The search for biomarkers and treatments in Chagas disease: insights from TGF-beta studies and immunogenetics

International audienceThe anti-inflammatory cytokine transforming growth factor beta (TGF-β) 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-β roles and its intracellular signaling pathways, and an update of what is known about TGF-β and Chagas disease. In in vitro assays, TGF-β increases during T. cruzi infection and modulates heart cells invasion by the parasite fostering its intracellular parasite cycle. TGF-β modulates host immune response and inflammation, increases heart fibrosis, stimulates remodeling, and slows heart conduction via gap junction modulation. TGF-β signaling inhibitors reverts these effects opening a promising therapeutic approach in pre-clinical studies. CD patients with higher TGF-β1 serum level show a worse clinical outcome, implicating a predictive value of serum TGF-β as a surrogate biomarker of clinical relevance. Moreover, pre-clinical studies in chronic T. cruzi infected mice proved that inhibition of TGF-β 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-β 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-β1 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-β1 gene, leading to the conclusion that 509 C>T and +10 T>C TGF-β1 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-β as a CD biomarker

Macrophage P2X7 Receptor Function Is Reduced during Schistosomiasis: Putative Role of TGF- β

Schistosomiasis is a chronic inflammatory disease whose macrophages are involved in immunopathology modulation. Although P2X7 receptor signaling plays an important role in inflammatory responses mediated by macrophages, no reports have examined the role of P2X7 receptors in macrophage function during schistosomiasis. Thus, we evaluated P2X7 receptor function in peritoneal macrophages during schistosomiasis using an ATP-induced permeabilization assay and measurements of the intracellular Ca2+ concentration. ATP treatment induced significantly less permeabilization in macrophages from S. mansoni-infected mice than in control cells from uninfected animals. Furthermore, P2X7-mediated increases in intracellular Ca2+ levels were also reduced in macrophages from infected mice. TGF-β1 levels were increased in the peritoneal cavity of infected animals, and pretreatment of control macrophages with TGF-β1 reduced ATP-induced permeabilization, mimicking the effect of S. mansoni infection. Western blot and qRT-PCR data showed no difference in P2X7 protein and mRNA between uninfected, infected, and TGF-β1-treated groups. However, immunofluorescence analysis revealed reduced cell surface localization of P2X7 receptors in macrophages from infected and TGF-β1-treated mice compared to controls. Therefore, our data suggest that schistosomiasis reduces peritoneal macrophage P2X7 receptor signaling. This effect is likely due to the fact that infected mice have increased levels of TGF-β1, which reduces P2X7 receptor cell surface expression

Macrophage P2X7 Receptor Function Is Reduced during Schistosomiasis: Putative Role of TGF-1

Made available in DSpace on 2015-05-19T13:26:07Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) mariana_waghabietal_IOC-2014.pdf: 2680030 bytes, checksum: 57b4445d0f2cd115349999c37718bade (MD5) Previous issue date: 2014Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Imunofisiologia. Instituto de Ciências Biomédicas. Laboratório de Farmacologia Molecular e Bioquímica. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Investigação Translacional em Saúde e Meio Ambiente da Amazônia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Imunofisiologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Imunofisiologia. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Investigação Translacional em Saúde e Meio Ambiente da Amazônia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Ciências Biomédicas. Laboratório de Farmacologia Bioquímica e Molecular. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Imunofisiologia. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Investigação Translacional em Saúde e Meio Ambiente da Amazônia. Rio de Janeiro, RJ, Brasil.Schistosomiasis is a chronic inflammatory disease whose macrophages are involved in immunopathology modulation. Although P2X7 receptor signaling plays an important role in inflammatory responses mediated by macrophages, no reports have examined the role of P2X7 receptors in macrophage function during schistosomiasis.Thus, we evaluated P2X7 receptor function in peritoneal macrophages during schistosomiasis using an ATP-induced permeabilization assay and measurements of the intracellular Ca2+ concentration. ATP treatment induced significantly less permeabilization in macrophages from S. mansoni-infected mice than in control cells from uninfected animals. Furthermore, P2X7-mediated increases in intracellular Ca2+ levels were also reduced in macrophages from infected mice. TGF-1 levels were increased in the peritoneal cavity of infected animals, and pretreatment of control macrophages with TGF-1 reduced ATP-induced permeabilization, mimicking the effect of S. mansoni infection.Western blot and qRT-PCR data showed no difference in P2X7 protein andmRNAbetween uninfected, infected, and TGF-1-treated groups. However, immunofluorescence analysis revealed reduced cell surface localization of P2X7 receptors in macrophages from infected and TGF-1-treated mice compared to controls. Therefore, our data suggest that schistosomiasis reduces peritoneal macrophage P2X7 receptor signaling.This effect is likely due to the fact that infected mice have increased levels of TGF-1, which reduces P2X7 receptor cell surface expression

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

International audienceTransforming 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

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

Submitted by Sandra Infurna ([email protected]) on 2020-03-26T20:31:17Z No. of bitstreams: 1 HeleneSBarbosa_PCVieira_etal-IOC_2019.pdf: 2526535 bytes, checksum: 4801e366dc3bc1fcea874e83869d4711 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2020-03-26T20:43:59Z (GMT) No. of bitstreams: 1 HeleneSBarbosa_PCVieira_etal-IOC_2019.pdf: 2526535 bytes, checksum: 4801e366dc3bc1fcea874e83869d4711 (MD5)Made available in DSpace on 2020-03-26T20:43:59Z (GMT). No. of bitstreams: 1 HeleneSBarbosa_PCVieira_etal-IOC_2019.pdf: 2526535 bytes, checksum: 4801e366dc3bc1fcea874e83869d4711 (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Estrutural. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Inovação em Terapias, Ensino e Bioprodutos. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Ciências Biomédicas. Laboratório de Embriologia de Vertebrados. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Ciências Biomédicas. Laboratório de Embriologia de Vertebrados. Rio de Janeiro, RJ, Brasil.Sorbonne Université. INSERM, Institut de Myologie, Myology Research Center. UMRS974, Paris, France.Sorbonne Université. INSERM, Institut de Myologie, Myology Research Center. UMRS974, Paris, France.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Estrutural. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Estrutural. Rio de Janeiro, RJ, Brasil.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-β1 was consistently decreased. Inhibition of the IL-6 receptor or supplementation with recombinant TGF-β 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/β-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 β-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

Data from: Cruzipain activates latent TGF-β 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-β signaling pathway by T. cruzi. Here, we tested the hypothesis that cruzipain could be an important activator of latent TGF-β and thereby trigger TGF-β-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-β 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-β 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-β 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-β antibody. Taken together, these results demonstrate that the activities of cruzipain and TGF-β 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-β activation

DESENVOLVIMENTO DE APTÂMEROS DE DNA CONTRA ACINETOBACTER BAUMANII MULTIDROGAS RESISTENTES

Introdução/Objetivo: As infecções causadas por agentes multirresistentes são um problema de saúde mundial, com altas taxas de mortalidade. A identificação rápida dessas infecções é essencial, devido à sua natureza contagiosa, dificuldade de tratamento e custos hospitalares. Para abordar esse desafio, o desenvolvimento de métodos de detecção rápidos e acessíveis, bem como terapias alternativas, são necessários. Uma solução promissora é o uso de aptâmeros, que são moléculas capazes de interagir com bactérias. Esses aptâmeros têm potencial para reconhecer agentes infecciosos e/ou inibir suas funções. O objetivo desse estudo foi selecionar e identificar aptâmeros capazes de se ligar a células de A. baumannii multirresistentes. Métodos: Uma cepa de A. baumannii isolada de amostra clínica foi submetida ao protocolo de cell-SELEX. O sucesso das rodadas de seleção foi acompanhado por RT-PCR (curva de melting comparada com controle positivo) e citometria de fluxo com aptâmeros marcados com FAM (deslocamento de pico de fluorescência indicando ligação dos aptâmeros às células). Os aptâmeros foram identificados por sequenciamento utilizando a plataforma illumina. Resultados: Primeiramente, otimizamos uma metodologia interna, previamente descrita, baseada em cell-SELEX, para identificação de aptâmeros com execução rápida e baixo custo. Foram realizadas 15 rodadas de cell-SELEX, sendo duas negativas utilizando células de K. pneumoniae. Os ensaios de citometria de fluxo revelaram que após a 15ª rodada além dos aptâmeros selecionados se ligarem à célula alvo, eles também apresentaram preferência de ligação quando comparados com o controle negativo. O sequenciamento revelou as 10 sequencias mais frequentemente encontradas após a seleção na 13ª e 15ª rodada, sugerindo os aptâmeros mais selecionados para ligação às células alvo e provavelmente os melhores candidatos. Os aptâmeros que se mantiveram nas duas rodadas e que aumentaram o número de cópias na 15ª com relação à 13ª foram selecionados. A avaliação inicial da estrutura tridimensional revelou que 3 aptâmeros apresentavam estruturas similares, indicando uma possível convergência na seleção. Testes de confirmação da especificidade e sensibilidade para cada novo aptâmero identificado estão sendo realizados. Conclusão: Esses dados indicam que em breve a tecnologia baseada em aptâmero pode se tornar uma alternativa tangível às abordagens tradicionais ao diagnóstico e terapia de bactérias multirresistentes como A. baumanni

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

International audienceChronic 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-β (TGF-β), a key cytokine controlling extracellular matrix synthesis and degradation. TGF-β 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-β 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-β 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 10 4 parasites from the Y strain and C57BL/6 mice infected with 10 2 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-β 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-β neutralization

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

International audienceChronic 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-β (TGF-β), a key cytokine controlling extracellular matrix synthesis and degradation. TGF-β 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-β 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-β 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 10 4 parasites from the Y strain and C57BL/6 mice infected with 10 2 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-β 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-β neutralization