MARILDA SAVOIA NASCIMENTO
Projetos de Pesquisa
Unidades Organizacionais
Departamento de Patologia, Faculdade de Medicina
LIM/49 - Laboratório de Protozoologia, Hospital das Clínicas, Faculdade de Medicina
LIM/49 - Laboratório de Protozoologia, Hospital das Clínicas, Faculdade de Medicina
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- Disease Tolerance and Pathogen Resistance Genes May Underlie Trypanosoma cruzi Persistence and Differential Progression to Chagas Disease Cardiomyopathy(2018) CHEVILLARD, Christophe; NUNES, Joao Paulo Silva; FRADE, Amanda Farage; ALMEIDA, Rafael Ribeiro; PANDEY, Ramendra Pati; NASCIMENTO, Marilda Savoia; KALIL, Jorge; CUNHA-NETO, EdecioChagas disease is caused by infection with the protozoan Trypanosoma cruzi and affects over 8 million people worldwide. In spite of a powerful innate and adaptive immune response in acute infection, the parasite evades eradication, leading to a chronic persistent infection with low parasitism. Chronically infected subjects display differential patterns of disease progression. While 30% develop chronic Chagas disease cardiomyopathy (CCC)-a severe inflammatory dilated cardiomyopathy-decades after infection, 60% of the patients remain disease-free, in the asymptomatic/indeterminate (ASY) form, and 10% develop gastrointestinal disease. Infection of genetically deficient mice provided a map of genes relevant for resistance to T. cruzi infection, leading to the identification of multiple genes linked to survival to infection. These include pathogen resistance genes (PRG) needed for intracellular parasite destruction, and genes involved in disease tolerance (protection against tissue damage and acute phase death-DTG). All identified DTGs were found to directly or indirectly inhibit IFN-gamma production or Th1 differentiation. We hypothesize that the absolute need for DTG to control potentially lethal IFN-gamma PRG activity leads to T. cruzi persistence and establishment of chronic infection. IFN-gamma production is higher in CCC than ASY patients, and is the most highly expressed cytokine in CCC hearts. Key DTGs that downmodulate IFN-gamma, like IL-10, and Ebi3/IL27p28, are higher in ASY patients. Polymorphisms in PRG and DTG are associated with differential disease progression. We thus hypothesize that ASY patients are disease tolerant, while an imbalance of DTG and IFN-gamma PRG activity leads to the inflammatory heart damage of CCC.
conferenceObject Repurposed drugs acting on host mechanisms of T. cruzi invasion synergize with Benznidazole: New therapies for Chagas disease(2019) PANDEY, R. P.; NASCIMENTO, M. Savoia; BARRIOS, L.; GIBALDI, D.; LANNES-VIEIRA, J.; KALIL, J.; CUNHA-NETO, E.- The oral transmission of chagas disease in Brazil: New food supplies and travel experience(2019) FUJITA, Dennis Minoru; NASCIMENTO, Marilda Savoia; ANDRADE JUNIOR, Heitor Franco de
- High sensitivity and reproducibility of in-house ELISAs using different genotypes of Trypanosoma cruzi(2019) E, Zuleima Caballero; CORREA, Ricardo; NASCIMENTO, Marilda S.; VILLARREAL, Alcibiades; LLANES, Alejandro; KESPER JR., NorivalThe adequate choice of Trypanosoma cruzi strains as antigen source for the diagnosis of Chagas disease is still controversial due to differences in terms of accuracy reported between different diagnostic tests. In this study was determined if the genetic variability between different genotypes of T. cruzi (TcI, TcII and TcIV) affect the final diagnosis of Chagas disease. The sensitivity and specificity index of in-house ELISA tests prepared with different T. cruzi strains were evaluated with chagasic and non-chagasic control sera and using the TESA-blot as a reference test. The results of this study revealed that the sensitivity index did not vary, with percentages of 100% for all strains in both tests. However, the specificity index for ELISA tests showed differences between 92% and 98%, but were reduced to 78%-89% when Leishmania-positive sera were included. All ELISAs and TESA-blot prepared with different antigens and the recombinant Wiener test were challenged in an endemic community for Chagas disease in Panama. Both ELISAs and TESA-blot recognized the same positive sera, corroborating the sensitivity indexes (100%) found with the control sera. The TESA-blot maintained the specificity index of 100% and did not display false positives. However, the recombinant Wiener test decreased its sensitivity to 81.25%.
conferenceObject POTENTIATION OF BENZNIDAZOLE EFFECT BY COADMINISTRATION OF REPURPOSED DRUGS ACTING IN THE INVASION OF HOST CELLS BY TRYPANOSOMA CRUZI(2017) PANDEY, Ramendra P.; SAVOIA, Marilda; SABINO, Ester; KALIL, Jorge; CUNHA-NETO, Edecio- New Approaches for the Treatment of Chagas Disease(2021) PANDEY, Ramendra Pati; NASCIMENTO, Marilda Savoia; MOORE, Catrin E.; RAJ, V. Samuel; KALIL, Jorge; CUNHA-NETO, EdecioChagas disease, caused by the protozoan Trypanosoma cruzi is a neglected tropical disease with high prevalence (5.7 million in Latin America, WHO 2015), significant burden, and significant morbimortality mostly due to severe heart disorders during the chronic phase of infection. Chagas disease is endemic in Latin America, and medical care for the disease is the major expense for Brazil's Universal Healthcare System (Sistema Unico de Saude (SUS). The efficacy of the available drugs benznidazole and nifurtimox are low for the chronic phase of Chagas disease, the phase in which most patients are diagnosed, and there are frequent side effects, and drug resistance occurs. The rapid deployment of new drug regimens that are effective for the chronic phase treatment is low-cost and less toxic than the currently available therapy, which is a global priority. Repurposing drugs already in clinical use with other combinations would be the fastest and safest strategy for treating Chagas disease patients. We hypothesize that the combined treatment using repurposing drugs with benznidazole will be more efficacious than benznidazole alone. This needs to be tested further both in vitro and in animal models to understand the efficacy of the treatment before performing human clinical trials. We further hypothesize that producing nanoparticle formulation of the drugs can reduce their toxicity and improve therapeutic use.
- Vimentin and Anti Vimentin Antibodies in Chagas' Disease(2018) NASCIMENTO, Marilda Savoia; STOLF, Anna Maria Simonsen; ANDRADE JUNIOR, Heitor Franco de; PANDEY, Ramendra Pati; UMEZAWA, Eufrosina SetsuBackground: Vimentin is a main structural protein of the cell, a component of intermediate cell filaments and immersed in cytoplasm. Vimentin is mimicked by some bacterial proteins and anti-vimentin antibodies occur in autoimmune cardiac disease, as rheumatic fever. In this work we studied vimentin distribution on LLC-MK2 cells infected with T. cruzi and anti-vimentin antibodies in sera from several clinical pictures of Chagas' disease or American Trypanosomiasis, in order to elucidate any vimentin involvement in the humoral response of this pathology. Objective: We standardized an indirect immunofluorescence assay (IFI) to determine sub cellular expression in either parasites and host cells, and ELISA to evaluate anti-vimentin antibodies in sera fron chagasic patients. Methods: We analyzed the distribution of vimentin in culture cells using indirect fluorescent assays, using as external controls anti-T. cruzi sera, derived from chronic infected patients for identification of the parasites in the same model. After infection and growth of T.cruzi amastigotes, those cells express larger amounts of vimentin, with heavy staining of cytoplasm outside the parasitophorous vacuole and some particle shadowing patterns, suggesting that vimentin are associated with cell cytoplasm. Anti-vimentin antibodies were present in most American trypanosomiasis samples, but notably, they are much more present in acute (76, 9%) or clinical defined syndromes, especially cardiac disease (87, 9%). Paradoxically, they were relatively infrequent in asymptomatic (25%) infected patients, which had a clearly positive serological reaction to parasite antigens, but had low frequency of anti-vimentin antibodies, similar to controls (2,5%). Conclusion: Our current data revealed that anti-vimentin antibodies induced during T. cruzi infection could be a marker of active disease in the host and its levels could also justify drug therapy in American Trypanosomiasis chronic infection, as a large group of asymptomatic patients would be submitted to treatment with frequent adverse reactions of the available drugs. Anti-vimentin antibodies could be a marker of cardiac muscle cell damage, appearing in American Trypanosomiasis patients during active muscle cell damage.
- Drug Repurposing in Chagas Disease: Chloroquine Potentiates Benznidazole Activity against Trypanosoma cruzi In Vitro and In Vivo(2022) PANDEY, Ramendra P.; NASCIMENTO, Marilda Savoia; FRANCO, Caio Haddad; BORTOLUCI, Karina; SILVA, Marcelo Nunes; ZINGALES, Bianca; GIBALDI, Daniel; BARRIOS, Leda Castano; LANNES-VIEIRA, Joseli; CARISTE, Leonardo Moro; VASCONCELOS, Jose Ronnie; MORAES, Carolina Borsoi; FREITAS-JUNIOR, Lucio H.; KALIL, Jorge; ALCANTARA, Laura; CUNHA-NETO, EdecioDrug combinations and drug repurposing have emerged as promising strategies to develop novel treatments for infectious diseases, including Chagas disease. In this study, we aimed to investigate whether the repurposed drugs chloroquine (CQ) and colchicine (COL), known to inhibit Trypanosoma cruzi infection in host cells, could boost the anti-T. cruzi effect of the trypanocidal drug benznidazole (BZN), increasing its therapeutic efficacy while reducing the dose needed to eradicate the parasite. The combination of BZN and COL exhibited cytotoxicity to infected cells and low antiparasitic activity. Conversely, a combination of BZN and CQ significantly reduced T. cruzi infection in vitro, with no apparent cytotoxicity. This effect seemed to be consistent across different cell lines and against both the partially BZN-resistant Y and the highly BZN-resistant Colombiana strains. In vivo experiments in an acute murine model showed that the BZN+CQ combination was eight times more effective in reducing T. cruzi infection in the acute phase than BZN monotherapy. In summary, our results demonstrate that the concomitant administration of CQ and BZN potentiates the trypanocidal activity of BZN, leading to a reduction in the dose needed to achieve an effective response. In a translational context, it could represent a higher efficacy of treatment while also mitigating the adverse effects of high doses of BZN. Our study also reinforces the relevance of drug combination and repurposing approaches in the field of Chagas disease drug discovery.
- STING Signaling Drives Production of Innate Cytokines, Generation of CD8(+) T Cells and Enhanced Protection Against Trypanosoma cruzi Infection(2022) VIEIRA, Raquel de Souza; NASCIMENTO, Marilda Savoia; NORONHA, Isau Henrique; VASCONCELOS, Jose Ronnie Carvalho; BENVENUTI, Luiz Alberto; BARBER, Glen N.; CAMARA, Niels Olsen Saraiva; KALIL, Jorge; CUNHA-NETO, Edecio; ALMEIDA, Rafael RibeiroA variety of signaling pathways are involved in the induction of innate cytokines and CD8(+) T cells, which are major players in protection against acute Trypanosoma cruzi infection. Previous data have demonstrated that a TBK-1/IRF3-dependent signaling pathway promotes IFN-beta production in response to Trypanosoma cruzi, but the role for STING, a main interactor of these proteins, remained to be addressed. Here, we demonstrated that STING signaling is required for production of IFN-beta, IL-6, and IL-12 in response to Trypanosoma cruzi infection and that STING absence negatively impacts activation of IRF-dependent pathways in response to the parasite. We reported no significant activation of IRF-dependent pathways and cytokine expression in RAW264.7 macrophages in response to heat-killed trypomastigotes. In addition, we showed that STING is essential for T. cruzi DNA-mediated induction of IFN-beta, IL-6, and IL-12 gene expression in RAW264.7 macrophages. We demonstrated that STING-knockout mice have significantly higher parasitemia from days 5 to 8 of infection and higher heart parasitism at day 13 after infection. Although we observed similar heart inflammatory infiltrates at day 13 after infection, IFN-beta, IL-12, CXCL9, IFN-gamma, and perforin gene expression were lower in the absence of STING. We also showed an inverse correlation between parasite DNA and the expression of CXCL9, IFN-gamma, and perforin genes in the hearts of infected animals at day 13 after infection. Finally, we reported that STING signaling is required for splenic IFN-beta and IL-6 expression early after infection and that STING deficiency results in lower numbers of splenic parasite-specific IFN-gamma and IFN-gamma/perforin-producing CD8(+) T cells, indicating a pivotal role for STING signaling in immunity to Trypanosoma cruzi.
- Integration of miRNA and gene expression profiles suggest a role for miRNAs in the pathobiological processes of acute Trypanosoma cruzi infection(2017) FERREIRA, Ludmila Rodrigues Pinto; FERREIRA, Frederico Moraes; LAUGIER, Laurie; CABANTOUS, Sandrine; NAVARRO, Isabela Cunha; CANDIDO, Darlan da Silva; RIGAUD, Vagner Carvalho; REAL, Juliana Monte; PEREIRA, Glaucia Vilar; PEREIRA, Isabela Resende; RUIVO, Leonardo; PANDEY, Ramendra Pati; SAVOIA, Marilda; KALIL, Jorge; LANNES-VIEIRA, Joseli; NAKAYA, Helder; CHEVILLARD, Christophe; CUNHA-NETO, EdecioChagas disease, caused by the parasite Trypanosoma cruzi, is endemic in Latin America. Its acute phase is associated with high parasitism, myocarditis and profound myocardial gene expression changes. A chronic phase ensues where 30% develop severe heart lesions. Mouse models of T. cruzi infection have been used to study heart damage in Chagas disease. The aim of this study was to provide an interactome between miRNAs and their targetome in Chagas heart disease by integrating gene and microRNA expression profiling data from hearts of T. cruzi infected mice. Gene expression profiling revealed enrichment in biological processes and pathways associated with immune response and metabolism. Pathways, functional and upstream regulator analysis of the intersections between predicted targets of differentially expressed microRNAs and differentially expressed mRNAs revealed enrichment in biological processes and pathways such as IFN gamma, TNF alpha, NF-kappa B signaling signatures, CTL-mediated apoptosis, mitochondrial dysfunction, and Nrf2-modulated antioxidative responses. We also observed enrichment in other key heart disease-related processes like myocarditis, fibrosis, hypertrophy and arrhythmia. Our correlation study suggests that miRNAs may be implicated in the pathophysiological processes taking place the hearts of acutely T. cruzi-infected mice.