RESUMO
Trypanosoma cruzi cruzipain (Cz) bears a C-terminal domain (C-T) that contains sulfated epitopes "sulfotopes" (GlcNAc6S) on its unique N-glycosylation site. The effects of in vivo exposure to GlcNAc6S on heart tissue ultrastructure, immune responses, and along the outcome of infection by T. cruzi, were evaluated in a murine experimental model, BALB/c, using three independent strategies. First, mice were pre-exposed to C-T by immunization. C-T-immunized mice (C-TIM) showed IgG2a/IgG1 <1, induced the production of cytokines from Th2, Th17, and Th1 profiles with respect to those of dC-TIM, which only induced IL-10 respect to the control mice. Surprisingly, after sublethal challenge, both C-TIM and dC-TIM showed significantly higher parasitemia and mortality than the control group. Second, mice exposed to BSA-GlcNAc6S as immunogen (BSA-GlcNAc6SIM) showed: severe ultrastructural cardiac alterations while BSA-GlcNAcIM conserved the regular tissue architecture with slight myofibril changes; a strong highly specific humoral-immune-response reproducing the IgG-isotype-profile obtained with C-TIM; and a significant memory-T-cell-response demonstrating sulfotope-immunodominance with respect to BSA-GlcNAcIM. After sublethal challenge, BSA-GlcNAc6SIM showed exacerbated parasitemias, despite elevated IFN-γ levels were registered. In both cases, the abrogation of ultrastructural alterations when using desulfated immunogens supported the direct involvement of sulfotopes and/or indirect effect through their specific antibodies, in the induction of tissue damage. Finally, a third strategy using a passive transference of sulfotope-specific antibodies (IgG-GlcNAc6S) showed the detrimental activity of IgG-GlcNAc6S on mice cardiac tissue, and mice treated with IgG-GlcNAc6S after a sublethal dose of T. cruzi, surprisingly reached higher parasitemias than control groups. These findings confirmed the indirect role of the sulfotopes, via their IgG-GlcNAc6S, both in the immunopathogenicity as well as favoring T. cruzi infection.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Animais , Antígenos de Protozoários , Cisteína Endopeptidases , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de ProtozoáriosRESUMO
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.
Assuntos
Doença de Chagas/tratamento farmacológico , Nanopartículas/administração & dosagem , Nitroimidazóis/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Doença Aguda/terapia , Animais , Modelos Animais de Doenças , Feminino , CamundongosRESUMO
This study shows the effects of tamoxifen, a known estrogen receptor antagonist used in the treatment of breast cancer, on the sphingolipid pathway of Trypanosoma cruzi, searching for potential chemotherapeutic targets. A dose-dependent epimastigote growth inhibition at increasing concentration of tamoxifen was determined. In blood trypomastigotes, treatment with 10⯵M showed 90% lysis, while 86% inhibition of intracellular amastigote development was obtained using 50⯵M. Lipid extracts from treated and non-treated metabolically labelled epimastigotes evidenced by thin layer chromatography different levels of sphingolipids and MALDI-TOF mass spectrometry analysis assured the identity of the labelled species. Comparison by HPLC-ESI mass spectrometry of lipids, notably exhibited a dramatic increase in the level of ceramide in tamoxifen-treated parasites and a restrained increase of ceramide-1P and sphingosine, indicating that the drug is acting on the enzymes involved in the final breakdown of ceramide. The ultrastructural analysis of treated parasites revealed characteristic morphology of cells undergoing an apoptotic-like death process. Flow cytometry confirmed cell death by an apoptotic-like machinery indicating that tamoxifen triggers this process by acting on the parasitic sphingolipid pathway.
Assuntos
Antiprotozoários/farmacologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Esfingolipídeos/antagonistas & inibidores , Tamoxifeno/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Ceramidas/antagonistas & inibidores , Ceramidas/biossíntese , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Modelos Animais de Doenças , Reposicionamento de Medicamentos , Antagonistas de Estrogênios/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Esfingolipídeos/biossíntese , Esfingosina/antagonistas & inibidores , Esfingosina/biossíntese , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismoRESUMO
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.
Assuntos
Doença de Chagas/tratamento farmacológico , Nanopartículas/uso terapêutico , Nitroimidazóis/uso terapêutico , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/efeitos dos fármacos , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Linhagem Celular , Doença de Chagas/parasitologia , Chlorocebus aethiops , DNA de Protozoário/sangue , DNA de Protozoário/genética , Modelos Animais de Doenças , Portadores de Fármacos/uso terapêutico , Feminino , Coração/parasitologia , Inflamação/parasitologia , Camundongos , Camundongos Endogâmicos C3H , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma cruzi/imunologia , Células VeroRESUMO
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.
Assuntos
Doença de Chagas/tratamento farmacológico , Estágios do Ciclo de Vida/efeitos dos fármacos , Nanopartículas/administração & dosagem , Nitroimidazóis/farmacologia , Parasitemia/tratamento farmacológico , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Doença de Chagas/mortalidade , Doença de Chagas/parasitologia , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Portadores de Fármacos , Feminino , Estágios do Ciclo de Vida/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/parasitologia , Nanopartículas/química , Nanopartículas/ultraestrutura , Nitroimidazóis/química , Parasitemia/mortalidade , Parasitemia/parasitologia , Tamanho da Partícula , Poloxâmero/química , Cultura Primária de Células , Análise de Sobrevida , Tripanossomicidas/química , Trypanosoma cruzi/crescimento & desenvolvimento , Células VeroRESUMO
In order to investigate the involvement of sulfated groups in the Trypanosoma cruzi host-parasite relationship, we studied the interaction between the major cysteine proteinase of T. cruzi, cruzipain (Cz), a sulfate-containing sialylated molecule and the sialic acid-binding immunoglobulin like lectin-E (Siglec-E). To this aim, ELISA, indirect immunofluorescence assays and flow cytometry, using mouse Siglec-E-Fc fusion molecules and glycoproteins of parasites, were performed. Competition assays verified that the lectins, Maackia amurensis II (Mal II) and Siglec-E-Fc, compete for the same binding sites. Taking into account that Mal II binding remains unaltered by sulfation, we established this lectin as sialylation degree control. Proteins of an enriched microsomal fraction showed the highest binding to Siglec-E as compared with those from the other parasite subcellular fractions. ELISA assays and the affinity purification of Cz by a Siglec-E column confirmed the interaction between both molecules. The significant decrease in binding of Siglec-E-Fc to Cz and to its C-terminal domain (C-T) after desulfation of these molecules suggests that sulfates contribute to the interaction between Siglec-E-Fc and these glycoproteins. Competitive ELISA assays confirmed the involvement of sulfated epitopes in the affinity between Siglec-E and Cz, probably modified by natural protein environment. Interestingly, data from flow cytometry of untreated and chlorate-treated parasites suggested that sulfates are not primary receptors, but enhance the binding of Siglec-E to trypomastigotic forms. Altogether, our findings support the notion that sulfate-containing sialylated glycoproteins interact with Siglec-E, an ortholog protein of human Siglec-9, and might modulate the immune response of the host, favoring parasitemia and persistence of the parasite.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos de Protozoários/metabolismo , Cisteína Endopeptidases/metabolismo , Interações Hospedeiro-Patógeno , Fatores Imunológicos/metabolismo , Sulfatos/metabolismo , Trypanosoma cruzi/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Ligação Proteica , Proteínas de Protozoários , CoelhosRESUMO
Sulfation, a post-translational modification which plays a key role in various biological processes, is inhibited by competition with chlorate. In Trypanosoma cruzi, the agent of Chagas' disease, sulfated structures have been described as part of glycolipids and we have reported sulfated high-mannose type oligosaccharides in the C-T domain of the cruzipain (Cz) glycoprotein. However, sulfation pathways have not been described yet in this parasite. Herein, we studied the effect of chlorate treatment on T. cruzi with the aim to gain some knowledge about sulfation metabolism and the role of sulfated molecules in this parasite. In chlorate-treated epimastigotes, immunoblotting with anti-sulfates enriched Cz IgGs (AS-enriched IgGs) showed Cz undersulfation. Accordingly, a Cz mobility shift toward higher isoelectric points was observed in 2D-PAGE probed with anti-Cz antibodies. Ultrastructural membrane abnormalities and a significant decrease of dark lipid reservosomes were shown by electron microscopy and a significant decrease in sulfatide levels was confirmed by TLC/UV-MALDI-TOF-MS analysis. Altogether, these results suggest T. cruzi sulfation occurs via PAPS. Sulfated epitopes in trypomastigote and amastigote forms were evidenced using AS-enriched IgGs by immunoblotting. Their presence on trypomastigotes surface was demonstrated by flow cytometry and IF with Cz/dCz specific antibodies. Interestingly, the percentage of infected cardiac HL-1 cells decreased 40% when using chlorate-treated trypomastigotes, suggesting sulfates are involved in the invasion process. The same effect was observed when cells were pre-incubated with dCz, dC-T or an anti-high mannose receptor (HMR) antibody, suggesting Cz sulfates and HMR are also involved in the infection process by T. cruzi.
Assuntos
Cloratos/metabolismo , Cisteína Endopeptidases/metabolismo , Endocitose/efeitos dos fármacos , Glicoconjugados/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Sulfatos/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Animais , Linhagem Celular , Eletroforese em Gel Bidimensional , Humanos , Immunoblotting , Ponto Isoelétrico , Microscopia Eletrônica , Miócitos Cardíacos/parasitologia , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários , Coelhos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/fisiologiaRESUMO
The efficacy of specific chemotherapy in congenital Chagas disease before the first year of life ranges between 90 and 100%. Between this age and 15 years of age, the efficacy decreases to around 60%. Therefore, early infection detection is a priority in vertical transmission. The aim of this work was to assess whether polymerase chain reaction (PCR) plays a predictive role in the diagnosis of congenital Chagas disease as compared to conventional parasitological and serological methods. To this end, we studied a total of 468 children born to Trypanosoma cruzi seroreactive mothers came from Argentina, Bolivia and Paraguay, who lived in the city of Buenos Aires and suburban areas (Argentina), a non-endemic area of this country. These children were assessed by PCR from 2004 to 2009 with the specific primers Tcz1 and Tcz2, and 121 and 122. PCR allowed detecting 49 T. cruzi-positive children. Eight of these 49 children were excluded from the analysis: six because they did not complete follow-up and two because the first control was performed after 12 months of age. Parasitological methods allowed detecting 25 positive children, 7 of whom had been earlier diagnosed by PCR (1.53±2.00 vs. 6.71±1.46 months; p=0.0002). Serological methods allowed detecting 16 positive children, 12 of whom had been earlier diagnosed by PCR (1.46±1.48 vs. 11.77±4.40 months; p<0.0001). None of the children negative by PCR was positive by serological or parasitological methods. This study shows that PCR allows early diagnosis in congenital Chagas disease. At present, an early positive PCR is not indicative for treatment. However, a positive PCR would alert the health system to search only those infected infants diagnosed by early PCR and thus generate greater efficiency in the diagnosis and treatment of congenital T. cruzi infection.
Assuntos
Doença de Chagas/congênito , Doença de Chagas/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Parasitologia/métodos , Reação em Cadeia da Polimerase/métodos , Trypanosoma cruzi/isolamento & purificação , Adulto , Argentina , Diagnóstico Precoce , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Gravidez , Testes Sorológicos/métodos , Trypanosoma cruzi/genética , Adulto JovemRESUMO
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.
Assuntos
Doença de Chagas/imunologia , Linfócitos T/imunologia , Trypanosoma cruzi/imunologia , Adolescente , Adulto , Ligante de CD40/análise , Criança , Pré-Escolar , ELISPOT , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Interferon gama/metabolismo , Interleucina-2/metabolismo , Masculino , Pessoa de Meia-Idade , Linfócitos T/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The trypanocidal activity of catechins on Trypanosoma cruzi bloodstream trypomastigotes has been previously reported. Herein, we present the effect of epigallocatechin gallate (EGCg) on parasitemia and survival in a murine model of acute Chagas' disease as well as on the epimastigote form of the parasite. Upon intraperitoneal administration of daily doses of 0.8 mg/kg/day of EGCg for 45 days, mice survival rates increased from 11% to 60%, while parasitemia diminished to 50%. No side effects were observed in EGCg-treated animals. Fifty percent inhibition of epimastigotes growth was achieved with 311 microM EGCg 120 h after drug addition. No lysis, total culture growth inhibition or morphological changes were observed upon addition of 1-3mM EGCg at 24 h. This treatment also produced oligosomal fragmentation of epimastigotes DNA, suggesting a programmed cell death (PCD)-like process. All these findings point out EGCg as a potential new lead compound for chemotherapy of Chagas' disease.