Assessing the effect of antimony pressure on trypanothione reductase activity in Leishmania (Viannia) braziliensis.

Leishmania parasites have an oxidative and chemical defense mechanism called trypanothione system (T[SH]2), the most abundant thiol system in trypanosomatids. This system has a central role in processing pentavalent antimony and resistance has been related to a better capacity to metabolize it through the activation of T[SH]2 enzymatic cascade. A biochemical approach was applied to assess the effect of trivalent (SbIII) and pentavalent antimony (SbV) on Trypanothione Reductase (TR) activity of two Leishmania (Viannia) braziliensis clinical isolates, which were labeled as responder (R) and non-responder (NR) after patient treatment with Glucantime®. Both isolates were characterized based on in vitro susceptibility to SbIII and SbV and trypanothione reductase (TR) activity. SbIII and SbV discriminated susceptibility profiles in all parasite forms, since isolate NR had significantly higher EC50 values than isolate R. Differences were observed in TR activity between promastigotes, axenic amastigotes and intracellular amastigotes: R (0.439 ± 0.009, 0.103 ± 0.01 and 0.185 ± 0.01AU.min-1.µg of protein-1) and NR (1.083 ± 0.04, 0.914 ± 0.04 and 0.343 ± 0.04 AU. min-1.µg of protein-1), respectively. Incubation with SbIII and SbV using each form EC50 value caused a time-dependent differential effect on TR activity suggesting that oxidative defense is related to the antimony susceptibility phenotype. Data gathered here shows a biochemical approach able to discriminate two L. (V.) braziliensis clinical isolates measurements TR activity of promastigotes, axenic amastigotes and intracellular amastigotes.

Synthesis, structure-activity relationship and trypanocidal activity of pyrazole-imidazoline and new pyrazole-tetrahydropyrimidine hybrids as promising chemotherapeutic agents for Chagas disease.

Drug therapy for Chagas disease remains a major challenge as potential candidate drugs have failed clinical trials. Currently available drugs have limited efficacy and induce serious side effects. Thus, the discovery of new drugs is urgently needed in the fight against Chagas' disease. Here, we synthesized and evaluated the biological effect of pyrazole-imidazoline (1a-i) and pyrazole-tetrahydropyrimidine (2a-i) derivatives against relevant clinical forms of Trypanosoma cruzi. The structure-activity relationship (SAR), drug-target search, physicochemical and ADMET properties of the major active compounds in vitro were also assessed in silico. Pyrazole derivatives showed no toxicity in Vero cells and also no cardiotoxicity. Phenotypic screening revealed two dichlorinated pyrazole-imidazoline derivatives (1c and 1d) with trypanocidal activity higher than that of benznidazole (Bz) against trypomastigotes; these were also the most potent compounds against intracellular amastigotes. Replacement of imidazoline with tetrahydropyrimidine in the pyrazole compounds completely abolished the trypanocidal activity of series 2(a-i) derivatives. The physicochemical and ADMET properties of the compounds predicted good permeability, good oral bioavailability, no toxicity and mutagenicity of 1c and 1d. Pyrazole nucleus had high frequency hits for cruzipain in drug-target search and structure activity relationship (SAR) analysis of pyrazole-imidazoline derivatives revealed enhanced activity when chlorine atom was inserted in meta-positions of the benzene ring. Additionally, we found evidence that both compounds (1c and 1d) have the potential to interact non-covalently with the active site of cruzipain and also inhibit the cysteine proteinase activity of T. cruzi. Collectively, the data presented here reveal pyrazole derivatives with promise for further optimization in the therapy of Chagas disease.

"Survey of Wellbeing of Young Children (SWYC)": how does it fit for screening developmental delay in Brazilian children aged 4 to 58 months?

OBJECTIVE: To replicate the original normative study of the SWYC's Milestones Questionnaires for children in Brazil. Our goals were to compare the performance of Brazilian and North American children using this screening tool and to verify the reliability and validity of the Brazilian version. STUDY DESIGN AND SETTING: Cross-sectional study with children aged 1-65 months and their guardians, recruited in southern Brazil. Parents were interviewed using the Developmental Milestones questionnaire, which contains 10 questions about cognitive, motor, social, and language abilities. Item response theory was used to examine item validity. RESULTS: We interviewed 415 parents. SWYC provided the most information on the children's development between 10 and 30 months. The performance of Brazilian and North American children was quite similar when children are younger than 36 months old. Above 36 months, North American children performed almost all items earlier than Brazilians. Convergent validity was 0.73 and internal consistency 0.97. CONCLUSION: The Brazilian version of the Developmental Milestones questionnaire presented acceptable measurement qualities that support the SWYCs potential as a developmental screening tool. As we found important differences between North American and Brazilian children in achieving the milestones, especially among the oldest children, additional normative studies are needed.

Fasting: a major limitation for resistance exercise training effects in rodents

Protocols that mimic resistance exercise training (RET) in rodents present several limitations, one of them being the electrical stimulus, which is beyond the physiological context observed in humans. Recently, our group developed a conditioning system device that does not use electric shock to stimulate rats, but includes fasting periods before each RET session. The current study was designed to test whether cumulative fasting periods have some influence on skeletal muscle mass and function. Three sets of male Wistar rats were used in the current study. The first set of rats was submitted to a RET protocol without food restriction. However, rats were not able to perform exercise properly. The second and third sets were then randomly assigned into three experimental groups: 1) untrained control rats, 2) untrained rats submitted to fasting periods, and 3) rats submitted to RET including fasting periods before each RET session. While the second set of rats performed a short RET protocol (i.e., an adaptation protocol for 3 weeks), the third set of rats performed a longer RET protocol including overload (i.e., 8 weeks). After the short-term protocol, cumulative fasting periods promoted loss of weight (P<0.001). After the longer RET protocol, no difference was observed for body mass, extensor digitorum longus (EDL) morphology or skeletal muscle function (P>0.05 for all). Despite no effects on EDL mass, soleus muscle displayed significant atrophy in the fasting experimental groups (P<0.01). Altogether, these data indicate that fasting is a major limitation for RET in rats.

Fasting: a major limitation for resistance exercise training effects in rodents.

Protocols that mimic resistance exercise training (RET) in rodents present several limitations, one of them being the electrical stimulus, which is beyond the physiological context observed in humans. Recently, our group developed a conditioning system device that does not use electric shock to stimulate rats, but includes fasting periods before each RET session. The current study was designed to test whether cumulative fasting periods have some influence on skeletal muscle mass and function. Three sets of male Wistar rats were used in the current study. The first set of rats was submitted to a RET protocol without food restriction. However, rats were not able to perform exercise properly. The second and third sets were then randomly assigned into three experimental groups: 1) untrained control rats, 2) untrained rats submitted to fasting periods, and 3) rats submitted to RET including fasting periods before each RET session. While the second set of rats performed a short RET protocol (i.e., an adaptation protocol for 3 weeks), the third set of rats performed a longer RET protocol including overload (i.e., 8 weeks). After the short-term protocol, cumulative fasting periods promoted loss of weight (P<0.001). After the longer RET protocol, no difference was observed for body mass, extensor digitorum longus (EDL) morphology or skeletal muscle function (P>0.05 for all). Despite no effects on EDL mass, soleus muscle displayed significant atrophy in the fasting experimental groups (P<0.01). Altogether, these data indicate that fasting is a major limitation for RET in rats.

Trypanosoma cruzi heparin-binding proteins present a flagellar membrane localization and serine proteinase activity.

Heparin-binding proteins (HBPs) play a key role in Trypanosoma cruzi-host cell interactions. HBPs recognize heparan sulfate (HS) at the host cell surface and are able to induce the cytoadherence and invasion of this parasite. Herein, we analysed the biochemical properties of the HBPs and also evaluated the expression and subcellular localization of HBPs in T. cruzi trypomastigotes. A flow cytometry analysis revealed that HBPs are highly expressed at the surface of trypomastigotes, and their peculiar localization mainly at the flagellar membrane, which is known as an important signalling domain, may enhance their binding to HS and elicit the parasite invasion. The plasmon surface resonance results demonstrated the stability of HBPs and their affinity to HS and heparin. Additionally, gelatinolytic activities of 70 kDa, 65·8 kDa and 59 kDa HBPs over a broad pH range (5·5-8·0) were revealed using a zymography assay. These proteolytic activities were sensitive to serine proteinase inhibitors, such as aprotinin and phenylmethylsulfonyl fluoride, suggesting that HBPs have the properties of trypsin-like proteinases.

Trypanosoma cruzi heparin-binding proteins mediate the adherence of epimastigotes to the midgut epithelial cells of Rhodnius prolixus.

Heparin-binding proteins (HBPs) have been demonstrated in both infective forms of Trypanosoma cruzi and are involved in the recognition and invasion of mammalian cells. In this study, we evaluated the potential biological function of these proteins during the parasite-vector interaction. HBPs, with molecular masses of 65·8 kDa and 59 kDa, were isolated from epimastigotes by heparin affinity chromatography and identified by biotin-conjugated sulfated glycosaminoglycans (GAGs). Surface plasmon resonance biosensor analysis demonstrated stable receptor-ligand binding based on the association and dissociation values. Pre-incubation of epimastigotes with GAGs led to an inhibition of parasite binding to immobilized heparin. Competition assays were performed to evaluate the role of the HBP-GAG interaction in the recognition and adhesion of epimastigotes to midgut epithelial cells of Rhodnius prolixus. Epithelial cells pre-incubated with HBPs yielded a 3·8-fold inhibition in the adhesion of epimastigotes. The pre-treatment of epimastigotes with heparin, heparan sulfate and chondroitin sulfate significantly inhibited parasite adhesion to midgut epithelial cells, which was confirmed by scanning electron microscopy. We provide evidence that heparin-binding proteins are found on the surface of T. cruzi epimastigotes and demonstrate their key role in the recognition of sulfated GAGs on the surface of midgut epithelial cells of the insect vector.

A novel xyloglucan film-based biosensor for toxicity assessment of ricin in castor seed meal.

Oil from the seed of the castor plant (Ricinus communis L.) is an important commodity for a number of industries, ranging from pharmaceuticals to renewable energy resources. However, the seed and subsequent seed meal contain ricin (RCA60), a potent cytotoxin, making it an unusable product for animal feed. In order to investigate the efficiency of reducing the toxicity of the seed meal, a biosensor is proposed by exploring the lectin-carbohydrate binding. A gold electrode was assembled with a film of Xyloglucan (XG) extracted from Hymenaea courbaril L. The analytical response to RCA60 was obtained using a polyclonal antibody against RCA60 conjugated to peroxidase. The current responses were generated by reaction with H2O2 and amplified with hydroquinone as chemical mediator. Voltammetric studies showed that the XG film was tightly bound to the gold electrode. This biosensor allows discriminate lectins in native and denatured forms. The limit of detection of native RCA60 was 2.1 µg mL(-1). This proposed biosensor showed to be a potential and accurate method for toxicity assessment of the ricin in castor seed meal by simple polysaccharide film-electrode strategy.

Trypanosoma cruzi: in vitro activity of Epoxy-alpha-Lap, a derivative of alpha-lapachone, on trypomastigote and amastigote forms.

Chagas disease is an endemic parasitic infection caused by Trypanosomacruzi that affects 18-20 million people in Central and South America. Recently we described the Epoxy-alpha-Lap, an oxyran derivative of alpha-lapachone, which presents a low toxicity profile and a high inhibitory activity against T.cruzi epimastigotes forms, the non-infective form of this parasite. In this work we described the trypanocidal effects of Epoxy-alpha-Lap on extracellular (trypomastigote) and intracellular (amastigote) infective forms of two T. cruzi strains (Y and Colombian) known by their different infective profile. Our results showed that Epoxy-alpha-Lap is lethal to trypomastigote Y and Colombian strains (97% and 84%, respectively). Interestingly, Epoxy-alpha-Lap also showed a trypanocidal effect in human macrophage infected with T. cruzi Y (85.6%) and Colombian (71.9%) strains amastigote forms. Similar effects were observed on T. cruzi amastigote infected Vero cells (96.4% and 95.0%, respectively). Our results pointed Epoxy-alpha-Lap as a potential candidate for Chagas disease chemotherapy since it presents trypanocidal activity on all T. cruzi forms with low) toxicity profile.

Heparin binding proteins from Leishmania (Viannia) braziliensis promastigotes.

We have examined the heparin binding proteins from Leishmania (Viannia) braziliensis promastigotes (HBP-Lb) by chromatography assays. The proposed strategy to isolate an enriched fraction of the HBP-Lb consisted of an association of the Triton X-114 method with affinity chromatography in heparin-Sepharose 4B column. SDS-PAGE analysis of the eluted proteins showed two main protein bands (65.0 and 54.5 kDa), while a single protein band was observed in native electrophoresis gel. The hemagglutination property of HBP-Lb over rabbit erythrocytes was confirmed up to 6.3+/-0.5 microg of protein mL(-1). Additionally, we have assayed the potential of HBP-Lb labeled with sulfo-NHS-LC-biotin in binding to nitrocellulose-immobilized gut proteins extracted of Lutzomyia intermedia and Lutzomyia whitmani. The results indicated a similar profile of five ligands (67.0, 62.1, 59.5, 56.0 and 47.5 kDa) in both studied Lutzomyia species. This is the first direct description of this class of protein in L. (V.) braziliensis with a suggestion of its biological activity in the interaction of Leishmania with Lutzomyia gut cells, which maybe a crucial step during this parasite's life cycle.

The main proteinases in Dermatobia hominis second and third instars larvae are serine-proteinases.

We performed a combination of proteinase assay, either in solution or immobilized in sodium dodecyl sulfate-polyacrylamide gel copolymerized with gelatin, to detect and quantify proteinases of Dermatobia hominis second (L2) and third (L3) instar larvae. In the quantitative assay, we examined proteinase activity by hydrolysis of a panel of peptide bonds specific for the main proteinase classes. We verified that the pGlu-Phe-Leu p-nitroanilide substrate was hydrolyzed by crude extracts of L2 (3.0+/-0.2 nmol h(-1)mg of protein(-1)) and L3 (7.7+/-0.1 nmol h(-1)mg of protein(-1)) and that both activities were partially inhibited by trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane, 15% and 3%, respectively. Also, we demonstrated that the Nalpha-p-Tosyl-l-Arg methyl ester substrate was hydrolyzed by crude extracts of L2 (117+/-24 nmol h(-1)mg of protein(-1)) and L3 (111+/-10 nmol h(-1)mg of protein(-1)), suggesting a predominance of esterase activity in the crude larval preparation. Interestingly, the specific activity of serine-proteinases was totally inhibited by phenylmethylsulphonyl fluoride in the L3 crude extract, while only 10% of this enzyme class activity was inhibited in the L2 crude extract. The results of the qualitative assays with substrate gels suggested that L2 and L3 larvae express serine-proteinases with similar (13 and 22 kDa) and distinct (50 kDa in L2 and 30 kDa in L3) relative molecular masses. These findings contribute to the biochemical characterization of D. hominis L2 and L3 larvae.

Trypanosoma cruzi: attachment to perimicrovillar membrane glycoproteins of Rhodnius prolixus.

Studies were carried out to identify proteins involved in the interface of Trypanosoma cruzi with the perimicrovillar membranes (PMM) of Rhodnius prolixus. Video microscopy experiments demonstrated high level of adhesion of T. cruzi Dm 28c epimastigotes to the surface of posterior midgut cells of non-treated R. prolixus. The parasites however were unable to attach to gut cells obtained from decapitated or azadirachtin-treated insects. The influence of carbohydrates on the adhesion to insect midgut was confirmed by inhibition of parasite attachment after midgut incubation with N-acetylgalactosamine, N-acetylmannosamine, N-acetylglucosamine, D-galactose, D-mannose or sialic acid. We observed that hydrophobic proteins in the surface of epimastigotes bind to polypeptides with 47.7, 45.5, 44, 43, 40.5, 36, 31 and 13kDa from R. prolixus PMM and that pre-incubation of lectins specifically inhibited binding to 31, 40.5, 44 and 45.5kDa proteins. We suggest that glycoproteins from PMM and hydrophobic proteins from epimastigotes are important for the adhesion of the parasite to the posterior midgut cells of the vector.

Leishmania amazonensis: early proteinase activities during promastigote-amastigote differentiation in vitro.

Leishmania proteinase activity is known as parasite differentiation marker, and has been considered relevant for leishmanial survival and virulence. These properties suggest that Leishmania proteinases can be promising targets for development of anti-leishmania drugs. Here, we analyze the activities of four proteinases during the early phase of the Leishmania amazonensis promastigotes differentiation into amastigotes induced by heat shock. We have examined activities of cysteine-, metallo-, serine-, and aspartic-proteinase by hydrolysis of specific chromogenic substrates at pH 5.0 and at the optimal pH for each enzyme. Our results show that metallo-, serine-, and aspartic-proteinases activities were down-regulated during the shock-induced transformation of promastigotes into amastigotes. In contrast, cysteine-proteinase activity increased concomitantly with the promastigote differentiation. Immunocytochemical localization using two anti-cysteine-proteinase monospecific rabbit antibodies detected the enzyme in several cell compartments of both parasite stages. Our results show different proteinase activity modulation and expression during the early phases of the shock-induced parasite transformation.

Th1 and Th2 immunological profile induced by cysteine proteinase in murine leishmaniasis.

This study evaluated the immune response to three synthetic peptides (pI, VMVEQVICFD; pII, VGGGLCFE; pIII, PYFLGSIMNTCHYT) from the COOH-terminal region of Leishmania amazonensis cysteine proteinases, in BALB/c- and CBA-infected mice with this parasite. Only BALB/c mice, previously inoculated with pI, showed a distinct exacerbation of the lesion. Blastogenesis assays were performed with lymph node cells from the group of mice infected with L. amazonensis, but not inoculated with the peptides, and we detected lymphoproliferative responses in BALB/c and CBA mice with a 5.0x and 2.5x stimulation index, respectively. Cell phenotypes were evaluated and in both mouse strains CD8(+)cells proliferated more extensively than CD4(+)cells. INF-gamma and nitric oxide were detected only in supernatants obtained from CBA mouse lymph node cell cultures, whereas IL-4 was detected in supernatant cultures from both strains of mice. Our results indicate a preferential stimulation of CD8(+)T-cell subsets by the pI cysteine proteinase peptide and the induction of both Th1 and Th2 phenotypes during L. amazonensis infections in both BALB/c and CBA mice. We suggest that the pI peptide from the COOH-terminal region of the cysteine proteinase induces immune responses different from those elicited by the whole molecule.

Subcellular localization of an extracellular serine protease in Leishmania (Leishmania) amazonensis.

Extracellular proteolytic activity was detected in a Leishmania ( L.) amazonensis culture supernatant and a 56-kDa protein was purified using (NH4)2SO4 precipitation followed by affinity chromatography on aprotinin-agarose. A rabbit serum obtained against the 56-kDa extracellular serine protease was used in order to analyze its location in L. ( L.) amazonensis parasites. Immunocytochemistry studies revealed that the enzyme is mainly found in the flagellar pocket and cytoplasmic vesicles of promastigote forms, whereas in amastigotes, it is located in electron-dense structures resembling megasomes. These results indicate that the 56-kDa serine protease is released into the extracellular environment through the flagellar pocket; and its intracellular location suggests either a correlated enzymatic activity or intracellular trafficking.

The use of metal chelate affinity chromatography on the isolation of Leishmania chagasi promastigote hydrophobic proteinases.

In this work, we have assessed the possibility of isolating metalloproteinase fractions from infective Leishmania chagasi promastigotes. Our strategy was the association of the Triton X-114 method with iminodiacetic chromatography enriched with Zn2+. Thus, by using acid conditions, it was possible to isolate two fractions containing two polypeptides, 59 and 63 kDa. The enzymatic activity assay indicated that the two fractions and the two polypeptides had proteinase activities. In addition, it was proposed that those proteinase activities were affected by the presence of 1,10-phenanthroline, a metalloproteinase inhibitor. With this gentle chromatography strategy proposed it is possible to obtain metalloproteinases from L. chagasi in folding that preserve the enzyme activity. This is important for further studies on pathological complications observed in visceral leishmaniasis.

Effect of Trypanosoma cruzi released antigens binding to non-infected cells on anti-parasite antibody recognition and expression of extracellular matrix components.

It has been proposed that antigens released by Trypanosoma cruzi sensitize vertebrate cells leading to their destruction by the immune response raised against the parasite. Here, we characterized antigens released by trypomastigotes of T. cruzi that bind to non-infected cells and investigated biological consequences of this adsorption. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of antigens released by [(35)S]-methionine-labeled parasites revealed the presence of polypeptides mainly ranging from 85 to 170 kDa that were specifically recognized by sera from chronically T. cruzi infected rabbits. Polypeptides of 85-110 and 160-170 kDa bound to non-infected epithelial, fibroblast and muscle mammalian cell lines, which thus became targets for anti-T. cruzi antibody binding. Cysteine-proteinase, but not trans-sialidase, was detected among the cell-bound antigens, and purified cysteine-proteinase was adsorbed to non-infected cells. Immunoelectron microscopic studies showed that parasite antigens were mainly released as membrane vesicles that adhered to membrane microvilli and were internalized by mammalian cells. We provide evidence that adsorption of parasite antigens induced an increase in expression of extracellular matrix (ECM) components (fibronectin, laminin and type I collagen) by sensitized cells. Thus, our data reinforce the idea that in vivo T. cruzi released antigens might be involved in the establishment of inflammation, sensitizing non-infected host cells and triggering an immune response against parasite antigens. Further, our data showed that antigen sensitization modulates biological cell functions as ECM expression that could mediate cell-cell or parasite-host cell interactions, contributing to the establishment of inflammation.

The use of primary stenting or balloon percutaneous transluminal coronary angioplasty for the treatment of acutely occluded saphenous vein grafts. Results from the Brazilian National Registry--CENIC. Central Nacional de Intervenções Cardiovasculares.

OBJECTIVE: We conducted a comparative analysis of the in-hospital outcomes of patients who underwent primary percutaneous transluminal angioplasty (PTCA) or stent implantation because of an acute myocardial infarction (AMI) related to an acute vein graft occlusion. METHODS: Since 1991 the Brazilian Society of Hemodynamic and Interventional Cardiology has maintained a large database (CENIC). From these, we selected all consecutive patients, who underwent primary PTCA or stenting in the first 24 hours of AMI, with the target vessel being an occluded vein graft. Immediate results and major coronary events occurring up until hospital discharge were analyzed. RESULTS: During this period, 5,932 patients underwent primary PTCA or stenting; 158 (3%) of the procedures were performed because of an acute vein graft occlusion. Stenting was performed in 74 (47%) patients. Patients treated with stents had a higher success rate and lower mean residual stenosis compared with those who underwent primary balloon PTCA. The incidence of reinfarction and death were similar for stenting and balloon PTCA. CONCLUSION: Primary percutaneous treatment of AMI related to acute vein graft occlusion is still an uncommon practice. Primary stenting improved luminal diameter and offered higher rates of success; however, this strategy did not reduce the in-hospital reinfarction and death rate, compared with that occurring with PTCA treatment.

Facilitation of latent inhibition by the atypical antipsychotic risperidone.

The action of the atypical antipsychotic risperidone on latent inhibition (LI), an animal model of schizophrenia, was investigated. The parameters of the procedure were set at values insufficient to generate LI in control rats. On the first day, rats administered 0.5, 1.0, or 2.0 mg/kg ip risperidone or vehicle were preexposed (PE) to 10 tone presentations. On the second day, they were again injected with drug or vehicle and then submitted to two conditioned stimulus (CS; tone)-unconditioned stimulus (US; shock) pairings. On the third day, suppression of their drinking response under the CS was measured. Nonpreexposed (NPE) animals were submitted to the same procedure except for the tone preexposure. On the suppression test, LI was not observed in control rats as well as in animals given 0.5 mg/kg risperidone. Animals given 1.0 and 2.0 mg risperidone, however, displayed an LI effect. The facilitation of LI by risperidone gives additional support to the LI paradigm as an animal model of schizophrenia.

A strategy for the identification of T-cell epitopes on Leishmania cysteine proteinases.

In this study computational analysis was used to compile sequence alignments, construct a dendrogram and calculate physical data in order to predict potential T-cell epitopes of the Leishmania cysteine proteinase. Using multiple alignment of human and Leishmania proteinase sequences deposited on data bank sequences, it was possible to predict that the extreme C-terminus of cysteine proteinase (Cyspep, 355-444) contained three peptides (pI 361-370, pII 415-422 and pIII 431-444) with charge score, hydrophobicity and isoelectric points compatible for human leucocyte-associated antigen (HLA) class II binding. The prediction was confirmed in vitro through the ability of synthetic peptides corresponding to the predicted regions to stimulate peripheral blood mononuclear cells of patients with leishmaniasis.