RESUMO
Chagas heart disease (CHD), caused by the protozoan parasite Trypanosoma cruzi, consists of a progressive myocarditis which may lead to congestive heart failure or sudden death. Previous work from our laboratory has demonstrated that the experimental infection of mice with T. cruzi positively modulates the expression of CD40 by myocardial cells, whose ligation potentiates IFN-γ-induced IL-6 production. Herein, we investigate the role of the CD40/CD40L interaction during T. cruzi infection using a CD40-targeted peptide and evaluating parasitological, histopathological and serological parameters. To reproduce acute and chronic phases of theT. cruzi infection, we used two experimental models: Balb/c mice infected with RA strain of T. cruzi (Balb/c-RA) and C3H/HeN mice infected with Sylvio X-10/4 parasites (C3H/HeN-Sylvio), respectively. Balb/c-RA treated with CD40-tageted peptide since day 0 post infection (pi), were unable to control the acute infection dying within 23-26 days pi with marked tissue damage. In contrast, treatment of C3H/HeN-Sylvio treated with CD40-targeted peptide starting on day 30 pi resulted in amelioration of myocardial and skeletal muscle damage. Altogether, our results indicate a dual role of CD40/CD40L dyad in the control of T.cruzi infection as well as the associated pathology, depending on the timing of treatment initiation.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Animais , Camundongos , Camundongos Endogâmicos C3H , Ligante de CD40 , Antígenos CD40 , Camundongos Endogâmicos BALB CRESUMO
Multiple perturbations of the immune response affecting a range of cells have been reported in Trypanosoma cruzi-infected individuals and associated to clinical manifestations of chronic Chagas disease. There is a paucity of knowledge about the role of T follicular helper (Tfh) cells in this infection. Here, we sought to characterize circulating Tfh (cTfh) cells in chronic Chagas disease patients and to identify potential associations with disease severity in humans. cTfh cells were characterized by flow cytometry in freshly isolated PBMCs from 7 T. cruzi-infected asymptomatic patients (ASYMP), 5 patients with chronic chagasic dilated cardiomyopathy (CCC) and 8 healthy controls, using antibodies against chemokine receptors CXCR5, CXCR3, CCR6, and CCR7. Our results showed significant expansion of CD4+CD45RO+CXCR5+CCR6+ cells in ASYMP and CCC patients, along with a contraction of CD4+CD45RO+CXCR5+CXCR3-CCR6- (cTfh2) cells. ASYMP patients further exhibited decreased CD4+CD45RO+CXCR5+CXCR3+CCR6- (cTfh1) cells and expanded CD4+CD45RO+CXCR5+CXCR3-CCR6+ (cTfh17) cells while CCC patients exhibited significantly increased frequencies of CD4+CD45RO+CXCR5+CCR7+ cells. Linear regression analysis revealed a positive trend of CD4+CD45RO+CXCR5+CXCR3+CCR6+ (cTfh1/17) cells and negative trends of cTfh1 and cTfh2 cells as disease was more severe. There was no correlation between the frequencies of cTfh cells and circulating CD19+IgD-IgG+ cells or serum levels of T. cruzi-specific IgG. These results demonstrate that the cTfh compartment of humans chronically infected with T. cruzi comprises expanded CCR6-expressing cells and reduced cTfh2 cells. The association of discrete phenotypic changes in cTfh subsets with different clinical forms suggests the potential contribution of T follicular helper cells to Chagas heart disease progression.
Assuntos
Doença de Chagas , Células T Auxiliares Foliculares , Linfócitos T CD4-Positivos , Humanos , Receptores CXCR5 , Linfócitos T Auxiliares-IndutoresRESUMO
The purpose of this study was to evaluate the efficacy of imiquimod-containing nanovesicles prepared with lipids extracted from the hyperhalophile archaebacterium Halorubrum tebenquichense (nanoARC-IMQ) to induce protection against Trypanosoma cruzi infection. The therapeutic efficacy of archaeolipid nanovesicles was assessed in an experimental murine model of acute infection with T. cruzi. The administration of nanoARQ-IMQ prevented mortality as compared to infected untreated animals, reduced parasitemia levels and diminished myocardial and musculoskeletal lesions in mice infected with a lethal strain of T. cruzi. Our findings suggest that the immunotherapy with nanoARC-IMQ has potential to limit the progression of Chagas disease.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Doença de Chagas/terapia , Imiquimode/uso terapêutico , Imunoterapia , Nanopartículas/uso terapêutico , Adjuvantes Imunológicos/química , Animais , Doença de Chagas/patologia , Imiquimode/química , Lipídeos/química , Masculino , Camundongos , Camundongos Endogâmicos C3H , Nanopartículas/química , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Leishmania parasites cause a broad spectrum of clinical manifestations in humans and the available clinical treatments are far from satisfactory. Since these pathogens require large amounts of NADPH to maintain intracellular redox homeostasis, oxidoreductases that catalyze the production of NADPH are considered as potential drug targets against these diseases. In the sequenced genomes of most Leishmania spp. two putative malic enzymes (MEs) with an identity of about 55% have been identified. In this work, the ME from L. major (LmjF24.0770, Lmj_ME-70) and its less similar homolog from L. mexicana (LmxM.24.0761, Lmex_ME-61) were cloned and functionally characterized. Both MEs specifically catalyzed NADPH production, but only Lmex_ME-61 was activated by l-aspartate. Unlike the allosterically activated human ME, Lmex_ME-61 exhibited typical hyperbolic curves without any sign of cooperativity in the absence of l-aspartate. Moreover, Lmex_ME-61 and Lmj_ME-70 differ from higher eukaryotic homologs in that they display dimeric instead of tetrameric molecular organization. Homology modeling analysis showed that Lmex_ME-61 and Lmj_ME-70 notably differ in their surface charge distribution; this feature encompasses the coenzyme binding pockets as well. However, in both isozymes, the residues directly involved in the coenzyme binding exhibited a good degree of conservation. Besides, only Lmex_ME-61 and its closest homologs were immunodetected in cell-free extracts from L. mexicana, L. amazonensis and L. braziliensis promastigotes. Our findings provide a first glimpse into the biochemical properties of leishmanial MEs and suggest that MEs could be potentially related to the metabolic differences among the species of Leishmania parasites.
Assuntos
Leishmania major/enzimologia , Leishmania mexicana/enzimologia , Malato Desidrogenase (NADP+)/genética , Malato Desidrogenase (NADP+)/metabolismo , Ácido Aspártico/metabolismo , Sítios de Ligação , Clonagem Molecular , Coenzimas/metabolismo , Biologia Computacional , Expressão Gênica , Leishmania major/genética , Leishmania mexicana/genética , Malato Desidrogenase (NADP+)/química , Modelos Moleculares , NADP/metabolismo , Multimerização ProteicaRESUMO
BACKGROUND: Leishmaniasis and Chagas disease are life-threatening illnesses caused by the protozoan parasites Leishmania spp. and Trypanosoma cruzi, respectively. They are known as "neglected diseases" due to the lack of effective drug treatments and the scarcity of research work devoted to them. Therefore, the development of novel and effective drugs is an important and urgent need. Natural products are an important source of bioactive molecules for the development of new drugs. In this study, we evaluated the activity of enhydrin, uvedalin and polymatin B, three sesquiterpene lactones (STLs) isolated from Smallanthus sonchifolius, on Leishmania mexicana (MNYC/BZ/62/M) and Trypanosoma cruzi (Dm28c). In addition, the in vivo trypanocidal activity of enhydrin and uvedalin and the effects of these STLs on parasites' ultrastructure were evaluated. METHODS: The inhibitory effect of the three STLs on the growth of L. mexicana amastigotes and promastigotes as well as T. cruzi epimastigotes was evaluated in vitro. The changes produced by the STLs on the ultrastructure of parasites were examined by transmission electron microscopy (TEM). Enhydrin and uvedalin were also studied in a murine model of acute T. cruzi infection (RA strain). Serum activities of the hepatic enzymes alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase were used as biochemical markers of hepatotoxicity. RESULTS: The three compounds exhibited leishmanicidal activity on both parasite forms with IC50 values of 0.42-0.54 µg/ml for promastigotes and 0.85-1.64 µg/ml for intracellular amastigotes. Similar results were observed on T. cruzi epimastigotes (IC50 0.35-0.60 µg/ml). The TEM evaluation showed marked ultrastructural alterations, such as an intense vacuolization and mitochondrial swelling in both L. mexicana promastigotes and T. cruzi epimastigotes exposed to the STLs. In the in vivo study, enhydrin and uvedalin displayed a significant decrease in circulating parasites (50-71%) and no signs of hepatotoxicity were detected. CONCLUSIONS: Enhydrin, uvedalin and polymatin B possess significant leishmanicidal and trypanocidal activity on different parasite stages. These results show that these compounds may provide valuable leads for the development of new drugs against these neglected parasitic diseases.
Assuntos
Lactonas/farmacologia , Leishmania mexicana/efeitos dos fármacos , Sesquiterpenos de Germacrano/química , Sesquiterpenos/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Asteraceae/química , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Modelos Animais de Doenças , Lactonas/administração & dosagem , Lactonas/efeitos adversos , Lactonas/uso terapêutico , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/ultraestrutura , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Fígado/efeitos dos fármacos , Camundongos , Microscopia Eletrônica de Transmissão , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Sesquiterpenos/administração & dosagem , Sesquiterpenos/efeitos adversos , Sesquiterpenos/química , Sesquiterpenos/uso terapêutico , Sesquiterpenos de Germacrano/farmacologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/ultraestruturaRESUMO
The inflammatory response in the myocardium is an important aspect of the pathogenesis of Chagas' heart disease raised by Trypanosoma cruzi. CD40, a transmembrane type I receptor belonging to the tumor necrosis factor receptor (TNFR) family, is expressed in a broad spectrum of cell types and is crucial in several inflammatory and autoimmune diseases. Activation of CD40 through ligation to CD40L (CD154) induces multiple effects, including the secretion of proinflammatory molecules. In the present study, we examined the ability of T. cruzi to trigger the expression of CD40 in cardiac myocytes in vitro and in a murine model of chagasic cardiomyopathy. Our results indicate, for the first time, that T. cruzi is able to induce the expression of CD40 in HL-1 murine cardiomyocytes. Moreover, ligation of CD40 receptor upregulated interleukin-6 (IL-6), associated with inflammation. Furthermore, the induction of this costimulatory molecule was demonstrated in vivo in myocardium of mice infected with T. cruzi. This suggests that CD40-bearing cardiac muscle cells could interact with CD40L-expressing lymphocytes infiltrating the heart, thus contributing to inflammatory injury in chagasic cardiomyopathy.
Assuntos
Antígenos CD40/metabolismo , Cardiomiopatia Chagásica/parasitologia , Interleucina-6/metabolismo , Miócitos Cardíacos/imunologia , Trypanosoma cruzi/fisiologia , Animais , Antígenos CD40/genética , Células Cultivadas , Cardiomiopatia Chagásica/patologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Interferon gama/farmacologia , Camundongos , Camundongos Endogâmicos C3H , Miocárdio/patologia , Miócitos Cardíacos/parasitologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Trypanosoma cruzi/imunologiaRESUMO
In this work, the in vitro anti-Leishmania activity of photodynamic liposomes made of soybean phosphatidylcholine, sodium cholate, total polar archaeolipids (TPAs) extracted from the hyperhalophile archaea Halorubrum tebenquichense and the photosensitizer zinc phthalocyanine (ZnPcAL) was compared to that of ultradeformable photodynamic liposomes lacking TPAs (ZnPcUDLs). We found that while ZnPcUDLs and ZnPcALs (130 nm mean diameter and -35 mV zeta potential) were innocuous against promastigotes, a low concentration (0.01 µM ZnPc and 7.6 µM phospholipids) of ZnPcALs irradiated at a very low-energy density (0.2 J/cm(2)) eliminated L. braziliensis amastigotes from J774 macrophages, without reducing the viability of the host cells. In such conditions, ZnPcALs were harmless for J774 macrophages, HaCaT keratinocytes, and bone marrow-derived dendritic cells. Therefore, topical photodynamic treatment would not likely affect skin-associated lymphoid tissue. ZnPcALs were extensively captured by macrophages, but ZnPcUDLs were not, leading to 2.5-fold increased intracellular delivery of ZnPc than with ZnPcUDLs. Despite mediating low levels of reactive oxygen species, the higher delivery of ZnPc and the multiple (caveolin- and clathrin-dependent plus phagocytic) intracellular pathway followed by ZnPc would have been the reason for the higher antiamastigote activity of ZnPcALs. The leishmanicidal activity of photodynamic liposomal ZnPc was improved by TPA-containing liposomes.
Assuntos
Antiprotozoários/farmacologia , Éteres de Glicerila/farmacologia , Indóis/farmacologia , Leishmania/efeitos dos fármacos , Leishmania/efeitos da radiação , Lipossomos/farmacologia , Compostos Organometálicos/farmacologia , Animais , Antiprotozoários/química , Antiprotozoários/farmacocinética , Antiprotozoários/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Éteres de Glicerila/química , Éteres de Glicerila/farmacocinética , Éteres de Glicerila/toxicidade , Humanos , Indóis/química , Indóis/farmacocinética , Indóis/toxicidade , Isoindóis , Lipossomos/química , Lipossomos/farmacocinética , Lipossomos/toxicidade , Macrófagos/metabolismo , Camundongos , Compostos Organometálicos/química , Compostos Organometálicos/farmacocinética , Compostos Organometálicos/toxicidade , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Compostos de ZincoRESUMO
Clinical symptoms of chronic Chagas disease occur in around 30% of the individuals infected with Trypanosoma cruzi and are characterized by heart inflammation and dysfunction. The pathogenesis of chronic chagasic cardiomyopathy (CCC) is not completely understood yet, partially because disease evolution depends on complex host-parasite interactions. Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine that promotes numerous pathophysiological processes. In the current study, we investigated the link between MIF and CCC progression.Immunohistochemical analysis demonstrated MIF overexpression in the hearts from chronically T. cruzi-infected mice, particularly those showing intense inflammatory infiltration. We also found that MIF exogenously added to parasite-infected murine macrophage cultures is capable of enhancing the production of TNF-α and reactive oxygen species, both with pathogenic roles in CCC. Thus, the integrated action of MIF and other cytokines and chemokines may account for leukocyte influx to the infected myocardium, accompanied by enhanced local production of multiple inflammatory mediators. We further examined by ELISA the level of MIF in the sera from chronic indeterminate and cardiomyopathic chagasic patients, and healthy subjects. CCC patients displayed significantly higher MIF concentrations than those recorded in asymptomatic T. cruzi-infected and uninfected individuals. Interestingly, increased MIF levels were associated with severe progressive Chagas heart disease, in correlation with elevated serum concentration of high sensitivity C-reactive protein and also with several echocardiographic indicators of left ventricular dysfunction, one of the hallmarks of CCC. Our present findings represent the first evidence that enhanced MIF production is associated with progressive cardiac impairment in chronic human infection with T. cruzi, strengthening the relationship between inflammatory response and parasite-driven pathology. These observations contribute to unravel the elements involved in the pathogenesis of CCC and may also be helpful for the design of novel therapies aimed to control long-term morbidity in chagasic patients.
Assuntos
Cardiomiopatias/sangue , Doença de Chagas/sangue , Fatores Inibidores da Migração de Macrófagos/sangue , Animais , Cardiomiopatias/complicações , Cardiomiopatias/patologia , Linhagem Celular , Doença de Chagas/complicações , Doença Crônica , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Archaeosomes (ARC), vesicles made from lipids extracted from Archaea, display strong adjuvant properties. In this study, we evaluated the ability of the highly stable ARC formulated from total polar lipids of a new Halorubrum tebenquichense strain found in Argentinean Patagonia, to act as adjuvant for soluble parasite antigens in developing prophylactic vaccine against the intracellular protozoan T. cruzi, the etiologic agent of Chagas disease. We demonstrated for the first time that C3H/HeN mice subcutaneously immunized with trypanosomal antigens entrapped in these ARC (ARC-TcAg) rapidly developed higher levels of circulating T. cruzi antibodies than those measured in the sera from animals receiving the antigen alone. Enhanced humoral responses elicited by ARC-TcAg presented a dominant IgG2a antibody isotype, usually associated with Th1-type immunity and resistance against T. cruzi. More importantly, ARC-TcAg-vaccinated mice displayed reduced parasitemia during early infection and were protected against an otherwise lethal challenge with the virulent Tulahuén strain of the parasite. Our findings suggest that, as an adjuvant, H. tebenquichense-derived ARC may hold great potential to develop a safe and helpful vaccine against this relevant human pathogen.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Doença de Chagas/prevenção & controle , Halorubrum/química , Lipossomos/administração & dosagem , Lipídeos de Membrana/administração & dosagem , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/imunologia , Adjuvantes Imunológicos/isolamento & purificação , Animais , Anticorpos Antiprotozoários/sangue , Argentina , Doença de Chagas/imunologia , Modelos Animais de Doenças , Feminino , Imunoglobulina G/sangue , Injeções Subcutâneas , Lipossomos/isolamento & purificação , Lipídeos de Membrana/isolamento & purificação , Camundongos Endogâmicos C3H , Parasitemia/imunologia , Parasitemia/prevenção & controle , Análise de Sobrevida , Células Th1/imunologia , Resultado do TratamentoRESUMO
OBJECTIVE: The presence of autoantibodies with adrenergic and cholinergic activity, capable of triggering neurotransmitter receptor-mediated effects, has been associated with pathogenesis in T. cruzi-infected hosts. The goal of this study was to investigate the production of anti-M2 muscarinic receptor autoantibodies (Anti-M2R AAbs) as well as the IFN-γ profile in children at the early stage of Chagas disease, and to examine whether trypanocidal chemotherapy with benzonidazole (BZ) could modify both response patterns. METHODS: This study comprised 30 T. cruzi-infected children (mean age: 13.8 years) and 19 uninfected controls (mean age: 12.7 years). Infected patients were treated with BZ and followed-up. Blood samples collected at diagnosis-T0, end of treatment-T1, and six months later-T2 were analysed by ELISA for detection of Anti-M2R AAbs and circulating levels of IFN-γ. RESULTS: At T0, anti-M2R AAbs were demonstrated in 56.7% of T. cruzi-infected patients, whereas uninfected controls were 100% negative. The average age of Anti-M2R AAbs(+) patients was higher than that from negative population. Infected children also displayed significantly stronger serum IFN-γ responses than controls. Upon BZ treatment, a significant linear decreasing trend in Anti-M2R AAb reactivity was recorded throughout the follow-up, with 29.7-88.1% decrease at T2. IFN-γ circulating levels also declined by T2. CONCLUSION: Anti-M2R AAbs and IFN-γ raise early during chagasic infection in children and are downmodulated by BZ therapy. These findings reinforce the usefulness of early BZ treatment not only to eliminate the parasite but also to reduce potentially pathogenic immune responses.