Unlocking Overexpressed Membrane Proteins to Guide Breast Cancer Precision Medicine.

Breast cancer (BC) is a prevalent form of cancer affecting women worldwide. However, the effectiveness of current BC drugs is limited by issues such as systemic toxicity, drug resistance, and severe side effects. Consequently, there is an urgent need for new therapeutic targets and improved tumor tracking methods. This study aims to address these challenges by proposing a strategy for identifying membrane proteins in tumors that can be targeted for specific BC therapy and diagnosis. The strategy involves the analyses of gene expressions in breast tumor and non-tumor tissues and other healthy tissues by using comprehensive bioinformatics analysis from The Cancer Genome Atlas (TCGA), UALCAN, TNM Plot, and LinkedOmics. By employing this strategy, we identified four transcripts (LRRC15, EFNA3, TSPAN13, and CA12) that encoded membrane proteins with an increased expression in BC tissue compared to healthy tissue. These four transcripts also demonstrated high accuracy, specificity, and accuracy in identifying tumor samples, as confirmed by the ROC curve. Additionally, tissue microarray (TMA) analysis revealed increased expressions of the four proteins in tumor tissues across all molecular subtypes compared to the adjacent breast tissue. Moreover, the analysis of human interactome data demonstrated the important roles of these proteins in various cancer-related pathways. Taken together, these findings suggest that LRRC15, EFNA3, TSPAN13, and CA12 can serve as potential biomarkers for improving cancer diagnosis screening and as suitable targets for therapy with reduced side effects and enhanced efficacy.

Aptamer-Based Recognition of Breast Tumor Cells: A New Era for Breast Cancer Diagnosis.

Breast cancer is one of the leading causes of death among women worldwide and can be classified into four major distinct molecular subtypes based on the expression of specific receptors. Despite significant advances, the lack of biomarkers for detailed diagnosis and prognosis remains a major challenge in the field of oncology. This study aimed to identify short single-stranded oligonucleotides known as aptamers to improve breast cancer diagnosis. The Cell-SELEX technique was used to select aptamers specific to the MDA-MB-231 tumor cell line. After selection, five aptamers demonstrated specific recognition for tumor breast cell lines and no binding to non-tumor breast cells. Validation of aptamer specificity revealed recognition of primary and metastatic tumors of all subtypes. In particular, AptaB4 and AptaB5 showed greater recognition of primary tumors and metastatic tissue, respectively. Finally, a computational biology approach was used to identify potential aptamer targets, which indicated that CSKP could interact with AptaB4. These results suggest that aptamers are promising in breast cancer diagnosis and treatment due to their specificity and selectivity.

Next Generation of Ovarian Cancer Detection Using Aptamers.

Ovarian cancer is among the seven most common types of cancer in women, being the most fatal gynecological tumor, due to the difficulty of detection in early stages. Aptamers are important tools to improve tumor diagnosis through the recognition of specific molecules produced by tumors. Here, aptamers and their potential targets in ovarian cancer cells were analyzed by in silico approaches. Specific aptamers were selected by the Cell-SELEX method using Caov-3 and OvCar-3 cells. The five most frequent aptamers obtained from the last round of selection were computationally modeled. The potential targets for those aptamers in cells were proposed by analyzing proteomic data available for the Caov-3 and OvCar-3 cell lines. Overexpressed proteins for each cell were characterized as to their three-dimensional model, cell location, and electrostatic potential. As a result, four specific aptamers for ovarian tumors were selected: AptaC2, AptaC4, AptaO1, and AptaO2. Potential targets were identified for each aptamer through Molecular Docking, and the best complexes were AptaC2-FXYD3, AptaC4-ALPP, AptaO1-TSPAN15, and AptaO2-TSPAN15. In addition, AptaC2 and AptaO1 could detect different stages and subtypes of ovarian cancer tissue samples. The application of this technology makes it possible to propose new molecular biomarkers for the differential diagnosis of epithelial ovarian cancer.

In Chagas disease, transforming growth factor beta neutralization reduces Trypanosoma cruzi infection and improves cardiac performance.

Chronic Chagasic cardiomyopathy (CCC), a progressive inflammatory and fibrosing disease, is the most prominent clinical form of Chagas disease, a neglected tropical disease caused by Trypanosoma cruzi infection. During CCC, the parasite remains inside the cardiac cells, leading to tissue damage, involving extensive inflammatory response and irregular fibrosis. Among the fibrogenic factors is transforming growth factor-ß (TGF-ß), a key cytokine controlling extracellular matrix synthesis and degradation. TGF-ß is involved in CCC onset and progression, with increased serum levels and activation of its signaling pathways in the cardiac tissue, which crucially contributes to fibrosis. Inhibition of the TGF-ß signaling pathway attenuates T. cruzi infection and prevents cardiac damage in an experimental model of acute Chagas disease. The aim of this study was to investigate the effect of TGF-ß neutralization on T. cruzi infection in both in vitro and in vivo pre-clinical models, using the 1D11 monoclonal antibody. To this end, primary cultures of cardiac cells were infected with T. cruzi trypomastigote forms and treated with 1D11. For in vivo studies, 1D11 was administered in different schemes for acute and chronic phase models (Swiss mice infected with 104 parasites from the Y strain and C57BL/6 mice infected with 102 parasites from the Colombian strain, respectively). Here we show that the addition of 1D11 to cardiac cells greatly reduces cardiomyocyte invasion by T. cruzi and the number of parasites per infected cell. In both acute and chronic experimental models, T. cruzi infection altered the electrical conduction, decreasing the heart rate, increasing the PR interval and the P wave duration. The treatment with 1D11 reduced cardiac fibrosis and reversed electrical abnormalities improving cardiac performance. Taken together, these data further support the major role of the TGF-ß signaling pathways in T. cruzi-infection and their biological consequences on parasite/host interactions. The therapeutic effects of the 1D11 antibody are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas' heart disease by TGF-ß neutralization.

Increased angiogenesis parallels cardiac tissue remodelling in experimental acute Trypanosoma cruzi infection.

BACKGROUND: Angiogenesis has been implicated in tissue injury in several noninfectious diseases, but its role in Chagas disease (CD) physiopathology is unclear. OBJECTIVES: The present study aimed to investigate the effect of Trypanosoma cruzi infection on cardiac angiogenesis during the acute phase of experimental CD. METHODS: The signalling pathway involved in blood vessel formation and cardiac remodelling was evaluated in Swiss Webster mice infected with the Y strain of T. cruzi. The levels of molecules involved in the regulation of angiogenesis, such as vascular endothelial growth factor-A (VEGF-A), Flk-1, phosphorylated extracellular-signal-regulated protein kinase (pERK), hypoxia-inducible factor-1α (HIF-1α), CD31, α-smooth muscle actin (α-SMA) and also the blood vessel growth were analysed during T. cruzi infection. Hearts were analysed using conventional histopathology, immunohistochemistry and western blotting. FINDINGS: In this study, our data demonstrate that T. cruzi acute infection in mice induces exacerbated angiogenesis in the heart and parallels cardiac remodelling. In comparison with noninfected controls, the cardiac tissue of T. cruzi-infected mice presented higher levels of (i) HIF-1α, VEGF-A, Flk-1 and pERK; (ii) angiogenesis; (iii) α-SMA+ cells in the tissue; and (iv) collagen -1 deposition around blood vessels and infiltrating throughout the myocardium. MAIN CONCLUSIONS: We observed cardiac angiogenesis during acute experimental T. cruzi infection parallels cardiac inflammation and remodelling.

Suppression of TGF-ß/Smad2 signaling by GW788388 enhances DENV-2 clearance in macrophages.

Dengue fever, caused by the dengue virus (DENV-1, -2, -3, and -4), affects millions of people in the tropical and subtropical regions worldwide. Severe dengue is correlated with high viraemia and cytokine storm, such as high levels of transforming growth factor-ß1 (TGF-ß1) in the patient's serum. Here, the TGF-ß1 signaling was investigated in the context of in vitro viral clearance. Macrophages were infected with DENV-2 at MOI 5 and treated with the TGF-ß receptor 1 and 2 inhibitor, GW788388. TGF-ß1 expression, signal transduction and viral load were evaluated 48 h after DENV-2 infection by enzyme-linked immunoassay, immunofluorescence, and RT-qPCR assays. Total TGF-ß1 level was reduced in 15% after DENV-2 infection, but the secretion of its biologically active form increased threefold during infection, which was followed by the phosphorylation of Smad2 protein. Phosphorylation of Smad2 was reduced by treatment with GW788388 and it was correlated with reduced cytokine production. Importantly, treatment led to a dose-dependent reduction in viral load, ranging from 6.6 × 105 RNA copies/ml in untreated cultures to 2.3 × 103 RNA copies/ml in cultures treated with 2 ng/ml of GW788388. The anti-TGF-ß1 antibody treatment also induced a significant reduction in viral load to 1.6 × 103 RNA copies/ml. On the other hand, the addition of recombinant TGF-ß1 in infected cultures promoted an increase in viral load to 7.0 × 106 RNA copies/ml. These results support that TGF-ß1 plays a significant role in DENV-2 replication into macrophages and suggest that targeting TGF-ß1 may represent an alternative therapeutic strategy to be explored in dengue infection.

Transforming growth factor-ß as a therapeutic target for the cardiac damage of Chagas disease.

Transforming growth factor beta (TGF-ß) is deeply involved on the pathogenesis of Chagas disease. Our group has been investigating the participation of this pleiotropic cytokine in different aspects of Chagas disease over the last 20 years. Important observations have been made, such as: (i) the ability of Trypanosoma cruzi in activating latent TGF-ß; (ii) the potential involvement of TGF-ß pathway on T. cruzi invasion of host cells; (iii) association of TGF-ß with parasite intracellular replication; (iv) cardiac fibrosis development and maintenance; (v) disruption of Connexin-43 plaque structures and (vi) inflammation and immune response. In this perspective article we intend to discuss the advances of the potential use of new therapies targeting TGF-ß to treat the cardiac alterations of Chagas disease-affected patients.

Increased angiogenesis parallels cardiac tissue remodelling in experimental acute Trypanosoma cruzi infection

BACKGROUND Angiogenesis has been implicated in tissue injury in several noninfectious diseases, but its role in Chagas disease (CD) physiopathology is unclear. OBJECTIVES The present study aimed to investigate the effect of Trypanosoma cruzi infection on cardiac angiogenesis during the acute phase of experimental CD. METHODS The signalling pathway involved in blood vessel formation and cardiac remodelling was evaluated in Swiss Webster mice infected with the Y strain of T. cruzi. The levels of molecules involved in the regulation of angiogenesis, such as vascular endothelial growth factor-A (VEGF-A), Flk-1, phosphorylated extracellular-signal-regulated protein kinase (pERK), hypoxia-inducible factor-1α (HIF-1α), CD31, α-smooth muscle actin (α-SMA) and also the blood vessel growth were analysed during T. cruzi infection. Hearts were analysed using conventional histopathology, immunohistochemistry and western blotting. FINDINGS In this study, our data demonstrate that T. cruzi acute infection in mice induces exacerbated angiogenesis in the heart and parallels cardiac remodelling. In comparison with noninfected controls, the cardiac tissue of T. cruzi-infected mice presented higher levels of (i) HIF-1α, VEGF-A, Flk-1 and pERK; (ii) angiogenesis; (iii) α-SMA+ cells in the tissue; and (iv) collagen -1 deposition around blood vessels and infiltrating throughout the myocardium. MAIN CONCLUSIONS We observed cardiac angiogenesis during acute experimental T. cruzi infection parallels cardiac inflammation and remodelling.

Transforming growth factor-ß as a therapeutic target for the cardiac damage of Chagas disease

Transforming growth factor beta (TGF-β) is deeply involved on the pathogenesis of Chagas disease. Our group has been investigating the participation of this pleiotropic cytokine in different aspects of Chagas disease over the last 20 years. Important observations have been made, such as: (i) the ability of Trypanosoma cruzi in activating latent TGF-β; (ii) the potential involvement of TGF-β pathway on T. cruzi invasion of host cells; (iii) association of TGF-β with parasite intracellular replication; (iv) cardiac fibrosis development and maintenance; (v) disruption of Connexin-43 plaque structures and (vi) inflammation and immune response. In this perspective article we intend to discuss the advances of the potential use of new therapies targeting TGF-β to treat the cardiac alterations of Chagas disease-affected patients.

The Search for Biomarkers and Treatments in Chagas Disease: Insights From TGF-Beta Studies and Immunogenetics.

The anti-inflammatory cytokine transforming growth factor beta (TGF-ß) plays an important role in Chagas disease (CD), a potentially life-threatening illness caused by Trypanosoma cruzi. In this review we revisited clinical studies in CD patients combined with in vitro and in vivo experiments, presenting three main sections: an overview of epidemiological, economic, and clinical aspects of CD and the need for new biomarkers and treatment; a brief panorama of TGF-ß roles and its intracellular signaling pathways, and an update of what is known about TGF-ß and Chagas disease. In in vitro assays, TGF-ß increases during T. cruzi infection and modulates heart cells invasion by the parasite fostering its intracellular parasite cycle. TGF-ß modulates host immune response and inflammation, increases heart fibrosis, stimulates remodeling, and slows heart conduction via gap junction modulation. TGF-ß signaling inhibitors reverts these effects opening a promising therapeutic approach in pre-clinical studies. CD patients with higher TGF-ß1 serum level show a worse clinical outcome, implicating a predictive value of serum TGF-ß as a surrogate biomarker of clinical relevance. Moreover, pre-clinical studies in chronic T. cruzi infected mice proved that inhibition of TGF-ß pathway improved several cardiac electric parameters, reversed the loss of connexin-43 enriched intercellular plaques, reduced fibrosis of the cardiac tissue, restored GATA-6 and Tbox-5 transcription, supporting cardiac recovery. Finally, TGF-ß polymorphisms indicate that CD immunogenetics is at the base of this phenomenon. We searched in a Brazilian population five single-nucleotide polymorphisms (-800 G>A rs1800468, -509 C>T rs1800469, +10 T>C rs1800470, +25 G>C rs1800471, and +263 C>T rs1800472), showing that CD patients frequently express the TGF-ß1 gene genotypes CT and TT at position -509, as compared to noninfected persons; similar results were observed with genotypes TC and CC at codon +10 of the TGF-ß1 gene, leading to the conclusion that 509 C>T and +10 T>C TGF-ß1 polymorphisms are associated with Chagas disease susceptibility. Studies in genetically different populations susceptible to CD will help to gather new insights and encourage the use of TGF-ß as a CD biomarker.

Inhibition of Brazilian ZIKV strain replication in primary human placental chorionic cells and cervical cells treated with nitazoxanide

ABSTRACT Zika virus (ZIKV) infection during pregnancy is associated with a congenital syndrome. Although the virus can be detected in human placental tissue and sexual transmission has been verified, it is not clear how the virus reaches the fetus. Despite the emerging severity caused by ZIKV infection, no specific prophylactic and/or therapeutic treatment is available. The aim of the present study was to evaluate the effectiveness antiviral of nitazoxanide (NTZ) in two important congenital transmission targets: (i) a primary culture of human placental chorionic cells, and (ii) human cervical epithelial cells (C33-A) infected with Brazilian ZIKV strain. Initially, NTZ activity was screened in ZIKV infected Vero cells under different treatment regimens with non-toxic drug concentrations for 48 h. Antiviral effect was found only when the treatment was carried out after the viral inoculum. A strong effect against the dengue virus serotype 2 (DENV-2) was also observed suggesting the possibility of treating other Flaviviruses. Additionally, it was shown that the treatment did not reduce the production of infectious viruses in insect cells (C6/36) infected with ZIKV, indicating that the activity of this drug is also related to host factors. Importantly, we demonstrated that NTZ treatment in chorionic and cervical cells caused a reduction of infected cells in a dose-dependent manner and decreased viral loads in up to 2 logs. Pre-clinical in vitro testing evidenced excellent therapeutic response of infected chorionic and cervical cells and point to future NTZ activity investigation in ZIKV congenital transmission models with the perspective of possible repurposing of NTZ to treat Zika fever, especially in pregnant women.

Insights into the proteomic profile and gene expression of Lutzomyia longipalpis-derived Lulo cell line.

BACKGROUND: Lutzomyia longipalpis-derived cell line (Lulo) has been suggested as a model for studies of interaction between sandflies and Leishmania. OBJECTIVES: Here, we present data of proteomic and gene expression analyses of Lulo cell related to interactions with Leishmania (Viannia) braziliensis. METHODS: Lulo cell protein extracts were analysed through a combination of two-dimensional gel electrophoresis and mass spectrometry and resulting spots were further investigated in silico to identify proteins using Mascot search and, afterwards, resulting sequences were applied for analysis with VectorBase. RESULTS: Sixty-four spots were identified showing similarities to other proteins registered in the databases and could be classified according to their biological function, such as ion-binding proteins (23%), proteases (14%), cytoskeletal proteins (11%) and interactive membrane proteins (9.5%). Effects of interaction with L. (V.) braziliensis with the expression of three genes (enolase, tubulin and vacuolar transport protein) were observed after an eight-hour timeframe and compared to culture without parasites, and demonstrated the impact of parasite interaction with the expression of the following genes: LLOJ000219 (1.69-fold), LLOJ000326 (1.43-fold) and LLOJ006663 (2.41-fold). CONCLUSIONS: This set of results adds relevant information regarding the usefulness of the Lulo cell line for studies with Leishmania parasites that indicate variations of protein expression.

Inhibition of Brazilian ZIKV strain replication in primary human placental chorionic cells and cervical cells treated with nitazoxanide.

Zika virus (ZIKV) infection during pregnancy is associated with a congenital syndrome. Although the virus can be detected in human placental tissue and sexual transmission has been verified, it is not clear how the virus reaches the fetus. Despite the emerging severity caused by ZIKV infection, no specific prophylactic and/or therapeutic treatment is available. The aim of the present study was to evaluate the effectiveness antiviral of nitazoxanide (NTZ) in two important congenital transmission targets: (i) a primary culture of human placental chorionic cells, and (ii) human cervical epithelial cells (C33-A) infected with Brazilian ZIKV strain. Initially, NTZ activity was screened in ZIKV infected Vero cells under different treatment regimens with non-toxic drug concentrations for 48 h. Antiviral effect was found only when the treatment was carried out after the viral inoculum. A strong effect against the dengue virus serotype 2 (DENV-2) was also observed suggesting the possibility of treating other Flaviviruses. Additionally, it was shown that the treatment did not reduce the production of infectious viruses in insect cells (C6/36) infected with ZIKV, indicating that the activity of this drug is also related to host factors. Importantly, we demonstrated that NTZ treatment in chorionic and cervical cells caused a reduction of infected cells in a dose-dependent manner and decreased viral loads in up to 2 logs. Pre-clinical in vitro testing evidenced excellent therapeutic response of infected chorionic and cervical cells and point to future NTZ activity investigation in ZIKV congenital transmission models with the perspective of possible repurposing of NTZ to treat Zika fever, especially in pregnant women.

Effect of Posaconazole in an in vitro model of cardiac fibrosis induced by Trypanosoma cruzi.

Posaconazole (POS) is an inhibitor of ergosterol biosynthesis in clinical use for treating invasive fungal infections. POS has potent and selective anti-Trypanosoma cruzi activity and has been evaluated as a possible treatment for Chagas disease. Microtissues are a 3D culture system that has been shown to reproduce better tissue architecture and functionality than cell cultures in monolayer (2D). It has been used to evaluate chemotropic response as in vitro disease models. We previously developed an in vitro model that reproduces aspects of cardiac fibrosis observed in Chagas cardiomyopathy, using microtissues formed by primary cardiac cells infected by the T. cruzi, here called T. cruzi fibrotic cardiac microtissue (TCFCM). We also showed that the treatment of TCFCM with a TGF-ß pathway inhibitor reduces fibrosis. Here, we aimed to evaluate the effect of POS in TCFCM, observing parasite load and molecules involved in fibrosis. To choose the concentration of POS to be used in TCFCM we first performed experiments in a monolayer of primary cardiac cell cultures and, based on the results, TCFCM was treated with 5 nM of POS for 96 h, starting at 144 h post-infection. Our previous studies showed that at this time the TCFCM had established fibrosis, resulting from T. cruzi infection. Treatment with POS of TCFCM reduced 50 % of parasite load as observed by real-time PCR and reduced markedly the fibrosis as observed by western blot and immunofluorescence, associated with a strong reduction in the expression of fibronectin and laminin (45 % and 54 %, respectively). POS treatment also changed the expression of proteins involved in the regulation of extracellular matrix proteins (TGF-ß and TIMP-4, increased by 50 % and decreased by 58 %, respectively) in TCFCM. In conclusion, POS presented a potent trypanocidal effect both in 2D and in TCFCM, and the reduction of the parasite load was associated with a reduction of fibrosis in the absence of external immunological effectors.

Impact of pharmaceutical care on the quality of life of patients with heart failure due to chronic Chagas disease: Randomized clinical trial.

AIMS: Chronic Chagas disease (ChD) has high morbimortality and loss in quality of life due to heart failure (HF). Pharmaceutical care (PC) optimizes clinical treatment and can improve quality of life in HF. We evaluated if PC improves quality of life of patients with ChD and HF. METHODS: Single-blinded, randomized, controlled trial that assigned adult patients with ChD and HF (81 patients; 61 ± 11 years; 48% male) to PC (n = 40) or standard care (n = 41). Quality of life according to SF-36 and Minnesota living with HF questionnaires, incidence of drug-related problems (DRPs), and adherence to medical treatment were determined at baseline and at every 3 months for 1 year. Intention-to-treat analyses were performed by mixed linear model to verify the treatment effect on the changes of these variables throughout the intervention period. RESULTS: Relative changes from baseline to 1 year of follow-up of the domains physical functioning (+16.6 vs -8.5; P < .001), role-physical (+34.0 vs +5.2; P = .01), general health (+19.4 vs -6.1; P < .001), vitality (+11.5 vs. -5.8; P = .003), social functioning (+7.5 vs -13.3; P = .002), and mental health (+9.0 vs -3.7; P = .006) of the SF-36 questionnaire and the Minnesota living with HF questionnaire score (-12.7 vs +4.8; P < .001) were superior in the PC group than in the standard care group. Adherence to medical treatment increased as early as after 3 months of follow-up and DRPs incidence decreased after 6 months of follow-up only in the PC group. CONCLUSIONS: Patients with ChD and HF who received PC presented improved quality of life, decrease in DRP frequency, and increase in medication adherence.

Toxoplasma gondii Impairs Myogenesis in vitro, With Changes in Myogenic Regulatory Factors, Altered Host Cell Proliferation and Secretory Profile.

Toxoplasma gondii is the causative agent of toxoplasmosis, a parasitic disease with a wide global prevalence. The parasite forms cysts in skeletal muscle cells and neurons, although no evident association with inflammatory infiltrates has been typically found. We studied the impact of T. gondii infection on the myogenic program of mouse skeletal muscle cells (SkMC). The C2C12 murine myoblast cell line was infected with T. gondii tachyzoites (ME49 strain) for 24 h followed by myogenic differentiation induction. T. gondii infection caused a general decrease in myotube differentiation, fusion and maturation, along with decreased expression of myosin heavy chain. The expression of Myogenic Regulatory Factors Myf5, MyoD, Mrf4 and myogenin was modulated by the infection. Infected cultures presented increased proliferation rates, as assessed by Ki67 immunostaining, whereas neither host cell lysis nor apoptosis were significantly augmented in infected dishes. Cytokine Bead Array indicated that IL-6 and MCP-1 were highly increased in the medium from infected cultures, whereas TGF-ß1 was consistently decreased. Inhibition of the IL-6 receptor or supplementation with recombinant TGF-ß failed to reverse the deleterious effects caused by the infection. However, conditioned medium from infected cultures inhibited myogenesis in C2C12 cells. Activation of the Wnt/ß-catenin pathway was impaired in T. gondii-infected cultures. Our data indicate that T. gondii leads SkMCs to a pro-inflammatory phenotype, leaving cells unresponsive to ß-catenin activation, and inhibition of the myogenic differentiation program. Such deregulation may suggest muscle atrophy and molecular mechanisms similar to those involved in myositis observed in human patients.

TGF-ß inhibitor therapy decreases fibrosis and stimulates cardiac improvement in a pre-clinical study of chronic Chagas' heart disease.

TGF-ß involvement in Chagas disease cardiomyopathy has been clearly demonstrated. The TGF-ß signaling pathway is activated in the cardiac tissue of chronic phase patients and is associated with an increase in extracellular matrix protein expression. The aim of this study was to investigate the effect of GW788388, a selective inhibitor of TßR1/ALK5, on cardiac function in an experimental model of chronic Chagas' heart disease. To this end, C57BL/6 mice were infected with Trypanosoma cruzi (102 parasites from the Colombian strain) and treated orally with 3mg/kg GW788388 starting at 120 days post-infection (dpi), when 100% of the infected mice show cardiac damage, and following three distinct treatment schedules: i) single dose; ii) one dose per week; or iii) three doses per week during 30 days. The treatment with GW788388 improved several cardiac parameters: reduced the prolonged PR and QTc intervals, increased heart rate, and reversed sinus arrhythmia, and atrial and atrioventricular conduction disorders. At 180 dpi, 30 days after treatment interruption, the GW3x-treated group remained in a better cardiac functional condition. Further, GW788388 treatment reversed the loss of connexin-43 enriched intercellular plaques and reduced fibrosis of the cardiac tissue. Inhibition of the TGF-ß signaling pathway reduced TGF-ß/pSmad2/3, increased MMP-9 and Sca-1, reduced TIMP-1/TIMP-2/TIMP-4, and partially restored GATA-6 and Tbox-5 transcription, supporting cardiac recovery. Moreover, GW788388 administration did not modify cardiac parasite load during the infection but reduced the migration of CD3+ cells to the heart tissue. Altogether, our data suggested that the single dose schedule was not as effective as the others and treatment three times per week during 30 days seems to be the most effective strategy. The therapeutic effects of GW788388 are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas' heart disease by TGF-ß inhibitors.

TGF-ß Polymorphisms Are a Risk Factor for Chagas Disease.

Transforming growth factor ß1 (TGF-ß1) is an important mediator in Chagas disease. Furthermore, patients with higher TGF-ß1 serum levels show a worse clinical outcome. Gene polymorphism may account for differences in cytokine production during infectious diseases. We tested whether TGFB1 polymorphisms could be associated with Chagas disease susceptibility and severity in a Brazilian population. We investigated five single-nucleotide polymorphisms (-800 G>A, -509 C>T, +10 T>C, +25 G>C, and +263 C>T). 152 patients with Chagas disease (53 with the indeterminate form and 99 with the cardiac form) and 48 noninfected subjects were included. Genotypes CT and TT at position -509 of the TGFB1 gene were more frequent in Chagas disease patients than in noninfected subjects. Genotypes TC and CC at codon +10 of the TGFB1 gene were also more frequent in Chagas disease patients than in noninfected subjects. We found no significant differences in the distribution of the studied TGFB1 polymorphisms between patients with the indeterminate or cardiac form of Chagas disease. Therefore, -509 C>T and +10 T>C TGFB1 polymorphisms are associated with Chagas disease susceptibility in a Brazilian population.

Proteins involved on TGF-ß pathway are up-regulated during the acute phase of experimental Chagas disease.

Studies developed by our group in the last years have shown the involvement of TGF-ß in acute and chronic Chagas heart disease, with elevated plasma levels and activated TGF-ß cell signaling pathway as remarkable features of patients in the advanced stages of this disease, when high levels of cardiac fibrosis is present. Imbalance in synthesis and degradation of extracellular matrix components is the basis of pathological fibrosis and TGF-ß is considered as one of the key regulators of this process. In the present study, we investigated the activity of the TGF-ß signaling pathway, including receptors and signaling proteins activation in the heart of animals experimentally infected with Trypanosoma cruzi during the period that mimics the acute phase of Chagas disease. We observed that T. cruzi-infected animals presented increased expression of TGF-ß receptors. Overexpression of receptors was followed by an increased phosphorylation of Smad2/3, p38 and ERK. Furthermore, we correlated these activities with cellular factors involved in the fibrotic process induced by TGF-ß. We observed that the expression of collagen I, fibronectin and CTGF were increased in the heart of infected animals on day 15 post-infection. Correlated with the increased TGF-ß activity in the heart, we found that serum levels of total TGF-ß were significantly higher during acute infection. Taken together, our data suggest that the commitment of the heart associates with increased activity of TGF-ß pathway and expression of its main components. Our results, confirm the importance of this cytokine in the development and maintenance of cardiac damage caused by T. cruzi infection.

Cruzipain Activates Latent TGF-ß from Host Cells during T. cruzi Invasion.

Several studies indicate that the activity of cruzipain, the main lysosomal cysteine peptidase of Trypanosoma cruzi, contributes to parasite infectivity. In addition, the parasitic invasion process of mammalian host cells is described to be dependent on the activation of the host TGF-ß signaling pathway by T. cruzi. Here, we tested the hypothesis that cruzipain could be an important activator of latent TGF-ß and thereby trigger TGF-ß-mediated events crucial for the development of Chagas disease. We found that live epimastigotes of T. cruzi, parasite lysates and purified cruzipain were able to activate latent TGF-ß in vitro. This activation could be inhibited by the cysteine peptidase inhibitor Z-Phe-Ala-FMK. Moreover, transfected parasites overexpressing chagasin, a potent endogenous cruzipain inhibitor, prevented latent TGF-ß activation. We also observed that T. cruzi invasion, as well as parasite intracellular growth, were inhibited by the administration of Z-Phe-Ala-FMK or anti-TGF-ß neutralizing antibody to Vero cell cultures. We further demonstrated that addition of purified cruzipain enhanced the invasive activity of trypomastigotes and that this effect could be completely inhibited by addition of a neutralizing anti-TGF-ß antibody. Taken together, these results demonstrate that the activities of cruzipain and TGF-ß in the process of cell invasion are functionally linked. Our data suggest that cruzipain inhibition is an interesting chemotherapeutic approach for Chagas disease not only because of its trypanocidal activity, but also due to the inhibitory effect on TGF-ß activation.