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Parasitic neglected diseases are in dire need of new drugs either to replace old drugs rendered ineffective because of resistance development, to cover clinical needs that had never been addressed or to tackle other associated problems of existing drugs such as high cost, difficult administration, restricted coverage or toxicity. The availability of transgenic parasites expressing reporter genes facilitates the discovery of new drugs through high throughput screenings, but also by allowing rapid screening in animal models of disease. Taking advantage of these, we propose an alternative pathway of drug development for neglected diseases, going from high throughput screening directly into in vivo testing of the top ranked compounds selected by medicinal chemistry. Rapid assessment animal models allow for identification of compounds with bona fide activity in vivo early in the development chain, constituting a solid basis for further development and saving valuable time and resources., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Large quantities of biomedical data are being produced at a rapid pace for a variety of organisms. With ontologies proliferating, data is increasingly being stored using the RDF data model and queried using RDF based querying languages. While existing systems facilitate the querying in various ways, the scientist must map the question in his or her mind to the interface used by the systems. The field of natural language processing has long investigated the challenges of designing natural language based retrieval systems. Recent efforts seek to bring the ability to pose natural language questions to RDF data querying systems while leveraging the associated ontologies. These analyze the input question and extract triples (subject, relationship, object), if possible, mapping them to RDF triples in the data. However, in the biomedical context, relationships between entities are not always explicit in the question and these are often complex involving many intermediate concepts. Results: We present a new framework, OntoNLQA, for querying RDF data annotated using ontologies which allows posing questions in natural language. OntoNLQA offers five steps in order to answer natural language questions. In comparison to previous systems, OntoNLQA differs in how some of the methods are realized. In particular, it introduces a novel approach for discovering the sophisticated semantic associations that may exist between the key terms of a natural language question, in order to build an intuitive query and retrieve precise answers. We apply this framework to the context of parasite immunology data, leading to a system called AskCuebee that allows parasitologists to pose genomic, proteomic and pathway questions in natural language related to the parasite, Trypanosoma cruzi. We separately evaluate the accuracy of each component of OntoNLQA as implemented in AskCuebee and the accuracy of the whole system. AskCuebee answers 68% of the questions in a corpus of 125 questions, and 60% of the questions in a new previously unseen corpus. If we allow simple corrections by the scientists, this proportion increases to 92%. Conclusions: We introduce a novel framework for question answering and apply it to parasite immunology data. Evaluations of translating the questions to RDF triple queries by combining machine learning, lexical similarity matching with ontology classes, properties and instances for specificity, and discovering associations between them demonstrate that the approach performs well and improves on previous systems. Subsequently, OntoNLQA offers a viable framework for building question answering systems in other biomedical domains. [ABSTRACT FROM AUTHOR]

Background: Trypanosoma cruzi is a protozoan parasite that causes severe disease in millions of habitants of developing countries. Currently there is no vaccine to prevent this disease and the available drugs have the consequences of side effects. Live vaccines are likely to be more effective in inducing protection than recombinant proteins or DNA vaccines; however, safety problems associated to their use have been pointed out. In recent years, increasing knowledge on the molecular genetics of Trypanosomes has allowed the identification and elimination of genes that may be necessary for parasite infectivity and survival. In this sense, targeted deletion or disruption of specific genes in the parasite genome may protect against such reversion to virulent genotypes. Methods and Findings: By targeted gene disruption we generated monoallelic mutant parasites for the dhfr-ts gene in a T. cruzi strain that has been shown to be naturally attenuated. In comparison to T. cruzi wild type epimastigotes, impairment in growth of dhfr-ts+/− mutant parasites was observed and mutant clones displayed decreased virulence in mice. Also, a lower number of T. cruzi-specific CD8+ T cells, in comparison to those induced by wild type parasites, was detected in mice infected with mutant parasites. However, no remarkable differences in the protective effect of TCC wild type versus TCC mutant parasites were observed. Mice challenged with virulent parasites a year after the original infection with the mutant parasites still displayed a significant control over the secondary infection. Conclusion: This study indicates that it is possible to generate genetically attenuated T. cruzi parasites able to confer protection against further T. cruzi infections. Author Summary: Chagas disease is the clinical manifestation of the infection produced by the flagellate parasite Trypanosoma cruzi and currently there is no vaccine to prevent this disease. Therefore, different approaches or alternatives are urgently needed. Vaccination with live attenuated parasites has been used effectively in mice to reduce parasitemia and histological damage. However, the use of live parasites as inmunogens is controversial due to the risk of reversion to a virulent phenotype. In this work we genetically manipulated a naturally attenuated strain of T. cruzi in order to produce parasites with impaired replication and infectivity, using the mutation as a safety device against reversion to virulence. We show that genetically modified parasites display a lower proliferation rate in vitro and induced almost undetectable levels of T. cruzi specific CD8+ T cells when injected in mice. Furthermore, the immune response induced by these live mutant parasites confers protection against a subsequent virulent infection even a year after the original immunization. [ABSTRACT FROM AUTHOR]

Background: Trypanosoma cruzi is a protozoan parasite that causes severe disease in millions of habitants of developing countries. Currently there is no vaccine to prevent this disease and the available drugs have the consequences of side effects. Live vaccines are likely to be more effective in inducing protection than recombinant proteins or DNA vaccines; however, safety problems associated to their use have been pointed out. In recent years, increasing knowledge on the molecular genetics of Trypanosomes has allowed the identification and elimination of genes that may be necessary for parasite infectivity and survival. In this sense, targeted deletion or disruption of specific genes in the parasite genome may protect against such reversion to virulent genotypes. Methods and Findings: By targeted gene disruption we generated monoallelic mutant parasites for the dhfr-ts gene in a T. cruzi strain that has been shown to be naturally attenuated. In comparison to T. cruzi wild type epimastigotes, impairment in growth of dhfr-ts+/- mutant parasites was observed and mutant clones displayed decreased virulence in mice. Also, a lower number of T. cruzi-specific CD8+ T cells, in comparison to those induced by wild type parasites, was detected in mice infected with mutant parasites. However, no remarkable differences in the protective effect of TCC wild type versus TCC mutant parasites were observed. Mice challenged with virulent parasites a year after the original infection with the mutant parasites still displayed a significant control over the secondary infection. Conclusion: This study indicates that it is possible to generate genetically attenuated T. cruzi parasites able to confer protection against further T. cruzi infections. [ABSTRACT FROM AUTHOR]

Background: The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential. Methods: In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC50 lower than that of BZ. Findings: This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (,2 weeks) assay mirrored that of a 40 day treatment course. Conclusion: These methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo. [ABSTRACT FROM AUTHOR]

Background: Trypanosoma cruzi, a kinetoplastid protozoan parasite that causes Chagas disease, infects approximately 15 million people in Central and South America. In contrast to the substantial in silico studies of the T. cruzi genome, transcriptome, and proteome, only a few genes have been experimentally characterized and validated, mainly due to the lack of facile methods for gene manipulation needed for reverse genetic studies. Current strategies for gene disruption in T. cruzi are tedious and time consuming. In this study we have compared the conventional multi-step cloning technique with two knockout strategies that have been proven to work in other organisms, one-step-PCR- and Multisite Gateway-based systems. Results: While the one-step-PCR strategy was found to be the fastest method for production of knockout constructs, it does not efficiently target genes of interest using gene-specific sequences of less than 80 nucleotides. Alternatively, the Multisite Gateway based approach is less timeconsuming than conventional methods and is able to efficiently and reproducibly delete target genes. Conclusion: Using the Multisite Gateway strategy, we have rapidly produced constructs that successfully produce specific gene deletions in epimastigotes of T. cruzi. This methodology should greatly facilitate reverse genetic studies in T. cruzi. [ABSTRACT FROM AUTHOR]

CD8+ T cells are crucial for control of a number of medically important protozoan parasites, including Trypanosoma cruzi, the agent of human Chagas disease. Yet, in contrast to the wealth of information from viral and bacterial infections, little is known about the antigen specificity or the general development of effector and memory T-cell responses in hosts infected with protozoans. In this study we report on a wide-scale screen for the dominant parasite peptides recognized by CD8+ T cells in T. cruzi-infected mice and humans. This analysis demonstrates that in both hosts the CD8+ T-cell response is highly focused on epitopes encoded by members of the large trans-sialidase family of genes. Responses to a restricted set of immunodominant peptides were especially pronounced in T. cruzi-infected mice, with more than 30% of the CD8+ T-cell response at the peak of infection specific for two major groups of trans-sialidase peptides. Experimental models also demonstrated that the dominance patterns vary depending on the infective strain of T. cruzi, suggesting that immune evasion may be occurring at a population rather than single-parasite level. [ABSTRACT FROM AUTHOR]