RESUMEN
A novel assay was developed for Daudi cells in which the antiviral (AV) and antiproliferative (AP) activities of interferon (IFN) can be measured simultaneously. Using this novel assay, conditions allowing IFN AV protection but no growth inhibition were identified and selected. Daudi cells were treated under these conditions, and gene expression microarray analyses were performed. The results of the analysis identified 25 genes associated with IFN-α AV activity. Upregulation of 23 IFN-induced genes was confirmed by using reverse transcription-PCR. Of 25 gene products, 17 were detected by Western blotting at 24 h. Of the 25 genes, 10 have not been previously linked to AV activity of IFN-α. The most upregulated gene was IFIT3 (for IFN-induced protein with tetratricopeptide repeats 3). The results from antibody neutralizing experiments suggested an association of the identified genes with IFN-α AV activity. This association was strengthened by results from IFIT3-small interfering RNA transfection experiments showing decreased expression of IFIT3 and a reduction in the AV activity induced by IFN-α. Overexpression of IFIT3 resulted in a decrease of virus titer. Transcription of AV genes after the treatment of cells with higher concentrations of IFN having an AP effect on Daudi cells suggested pleiotropic functions of identified gene products.
Asunto(s)
Antivirales/farmacología , Interferón Tipo I/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Virus Maus Elberfeld/efectos de los fármacos , Virus Maus Elberfeld/patogenicidad , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Recombinantes , Regulación hacia Arriba/efectos de los fármacos , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacos , Virus de la Estomatitis Vesicular Indiana/patogenicidad , Virosis/tratamiento farmacológico , Virosis/genéticaRESUMEN
Homogeneous human lymphoblastoid interferon with an apparent molecular size of 18,500 daltons was characterized by its amino acid composition. Analysis of the amino terminal sequence by Edman degradation indicates that the sequence is unique.
Asunto(s)
Interferones , Linfocitos/análisis , Secuencia de Aminoácidos , Aminoácidos/análisis , Línea Celular , HumanosRESUMEN
Suppression and/or cytotoxicity are believed to play an important role in the defense against Epstein-Barr virus (EBV) infection. To analyze the role of suppressor T cells in relation to EBV, we sought to clone and study these T cells. Analysis of 152 T cell clones derived from the peripheral blood of two patients with acute EBV-induced infectious mononucleosis (IM) yielded 11 highly suppressive clones that had no cytotoxic activity for the natural killer sensitive K562 cell line, an autologous EBV-infected cell line, or an allogeneic EBV-infected B cell line. Four of six suppressor T cell clones also profoundly inhibited EBV-induced immunoglobulin production, and five of five clones delayed the outgrowth of immortalized cells. These results indicate that during acute IM, suppressor T cells capable of inhibiting B cell activation in the absence of cytotoxicity can be identified, and may play a key role in the control of EBV infection.
Asunto(s)
Mononucleosis Infecciosa/inmunología , Linfocitos T Reguladores/inmunología , Formación de Anticuerpos , Linfocitos B/inmunología , Linfocitos B/microbiología , Herpesvirus Humano 4/inmunología , Humanos , Interferón Tipo I/biosíntesis , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Macrophages are uniquely responsive to bacterial lipopolysaccharide (LPS) for activation of a number of host defense functions and production of bioactive mediators. One potentially important mediator produced by LPS-stimulated macrophages is interferon (IFN-alpha/beta). In contrast to murine observations, we have observed that freshly isolated human monocytes, purified by counter-current centrifugal elutriation, do not produce interferon in response to LPS. This is not due to a lack of response to LPS, as assessed by the induction of other monokines, or to an incapacity for IFN production, since IFN was inducible by poly-I,C treatment of monocytes in the absence of any other exogenous stimulus. However, human monocytes can be primed for the production of IFN in response to LPS if they are cultured in the presence of either granulocyte-macrophage colony stimulating factor (GM-CSF) or interferon-gamma (IFN-gamma). The IFN secreted is of the alpha subtype. Monocytes primed with GM-CSF or IFN-gamma also maintained LPS responses for production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1). M-CSF did not prime monocytes for LPS-induced IFN production, although it did enhance production of TNF-alpha and promoted monocyte survival. Northern analysis indicated that the induction of IFN-alpha by LPS was regulated primarily at the mRNA level. The highly regulated production of IFN-alpha by monocytes/macrophages has important implications for autocrine action of interferons in the activation and differentiation of these cells.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interferón Tipo I/genética , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Monocitos/fisiología , Animales , Línea Celular , Células Cultivadas , Expresión Génica/efectos de los fármacos , Humanos , Interferón Tipo I/biosíntesis , Interferón Tipo I/farmacología , Interleucina-1/biosíntesis , Interleucina-1/farmacología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacosRESUMEN
The occurrence of Ebola virus (EBOV) in West Africa during 2013-2015 is unprecedented. Early reports suggested that in this outbreak EBOV is mutating twice as fast as previously observed, which indicates the potential for changes in transmissibility and virulence and could render current molecular diagnostics and countermeasures ineffective. We have determined additional full-length sequences from two clusters of imported EBOV infections into Mali, and we show that the nucleotide substitution rate (9.6 × 10(-4) substitutions per site per year) is consistent with rates observed in Central African outbreaks. In addition, overall variation among all genotypes observed remains low. Thus, our data indicate that EBOV is not undergoing rapid evolution in humans during the current outbreak. This finding has important implications for outbreak response and public health decisions and should alleviate several previously raised concerns.
Asunto(s)
Ebolavirus/genética , Fiebre Hemorrágica Ebola/virología , Tasa de Mutación , Secuencia de Bases , Brotes de Enfermedades , Ebolavirus/clasificación , Ebolavirus/aislamiento & purificación , Genotipo , Fiebre Hemorrágica Ebola/epidemiología , Humanos , Malí/epidemiología , Datos de Secuencia Molecular , FilogeniaRESUMEN
There are four general assay methods used to quantify a drug/biologic in a preparation, including: (1) in vivo bioassays; (2) in vitro bioassays; (3) immunoassays; and (4) receptor assays. The cell receptor assay is used to evaluate the first step in the molecular action of the drug biologic, its interaction with a specific cellular receptor. Subsequently, the drug biologic must initiate other events, such as internalisation, signal transduction, and/or alterations of one or more cellular constituents in order to elicit its biological effect. Major factors to consider in cell receptor assay development include: (1) establishment of a reference standard preparation; (2) labelling; purifying and characterisation of the biologic drug; (3) cell receptor source; (4) methodology, e.g. separation of bound and free, and other factors affecting accuracy and reproducibility; (5) ligand specificity; and (6) correlation with bioactivity. It should be emphasised that cell receptor binding cannot be assumed to correlate with biological activity because of the requirement that subsequent steps must take place prior to achieving the final response. Chemically altered drugs biologics may bind to a specific cell receptor without eliciting a biological activity. Thus, utilisation of a cell receptor assay requires careful evaluation at both the chemical and biological levels prior to its acceptance as a measure of potency.
Asunto(s)
Bioensayo , Receptores de Droga/efectos de los fármacos , Animales , Células Cultivadas , HumanosAsunto(s)
Receptores de Superficie Celular/fisiología , Animales , Transporte Biológico , ADN/biosíntesis , Endocitosis , Humanos , Interferones/metabolismo , Ensayo de Unión Radioligante , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/aislamiento & purificación , Receptores de InterferónAsunto(s)
Factores de Crecimiento de Célula Hematopoyética/uso terapéutico , Legislación de Medicamentos , Animales , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Humanos , Proteínas Recombinantes/uso terapéutico , Estados Unidos , United States Food and Drug AdministrationAsunto(s)
Linfoma de Burkitt/inmunología , Interferones/aislamiento & purificación , Secuencia de Aminoácidos , Línea Celular , Células Cultivadas , Cromatografía de Afinidad/métodos , Cromatografía en Gel/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Interferones/farmacología , Masculino , Piel , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacosAsunto(s)
Anticuerpos Monoclonales/metabolismo , Interferón Tipo I/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes/metabolismo , Complejo Antígeno-Anticuerpo , Línea Celular , Humanos , Radioisótopos de Yodo , Cinética , Técnica de Dilución de Radioisótopos , Receptores Inmunológicos/inmunología , Receptores de InterferónAsunto(s)
Transfusión Sanguínea/instrumentación , Filtración/instrumentación , Hipotensión/etiología , Leucaféresis/instrumentación , Reacción a la Transfusión , Sistemas de Registro de Reacción Adversa a Medicamentos , Humanos , Guías de Práctica Clínica como Asunto , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Other relevant areas of research regarding the structure and function of IFNs include the study of IFN fragments and hybrid IFN molecules prepared by recombinant DNA technology, and monoclonal antibodies against IFNs have also made major contributions to our knowledge of IFN structure and function. However, these topics have been addressed in a number of recent reviews (Zoon and Wetzel, 1984; Langer and Pestka, 1985). We are now awaiting the crystal structure of several human IFNs. Once this knowledge has been acquired, we can proceed to envisage at the three-dimensional level how these molecules interact with their receptors to produce their numerous biological activities.
Asunto(s)
Interferones , Secuencia de Aminoácidos , Humanos , Interferón Tipo I/fisiología , Interferón gamma/fisiología , Interferones/fisiología , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
Cell envelopes of Haemophilus influenzae have been prepared by breakage in a French pressure cell followed by differential centrifugation. The envelope fraction may be resolved into an inner-membrane (light) and an outer-membrane (heavy) fraction on density gradients. Envelopes from competent cells possess elevated levels of lipopolysaccharide with a composition different from that of log-phase cell envelopes. Three apparently new polypeptides have been observed in envelopes from competent cells by gel electrophoresis in sodium dodecyl sulfate; additional quantitative alterations in the profiles of membrane polypeptides also company the development of the capacity to transport deoxyribonucleic acid. Most of the polypeptide changes are confined to the outer membrane; one new polypeptide is associated with the inner cytoplasmic membrane of competent cells. Protein synthesis during competence developement is rquired for the change in lipopolysaccharides and in the envelope polypeptides to occur.