Trypanosoma cruzi Secreted Cyclophilin TcCyP19 as an Early Marker for Trypanocidal Treatment Efficiency.

Cyclophilins (CyPs) are a family of enzymes involved in protein folding. Trypanosoma cruzi, the causative agent of Chagas disease, has a 19-kDa cyclophilin, TcCyP19, that was found to be secreted in parasite stages of the CL Brener clone and recognized by sera from T. cruzi-infected mice and patients. The levels of specific antibodies against TcCyP19 in T. cruzi-infected mice and subjects before and after drug treatment were measured by an in-house enzyme linked immunosorbent assay (ELISA). Mice in the acute and chronic phase of infection, with successful trypanocidal treatments, showed significantly lower anti-TcCyP19 antibody levels than untreated mice. In children and adults chronically infected with T. cruzi, a significant decrease in the anti-TcCyP19 titers was observed after 12 months of etiological treatment. This decrease was maintained in adult chronic patients followed-up 30-38 months post-treatment. These results encourage further studies on TcCyP19 as an early biomarker of trypanocidal treatment efficiency.

Effectiveness of the repurposed drug isotretinoin in an experimental murine model of Chagas disease.

Benznidazole and nifurtimox are the drugs currently used for the treatment of Chagas disease, however its side effects may affect patient adherence. In the search for new alternative therapies, we previously identified isotretinoin (ISO), an FDA-approved drug widely used for the treatment of severe acne through a drug repurposing strategy. ISO shows a strong activity against Trypanosoma cruzi parasites in the nanomolar range, and its mechanism of action is through the inhibition of T. cruzi polyamine and amino acid transporters from the Amino Acid/Auxin Permeases (AAAP) family. In this work, a murine model of chronic Chagas disease (C57BL/6 J mice), intraperitoneally infected with T. cruzi Nicaragua isolate (DTU TcI), were treated with different oral administrations of ISO: daily doses of 5 mg/kg/day for 30 days and weekly doses of 10 mg/kg during 13 weeks. The efficacy of the treatments was evaluated by monitoring blood parasitemia by qPCR, anti-T. cruzi antibodies by ELISA, and cardiac abnormalities by electrocardiography. No parasites were detected in blood after any of the ISO treatments. The electrocardiographic study of the untreated chronic mice showed a significant decrease in heart rate, while in the treated mice this negative chronotropic effect was not observed. Atrioventricular nodal conduction time in untreated mice was significantly longer than in treated animals. Mice treated even with ISO 10 mg/kg dose every 7 days, showed a significant reduction in anti-T. cruzi IgG levels. In conclusion, the intermittent administration of ISO 10 mg/kg would improve myocardial compromise during the chronic stage.

In vitro studies and preclinical evaluation of benznidazole microparticles in the acute Trypanosoma cruzi murine model.

Chagas disease is a serious parasitic infection caused by Trypanosoma cruzi. Unfortunately, the current chemotherapeutic tools are not enough to combat the infection. The aim of this study was to evaluate the trypanocidal activity of benznidazole-loaded microparticles during the acute phase of Chagas infection in an experimental murine model. Microparticles were prepared by spray-drying using copolymers derived from esters of acrylic and methacrylic acids as carriers. Dissolution efficiency of the formulations was up to 3.80-fold greater than that of raw benznidazole. Stability assay showed no significant difference (P > 0.05) in the loading capacity of microparticles for 3 years. Cell cultures showed no visible morphological changes or destabilization of the cell membrane nor haemolysis was observed in defibrinated human blood after microparticles treatment. Mice with acute lethal infection survived 100% after 30 days of treatment with benznidazole microparticles (50 mg kg-1 day-1). Furthermore, no detectable parasite load measured by quantitative polymerase chain reaction and lower levels of T. cruzi-specific antibodies by enzyme-linked immunosorbent assay were found in those mice. A significant decrease in the inflammation of heart tissue after treatment with these microparticles was observed, in comparison with the inflammatory damage observed in both infected mice treated with raw benznidazole and untreated infected mice. Therefore, these polymeric formulations are an attractive approach to treat Chagas disease.

Distinct Treatment Outcomes of Antiparasitic Therapy in Trypanosoma cruzi-Infected Children Is Associated With Early Changes in Cytokines, Chemokines, and T-Cell Phenotypes.

Background: In contrast to adults, Trypanosoma cruzi-infected children have more broadly functional Trypanosoma cruzi-specific T cells, and the total T-cell compartment exhibits fewer signs of immune exhaustion. However, not much is known about the link between immunocompetence and the treatment efficacy for human Chagas disease. Methods: Using cytokine enzyme-linked immunosorbent spot (ELISPOT) polychromatic flow cytometry, cytometric bead assay, multiplex serological assays and quantitative PCR, we evaluated T. cruzi-specific T-cell and antibody immune responses, T-cell phenotypes and parasitemia in children in the early chronic phase of Chagas disease undergoing anti-Trypanosoma cruzi treatment. Results: Treatment with benznidazole or nifurtimox induced a decline in T. cruzi-specific IFN-γ- and IL-2-producing cells and proinflammatory cytokines and chemokines. T-cell responses became detectable after therapy in children bearing T-cell responses under background levels prior to treatment. The total frequencies of effector, activated and antigen-experienced T cells also decreased following anti-T. cruzi therapy, along with an increase in T cells expressing the receptor of the homeostatic cytokine IL-7. Posttreatment changes in several of these markers distinguished children with a declining serologic response suggestive of successful treatment from those with sustained serological responses in a 5-year follow-up study. A multivariate analysis demonstrated that lower frequency of CD4+CD45RA-CCR7-CD62L- T cells prior to drug therapy was an independent indicator of successful treatment. Conclusions: These findings further validate the usefulness of alternative metrics to monitor treatment outcomes. Distinct qualitative and quantitative characteristics of T cells prior to drug therapy may be linked to treatment efficacy.

Elucidating the impact of low doses of nano-formulated benznidazole in acute experimental Chagas disease.

BACKGROUND: Chagas disease is a neglected parasitic infection caused by the protozoan Trypanosoma cruzi (T. cruzi) that affects more than 6 million people, mainly in Latin America. Benznidazole is still the drug of choice in many countries to treat it in spite of its dosage regimen and adverse side effects such as such as allergic dermatitis, peripheral neuropathy and anorexia. Thus, novel, safer, and more efficacious treatments for such neglected infection are urgently required. METHODOLOGY: In this study, the efficacy of orally administered low doses of benznidazole (BNZ) nanoparticles was evaluated during the acute phase in mice infected with T. cruzi Nicaragua (TcN) that were immunosuppressed during the chronic stage of the disease. Moreover, the production of T. cruzi-specific antibodies, cardiac tissue inflammation and reactive oxygen species generation by Vero cells treated with both BNZ nanoparticles (BNZ-nps) and raw BNZ (R-BNZ) were also evaluated. PRINCIPAL FINDINGS: T. cruzi infected mice treated with 10, 25 or 50 mg/kg/day of BNZ-nps survived until euthanasia (92 days post infection (dpi)), while only 15% of infected untreated mice survived until the end of the experiment. PCR analysis of blood samples taken after induction of immunosuppression showed that a dosage of 25 mg/kg/day rendered 40% of the mice PCR-negative. The histological analysis of heart tissue showed a significant decrease in inflammation after treatments with 25 and 50 mg/kg/day, while a similar inflammatory damage was observed in both infected mice treated with R-BNZ (50 mg/kg/day) and untreated mice. In addition, only BNZ-nps treated mice led to lower levels of T. cruzi-specific antibodies to 50-100%. Finally, mammalian Vero cells treated with BNZ-nps or R-BNZ lead to a significant increase in ROS production. CONCLUSIONS: Based on these findings, this research highlights the in-vitro/in-vivo efficacy of nanoformulated BNZ against T. cruzi acute infections in immunosuppressed and non-immunosuppressed mice and provides further evidence for the optimization of dosage regimens to treat Chagas disease.

Promising Efficacy of Benznidazole Nanoparticles in Acute Trypanosoma cruzi Murine Model: In-Vitro and In-Vivo Studies.

The aim of this study was to evaluate the effectiveness of benznidazole nanoparticles (BNZ-nps) on trypomastigote forms and on intracellular infection in mammalian cells and primary cardiac myocyte cells. Its effectiveness was also evaluated on acute Trypanosoma cruzi Nicaragua mice infection. Trypomastigotes from culture were treated with different concentrations of BNZ-nps to determine the drug concentration that lyses 50% of trypomastigotes (LC50). Infected mammalian cells were incubated with different concentrations of BNZ-nps to determine the percentage of amastigote inhibition. C3H/HeN mice with lethal acute infection were treated with 10, 25, and 50 mg/kg/day of BNZ-nps for 30 and 15 days to control the survival rate of animals. BNZ-nps having a mean particle size of 63.3 nm, a size distribution of 3.35, and a zeta potential of -18.30 were successfully prepared using poloxamer 188 as a stabilizer. BNZ-nps 25 and 50 µg/mL showed no significant differences in the percentage of inhibition of infected mammalian cells. Infected mice treated with BNZ-nps (50, 25, and 10 mg/kg/day) for 30 days and with BNZ-nps (50 and 25 mg/kg/day) for 15 days presented a 100% survival, whereas the animals treated with 10 mg/kg/day for 15 days of BNZ-nps showed a 70% survival rate. The results obtained demonstrate, for the first time, that benznidazole nanoparticles are a useful and attractive approach to treat Chagas disease in infected mice.

Polyfunctional T cell responses in children in early stages of chronic Trypanosoma cruzi infection contrast with monofunctional responses of long-term infected adults.

BACKGROUND: Adults with chronic Trypanosoma cruzi exhibit a poorly functional T cell compartment, characterized by monofunctional (IFN-γ-only secreting) parasite-specific T cells and increased levels of terminally differentiated T cells. It is possible that persistent infection and/or sustained exposure to parasites antigens may lead to a progressive loss of function of the immune T cells. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, the quality and magnitude of T. cruzi-specific T cell responses were evaluated in T. cruzi-infected children and compared with long-term T. cruzi-infected adults with no evidence of heart failure. The phenotype of CD4(+) T cells was also assessed in T. cruzi-infected children and uninfected controls. Simultaneous secretion of IFN-γ and IL-2 measured by ELISPOT assays in response to T. cruzi antigens was prevalent among T. cruzi-infected children. Flow cytometric analysis of co-expression profiles of CD4(+) T cells with the ability to produce IFN-γ, TNF-α, or to express the co-stimulatory molecule CD154 in response to T. cruzi showed polyfunctional T cell responses in most T. cruzi-infected children. Monofunctional T cell responses and an absence of CD4(+)TNF-α(+)-secreting T cells were observed in T. cruzi-infected adults. A relatively high degree of activation and differentiation of CD4(+) T cells was evident in T. cruzi-infected children. CONCLUSIONS/SIGNIFICANCE: Our observations are compatible with our initial hypothesis that persistent T. cruzi infection promotes eventual exhaustion of immune system, which might contribute to disease progression in long-term infected subjects.

Combined treatment with benznidazole and allopurinol in mice infected with a virulent Trypanosoma cruzi isolate from Nicaragua.

We evaluated the effect of chemotherapy with a sequential combined treatment of a low dose of benznidazole and allopurinol, in different schedules of administration, in experimental models of acute and chronic Trypanosoma cruzi infection. Mice were infected with Nicaragua T. cruzi isolate, a virulent parasite from an endemic area of Nicaragua, genotyped as TcI (Grosso et al. 2010). We assessed survival rate, IgG levels, histopathological studies and quantified parasitaemia. A 15% survival rate was recorded in untreated mice during the acute phase of T. cruzi infection. Allopurinol administered immediately after benznidazole treatment was able to reduce parasitaemia and attenuate tissue damage by reducing inflammation. Trypanosoma cruzi-specific antibodies also decreased in 40-50% of the treated mice. The addition of allopurinol during the chronic phase showed the highest beneficial effect, not only by reducing parasitaemia but also by lowering the degree of inflammation and fibrosis.

Trypanosoma cruzi: biological characterization of a isolate from an endemic area and its susceptibility to conventional drugs.

We describe some biological and molecular characteristics of a Trypanosoma cruzi isolate derived from a Triatomine captured in Nicaragua. PCR based typification showed that this isolate, named Nicaragua, belonged to the lineage Tc I. Nicaragua infected culture cells were treated with allopurinol, showing different behavior according to the cellular compartment, being cardiomyocyte primary cultures more resistant to this drug. The course of the infection in a mice experimental model and its susceptibility to benznidazole and allopurinol was analyzed. In benznidazole treatment, mice reverted the high lethal effect of parasites during the acute infection, however, a few parasites were detected in the heart of 88% of mice 1 year post-infection. Since T. cruzi is a heterogeneous species population it is important to study and characterize different parasites actually circulating in humans in endemic areas. In this work we show that T. cruzi Nicaragua isolate, is sensitive to early benznidazole treatment.

Trypanosoma cruzi-specific immune responses in subjects from endemic areas of Chagas disease of Argentina.

Trypanosoma cruzi-specific immune responses were evaluated in a total of 88 subjects living in areas endemic of Chagas disease of Argentina by IFN-gamma ELISPOT assays and immunoblotting. Positive T. cruzi antigen-induced IFN-gamma responses were detected in 42% of subjects evaluated (15/26 positive by conventional serology and 22/62 seronegative subjects). Using immunoblotting, T. cruzi-specific IgG reactivity was detected in all seropositive subjects and in 11% (7/61) of subjects negative by conventional serology. Measurements of T cell responses and antibodies by immunoblotting, in conjunction with conventional serology, might enhance the capability of detection of exposure to T. cruzi in endemic areas.

In vitro anti-parasitic activity of Cyclosporin A analogs on Trypanosoma cruzi.

Cyclosporin A (CsA) nonimmunosuppressive analogs were evaluated against Trypanosoma cruzi and on TcCyP19, a cyclophilin of 19 kDa. Two out of eight CsA analogs, H-7-94 and F-7-62 showed the best anti-parasitic effects on all in vitro assays. Their IC(50) values were 0.82 and 3.41 microM, respectively, compared to CsA IC(50) value 5.39 microM on epimastigote proliferation; and on trypomastigote lysis their IC(50) values were 0.97 and 2.66 microM compared to CsA IC(50) value 7.19 microM. H-7-94 and F-7-62 were also more effective than CsA in inhibiting trypomastigote infection. The enzymatic activity of TcCyP19 was inhibited by all CsA derivatives, suggesting this target is involved in the trypanocidal effects observed.

Respuesta de las celulas musculares cardiacas a la infeccion con Trypanosoma cruzi / Myocardial cell response to Trypanosoma cruzi infection

El objetivo de este estudio es determinar la capacidad de respuesta de la célula muscular cardíaca a la infección por Trypanosoma cruzi. Se investigó el rol de la multiplicación de las células musculares en la remodelación cardíaca, y su capacidad de producción de óxido nítrico y control del parasitismo intracelular. Para ello, se determinó la presencia del antígeno nuclear de proliferación celular (PCNA) en los miocitos cardíacos re rata Wistar infectadas experimentalmente con T. cruzi, determinándose un aumento significativo del número de núcleos PCNA+ en los animales infectados agudos y crónicos. La capacidad de control del parasitismo intracelular y de producción de óxido nítrico fueron evaluados en cultivos primarios de miocitos cardíacos de ratas neonatas, estimulados con citoquinas in vitro. El análisis del parasitismo mostró que el número de miocitos cardíacos con amastigotes intracelulares fue menor en los cultivos estimulados con IL-1b, TNF-a e IFN-g. La producción de óxido nítrico fue mayor en los sobrenadantes de los cultivos celulares estimulados con citoquinas, tanto en los infectados como en los controles. Estos datos demuestran que la célula muscular cardíaca participa activamente en la respuesta del organismo durante la infección con T. cruzi. (AU)

Respuesta de las celulas musculares cardiacas a la infeccion con Trypanosoma cruzi / Myocardial cell response to Trypanosoma cruzi infection

El objetivo de este estudio es determinar la capacidad de respuesta de la célula muscular cardíaca a la infección por Trypanosoma cruzi. Se investigó el rol de la multiplicación de las células musculares en la remodelación cardíaca, y su capacidad de producción de óxido nítrico y control del parasitismo intracelular. Para ello, se determinó la presencia del antígeno nuclear de proliferación celular (PCNA) en los miocitos cardíacos re rata Wistar infectadas experimentalmente con T. cruzi, determinándose un aumento significativo del número de núcleos PCNA+ en los animales infectados agudos y crónicos. La capacidad de control del parasitismo intracelular y de producción de óxido nítrico fueron evaluados en cultivos primarios de miocitos cardíacos de ratas neonatas, estimulados con citoquinas in vitro. El análisis del parasitismo mostró que el número de miocitos cardíacos con amastigotes intracelulares fue menor en los cultivos estimulados con IL-1b, TNF-a e IFN-g. La producción de óxido nítrico fue mayor en los sobrenadantes de los cultivos celulares estimulados con citoquinas, tanto en los infectados como en los controles. Estos datos demuestran que la célula muscular cardíaca participa activamente en la respuesta del organismo durante la infección con T. cruzi.

Embryology of Triatoma infestans (Klug), (Hemiptera, Reduviidae), a Chagas' disease vector

This study reports the embryogenesis of T. infestans (Hemiptera, Reduviidae). Morphological parameters of growth sequences from oviposition until hatching (12-14 d 28 degrees C) were established. Five periods, as percent of time of development (TD), were characterized from oviposition until hatching. The most important morphological features were: 1) formation of blastoderm within 7% of TD; 2) germ band and gastrulation within 30% of TD; 3) nerve cord, limb budding, thoracic and abdominal segmentation and formation of body cavity within 50% of TD; 4) nervous system and blastokinesis end, and development of embryonic cuticle within 65% of TD; 5) differentiation of the mouth parts, fat body, and malpighian tubules during final stage and completion of embryo at day 12 to day 14 around hatching. These signals were chosen as appropriate morphological parameters which should enable the evaluation of embryologic modifications due to the action/s of different insecticides

Structures and properties of vitellin isolated from Triatominae species

Se realizaron estudios comparativos de los pesos moleculares composicion quimica e identidad inmunológica de la vitelina (VN) de distintas especies de triatominos. Se determinó que la VN es una lipoglicofosfoproteina con 12.76% de hidratos de carbono 12.53% de lipidos y 0.6% de fósforo unido a mas aproteica. La VN nativa muestra por filtración en gel una única banda de 310 KDa. La VN delipidizada muestra por PAGE SDS una banda principal de 175 KDa coincidente con la banda hallada en la vitellogenina (VG). La composición de aminoácidos fue similar en las cuatro especies ensayadas (AU)

Structures and properties of vitellin isolated from Triatominae species

Se realizaron estudios comparativos de los pesos moleculares composicion quimica e identidad inmunológica de la vitelina (VN) de distintas especies de triatominos. Se determinó que la VN es una lipoglicofosfoproteina con 12.76% de hidratos de carbono 12.53% de lipidos y 0.6% de fósforo unido a mas aproteica. La VN nativa muestra por filtración en gel una única banda de 310 KDa. La VN delipidizada muestra por PAGE SDS una banda principal de 175 KDa coincidente con la banda hallada en la vitellogenina (VG). La composición de aminoácidos fue similar en las cuatro especies ensayadas

Bioquímica del ciclo evolutivo del Triatoma infestans (vinchuca): X. Lipoproteinas hemolinfáticas de hembras / Biochemistry of the evolutive cycle of Triatoma infestans (venchuca): X. Hemolymph lipoproteins of females

En la hemolinfa de Triatoma infestans hembras se separaron por ultracentrifugación una lipoproteína de alta densidad, HDL y tres de muy alta densidad, VHDL-I, VHDL-f y VHDL-II. La proporción de lípidos es mayor en HDL y la de proteínas en VHDL-II. En todas las lipoproteínas se reconoció la presencia de 1,2 y 1,3-diacilgliceroles, triacilgliceroles, ácidos grasos libres, colesterol, ésteres de colesterol e hidrocarburos. Entre los fosfolípidos se reconocieron la fosfatidilcolina y fosfatidiletanolamina. Los lípidos mayoritarios fueron fosfolípidos, 1,2 y 1,3-diacilgliceroles e hidrocarburos. Para estudiar las correspondientes apoliproteínas se deslipidizaron las lipoproteínas. Se examinaron por electroforesis en gel de poliacrilamida con SDS en presencia de 2-mercaptoetanol. Las apo-HDL se separaron en 4 bandas polipeptídicas intensas de PM aproximados 19 000, 45 000, 86 000 y 200 000. Las apo-VHDL-I fraccionaron en 3 bandas polipeptídicas, una mayoritaria de PM 18 500 y dos débiles de 28 000 y 45 000. Las apo-VHDL-f que sólo aparecen en hembras se separaron en 10 bandas. Las mayoritarias correspondieron a los PM 58 000, 86 000 y 160 000. Las bandas de 58 000 y 160 000 presentaron reacción positiva de hidratos de carbono. En las apo-VHDL-II las subunidades proteicas más intensas correspondieron a las bandas de PM 86 000 y 160 000. Aparentemente, estas cuatro lipoproteínas tendrian dos cadenas polipeptídicas comunes (AU)

Bioquímica del ciclo evolutivo del Triatoma infestans (vinchuca): X. Lipoproteinas hemolinfáticas de hembras / Biochemistry of the evolutive cycle of Triatoma infestans (venchuca): X. Hemolymph lipoproteins of females

En la hemolinfa de Triatoma infestans hembras se separaron por ultracentrifugación una lipoproteína de alta densidad, HDL y tres de muy alta densidad, VHDL-I, VHDL-f y VHDL-II. La proporción de lípidos es mayor en HDL y la de proteínas en VHDL-II. En todas las lipoproteínas se reconoció la presencia de 1,2 y 1,3-diacilgliceroles, triacilgliceroles, ácidos grasos libres, colesterol, ésteres de colesterol e hidrocarburos. Entre los fosfolípidos se reconocieron la fosfatidilcolina y fosfatidiletanolamina. Los lípidos mayoritarios fueron fosfolípidos, 1,2 y 1,3-diacilgliceroles e hidrocarburos. Para estudiar las correspondientes apoliproteínas se deslipidizaron las lipoproteínas. Se examinaron por electroforesis en gel de poliacrilamida con SDS en presencia de 2-mercaptoetanol. Las apo-HDL se separaron en 4 bandas polipeptídicas intensas de PM aproximados 19 000, 45 000, 86 000 y 200 000. Las apo-VHDL-I fraccionaron en 3 bandas polipeptídicas, una mayoritaria de PM 18 500 y dos débiles de 28 000 y 45 000. Las apo-VHDL-f que sólo aparecen en hembras se separaron en 10 bandas. Las mayoritarias correspondieron a los PM 58 000, 86 000 y 160 000. Las bandas de 58 000 y 160 000 presentaron reacción positiva de hidratos de carbono. En las apo-VHDL-II las subunidades proteicas más intensas correspondieron a las bandas de PM 86 000 y 160 000. Aparentemente, estas cuatro lipoproteínas tendrian dos cadenas polipeptídicas comunes