Ifn-g induction by the tc13tul antigen from trypanosoma cruzi in naïve balb/c mice

Trypanosoma cruzi, the etiological agent of Chagas disease, releases factors which modulate the host immune responses, including Tc13 antigens. Regarding innate immune responses, Tc13 antigen from Tulahuén strain, Tc13Tul, has shown to induce B cell expansion and non-specific IgM production on cultures of splenocytes from naïve BALB/c mice. To obtain further information about this role, we evaluated Tc13Tul ability to induce cytokine secretion by in vitro and in vivo stimulation. In vitro Tc13Tul stimulation of splenocytes from naïve BALB/c mice induced higher IFN-g secretion than that induced by the control protein MBP (554 ± 140 pg/ml and 35.33 ± 18.56 pg/ml, respectively). Differences in neither IL-17 nor IL-4 were detected. Tc13Tul-induced IFN-? secretion was also observed in cultured naïve splenocytes from the LPS-resistant C3H/HeJ mouse strain. In vivo administration of Tc13Tul (or MBP as control) to naïve BALB/c mice (3 daily ip doses of 1 ?g/mouse/dose) increased non-specific IgG in sera. In addition, in vitro cultured splenocytes from Tc13Tul-inoculated mice secreted a higher basal level of non-specific IgM than controls and the in vitro Tc13Tul stimulation of these cells showed an additive effect on IgM secretion. Regarding Tc13Tul-induced IFN-g secretion, in vitro cultured splenocytes from Tc13Tul-inoculated mice have no differences in basal levels respect to controls; however, when splenocytes were in vitro stimulated with Tc13Tul, cells from Tc13Tul-inoculated mice showed higher IFN-g secretion than cells from MBP-inoculated mice (950 ± 265.2 pg/ml and 147.3 ± 55.48 pg/ml, respectively). Results indicate that Tc13Tul participation in the innate immune response against T. cruzi is mainly exerted in phenomena related to the evasion of the immune system, such as non-specific Ig production. In contrast, as IFN-g is an important factor involved in T. cruzi resistance, this may be considered a Tc13Tul effect in favor to the host.Fil: Tasso, Laura Mónica. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garavaglia, Patricia Andrea. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Esteva, Mónica Inés. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; ArgentinaFil: Bruballa, Andrea Cecilia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Albareda, María Cecilia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garcia, Gabriela Andrea. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaXXXI Reunión de la Sociedad Argentina de ProtozoologíaMar del PlataArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de Protozoologi

Effect of the Tc13Tul antigen from Trypanosoma cruzi on splenocytes from naïve mice

Trypanosoma cruzi, the etiological agent of Chagas disease, releases factors, including antigens from the trans-sialidase (TS) superfamily, which modulate the host immune responses. Tc13 antigens belong to group IV of TSs and are characterized by C-terminal EPKSA repeats. Here, we studied the effect of the Tc13 antigen from the Tulahuén strain, Tc13Tul, on primary cultures of splenocytes from naïve BALB/c mice. Recombinant Tc13Tul increased the percentage of viable cells and induced B (CD19+) lymphocyte proliferation. Tc13Tul stimulation also induced secretion of non-specific IgM and interferon-γ (IFN-γ). The same effects were induced by Tc13Tul on splenocytes from naïve C3H/HeJ mice. In vivo administration of Tc13Tul to naïve BALB/c mice increased non-specific IgG in sera. In addition, in vitro cultured splenocytes from Tc13Tul-inoculated mice secreted a higher basal level of non-specific IgM than controls and the in vitro Tc13Tul stimulation of these cells showed an enhanced effect on IgM and IFN-γ secretion. Our results indicate that Tc13Tul may participate in the early immunity in T. cruzi infection by favouring immune system evasion through B-cell activation and non-specific Ig secretion. In contrast, as IFN-γ is an important factor involved in T. cruzi resistance, this may be considered a Tc13Tul effect in favour of the host.Fil: Tasso, Laura Mónica. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán". Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bruballa, Andrea Cecilia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán". Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Garavaglia, Patricia Andrea. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán". Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben"; ArgentinaFil: Esteva, Mónica Inés. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán". Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben"; ArgentinaFil: Albareda, María Cecilia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán". Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garcia, Gabriela Andrea. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán". Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Trypanosoma cruzi: death phenotypes induced by ortho-naphthoquinone substrates of the aldo-keto reductase (TcAKR). Role of this enzyme in the mechanism of action of β-lapachone

Fil: Garavaglia, Patricia A ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Rubio, María Fernanda. Laboratorio de Biología Molecular y Apoptosis,Instituto de Investigaciones Médicas Alfredo Lanari (IDIM-CONICET),Universidad de Buenos Aires,Ciudad de Buenos Aires (1427); Argentina.Fil: Laverrière, Marc. Instituto de Investigaciones Biotecnológicas (IIB-INTECH),Universidad Nacional de General San Martín-CONICET,San Martín (1650),Prov. Buenos Aires; Argentina.Fil: Tasso, Laura Mónica. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Fichera, Laura E. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Cannata, Joaquín J B. Instituto de Investigaciones Biotecnológicas (IIB-INTECH),Universidad Nacional de General San Martín-CONICET,San Martín (1650),Prov. Buenos Aires; Argentina.Fil: García, Gabriela Andrea. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Several ortho-naphthoquinones (o-NQs) have trypanocidal activity against Trypanosoma cruzi, the aetiological agent of Chagas disease. Previously, we demonstrated that the aldo-keto reductase from this parasite (TcAKR) reduces o-NQs, such as β-lapachone (β-Lap) and 9,10-phenanthrenequinone (9,10-PQ), with concomitant reactive oxygen species (ROS) production. Recent characterization of TcAKR activity and expression in two T. cruzi strains, CL Brener and Nicaragua, showed that TcAKR expression is 2.2-fold higher in CL Brener than in Nicaragua. Here, we studied the trypanocidal effect and induction of several death phenotypes by β-Lap and 9,10-PQ in epimastigotes of these two strains. The CL Brener strain was more resistant to both o-NQs than Nicaragua, indicating that greater TcAKR activity is unlikely to be a major influence on o-NQ toxicity. Evaluation of changes in ROS production, mitochondrial membrane potential, phosphatidylserine exposure and monodansylcadaverine labelling evidenced that β-Lap and 9,10-PQ induce different death phenotypes depending on the combination of drug and T. cruzi strain analysed. To study whether TcAKR participates in o-NQ activation in intact parasites, β-Lap and 9,10-PQ trypanocidal effect was next evaluated in TcAKR-overexpressing parasites. Only β-Lap was more effective and induced greater ROS production in TcAKR-overexpressing epimastigotes than in controls, suggesting that TcAKR may participate in β-Lap activation

Identification and biochemical characterization of an ATP-dependent dihydroxyacetone kinase from Trypanosoma cruzi

Dihydroxyacetone (DHA) can be used as an energy source by many cell types; however, it is toxic at high concentrations. The enzyme dihydroxyacetone kinase (DAK) has shown to be involved in DHA detoxification and osmoregulation. Among protozoa of the genus Trypanosoma, T. brucei, which causes sleeping sickness, is highly sensitive to DHA and does not have orthologous genes to DAK. Conversely, T. cruzi, the etiological agent of Chagas Disease, has two putative ATP-dependent DAK (TcDAKs) sequences in its genome. Here we show that T. cruzi epimastigote lysates present a DAK specific activity of 27.1 nmol/min/mg of protein and that this form of the parasite is able to grow in the presence of 2 mM DHA. TcDAK gene was cloned and the recombinant enzyme (recTcDAK) was expressed in Escherichia coli. An anti-recTcDAK serum reacted with a protein of the expected molecular mass of 61 kDa in epimastigotes. recTcDAK presented maximal activity using Mg+2, showing a Km of 6.5 μM for DHA and a K0.5 of 124.7 μM for ATP. As it was reported for other DAKs, recTcDAK activity was inhibited by FAD with an IC50 value of 0.33 mM. In conclusion, TcDAK is the first DAK described in trypanosomatids confirming another divergent metabolism between T. brucei and T. cruzi.Fil: Viecenz, Juan Matías. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; ArgentinaFil: Garavaglia, Patricia Andrea. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; ArgentinaFil: Tasso, Laura Mónica. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Maidana, Cristina Graciela. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; ArgentinaFil: Bautista Cannata, Joaquín Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Garcia, Gabriela Andrea. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentin