Chagas' disease: an update on immune mechanisms and therapeutic strategies

The final decade of the 20th century was marked by an alarming resurgence in infectious diseases caused by tropical parasites belonging to the kinetoplastid protozoan order. Among the pathogenic trypanosomatids, some species are of particular interest due to their medical importance. These species include the agent responsible for Chagas' disease, Trypanosoma cruzi. Approximately 8 to 10 million people are infected in the Americas, and approximately 40 million are at risk. in the present review, we discuss in detail the immune mechanisms elicited during infection by T. cruzi and the effects of chemotherapy in controlling parasite proliferation and on the host immune system.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Instituto Nacional de Biologia Estrutural e QuImica Medicinal em Doencas Infecciosas (INBEQMeDI)Univ São Paulo, Inst Biomed Sci, Dept Parasitol, São Paulo, BrazilMackenzie Presbeterian Univ, Ctr Biomol Sci & Hlth, São Paulo, BrazilNatl Univ Rosario, Sch Med Sci, Inst Immunol, Rosario, Santa Fe, ArgentinaCSIC, Inst Parasitol & Biomed Lopez Neyra, Granada, SpainUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilFAPESP: 08/57596-4FAPESP: 07/08648-9CNPq: 473906/2008-2Web of Scienc

Efficacy of subcutaneous immunotherapy achieved with a whole-body extract of Pseudomyrmex ant. A case reported in a young woman, in Argentina

The aim of this work is to report for the first time a patient case with anaphylaxis by Pseudomyrmex acanthobius /favidulus ant sting, prepare an extract with the wholeant-body, to study their biochemical and immunological properties and to validate the efficacy of the subcutaneous immunotherapy with this non-commercial extract. An argentine woman patient, 19 years old, with background of allergic rhinitis and bronchial asthma in the childhood, suffered repeated episodes of anaphylaxis by non-identified insect’s stings and previous immunotherapies treatment. The entomologic analysis revealed that the aggressor insect was an ant belonging to Pseudomyrmex genera and acanthobius or favidulus species. High serum total levels of IgE (202 UI/ml) were measured by ELISA and a positive 6-mm wheal and flare reaction (extract dilution 1/100000) was revealed by in vivo intradermal test. A sample of the extract proteins fraction was analysed by SDS⁄PAGE. The silver stained protein bands ranged since 20 to 220 kDa. The patient serum immune-recognized allergenic extract proteins at approximately 160, 90, and a double band at 42/46 kDa prior to desensitization treatment by electro-blotting. Interestingly, post-specific-subcutaneous-immunotherapy the mentioned double band almost disappeared. After a 6-years-treatment, the total IgE serum values strongly decreased and the specific intradermal test was negative up to 1/10 extract dilution. The tolerance to the treatment was good. Altogether, our findings revealed that the immunotherapy performed with the whole-body-Pseudomyrmex ant allergenic extract was highly effective and the IgE specific-double protein allergenic extract bands (42/46 kDa) that disappeared after immunotherapy were responsible for the anaphylactic shock.Fil: Rodríguez Rodríguez, Raquel Milagros. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Irañeta, Silvia G.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Olgiati, María Laura. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Soprano, Luciana Lía. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mouchian, Krikor. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Alonso, Angel. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Duschak, Vilma Gladys. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Input of NAcGlc6SO3 epitopes (sulfotopes) present in Trypanosoma cruzi glycoproteins, and their specific antibodies, in the infection and immune pathogenesis of experimental Chagas disease

Background: Trypanosoma cruzi, the causative agent of Chagas disease contains a major antigen, cruzipain (Cz). The C-terminal domain (C-T) of this glycoprotein bears N-linked high mannose type sulfated oligosaccharide chains and is responsible for most antibodiesinnaturalandexperimentalinfections.Miceimmunization with C-T has shown that sulfate moieties of Cz molecule are targets for specific immune responses and responsible for cardiac ultrastructural abnormalities in absence of infection. Methods & Materials: After the molecular characterization of these sufotopes, BALB/c mice were immunized with Cz/C-T, prior and after desulfation treatment, and with NAcGlc6SO3-BSA, to be furthersublethallychallengedwithtrypomastigotestoinvestigate whether they are involved in immunepathogenesis and/or infection of experimental Chagas disease. Results: C-T-immunized mice showed low IL-4 levels and elevated IFN- concentration by capture ELISA using C-T as stimulus and a cytokines profile compatible with a mixed response showing: Th2 tendency with excessively high IFN and raised IL-17 levels. By contrast, dC-T-immunized-mice presented undetectable IL-4 levels, low IFN- level and a cytokines profile like that of control but with a significantly elevated IL-10 value. In addition, ultrastructuralcardiacalterationsandmainimmunorecognitionof fibrils and mitochondria were observed in C-T-immunized mice bothconfrontedwithpolyclonalanti-CzandmyosinadsorbedantiCz sera. After sublethal challenge, elevated parasitaemias were observed. Mortality was 20 and 80% in C-T and dC-T immunized mice, respectively and mice from dC-T group that survived presentedseveremusclealterations.BSA-NAcGlc6SO3-immunized mice mounted a predominant IgG1and IgG2b immune response followed by IgG2a, demonstrating the immunodominance of the sulfotope and a vigorous mice memory T cells response, similarly toC-T-immunizedmice.Aftersublethalinfection,miceimmunized with the sulfotope displayed excessively elevated parasitemias, similarIFN-levelsandsignificantlowermortalitypercentagethan those from BSA-NAcGlc control group. Furthermore, mice treated by passive transference of sulfate-specific IgGs purified from sera of BSA-NAcGlc6SO3-immunized mice, exhibited ultrastructural alterations in cardiac tissue. After challenge, those treated with sulfate-specific IgGs presented higher parasitemias than controls Conclusion:Altogether,thesefindingshavedemonstratedthat sulfotopes and their specific antibodies display a dual role, participating in the host-tissue immunopathogenicity of experimental Chagas disease and favoring the infection by T. cruziFil: Soprano, Luciana Lía. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ferrero, Maximiliano Ruben. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Olgiati, María Laura. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Landoni, Malena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Garcia, Gabriela Andrea. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Esteva, Mónica Inés. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; ArgentinaFil: Couto, Alicia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Duschak, Vilma Gladys. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina18th International Congress of Infectious diseasesBuenos AiresArgentinaInternational Society of Infectious Disease

Gamma-glutamylcysteine synthetase and tryparedoxin 1 exert high control on the antioxidant system in Trypanosoma cruzi contributing to drug resistance and infectivity

Trypanothione (T(SH)2) is the main antioxidant metabolite for peroxide reduction in Trypanosoma cruzi; therefore, its metabolism has attracted attention for therapeutic intervention against Chagas disease. To validate drug targets within the T(SH)2 metabolism, the strategies and methods of Metabolic Control Analysis and kinetic modeling of the metabolic pathway were used here, to identify the steps that mainly control the pathway fluxes and which could be appropriate sites for therapeutic intervention. For that purpose, gamma-glutamylcysteine synthetase (γECS), trypanothione synthetase (TryS), trypanothione reductase (TryR) and the tryparedoxin cytosolic isoform 1 (TXN1) were separately overexpressed to different levels in T. cruzi epimastigotes and their degrees of control on the pathway flux as well as their effect on drug resistance and infectivity determined. Both experimental in vivo as well as in silico analyses indicated that γECS and TryS control T(SH)2 synthesis by 60–74% and 15–31%, respectively. γECS overexpression prompted up to a 3.5-fold increase in T(SH)2 concentration, whereas TryS overexpression did not render an increase in T(SH)2 levels as a consequence of high T(SH)2 degradation. The peroxide reduction flux was controlled for 64–73% by TXN1, 17–20% by TXNPx and 11–16% by TryR. TXN1 and TryR overexpression increased H2O2 resistance, whereas TXN1 overexpression increased resistance to the benznidazole plus buthionine sulfoximine combination. γECS overexpression led to an increase in infectivity capacity whereas that of TXN increased trypomastigote bursting. The present data suggested that inhibition of high controlling enzymes such as γECS and TXN1 in the T(SH)2 antioxidant pathway may compromise the parasite's viability and infectivity.</p

Advancing Drug Innovation for Neglected Diseases—Criteria for Lead Progression

The current drug R&D pipeline for most neglected diseases remains weak, and unlikely to support registration of novel drug classes that meet desired target product profiles in the short term. This calls for sustained investment as well as greater emphasis in the risky upstream drug discovery. Access to technologies, resources, and strong management as well as clear compound progression criteria are factors in the successful implementation of any collaborative drug discovery effort. We discuss how some of these factors have impacted drug discovery for tropical diseases within the past four decades, and highlight new opportunities and challenges through the virtual North–South drug discovery network as well as the rationale for greater participation of institutions in developing countries in product innovation. A set of criteria designed to facilitate compound progression from screening hits to drug candidate selection is presented to guide ongoing efforts

MDL28170, a Calpain Inhibitor, Affects Trypanosoma cruzi Metacyclogenesis, Ultrastructure and Attachment to Rhodnius prolixus Midgut

BACKGROUND: Trypanosoma cruzi is the etiological agent of Chagas' disease. During the parasite life cycle, many molecules are involved in the differentiation process and infectivity. Peptidases are relevant for crucial steps of T. cruzi life cycle; as such, it is conceivable that they may participate in the metacyclogenesis and interaction with the invertebrate host. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we have investigated the effect of the calpain inhibitor MDL28170 on the attachment of T. cruzi epimastigotes to the luminal midgut surface of Rhodnius prolixus, as well as on the metacyclogenesis process and ultrastructure. MDL28170 treatment was capable of significantly reducing the number of bound epimastigotes to the luminal surface midgut of the insect. Once the cross-reactivity of the anti-Dm-calpain was assessed, it was possible to block calpain molecules by the antibody, leading to a significant reduction in the capacity of adhesion to the insect guts by T. cruzi. However, the antibodies were unable to interfere in metacyclogenesis, which was impaired by the calpain inhibitor presenting a significant reduction in the number of metacyclic trypomastigotes. The calpain inhibitor also promoted a direct effect against bloodstream trypomastigotes. Ultrastructural analysis of epimastigotes treated with the calpain inhibitor revealed disorganization in the reservosomes, Golgi and plasma membrane disruption. CONCLUSIONS/SIGNIFICANCE: The presence of calpain and calpain-like molecules in a wide range of organisms suggests that these proteins could be necessary for basic cellular functions. Herein, we demonstrated the effects of MDL28170 in crucial steps of the T. cruzi life cycle, such as attachment to the insect midgut and metacyclogenesis, as well as in parasite viability and morphology. Together with our previous findings, these results help to shed some light on the functions of T. cruzi calpains. Considering the potential roles of these molecules on the interaction with both invertebrate and vertebrate hosts, it is interesting to improve knowledge on these molecules in T. cruzi

Significance of Cuscutain, a cysteine protease from Cuscuta reflexa, in host-parasite interactions

<p>Abstract</p> <p>Background</p> <p>Plant infestation with parasitic weeds like <it>Cuscuta reflexa </it>induces morphological as well as biochemical changes in the host and the parasite. These modifications could be caused by a change in protein or gene activity. Using a comparative macroarray approach <it>Cuscuta </it>genes specifically upregulated at the host attachment site were identified.</p> <p>Results</p> <p>One of the infestation specific <it>Cuscuta </it>genes encodes a cysteine protease. The protein and its intrinsic inhibitory peptide were heterologously expressed, purified and biochemically characterized. The haustoria specific enzyme was named cuscutain in accordance with similar proteins from other plants, e.g. papaya. The role of cuscutain and its inhibitor during the host parasite interaction was studied by external application of an inhibitor suspension, which induced a significant reduction of successful infection events.</p> <p>Conclusions</p> <p>The study provides new information about molecular events during the parasitic plant - host interaction. Inhibition of cuscutain cysteine proteinase could provide means for antagonizing parasitic plants.</p