RESUMEN
Apoptosis has been found to help in the defence against pathogens. Infection with the obligate intracellular parasite Toxoplasma gondii is known to trigger host-cell apoptosis. When using a T. gondii-infected macrophage cell line, J774A.1, treatment with IFN-gamma significantly enhanced apoptosis in noninfected bystander cells while parasitized cells became relatively resistant. Infection and IFN-gamma treatment activated the expression of inducible nitric oxide synthase (iNOS), and the production of nitric oxide (NO) and treatment of cells with an iNOS inhibitor, N(G)-monomethlyl-L-arginine acetate (L-NMMA) reduced the apoptosis frequency. However, the reversal was only partial suggesting that not only NO, but also other, as of yet, unknown factors are induced. Finally, we studied the effect in vivo by infecting mice with either a virulent or an avirulent strain. Challenge with the virulent strain lead to a higher parasite burden, induced host-cell apoptosis in peritoneal cells, and produced higher levels of IFN-gamma and NO. Moreover, treatment of mice with a NO synthase inhibitor, aminoguanidine, partially inhibited the host-cell apoptosis induced by the parasite infection. Altogether, our findings indicate that apoptosis in bystander host cells is due to the secretion of NO and other soluble factors released by parasite-infected cells.
Asunto(s)
Apoptosis , Interferón gamma/biosíntesis , Macrófagos/fisiología , Óxido Nítrico/biosíntesis , Toxoplasma/patogenicidad , Animales , Línea Celular , Femenino , Interacciones Huésped-Parásitos , Activación de Macrófagos , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/metabolismo , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis/inmunología , Toxoplasmosis/parasitologíaRESUMEN
SUMMARYThe dense granule antigen 4 (GRA4) is known as an immundominant antigen of Toxoplasma gondii and, therefore, is considered as a vaccine candidate. For further evaluation of its vaccine effect, a recombinant plasmid and vaccinia virus, both expressing GRA4, were constructed, and a heterologous prime-boost vaccination regime was performed in a mouse model. The mice immunized with the heterologous prime-boost vaccination regime showed a high level of specific antibody response against GRA4 and a significantly high level of gamma interferon (IFN-gamma) production and survived completely against a subsequent challenge infection with a lethal dose of T. gondii. In addition, the formation of cysts was inhibited in the mice vaccinated with the heterologous prime-boost vaccination regime. These results demonstrate that the heterologous prime-boost vaccination regime using DNA and a vaccinia virus, both expressing GRA4, could induce both humoral and cellular immune responses and provide effective protection against lethal acute and chronic T. gondii infections in mice.
Asunto(s)
Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Toxoplasmosis/prevención & control , Vacunas de ADN/inmunología , Virus Vaccinia/genética , Animales , Anticuerpos Antiprotozoarios/sangre , Formación de Anticuerpos/inmunología , Encéfalo/parasitología , Chlorocebus aethiops , Femenino , Inmunidad Celular/inmunología , Interferón gamma/análisis , Ratones , Ratones Endogámicos C57BL , Proteínas Protozoarias/genética , Factores de Tiempo , Toxoplasma/genética , Células VeroRESUMEN
The acrosome reaction (AR) is a fundamental event for fertilization, which is induced in concert with acrosome reaction-inducing substance (ARIS) and asterosap, both of which are components of starfish egg jelly (EJ). During the AR, a spermatozoon undergoes a series of physiological changes, such as in intracellular cGMP concentration ([cGMP]i), pHi and intracellular Ca2+ concentration ([Ca2+]i). Affinity purification of cGMP-binding protein resulted in the isolation of a regulatory subunit of the cAMP-dependent protein kinase A (PKA), suggesting the involvement of a cAMP-dependent pathway in the AR. By using a cAMP enzyme immunoassay, [cAMP]i was found to increase in starfish spermatozoa when stimulated with ARIS and asterosap. ARIS could also increase the [cAMP]i in the presence of high pH seawater. Pretreatment of spermatozoa with two specific and cell-permeable PKA inhibitors, H89 and KT5720, prevented the induction of the AR in a concentration-dependent manner. These results suggest that PKA activity participates in the induction of the AR with ARIS and asterosap. To investigate this, we have cloned a gene that encodes a regulatory subunit of PKA that had been identified in starfish spermatozoa.
Asunto(s)
Reacción Acrosómica/fisiología , Asterias/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Glicoproteínas/fisiología , Secuencia de Aminoácidos , Animales , Asterias/genética , Secuencia de Bases , Clonación Molecular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , GMP Cíclico/metabolismo , ADN Complementario/genética , Activación Enzimática , Femenino , Fertilización/fisiología , Concentración de Iones de Hidrógeno , Péptidos y Proteínas de Señalización Intercelular , Masculino , Datos de Secuencia Molecular , Filogenia , Subunidades de Proteína , Agua de Mar , Homología de Secuencia de AminoácidoRESUMEN
201Tl myocardial SPECT is known for better sensitivity, specificity, and accuracy than planar images in detecting coronary artery disease and diagnosing myocardial viability. SPECT images are also superior to planar images in diagnostic sensitivity and anatomical orientation. However, as limitation of the spatial resolution of the machine, we often encounter poor SPECT plower image quality in patients with decreased wall thickness. To test the accuracy of SPECT images in patients with marked thinning of the left ventricular wall, as occurs in dilated cardiomyopathy, we performed a experimental study using myocardial phantom with 7 mm wall thickness. Tomographic image of the phantom images were rather heterogeneous, though no artificial defect was located. Dilated cardiomyopathy is thought to be characterized by patchy defects in the left ventricle. Careful attention should be given to elucidating myocardial perfusion in patients with a thin left ventricle wall, as there are technical limitations in addition to clinical features.