The Bisindole Alkaloid Caulerpin, from Seaweeds of the Genus Caulerpa, Attenuated Colon Damage in Murine Colitis Model.

Caulerpin (CLP), an alkaloid from algae of the genus Caulerpa, has shown anti-inflammatory activity. Therefore, this study aimed to analyze the effect of CLP in the murine model of peritonitis and ulcerative colitis. Firstly, the mice were submitted to peritonitis to evaluate which dose of CLP (40, 4, or 0.4 mg/kg) could decrease the inflammatory infiltration in the peritoneum. The most effective doses were 40 and 4 mg/kg. Then, C57BL/6 mice were submitted to colitis development with 3% dextran sulfate sodium (DSS) and treated with CLP at doses of 40 and 4 mg/kg. The disease development was analyzed through the disease activity index (DAI); furthermore, colonic tissue samples were submitted to histological analysis, NFκB determination, and in vitro culture for cytokines assay. Therefore, CLP at 4 mg/kg presented the best results, triggering improvement of DAI and attenuating the colon shortening and damage. This dose was able to reduce the TNF-α, IFN-γ, IL-6, IL-17, and NFκB p65 levels, and increased the levels of IL-10 in the colon tissue. Thus, CLP mice treatment at a dose of 4 mg/kg showed promising results in ameliorating the damage observed in the ulcerative colitis.

Apoptosis in human liver carcinoma caused by gold nanoparticles in combination with carvedilol is mediated via modulation of MAPK/Akt/mTOR pathway and EGFR/FAAD proteins.

In cancers, apoptosis signaling pathways and cell survival and growth pathways responsible for resistance to conventional treatments, such as Pi3K/Akt/mTOR and mitogen-activated protein kinase (MAPK) become dysregulated. Recently, alternative treatments to promote tumor cell death have become important. The present study reports on the antitumor and cytoprotective action of gold nanoparticles (GNPs) and carvedilol in combination and in isolated application. Apoptosis was analyzed by FITC/propidium iodide staining flow cytometry; caspase-3, caspase-8, Bcl-2 and MAPK/ERK activity by immunofluorescence microscopy; gene expression of proteins related to cell death as Akt, mTOR, EGFR, MDR1, survivin, FADD and Apaf, by the real-time PCR; and western blot analysis for MAPK/ERK, Akt and mTOR. Oxidative stress evaluation was performed by reduced glutathione (GSH) and malondialdehyde (MDA) levels. Intracellular GNPs targets were identified by transmission electron microscopy. After exposure to a combination of GNPs (6.25 µg/ml) and carvedilol (3 µM), death as promoted by apoptosis was detected using flow cytometry, for expression of pro-apoptotic proteins FADD, caspase-3, caspase-8 and sub-regulation of anti-apoptotic MAPK/ERK, Akt, mTOR, EGFR and MDR1 resistance. Non-tumor cell cytoprotection with GSH elevation and MDA reduction levels was detected. GNPs were identified within the cell near to the nucleus when combined with carvedilol. The combination of GNP and carvedilol promoted downregulation of anti-apoptotic and drug resistance genes, over-regulation of pro-apoptotic proteins in tumor cells, as well as cytoprotection of non-tumor cells with reduction of apoptosis and oxidative stress.

The use of a heterogeneously controlled mouse population reveals a significant correlation of acute phase parasitemia with mortality in Chagas disease.

Chagas disease develops upon infection with the protozoan parasite Trypanosoma cruzi and undergoes an acute phase characterized by massive parasite replication and the presence of parasites in the blood. This condition is known as acute phase parasitemia. This initial stage may result in a cure, in the development of the chronic stages of the disease or in the death of the infected host. Despite intensive investigation related to the characterization of the acute and chronic phases of the disease, the cause-effect relationship of acute phase parasitemia to the outcome of the disease is still poorly understood. In this study, we artificially generated a heterogeneously controlled mouse population by intercrossing F1 mice obtained from a parental breeding of highly susceptible A/J with highly resistant C57BL/6 mouse strains. This F2 population was infected and used to assess the correlation of acute phase parasitemia with the longevity of the animals. We used nonparametric statistical analyses and found a significant association between parasitemia and mortality. If males and females were evaluated separately, we found that the former were more susceptible to death, although parasitemia was similar in males and females. In females, we found a strong negative correlation between parasitemia and longevity. In males, however, additional factors independent of parasitemia may favor mouse mortality during the development of the disease. The correlations of acute phase parasitemia with mortality reported in this study may facilitate an appropriate prognostic approach to the disease in humans. Moreover, these results illustrate the complexity of the mammalian genetic traits that regulate host resistance during Chagas disease.

Regulation of the immune response during infectious myocarditis.

Infectious myocarditis (IM) is a commonly undiagnosed condition that may cause several heart diseases, including dilated cardiomyopathy and chronic heart failure. The understanding of the physiopathology of myocardial inflammation is crucial for a timely diagnosis and for the control of the tissue damage, which may occur in some cases of IM. Of note, some experimental studies suggest that dilated cardiomyopathy could be a consequence of untreated IM. However, further research is required to address the molecular mechanisms that may link these two clinical entities. Here we review the mechanisms involved in the regulation at different levels of the immune response during IM, with a special focus on diagnostic and therapeutic perspectives of molecules that have been linked to the development of IM and the resulting chronic heart diseases.

Haeme oxygenase activity protects the host against excessive cardiac inflammation during experimental Trypanosoma cruzi infection.

The infection with Trypanosoma cruzi induces a robust cardiac inflammation that plays a pathogenic role in the development of Chagas heart disease. In this study, we aimed at investigating the effects of Haem Oxygenase (HO) during experimental infection by T. cruzi in BALB/c and C57BL/6 mice. HO has recently emerged as a key factor modulating the immune response in diverse models of inflammatory diseases. In mice with two different genetic backgrounds, the pharmacologic inhibition of HO activity with zinc-protoporphyrin IX (ZnPPIX) induced enhanced myocarditis and reduced parasitaemia, which was accompanied by an amplified production of nitric oxide and increased influx of CD4(+), CD8(+) and IFN-γ(+) cells to the myocardium in comparison with the control group. Conversely, treatment with haemin (an activator of HO) lead to a decreased number of intracardiac CD4(+) (but not CD8(+)) cells compared to the control group. The mechanism involved in these observations is a modulation of the induction of regulatory T cells, because the stimulation or inhibition of HO was parallelled by an enhanced or reduced frequency of regulatory T cells, respectively. Hence, HO may be involved in the regulation of heart tissue inflammation and could be a potential target in conceiving future therapeutic approaches for Chagas disease.

Naturally Leishmania infantum-infected dogs display an overall impairment of chemokine and chemokine receptor expression during visceral leishmaniasis.

Dogs are the primary reservoir for Leishmania parasites. The immune response induced by Leishmania infantum infection in these animals has not been completely elucidated, and few studies have investigated the relationship between the expression levels of chemokines and chemokine receptors and the clinical status of dogs with canine visceral leishmaniasis (CVL). The aim of this study was to correlate the clinical status of naturally L. infantum-infected dogs (from rural areas of Mossoró city, State of Rio Grande do Norte, Brazil) with the expression levels of chemokines (ccl1, ccl2, ccl3, ccl4, ccl5, ccl17, ccl20, ccl24, ccl26, cxcl9, cxcl10) and chemokine receptors (cxcr3, ccr3, ccr4, ccr5, ccr6, ccr8) in the liver and spleen determined using real-time PCR. Twenty-one dogs were clinically evaluated and classified as asymptomatic (n=11) or symptomatic (n=10). Splenomegaly, weight loss and onychogryphosis were the most pronounced symptoms. In the liver, the mRNA expression levels of ccl1, ccl17, ccl26, ccr3, ccr4, ccr5, ccr6, and ccr8 were lower in symptomatic animals than in asymptomatic animals. Compared with uninfected animals, symptomatic dogs had lower expression levels of almost all molecules analyzed. Moreover, high clinical scores were negatively correlated with ccr5 and ccr6 expression and positively correlated with cxcl10 expression. We conclude that the impairment of the expression of chemokines and chemokine receptors results in deficient leukocyte migration and hampers the immune response, leading to the development of disease.

Protein deficiency alters CX3CL1 and endothelin-1 in experimental Trypanosoma cruzi-induced cardiomyopathy.

OBJECTIVE: Chagas heart disease is developed as a result of the infection with Trypanosoma cruzi. Protein malnutrition contributes to secondary immunodeficiency. The aim of this study was to investigate the role of a low protein diet on the production of endothelin-1 and CX3CL1 in blood and cardiac tissue samples in an experimental model with T. cruzi infection. METHODS: Fisher rats were submitted to low protein (6%) and normal protein (15%) diets and then infected with the Y strain of T. cruzi. At days 15 and 120, parasites and immune cells were evaluated. RESULTS: The low protein diet reduced body weight and circulating serum proteins, but promoted elevation of CX3CL1 and endothelin-1 levels in infected animals, which were unable to control blood parasitemia replication. In heart tissue, the low protein diet reduced cardiac CX3CL1, endothelin-1 and leucocyte infiltration in the acute phase, in particular CD68 and CD163 macrophage phenotypes. CONCLUSION: Together, these results highlight the participation of endothelin-1 and CX3CL1 in the inflammatory process of Chagas diesease, both being mediators partially controlled by the host nutritional status.

Caracterização clínica e laboratorial de cães naturalmente infectados com Trypanosoma cruzi no semiárido nordestino / Clinical and laboratorial characterization of naturally infected Trypanosoma cruzi dogs in the northeastern semi-arid

Objetivou-se, com este estudo, evidenciar os sinais clínicos e laboratoriais desta enfermidade para auxiliar na caracterização da doença de forma natural na área semi-árida da região nordeste. Foram avaliados 10 cães positivos para Trypanosoma cruzi, identificados mediante análises sorológicas de reação de imunofluorescência indireta (RIFI) e enzyme linked immunosorbent assay (ELISA); análise molecular pela Reação em Cadeia Polimerase (PCR), microscopia direta e hemocultura. Os cães chagásicos foram submetidos à avaliação física, verificação da pressão arterial, exames eletrocardiográficos, radiográficos, hematológicos (eritrograma e leucograma) e bioquímicos (ureia, creatinina, ALT, AST, PT, albumina, globulina, CK, CK-MB e cTnI). O exame físico e os valores das pressões arteriais dos cães apresentaram dentro dos parâmetros de normalidade, enquanto que na eletrocardiografia observou-se FC normal com ritmo sinusal, com exceção de um cão, que apresentou taquicardia sinusal (168 bat/min). No ECG de oito cães houve aumento da duração de P (47±6,5ms) sugestivo de aumento atrial, não confirmado radiograficamente. Foi observado supradesnivelamento do segmento ST em um cão. Nos resultados hematológicos constatou-se trombocitopenia (187,4x10³ ±137,2x10³) e anemia (5,0x10(6) ±1,39x10(6)/uL). Os valores médios da hemoglobina (11±2,7g/dL) e do hematócrito (34±10,5%) estavam abaixo dos limites de normalidade. A série branca apresentou-se dentro dos limites de normalidade, com exceção da eosinofilia observada em três cães. Individualmente, registrou-se em dois cães, leucocitose, linfocitose e neutrofilia. Na avaliação bioquímica, registrou-se hiperproteinemia (7,2±0,9g/dL), hipoalbuminemia (2,2±0,4g/dL), hiperglobulinemia (5,1±1,0g/dL) e aumento da CK (196±171U/L). Não houve alteração nas enzimas ALT e AST. A isoenzima CK-MB e o cTnI alteraram somente em três cães. Os cães infectados naturalmente no semiárido nordestino apresentam características relacionáveis à forma crônica indeterminada, ou seja, cães assintomáticos. A identificação dos cães infectados naturalmente sem características patognomônicas da doença de Chagas ressalta a importância desta enfermidade no processo diagnóstico com as demais que manifestam perfis inespecíficos associados ou não às doenças cardiovasculares.

This study aimed to evidence the clinical and laboratorial signs of this disease to help characterize this illness in a natural way in the semiarid in the northeastern region. We evaluated 10 positive for Trypanosoma cruzi dogs, that were identified by serological analysis of immunofluorescence assay (RIFI) and enzyme linked immunosorbent assay (ELISA); molecular analysis by polymerase chain reaction (PCR), direct microscopy and blood culture. The chagasic dogs underwent physical examination, electrocardiographic, radiographic, blood pressure, hematology (erythrocyte and leukocyte count) and biochemical exams (urea, creatinine, ALT, AST, PT, albumin, globulin, CK, CK-MB, and cTnl). The physical examination and the blood pressure were presented within the normal range, while in the electrocardiography the FC was observed as normal with a sinus rhythm, with the exception of one dog that presented a sinus tachycardia (168 bat/min). In the ECG of eight dogs there was increase of duration of P (47±6.5ms) suggestive to atrial enlargement, not confirmed in the radiography. A supraunlevelling was observed in the ST segment in one dog. In the hematological results, thrombocytopenia (187.4x10³ ±137.2x10³) and anemia (5.0x10(6) ±1.39x10(6)/ul) were noted. The mean hemoglobin (11 ±2.7g/dL), hematocrit (34±10.5%) were below normal limits. The white series were within normal variation, with the exception of eosinophilia observed in three dogs. Individually, there were two dogs which registered leukocytosis, lymphocytosis and neutrophilia. In the biochemical evaluation there was hyperproteinemia PT=7.2 ±0.9g/dL, hypoalbuminemia (2.2±0.4g/dL), hyperglobulinemia (5.1±1.0g/dL), increased of CK (196+171 U/L) and there was no alteration on ALT and AST enzymes. The CK-MB isoenzymes and cTnI did not change, except in three dogs. We conclude that dogs naturally infected in the northeastern semiarid present characteristics related to indeterminate chronic form (asymptomatic dogs) and that the identification of the naturally infected dogs with no pathognomonic characteristics of the Chagas disease underscores the importance of this illness in the diagnostic process with the other profiles that show nonspecific or not associated to cardiovascular disease.

Current status of Chagas disease chemotherapy.

Chagas disease affects 7.7 million people and 28 million people are at risk of acquiring the disease in 15 endemic countries of Latin America. Benznidazole and nifurtimox are drugs that have been used to treat the disease. However, both drugs induce severe side effects. Treatment with benznidazole has been recommended for the acute phase (0-4 months after infection), recent chronic phase (children 0-14 years of age, treated 4 months after infection) and congenital infection. Average cure rates for Chagas disease patients obtained from clinical trials were 97.9% (congenital infection, treatment performed 0-6 months of age), 71.5% (acute phase), 57.6% (recent chronic phase, children 0-13 years of age) and 5.9% (late chronic phase, great majority of patients between 15 and 69 years of age). Clinical evidence about the capacity of antiparasitic treatment to avoid, stop or revert heart pathology in indeterminate and cardiac chronic patients is contradictory. The investigation of novel therapeutic strategies against Chagas disease remains a priority in the research of tropical diseases. Unfortunately, Chagas disease remains neglected in the formulation of strategies toward control of this disease. This article focuses on current therapeutic approaches to Chagas disease.

Regulation of Trypanosoma cruzi-induced myocarditis by programmed death cell receptor 1.

Trypanosoma cruzi infection causes intense myocarditis, leading to cardiomyopathy and severe cardiac dysfunction. Protective adaptive immunity depends on balanced signaling through a T cell receptor and coreceptors expressed on the T cell surface. Such coreceptors can trigger stimulatory or inhibitory signals after binding to their ligands in antigen-presenting cells (APC). T. cruzi modulates the expression of coreceptors in lymphocytes after infection. Deregulated inflammation may be due to unbalanced expression of these molecules. Programmed death cell receptor 1 (PD-1) is a negative T cell coreceptor that has been associated with T cell anergy or exhaustion and persistent intracellular infections. We aimed to study the role of PD-1 during T. cruzi-induced acute myocarditis in mice. Cytometry assays showed that PD-1 and its ligands are strongly upregulated in lymphocytes and APC in response to T. cruzi infection in vivo and in vitro. Lymphocytes infiltrating the myocardium exhibited high levels of expression of these molecules. An increased cardiac inflammatory response was found in mice treated with blocking antibodies against PD-1, PD-L1, and to a lesser extent, PD-L2, compared to that found in mice treated with rat IgG. Similar results in PD-1(-/-) mice were obtained. Moreover, the PD-1 blockade/deficiency led to reduced parasitemia and tissue parasitism but increased mortality. These results suggest the participation of a PD-1 signaling pathway in the control of acute myocarditis induced by T. cruzi and provide additional insight into the regulatory mechanisms in the pathogenesis of Chagas' disease.

Increased type 1 chemokine expression in experimental Chagas disease correlates with cardiac pathology in beagle dogs.

Chemokines and chemokine receptors interaction have presented important role in leukocyte migration to specific immune reaction sites. Recently, it has been reported that chemokine receptors CXC (CXCR3) and CC (CCR5) were preferentially expressed on Th1 cells while CCR3 and CCR4 were preferentially expressed on Th2 cells. This study evaluated the mRNA expression of type 1 and type 2 chemokine and chemokine receptors in the cardiac tissue of Beagle dogs infected with distinct genetic groups of Trypanosoma cruzi (Y, Berenice-78 and ABC strains) during acute and chronic phases. To analyze the correlation between chemokine and chemokine receptors expression and the development of heart pathology, the chronic infected animals were divided into groups, according to the parasite strain and based on the degree of heart damage: cardiac and indeterminate form of Chagas disease. Our results indicated that cardiac type1/2 chemokines and their receptors were partially dependent on the genetic diversity of parasites as well as the polarization of clinical forms. Also, dogs presenting cardiac form showed lower heart tissue mRNA expression of CCL24 (type 2) and higher expression of CCL5, CCL4 and CXCR3 (type 1) when compared with those with indeterminate form of disease. Together, these data reinforce a close-relation between T. cruzi genetic population and the host specific type 1 immune response and, for the first time, we show the distribution of type 1/2 chemokines associated with the development of cardiac pathology using dogs, a well similar model to study human Chagas disease.

'Click chemistry' synthesis of a library of 1,2,3-triazole-substituted galactose derivatives and their evaluation against Trypanosoma cruzi and its cell surface trans-sialidase.

Trypanosoma cruzi trans-sialidase (TcTS) plays a key role in the recognition and invasion of host cells and in enabling the parasite to escape the human immune response. To explore this potential drug target, we have synthesized a small library of substrate analogues based on 1,4-disubstituted 1,2,3-triazole derivatives of galactose modified at either the C-1 or C-6 positions. This was achieved by coupling the appropriate azido-sugars with a panel of 23 structurally diverse terminal alkynes by using the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction, giving a library of 46 derivatives in good to excellent yield and with complete regioselectivity. The sugar triazoles showed weak inhibition towards TcTS-catalyzed hydrolysis of 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid in vitro (<40% inhibition at 1mM concentration); many of the compounds assessed proved to be acceptor substrates for the enzyme. Despite this modest inhibitory activity, in vitro trypanocidal activity assays against the trypomastigote form of T. cruzi Y strain revealed several compounds active in the low 100s of muM range. Further assessment of these compounds against cultured mouse spleen cells suggests a specific mode of anti-parasite action rather than a generic cytotoxic effect.

Nitric oxide donor trans-[RuCl([15]aneN)NO] as a possible therapeutic approach for Chagas' disease.

BACKGROUND AND PURPOSE: Benznidazole (Bz) is the therapy currently available for clinical treatment of Chagas' disease. However, many strains of Trypanosoma cruzi parasites are naturally resistant. Nitric oxide (NO) produced by activated macrophages is crucial to the intracellular killing of parasites. Here, we investigate the in vitro and in vivo activities against T. cruzi, of the NO donor, trans-[RuCl([15]aneN(4))NO](2+). EXPERIMENTAL APPROACH: Trans-[RuCl([15]aneN(4))NO](2+)was incubated with a partially drug-resistant T. cruzi Y strain and the anti-proliferative (epimastigote form) and trypanocidal activities (trypomastigote and amastigote) evaluated. Mice were treated during the acute phase of Chagas' disease. The anti-T. cruzi activity was evaluated by parasitaemia, survival rate, cardiac parasitism, myocarditis and the curative rate. KEY RESULTS: Trans-[RuCl([15]aneN(4))NO](2+) was 10- and 100-fold more active than Bz against amastigotes and trypomastigotes respectively. Further, trans-[RuCl([15]aneN(4))NO](2+) (0.1 mM) induced 100% of trypanocidal activity (trypomastigotes forms) in vitro. Trans-[RuCl([15]aneN(4))NO](2+) induced permanent suppression of parasitaemia and 100% survival in a murine model of acute Chagas' disease. When the drugs were given alone, parasitological cures were confirmed in only 30 and 40% of the animals treated with the NO donor (3.33 micromol.kg(-1).day(-1)) and Bz (385 micromol.kg(-1).day(-1)), respectively, but when given together, 80% of the animals were parasitologically cured. The cured animals showed an absence of myocarditis and a normalisation of cytokine production in the sera. In addition, no in vitro toxicity was observed at the tested doses. CONCLUSIONS AND IMPLICATIONS: These findings indicate that trans-[RuCl([15]aneN(4))NO](2+)is a promising lead compound for the treatment of human Chagas' disease.

Cutting edge: nucleotide-binding oligomerization domain 1-dependent responses account for murine resistance against Trypanosoma cruzi infection.

An effective innate immune recognition of the intracellular protozoan parasite Trypanosoma cruzi is critical for host resistance against Chagas disease, a severe and chronic illness that affects millions of people in Latin America. In this study, we evaluated the participation of nucleotide-binding oligomerization domain (Nod)-like receptor proteins in host response to T. cruzi infection and found that Nod1-dependent, but not Nod2-dependent, responses are required for host resistance against infection. Bone marrow-derived macrophages from Nod1(-/-) mice showed an impaired induction of NF-kappaB-dependent products in response to infection and failed to restrict T. cruzi infection in presence of IFN-gamma. Despite normal cytokine production in the sera, Nod1(-/-) mice were highly susceptible to T. cruzi infection, in a similar manner to MyD88(-/-) and NO synthase 2(-/-) mice. These studies indicate that Nod1-dependent responses account for host resistance against T. cruzi infection by mechanisms independent of cytokine production.

The effects of nitric oxide on the immune system during Trypanosoma cruzi infection.

Trypanosoma cruzi infection triggers substantial production of nitric oxide (NO), which has been shown to have protective and toxic effects on the host's immune system. Sensing of trypomastigotes by phagocytes activates the inducible NO-synthase (NOS2) pathway, which produces NO and is largely responsible for macrophage-mediated killing of T. cruzi. NO is also responsible for modulating virtually all steps of innate and adaptive immunity. However, NO can also cause oxidative stress, which is especially damaging to the host due to increased tissue damage. The cytokines IFN-gamma and TNF-alpha, as well as chemokines, are strong inducers of NOS2 and are produced in large amounts during T. cruzi acute infection. Conversely, TGF-beta and IL-10 negatively regulate NO production. Here we discuss the recent evidence describing the mechanisms by which NO is able to exert its antimicrobial and immune regulatory effects, the mechanisms involved in the oxidative stress response during infection and the implications of NO for the development of therapeutic strategies against T. cruzi.

The effects of nitric oxide on the immune system during Trypanosoma cruzi infection

Trypanosoma cruzi infection triggers substantial production of nitric oxide (NO), which has been shown to have protective and toxic effects on the host's immune system. Sensing of trypomastigotes by phagocytes activates the inducible NO-synthase (NOS2) pathway, which produces NO and is largely responsible for macrophage-mediated killing of T. cruzi. NO is also responsible for modulating virtually all steps of innate and adaptive immunity. However, NO can also cause oxidative stress, which is especially damaging to the host due to increased tissue damage. The cytokines IFN-³ and TNF-±, as well as chemokines, are strong inducers of NOS2 and are produced in large amounts during T. cruzi acute infection. Conversely, TGF-² and IL-10 negatively regulate NO production. Here we discuss the recent evidence describing the mechanisms by which NO is able to exert its antimicrobial and immune regulatory effects, the mechanisms involved in the oxidative stress response during infection and the implications of NO for the development of therapeutic strategies against T. cruzi.

Influence of Ecto-nucleoside triphosphate diphosphohydrolase activity on Trypanosoma cruzi infectivity and virulence.

BACKGROUND: The protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family) from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models. METHODOLOGY/PRINCIPAL FINDINGS: We followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes) obtained during sequential sub-cultivation in mammalian cells. ATPase/ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin), or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites. CONCLUSIONS/SIGNIFICANCE: Our results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target candidates in chemotherapy of Chagas disease.

Immunological imbalance between IFN-gamma and IL-10 levels in the sera of patients with the cardiac form of Chagas disease.

The immune response is crucial for protection against disease; however, immunological imbalances can lead to heart and digestive tract lesions in chagasic patients. Several studies have evaluated the cellular and humoral immune responses in chagasic patients in an attempt to correlate immunological findings with clinical forms of Chagas disease. Moreover, immunoglobulins and cytokines are important for parasitic control and are involved in lesion genesis. Here, cytokine and IgG isotype production were studied, using total epimastigote antigen on sera of chagasic patients with indeterminate (IND, n = 27) and cardiac (CARD, n = 16) forms of the disease. Samples from normal,uninfected individuals (NI, n = 30) were use as controls. The results showed that sera from both IND and CARD patients contained higher levels of Trypanosoma cruzi-specific IgG1 (IgG1) antibodies than sera from NI. No difference in IgG2 production levels was observed between NI, IND and CARD patients, nor was a difference in IL-10 and IFN-gamma production detected in the sera of IND, CARD and NI patients. However, IND patients displayed a positive correlation between IL-10 and IFN-gamma levels in serum, while CARD patients showed no such correlation, indicating an uncontrolled inflammatory response in CARD patients. These findings support the hypothesis that a lack of efficient regulation between IFN-gamma and IL-10 productions in CARD patients may lead to cardiac immunopathology.

Development of chronic cardiomyopathy in canine Chagas disease correlates with high IFN-gamma, TNF-alpha, and low IL-10 production during the acute infection phase.

When infected with Trypanosoma cruzi, Beagle dogs develop symptoms similar to those of Chagas disease in human beings, and could be an important experimental model for a better understanding of the immunopathogenic mechanisms involved in chronic chagasic infection. This study evaluates IL-10, IFN-gamma and TNF-alpha production in the sera, culture supernatant, heart and cervical lymph nodes and their correlation with cardiomegaly, cardiac inflammation and fibrosis in Beagle dogs infected with T. cruzi. Pathological analysis showed severe splenomegaly, lymphadenopathy and myocarditis in all infected dogs during the acute phase of the disease, with cardiomegaly, inflammation and fibrosis observed in 83% of the animals infected by T. cruzi during the chronic phase. The data indicate that infected animals producing IL-10 in the heart during the chronic phase and showing high IL-10 production in the culture supernatant and serum during the acute phase had lower cardiac alterations (myocarditis, fibrosis and cardiomegaly) than those with high IFN-gamma and TNF-alpha levels. These animals produced low IL-10 levels in the culture supernatant and serum during the acute phase and did not produce IL-10 in the heart during the chronic phase of the disease. Our findings showed that Beagle dogs are a good model for studying the immunopathogenic mechanism of Chagas disease, since they reproduce the clinical and immunological findings described in chagasic patients. The data suggest that the development of the chronic cardiac form of the disease is related to a strong Th1 response during the acute phase of the disease, while the development of the indeterminate form results from a blend of Th1 and Th2 responses soon after infection, suggesting that the acute phase immune response is important for the genesis of chronic cardiac lesions.

Immunological imbalance between IFN-³ and IL-10 levels in the sera of patients with the cardiac form of Chagas disease

The immune response is crucial for protection against disease; however, immunological imbalances can lead to heart and digestive tract lesions in chagasic patients. Several studies have evaluated the cellular and humoral immune responses in chagasic patients in an attempt to correlate immunological findings with clinical forms of Chagas disease. Moreover, immunoglobulins and cytokines are important for parasitic control and are involved in lesion genesis. Here, cytokine and IgG isotype production were studied, using total epimastigote antigen on sera of chagasic patients with indeterminate (IND, n = 27) and cardiac (CARD, n = 16) forms of the disease. Samples from normal, uninfected individuals (NI, n = 30) were use as controls. The results showed that sera from both IND and CARD patients contained higher levels of Trypanosoma cruzi-specific IgG1 (IgG1) antibodies than sera from NI. No difference in IgG2 production levels was observed between NI, IND and CARD patients, nor was a difference in IL-10 and IFN-³ production detected in the sera of IND, CARD and NI patients. However, IND patients displayed a positive correlation between IL-10 and IFN-³ levels in serum, while CARD patients showed no such correlation, indicating an uncontrolled inflammatory response in CARD patients. These findings support the hypothesis that a lack of efficient regulation between IFN-³ and IL-10 productions in CARD patients may lead to cardiac immunopathology.