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Item Amlodipine increases the therapeutic potential of ravuconazole upon Trypanosoma cruzi infection.(2020) Machado, Yara Almeida; Bahia, Maria Terezinha; Caldas, Ivo Santana; Mazzeti, Ana Lia; Novaes, Rômulo Dias; Vilas Boas, Breno Raimundo; Santos, Lorena Júnia de Souza; Martins Filho, Olindo Assis; Marques, Marcos José; Diniz, Lívia de FigueiredoMining existing agents that enhance the therapeutic potential of ergosterol biosynthesis inhibitors (EBI) is a promising approach to improve Chagas disease chemotherapy. In this study, we evaluated the effect of ravuconazole, an EBI, combined with amlodipine, a calcium channel blocker, upon Trypanosoma cruzi experimental infection. In vitro assays confirmed the trypanocidal activity of both compounds in monotherapy and demonstrated an additive effect (sum of the fractional inhibitory concentration [FIC] 0.5) of the combined treatment without additional toxicity to host cells. In vivo experiments, using a murine model of the T. cruzi Y strain in a short-term protocol, demonstrated that amlodipine, although lacking trypanocidal activity, dramatically increased the antiparasitic activity of underdosing ravuconazole regimens. Additional analysis using long-term treatment (20 days) showed that parasitemia relapse until 60 days after treatment was significatively lower in mice treated with the combination (4 out of 14 mice) than ravuconazole monotherapy (10 out of 14 mice), even in the presence of immunosuppressant pressure. Furthermore, the combined therapy was well tolerated and protected the mice from mortality. The treatments also impacted on the cellular and humoral immune response of infected animals, inducing a reduction of serum cytokine levels in all ravuconazole-treated mice. Our findings demonstrate that amlodipine is efficacious in enhancing the antiparasitic activity of ravuconazole in an experimental model of T. cruzi infection and indicates a potential strategy to be explored in Chagas disease treatment.Item Anti-adrenergic and muscarinic receptor autoantibodies in a canine model of Chagas disease and their modulation by benznidazole.(2014) Daliry, Anissa; Caldas, Ivo Santana; Diniz, Lívia de Figueiredo; Torres, Rosália Morais; Silva, André Talvani Pedrosa da; Bahia, Maria Terezinha; Carvalho, Antônio Carlos Campos deAnti-adrenergic and muscarinic receptor autoantibodies in a canine model of Chagas disease and their modulation by benznidazole.Item Antitrypanosomal activity of fexinidazole metabolites, potential new drug candidates for Chagas disease.(2014) Bahia, Maria Terezinha; Nascimento, Álvaro Fernando da Silva do; Mazzeti, Ana Lia; Marques, Luiz F.; Gonçalves, Karolina Ribeiro; Mota, Ludmilla Walter Reis; Diniz, Lívia de Figueiredo; Caldas, Ivo Santana; Silva, André Talvani Pedrosa da; Shackleford, David M.; Koltun, Maria; Saunders, Jessica; White, Karen L.; Scandale, Ivan; Charman, Susan A.; Chatelain, EricThis study was designed to verify the in vivo efficacy of sulfoxide and sulfone fexinidazole metabolites following oral administration in a murine model of Chagas disease. Female Swiss mice infected with the Y strain of Trypanosoma cruzi were treated orally once per day with each metabolite at doses of 10 to 100 mg/kg of body weight for a period of 20 days. Parasitemia was monitored throughout, and cures were detected by parasitological and PCR assays. The results were compared with those achieved with benznidazole treatment at the same doses. Fexinidazole metabolites were effective in reducing the numbers of circulating parasites and protecting mice against death, compared with untreated mice, but without providing cures at daily doses of 10 and 25 mg/kg. Both metabolites were effective in curing mice at 50 mg/kg/day (30% to 40%) and 100 mg/kg/day (100%). In the benznidazole- treated group, parasitological cure was detected only in animals treated with the higher dose of 100 mg/kg/day (80%). Single-dose pharmacokinetic parameters for each metabolite were obtained from a parallel group of uninfected mice and were used to estimate the profiles following repeated doses. Pharmacokinetic data suggested that biological efficacy most likely resides with the sulfone metabolite (or subsequent reactive metabolites formed following reduction of the nitro group) following administration of either the sulfoxide or the sulfone and that prolonged plasma exposure over the 24-h dosing window is required to achieve high cure rates. Fexinidazole metabolites were effective in treating T. cruzi in a mouse model of acute infection, with cure rates superior to those achieved with either fexinidazole itself or benznidazole.Item Association between nutritional status, C-reactive protein, adiponectin and HOMA-AD in Brazilian children.(2014) Domingos, Ana Luiza Gomes; Coelho, George Luiz Lins Machado; Volp, Ana Carolina Pinheiro; Oliveira, Fernando Luiz Pereira de; Caldas, Ivo Santana; Freitas, Silvia Nascimento deIntroduction: In children, the presence of obesity is a major risk factor for the occurrence of cardiovascular diseases on the adulthood. Objective: To evaluate the association of anthropometry, body composition, clinical variables and biochemical profile with C-reactive protein and adiponectin levels, and insulin resistance in children in the municipality of Nova Era, Brazil. Methods: Nested case-control study following a crosssectional study. We evaluated 178 children, 57 of them classified as obese and 121 as normal-weight from a population of 1024 schoolchildren 6 to 10 years old: Blood samples were collected after 12-hour fast to obtain serum and plasma. We collected anthropometric and body composition measures, systolic and diastolic blood pressure data. Sexual maturation was assessed according to the stage of sexual development. We performed Student’s t-test, Mann-Whitney U test, Pearson’s correlation, Spearman’s test and multiple linear regression analysis. Independent variables with p < 0.05 were included in the multiple regression model. Residual analysis was performed to assess model validity. Results: Among obese children, C-reactive protein levels were associated with triacylglycerol levels and body fat percentage estimated by skinfold thickness (R2 adjusted = 27.6%, p < 0.001). Adiponectin was associated with HOMA-IR, HOMAAD and body fat percentage estimated by skinfold thickness (R2 adjusted = 75.5%, p < 0.001). HOMA-AD index was associated with HOMA-IR, adiponectin, systolic blood pressure and weight (R2 adjusted = 90.7%, p < 0.001). Conclusion: Significant associations were found between body composition, anthropometry, clinical variables, biochemical profile and adiponectin and C-reactive protein levels and insulin resistance in obese and normal-weight children.Item Avaliação da eficácia do tratamento específico com Benznidazol na progressão da doença de Chagas Experimental e a correlação entre a eficácia do tratamento nos modelos canino e murino.(Programa de Pós-Graduação em Ciências Biológicas. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto., 2008) Caldas, Ivo Santana; Bahia, Maria TerezinhaNeste trabalho foi avaliado o benefício do tratamento precoce da doença de Chagas experimental na diminuição das lesões cardíacas de cães e camundongos inoculados com cepas sensíveis (Be-78), parcialmente sensíveis (Y, ABC e AAS) e resistentes (VL10) ao Benznidazol. Em adição, foi avaliada a influência do tratamento específico na evolução dos anticorpos da classe IgG e das subclasses IgG1 e IgG2 (cães) e IgG1, IgG2a e IgG2b (camundongos) na tentativa de identificar um novo marcador sorológico para ser utilizado no controle de cura da doença de Chagas. O tratamento com Benznidazol previniu a inflamação e a fibrose no miocárdio de todos os cães e camundongos experimentalmente infectados que alcançaram a cura parasitológica, exceto nos cães inoculados com a cepa Y do T.cruzi, que apresentaram fibrose. Entre os cães tratados e não curados o tratamento induziu a diferentes padrões de resposta: (1) não foi observada redução da inflamação e nem da fibrose no tecido cardíaco dos animais inoculados com as cepas ABC e VL10, (2) redução das lesões cardíacas dos cães não curados inoculados com a cepa AAS. De forma diferente, o tratamento específico não foi capaz de diminuir a intensidade de inflamação no miocárdio de nenhum camundongo tratado e não curado inoculado com as cepas ABC, AAS e VL10, entretanto preveniu a fibrose entre os animais inoculados com a cepa VL10. Os resultados da dosagem de anticorpos específicos mostraram que a intensidade da inflamação no miocárdio dos animais inoculados com as diferentes cepas do T. cruzi e não tratados pode ser correlacionada com os níveis de IgG1 nos dois modelos experimentais usados neste estudo. Foi observado que quanto maior é o infiltrado inflamatório no miocárdio de camundongos e cães inoculados com as cepas Be-78, Y, ABC, AAS e VL10 do T.cruzi, maiores são os níveis de IgG1 detectados no soro. O tratamento específico induziu uma redução nos níveis de todas as classes e subclasses de imunoglobulinas avaliadas. Entretanto a negativação da sorologia foi observada apenas nos animais tratados e curados, nos tratados que não alcançaram a cura parasitológica os níveis de todas as classes e subclasses de imunoglobulinas avaliadas atingiram níveis próximos ao observado nos soros dos animais controle infectado.Item Benznidazole alters the pattern of Cyclophosphamide-induced reactivation in experimental Trypanosoma cruzi-dependent lineage infection.(2010) Santos, Daniela Maria dos; Martins, Tassiane Assíria Fontes; Caldas, Ivo Santana; Diniz, Lívia de Figueiredo; Coelho, George Luiz Lins Machado; Carneiro, Cláudia Martins; Oliveira, Riva de Paula; Silva, André Talvani Pedrosa da; Lana, Marta de; Bahia, Maria TerezinhaThe factors involved in the reactivation of chronic Chagas disease infection are not clear enough and may be related to host immune unbalance and/or parasite genetic diversity. To evaluate the role of the Trypanosoma cruzi genetic background in the Chagas disease reactivation, we inoculated Cyclophosphamide-immunos upressed (CyI) Swiss mice with clonal stocks from T. cruzi I (Cuica cl1, P209 cl1, Gamba cl1, SP104 cl1), T. cruzi II (IVV cl4, MVB cl8) and T. cruzi (Bug2148 cl1, MN cl2) lineages. We used the parasitemia as the parameter for Chagas disease reactivation and observed that CyI animals infected with T. cruzi stocks showed no reactivation and those infected with T. cruzi II stocks showed only 5% of reactivation. In contrast, immunosuppressed mice infected with stocks from T. cruzi I lineage showed 77.5 and 51.25% reactivation of the infection when Cyclophosphamide treatment was performed 60 and 180 days after inoculation, respectively. Next, we evaluated the efficacy of the Benznidazole (Bz) pre-treatment in reducing or preventing the recurrence of the infection in these CyI animals. In general, the percentage of the parasite recurrence was not altered among the CyI mice that received the Bz pretreatment during the acute phase of the infection. Interestingly, when pre-Bz treatment was performed during the chronic phase, we observed two different patterns of response: (i) an increased protection among the animals inoculated with the SP104 cl1 (genotype 19) and Cuica cl1 (genotype 20) stocks; (ii) an increased percentage of parasitemia reactivation among mice inoculated with Gamba cl1 (genotype 19) and P209 cl1 (genotype 20) T. cruzi stocks. Our results corroborate our hypothesis by showing that the T. cruzi genetic background in combination with specific Bz treatment has an important role in the Chagas disease reactivation in immunosuppressed animals.Item Benznidazole and Posaconazole in experimental Chagas disease: positive interaction in concomitant and sequential treatments.(2013) Diniz, Lívia de Figueiredo; Urbina, Julio Alberto; Andrade, Isabel Mayer de; Mazzeti, Ana Lia; Martins, Tassiane Assíria Fontes; Caldas, Ivo Santana; Silva, André Talvani Pedrosa da; Ribeiro, Isabela; Bahia, Maria TerezinhaBackground: Current chemotherapy for Chagas disease is unsatisfactory due to its limited efficacy, particularly in the chronic phase, with frequent side effects that can lead to treatment discontinuation. Combined therapy is envisioned as an ideal approach since it may improve treatment efficacy whilst decreasing toxicity and the likelihood of resistance development. We evaluated the efficacy of posaconazole in combination with benznidazole on Trypanosoma cruzi infection in vivo. Methods and Findings: Benznidazole and posaconazole were administered individually or in combination in an experimental acute murine infection model. Using a rapid treatment protocol for 7 days, the combined treatments were more efficacious in reducing parasitemia levels than the drugs given alone, with the effects most evident in combinations of sub-optimal doses of the drugs. Subsequently, the curative action of these drug combinations was investigated, using the same infection model and 25, 50, 75 or 100 mg/kg/day (mpk) of benznidazole in combination with 5, 10 or 20 mpk of posaconazole, given alone or concomitantly for 20 days. The effects of the combination treatments on parasitological cures were higher than the sum of such effects when the drugs were administered separately at the same doses, indicating synergistic activity. Finally, sequential therapy experiments were carried out with benznidazole or posaconazole over a short interval (10 days), followed by the second drug administered for the same period of time. It was found that the sequence of benznidazole (100 mpk) followed by posaconazole (20 mpk) provided cure rates comparable to those obtained with the full (20 days) treatments with either drug alone, and no cure was observed for the short treatments with drugs given alone. Conclusions: Our data demonstrate the importance of investigating the potential beneficial effects of combination treatments with marketed compounds, and showed that combinations of benznidazole with posaconazole have a positive interaction in murine models of Chagas disease.Item Benznidazole therapy during acute phase of chagas disease reduces parasite load but does not prevent chronic cardiac lesions.(2008) Caldas, Ivo Santana; Silva, André Talvani Pedrosa da; Caldas, Sérgio; Carneiro, Cláudia Martins; Lana, Marta de; Guedes, Paulo Marcos da Matta; Bahia, Maria TerezinhaThe goals of this study were to evaluate the efficacy of benznidazole (Bz) treatment in decreasing of the parasitic load during the acute phase of experimental Chagas disease and to analyze its influence in the development of cardiac chronic alterations in mice inoculated with drug-resistant Trypanosoma cruzi strains. Our results showed that the early Bz treatment (started at 4th day of infection) was efficient in reducing the parasite load in animals from both acute and chronic phase of the infection. Moreover, this reduction in the parasite load could not be associated with the intensity of the cardiac chronic lesions. The histopathological evaluation of cardiac tissue of Bz-treated mice showed three different patterns of response: (1) presence of a small number of inflammatory cells and fibrotic area similar to noninfected mice; (2) similar intensity of inflammatory infiltrate and smaller fibrotic area in relation to nontreated animals; (3) similar intensity of inflammatory infiltrated and fibrosis area among the Bz-treated and nontreated animals. Each specific pattern was obtained with different T. cruzi strain, suggesting that the pattern of the heart lesions in chronic phase of Bz-treated animals was T. cruzi strain dependent but not related with drug resistance levels.Item Benznidazole/Itraconazole combination treatment enhances anti-Trypanosoma cruzi activity in experimental Chagas disease.(2015) Martins, Tassiane Assíria Fontes; Diniz, Lívia de Figueiredo; Mazzeti, Ana Lia; Nascimento, Álvaro Fernando da Silva do; Caldas, Sérgio; Caldas, Ivo Santana; Andrade, Isabel Mayer de; Ribeiro, Isabela; Bahia, Maria TerezinhaThe nitroheterocyclic drugs nifurtimox and benznidazole are first-line drugs available to treat Chagas disease; however, they have limitations, including long treatment courses and toxicity. Strategies to overcome these limitations include the identification of new drugs with specific target profiles, re-dosing regimens for the current drugs, drug repositioning and combination therapy. In this work, we evaluated combination therapy as an approach for optimization of the current therapeutic regimen for Chagas disease. The curative action of benznidazole/itraconazole combinations was explored in an established infection of the mice model with the T. cruzi Y strain. The activities of the benznidazole/itraconazole combinations were compared with the results from those receiving the same dosage of each individual drug. The administration of benznidazole/itraconazole in combination eliminated parasites from the blood more efficiently than each drug alone. Here, there was a significant reduction of the number of treatment days (number of doses) necessary to induce parasitemia suppression with the benznidazole/itraconazole combination, as compared to each compound administered alone. These results clearly indicate the enhanced effects of these drugs in combination, particularly at the dose of 75 mg/kg, as the effects observed with the drug combinations were four times more effective than those of each drug used alone. Moreover, benznidazole/itraconazole treatment was shown to prevent or decrease the typical lesions associated with chronic experimental Chagas disease, as illustrated by similar levels of inflammatory cells and fibrosis in the cardiac muscle tissue of healthy and treated mice. These results emphasize the importance of exploring the potential of combination treatments with currently available compounds to specifically treat Chagas disease.Item Canine visceral leishmaniasis in the Krenak indigenous community, Resplendor, Minas Gerais State, Brazil, 2007.(2011) Antônio, Eloiza Gonçalves; Malacco, Marcos Aurélio Fulgêncio; Gontijo, Célia Maria Ferreira; Moreira, Eliana Furtado; Caldas, Ivo Santana; Pena, João Luiz; Coelho, George Luiz Lins MachadoItem Correlação entre a carga parasitária do Trypanosoma cruzi no tecido cardíaco e detecção de autoanticorpos no soro de cães infectados com a patologia da doença de chagas experimental.(2015) Menezes, Ana Paula de Jesus; Caldas, Ivo Santana; Caldas, Sérgio; Bahia, Maria Terezinha; Roatt, Bruno Mendes; Marques, Marcos JoséNa cardiomiopatia Chagásica Crônica a presença do parasito parece ser essencial para a perpetuação da inflamação, entretanto devido à intensidade das lesões com escassos parasitos, o fenômeno de autoimunidade é apontado como importante na indução da miocardite. Dessa forma, o uso de drogas tripanocidas pode alterar a patogênese ao diminuir o parasitismo. No Brasil a droga indicada, o Benznidazol (Bz), gera cura parasitológica somente no tratamento realizado na fase aguda da infecção.O objetivo deste estudo foi quantificar o parasitismo no tecido cardíaco e avaliar a presença de autoanticorpos no soro de cães inoculados com diferentes cepas do T. cruzi e correlacionar a carga parasitária e a autoimunidade com a intensidade das lesões cardíacas. Sessenta cães sem raça definida, nascidos e mantidos no canil da UFOP foram infectados com 2,0x103 tripomastigotas metacíclicos/Kg das cepas Berenice-78, AAS e VL-10 do T.cruzi, e após a confirmação da infecção, metade dos cães foi tratada com Bz e a outra metade mantida sem tratamento. Noventa dias após a infecção, 30 animais foram eutanasiados na fase aguda, sendo os outros 30 na fase crônica da infecção. O átrio direito foi seccionado para análise da intensidade do infiltrado inflamatório por H&E, e utilizado para a quantificação do T.cruzi por qPCR. Além da detecção de autoanticorpos por immunoblotting no soro. Todos os grupos experimentais apresentaram miocardite intensa tanto na fase aguda, quanto na fase crônica. A quantificação do DNA do parasito nos animais infectados e não tratados variou de 5,0x102 a 2,6x106cópias/μL na fase aguda e na fase crônica o pico de quantificação foi de 4,9x105 cópias/μL. O tratamento com Bz reduziu significativamente o parasitismo cardíaco entre os animais infectados com a cepa VL-10 e Berenice-78(p<0.05) durante a fase aguda da infecção. Entretanto,na fase crônica não foi houve redução da carga parasitária entre os cães infectados pela cepa VL-10, não sendo possível avaliar a redução entre as outras duas cepas. A análise entre inflamação cardíaca e o parasitismo revelou uma correlação positiva entre o aumento da quantidade de DNA do parasito com o aumento da inflamação. Em relação à autoimunidade, foi observada maior produção de autoanticorpos durante a fase aguda em relação à fase crônica sem correlação com a miocardite.Item Development of chronic cardiomyopathy in canine Chagas disease correlates with high IFN-g, TNF-a, and low IL-10 production during the acute infection phase.(2009) Guedes, Paulo Marcos da Matta; Veloso, Vanja Maria; Afonso, Luís Carlos Crocco; Caliari, Marcelo Vidigal; Carneiro, Cláudia Martins; Diniz, Lívia de Figueiredo; Silva, Eduardo de Almeida Marques da; Caldas, Ivo Santana; Matta, Maria Adelaide do Valle; Souza, Sheler Martins de; Lana, Marta de; Chiari, Egler; Galvão, Lúcia Maria da Cunha; Bahia, Maria TerezinhaWhen infected with Trypanosoma cruzi, Beagle dogs develop symptoms similar to those of Chagas disease in human beings, and could be an important experimental model for a better understanding of the immunopathogenic mechanisms involved in chronic chagasic infection. This study evaluates IL-10, IFN-g and TNF-a production in the sera, culture supernatant, heart and cervical lymph nodes and their correlation with cardiomegaly, cardiac inflammation and fibrosis in Beagle dogs infected with T. cruzi. Pathological analysis showed severe splenomegaly, lymphadenopathy and myocarditis in all infected dogs during the acute phase of the disease, with cardiomegaly, inflammation and fibrosis observed in 83% of the animals infected by T. cruzi during the chronic phase. The data indicate that infected animals producing IL-10 in the heart during the chronic phase and showing high IL-10 production in the culture supernatant and serum during the acute phase had lower cardiac alterations (myocarditis, fibrosis and cardiomegaly) than those with high IFN-g and TNF-a levels. These animals produced low IL-10 levels in the culture supernatant and serum during the acute phase and did not produce IL-10 in the heart during the chronic phase of the disease. Our findings showed that Beagle dogs are a good model for studying the immunopathogenic mechanism of Chagas disease, since they reproduce the clinical and immunological findings described in chagasic patients. The data suggest that the development of the chronic cardiac form of the disease is related to a strong Th1 response during the acute phase of the disease, while the development of the indeterminate form results from a blend of Th1 and Th2 responses soon after infection, suggesting that the acute phase immune response is important for the genesis of chronic cardiac lesions.Item Effects of ravuconazole treatment on parasite load and immune response in dogs experimentally infected with trypanosoma cruzi.(2010) Diniz, Lívia de Figueiredo; Caldas, Ivo Santana; Guedes, Paulo Marcos da Matta; Crepalde, Geovam Pereira; Lana, Marta de; Carneiro, Cláudia Martins; Silva, André Talvani Pedrosa da; Urbina, Julio Alberto; Bahia, Maria TerezinhaIn this work, we investigated the in vivo activity of ravuconazole against the Y and Berenice-78 Trypanosoma cruzi strains using acutely infected dogs as hosts. Ravuconazole was well tolerated, as no significant side effects were observed during the treatment using 6.0 mg/kg twice a day (12 mg/kg/day) for up to 90 days. In all treated animals, parasitemia was permanently suppressed by the first day of treatment, independently of the parasite strain. Cultures of blood obtained posttreatment were negative for 90% of the animals, confirming that the drug induced a marked reduction in the parasite load. The results of PCR tests for T. cruzi in blood performed 1 month posttreatment were consistently negative for three of five and two of five animals infected with the Y and Berenice-78 strains, respectively. All ravuconazole-treated dogs consistently had negative serological test results during and until 30 days after treatment, regardless of the therapeutic scheme used. However, after the end of treatment, an increase in specific antibody levels was observed in all treated animals, although the antibody levels were always significantly lower than those of the nontreated control dogs. Despite being unable to induce a parasitological cure, ravuconazole treatment led to significant reductions in the levels of gamma interferon expression and lesions in cardiac tissues in animals infected with the Y strain, while the level of interleukin-10 mRNA expression increased. We conclude that ravuconazole has potent suppressive but not curative activity in the canine model of acute Chagas' disease, probably due to its unfavorable pharmacokinetic properties (half-life, 8.8 h). The longer half-life of ravuconazole in humans (4 to 8 days) makes it a promising drug for assessment for use as chemotherapy in human Chagas' disease.Item Eficácia da combinação de ravuconazol ou seu pró-fármaco E1224 com o metabólito sulfona do fexinidazol no tratamento da infecção experimental por Trypanosoma cruzi.(2014) Mota, Ludmilla Walter Reis; Diniz, Lívia de Figueiredo; Bahia, Maria Terezinha; Caldas, Ivo Santana; Soares, Rodrigo Pedro Pinto; Diniz, Lívia de FigueiredoO objetivo do presente estudo foi avaliar o efeito do tratamento com o derivado sulfona do fexinidazol (SfN) em combinação com o E1224 (pró-fármaco do ravuconazol- Rv) na infecção experimental por T. cruzi. Após a determinação dos valores de IC-50 de SfN e Rv sobre a infecção de células H9c2 pela cepa Y, foi determinada a natureza da interação entre esses fármacos in vitro. As células infectadas foram incubadas com os diferentes fármacos isoladamente ou combinados em proporções fixas. SfN e Rv não apresentaram interação in vitro na eliminação de formas amastigotas, resultando em efeito aditivo (média de ΣFIC= 1,08±0,7). Adicionalmente, as combinações de fármacos não induziram citotoxicidade em células H9c2 na ausência de infecção. A partir desses dados foi investigado o efeito in vivo das combinações usando modelo murino de infecção aguda. Nessa etapa camundongos Swiss fêmeas foram inoculados com formas tripomastigotas sanguíneas da cepa Y de T. cruzi. Após a detecção da parasitemia os animais foram tratados com doses subótimas de E1224 (2,5 e 5 mg por Kg de peso corporal-mg/Kg) e fex-sulfona (25 e 50mg/Kg), isoladamente ou em combinação por 20 dias. Foi também avaliado o efeito do tratamento na resposta imune humoral, por meio da detecção de anticorpos IgG anti-T-cruzi aos 180 dias após o tratamento. Os resultados mostraram que todos os tratamentos foram bem tolerados e eficientes em proteger os animais da mortalidade que acometeu 100% dos animais infectados e não tratados. A subdoses de SfN e E1224 quando combinadas foram mais eficientes em induzir cura do que quando usadas em monoterapia. Nos grupos tratados com E1224 nas doses de 2,5 e 5 mg/Kg, foi observada cura em 2/7 (28,5%) e 3/7 (42,8%) dos animais, respectivamente. No caso dos tratamentos com SfN, foi observada reativação natural da parasitemia em todos os animais. De forma interessante, nos grupos de animais tratados com ambas as combinações, 85,7% de cura foi observada, percentual próximo do obtido com a utilização de doses ótimas de cada fármaco. Esses resultados demonstraram o benefício resultante da administração combinada de SfN e E1224 in vivo e indicaram que combinações aditivas in vitro devem ser submetidas a avaliações in vivo.Item Fexinidazole : a potential new drug candidate for Chagas disease.(2012) Bahia, Maria Terezinha; Andrade, Isabel Mayer de; Martins, Tassiane Assíria Fontes; Nascimento, Álvaro Fernando da Silva do; Diniz, Lívia de Figueiredo; Caldas, Ivo Santana; Silva, André Talvani Pedrosa da; Trunz, Bernadette Bourdin; Torreele, Els; Ribeiro, IsabelaBackground: New safe and effective treatments for Chagas disease (CD) are urgently needed. Current chemotherapy options for CD have significant limitations, including failure to uniformly achieve parasitological cure or prevent the chronic phase of CD, and safety and tolerability concerns. Fexinidazole, a 2-subsituted 5-nitroimidazole drug candidate rediscovered following extensive compound mining by the Drugs for Neglected Diseases initiative and currently in Phase I clinical study for the treatment of human African trypanosomiasis, was evaluated in experimental models of acute and chronic CD caused by different strains of Trypanosoma cruzi. Methods and Findings: We investigated the in vivo activity of fexinidazole against T. cruzi, using mice as hosts. The T. cruzi strains used in the study were previously characterized in murine models as susceptible (CL strain), partially resistant (Y strain), and resistant (Colombian and VL-10 strains) to the drugs currently in clinical use, benznidazole and nifurtimox. Our results demonstrated that fexinidazole was effective in suppressing parasitemia and preventing death in infected animals for all strains tested. In addition, assessment of definitive parasite clearance (cure) through parasitological, PCR, and serological methods showed cure rates of 80.0% against CL and Y strains, 88.9% against VL-10 strain, and 77.8% against Colombian strain among animals treated during acute phase, and 70% (VL-10 strain) in those treated in chronic phase. Benznidazole had a similar effect against susceptible and partially resistant T. cruzi strains. Fexinidazole treatment was also shown to reduce myocarditis in all animals infected with VL-10 or Colombian resistant T. cruzi strains, although parasite eradication was not achieved in all treated animals at the tested doses. Conclusions: Fexinidazole is an effective oral treatment of acute and chronic experimental CD caused by benznidazolesusceptible, partially resistant, and resistant T. cruzi. These findings illustrate the potential of fexinidazole as a drug candidate for the treatment of human CD.Item Genetic modulation in Be-78 and Y Trypanosoma cruzi strains after long-term infection in Beagle dogs revealed by molecular markers.(2012) Veloso, Vanja Maria; Guedes, Paulo Marcos da Matta; Lana, Marta de; Martins, Helen Rodrigues; Carneiro, Cláudia Martins; Câmara, Antônia Cláudia Jácome da; D’Ávila, Daniella Alchaar; Caldas, Ivo Santana; Galvão, Lúcia Maria da Cunha; Chiari, Egler; Bahia, Maria TerezinhaThe genetic profile of Trypanosoma cruzi was evaluated in parasite populations isolated from Beagle dogs experimentally infected with Be-78 and Y strains that present distinct biological and genetic characteristics. Molecular characterization of the isolates obtained 30 days and 2 years after infection was carried out. For typing MLEE, sequence polymorphisms of the mitochondrial cytochrome oxidase subunit II gene (COII) and RAPD profiles were used. The profiles of MLEE were the same for the parental Be-78 strains as their respective isolates. However, changes of MLEE profile were observed in two T. cruzi isolates from dogs inoculated with Y strain. Changes in the mitochondrial DNA (COII) and RAPD profiles of the Y strain were also observed. The dendogram constructed by UPGMA with RAPD results indicated two major branches. Global data show that the genetic modulation in polyclonal strains during the long-term infection occurred and was strain-dependent. This study still suggests that each host (here each dog) harbors a determinate T. cruzi population that may change or be modulated throughout long-term infection. This might to hinder the observation of correlation between the genetics of T. cruzi and their biological properties and behavior in different host species due to the complexity of the parasite-host interaction in which probably the genetic background of both should be considered.Item Hematological alterations during experimental canine infection by Trypanosoma cruzi.(2012) Guedes, Paulo Marcos da Matta; Veloso, Vanja Maria; Mineo, Tiago Wilson Patriarca; Silva, Juliana Santiago; Crepalde, Geovam Pereira; Caldas, Ivo Santana; Nascimento, Manuela Sales Lima; Lana, Marta de; Chiari, Egler; Galvão, Lúcia Maria da Cunha; Bahia, Maria TerezinhaPara confirmar que cães Beagle são um bom modelo para doença de Chagas, foram avaliadas as alterações hematológicas durante as fases aguda e crônica em cães Beagle infectados com as cepas Y, Berenice-78 (Be-78) e ABC de Trypanosoma cruzi, correlacionando os sinais clínicos com a curva de parasitemia. Foi demonstrado que a fase aguda da infecção foi marcada por letargia e perda de apetite. Simultaneamente, observou-se anemia, leucocitose e linfocitose. Ainda, foram descritas alterações hematológicas e sinais clínicos positivamente correlacionados com a parasitemia durante a infecção experimental com as três cepas de T. cruzi estudadas, demonstrando que a infecção em cães Beagle constitui um modelo fidedigno para a doença de Chagas.Item IgG isotype profile is correlated with cardiomegaly in Beagle dogs infected with distinct Trypanosoma cruzi strains.(2008) Guedes, Paulo Marcos da Matta; Veloso, Vanja Maria; Gollob, Kenneth John; Afonso, Luís Carlos Crocco; Caldas, Ivo Santana; Vianna, Priscila; Lana, Marta de; Chiari, Egler; Bahia, Maria Terezinha; Galvão, Lúcia Maria da CunhaA systematic study following infection by various strains of the protozoan parasite, Trypanosoma cruzi, and the simultaneous monitoring of the humoral immune response together with the elicited cellular response, could add greatly to our understanding of differences between strains of this important human pathogen. In that sense, acute and chronic infections with distinct T. cruzi strains (Y, Berenice-78 and ABC) in Beagle dogs were studied through a longitudinal evaluation of immunoglobulin G (IgG), IgG1 and IgG2 isotypes (by ELISA and flow cytometry (FC)), as well as measurements of peripheral blood mononuclear cell (PBMC) proliferation over a 100-week period, and their correlation with cardiomegaly. Our results show that infected animals presenting cardiomegaly showed lower or absent levels of IgG1 during the chronic phase of the infection, when compared to those that did not show an increase in heart weight. In that manner, our results suggest that IgG1 could be used as a marker for cardiac pathogenicity in Chagas disease.Item Impacto do tratamento com Benznidazol na evolução da doença de Chagas experimental - análise imunopatológica e funcional.(Programa de Pós-Graduação em Ciências Biológicas. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto., 2011) Caldas, Ivo Santana; Bahia, Maria TerezinhaConsiderando as controvérsias sobre a eficácia do tratamento específico com Benznidazol (Bz) na prevenção ou redução das lesões cardíacas crônicas, este projeto propôs avaliar a influência do tratamento específico na evolução da cardiopatia chagásica, utilizando como modelo experimental cães infectados por cepas do Trypanosoma cruzi sensíveis e resistentes ao Bz. As avaliações imunopatológicas e clínicas foram realizadas utilizando: (i) a avaliação da área ocupada pela inflamação e pela fibrose no músculo cardíaco (ii) avaliação dos níveis de expressão mRNA para citocinas no tecido cardíaco; (iii) avaliação da resposta proliferativa e morte por apoptose de células mononucleares do sangue periférico (CMSP) e quantificação da produção de citocinas no sobrenadante das CMSP dos animais ao longo da infecção; (iv) avaliação das possíveis interferências do tratamento com Bz sobre a evolução clínica da cardiopatia experimental, através da detecção das alterações eletrocardiográficas, em cães chagásicos tratados e não tratados. Nossos resultados mostraram que o tratamento específico induziu 100% de cura nos animais infectados pela cepa Berenice-78, e que não houve cura parasitológica entre os infectados pelas cepas VL-10 e AAS. O tratamento específico, de maneira geral, preveniu as lesões cardíacas dos animais curados, bem como as alterações eletrocardiográficas. De forma interessante, entre os animais não curados foi observado um padrão de resposta variável de acordo com a população do T. cruzi. Nos animais infectados pela cepa AAS foi detectada redução significativa do processo inflamatório e da fibrose no tecido cardíaco acompanhado de ausência de alterações eletrocardiográficas. De forma diferente, entre os animais infectados pela cepa VL-10, o tratamento não foi eficaz em reduzir tanto as lesões cardíacas quanto as alterações eletrocardiográficas. Os parâmetros imunológicos avaliados durante as fases aguda e crônica da infecção foram marcadamente influenciados tanto pela cepa do parasito quanto pelo tratamento específico. Foi possível correlacionar a intensidade das lesões cardíacas e a produção de imunoglobulinas (IgG, IgG1, IgG2) no soro e de citocinas (TNF-, IFN- e IL-10) no sobrenadante de CMSP, além da expressão de mRNA dessas citocinas no tecido cardíaco dos animais submetidos ou não à quimioterapia com o Bz. De forma geral, nossos resultados contribuirão para uma melhor compreensão do benefício do tratamento específico na evolução clínica e nos mecanismos imunopatológicos envolvidos na doença de Chagas.Item Increased type 1 chemokine expression in experimental Chagas disease correlates with cardiac pathology in Beagle dogs.(2010) Guedes, Paulo Marcos da Matta; Veloso, Vanja Maria; Silva, André Talvani Pedrosa da; Diniz, Lívia de Figueiredo; Caldas, Ivo Santana; Matta, Maria Adelaide do Valle; Silva, Juliana Santiago; Chiari, Egler; Galvão, Lúcia Maria da Cunha; Silva, João Santana da; Bahia, Maria TerezinhaChemokines and chemokine receptors interaction have presented important role in leukocyte migration to specific immune reaction sites. Recently, it has been reported that chemokine receptors CXC (CXCR3) and CC (CCR5) were preferentially expressed on Th1 cells while CCR3 and CCR4 were preferentially expressed on Th2 cells. This study evaluated the mRNA expression of type 1 and type 2 chemokine and chemokine receptors in the cardiac tissue of Beagle dogs infected with distinct genetic groups of Trypanosoma cruzi (Y, Berenice- 78 and ABC strains) during acute and chronic phases. To analyze the correlation between chemokine and chemokine receptors expression and the development of heart pathology, the chronic infected animals were divided into groups, according to the parasite strain and based on the degree of heart damage: cardiac and indeterminate form of Chagas disease. Our results indicated that cardiac type1/2 chemokines and their receptors were partially dependent on the genetic diversity of parasites as well as the polarization of clinical forms. Also, dogs presenting cardiac form showed lower heart tissue mRNA expression of CCL24 (type 2) and higher expression of CCL5, CCL4 and CXCR3 (type 1) when compared with those with indeterminate form of disease. Together, these data reinforce a close-relation between T. cruzi genetic population and the host specific type 1 immune response and, for the first time, we show the distribution of type 1/2 chemokines associated with the development of cardiac pathology using dogs, a well similar model to study human Chagas disease.