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1.
Front Microbiol ; 9: 3320, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30697201

RESUMO

Trypanosoma cruzi (Tc) infection causes Chagas disease (ChD) presented by dilated cardiomyopathy and heart failure. During infection, oxidative and nitrosative stresses are elicited by the immune cells for control the pathogen; however, excess nitric oxide and superoxide production can result in cysteine S-nitrosylation (SNO) of host proteins that affects cellular homeostasis and may contribute to disease development. To identify the proteins with changes in SNO modification levels as a hallmark of ChD, we obtained peripheral blood mononuclear cells (PBMC) from seronegative, normal healthy (NH, n = 30) subjects, and from seropositive clinically asymptomatic (ChD CA, n = 25) or clinically symptomatic (ChD CS, n = 28) ChD patients. All samples were treated (Asc+) or not-treated (Asc-) with ascorbate (reduces nitrosylated thiols), labeled with the thiol-labeling BODIPY FL-maleimide dye, resolved by two-dimensional electrophoresis (total 166 gels), and the protein spots that yielded significant differences in abundance or SNO level at p-value of ≤ 0.05 t-test/Welch/BH were identified by MALDI-TOF/TOF MS or OrbiTrap LC-MS/MS. Targeted analysis of a new cohort of PBMC samples (n = 10-14/group) was conducted to verify the differential abundance/SNO levels of two of the proteins in ChD (vs. NH) subjects. The multivariate adaptive regression splines (MARS) modeling, comparing differences in relative SNO level (Asc-/Asc+ ratio) of the protein spots between any two groups yielded SNO biomarkers that exhibited ≥90% prediction success in classifying ChD CA (582-KRT1 and 884-TPM3) and ChD CS (426-PNP, 582-KRT1, 486-ALB, 662-ACTB) patients from NH controls. Ingenuity Pathway Analysis (IPA) of the SNO proteome dataset normalized to changes in protein abundance suggested the proteins belonging to the signaling networks of cell death and the recruitment and migration of immune cells were most affected in ChD CA and ChD CS (vs. NH) subjects. We propose that SNO modification of the select panel of proteins identified in this study have the potential to identify ChD severity in seropositive individuals exposed to Tc infection.

2.
Sci Rep ; 7: 44451, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28294160

RESUMO

The exovesicles (EVs) are involved in pathologic host-parasite immune associations and have been recently used as biomarkers for diagnosis of infectious diseases. The release of EVs by Trypanosoma cruzi, the causative agent of Chagas disease, has recently been described, with different protein cargoes including the MASP multigene family of proteins MASPs are specific to this parasite and characterized by a conserved C-terminal (C-term) region and an N-terminal codifying for a signal peptide (SP). In this investigation, we identified immature MASP proteins containing the MASP SP in EVs secreted by the infective forms of the parasite. Those EVs are responsible for the formation of immune complexes (ICs) containing anti-MASP SP IgGs in patients with different (cardiac, digestive and asymptomatic) chronic Chagas disease manifestations. Moreover, purified EVs as well as the MASP SP inhibit the action of the complement system and also show a significant association with the humoral response in patients with digestive pathologies. These findings reveal a new route for the secretion of MASP proteins in T. cruzi, which uses EVs as vehicles for immature and misfolded proteins, forming circulating immune complexes. Such complexes could be used in the prognosis of digestive pathologies of clinical forms of Chagas disease.


Assuntos
Complexo Antígeno-Anticorpo/isolamento & purificação , Doença de Chagas/imunologia , Vesículas Extracelulares/imunologia , Serina Proteases Associadas a Proteína de Ligação a Manose/isolamento & purificação , Animais , Antígenos de Protozoários/imunologia , Biomarcadores/metabolismo , Doença de Chagas/diagnóstico , Doença de Chagas/parasitologia , Vesículas Extracelulares/genética , Interações Hospedeiro-Parasita/imunologia , Humanos , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Sinais Direcionadores de Proteínas/genética , Trypanosoma cruzi/imunologia , Trypanosoma cruzi/patogenicidade
3.
J Innate Immun ; 9(2): 203-216, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27902980

RESUMO

BACKGROUND: Chronic inflammation and oxidative stress are hallmarks of chagasic cardiomyopathy (CCM). In this study, we determined if microparticles (MPs) generated during Trypanosoma cruzi (Tc) infection carry the host's signature of the inflammatory/oxidative state and provide information regarding the progression of clinical disease. METHODS: MPs were harvested from supernatants of human peripheral blood mononuclear cells in vitro incubated with Tc (control: LPS treated), plasma of seropositive humans with a clinically asymptomatic (CA) or symptomatic (CS) disease state (vs. normal/healthy [NH] controls), and plasma of mice immunized with a protective vaccine before challenge infection (control: unvaccinated/infected). Macrophages (mφs) were incubated with MPs, and we probed the gene expression profile using the inflammatory signaling cascade and cytokine/chemokine arrays, phenotypic markers of mφ activation by flow cytometry, cytokine profile by means of an ELISA and Bioplex assay, and oxidative/nitrosative stress and mitotoxicity by means of colorimetric and fluorometric assays. RESULTS: Tc- and LPS-induced MPs stimulated proliferation, inflammatory gene expression profile, and nitric oxide (∙NO) release in human THP-1 mφs. LPS-MPs were more immunostimulatory than Tc-MPs. Endothelial cells, T lymphocytes, and mφs were the major source of MPs shed in the plasma of chagasic humans and experimentally infected mice. The CS and CA (vs. NH) MPs elicited >2-fold increase in NO and mitochondrial oxidative stress in THP-1 mφs; however, CS (vs. CA) MPs elicited a more pronounced and disease-state-specific inflammatory gene expression profile (IKBKB, NR3C1, and TIRAP vs. CCR4, EGR2, and CCL3), cytokine release (IL-2 + IFN-γ > GCSF), and surface markers of mφ activation (CD14 and CD16). The circulatory MPs of nonvaccinated/infected mice induced 7.5-fold and 40% increases in ∙NO and IFN-γ production, respectively, while these responses were abolished when RAW264.7 mφs were incubated with circulatory MPs of vaccinated/infected mice. CONCLUSION: Circulating MPs reflect in vivo levels of an oxidative, nitrosative, and inflammatory state, and have potential utility in evaluating disease severity and the efficacy of vaccines and drug therapies against CCM.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Doença de Chagas/imunologia , Inflamação/imunologia , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Trypanosoma cruzi/imunologia , Vacinas/imunologia , Animais , Doenças Assintomáticas , Linhagem Celular , Micropartículas Derivadas de Células/imunologia , Citocinas/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação/genética , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Estresse Oxidativo , Vacinação
4.
Infect Immun ; 84(6): 1842-1856, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27068090

RESUMO

Trypanosoma cruzi species is categorized into six discrete typing units (TcI to TcVI) of which TcI is most abundantly noted in the sylvatic transmission cycle and considered the major cause of human disease. In our study, the TcI strains Colombiana (COL), SylvioX10/4 (SYL), and a cultured clone (TCC) exhibited different biological behavior in a murine model, ranging from high parasitemia and symptomatic cardiomyopathy (SYL), mild parasitemia and high tissue tropism (COL), to no pathogenicity (TCC). Proteomic profiling of the insect (epimastigote) and infective (trypomastigote) forms by two-dimensional gel electrophoresis/matrix-assisted laser desorption ionization-time of flight mass spectrometry, followed by functional annotation of the differential proteome data sets (≥2-fold change, P < 0.05), showed that several proteins involved in (i) cytoskeletal assembly and remodeling, essential for flagellar wave frequency and amplitude and forward motility of the parasite, and (ii) the parasite-specific antioxidant network were enhanced in COL and SYL (versus TCC) trypomastigotes. Western blotting confirmed the enhanced protein levels of cytosolic and mitochondrial tryparedoxin peroxidases and their substrate (tryparedoxin) and iron superoxide dismutase in COL and SYL (versus TCC) trypomastigotes. Further, COL and SYL (but not TCC) were resistant to exogenous treatment with stable oxidants (H2O2 and peroxynitrite [ONOO(-)]) and dampened the intracellular superoxide and nitric oxide response in macrophages, and thus these isolates escaped from macrophages. Our findings suggest that protein expression conducive to increase in motility and control of macrophage-derived free radicals provides survival and persistence benefits to TcI isolates of T. cruzi.


Assuntos
Antioxidantes/metabolismo , Doença de Chagas/genética , Estágios do Ciclo de Vida/genética , Macrófagos/metabolismo , Proteínas de Protozoários/genética , Trypanosoma cruzi/patogenicidade , Animais , Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Humanos , Peróxido de Hidrogênio/farmacologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Parasitemia/genética , Parasitemia/metabolismo , Parasitemia/parasitologia , Peroxidases/genética , Peroxidases/metabolismo , Ácido Peroxinitroso/farmacologia , Proteínas de Protozoários/metabolismo , Índice de Gravidade de Doença , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimento
5.
PLoS Negl Trop Dis ; 7(8): e2364, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23951383

RESUMO

BACKGROUND: We investigated the pathological and diagnostic role of selected markers of inflammation, oxidant/antioxidant status, and cellular injury in human Chagas disease. METHODS: Seropositive/chagasic subjects characterized as clinically-symptomatic or clinically-asymptomatic (n = 116), seronegative/cardiac subjects (n = 102), and seronegative/healthy subjects (n = 45) were analyzed for peripheral blood biomarkers. RESULTS: Seropositive/chagasic subjects exhibited an increase in sera or plasma levels of myeloperoxidase (MPO, 2.8-fold), advanced oxidation protein products (AOPP, 56%), nitrite (5.7-fold), lipid peroxides (LPO, 12-17-fold) and malondialdehyde (MDA, 4-6-fold); and a decline in superoxide dismutase (SOD, 52%) and glutathione (GSH, 75%) contents. Correlation analysis identified a significant (p<0.001) linear relationship between inflammatory markers (AOPP/nitrite: r = 0.877), inflammation and antioxidant/oxidant status (AOPP/glutathione peroxidase (GPX): r = 0.902, AOPP/GSH: r = 0.806, Nitrite/GPX: 0.773, Nitrite/LPO: 0.805, MDA/MPO: 0.718), and antioxidant/oxidant levels (GPX/MDA: r = 0.768) in chagasic subjects. Of these, MPO, LPO and nitrite biomarkers were highly specific and sensitive for distinguishing seropositive/chagasic subjects from seronegative/healthy controls (p<0.001, training and fitting AUC/ROC >0.95). The MPO (r = 0.664) and LPO (r = 0.841) levels were also correlated with clinical disease state in chagasic subjects (p<0.001). Seronegative/cardiac subjects exhibited up to 77% decline in SOD, 3-5-fold increase in LPO and glutamate pyruvate transaminase (GPT) levels, and statistically insignificant change in MPO, AOPP, MDA, GPX, GSH, and creatine kinase (CK) levels. CONCLUSIONS: The interlinked effects of innate immune responses and antioxidant/oxidant imbalance are major determinants of human Chagas disease. The MPO, LPO and nitrite are excellent biomarkers for diagnosing seropositive/chagasic subjects, and MPO and LPO levels have potential utility in identifying clinical severity of Chagas disease.


Assuntos
Antioxidantes/análise , Biomarcadores/sangue , Doença de Chagas/imunologia , Doença de Chagas/patologia , Imunidade Inata , Oxidantes/sangue , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
6.
PLoS One ; 7(1): e28449, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22238578

RESUMO

Trypanosoma cruzi elicits reactive oxygen species (ROS) of inflammatory and mitochondrial origin in infected hosts. In this study, we examined ROS-induced oxidative modifications in the heart and determined whether the resultant oxidized cardiac proteins are targets of immune response and of pathological significance in Chagas disease. Heart biopsies from chagasic mice, rats and human patients exhibited, when compared to those from normal controls, a substantial increase in protein 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), carbonyl, and 3-nitrotyrosine (3-NT) adducts. To evaluate whether oxidized proteins gain antigenic properties, heart homogenates or isolated cardiomyocytes were oxidized in vitro and one- or two-dimensional gel electrophoresis (2D-GE)/Western blotting (WB) was performed to investigate the proteomic oxidative changes and recognition of oxidized proteins by sera antibodies in chagasic rodents (mice, rats) and human patients. Human cardiomyocytes exhibited LD(50) sensitivity to 30 µM 4-HNE and 100 µM H(2)O(2) at 6 h and 12 h, respectively. In vitro oxidation with 4-HNE or H(2)O(2) resulted in a substantial increase in 4-HNE- and carbonyl-modified proteins that correlated with increased recognition of cardiac (cardiomyocytes) proteins by sera antibodies of chagasic rodents and human patients. 2D-GE/Western blotting followed by MALDI-TOF-MS/MS analysis to identify cardiac proteins that were oxidized and recognized by human chagasic sera yielded 82 unique proteins. We validated the 2D-GE results by enzyme-linked immunosorbent assay (ELISA) and WB and demonstrated that oxidation of recombinant titin enhanced its immunogenicity and recognition by sera antibodies from chagasic hosts (rats and humans). Treatment of infected rats with phenyl-α-tert-butyl nitrone (PBN, antioxidant) resulted in normalized immune detection of cardiac proteins associated with control of cardiac pathology and preservation of heart contractile function in chagasic rats. We conclude that ROS-induced, cardiac-oxidized antigens are targets of immune recognition by antibodies and molecular determinants for pathogenesis during Chagas disease.


Assuntos
Autoanticorpos/imunologia , Doença de Chagas/etiologia , Doença de Chagas/imunologia , Miocárdio/imunologia , Espécies Reativas de Oxigênio/imunologia , Animais , Afinidade de Anticorpos , Antígenos/imunologia , Antígenos/metabolismo , Cardiomiopatia Chagásica/etiologia , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/metabolismo , Cardiomiopatia Chagásica/patologia , Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Ensaios Clínicos como Assunto , Coração/parasitologia , Humanos , Camundongos , Camundongos Endogâmicos C3H , Miocárdio/metabolismo , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/parasitologia , Oxirredução , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma cruzi/imunologia
7.
Free Radic Res ; 39(6): 621-8, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16036340

RESUMO

We evaluated whether nutritional vitamin A deficiency generates oxidative stress and inflammation in aorta. Wistar male rats (21 days old) were given free access to a control (8 mg retinol as retinyl palmitate/kg) or a vitamin A- deficient diet for three months. One group of deficient animals was fed with the control diet fifteen days before sacrifice. Thiobarbituric acid-reactive substances (TBARS) and nitrite concentration where both analyzed in serum and aorta. Aorta Copper-Zinc Superoxide dismutase (CuZnSOD), Glutathion peroxidase (GPx) and Catalase (CAT) activities were measured. In addition, binding activity of the nuclear factor- kB (NF-kB), inducible and endothelial Nitric Oxide synthase (iNOS and eNOS, respectively) and Ciclooxygenase-2 (COX-2) expressions were determinated in aorta. Rats fed the vitamin A- deficient diet were characterized by sub-clinical plasma retinol concentration and showed increased serum and aorta concentrations of TBARS compared to controls. Lower than control activities of CuZnSOD, GPx, and CAT were observed in aorta of the vitamin A- deficient group. The binding activity of NF- kB was higher in vitamin A- deficient animals than controls. In addition, NO production evaluated as nitrite concentration increased in aorta and serum, associated with a higher expression of iNOS, eNOS and COX-2 in aorta of vitamin A-deficient rats. The incorporation of vitamin A into the diet of vitamin A-deficient rats reverted the changes observed in TBARS level, CuZnSOD and GPx activities, nitrite concentration and also, iNOS, eNOS and COX-2 expression. Prooxidant environment and inflammation are induced by vitamin A deficiency in rat aorta.


Assuntos
Antioxidantes/metabolismo , Aorta/metabolismo , Aorta/patologia , Deficiência de Vitamina A/metabolismo , Deficiência de Vitamina A/patologia , Animais , Aorta/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Inflamação/metabolismo , Inflamação/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , NF-kappa B/metabolismo , Nitratos/metabolismo , Oxirredução/efeitos dos fármacos , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Vitamina A/sangue
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