RESUMEN

Acute promyelocytic leukemiais a subtype of acute my eloid leukemia characterized by the presence of the PML:RAR aonco proteindueto a specific geneticalteration, translocation (15;17), which avoids my eloid differentiation, generating the accumulation of leukemic promyelocytes with posterior alteration of hemostasis. It presents with symptoms related to pancytopenia and thrombohemorrhagic coagulopathy, and there are few report sof cases with vasculitis secondary to promyelocytic leukemia. The clinical case aimed to describe a particular form of presentation of acute promyelocytic leukemia with vasculitis.

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La leucemia promielocítica aguda es un subtipo de leucemia mieloide aguda caracterizada por la presencia de la oncoproteína PML-RARa debido a una alteracióngenéticaespecífica, la translocación (15;17), que impide la diferenciación mieloide generando la acumulación de promielocitos leucémicos y una alteración compleja de la hemostasia. Se presenta con síntomas relacionados a pancitopenia y coagulopatía trombo-hemorrágica existiendo escasos reportes de casos de vasculitis asociada a leucemia. El caso clínico descrito representa una forma particular de presentación de una leucemia promielocítica aguda con vasculitis.

Asunto(s)

Leucemia Promielocítica Aguda , Tretinoina , Vasculitis

RESUMEN

Neospora caninum is the etiological agent of neosporosis, a worldwide infectious disease recognized as the major cause of abortions and reproductive failures in livestock, responsible for significant economic losses in cattle industries. Currently, there are not cost-effective control options for this pathology, and the development of a vaccine involving new and integrated approaches is highly recommended. In this study, we evaluated the immunogenic and protective efficacy, as well as the potential DIVA (Differentiation of Infected from Vaccinated Animals) character of a recombinant subunit vaccine composed by the major surface antigen from N. caninum (NcSAG1) and the carrier/adjuvant heat shock protein 81.2 from Arabidopsis thaliana (AtHsp81.2) in a mouse model of congenital neosporosis. BALB/c female mice were intraperitoneal (i.p.) immunized with a mixture of equimolar quantities of rNcSAG1 and rAtHSP81.2 or each protein alone (rNcSAG1 or rAtHsp81.2). The vaccine containing a mixture of rNcSAG1 and rAtHsp81.2 significantly enhanced the production of specific anti-rNcSAG1 total IgG (tIgG), IgG1 and IgG2a antibodies in immunized mice when compared to control groups (non-vaccinated and rAtHsp81.2 immunized mice) as well as to the group of mice immunized only with the antigen (rNcSAG1). In addition, partial protection against vertical transmission and improvement of the offspring survival time was observed in this group. On the other hand, rAtHsp81.2 induced the production of specific anti-rAtHsp81.2 tIgG, allowing us to differentiate vaccinated from infected mice. Despite further experiments have to be made in cattle to test the capability of this vaccine formulation to differentiate vaccinated from infected animals in the field, our results suggest that the formulation composed by rNcSAG1 and rAtHsp81.2 could serve as a basis for the development of a new vaccine approach against bovine neosporosis.

Asunto(s)

Antígenos de Protozoos/inmunología , Coccidiosis/prevención & control , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Complicaciones Parasitarias del Embarazo/prevención & control , Vacunas Antiprotozoos/inmunología , Animales , Anticuerpos Antiprotozoarios , Coccidiosis/parasitología , Femenino , Inmunoglobulina G , Ratones , Ratones Endogámicos BALB C , Neospora/inmunología , Embarazo , Vacunación , Vacunas Sintéticas/inmunología

RESUMEN

Previously, we showed that transplastomic tobacco plants expressing the LiHsp83-SAG1 fusion protein displayed a chlorotic phenotype and growth retardation, while plants expressing the SAG1 and GRA4 antigens alone did not. We conducted a comprehensive examination of the metabolic and photosynthetic parameters that could be affecting the normal growth of LiHsp83-SAG1 plants in order to understand the origin of these pleiotropic effects. These plants presented all photosynthetic pigments and parameters related to PSII efficiency significantly diminished. However, the expression of CHLI, RSSU and LHCa/b genes did not show significant differences between LiHsp83-SAG1 and control plants. Total protein, starch, and soluble sugar contents were also greatly reduced in LiHsp83-SAG1 plants. Since Hsp90 s are constitutively expressed at much higher concentrations at high temperatures, we tested if the fitness of LiHsp83-SAG1 over-expressing LiHsp83 would improve after heat treatment. LiHsp83-SAG1 plants showed an important alleviation of their phenotype and an evident recovery of the PSII function. As far as we know, this is the first report where it is demonstrated that a transplastomic line performs much better at higher temperatures. Finally, we detected that LiHsp83-SAG1 protein could be binding to key photosynthesis-related proteins at 37 °C. Our results suggest that the excess of this molecular chaperone could benefit the plant in a possible heat shock and prevent the expected denaturation of proteins. However, the LiHsp83-SAG1 protein content was weakly decreased in heat-treated plants. Therefore, we cannot rule out that the alleviation observed at 37 °C may be partially due to a reduction of the levels of the recombinant protein.

Asunto(s)

Antígenos de Protozoos/metabolismo , Proteínas de Choque Térmico/metabolismo , Leishmania infantum/metabolismo , Fotosíntesis , Plantas Modificadas Genéticamente/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Toxoplasma/metabolismo , Clorofila/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Perfilación de la Expresión Génica , Calor , Inmunoprecipitación , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/parasitología , Nicotiana

RESUMEN

BACKGROUND: The 90-kDa heat-shock protein (Hsp90) from Nicotiana benthamiana (NbHsp90.3) is a promising adjuvant, especially for those vaccines that require a T cell-mediated immune response. Toxoplasma gondii SAG1 is considered one of the most important antigens for the development of effective subunit vaccines. Some epitopes located in the SAG1 C-terminus region have showed a strong humoral and cellular immune response. In the present study, we aimed to assess the efficacy of NbHsp90.3 as carrier/adjuvant of SAG1-derived peptide (SAG1HC) in a T. gondii infection murine model. METHODS: In the present study, C57BL/6 mice were intraperitoneal immunized with the NbHsp90.3-SAG1HC fusion protein (NbHsp90.3-SAG1HC group), mature SAG1 (SAG1m group), NbHsp90.3 (NbHsp90.3 group) or PBS buffer 1× (PBS group). The levels of IgG antibodies and the cytokine profile were determined by ELISA. Two weeks after the last immunization, all mice were orally challenged with 20 cysts of T. gondii Me49 strain and the number of brain cysts was determined. In addition, both humoral and cellular immune responses were also evaluated during the acute and chronic phase of T. gondii infection by ELISA. RESULTS: The characterization of the immune response generated after vaccination with NbHsp90.3 as an adjuvant showed that NbHsp90.3-SAG1HC-immunized mice produced antibodies that were able to recognize not only rSAG1m but also the native SAG1 present in the total lysate antigen extract (SAG1TLA) from T. gondii tachyzoites, while control groups did not. Furthermore, anti-rSAG1m IgG2a/2b antibodies were significantly induced. In addition, only the spleen cell cultures from NbHsp90.3-SAG1HC-immunized mice showed a significantly increased production of IFN-γ. During the chronic phase of T. gondii infection, the antibodies generated by the infection were unable to detect the recombinant protein, but they did react with TLA extract. In addition, splenocytes from all groups showed a high production of IFN-γ when stimulated with rGRA4, but only those from NbHsp90.3-SAG1HC group stimulated with rSAG1m showed high production of IFN-γ. Finally, NbHsp90.3-SAG1HC-immunized mice exhibited a significant reduction in the cyst load (56%) against T. gondii infection. CONCLUSIONS: We demonstrated that NbHsp90.3 enhances the humoral and cell-mediated immune response through a Th1 type cytokine production. Mice vaccinated with NbHsp90.3-SAG1HC exhibited a partial protection against T. gondii infection and it was correlated with the induction of memory immune response. We developed and validated a vaccine formulation which, to our knowledge, for the first time includes the NbHsp90.3 protein covalently fused to a peptide from T. gondii SAG1 protein that contains T- and B-cell epitopes.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Antígenos de Protozoos/inmunología , Chaperonina 60/inmunología , Nicotiana/química , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Citocinas/inmunología , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , Femenino , Inmunidad Celular , Inmunidad Humoral , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos C57BL , Vacunas Antiprotozoos/administración & dosificación , Toxoplasma , Toxoplasmosis Animal/prevención & control

RESUMEN

Chloroplast transformation technology has emerged as an alternative platform offering many advantages over nuclear transformation. SAG1 is the main surface antigen of the intracellular parasite Toxoplasma gondii and a promising candidate to produce an anti-T. gondii vaccine. The aim of this study was to investigate the expression of SAG1 using chloroplast transformation technology in tobacco plants. In order to improve expression in transplastomic plants, we also expressed the 90-kDa heat shock protein of Leishmania infantum (LiHsp83) as a carrier for the SAG1 antigen. SAG1 protein accumulation in transplastomic plants was approximately 0.1-0.2 µg per gram of fresh weight (FW). Fusion of SAG1 to LiHsp83 significantly increased the level of SAG1 accumulation in tobacco chloroplasts (by up to 500-fold). We also evaluated the functionality of the chLiHsp83-SAG1. Three human seropositive samples reacted with SAG1 expressed in transplastomic chLiHsp83-SAG1 plants. Oral immunization with chLiHsp83-SAG1 elicited a significant reduction of the cyst burden that correlated with an increase of SAG1-specific antibodies. We propose the fusion of foreign proteins to LiHsp83 as a novel strategy to increase the expression level of the recombinant proteins using chloroplast transformation technology, thus addressing one of the current challenges for this approach in antigen protein production.

Asunto(s)

Antígenos de Protozoos/metabolismo , Cloroplastos/genética , Proteínas de Choque Térmico/genética , Leishmania infantum/metabolismo , Nicotiana/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Vacunas Antiprotozoos/biosíntesis , Animales , Anticuerpos Antiprotozoarios/inmunología , Anticuerpos Antiprotozoarios/metabolismo , Antígenos de Protozoos/genética , Cloroplastos/metabolismo , Femenino , Humanos , Inmunoglobulina G/metabolismo , Inmunoglobulina M/metabolismo , Leishmania infantum/genética , Ratones , Vacunas Antiprotozoos/administración & dosificación , Vacunas Antiprotozoos/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Nicotiana/genética , Transformación Genética , Vacunación