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One new (compound 3) along with two previously known ursane type triterpenoids (compounds 1 and 2) were purified by chromatographic techniques from ethyl acetate extract of aerial parts of Potentilla atrosanguniea and characterized by HRMS, 1 D and 2 D-NMR. Compounds 1 (ursolic acid), 2 (euscaphic acid) and 3 (3α,20α-dihydroxy 2-oxo-urs-12-en-28-oic acid) were tested for their antiproliferative activity along with standard bazedoxifene. Compounds 1 and 3 were found to be of higher activity (3.71 and 6.05 μg/mL) as compared to compound 2 and bazedoxifene (IC50: 24.53 and 17.87 μg/mL). Anti-estrogenic activity of three compounds on breast cancer (BC) were studied in vitro by accessing their antiproliferative activity and binding with estrogen receptor alpha (ER-α). All three compounds have effective binding affinity towards ER-α and decreased cell growth by downregulating the expression of mRNA and its translational protein as tested by semi-qRT-PCR and western blotting. In terms of effectiveness compounds 1 and 3 were found more active due to their antiproliferative, and antiestrogenic activity as compared to standard bazedoxifene.

An imbalance between the production of oxygen and nitrogen free radicals and their degradation by the antioxidant system are the major causative factors for the wheat intolerance diseases. In the present study, we have examined the wheat gliadin protein-induced oxidative and nitrosative stress and downstream responses in the human intestinal cell lines viz. HCT-116 and HT-29. The role of phytochemical curcumin was investigated to alleviate the gliadin associated cellular damages. The focus of the study was to identify the role of key DNA repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1) in gliadin protein-induced toxicity in the intestine, which may be crucial for establishing the gut-associated diseases. Reactive oxygen species (ROS); reactive nitrogen species (RNS); mitochondrial ROS; mitochondrial trans-membrane potential; protein carbonylation; lipid peroxidation; and the oxidized DNA base damage was estimated in HCT-116 and HT-29 cells after 24 h treatment of 160 µg/ml of gliadin, 10 µM of curcumin and its combination. In addition, the transcriptional expression and enzymatic activities of antioxidants (SOD; Catalase; and GSH) were measured in the in these cells. Furthermore, the cross-talk between the nuclear factor erythroid 2-related factor-2 (Nrf-2) and the multifunctional enzyme APE1 was analyzed by the immunofluorescent based imaging and co-immunoprecipitation assays. The endonuclease activity of APE1 and the DNA-protein interaction of NRF-2 with ARE was analyzed by using electrophoretic mobility shift assay (EMSA) with the nuclear lysates of HCT-116 and HT-29 cells. Results suggest that 3 h pre-treatment of curcumin followed by the treatment of gliadin protein for 24 h time protect the HCT-116 and HT-29 cells via (1) decreasing the ROS, RNS, oxidative stress, mitochondrial ROS, recuperate mitochondrial trans-membrane potential; (2) reestablishing the cellular antioxidant defence systems; (3) enhancing the DNA-repair via APE1 and which further activates the ARE elements via activation of Nrf-2. In conclusion, wheat gliadin induces the oxidative/nitrosative stress, mitochondrial damage and damages the cellular biomolecules; hence is associated with the disease pathogenesis and tissue damage in wheat intolerance diseases. The gliadin induced stress and its consequences are significantly reduced by the pre-treatment of curcumin via DNA repair pathways and oxidative stress which is evident through the interaction between two essential proteins of these pathways APE1 and Nrf-2 hence suggesting the role of curcumin based management of wheat intolerance diseases like celiac disease.

In the present study, we have designed and synthesized indole derivatives by coalescing the indole nucleus with chromene carbonitrile and dihydropyridine nucleus. Two compounds 5c and 6d were selected from series I and II after sequential combinatorial library generation, docking, absorption, distribution, metabolism and excretion (ADME) filtering, anti-proliferative activity, cytotoxicity, and ER-α competitor assay kit by utilizing estrogen receptor-α (ER-α) dominant T47D BC cells line and PBMCs (Peripheral Blood Mononuclear Cells). Cell imaging experiment suggested that both the compounds successfully cross cellular biomembrane and accumulate in nuclear, cytoplasmic and plasma membrane region. Semiquantitative RT-PCR and Western blotting experiments further supported that both compounds reduced the expression of mRNA and receptor protein of ER-α, thereby preventing downstream transactivation and signaling pathway in T47D cells line. Current findings imply that 5c and 6d represent novel ER-α antagonists and may be used in the development of chemotherapy for the management of BC.

Cystatin 9 (CST9) is a member of the type 2 cysteine protease inhibitor family, which has been shown to have immunomodulatory effects that restrain inflammation, but its functions against bacterial infections are unknown. Here, we report that purified human recombinant (r)CST9 protects against the deadly bacterium Francisella tularensis (Ft) in vitro and in vivo. Macrophages infected with the Ft human pathogen Schu 4 (S4), then given 50 pg of rCST9 exhibited significantly decreased intracellular bacterial replication and increased killing via preventing the escape of S4 from the phagosome. Further, rCST9 induced autophagy in macrophages via the regulation of the mammalian target of rapamycin (mTOR) signaling pathways. rCST9 promoted the upregulation of macrophage proteins involved in antiinflammation and antiapoptosis, while restraining proinflammatory-associated proteins. Interestingly, the viability and virulence of S4 also was decreased directly by rCST9. In a mouse model of Ft inhalation, rCST9 significantly decreased organ bacterial burden and improved survival, which was not accompanied by excessive cytokine secretion or subsequent immune cell migration. The current report is the first to show the immunomodulatory and antimicrobial functions of rCST9 against Ft. We hypothesize that the attenuation of inflammation by rCST9 may be exploited for therapeutic purposes during infection.

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

Trypanosoma cruzi, the aetiological agent of Chagas disease, invades nucleated mammalian cells including macrophages. In this study, we investigated the crosstalk between T. cruzi-induced immune activation of reactive oxygen species (ROS) and pro-inflammatory responses, and their role in myocardial pathology. Splenocytes of infected mice (C3H/HeN) responded to Tc-antigenic stimulus by more than a two-fold increase in NADPH oxidase (NOX) activity, ROS generation, cytokine production (IFN-γ > IL-4 > TNFα > IL1-β≈ IL6), and predominant expansion of CD4+ and CD8+ T cells. Inhibition of NOX, but not of myeloperoxidase and xanthine oxidase, controlled the ROS (>98%) and cytokine (70–89%) release by Tc-stimulated splenocytes of infected mice. Treatment of infected mice with apocynin (NOX inhibitor) in drinking water resulted in a 50–90% decline in endogenous NOX/ROS and cytokine levels, and splenic phagocytes' proliferation. The splenic percentage of T cells was maintained, though more than a 40% decline in splenic index (spleen weight/body weight) indicated decreased T-cell proliferation in apocynin-treated/infected mice. The blood and tissue parasite burden were significantly increased in apocynin-treated/infected mice, yet acute myocarditis, ie inflammatory infiltrate consisting of macrophages, neutrophils, and CD8+ T cells, and tissue oxidative adducts (eg 8-isoprostanes, 3-nitrotyrosine, and 4-hydroxynonenal) were diminished in apocynin-treated/infected mice. Consequently, hypertrophy (increased cardiomyocytes' size and β-MHC, BNP, and ANP mRNA levels) and fibrosis (increased collagen, glycosaminoglycans, and lipid contents) of the heart during the chronic phase were controlled in apocynin-treated mice. We conclude that NOX/ROS is a critical regulator of the splenic response (phagocytes, T cells, and cytokines) to T. cruzi infection, and bystander effects of heart-infiltrating phagocytes and CD8+ T cells resulting in cardiac remodelling in chagasic mice. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

ObjectivesThe purpose of this study was to determine the pathological importance of oxidative stress–induced injurious processes in chagasic heart dysfunction.BackgroundTrypanosoma cruzi–induced inflammatory pathology and a feedback cycle of mitochondrial dysfunction and oxidative stress may contribute to Chagas disease.MethodsSprague-Dawley rats were infected with T. cruziand treated with phenyl-α-tert-butylnitrone (PBN), an antioxidant, and/or benzonidazole (BZ), an antiparasitic agent. We monitored myocardial parasite burden, oxidative adducts, mitochondrial complex activities, respiration, and adenosine triphosphate synthesis rates, and inflammatory and cardiac remodeling responses during disease development. The cardiac hemodynamics was determined for all rats.ResultsBenzonidazole (not PBN) decreased the parasite persistence and immune adverse events (proinflammatory cytokine expression, β-nicotinamide adenine dinucleotide phosphate oxidase and myeloperoxidase activities, and inflammatory infiltrate) in chronically infected hearts. PBN ± BZ (not BZ alone) decreased the mitochondrial reactive oxygen species level, oxidative adducts (malonyldialdehyde, 4-hydroxynonenal, carbonyls), hypertrophic gene expression (atrial natriuretic peptide, B-type natriuretic peptide, α-skeletal actin), and collagen deposition and preserved the respiratory chain efficiency and energy status in chronically infected hearts. Subsequently, LV dysfunction was prevented in PBN ± BZ-treated chagasic rats.ConclusionsBZ treatment after the acute stage decreased the parasite persistence and inflammatory pathology. Yet, oxidative adducts, mitochondrial dysfunction, and remodeling responses persisted and contributed to declining cardiac function in chagasic rats. Combination treatment (PBN + BZ) was beneficial in arresting the T. cruzi–induced inflammatory and oxidative pathology and chronic heart failure in chagasic rats.

In this study, we investigated whether inflammatory responses contribute to oxidative/nitrosative stress in patients with Chagas' disease. We used three tests (enzyme-linked immunosorbent assay, immuno-flow cytometry, and STAT-PAK immunochromatography) to screen human serum samples ( n = 1,481) originating from Chiapas, Mexico, for Trypanosoma cruzi -specific antibodies. We identified 121 subjects who were seropositive for T. cruzi- specific antibodies, a finding indicative of an 8.5% seroprevalence in the rural population from Chiapas. Seropositive and seronegative subjects were examined for plasma levels of biomarkers of inflammation, i.e., myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), and xanthine oxidase (XOD), as well as for oxidative (advanced oxidation protein products [AOPPs]) and nitrosative (3-nitrotyrosine [3NT]) biomarkers. The seropositive subjects exhibited a significant increase in MPO activity and protein level, the indicator of neutrophil activation. Subsequently, a corresponding increase in AOPP contents, formed by MPO-dependent hypochlorous acid and chloramine formation, was noted in seropositive subjects. The plasma level of 3NT was significantly increased in seropositive subjects, yet we observed no change in XOD activity (O 2 − source) and nitrate/nitrite contents (denotes iNOS activation and NO production), which implied that direct peroxynitrite formation does not contribute to increased nitrosative damage in chagasic subjects. Instead, a positive correlation between increased MPO activity and protein 3NT formation was observed, which suggested to us that MPO-dependent formation of nitrylchloride that occurs in the presence of physiological NO and O 2 − concentrations contributes to protein nitration. Overall, our data demonstrate that T. cruzi- induced neutrophil activation is pathological and contributes to MPO-mediated collateral protein oxidative and nitrosative damage in human patients with Chagas' disease. Therapies capable of suppressing MPO activity may be useful in controlling the inflammation and oxidative/nitrosative pathology in chagasic cardiomyopathy.

Oxidative/nitrosative stress may be important in the pathology of Chagas' disease. Experimental animals infected by Trypanosoma cruzi showed an early rise in myocardial and peripheral protein-3-nitrotyrosine (3NT) and protein-carbonyl formation that persisted during the chronic stage of disease. In comparison, experimental chronic ethanol-induced cardiomyopathy was slow to develop and presented with a moderate increase in oxidative stress and minimal to no nitrosative stress after long-term alcohol feeding of animals. The oxidative stress in both chagasic animals and animals with ethanol-induced cardiomyopathy correlated with the persistence of reactive oxygen species-producing inflammatory intermediates. Protein-3NT formation in T. cruzi-infected animals was associated with enhanced nitric oxide expression (inferred by nitrite/nitrate levels) and myeloperoxidase activity, suggesting that both peroxynitrite- and myeloperoxidase-mediated pathways contribute to increased protein nitration in Chagas' disease. We used one- and two-dimensional gel electrophoresis and Western blot analysis to identify disease-specific plasma proteins that were 3NT-modified in T. cruzi-infected animals. Nitrated protein spots (56 in total) were sequenced by matrix-assisted laser desorption ionization/time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry and identified by a homology search of public databases. Clustering of 3NT-modified proteins according to their functional characteristics revealed that the nitration of immunoglobulins, apolipoprotein isoforms, and other proteins might perturb their functions and be important in the pathology of Chagas' disease. We also showed that nitrated peptides derived from titin and alpha-actin were released into the plasma of patients with Chagas' disease. Such modified proteins may be useful biomarkers of Chagas' disease.

In this study, we examined the tissue specificity of inflammatory and oxidative responses and mitochondrial dysfunction in mice infected by Trypanosoma cruzi. In acute mice, parasite burden and associated inflammatory infiltrate was detected in all tissues (skeletal-muscle>heart>stomach>colon). The extent of oxidative damage and mitochondrial decay was in the order of heart>stomach>skeletal-muscle>colon. In chronic mice, a low level of parasite burden and inflammation continued in all tissues; however, oxidant overload and mitochondrial inefficiency mainly persisted in the heart tissue (also detectable in stomach). Further, we noted an unvaryingly high degree of oxidative stress, compromised antioxidant status, and decreased mitochondrial respiratory complex activities in peripheral-blood of infected mice. A pair-wise log analysis showed a strong positive correlation in the heart-versus-blood (but not other tissues) levels of oxidative stress markers (malonylaldehyde, glutathione-disulfide), antioxidants (superoxide-dismutase, MnSOD, catalase), and mitochondrial inhibition of respiratory complexes (CI/CIII) in infected mice. Conclusions: T.cruzi-induced acute inflammatory and oxidative responses are widespread in different muscle tissues. Antioxidant/oxidant status and mitochondrial function are consistently attenuated in the heart, and reflected in the peripheral-blood of T.cruzi-infected mice. Our results provide an impetus to investigate the peripheral-blood oxidative responses in relation to clinical severity of heart disease in chagasic human patients.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by endogenous and exogenous agents. APE1 acts as a reductive activator of many transcription factors (TFs) and has also been named redox effector factor 1, Ref-1. For example, APE1 activates activator protein-1, nuclear factor kappa B, hypoxia-inducible factor 1α, paired box gene 8, signal transducer activator of transcription 3 and p53, which are involved in apoptosis, inflammation, angiogenesis and survival pathways. APE1/Ref-1 maintains cellular homeostasis (redox) via the activation of TFs that regulate various physiological processes and that crosstalk with redox balancing agents (for example, thioredoxin, catalase and superoxide dismutase) by controlling levels of reactive oxygen and nitrogen species. The efficiency of APE1/Ref-1's function(s) depends on pairwise interaction with participant protein(s), the functions regulated by APE1/Ref-1 include the BER pathway, TFs, energy metabolism, cytoskeletal elements and stress-dependent responses. Thus, APE1/Ref-1 acts as a ‘hub-protein' that controls pathways that are important for cell survival. In this review, we will discuss APE1/Ref-1's versatile nature in various human etiologies, including neurodegeneration, cancer, cardiovascular and other diseases that have been linked with alterations in the expression, subcellular localization and activities of APE/Ref-1. APE1/Ref-1 can be targeted for therapeutic intervention using natural plant products that modulate the expression and functions of APE1/Ref-1. In addition, studies focusing on translational applications based on APE1/Ref-1-mediated therapeutic interventions are discussed.

Background We utilized genetically modified mice equipped with a variable capacity to scavenge mitochondrial and cellular reactive oxygen species to investigate the pathological significance of oxidative stress in C hagas disease. Methods and Results C57 BL /6 mice (wild type, Mn SOD tg , Mn SOD +/− , GP x1 −/− ) were infected with Trypanosoma cruzi and harvested during the chronic disease phase. Chronically infected mice exhibited a substantial increase in plasma levels of inflammatory markers (nitric oxide, myeloperoxidase), lactate dehydrogenase, and myocardial levels of inflammatory infiltrate and oxidative adducts (malondialdehyde, carbonyls, 3‐nitrotyrosine) in the order of wild type=Mn SOD +/− > GP x1 −/− >Mn SOD tg . Myocardial mitochondrial damage was pronounced and associated with a >50% decline in mitochondrial DNA content in chronically infected wild‐type and GP x1 −/− mice. Imaging of intact heart for cardiomyocytes and collagen by the nonlinear optical microscopy techniques of multiphoton fluorescence/second harmonic generation showed a significant increase in collagen (>10‐fold) in chronically infected wild‐type mice, whereas GP x1 −/− mice exhibited a basal increase in collagen that did not change during the chronic phase. Chronically infected Mn SOD tg mice exhibited a marginal decline in mitochondrial DNA content and no changes in collagen signal in the myocardium. P47 phox−/− mice lacking phagocyte‐generated reactive oxygen species sustained a low level of myocardial oxidative stress and mitochondrial DNA damage in response to Trypanosoma cruzi infection. Yet chronically infected p47 phox−/− mice exhibited increase in myocardial inflammatory and remodeling responses, similar to that noted in chronically infected wild‐type mice. Conclusions Inhibition of oxidative burst of phagocytes was not sufficient to prevent pathological cardiac remodeling in Chagas disease. Instead, enhancing the mitochondrial reactive oxygen species scavenging capacity was beneficial in controlling the inflammatory and oxidative pathology and the cardiac remodeling responses that are hallmarks of chronic Chagas disease.

Background Trypanosoma cruzi, the etiologic agent of Chagas Disease, is a major vector borne health problem in Latin America and an emerging infectious disease in the United States. Methods We tested the efficacy of a multi-component DNA-prime/DNA-boost vaccine (TcVac1) against experimental T. cruzi infection in a canine model. Dogs were immunized with antigen-encoding plasmids and cytokine adjuvants, and two weeks after the last immunization, challenged with T. cruzi trypomastigotes. We measured antibody responses by ELISA and haemagglutination assay, parasitemia and infectivity to triatomines by xenodiagnosis, and performed electrocardiography and histology to assess myocardial damage and tissue pathology. Results Vaccination with TcVac1 elicited parasite-and antigen-specific IgM and IgG (IgG2>IgG1) responses. Upon challenge infection, TcVac1-vaccinated dogs, as compared to non-vaccinated controls dogs, responded to T. cruzi with a rapid expansion of antibody response, moderately enhanced CD8+ T cell proliferation and IFN-γ production, and suppression of phagocytes’ activity evidenced by decreased myeloperoxidase and nitrite levels. Subsequently, vaccinated dogs controlled the acute parasitemia by day 37 pi (44 dpi in non-vaccinated dogs), and exhibited a moderate decline in infectivity to triatomines. TcVac1-immunized dogs did not control the myocardial parasite burden and electrocardiographic and histopatholgic cardiac alterations that are the hallmarks of acute Chagas disease. During the chronic stage, TcVac1-vaccinated dogs exhibited a moderate decline in cardiac alterations determined by EKG and anatomo-/histo-pathological analysis while chronically-infected/non-vaccinated dogs continued to exhibit severe EKG alterations. Conclusions Overall, these results demonstrated that TcVac1 provided a partial resistance to T. cruzi infection and Chagas disease, and provide an impetus to improve the vaccination strategy against Chagas disease., Author Summary Immunization of dogs with DNA-prime/DNA-boost vaccine (TcVac1) enhanced the Trypanosoma cruzi-specific type 1 antibody and CD8+ T cell responses that resulted in an early control of acute parasitemia and a moderate decline in pathological symptoms during chronic phase. Further improvement of vaccine-induced immunity would be required to achieve clinical and epidemiological benefits and prevent transmission of parasites from vaccinated/infected dogs to triatomines.

Trypanosoma cruzi, the aetiological agent of Chagas disease, invades nucleated mammalian cells including macrophages. In this study, we investigated the crosstalk between T. cruzi-induced immune activation of reactive oxygen species (ROS) and pro-inflammatory responses, and their role in myocardial pathology. Splenocytes of infected mice (C3H/HeN) responded to Tc-antigenic stimulus by more than a two-fold increase in NADPH oxidase (NOX) activity, ROS generation, cytokine production (IFN-γ > IL-4 > TNFα > IL1-β ≈ IL6), and predominant expansion of CD4+ and CD8+ T cells. Inhibition of NOX, but not of myeloperoxidase and xanthine oxidase, controlled the ROS (>98%) and cytokine (70–89%) release by Tc-stimulated splenocytes of infected mice. Treatment of infected mice with apocynin (NOX inhibitor) in drinking water resulted in a 50–90% decline in endogenous NOX/ROS and cytokine levels, and splenic phagocytes’ proliferation. The splenic percentage of T cells was maintained, though more than a 40% decline in splenic index (spleen weight/body weight) indicated decreased T-cell proliferation in apocynin-treated/infected mice. The blood and tissue parasite burden were significantly increased in apocynin-treated/infected mice, yet acute myocarditis, ie inflammatory infiltrate consisting of macrophages, neutrophils, and CD8+ T cells, and tissue oxidative adducts (eg 8-isoprostanes, 3-nitrotyrosine, and 4-hydroxynonenal) were diminished in apocynin-treated/infected mice. Consequently, hypertrophy (increased cardiomyocytes’ size and β-MHC, BNP, and ANP mRNA levels) and fibrosis (increased collagen, glycosaminoglycans, and lipid contents) of the heart during the chronic phase were controlled in apocynin-treated mice. We conclude that NOX/ROS is a critical regulator of the splenic response (phagocytes, T cells, and cytokines) to T. cruzi infection, and bystander effects of heart-infiltrating phagocytes and CD8+ T cells resulting in cardiac remodelling in chagasic mice.

Oxidative/nitrosative stress may be important in the pathology of Chagas' disease. Experimental animals infected by Trypanosoma cruzi showed an early rise in myocardial and peripheral protein-3-nitrotyrosine (3NT) and protein-carbonyl formation that persisted during the chronic stage of disease. In comparison, experimental chronic ethanol-induced cardiomyopathy was slow to develop and presented with a moderate increase in oxidative stress and minimal to no nitrosative stress after long-term alcohol feeding of animals. The oxidative stress in both chagasic animals and animals with ethanol-induced cardiomyopathy correlated with the persistence of reactive oxygen species-producing inflammatory intermediates. Protein-3NT formation in T. cruzi-infected animals was associated with enhanced nitric oxide expression (inferred by nitrite/nitrate levels) and myeloperoxidase activity, suggesting that both peroxynitrite- and myeloperoxidase-mediated pathways contribute to increased protein nitration in Chagas' disease. We used one- and two-dimensional gel electrophoresis and Western blot analysis to identify disease-specific plasma proteins that were 3NT-modified in T. cruzi-infected animals. Nitrated protein spots (56 in total) were sequenced by matrix-assisted laser desorption ionization/time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry and identified by a homology search of public databases. Clustering of 3NT-modified proteins according to their functional characteristics revealed that the nitration of immunoglobulins, apolipoprotein isoforms, and other proteins might perturb their functions and be important in the pathology of Chagas' disease. We also showed that nitrated peptides derived from titin and alpha-actin were released into the plasma of patients with Chagas' disease. Such modified proteins may be useful biomarkers of Chagas' disease.

Background Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America and an emerging infectious disease in the US and Europe. We have shown TcG1, TcG2, and TcG4 antigens elicit protective immunity to T. cruzi in mice and dogs. Herein, we investigated antigenicity of the recombinant proteins in humans to determine their potential utility for the development of next generation diagnostics for screening of T. cruzi infection and Chagas disease. Methods and Results Sera samples from inhabitants of the endemic areas of Argentina-Bolivia and Mexico-Guatemala were analyzed in 1st-phase for anti-T. cruzi antibody response by traditional serology tests; and in 2nd-phase for antibody response to the recombinant antigens (individually or mixed) by an ELISA. We noted similar antibody response to candidate antigens in sera samples from inhabitants of Argentina and Mexico (n = 175). The IgG antibodies to TcG1, TcG2, and TcG4 (individually) and TcGmix were present in 62–71%, 65–78% and 72–82%, and 89–93% of the subjects, respectively, identified to be seropositive by traditional serology. Recombinant TcG1- (93.6%), TcG2- (96%), TcG4- (94.6%) and TcGmix- (98%) based ELISA exhibited significantly higher specificity compared to that noted for T. cruzi trypomastigote-based ELISA (77.8%) in diagnosing T. cruzi-infection and avoiding cross-reactivity to Leishmania spp. No significant correlation was noted in the sera levels of antibody response and clinical severity of Chagas disease in seropositive subjects. Conclusions Three candidate antigens were recognized by antibody response in chagasic patients from two distinct study sites and expressed in diverse strains of the circulating parasites. A multiplex ELISA detecting antibody response to three antigens was highly sensitive and specific in diagnosing T. cruzi infection in humans, suggesting that a diagnostic kit based on TcG1, TcG2 and TcG4 recombinant proteins will be useful in diverse situations., Author Summary Chagas disease is the most common cause of congestive heart failure related deaths among young adults in the endemic areas of South and Central America and Mexico. Diagnosis and treatment of T. cruzi infection has remained difficult and challenging after 100 years of its identification. In >95% of human cases, T. cruzi infection remains undiagnosed until several years later when chronic evolution of progressive disease results in clinical symptoms associated with cardiac damage. Diagnosis generally depends on the measurement of T. cruzi–specific antibodies that can result in false positives. A conclusive diagnosis of T. cruzi infection thus often requires multiple serological tests, in combination with epidemiological data and clinical symptoms. In this study, we investigated the antibody response to TcG1, TcG2, and TcG4 in clinically characterized chagasic patients. These antigens were identified as vaccine candidates and shown to elicit protective immunity to T. cruzi and Chagas disease in experimental animals. Our data show the serology test developed using the TcGmix (multiplex ELISA) is a significantly better alternative to epimastigote extracts currently used in T. cruzi serodiagnosis or the trypomastigote lysate used in this study for comparison purposes.