RESUMEN
In April of 2013, letters addressed to the President of United States and other government officials were intercepted and found to be contaminated with ricin, heightening awareness about the need to evaluate laboratory methods for detecting ricin. This study evaluated commercial DNA purification methods for isolating Ricinus communis DNA as measured by real-time polymerase chain reaction (PCR). Four commercially available DNA purification methods (two automated, MagNA Pure compact and MagNA Pure LC, and two manual, MasterPure complete DNA and RNA purification kit and QIAamp DNA blood mini kit) were evaluated. We compared their ability to purify detectable levels of R. communis DNA from four different sample types, including crude preparations of ricin that could be used for biological crimes or acts of bioterrorism. Castor beans, spiked swabs, and spiked powders were included to simulate sample types typically tested during criminal and public health investigations. Real-time PCR analysis indicated that the QIAamp kit resulted in the greatest sensitivity for ricin preparations; the MasterPure kit performed best with spiked powders. The four methods detected equivalent levels by real-time PCR when castor beans and spiked swabs were used. All four methods yielded DNA free of PCR inhibitors as determined by the use of a PCR inhibition control assay. This study demonstrated that DNA purification methods differ in their ability to purify R. communis DNA; therefore, the purification method used for a given sample type can influence the sensitivity of real-time PCR assays for R. communis.
Asunto(s)
ADN de Plantas/aislamiento & purificación , Ricinus/genética , Manejo de Especímenes/métodos , Automatización de Laboratorios , Ricinus communis/química , Sustancias para la Guerra Química/análisis , Extractos Vegetales/genética , Extractos Vegetales/aislamiento & purificación , Polvos/química , Reacción en Cadena en Tiempo Real de la Polimerasa , Ricina/análisisRESUMEN
Immunity often depends on proper cell fate choice by helper T lymphocytes. A naive cell, with minimal expression of IFN-gamma and IL-4, must give rise to progeny expressing high levels of either one, but not both, of those cytokines to defend against protozoan and helminthic pathogens, respectively. In the present study, we show that inactivation of the Mbd2 gene, which links DNA methylation and repressed chromatin, results in enhanced resistance to the protozoan parasite Leishmania major but impaired immunity to the intestinal helminth Trichuris muris. Helper T cells from methyl CpG-binding domain protein-2-deficient mice exhibit exuberant patterns of cytokine expression despite appropriate silencing of genes encoding the lineage-specifying factors T-bet and GATA-3. These results suggest that gene silencing can facilitate the ability of a progenitor cell to give rise to appropriately differentiated daughter cells in vivo. These findings also point to novel pathways that could participate in genetic control of resistance to infection and autoimmunity.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Silenciador del Gen , Predisposición Genética a la Enfermedad , Animales , Autoinmunidad , Metilación de ADN , Proteínas de Unión al ADN/genética , Inmunidad Innata , Interferón gamma/biosíntesis , Leishmania major , Leishmaniasis Cutánea/genética , Leishmaniasis Cutánea/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas de Dominio T Box , Factores de Transcripción/fisiología , Tricuriasis/inmunologíaRESUMEN
The stability of helper T cell fates is not well understood. Using conditional introduction of dominant-negative factors, we now show that T-bet and GATA-3 are far more critical in establishment than maintenance of IFN-gamma and IL-4 activity during Th1 and Th2 maturation, respectively. We also show that a genetic interaction between T-bet and its target Hlx seems to be required for Th1 maturation, but that Hlx may also be dispensable for maintenance of a transcriptionally permissive ifng gene. In parallel to progressive activator independence in the permissive lineage, the ifng gene becomes more recalcitrant to switching as the forbidden lineage matures. T-bet plus Hlx can disrupt ifng silencing when introduced into developing Th2 cells, but they fail to perturb ifng silencing in mature Th2 cells. In contrast, a hypermorphic allele of T-bet can reverse silencing of the ifng gene in mature Th2 cells. These results suggest that signature gene activity of helper T cells is initially plastic but later becomes epigenetically fixed and offer an initial strategy for inducing mature cells to switch their fate.
Asunto(s)
Interferón gamma/genética , Interleucina-4/genética , Células TH1/fisiología , Células Th2/fisiología , Transactivadores/fisiología , Animales , Factor de Transcripción GATA3/fisiología , Proteínas de Homeodominio/fisiología , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Dominio T Box , Factores de Transcripción/fisiologíaRESUMEN
Activated CD8+ T cells play a critical role in host defense against viruses, intracellular microbes, and tumors. It is not clear if a key regulatory transcription factor unites the effector functions of CD8+ T cells. We now show that Eomesodermin (Eomes), a paralogue of T-bet, is induced in effector CD8+ T cells in vitro and in vivo. Ectopic expression of Eomes was sufficient to invoke attributes of effector CD8+ T cells, including interferon-gamma (IFN-gamma), perforin, and granzyme B. Loss-of-function analysis suggests Eomes may also be necessary for full effector differentiation of CD8+ T cells. We suggest that Eomesodermin is likely to complement the actions of T-bet and act as a key regulatory gene in the development of cell-mediated immunity.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Proteínas de Dominio T Box/fisiología , Secuencia de Aminoácidos , Animales , Infecciones por Arenaviridae/inmunología , Secuencia de Bases , Linfocitos T CD8-positivos/fisiología , Diferenciación Celular , Citotoxicidad Inmunológica , Regulación de la Expresión Génica , Granzimas , Interferón gamma/biosíntesis , Activación de Linfocitos , Virus de la Coriomeningitis Linfocítica/inmunología , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Perforina , Proteínas Citotóxicas Formadoras de Poros , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/genética , Proteínas de Dominio T Box/química , Proteínas de Dominio T Box/genética , Células Th2/inmunología , Células Th2/fisiología , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
The quality of the helper T cell response against antigen can determine the outcomes of infectious, inflammatory, and autoimmune diseases. Mature Th1 and Th2 cell subsets are thought to arise from a common naive progenitor. In these precursor cells, effector cytokine genes appear to exist in a restrictive structure, which is determined by methylation of cytosine bases and higher-order structure of chromatin. The restrictive gene structures appear to be plastic, giving way to more active structures in some daughter cells. Some genetic loci, which are active in naive cells, however, become silenced during terminal differentiation. Both the derepression of silent loci and the silencing of active loci appear to be linked to the process of DNA replication. Future investigation will be directed toward understanding the way in which patterns of gene expression are altered or transmitted during the cell division of helper T lymphocytes.
Asunto(s)
Diferenciación Celular/genética , Regulación de la Expresión Génica/inmunología , Subgrupos de Linfocitos T/fisiología , Linfocitos T Colaboradores-Inductores/fisiología , Animales , Diferenciación Celular/inmunología , Citocinas/genética , Citocinas/inmunología , HumanosRESUMEN
How a single cell gives rise to progeny with differing fates remains poorly understood. We examined cells lacking methyl CpG binding domain protein-2 (MBD2), a molecule that has been proposed to link DNA methylation to silent chromatin. Helper T cells from Mbd2(-/-) mice exhibit disordered differentiation. IL-4, the signature of a restricted set of progeny, is expressed ectopically in Mbd2(-/-) parent and daughter cells. Loss of MBD2-mediated silencing renders the normally essential activator, Gata-3, dispensable for IL-4 induction. Gata-3 and MBD2 act in competition, wherein each factor independently, and quantitatively, regulates the binary choice of whether heritable IL-4 expression is established. Gata-3 functions, in part, to displace MBD2 from methylated DNA. These results suggest that activating and silencing signals integrate to provide spatially and temporally restricted patterns of gene activity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Transactivadores/metabolismo , Animales , Unión Competitiva , Diferenciación Celular , Línea Celular , Linaje de la Célula , Cromatina/metabolismo , Islas de CpG/genética , Metilación de ADN , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Citometría de Flujo , Factor de Transcripción GATA3 , Eliminación de Gen , Interleucina-4/genética , Interleucina-4/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/metabolismo , Transactivadores/deficiencia , Transactivadores/genéticaRESUMEN
Type 1 helper T (T(H)1) cells are essential for cellular immunity, but their ontogeny, maturation and durability remain poorly understood. By constructing a dominant-negative form of T-bet, we were able to determine the role played by this lineage-inducing trans-activator in the establishment and maintenance of heritable T(H)1 gene expression. Optimal induction of interferon-gamma (IFN-gamma) expression required genetic interaction between T-bet and its target, the homeoprotein Hlx. In fully mature T(H)1 cells, reiteration of IFN-gamma expression and stable chromatin remodeling became relatively independent of T-bet activity and coincided with demethylation of DNA. In contrast, some lineage attributes, such as expression of IL-12R beta 2 (interleukin 12 receptor beta 2), required ongoing T-bet activity in mature T(H)1 cells and their progeny. These findings suggest that heritable states of gene expression might be maintained by continued expression of the inducing factor or by a mechanism that confers a stable imprint of the induced state.