Importance of CD40/CD40 dyad in the course of infection with Trypanosoma cruzi: Impact of its inhibition.

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.

Circulating T Follicular Helper Cell Abnormalities Associated to Different Clinical Forms of Chronic Chagas Disease.

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.

Imiquimod-loaded nanoarchaeosomes as a promising immunotherapy against Trypanosoma cruzi infection.

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.

Germacranolide-type sesquiterpene lactones from Smallanthus sonchifolius with promising activity against Leishmania mexicana and Trypanosoma cruzi.

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.

MIF-driven activation of macrophages induces killing of intracellular Trypanosoma cruzi dependent on endogenous production of tumor necrosis factor, nitric oxide and reactive oxygen species.

The proinflammatory cytokine macrophage migration inhibitory factor (MIF) is a key player in innate immunity. MIF has been considered critical for controlling acute infection by the protozoan Trypanosoma cruzi, but the underlying mechanisms are poorly understood. Our study aimed to analyze whether MIF could favor microbicidal activity of the macrophage, a site where T. cruzi grows and the initial effector cell against this parasite. Using murine macrophages infected in vitro, we examined the effect of MIF on their parasiticidal ability and attempted to identify inflammatory agents involved in MIF-induced protection. Our findings show that MIF is readily secreted from peritoneal macrophages upon T. cruzi infection. MIF activates both primary and J774 phagocytes boosting the endogenous production of tumor necrosis factor-alpha via mitogen-activated protein kinase p38 signaling, as well as the release of nitric oxide and reactive oxygen species, leading to enhanced pathogen elimination. MIF can also potentiate the effect of interferon-gamma on T. cruzi killing by J774 and mouse peritoneal macrophages, rendering these cells more competent in reducing intracellular parasite burden. The present results unveil a novel innate immune pathway that contributes to host defense and broaden our understanding of the regulation of inflammatory mediators implicated in early parasite containment that is decisive for resistance to T. cruzi infection.

Elevated serum levels of macrophage migration inhibitory factor are associated with progressive chronic cardiomyopathy in patients with Chagas disease.

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.

Archaeosomes display immunoadjuvant potential for a vaccine against Chagas disease.

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.

Benzonidazole therapy modulates interferon-γ and M2 muscarinic receptor autoantibody responses in Trypanosoma cruzi-infected children.

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.

Archaeosomes made of Halorubrum tebenquichense total polar lipids: a new source of adjuvancy.

BACKGROUND: Archaeosomes (ARC), vesicles prepared from total polar lipids (TPL) extracted from selected genera and species from the Archaea domain, elicit both antibody and cell-mediated immunity to the entrapped antigen, as well as efficient cross priming of exogenous antigens, evoking a profound memory response. Screening for unexplored Archaea genus as new sources of adjuvancy, here we report the presence of two new Halorubrum tebenquichense strains isolated from grey crystals (GC) and black mood (BM) strata from a littoral Argentinean Patagonia salt flat. Cytotoxicity, intracellular transit and immune response induced by two subcutaneous (sc) administrations (days 0 and 21) with BSA entrapped in ARC made of TPL either form BM (ARC-BM) and from GC (ARC-GC) at 2% w/w (BSA/lipids), to C3H/HeN mice (25 microg BSA, 1.3 mg of archaeal lipids per mouse) and boosted on day 180 with 25 microg of bare BSA, were determined. RESULTS: DNA G+C content (59.5 and 61.7% mol BM and GC, respectively), 16S rDNA sequentiation, DNA-DNA hybridization, arbitrarily primed fingerprint assay and biochemical data confirmed that BM and GC isolates were two non-previously described strains of H. tebenquichense. Both multilamellar ARC mean size were 564 +/- 22 nm, with -50 mV zeta-potential, and were not cytotoxic on Vero cells up to 1 mg/ml and up to 0.1 mg/ml of lipids on J-774 macrophages (XTT method). ARC inner aqueous content remained inside the phago-lysosomal system of J-774 cells beyond the first incubation hour at 37 degrees C, as revealed by pyranine loaded in ARC. Upon subcutaneous immunization of C3H/HeN mice, BSA entrapped in ARC-BM or ARC-GC elicited a strong and sustained primary antibody response, as well as improved specific humoral immunity after boosting with the bare antigen. Both IgG1 and IgG2a enhanced antibody titers could be demonstrated in long-term (200 days) recall suggesting induction of a mixed Th1/Th2 response. CONCLUSION: We herein report the finding of new H. tebenquichense non alkaliphilic strains in Argentinean Patagonia together with the adjuvant properties of ARC after sc administration in mice. Our results indicate that archaeosomes prepared with TPL from these two strains could be successfully used as vaccine delivery vehicles.

In vitro activity of Etanidazole against the protozoan parasite Trypanosoma cruzi.

We investigated the in vitro action of an hydrosoluble 2-nitroimidazole, Etanidazole (EZL), against Trypanosoma cruzi, the etiologic agent of Chagas disease. EZL displayed lethal activity against isolated trypomastigotes as well as amastigotes of T. cruzi (RA strain) growing in Vero cells or J774 macrophages, without affecting host cell viability. Although not completely equivalent to Benznidazole (BZL), the reference drug for Chagas chemotherapy, EZL takes advantage in exerting its anti-T. cruzi activity for longer periods without serious toxic side effects, as those recorded in BZL-treated patients. Our present results encourage further experiments to study in depth the trypanocidal properties of this drug already licensed for use in human cancers.

In vitro activity of Etanidazole against the protozoan parasite Trypanosoma cruzi

We investigated the in vitro action of an hydrosoluble 2-nitroimidazole, Etanidazole (EZL), against Trypanosoma cruzi, the etiologic agent of Chagas disease. EZL displayed lethal activity against isolated trypomastigotes as well as amastigotes of T. cruzi (RA strain) growing in Vero cells or J774 macrophages, without affecting host cell viability. Although not completely equivalent to Benznidazole (BZL), the reference drug for Chagas chemotherapy, EZL takes advantage in exertingits anti-T. cruzi activity for longer periods without serious toxic side effects, as those recorded in BZL-treated patients. Our present results encourage further experiments to study in depth the trypanocidal properties of this drug already licensed for use in human cancers.

Use of a purified Trypanosoma cruzi antigen and CpG oligodeoxynucleotides for immunoprotection against a lethal challenge with trypomastigotes.

The crucial role played by Ag163B6/cruzipain, the major cystein proteinase of Trypanosoma cruzi, in the process of parasite internalization into mammalian cells and IgG hydrolysis, signals this antigen as a potential target for raising a protective immune response against Chagas' disease. On the other hand, synthetic oligodeoxynucleotides containing CpG-motifs (CpG-ODN) are capable of driving immunity toward a Th1 bias. Considering the importance of Th1 mechanisms in resistance against this intracellular parasite, we analyzed the ability of Ag163B6/cruzipain plus CpG-ODN to induce immunoprotection against a lethal challenge with trypomastigotes. Mice were immunized with Ag163B6+CpG-ODN showing high specific antibody titers, mostly IgG2a. Spleen cells from these mice strongly proliferated and presented significant increase of IL-2 and IFN-gamma concentrations in their supernatant upon antigen stimulation. Trypomastigote challenge rendered elevated parasitemia and mortality in all control groups, meanwhile Ag163B6+CpG-ODN mice displayed the lowest level of blood parasites and 100% survival to acute infection. Besides, we demonstrated that other parasite antigens introduced into mice when challenged, and consequently never seen before by the immune system, also elicited a Th1 immune response. Taken together, these results plus others provide the basis for the design of a multicomponent anti-T. cruzi vaccine which may ultimately be used not only to protect humans at risk of infection, but also may alleviate or prevent the pathogenic responses characteristic of chronic Chagas' disease by reducing or perhaps eliminating tissue parasites from infected patients.