In Vitro and In Vivo High-Throughput Assays for the Testing of Anti-Trypanosoma cruzi Compounds

The treatment of Trypanosoma cruzi infection (the cause of human Chagas disease) remains a significant challenge. Only two drugs, both with substantial toxicity, are available and the efficacy of these dugs is often questioned – in many cases due to the limitations of the methods for assessing efficacy rather than to true lack of efficacy. For these reasons relatively few individuals infected with T. cruzi actually have their infections treated. In this study, we report on innovative methods that will facilitate the discovery of new compounds for the treatment of T. cruzi infection and Chagas disease. Utilizing fluorescent and bioluminescent parasite lines, we have developed in vitro tests that are reproducible and facile and can be scaled for high-throughput screening of large compound libraries. We also validate an in vivo screening test that monitors parasite replication at the site of infection and determines the effectiveness of drug treatment in less than two weeks. More importantly, results in this rapid in vivo test show strong correlations with those obtained in long-term (e.g. 40 day or more) treatment assays. The results of this study remove one of the obstacles for identification of effective and safe compounds to treat Chagas disease

Knockout of the dhfr-ts Gene in Trypanosoma cruzi Generates Attenuated Parasites Able to Confer Protection against a Virulent Challenge

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

Integrated multivariate analysis of selected soil microbial properties and their relationships with mineral fertilization management in a conservation agriculture system

The effect of mineral fertilizer application on soil microbial community was investigated in a conservation agriculture system. The aim of this work was to evaluate how mineral fertilization with nitrogen (N), phosphorus (P), sulfur (S), and micronutrients (M) affects microbial community structure and function. A 10-year experiment, conducted on a typic Hapludoll using six mineral fertilizer treatments (control, CK; PS; NS; NP; NPS; and NPSM) was evaluated in central Argentina. Microbial community structure and function were characterized by phospholipid fatty acids and community-level physiological profiles, respectively. Soil microbial metabolic activity was determined by monitoring microbial respiration, fluorescein diacetate activity (FDA), dehydrogenase activity and phosphatase activity (PHA). NPS and NPSM treatments showed higher total microbial biomass and gram-positive and gram-negative bacteria, but lower fungal biomass than the remaining treatments. Fertilizer treatments without S (CK and NP) showed lower carbon source utilization and Shannon index than the other treatments. In addition, both FDA and PHA significantly increased under NPSM. An integrated PC analysis indicated that sensitive bioindicators were significantly associated with three carbon sources, one metabolic parameter, and six fatty acid bioindicators. These results provide information about the importance of balanced fertilization with P, N, S, and M in promoting microbial biomass, metabolic activity, and functional diversity.Fil: Verdenelli, Romina Aylen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinario de Biología Vegetal (p); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba; ArgentinaFil: Conforto, C. B.. Instituto Nacional de Tecnologi­a Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Instituto de Patologi­a Vegetal; ArgentinaFil: Perez Brandan, Cecilia Maria. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Chavarría, Diego Nicolás. Instituto Nacional de Tecnologia Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Instituto de Patologia Vegetal; ArgentinaFil: Rovea, A.. Consorcio Regional de Experimentacion Agrıcola ; ArgentinaFil: Vargas Gil, Silvina. Instituto Nacional de Tecnologia Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Instituto de Patologia Vegetal; ArgentinaFil: Meriles, Jose Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinario de Biología Vegetal (p); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba; Argentin

Betaglycan expression is transcriptionally up-regulated during skeletal muscle differentiation - Cloning of murine betaglycan gene promoter and its modulation by myoD, retinoic acid, and transforming growth factor-beta

Betaglycan is a membrane-anchored proteoglycan co-receptor that binds transforming growth factor beta (TGF-beta) via its core protein and basic fibroblast growth factor through its glycosaminoglycan chains. In this study we evaluated the expression of betaglycan during the C2C12 skeletal muscle differentiation. Betaglycan expression, as determined by Northern and Western blot, was up-regulated during the conversion of myoblasts to myotubes. The mouse betaglycan gene promoter was cloned, and its sequence showed putative binding sites for SP1, Smad3, Smad4, muscle regulatory factor elements such as MyoD and MEF2, and retinoic acid receptor. Transcriptional activity of the mouse betaglycan promoter reporter was also up-regulated in differentiating C2C12 cells. We found that MyoD, but not myogenin, stimulated this transcriptional activity even in the presence of high serum. Betaglycan promoter activity was increased by RA and inhibited by the three isoforms of TGF-beta. On the other hand, basic fibroblast growth factor, BMP-2, and hepatocyte growth factor/scatter factor, which are inhibitors of myogenesis, had little effect. In myotubes, up-regulated betaglycan was also detectable by TGF-beta affinity labeling and immunofluorescence microscopy studies. The latter indicated that betaglycan was localized both on the cell surface and in the ECM Forced expression of betaglycan in C2C12 myoblasts increases their responsiveness to TGF-beta2, suggesting that it performs a TGF-beta presentation function in this cell lineage. These results indicate that betaglycan expression is up-regulated during myogenesis and that MyoD and RA modulate its expression by a mechanism that is independent of myogenin

Plasmids used in the generation of fluorescent and bioluminescent <i>T. cruzi</i>.

<p>Schematic representation of (A) the pTrex-Neo-tdTomato plasmid, and (B) the pLew90β-GW/T7/PARP SAS/luciferase/Aldolase 3′UTR plasmid used for generation of the <i>T. cruzi</i> reporter lines.</p

<i>In vitro</i> amastigote growth assays using tdTomato parasites.

<p>(A) Amastigotes growth in Vero cells grown in 96 well plates over time in the presence of benznidazole (n = 8). (B) Comparison of IC50 calculations in response to EXO2-04 in 96 and 384 well plates at 3 days of treatment (n = 4). (C) Amastigote growth assay in 96 or 384 well plates using the Colombiana and TCC strain of <i>T. cruzi</i> expressing tdTomato fluorescent protein at 3 days of treatment (n = 8).</p

<i>In vitro</i> epimastigote growth assays using tdTomato parasites.

<p>(A) Epimastigotes growth over time in the presence of benznidazole at the indicated concentrations and comparison of measurement of drug inhibition of epimastigote growth by fluorescence and visual counting by hemacytometer. (B) Intra-assay analysis (left) showing the low variation among wells with the same drug concentration (n = 4). Inter-assay analysis (right) showing the low variation among IC₅₀ curves from individual assays. (C) IC₅₀ calculation in response to benznidazole and the EXO2 derivatives activity against epimastigotes after 3 days of treatment/culture.</p

Luminescent <i>T. cruzi</i> imaged at various times post-treatment.

<p>(A) Mice (10 per group) were infected in the footpad with 1×10⁵<i>T. cruzi</i> bioluminescent trypomastigotes. For all images shown the color scale ranges from blue (with a minimum set at 60 photons/s/cm²/sr) to red (maximum of 3000 photons/s/cm2/sr). (B) Quantification of luminescent signal from mice in panel A.</p

Fluorescent <i>T. cruzi</i>-tdTomato expressing parasites imaged post-treatment.

<p>Mice (10 per group) were infected in the hind foot pads with 2.5×10⁵<i>T. cruzi</i> tdTomato trypomastigotes and the images were taken every two days from day 1 to 11 post infection. (A) Images from days 5, 7 and 9 post infection. (B) Quantification of the fluorescent signal from mice in panel A at all imaging points.</p