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Miguel A Chávez-Fumagalli,
Vivian T Martins,
Miriam C S Testasicca,
Daniela P Lage, Lourena E Costa,
Paula S Lage,
Mariana C Duarte,
Henrique G Ker,
Tatiana G Ribeiro,
Fernando A A Carvalho,
Wiliam C B Régis,
Alexandre B Dos Reis,
Carlos A P Tavares,
Manuel Soto,
Ana Paula Fernandes,
Eduardo A F Coelho
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ABSTRACT: In Brazil, the percentage of infected dogs living in area where canine visceral leishmaniasis (CVL) is endemic range from 10 to 62%; however, the prevalence of infection in dogs is probably higher than figures reported from serological studies. In addition, problems with the occurrence of false positive or negative results have been reported in the serodiagnosis of CVL. The present work analyzed the potential of synthetic peptides mapped from hypothetical proteins to the improvement of serodiagnosis of L. infantum infection in dogs. From 26 identified leishmanial proteins; eight were selected, considering that no homologies could be encountered between these proteins and others from sequence trypanosomatides databases. The sequences of these proteins were mapped to identify linear B-cell epitopes, and seventeen peptides were synthesized and tested in enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of L. infantum infection in dogs. From these, three of them presented sensitivity and specificity higher than 75% and 90%, respectively, in order to differentiate L. infantum-infected animals from those T. cruzi-infected and healthy animals. Noting that, soluble Leishmania antigen (SLA) showed poor sensitivity (4%) and specificity (36%) in order to differentiate L. infantum-infected dogs from healthy and T. cruzi-infected dogs, respectively. Lately, the three selected peptides were combined in different mixtures and higher sensitivity and specificity were reached, even when the sera from T. cruzi-infected dogs were used. The study's findings suggest that these three peptides can constitute a potential tool for a more sensitive and specific serodiagnosis of L. infantum infection in dogs.
Clinical and vaccine immunology: CVI 04/2013; · 2.37 Impact Factor
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Vivian T Martins,
Miguel A Chávez-Fumagalli, Lourena E Costa,
Adriana M C Canavaci,
Paula S Lage,
Daniela P Lage,
Mariana C Duarte,
Diogo G Valadares,
Rubens D M Magalhães,
Tatiana G Ribeiro,
Ronaldo A P Nagem,
Wanderson D Darocha,
Wiliam C B Régis,
Manuel Soto,
Eduardo A F Coelho,
Ana Paula Fernandes,
Carlos A P Tavares
PLoS Neglected Tropical Diseases 04/2013; 7(4). · 4.69 Impact Factor
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Vivian T Martins,
Miguel A Chávez-Fumagalli, Lourena E Costa,
Adriana M C C Martins,
Paula S Lage,
Daniela P Lage,
Mariana C Duarte,
Diogo G Valadares,
Rubens D M Magalhães,
Tatiana G Ribeiro,
Ronaldo A P Nagem,
Wanderson D Darocha,
Wiliam C B Régis,
Manuel Soto,
Eduardo A F Coelho,
Ana Paula Fernandes,
Carlos A P Tavares
[show abstract]
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ABSTRACT: BACKGROUND: The present study aimed to evaluate a hypothetical Leishmania amastigote-specific protein (LiHyp1), previously identified by an immunoproteomic approach performed in Leishmania infantum, which showed homology to the super-oxygenase gene family, attempting to select a new candidate antigen for specific serodiagnosis, as well as to compose a vaccine against VL. METHODOLOGYPRINCIPAL FINDINGS: The LiHyp1 DNA sequence was cloned; the recombinant protein (rLiHyp1) was purified and evaluated for its antigenicity and immunogenicity. The rLiHyp1 protein was recognized by antibodies from sera of asymptomatic and symptomatic animals with canine visceral leishmaniasis (CVL), but presented no cross-reactivity with sera of dogs vaccinated with Leish-Tec, a Brazilian commercial vaccine; with Chagas' disease or healthy animals. In addition, the immunogenicity and protective efficacy of rLiHyp1 plus saponin was evaluated in BALB/c mice challenged subcutaneously with virulent L. infantum promastigotes. rLiHyp1 plus saponin vaccinated mice showed a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with the recombinant protein. Immunized and infected mice, as compared to the control groups (saline and saponin), showed significant reductions in the number of parasites found in the liver, spleen, bone marrow, and in the paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, produced mainly by CD4 T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 response could also be observed. CONCLUSIONSSIGNIFICANCE: The present study showed that this Leishmania oxygenase amastigote-specific protein can be used for a more sensitive and specific serodiagnosis of asymptomatic and symptomatic CVL and, when combined with a Th1-type adjuvant, can also be employ as a candidate antigen to develop vaccines against VL.
PLoS Neglected Tropical Diseases 03/2013; 7(3):e2148. · 4.69 Impact Factor
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Diogo G Valadares,
Mariana C Duarte,
Laura Ramírez,
Miguel A Chávez-Fumagalli,
Vivian T Martins, Lourena E Costa,
Paula S Lage,
Tatiana G Ribeiro,
Rachel O Castilho,
Ana Paula Fernandes,
Wiliam C B Régis,
Manuel Soto,
Carlos A P Tavares,
Eduardo A F Coelho
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ABSTRACT: The present study aimed to investigate the in vitro antileishmanial activity of five fractions obtained from Agaricus blazei water extract (AbM), namely, Fab1, Fab2, Fab3, Fab4, and Fab5; and use the selected leishmanicidal fraction to treat BALB/c mice infected with Leishmania chagasi. A curve dose-titration was performed to obtain the concentration to be test in infected animals. In this context, Fab5 fraction and AbM were used in the doses of 20 and 100mg/kg/day, respectively, with the product been administered once a day. The effect induced by a chemo-prophylactic regimen, based on the administration Fab5 fraction and AbM 5days before infection, and maintained for an additional 20days post-infection was compared to a therapeutic regimen, in which the compounds were administered from 0 to 20days of infection. Control animals were either treated with amphotericin B deoxycholate (AmpB) or received distilled water. All groups were followed up for 10weeks post-infection, when parasitological and immunological parameters were analyzed. The Fab5 presented the best results of in vitro leishmanicidal activity. In the in vivo experiments, the use of Fab5 or AbM, as compared to control groups, resulted in significant reduced parasite burdens in the liver, spleen, and draining lymph nodes of the infected animals, as compared to control groups. A Type 1 immune response was observed in the Fab5 or AbM treated animals. No significant toxicity was observed. The chemo-prophylactic regimen proved to be more effective to induce theses responses. In this context, the data presented in this study showed the potential of the purified Fab5 fraction of AbM as a therapeutic alternative to treat visceral leishmaniasis. In addition, it can be postulated that this fraction can be also employed in a chemo-prophylactic regimen associated or not with other therapeutic products.
Experimental Parasitology 07/2012; 132(2):228-36. · 2.12 Impact Factor
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Diogo G Valadares,
Mariana C Duarte,
Laura Ramírez,
Miguel A Chávez-Fumagalli,
Paula S Lage,
Vivian T Martins, Lourena E Costa,
Tatiana G Ribeiro,
Wiliam C B Régis,
Manuel Soto,
Ana Paula Fernandes,
Carlos A P Tavares,
Eduardo A F Coelho
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ABSTRACT: The development of therapeutic alternatives to treat leishmaniasis has received considerable attention. The present study aimed to investigate the efficacy of the Agaricus blazei Murill water extract (AbM) to treat BALB/c mice infected with Leishmania amazonensis. First, a dose-titration curve was performed. The most well-defined concentration able to induce the most effective results in the infected animals, considering a daily administration of the product, was that of 100 mg kg(-1) day(-1). In this context, the AbM was administered orally, beginning on day 0 up to 20 days postinfection. Additional animals were treated with amphotericin B (AmpB, 5 mg kg(-1) day(-1)) by peritoneal route for the same period of time, while the control group received distilled water. The animals were evaluated at 14 weeks post-infection, at which time the parasitological and immunological parameters were analyzed. Mice treated with the AbM presented a 60 % reduction in the inflammation of infected footpads as compared to untreated control-infected mice. Moreover, in the treated mice, as compared to the untreated controls, approximately 60 and 66 % reductions could be observed in the parasite burdens of the footpad and draining lymph nodes, respectively. In addition, no parasites could be detected in the spleen of treated mice at week 14 postinfection. These treated animals produced significantly higher levels of interferon gamma (IFN-γ) and nitric oxide (NO), higher levels of parasite-specific IgG2a isotype antibodies, and lower levels of interleukin (IL)-4, and IL-10 in the spleen and lymph node cell cultures than did the controls. Differences could be observed by comparing animals treated with AbM to those treated with AmpB, as indicated by a significant reduction in tissue parasitism, higher levels of IFN-γ and NO, and lower levels of IL-4 and IL-10, as well as by a decreased hepatic toxicity. In conclusion, the present study's data show that the A. blazei Murill water extract presents a high potential for the treatment of leishmaniasis, although additional studies on mice, as well as on other mammal hosts, are warranted in an attempt to determine this extract's true efficacy as compared to other known therapeutic products.
Parasitology Research 07/2012; 111(4):1807-16. · 2.15 Impact Factor
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Dulcilene M Oliveira,
Mariana Amália F Costa,
Miguel A Chavez-Fumagalli,
Diogo G Valadares,
Mariana C Duarte, Lourena E Costa,
Vivian T Martins,
Rosângela F Gomes,
Maria N Melo,
Manuel Soto,
Carlos Alberto P Tavares,
Eduardo Antonio F Coelho
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ABSTRACT: Experimental vaccines to protect against visceral leishmaniasis (VL) have been developed by using BALB/c mice infected with a large (10⁷ to 10⁸) inoculum of parasites. Remarkably, prior literature has reported that the poor protection observed is mainly due to the high susceptibility of this strain. To determine factors inherent to mice that might abrogate vaccine-induced efficacy, the present research sought to investigate the impact of the administration of different infective inoculums of Leishmania chagasi (syn. L. infantum) in BALB/c mice, evaluating subcutaneous and intravenous routes of administration as well as parasitological and immunological parameters over different periods of time. This study shows that the injection of a highly infective inoculum in mice, through both subcutaneous and intravenous routes, results in a sustained infection. The mice developed a high parasite load in the liver; however, these values diminished over time. This result did not corroborate with the parasite load in the bone marrow and brain and proved to be expressively different in the spleen and draining lymph nodes, where the values increased over time. Mice infected with a low dose of parasites (10³) showed a certain resistance against infection, based mainly on the IFN-γ and oxide nitric production. Considering all the elements, it could be concluded that the models employing high doses (10⁷) of L. chagasi in BALB/c mice can bring about an imbalance in the animals' immune response, thus allowing for the development of the disease at the expense of efficacy within the vaccine candidates.
Parasitology Research 09/2011; 110(3):1277-85. · 2.15 Impact Factor
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Diogo G Valadares,
Mariana C Duarte,
Jamil S Oliveira,
Miguel A Chávez-Fumagalli,
Vivian T Martins, Lourena E Costa,
João Paulo V Leite,
Marcelo M Santoro,
Wiliam C B Régis,
Carlos A P Tavares,
Eduardo A F Coelho
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ABSTRACT: Leishmaniasis is a major public health problem, and the alarming spread of parasite resistance underlines the importance of discovering new therapeutic products. The present study aims to investigate the in vitro leishmanicidal activity of an Agaricus blazei Murill mushroom extract as compared to different Leishmania species and stages. The water extract proved to be effective against promastigote and amastigote-like stages of Leishmania amazonensis, L. chagasi, and L. major, with IC(50) (50% inhibitory concentration) values of 67.5, 65.8, and 56.8 μg/mL for promastigotes, and 115.4, 112.3, and 108.4 μg/mL for amastigotes-like respectively. The infectivity of the three Leishmania species before and after treatment with the water extract was analyzed, and it could be observed that 82%, 57%, and 73% of the macrophages were infected with L. amazonensis, L. major, and L. chagasi, respectively. However, when parasites were pre-incubated with the water extract, and later used to infect macrophages, they were able to infect only 12.7%, 24.5%, and 19.7% of the phagocytic cells for L. amazonensis, L. chagasi, and L. major, respectively. In other experiments, macrophages were infected with L. amazonensis, L. chagasi, or L. major, and later treated with the aforementioned extract, presented reductions of 84.4%, 79.6%, and 85.3% in the parasite burden after treatment. A confocal microscopy revealed the loss of the viability of the parasites within the infected macrophages after treatment with the water extract. The applied extract presented a low cytotoxicity in murine macrophages and a null hemolytic activity in type O(+) human red blood cells. No nitric oxide (NO) production, nor inducible nitric oxide syntase expression, could be observed in macrophages after stimulation with the water extract, suggesting that biological activity may be due to direct mechanisms other than macrophage activation by means of NO production. In conclusion, the results demonstrate that the A. blazei Murill water extract can potentially be used as a therapeutic alternative on its own, or in association with other drugs, to treat Visceral and Cutaneous Leishmaniasis.
Parasitology International 06/2011; 60(4):357-63. · 2.13 Impact Factor
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Miguel A Chávez-Fumagalli,
Mariana A F Costa,
Dulcilene M Oliveira,
Laura Ramírez, Lourena E Costa,
Mariana C Duarte,
Vivian T Martins,
Jamil S Oliveira,
Carlos C Olortegi,
Pedro Bonay,
Carlos Alonso,
Carlos A P Tavares,
Manuel Soto,
Eduardo A F Coelho
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ABSTRACT: Leishmania chagasi and Leishmania amazonensis are the etiologic agents of different clinical forms of human leishmaniasis in South America. In an attempt to select candidate antigens for a vaccine protecting against different Leishmania species, the efficacy of vaccination using Leishmania ribosomal proteins and saponin as adjuvant was examined in BALB/c mice against challenge infection with both parasite species. Mice vaccinated with parasite ribosomal proteins purified from Leishmania infantum plus saponin showed a specific production of IFN-γ, IL-12 and GM-CSF after in vitro stimulation with L. infantum ribosomal proteins. Vaccinated mice showed a reduction in the liver and spleen parasite burdens after L. chagasi infection. After L. amazonensis challenge, vaccinated mice showed a decrease of the dermal pathology and a reduction in the parasite loads in the footpad and spleen. In both models, protection was correlated to an IL-12-dependent production of IFN-γ by CD4(+) and CD8(+) T cells that activate macrophages for the synthesis of NO. In the protected mice a decrease in the parasite-mediated IL-4 and IL-10 responses was also observed. In mice challenged with L. amazonensis, lower levels of anti-parasite-specific antibodies were detected. Thus, Leishmania ribosomal proteins plus saponin fits the requirements to compose a pan-Leishmania vaccine.
Microbes and Infection 11/2010; 12(12-13):967-77. · 3.10 Impact Factor
