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

Rapid suppression of parasitemia following drug-treatment is a poor indicator of drug efficacy and parasitological cure.

<p>(A) Evolution of parasitemia after infection with 1×10³ CL strain of <i>T. cruzi</i> on day 0 in untreated (▪), BZ-40 (▵), POS (○), NTLA-1 (▴), or BIS767 (□) treated mice. “BIS767, BZ-40, POS and NTLA-1” bars below x axis indicate period of treatments. (B) Parasitemias in untreated or treated mice at 120dpi, after administration of the immunosuppressant cyclophosphamide (cy) (days 105, 108, 111, 113 and 117).</p

The Acute Phase of Trypanosoma cruzi Infection Is Attenuated in 5-Lipoxygenase-Deficient Mice

In the present work we examine the contribution of 5-lipoxygenase- (5-LO-) derived lipid mediators to immune responses during the acute phase of Trypanosoma cruzi infection in 5-LO gene knockout (5-LO−/−) mice and wild-type (WT) mice. Compared with WT mice, the 5-LO−/− mice developed less parasitemia/tissue parasitism, less inflammatory cell infiltrates, and a lower mortality. This resistance of 5-LO−/− mice correlated with several differences in the immune response to infection, including reduced PGE2 synthesis; sustained capacity of splenocytes to produce high levels of interleukin (IL)-12 early in the infection; enhanced splenocyte production of IL-1β, IL-6, and IFN-γ; rapid T-cell polarization to secrete high quantities of IFN-γ and low quantities of IL-10; and greater numbers of CD8+CD44highCD62Llow memory effector T cells at the end of the acute phase of infection. The high mortality in WT mice was associated with increased production of LTB4/LTC4, T cell bias to produce IFN-γ, high levels of serum nitrite, and marked protein extravasation into the peritoneal cavity, although survival was improved by treatment with a cys-LT receptor 1 antagonist. These data also provide evidence that 5-LO-derived mediators negatively affect host survival during the acute phase of T. cruzi infection

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

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

<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

<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

Fluorescence evaluation of tdTomato parasites.

<p>(A) Microscope image showing the tdTomato expressing parasites in all the life stages. (B) The fluorescence intensity in epimastigotes was assessed using a CyAn flow cytometer (DakoCytomation) and analyzed with FlowJo software (Tree Star). No decrease in fluorescence intensity was observed in parasites cultured for >5 months with (red) and without antibiotic (blue). The background fluorescence of WT parasites (green) can also be observed.</p