for at least 6 days and induced an inflammatory infiltrate. However, mice expressing the IFN-alpha transgenes showed a marked reduction in the peak titers of virus replication, with less severe inflammation in the muscles compared with control mice that were inoculated with blank vectors. Moreover, mice expressing the IFN-alpha1 transgene had significantly lower CMV titers in the inoculated muscle than mice expressing either the IFN-alpha4 or the IFN-alpha9 transgenes. Furthermore, IFN-alpha/beta receptor knockout mice had markedly higher levels of CMV replication in the tibialis anterior muscles than the wild-type parental strain (129/Sv/Ev) following IFN-alpha1 transgene inoculation, suggesting that the protection observed is due to host cell-mediated IFN signaling. These data provide the first evidence indicating that there are in vivo differences in the antiviral efficacy of the IFN-alpha subtypes.
Asunto(s)
Antivirales/clasificación , Infecciones por Citomegalovirus/terapia , Interferón-alfa/clasificación , Muromegalovirus/inmunología , Transfección , Replicación Viral/efectos de los fármacos , Animales , Femenino , Inyecciones Intramusculares , Interferón-alfa/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Immunity to viral infections involves both innate and antigen-specific immune responses. The antiviral properties of interferons (IFNs) are part of the innate immune response. Low doses of type I IFNs (IFN-alpha and IFN-beta) administered daily (10 IU per mouse) by the oral route significantly reduced the early replication of murine cytomegalovirus (MCMV) in both the spleen and liver of MCMV-infected susceptible BALB/c mice. Significant inhibition of virus replication was observed for two different inoculum doses of virus (2 x 10(4) pfu per mouse [0.6 LD50] and 2 x 10(4.12) pfu per mouse [0.8 LD50]). Analysis of IFN retention, using [35S]-labeled IFN-alpha1 compared with the nonreceptor binding mutant IFN-alpha1 (R33M) administered orally to mice, revealed binding of wild-type IFN-alpha1 to several tissues. In particular, IFN was retained by tissues proximal to lymphoid regions, including the posterior nasal cavity, posterior tongue, small intestine, and rectum. These findings suggest that type I IFNs may inhibit MCMV replication by distal binding of the orally administered IFN to various tissues, which in turn augment the primary immune response to virus infection.
Asunto(s)
Muromegalovirus/fisiología , Replicación Viral , Administración Oral , Animales , Relación Dosis-Respuesta a Droga , Hígado/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Bazo/virología , Factores de TiempoRESUMEN
Acid-stable type I interferons belong to a multigene family. The biologic relevance of each subtype in vivo remains unknown. We have developed an experimental model in which muscles were transfected in situ with naked DNA plasmids encoding an IFN transgene to assess the roles of individual IFN subtypes in vivo. Murine IFN-alpha 9 gene was subcloned into several mammalian expression vectors. Adult C57BL/6 mice were injected bilaterally in regenerating tibialis anterior muscles with naked DNA 5 days after muscle injury to enhance DNA uptake and expression. In the muscles of mice given the IFN-alpha 9 plasmid constructs, acid-stable IFNs were detected by bioassay using reduction in cytopathic effect of encephalomyocarditis virus-infected L929 cells. In these same muscles, IFN-alpha 9 transcripts were identified by RT-PCR, indicating that transcription had occurred. Acid-stable IFNs were detected from days 7 to 28 post-DNA inoculation. Furthermore, these proteins were found in the sera of DNA-inoculated mice. Control groups of mice given the blank expression vectors did not produce detectable IFNs in muscle or sera as determined by bioassay, nor were transcripts detected by RT-PCR. This approach now allows investigation of the effector function of individual subtypes in various murine disease models.
Asunto(s)
Interferón-alfa/genética , Transfección , Animales , Femenino , Inyecciones Intramusculares , Interferón-alfa/sangre , Interferón-alfa/clasificación , Ratones , Ratones Endogámicos C57BL , Músculos/metabolismo , ARN Mensajero/análisisRESUMEN
Type I interferon (IFN) family has more than 10 structurally related subtypes of alpha IFN (IFNA) genes and a single beta IFN (IFNB) gene. This study examined the expression of MuIFNA1, A4, A5, A6, A9 and MuIFNB mRNAs in the livers and spleens of MCMV-infected mice at 2, 4 and 6 h post infection. The three strains of inbred mice studied were C57BL/6, B6.C-H-28c and BALB/c. B6.C-H-28c strain is a congenic strain of C57BL/6 genomic background. Detection of the specific mRNAs was by an established semiquantitative procedure based on reverse transcription and PCR amplification followed by differential hybridization to specific oligonucleotides. Similar expression patterns of the type I IFN mRNAs were found in C57BL/6 and B6.C-H-28c strains of mice. However, when the genotype was significantly different (BALB/c), a different expression pattern of IFN mRNAs was seen. Differences in the expression patterns of the type I IFN mRNAs was also seen between the livers and spleens of a given mouse strain. Thus, the present study indicates that mouse genotype appears to be a major determinant of the subtype response pattern seen in vivo and that tissue-type can influence the subtype response pattern seen within a given mouse genotype.
Asunto(s)
Infecciones por Citomegalovirus/genética , Interferón Tipo I/genética , Muromegalovirus/genética , Animales , Genotipo , Hígado/metabolismo , Ratones , Ratones Endogámicos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Bazo/metabolismoRESUMEN
Murine type I interferon levels present in mice sera following Newcastle disease virus infections are influenced by the If-1 locus. Sera interferon levels in C57BL/6 mice (If-1h allele) are 10- to 15-fold higher than in BALB/c mice (If-1(1) allele). The B6.C-H-28c strain, which carries BALB/c If-1(1) allele on C57BL/6 genomic background, has low interferon levels in sera. This study examined the expression of interferon alpha 1, alpha 4, alpha 5, alpha 6, alpha 9 and beta mRNAs at 7 hr after Newcastle disease virus infection of primary cells (splenocytes and mouse embryo fibroblasts) from C57BL/6, B6.C-H-28c and BALB/c mouse genotypes. Total RNA from these cells was reverse transcribed and all known type I interferon subtypes were amplified. The products were identified by differential hybridization to a panel of subtype specific oligonucleotides. The results show that the pattern of interferon subtypes examined in splenocytes did not differ between If-1h and If-1(1) allele carrying C57BL mice. However, when the genotype was different (BALB/c splenocytes) the pattern of type I interferon mRNAs seen was altered. This genotype-dependent expression was also seen in newcastle disease virus infected fibroblasts. Within a given mouse strain, there were also differences in the subtype response patterns detected in fibroblasts compared with those seen in splenocytes. In conclusion, the present study indicates that mouse genotype appears to be a major determinant of the subtype response pattern seen and tissue specific pattern differences are present within a given mouse genotype.
Asunto(s)
Fibroblastos/virología , Interferón Tipo I/genética , Virus de la Enfermedad de Newcastle/patogenicidad , Bazo/virología , Animales , Fibroblastos/inmunología , Hibridación in Situ/métodos , Interferón Tipo I/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos , Familia de Multigenes , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Bazo/citología , Bazo/inmunologíaRESUMEN
Murine AIDS (MAIDS) is caused by a defective retrovirus present in the LP-BM5 murine leukemia virus mixture. Strains of inbred mice differ in resistance to MAIDS development; some are susceptible (e.g., C57BL/6), while others are resistant (e.g., CBA and B10.BR). As an early block to viral replication in resistant mice has been demonstrated previously by PCR studies, we postulated that alpha/beta interferons (IFN-alpha/beta) may be involved in resistance to MAIDS. Susceptible C57BL/6 mice infected with LP-BM5 were treated with IFN-alpha/beta or Newcastle disease virus. Newcastle disease virus induces high endogenous IFN-alpha/beta production in mice. Both treatments delayed the development of MAIDS, as assessed by splenomegaly and T- and B-cell proliferation. In addition, an IFN-alpha/beta response was detected by reverse transcription-PCR and dot blotting 3, 6, and 9 h after LP-BM5 infection in resistant mice but not in susceptible mice. These results suggest that the ability to produce IFN-alpha/beta in response to LP-BM5 infection may contribute to host resistance to MAIDS.
Asunto(s)
Interferón-alfa/inmunología , Interferón beta/inmunología , Virus de la Leucemia Murina/inmunología , Síndrome de Inmunodeficiencia Adquirida del Murino/inmunología , Animales , Secuencia de Bases , Células Cultivadas , ADN Viral , Femenino , Inmunidad Innata/inmunología , Interferón-alfa/uso terapéutico , Interferón beta/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Bazo/citología , Bazo/inmunologíaRESUMEN
Excellent long-term survival (up to 1 yr) of donor skeletal muscle cells was demonstrated using a mouse Y-chromosome specific probe, following the transplantation of grafts of whole muscles from male "normal" C57B1/10Sn mice into dystrophic muscles of female host mice. After the transplantation of equivalent sliced muscle grafts there was extensive movement of the male donor cells and fusion with host myofibres. This contrasts with the extremely poor survival of isolated myoblasts after injection into the same mouse model for Duchenne muscular dystrophy. The use of sliced muscle grafts may therefore represent a potential alternative approach to myoblast transfer therapy.
Asunto(s)
Trasplante de Células/métodos , Músculo Esquelético/trasplante , Distrofia Muscular Animal/cirugía , Cromosoma Y , Animales , Movimiento Celular , Disección , Femenino , Supervivencia de Injerto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Músculo Esquelético/citología , Músculo Esquelético/fisiología , Trasplante HomólogoRESUMEN
The immature erythroid cell line J2E responds to erythropoietin (Epo) by proliferating and terminally differentiating into hemoglobin-synthesizing red blood cells. These cells produce a rapid, fatal erythroleukemia in mice characterized by hepatosplenomegaly and severe anemia. The aim of this study was to investigate the effects of murine interferons-alpha (MuIFN-alpha) on J2E cells in vitro and in vivo. Here we show that in culture MuIFN-alpha inhibited the Epo-induced proliferation of J2E cells but did not interfere with differentiation. When mice with J2E erythroleukemias were treated with MuIFNs in vivo, an extension of their life span was observed. Moreover, numerous necrotic lesions of infiltrating leukemic cells were detected in the spleens of these mice. Finally, ex vivo treatment of leukemic bone marrow cells with Epo and MuIFNs delayed mortality even further. It was concluded that MuIFNs (1) suppressed the proliferation of J2E cells in vitro but did not affect Epo-induced differentiation, and (2) inhibited the progress of erythroleukemias, especially in combination with Epo.
Asunto(s)
Eritropoyetina/antagonistas & inhibidores , Interferón-alfa/uso terapéutico , Leucemia Eritroblástica Aguda/terapia , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Resistencia a Medicamentos , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/patología , Femenino , Leucemia Eritroblástica Aguda/patología , Ratones , Necrosis , Bazo/efectos de los fármacos , Bazo/patología , Resultado del TratamientoRESUMEN
The J2E cell line is an immature erythroid line which terminally differentiates in response to erythropoietin (epo), producing mature, hemoglobin-synthesizing red blood cells. We have shown that when these cells were injected into mice a rapid and fatal erythroleukemia developed with symptoms of severe anemia and hepatosplenomegaly. Southern blotting demonstrated that the leukemic cells were the introduced J2E cells. In addition to spleen and liver, the bone marrow was a major site of leukemic cell infiltration, and when grown in vitro leukemic cells from bone marrow remained responsive to erythropoietin. We reasoned, therefore, that treatment of mice with this hormone should alleviate the erythroleukemia, but regular injections of epo in vivo failed to arrest the progress of the disease. However, when bone marrow from leukemic mice was exposed continuously to the hormone ex vivo, before reinfusion into naive recipients, a marked extension in life span was observed. It was concluded that ex vivo epo treatment could be used therapeutically for J2E cell erythroleukemias.
Asunto(s)
Eritropoyetina/uso terapéutico , Leucemia Eritroblástica Aguda/etiología , Leucemia Eritroblástica Aguda/terapia , Animales , Diferenciación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Eritroblastos/fisiología , Femenino , Leucemia Eritroblástica Aguda/patología , Ratones , Ratones Endogámicos , Trasplante de Neoplasias , Células Tumorales CultivadasRESUMEN
Monocyte derived cytokines (monokines) are important mediators in inflammatory diseases and cancer. Control of monokine expression is also a major therapeutic target in autoimmune inflammation. Whole blood cultures permit examination of monokine expression under conditions which emulate the in-vivo environment whilst avoiding many of the artefacts associated with monocyte separation and culture. Here we describe a system for measuring interleukin-1 beta, interleukin-1 alpha, interleukin-6 and tumour necrosis factor-alpha mRNA in stimulated human whole blood ex-vivo, which can be applied to specimens from treated patients. Oligodeoxyribonucleotide probes are designed to allow standardisation of hybridisation and washing procedures. Washing and reprobing of membranes in appropriate sequence permits measurement of each monokine mRNA and mRNA for glyceraldehyde-3-phosphate dehydrogenase in only 7 ml of lipopolysaccharide-stimulated human blood. The method has been used successfully in studies of dexamethasone and methotrexate action on lipopolysaccharide stimulated IL-beta gene expression.
Asunto(s)
Expresión Génica , Interleucina-6/biosíntesis , Monocitos/metabolismo , Monocinas/biosíntesis , ARN Mensajero/biosíntesis , Northern Blotting/métodos , Dexametasona/farmacología , Expresión Génica/efectos de los fármacos , Gliceraldehído-3-Fosfato Deshidrogenasas/biosíntesis , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Humanos , Interferón gamma/farmacología , Interleucina-1/biosíntesis , Interleucina-1/sangre , Interleucina-1/genética , Interleucina-6/sangre , Interleucina-6/genética , Lipopolisacáridos/farmacología , Metotrexato/farmacología , Datos de Secuencia Molecular , Monocinas/sangre , Monocinas/genética , ARN Mensajero/sangre , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Primary mouse embryo fibroblasts from 4 strains of mice (BALB/c, C57Bl/6, B6.C-H-28c and CBA) were infected with either Newcastle disease virus or murine cytomegalovirus. The time course of the total type I interferon response was assessed and the presence of individual subtypes determined. The total type I interferon produced was titrated using the cytopathic effect reduction assay and the relative levels of type I interferon subtypes expressed (alpha 1, alpha 4, alpha 5, alpha 6 and beta) were evaluated using a reverse transcription-polymerase chain reaction-based technique. In general, the patterns of type I interferon subtypes expressed appeared to be determined by the strain of mouse cells used rather than the inducing virus. However, the overall titre of type I interferons produced in response to a given virus was quite uniform across the strains of mice from which the mouse embryo fibroblasts were derived regardless of the subtype expression pattern. The latter observation fits the proposition that "cross-talk" or feedback between the type I interferon genes and their products is is occurring and that the inducer determines the level of response.
Asunto(s)
Fibroblastos/metabolismo , Fibroblastos/virología , Interferón Tipo I/biosíntesis , Interferón Tipo I/clasificación , Animales , Secuencia de Bases , Embrión de Pollo , Citomegalovirus/crecimiento & desarrollo , Estabilidad de Medicamentos , Embrión de Mamíferos , Femenino , Fibroblastos/fisiología , Genotipo , Concentración de Iones de Hidrógeno , Interferón Tipo I/genética , Interferón beta/biosíntesis , Interferón beta/clasificación , Interferón beta/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Virus de la Enfermedad de Newcastle/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa , ADN Polimerasa Dirigida por ARN , Activación ViralRESUMEN
The time course of expression of the skeletal muscle-specific regulatory genes MyoD and myogenin was studied in primary cultures of skeletal muscle from adult SJL/J and BALB/c mice. In situ detection of expression with MyoD and myogenin riboprobes and myogenin antibody showed that the onset of expression of these genes occurred earlier in cells from SJL/J mice. Probe-positive cells and myotubes were also more frequent in cultures from SJL/J mice than in BALB/c. The onset of expression of MyoD and myogenin was delayed in cultured cells relative to the time course seen following injury in vivo. Myogenin protein was demonstrated in replicating cells and all myogenin-positive cells expressed desmin. The observed strain-specific difference infers a greater intrinsic myogenicity of cells in SJL/J muscle in vitro and reflects the superior capacity for new muscle formation previously reported in SJL/J mice in vivo.
Asunto(s)
Músculos/química , Músculos/citología , Proteína MioD/análisis , Miogenina/análisis , Animales , Bromodesoxiuridina/metabolismo , Células Cultivadas , Colágeno , Desmina/análisis , Combinación de Medicamentos , Femenino , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/genética , Genes/genética , Laminina , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Microscopía Electrónica , Músculos/ultraestructura , Proteína MioD/genética , Miogenina/genética , Proteoglicanos , Especificidad de la Especie , Timidina/metabolismoRESUMEN
The development of a technique for identifying murine type I interferon messenger RNAs is described that involves the following essential steps: (a) the reverse transcription of total RNA extracts using oligo(dT)12-18 as a primer, (b) the amplification of any type I interferon cDNAs produced by polymerase chain reaction, and (c) the identification of interferon subtypes by hybridization of the polymerase chain reaction products to specific oligonucleotides. The technique was used to characterize the expression of the mouse interferon subtypes alpha 1, alpha 4, alpha 5, alpha 6, and beta in murine L929 cells that had been infected with Newcastle disease virus. The data derived from this study are in excellent agreement with earlier RNA protection experiments performed in the same system to characterize expression of the same genes. The present technique has advantages over those used previously, including superior sensitivity, speed, and far smaller input RNA requirements. The technique is not only applicable to other in vitro systems, but is appropriate for use in vivo.