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Melo M.F.,Laboratorio Of Biologia Molecular E Doencas Endemicas | Moreira O.C.,Laboratorio Of Biologia Molecular E Doencas Endemicas | Junior W.O.,University of Pernambuco | Britto C.,Laboratorio Of Biologia Molecular E Doencas Endemicas
Parasites and Vectors | Year: 2015

Background: Inconclusive results of serological diagnosis in Chagas disease have an important impact on blood banks worldwide, reflecting in the high number of discarded bags or in an increased transmission by blood transfusion. Molecular techniques such as qPCR have been used for diagnosis and to monitor Trypanosoma cruzi load in peripheral blood samples. A promising perspective refers to the possibility of parasite DNA detection in serum, taking advantage in using the same samples collected for serological screening. Methods: In order to evaluate the effectiveness of a qPCR strategy for detecting and quantifying T. cruzi DNA in serum, we selected 40 chronic Chagas disease patients presenting different clinical manifestations: Cardiac (23), Digestive (4), Mixed form [cardiodigestive] (7), and asymptomatic (6). Twenty seronegative individuals from non-endemic areas were included as controls. Samples were extracted using QIAamp DNA mini kit (QIAGEN) and qPCR was performed in a multiplex format with TaqMan probes for the nuclear satellite DNA of T. cruzi and for the human RNase P gene. In addition, DNA migration to serum during blood coagulation was assessed using a commercial exogenous control (Exo IPC, Applied Biosystems) in a separate qPCR reaction. Results: The comparative duplex qPCR analysis revealed that, even with an increase in Ct values, it was possible to detect all DNA targets in serum. In addition, the same linearity range for T. cruzi quantification (from 105 to 0.5 par. eq./mL) between serum, blood or culture samples (T. cruzi epimastigotes - Cl Brener strain) was found. When patient samples were evaluated, no significant differences in parasite load between the distinct clinical manifestations were found for both blood and serum samples. Moreover, median values of parasite burden were 1.125 and 1.230 par. eq./mL for serum and blood, respectively. Using serology as gold standard, we found 95% sensitivity for T. cruzi detection in serum and 97.5% for blood, and 100% specificity for both samples. Conclusions: Taken together, our data indicate the potential of using serum samples for molecular diagnosis and parasite load quantification by qPCR, suggesting its use in reference laboratories for the diagnosis of Chagas disease patients. © 2015 Melo et al.; licensee BioMed Central.


Souza-Silva F.,Laboratorio Of Biologia Molecular E Doencas Endemicas | Pereira B.A.S.,Laboratorio Of Biologia Molecular E Doencas Endemicas | Finkelstein L.C.,Laboratorio Of Imunoparasitologia | Zucolotto V.,University of Sao Paulo | And 2 more authors.
Journal of Molecular Recognition | Year: 2014

Peptides from the COOH-terminal extension of cysteine proteinase B from Leishmania (Leishmania) amazonensis (cyspep) can modulate immune responses in vertebrate hosts. With this hypothesis as base, we used the online analysis tool SYFPEITHI to predict seven epitopes from this region with potential to bind H2 proteins. We performed proliferation tests and quantified reactive T lymphocytes applying a cytometry analysis, using samples from draining lymph node of lesions from L. (L.) amazonensis-infected mice. To define reactivity of T cells, we used complexes of DimerX (H2 Db:Ig and H2 Ld:Ig) and the putative epitopes. Additionally, we applied surface plasmon resonance to verify real time interactions between the putative epitopes and DimerX proteins. Five peptides induced blastogenesis in BALB/c cells, while only two presented the same property in C57BL/6 mouse cells. In addition, our data indicate the existence of CD8+ T lymphocyte populations able to recognize each tested peptide in both murine strains. We observed an overlapping of results between the peptides that induced lymphocyte proliferation and those capable of binding to the DimerX in the surface plasmon resonance assays thus indicating that using these recombinant proteins in biosensing analyses is a promising tool to study real time molecular interactions in the context of major histocompatibility complex epitopes. The data gathered in this study reinforce the hypothesis that cyspep-derived peptides are important factors in the murine host infection by L. (L.) amazonensis. Copyright © 2014 John Wiley & Sons, Ltd.


PubMed | Laboratorio Of Biologia Molecular E Doencas Endemicas and University of Pernambuco
Type: | Journal: Parasites & vectors | Year: 2015

Inconclusive results of serological diagnosis in Chagas disease have an important impact on blood banks worldwide, reflecting in the high number of discarded bags or in an increased transmission by blood transfusion. Molecular techniques such as qPCR have been used for diagnosis and to monitor Trypanosoma cruzi load in peripheral blood samples. A promising perspective refers to the possibility of parasite DNA detection in serum, taking advantage in using the same samples collected for serological screening.In order to evaluate the effectiveness of a qPCR strategy for detecting and quantifying T. cruzi DNA in serum, we selected 40 chronic Chagas disease patients presenting different clinical manifestations: Cardiac (23), Digestive (4), Mixed form [cardiodigestive] (7), and asymptomatic (6). Twenty seronegative individuals from non-endemic areas were included as controls. Samples were extracted using QIAamp DNA mini kit (QIAGEN) and qPCR was performed in a multiplex format with TaqMan probes for the nuclear satellite DNA of T. cruzi and for the human RNase P gene. In addition, DNA migration to serum during blood coagulation was assessed using a commercial exogenous control (Exo IPC, Applied Biosystems) in a separate qPCR reaction.The comparative duplex qPCR analysis revealed that, even with an increase in Ct values, it was possible to detect all DNA targets in serum. In addition, the same linearity range for T. cruzi quantification (from 10(5) to 0.5 par. eq./mL) between serum, blood or culture samples (T. cruzi epimastigotes - Cl Brener strain) was found. When patient samples were evaluated, no significant differences in parasite load between the distinct clinical manifestations were found for both blood and serum samples. Moreover, median values of parasite burden were 1.125 and 1.230 par. eq./mL for serum and blood, respectively. Using serology as gold standard, we found 95% sensitivity for T. cruzi detection in serum and 97.5% for blood, and 100% specificity for both samples.Taken together, our data indicate the potential of using serum samples for molecular diagnosis and parasite load quantification by qPCR, suggesting its use in reference laboratories for the diagnosis of Chagas disease patients.


Pereira I.R.,Instituto Oswaldo Cruz Fiocruz | Vilar-Pereira G.,Instituto Oswaldo Cruz Fiocruz | Silva A.A.,Federal University of Fluminense | Moreira O.C.,Laboratorio Of Biologia Molecular E Doencas Endemicas | And 3 more authors.
Mediators of Inflammation | Year: 2014

Background. Chagas disease (CD) is characterized by parasite persistence and immunological unbalance favoring systemic inflammatory profile. Chronic chagasic cardiomyopathy, the main manifestation of CD, occurs in a TNF-enriched milieu and frequently progresses to heart failure. Aim of the Study. To challenge the hypothesis that TNF plays a key role in Trypanosoma cruzi-induced immune deregulation and cardiac abnormalities, we tested the effect of the anti-TNF antibody Infliximab in chronically T. cruzi-infected C57BL/6 mice, a model with immunological, electrical, and histopathological abnormalities resembling Chagas' heart disease. Results. Infliximab therapy did not reactivate parasite but reshaped the immune response as reduced TNF mRNA expression in the cardiac tissue and plasma TNF and IFNγ levels; diminished the frequency of IL-17A+ but increased IL-10+ CD4+ T-cells; reduced TNF+ but augmented IL-10+ Ly6C+ and F4/80+ cells. Further, anti-TNF therapy decreased cytotoxic activity but preserved IFNγ-producing VNHRFTLV-specific CD8+ T-cells in spleen and reduced the number of perforin+ cells infiltrating the myocardium. Importantly, Infliximab reduced the frequency of mice afflicted by arrhythmias and second degree atrioventricular blocks and decreased fibronectin deposition in the cardiac tissue. Conclusions. Our data support that TNF is a crucial player in the pathogenesis of Chagas' heart disease fueling immunological unbalance which contributes to cardiac abnormalities. © 2014 Isabela Resende Pereira et al.


da Gama Jaen Batista D.,Laboratorio Of Biologia Celular | Batista M.M.,Laboratorio Of Biologia Celular | de Oliveira G.M.,Laboratorio Of Biologia Celular | Britto C.C.,Laboratorio Of Biologia Molecular E Doencas Endemicas | And 4 more authors.
PLoS ONE | Year: 2011

Chagas disease caused by Trypanosoma cruzi is an important cause of mortality and morbidity in Latin America but no vaccines or safe chemotherapeutic agents are available. Combined therapy is envisioned as an ideal approach since it may enhance efficacy by acting upon different cellular targets, may reduce toxicity and minimize the risk of drug resistance. Therefore, we investigated the activity of benznidazole (Bz) in combination with the diamidine prodrug DB289 and in combination with the arylimidamide DB766 upon T. cruzi infection in vivo. The oral treatment of T.cruzi-infected mice with DB289 and Benznidazole (Bz) alone reduced the number of circulating parasites compared with untreated mice by about 70% and 90%, respectively. However, the combination of these two compounds decreased the parasitemia by 99% and protected against animal mortality by 100%, but without providing a parasitological cure. When Bz (p.o) was combined with DB766 (via ip route), at least a 99.5% decrease in parasitemia levels was observed. DB766+Bz also provided 100% protection against mice mortality while Bz alone provided about 87% protection. This combined therapy also reduced the tissular lesions induced by T. cruzi infection: Bz alone reduced GPT and CK plasma levels by about 12% and 78% compared to untreated mice group, the combination of Bz with DB766 resulted in a reduction of GPT and CK plasma levels of 56% and 91%. Cure assessment through hemocultive and PCR approaches showed that Bz did not provide a parasitological cure, however, DB766 alone or associated with Bz cured ≥13% of surviving animals.


PubMed | Federal University of Fluminense, Laboratorio Of Biologia Molecular E Doencas Endemicas and Instituto Oswaldo Cruz Fiocruz
Type: | Journal: Mediators of inflammation | Year: 2014

Chagas disease (CD) is characterized by parasite persistence and immunological unbalance favoring systemic inflammatory profile. Chronic chagasic cardiomyopathy, the main manifestation of CD, occurs in a TNF-enriched milieu and frequently progresses to heart failure.To challenge the hypothesis that TNF plays a key role in Trypanosoma cruzi-induced immune deregulation and cardiac abnormalities, we tested the effect of the anti-TNF antibody Infliximab in chronically T. cruzi-infected C57BL/6 mice, a model with immunological, electrical, and histopathological abnormalities resembling Chagas heart disease.Infliximab therapy did not reactivate parasite but reshaped the immune response as reduced TNF mRNA expression in the cardiac tissue and plasma TNF and IFN levels; diminished the frequency of IL-17A(+) but increased IL-10(+) CD4(+) T-cells; reduced TNF(+) but augmented IL-10(+) Ly6C(+) and F4/80(+) cells. Further, anti-TNF therapy decreased cytotoxic activity but preserved IFN-producing VNHRFTLV-specific CD8(+) T-cells in spleen and reduced the number of perforin(+) cells infiltrating the myocardium. Importantly, Infliximab reduced the frequency of mice afflicted by arrhythmias and second degree atrioventricular blocks and decreased fibronectin deposition in the cardiac tissue.Our data support that TNF is a crucial player in the pathogenesis of Chagas heart disease fueling immunological unbalance which contributes to cardiac abnormalities.


Pereira F.M.,Federal University of Rio de Janeiro | Santos-Mallet J.R.,Instituto Oswaldo Cruz IOC | Branquinha M.H.,Federal University of Rio de Janeiro | d'Avila-Levy C.M.,Laboratorio Of Biologia Molecular E Doencas Endemicas | Santos A.L.S.,Federal University of Rio de Janeiro
Microbes and Infection | Year: 2010

Monoxenous trypanosomatids usually have an invertebrate as the only host in their life cycles, however, they have been found repeatedly in plants and/or mammals. To succeed in colonizing a vertebrate host, the parasite must quickly adapt to drastic changes in the environment (e.g. temperature), which reflect the conditions found in the insect and mammalian hosts. Leishmanolysin is a metalloprotease ubiquitously distributed in trypanosomatids, playing a myriad of functions. In Herpetomonas samuelpessoai, an insect trypanosomatid, the leishmanolysin-like molecule was implicated in the nutrition and insect adhesion. Herein, we showed that leishmanolysin expression is equally expressed in H. samuelpessoai parasites submitted to insect (26 °C) and mammalian (37 °C) temperatures. Also, the parasites grown in both temperatures interacted at similar rates with macrophages. Finally, we showed that leishmanolysin is involved in crucial steps in the interaction of H. samuelpessoai cells with macrophages, since the treatment with either anti-leishmanolysin antibodies or metalloprotease inhibitor 1,10-phenanthroline significantly reduced the association index. Similarly, the treatment of the macrophages with purified leishmanolysin promoted a powerful reduction in the association index, suggesting the direct involvement of macrophage receptors. These results suggest that H. samuelpessoai leishmanolysin molecules are not modulated by temperature and are involved in the interaction with mammalian cells. © 2010 Institut Pasteur.


Pereira I.R.,Laboratorio Of Biologia Das Interacoes | Vilar-Pereira G.,Laboratorio Of Biologia Das Interacoes | Moreira O.C.,Laboratorio Of Biologia Molecular E Doencas Endemicas | Ramos I.P.,Federal University of Rio de Janeiro | And 4 more authors.
PLoS Neglected Tropical Diseases | Year: 2015

Chronic chagasic cardiomyopathy (CCC), the main clinical sign of Chagas disease, is associated with systemic CD8+ T-cell abnormalities and CD8-enriched myocarditis occurring in an inflammatory milieu. Pentoxifylline (PTX), a phosphodiesterase inhibitor, has immunoregulatory and cardioprotective properties. Here, we tested PTX effects on CD8+ T-cell abnormalities and cardiac alterations using a model of experimental Chagas’ heart disease. C57BL/6 mice chronically infected by the Colombian Trypanosoma cruzi strain and presenting signs of CCC were treated with PTX. The downmodulation of T-cell receptors on CD8+ cells induced by T. cruzi infection was rescued by PTX therapy. Also, PTX reduced the frequency of CD8+ T-cells expressing activation and migration markers in the spleen and the activation of blood vessel endothelial cells and the intensity of inflammation in the heart tissue. Although preserved interferon-gamma production systemically and in the cardiac tissue, PTX therapy reduced the number of perforin+ cells invading this tissue. PTX did not alter parasite load, but hampered the progression of heart injury, improving connexin 43 expression and decreasing fibronectin overdeposition. Further, PTX reversed electrical abnormalities as bradycardia and prolonged PR, QTc and QRS intervals in chronically infected mice. Moreover, PTX therapy improved heart remodeling since reduced left ventricular (LV) hypertrophy and restored the decreased LV ejection fraction. PTX therapy ameliorates critical aspects of CCC and repositioned CD8+ T-cell response towards homeostasis, reinforcing that immunological abnormalities are crucially linked, as cause or effect, to CCC. Therefore, PTX emerges as a candidate to treat the non-beneficial immune deregulation associated with chronic Chagas' heart disease and to improve prognosis. © 2015 Pereira et al.


PubMed | Laboratorio Of Biologia Das Interacoes, Federal University of Rio de Janeiro, Laboratorio Of Biologia Molecular E Doencas Endemicas and Laboratorio Of Hanseniase
Type: Journal Article | Journal: PLoS neglected tropical diseases | Year: 2015

Chronic chagasic cardiomyopathy (CCC), the main clinical sign of Chagas disease, is associated with systemic CD8+ T-cell abnormalities and CD8-enriched myocarditis occurring in an inflammatory milieu. Pentoxifylline (PTX), a phosphodiesterase inhibitor, has immunoregulatory and cardioprotective properties. Here, we tested PTX effects on CD8+ T-cell abnormalities and cardiac alterations using a model of experimental Chagas heart disease.C57BL/6 mice chronically infected by the Colombian Trypanosoma cruzi strain and presenting signs of CCC were treated with PTX. The downmodulation of T-cell receptors on CD8+ cells induced by T. cruzi infection was rescued by PTX therapy. Also, PTX reduced the frequency of CD8+ T-cells expressing activation and migration markers in the spleen and the activation of blood vessel endothelial cells and the intensity of inflammation in the heart tissue. Although preserved interferon-gamma production systemically and in the cardiac tissue, PTX therapy reduced the number of perforin+ cells invading this tissue. PTX did not alter parasite load, but hampered the progression of heart injury, improving connexin 43 expression and decreasing fibronectin overdeposition. Further, PTX reversed electrical abnormalities as bradycardia and prolonged PR, QTc and QRS intervals in chronically infected mice. Moreover, PTX therapy improved heart remodeling since reduced left ventricular (LV) hypertrophy and restored the decreased LV ejection fraction.PTX therapy ameliorates critical aspects of CCC and repositioned CD8+ T-cell response towards homeostasis, reinforcing that immunological abnormalities are crucially linked, as cause or effect, to CCC. Therefore, PTX emerges as a candidate to treat the non-beneficial immune deregulation associated with chronic Chagas heart disease and to improve prognosis.


Cortes L.M.,Laboratorio Of Biologia Molecular E Doencas Endemicas
Parasites & vectors | Year: 2011

Leishmania (Vianna) braziliensis, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) chagasi are important parasites in the scenario of leishmaniasis in Brazil. During the life cycle of these parasites, the promastigote forms adhere to the midgut epithelial microvillii of phlebotomine insects to avoid being secreted along with digestive products. Lulo cells are a potential model that will help to understand the features of this adhesion phenomenon. Here, we analyze the interaction between Leishmania spp. promastigotes and Lulo cells in vitro, specifically focusing on adhesion events occurring between three Leishmania species and this cell line. Confluent monolayers of Lulo cells were incubated with promastigotes and adhesion was assessed using both light microscopy and scanning electron microscopy. The results indicate that species from the subgenera Leishmania and Viannia have great potential to adhere to Lulo cells. The highest adherence rate was observed for L. (L.) chagasi after 24 h of incubation with Lulo cells (27.3 ± 1.8% of cells with adhered promastigotes), followed by L. (L.) amazonensis (16.0 ± 0.7%) and L. (V.) braziliensis (3.0 ± 0.7%), both after 48 h. In the ultrastructural analysis, promastigote adherence was also assessed by scanning electron microscopy, showing that, for parasites from both subgenera, adhesion occurs by both the body and the flagellum. The interaction of Lulo cells with Leishmania (L.) chagasi showed the participation of cytoplasmic projections from the former closely associating the parasites with the cells. We present evidence that Lulo cells can be useful in studies of insect-parasite interactions for Leishmania species.

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