Analysis of Single-Nucleotide Polymorphisms in the crt-o and mdr1 Genes of Plasmodium vivax among Chloroquine-Resistant Isolates from the Brazilian Amazon Region

Plasmodium vivax parasites with chloroquine resistance (CQR) are already circulating in the Brazilian Amazon. Complete single-nucleotide polymorphism (SNP) analyses of coding and noncoding sequences of the pvmdr1 and pvcrt-o genes revealed no associations with CQR, even if some mutations had not been randomly selected. In addition, striking differences in the topologies and numbers of SNPs in these transporter genes between P. vivax and P. falciparum reinforce the idea that mechanisms other than mutations may explain this virulent phenotype in P. vivax.

TESA-blot for the diagnosis of Chagas disease in dogs from co-endemic regions for Trypanosoma cruzi, Trypanosoma evansi and Leishmania chagasi

We standardized serodiagnosis of dogs infected with Trypanosoma cruzi using TESA (trypomastigote excreted-secreted antigen)-blot developed for human Chagas disease. TESA-blot showed 100% sensitivity and specificity. In contrast, ELISA using TESA (TESA-ELISA) or epimastigotes (epi-ELISA) as antigen yielded 100% sensitivity but specificity of 94.1% and 49.4%, respectively. When used in field studies in an endemic region for Chagas disease, visceral leishmaniasis and Trypanosoma evansi (Mato Grosso do Sul state, Central Brazil), positivities were 9.3% for TESA-blot, 10.7% for TESA-ELISA and 32% for epi-ELISA. Dogs from a non-endemic region for these infections (Rondonia state, western Amazonia) where T cruzi is enzootic showed positivity of 4.5% for TESA-blot and epi-ELISA and 6.8% for TESA-ELISA. Sera from urban dogs from Santos, Sao Paulo, where these diseases are absent, yielded negative results. TESA-blot was the only method that distinguished dogs infected with T cruzi from those infected with Leishmania chagasi and/or Trypanosoma evansi. (C) 2009 Published by Elsevier B.V.

Direct effect of Plasmodium vivax recombinant vaccine candidates AMA-1 and MSP-1(19) on the innate immune response

The recombinant apical membrane antigen 1 (AMA-1) and 19-kDa fragment of merozoite surface protein (MSP-1(19)) are the lead candidates for inclusion in a vaccine against blood stages of malaria due to encouraging protective studies in humans and animals. Despite the importance of an efficacious malaria vaccine, vaccine-related research has focused on identifying antigens that result in protective immunity rather than determining the nature of anti-malarial immune effector mechanisms. Moreover, emphasis has been placed on adaptive rather than innate immune responses. In this study, we investigated the effect of Plasmodium vivax vaccine candidates Pv-AMA-1 and Pv-MSP-1(19) on the immune response of malaria-naive donors. Maturation of dendritic cells is altered by Pv-AMA-1 but not Pv-MSP-1(19), as observed by differential expression of cell surface markers. In addition, Pv-AMA-1 induced an increased production of MIP-1 alpha/CCL3 and decreased production of TARC/CCL17 levels in both dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs). Finally, a significant pro-inflammatory response was elicited by Pv-AMA-1-stimulated PBMCs. These results suggest that the recombinant vaccine candidate Pv-AMA-1 may play a direct role on innate immune response and might be involved in parasite destruction. (C) 2007 Elsevier Ltd. All rights reserved.

Splenectomy Increases Mortality in Murine Trypanosoma cruzi Infection

The spleen is a secondary lymphoid organ that harbours a variety of cells such as T and B lymphocytes and antigen-presenting cells important to immune response development. In this study, we evaluated the impact of spleen removal in the immune response to experimental Trypanosoma cruzi infection. C57BL/6 mice were infected with Y strain of the parasite and infection was followed daily. Mice that underwent splenectomy had fewer parasites in peripheral blood at the peak of infection; however, mortality was increased. Histological analysis of heart and liver tissues revealed an increased number of parasites and inflammatory infiltrates at these sites. Spleen removal was associated with reduction in IFN-gamma and TNF-alpha production during infection as well as with a decrease in specific antibody secretion. Haematological disorders were also detected. Splenectomized mice exhibited severe anaemia and decreased bone marrow cell numbers. Our results indicate that spleen integrity is critical in T. cruzi infection for the immune response against the parasite, as well as for the control of bone marrow haematological function.

Plasmodium vivax recombinant vaccine candidate AMA-1 plays an important role in adaptive immune response eliciting differentiation of dendritic cells

The Apical Membrane Antigen-1 (AMA-1) is a well-characterized and functionally important merozoite protein and is currently considered a major candidate antigen for a malaria vaccine. Previously, we showed that AMA-1 has an influence on cellular immune responses of malaria-naive subjects, resulting in an alternative activation of monocyte-derived dendritic cells and induction of a pro-inflammatory response by stimulated PBMCs. Although there is evidence, from human and animal malaria model systems that cell-mediated immunity may contribute to both protection and pathogenesis, the knowledge on cellular immune responses in vivax malaria and the factors that may regulate this immunity are poorly understood. In the current work, we describe the maturation of monocyte-derived dendritic cells of P. vivax naturally infected individuals and the effect of P. vivax vaccine candidate Pv-AMA-1 on the immune responses of the same donors. We show that malaria-infected subjects present modulation of DC maturation, demonstrated by a significant decrease in expression of antigen-presenting molecules (CD1a, HLA-ABC and HLA-DR), accessory molecules (CD40, CD80 and CD86) and Fc gamma RI (CD64) receptor (P <= 0.05). Furthermore, Pv-AMA-1 elicits an upregulation of CD1a and HLA-DR molecules on the surface of monocyte-derived dendritic cells (P=0.0356 and P=0.0196, respectively), and it is presented by AMA-1-stimulated DCs. A significant pro-inflammatory response elicited by Pv-AMA-1-pulsed PBMCs is also demonstrated, as determined by significant production of TNF-alpha, IL-12p40 and IFN-gamma (P <= 0.05). Our results suggest that Pv-AMA-1 may partially revert DC down-modulation observed in infected subjects, and exert an important role in the initiation of pro-inflammatory immunity that might contribute substantially to protection. (c) 2009 Elsevier Ltd. All rights reserved.

Plasmodium vivax apical membrane antigen-1: comparative recognition of different domains by antibodies induced during natural human infection

The Apical Membrane Antigen-1 (AMA-1) of Plasmodium sp. has been suggested as a vaccine candidate against malaria. This protein seems to be involved in merozoite invasion and its extra-cellular portion contains three distinct domains: DI, DII, and DIII. Previously, we described that Plasmodium vivax AMA-1 (PvAMA-1) ectodomain is highly immunogenic in natural human infections. Here, we expressed each domain, separately or in combination (DI-II or DII-III), as bacterial recombinant proteins to map immunodominant epitopes within the PvAMA-1 ectodomain. IgG recognition was assessed by ELISA using sera of P. vivax-infected individuals collected from endemic regions of Brazil or antibodies raised in immunized mice. The frequencies of responders to recombinant proteins containing the DII were higher than the others and similar to the ones observed against the PvAMA-1 ectodomain. Moreover, ELISA inhibition assays using the PvAMA-1 ectodomain as substrate revealed the presence of many common epitopes within DI-II that are recognized by human immune antibodies. Finally, immunization of mice with the PvAMA-1 ectodomain induced high levels of antibodies predominantly to DI-II. Together, our results indicate that DII is particularly immunogenic during natural human infections, thus indicating that this region could be used as part of an experimental sub-unit vaccine to prevent vivax malaria. (C) 2008 Elsevier Masson SAS. All rights reserved.

Biophysical characterization of the recombinant merozoite surface protein-3 of Plasmodium vivax

Plasmodium vivax Merozoite Surface Protein-3 alpha and 3 beta are members of a family of related merozoite surface proteins that contain a central alanine-rich domain with heptad repeats that is predicted to form alpha-helical secondary and coiled-coil tertiary structures. Seven recombinant proteins representing different regions of MSP-3 alpha and MSP-3 beta of P. vivax were generated to investigate their structure. Circular dichroism spectra analysis revealed that some proteins are folded with a high degree of alpha-helices as secondary structure, whereas other products contain a high content of random coil. Using size exclusion chromatography, we found that the two smaller fragments of the MSP-3 alpha, named CC4 and CC5, predicted to form coiled-coil (CC) structures, eluted at volumes corresponding to molecular weights larger than their monomeric masses. This result suggests that both proteins are oligomeric molecules. Analytical ultracentrifugation experiments showed that the CC5 oligomers are elongated molecules. Together, these data may help to understand important aspects of P. vivax biology. (C) 2008 Elsevier B.V. All rights reserved.

Cruzain inhibition by hydroxymethylnitrofurazone and nitrofurazone: investigation of a new target in Trypanosoma cruzi

Nitrofurazone (NF) and its derivative, hydroxymethylnitrofurazone (NFOH), have presented antichagasic activity. NFOH has higher activity and lower mutagenicity. The aim of this work was to assess whether NF and its derivative NFOH would also be inhibitors of cruzain, besides their trypanothione reductase inhibitory activity. In vitro cruzain inhibition tests were performed for both compounds, and the 50% inhibitory concentration (IC(50)) for NF and NFOH presented values of 22.83 +/- 1.2 mu M and 10.55 +/- 0.81 mu M, respectively. AM1 semi-empirical molecular modeling studies were performed to understand the activity of the compounds, corroborating the observed cruzain inhibitory activity.

A recombinant vaccine based on domain II of Plasmodium vivax Apical Membrane Antigen 1 induces high antibody titres in mice

The Apical Membrane Antigen 1 (AMA-1) is considered a promising candidate for development of a malaria vaccine against asexual stages of Plasmodium. We recently identified domain II (DII) of Plasmodium vivax AMA-1 (PvAMA-1) as a highly immunogenic region recognised by IgG antibodies present in many individuals during patent infection with P. vivax. The present study was designed to evaluate the immunogenic properties of a bacterial recombinant protein containing PvAMA-1 DII. To accomplish this, the recombinant protein was administered to mice in the presence of each of the following six adjuvants: Complete/Incomplete Freund`s Adjuvant (CFA/IFA), aluminium hydroxide (Alum), Quil A, QS21 saponin, CpG-ODN 1826 and TiterMax. We found that recombinant DII was highly immunogenic in BALB/c mice when administered in the presence of any of the tested adjuvants. Importantly, we show that DII-specific antibodies recognised the native AMA-1 protein expressed on the surface of P. vivax merozoites isolated from the blood of infected patients. These results demonstrate that a recombinant protein containing PvAMA-1 DII is immunogenic when administered in different adjuvant formulations, and indicate that this region of the AMA-1 protein should continue to be evaluated as part of a subunit vaccine against vivax malaria. (C) 2010 Elsevier Ltd. All rights reserved.

On the Cytoadhesion of Plasmodium vivax-Infected Erythrocytes

Background. Plasmodium falciparum and Plasmodium vivax are responsible for most of the global burden of malaria. Although the accentuated pathogenicity of P. falciparum occurs because of sequestration of the mature erythrocytic forms in the microvasculature, this phenomenon has not yet been noted in P. vivax. The increasing number of severe manifestations of P. vivax infections, similar to those observed for severe falciparum malaria, suggests that key pathogenic mechanisms (eg, cytoadherence) might be shared by the 2 parasites. Methods. Mature P. vivax-infected erythrocytes (Pv-iEs) were isolated from blood samples collected from 34 infected patients. Pv-iEs enriched on Percoll gradients were used in cytoadhesion assays with human lung endothelial cells, Saimiri brain endothelial cells, and placental cryosections. Results. Pv-iEs were able to cytoadhere under static and flow conditions to cells expressing endothelial receptors known to mediate the cytoadhesion of P. falciparum. Although Pv-iE cytoadhesion levels were 10-fold lower than those observed for P. falciparum-infected erythrocytes, the strength of the interaction was similar. Cytoadhesion of Pv-iEs was in part mediated by VIR proteins, encoded by P. vivax variant genes (vir), given that specific antisera inhibited the Pv-iE-endothelial cell interaction. Conclusions. These observations prompt a modification of the current paradigms of the pathogenesis of malaria and clear the way to investigate the pathophysiology of P. vivax infections.

Crystal structure of Trypanosoma cruzi dihydroorotate dehydrogenase from Y strain

Trypanosoma cruzi is the etiological agent of Chagas` disease, a pathogenesis that affects millions of people in Latin America. Here, we report the crystal structure of dihydroorotate dehydrogenase (DHODH) from T cruzi strain Y solved at 2.2 angstrom resolution. DHODH is a flavin mononucleotide containing enzyme, which catalyses the oxidation Of L-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. Genetic studies have shown that DHODH is essential for T cruzi survival, validating the idea that this enzyme can be considered an attractive target for the development of antichagasic drugs. In our work, a detailed analysis of T cruzi DHODH crystal structure has allowed us to suggest potential sites to be further exploited for the design of highly specific inhibitors through the technology of structure-based drug design. (c) 2008 Elsevier Inc. All rights reserved.

Reduction of parasitism tissue by treatment of mice chronically infected with Trypanosoma cruzi with lignano lactones

The reduction of parasitism tissue upon treatment with two lignano lactones, namely (-)- cubebin (CUB) and (-)-hinokinin (HNK), was evaluated in the chronic phase of Chagas` disease by quantifying the enzyme beta-galactosidase expressed by the CL B5 clone strain of Trypanosoma cruzi. Tissue karyometry was also performed. Treatment with the assessed lignans led to a larger reduction in parasitism tissue in all evaluated organs, compared with benznidazole (BZN). Oral treatment with CUB or HNK was more effective. Karyometry results demonstrated that the infected control animals had increased nuclear area compared with uninfected controls, indicating cellular hypertrophy. Results also revealed that use of CUB or HNK was able to significantly prevent this increase, and a slight decrease in the nuclear area was observed, compared with mice treated with BZN. Taken together, these data demonstrate that CUB and HNK could be considered as potential compounds for the development of new drugs for treatment of Chagas` disease.

Trypanosoma cruzi: evaluation of (-)-cubebin derivatives activity in the messenger RNAs processing

No fully effective treatment has been developed since the discovery of Chagas` disease. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effective in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing, which is unusual RNA processing reaction, and it implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. Cubebin derivatives seem to provide treatments with less collateral effects than benznidazole and showed similar or better trypanocidal activities than benznidazole. Therefore, the cubebin derivatives ((-)-6,6`-dinitrohinokinin (DNH) and (-)-hinokinin (HQ)) interference in the mRNA processing was evaluated using T. cruzi permeable cells (Y and BOL (Bolivia) strains) following by RNase protection reaction. These substances seem to intervene in any step of the RNA transcription, promoting alterations in the RNA synthesis, even though the RNA processing mechanism still occurs. Furthermore, HQ presented better activity against the parasites than DNH, meaning that BOL strain seems to be more resistant than Y.

Trypanosoma cruzi: The effects of zinc supplementation in the immune response during the course of experimental disease

Zinc is an essential nutritional component required for normal development and maintenance of immune functions. The possible effects of zinc in upregulating the host immune response during the acute and chronic phases of experimental Chagas` disease were evaluated. In young, infected and Zn-supplemented animals, higher concentrations of IFN-gamma and NO were observed. During the chronic phase, decreased concentrations of NO and IFN-gamma were found for older infected animals that received Zn supplementation. For young animals, hearts from Zn-supplemented groups displayed reduced inflammatory infiltrate, heart weight and number of amastigote burdens. For older, infected and Zn-supplemented animals amastigote nests were absent with reduced inflammatory cell infiltrate. This study identifies a potentially novel therapeutic approach that could control the parasite load during acute phase of disease, consequently preventing the deleterious, parasite-elicited responses observed during chronic phase. (C) 2008 Elsevier Inc. All rights reserved.

Effects of repetitive stress during the acute phase of Trypanosoma cruzi infection on chronic Chagas` disease in rats

The effect of repetitive stress during acute infection with Trypanosoma cruzi (T. cruzi) on the chronic phase of ensuing Chagas` disease was the focus of this investigation. The aim of this study was to evaluate in Wistar rats the influence of repetitive stress during the acute phase of infection (7 days) with the Y strain of T. cruzi on the chronic phase of the infection (at 180 days). Exposure to ether vapor for 1min twice a day was used as a stressor. Repetitive stress enhanced the number of circulating parasites and cardiac tissue disorganization, from a moderate to a severe diffuse mononuclear inflammatory process and the presence of amastigote burden in the cardiac fibers. Immunological parameters revealed that repetitive stress triggered a reduced concanavalin A induced splenocyte proliferation in vitro with major effects on the late chronic phase. Serum interleukin-12 concentration decreased in both stressed and infected rats in the early phase of infection although it was higher on 180 days post-infection. These results suggest that repetitive stress can markedly impair the host`s immune system and enhance the pathological process during the chronic phase of Chagas` disease.