Different serological cross-reactivity of Trypanosoma rangeli forms in Trypanosoma cruzi-infected patients sera

<p>Abstract</p> <p>Background</p> <p>American Trypanosomiasis or Chagas disease is caused by <it>Trypanosoma cruzi </it>which currently infects approximately 16 million people in the Americas causing high morbidity and mortality. Diagnosis of American trypanosomiasis relies on serology, primarily using indirect immunofluorescence assay (IFA) with <it>T. cruzi </it>epimastigote forms. The closely related but nonpathogenic <it>Trypanosoma rangeli </it>has a sympatric distribution with <it>T. cruzi </it>and is carried by the same vectors. As a result false positives are frequently generated. This confounding factor leads to increased diagnostic test costs and where false positives are not caught, endangers human health due to the toxicity of the drugs used to treat Chagas disease.</p> <p>Results</p> <p>In the present study, serologic cross-reactivity between the two species was compared for the currently used epimastigote form and the more pathologically relevant trypomastigote form, using IFA and immunoblotting (IB) assays. Our results reveal an important decrease in cross reactivity when <it>T. rangeli </it>culture-derived trypomastigotes are used in IFA based diagnosis of Chagas disease. Western blot results using sera from both acute and chronic chagasic patients presenting with cardiac, indeterminate or digestive disease revealed similar, but not identical, antigenic profiles.</p> <p>Conclusion</p> <p>This is the first study addressing the serological cross-reactivity between distinct forms and strains of <it>T. rangeli </it>and <it>T. cruzi </it>using sera from distinct phases of the Chagasic infection. Several <it>T. rangeli</it>-specific proteins were detected, which may have potential as diagnostic tools.</p

Transsulfuration is an active pathway for cysteine biosynthesis in Trypanosoma rangeli

BACKGROUND: Cysteine, a sulfur-containing amino acid, plays an important role in a variety of cellular functions such as protein biosynthesis, methylation, and polyamine and glutathione syntheses. In trypanosomatids, glutathione is conjugated with spermidine to form the specific antioxidant thiol trypanothione (T[SH]2) that plays a central role in maintaining intracellular redox homeostasis and providing defence against oxidative stress. METHODS: We cloned and characterised genes coding for a cystathionine β-synthase (CβS) and cysteine synthase (CS), key enzymes of the transsulfuration and assimilatory pathways, respectively, from the hemoflagellate protozoan parasite Trypanosoma rangeli. RESULTS: Our results show that T. rangeli CβS (TrCβS), similar to its homologs in T. cruzi, contains the catalytic domain essential for enzymatic activity. Unlike the enzymes in bacteria, plants, and other parasites, T. rangeli CS lacks two of the four lysine residues (Lys26 and Lys184) required for activity. Enzymatic studies using T. rangeli extracts confirmed the absence of CS activity but confirmed the expression of an active CβS. Moreover, CβS biochemical assays revealed that the T. rangeli CβS enzyme also has serine sulfhydrylase activity. CONCLUSION: These findings demonstrate that the RTS pathway is active in T. rangeli, suggesting that this may be the only pathway for cysteine biosynthesis in this parasite. In this sense, the RTS pathway appears to have an important functional role during the insect stage of the life cycle of this protozoan parasite

SÍNTESE E CARACTERIZAÇÃO DO NÚCLEO PIRIDO[2,3-g]QUINOLÍNICO SUBSTITUÍDO E AVALIAÇÃO DE SUAS PROPRIEDADES CITOTÓXICAS

Segundo a Organização Mundial da Saúde, a leishmaniose e a tripanossomíase são problemas crescentes de saúde pública, sendo endêmicos particularmente na América Latina. Os fármacos utilizados no tratamento destas doenças foram desenvolvidos há muitas décadas, e apresentam pouca eficácia e fortes efeitos colaterais. As piridoquinolinas são compostos heterocíclicos diazaantracênicos que apresentam propriedades citotóxicas. A importância biológica das piridoquinolinas e o interesse em descobrir novos agentes leishmanicidas e tripanocidas estimularam a síntese de pirido[2,3–g]quinolinas substituídas, e a avaliação do seu potencial leishmanicida e tripanocida. Assim, este trabalho descreve a síntese de piridoquinolinas empregando a reação de termociclização do bis-aduto fenilenodiamínico do ácido de Meldrum. O 2,5-dimetil-1,4-bis[(2,2-dimetil-4,6-dioxo-1,3-dioxano-5-ilidenometil)amino]-benzeno foi obtido em bom rendimento pela condensação do ácido de Meldrum com a 2,5-dimetil-1,4-fenilenodiamina em ortoformato de trimetila. A termociclização em éter difenílico foi acompanhada pela eliminação espontânea de acetona e dióxido de carbono gerando o diazatriciclo, que foi clorado sem prévia purificação com oxicloreto de fósforo originando a 4,9-dicloro-5,10-dimetil-pirido[2,3-g]quinolina, em rendimentos baixos. A reação de substituição nucleofílica com o 2-amino-5-dietilaminopentano foi repetida várias vezes em diferentes condições reacionais, mas não se obteve o produto esperado, necessitando da busca de novos procedimentos e otimização das condições de reação. Os compostos obtidos foram caracterizados por técnicas espectroscópicas de IV e RMN 1H. A avaliação das propriedades leishmanicida e tripanocida foi feita para o composto 4,9-dicloro-5,10-dimetil-pirido[2,3-g]quinolina, porém o mesmo não apresentou qualquer atividade

Mannose-Binding Lectin Regulates Host Resistance and Pathology during Experimental Infection with Trypanosoma cruzi

Mannose-binding lectin (MBL) is a humoral pattern-recognition molecule important for host defense. Although recent genetic studies suggest an involvement of MBL/MASP2-associated pathways in Chagas’ disease, it is currently unknown whether MBL plays a role in host resistance to the intracellular protozoan Trypanosoma cruzi, the causative agent of Chagas’ disease. In this study we employed MBL−/− mice to assess the role of MBL in resistance to experimental infection with T. cruzi. T. cruzi infection enhanced tissue expression of MBL both at the mRNA and protein level. Similarly, symptomatic acute Chagas’ disease patients displayed increased serum concentrations of MBL compared to patients with indeterminate, asymptomatic forms of the disease. Furthermore, increased parasite loads in the blood and/or tissue were observed in MBL−/− mice compared to WT controls. This was associated with reduced systemic levels of IL-12/23p40 in MBL−/− mice. Importantly, MBL−/− mice infected with a cardiotropic strain of T. cruzi displayed increased myocarditis and cardiac fibrosis compared to WT controls. The latter was accompanied by elevated hydroxyproline content and mRNA levels of collagen-1 and -6 in the heart. These observations point to a previously unappreciated role for MBL in regulating host resistance and cardiac inflammation during infection with a major human pathogen

Recommendations from a Satellite Meeting (International Symposium to Commemorate the 90th Anniversary of the Discovery of Chagas Disease, April 11-16 1999, Rio de Janeiro, Brazil)

During this symposium the standardization of the nomenclature of Trypanosoma cruzi strains was discussed, in a parallel session, with a view to facilitating the use and understanding of a common nomenclature that would serve not only taxonomists but the general community of researchers working with T. cruzi. The diversity in the behavior and morphology of T. cruzi isolates was soon recognized after the discovery of Chagas disease. Since then a variety of biochemical and molecular techniques have revealed the great genetic diversity present in strains of this parasite. Different investigators have described this diversity by using various terms. Correlation between this diversity and the complex epidemiological and clinical manifestations of the disease has however been hindered by the lack of a common nomenclature. Recent studies have indicated a convergence among investigators regarding the clustering of strains of T. cruzi, into two principal groups. This consensus, together with the report of a meeting on the standardization of methods for T. cruzi classification held in Panama (unpublished document TDR/EPICHA-TCC/85.3 Geneva, World Health Organization, 1985), form the basis of the recommendations outlined in this document

Improving the serodiagnosis of canine Leishmania infantum infection in geographical areas of Brazil with different disease prevalence

Serodiagnosis of Leishmania infantum infection in dogs relies on the detection of antibodies against leishmanial crude extracts or parasitic defined antigens. The expansion of canine leishmaniasis from geographical areas of Brazil in which the infection is endemic to regions in which the disease is emerging is occurring. This fact makes necessary the analysis of the serodiagnostic capabilities of different leishmanial preparations in distinct geographical locations. In this article sera from dogs infected with Leishmania and showing the clinical form of the disease, were collected in three distinct Brazilian States and were tested against soluble leishmanial antigens or seven parasite individual antigens produced as recombinant proteins. We show that the recognition of soluble leishmanial antigens by sera from these animals was influenced by the geographical location of the infected dogs. Efficacy of the diagnosis based on this crude parasite preparation was higher in newly endemic regions when compared with areas of high disease endemicity. We also show that the use of three of the recombinant proteins, namely parasite surface kinetoplastid membrane protein of 11 kDa (KMP-11), and two members of the P protein family (P2a and P0), can improve the degree of sensitivity without adversely affecting the specificity of the diagnostic assays for canine leishmaniasis, independently of the geographical area of residence. In addition, sera from dogs clinically healthy but infected were also assayed with some of the antigen preparations. We demonstrate that the use of these proteins can help to the serodiagnosis of Leishmania infected animals with subclinical infections. Finally, we propose a diagnostic protocol using a combination of KMP-11, P2a y P0, together with total leishmanial extractsThis work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brazil) within the call“CNPq/MS/SCTIE/DECIT N° 32/2014 - Pesquisas sobre Leishmanioses”grant number reference 467389/2014-4. Institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMSO are also acknowledged. TC received scholarship from Fundação de Amparo a Pesquisa do Estado de Santa Catarina–FAPES

Characterising the KMP-11 and HSP-70 recombinant antigens' humoral immune response profile in chagasic patients

11 pages, 6 figures.-- The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2334/9/186/pre pubBackground: Antigen specificity and IgG subclass could be significant in the natural history of Chagas' disease. The relationship between the different stages of human Chagas' disease and the profiles of total IgG and its subclasses were thus analysed here; they were directed against a crude T. cruzi extract and three recombinant antigens: the T. cruzi kinetoplastid membrane protein-11 (rKMP-11), an internal fragment of the T. cruzi HSP-70 protein192-433, and the entire Trypanosoma rangeli HSP-70 protein. Methods: Seventeen Brazilian acute chagasic patients, 50 Colombian chronic chagasic patients (21 indeterminate and 29 cardiopathic patients) and 30 healthy individuals were included. Total IgG and its subtypes directed against the above-mentioned recombinant antigens were determined by ELISA tests. Results: The T. cruzi KMP-11 and T. rangeli HSP-70 recombinant proteins were able to distinguish both acute from chronic chagasic patients and infected people from healthy individuals. Specific antibodies to T. cruzi crude antigen in acute patients came from IgG3 and IgG4 subclasses whereas IgG1 and IgG3 were the prevalent isotypes in indeterminate and chronic chagasic patients. By contrast, the specific prominent antibodies in all disease stages against T. cruzi KMP-11 and T. rangeli HSP-70 recombinant antigens were the IgG1 subclass.This work was supported by Colciencias Research project No. 1203-333- 18692. IDF was supported by Colciencias and the Universidad Javeriana's Young Researcher 2008 Programme (Bogotá, Colombia). MCT and MCL were supported by P06-CTS-02242 Grant from PAI (Junta de Andalucia) and RICET-RD06/0021-0014, Spain. MS received financial support from the Brazilian agency - CNPq.Peer reviewe