Drug screening and development cascade for Chagas disease: an update of in vitro and in vivo experimental models

Chagas disease is a tropical neglected disease that affects millions of people worldwide, still demanding a more effective and safer therapy, especially in its chronic phase which lacks a treatment that promotes substantial parasitological cure. The technical note of Romanha and collaborators published in 2010 aimed establish a guideline with the set of minimum criteria and decision gates for the development of new agents against Trypanosoma cruzi with the focus on developing new antichagasic drugs. In this sense, the present review aims to update this technical note, bringing the state of the art and new advances on this topic in recent years.

Increased angiogenesis parallels cardiac tissue remodelling in experimental acute Trypanosoma cruzi infection

BACKGROUND Angiogenesis has been implicated in tissue injury in several noninfectious diseases, but its role in Chagas disease (CD) physiopathology is unclear. OBJECTIVES The present study aimed to investigate the effect of Trypanosoma cruzi infection on cardiac angiogenesis during the acute phase of experimental CD. METHODS The signalling pathway involved in blood vessel formation and cardiac remodelling was evaluated in Swiss Webster mice infected with the Y strain of T. cruzi. The levels of molecules involved in the regulation of angiogenesis, such as vascular endothelial growth factor-A (VEGF-A), Flk-1, phosphorylated extracellular-signal-regulated protein kinase (pERK), hypoxia-inducible factor-1α (HIF-1α), CD31, α-smooth muscle actin (α-SMA) and also the blood vessel growth were analysed during T. cruzi infection. Hearts were analysed using conventional histopathology, immunohistochemistry and western blotting. FINDINGS In this study, our data demonstrate that T. cruzi acute infection in mice induces exacerbated angiogenesis in the heart and parallels cardiac remodelling. In comparison with noninfected controls, the cardiac tissue of T. cruzi-infected mice presented higher levels of (i) HIF-1α, VEGF-A, Flk-1 and pERK; (ii) angiogenesis; (iii) α-SMA+ cells in the tissue; and (iv) collagen -1 deposition around blood vessels and infiltrating throughout the myocardium. MAIN CONCLUSIONS We observed cardiac angiogenesis during acute experimental T. cruzi infection parallels cardiac inflammation and remodelling.

Natural products from marine red and brown algae against Trypanosoma cruzi

ABSTRACT Various extracts obtained from the red alga Plocamium brasiliense (Greville Howe & Taylor), including a fraction containing crude 5-chloro-1-(E)-chlorovinyl-2,4-dibromo-1,5-dimethylcyclohexane (1) and another containing a mixture of halogenated monoterpenes (F), as well as atomaric acid meroditerpene (2) isolated from brown alga Stypopodium zonale (J. V. Lamouroux) Papenfuss, were evaluated for their activity against Trypanosoma cruzi. The cytotoxic and trypanosomicidal effects of these extracts were evaluated in Vero cells and clinically relevant forms of T. cruzi (amastigotes and trypomastigotes). All extracts from P. brasiliense presented low cytotoxicity and moderate trypanosomicidal effects, except for the hydroalcoholic extract. The crude 1 and F fractions had enhanced trypanocidal activity but showed low selectivity. Moreover, atomaric acid (2) was identified as a hit, demonstrating a potent trypanocidal effect reaching an IC50 <10 µM against two different DTU (Yand high selectivity index (<10). These results identify marine natural products as promising candidates against Chagas disease.

Trypanosoma janseni n. sp. (Trypanosomatida: Trypanosomatidae) isolated from Didelphis aurita (Mammalia: Didelphidae) in the Atlantic Rainforest of Rio de Janeiro, Brazil: integrative taxonomy and phylogeography within the Trypanosoma cruzi clade

BACKGROUND Didelphis spp. are a South American marsupial species that are among the most ancient hosts for the Trypanosoma spp. OBJECTIVES We characterise a new species (Trypanosoma janseni n. sp.) isolated from the spleen and liver tissues of Didelphis aurita in the Atlantic Rainforest of Rio de Janeiro, Brazil. METHODS The parasites were isolated and a growth curve was performed in NNN and Schneider's media containing 10% foetal bovine serum. Parasite morphology was evaluated via light microscopy on Giemsa-stained culture smears, as well as scanning and transmission electron microscopy. Molecular taxonomy was based on a partial region (737-bp) of the small subunit (18S) ribosomal RNA gene and 708 bp of the nuclear marker, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. Maximum likelihood and Bayesian inference methods were used to perform a species coalescent analysis and to generate individual and concatenated gene trees. Divergence times among species that belong to the T. cruzi clade were also inferred. FINDINGS In vitro growth curves demonstrated a very short log phase, achieving a maximum growth rate at day 3 followed by a sharp decline. Only epimastigote forms were observed under light and scanning microscopy. Transmission electron microscopy analysis showed structures typical to Trypanosoma spp., except one structure that presented as single-membraned, usually grouped in stacks of three or four. Phylogeography analyses confirmed the distinct species status of T. janseni n. sp. within the T. cruzi clade. Trypanosoma janseni n. sp. clusters with T. wauwau in a well-supported clade, which is exclusive and monophyletic. The separation of the South American T. wauwau + T. janseni coincides with the separation of the Southern Super Continent. CONCLUSIONS This clade is a sister group of the trypanosomes found in Australian marsupials and its discovery sheds light on the initial diversification process based on what we currently know about the T. cruzi clade.

Regulação e localização de RNA mensageiro durante miogênese de células musculares cardíacas e após infecção pelo Trypanosoma cruzi / Regulation and localization of messenger RNA during myogenesis of cardiac muscle cells after infection with Trypanosoma cruzi

Um sistema experimental in vitro foi utilizado para estudar eventos biológicos e moleculares envolvidos na interação Trypanosoma cruzi – cardiomiócitos. As análises de organização do citoesqueleto e de regulação e localização intracelular de RNA mensageiros (RNAm) forneceram elementos para um melhore entendimento do efeito citopatológico induzido nas celular do miocárdio pelo T.c cruzi. O padrão de expressão e de distribuição de componentes estruturais de células musculares foi analisado durante a miogênese cardíaca. Estudos ultra-estruturais e imunofluorescentes revelaram mudanças na organização do citoesqueleto em cardiomiócitos infectados. Um dos eventos mais marcantes foi a quebra das miofibrilas na região onde o parasita intracelular encontrava-se localizado. Microtúbulos e filamentos de desmina também encontravam-se destruídos com a progressão da infecção (48 e 72 h). Avaliamos se estas mudanças no citoesqueleto afetariam a regulação de RNA mensageiros de actina. Nossos dados revelaram uma expressão temporal e seqüencial de RNA mensageiros de isoformas de actina durante a miogênses cardíaca em células normais: ocorreu um aumento nos níveis de RNAm de a-actina cardíaca concomitante com uma diminuição nos níveis de RNAm de beta e gama actina durante a diferenciação de células musculares. A localização intracelular dos RNAm de actina também foi distinta. Mioblastos apresentaram o sinal de RNAm de beta-actina na periferia da célula, enquanto o RNAm de a-actina cardíaca após 72 h de infecção. Em contraste, o RNAm de beta menos actina aumentou 79 porcento após 48 h de infecção. Além disso, a infecção pelo T. cruzi delocalizou o RNAm de beta menos actina da periferia para a região perinuclear. Esta diminuição de RNAm de alfa menos actina cardíaca e aumento de beta menosactina sugere uma reativação do programa genético de células não musculares sob condições patológicas. A análise da distribuição espacial de poli (A) mais RNA e sua quantidade relativa em cardiomiócitos normais e infectados pelo T. cruzi revelaram porcento de redução no conteúdo citoplasmático de células infectadas, a qual foiconcomitante com a proliferação dos parasitas intracelulares.mudanças nos níveis de poli (A)mais RNA durante o ciclo intracelular do parasita também foram observadas. Um aumento no conteúdo de poli (A)mais RNA em formas amastigotas e subseqüente diminuição após diferenciação a formas tripomastigotas indicaram um aumento progressivo no conteúdo de RNA durante a proliferação do parasita intracelular. Além disso, investigamos a distribuição espacial do RNAm de actina e seu produto protéico no T. cruzi. Embora o RNAm de actina tenha sido visualizado no núcleo e citoplasma do parasita, não foi possível detectar a proteína (actina) usando métodos bioquímicos e imunofluorescentes.

Leishmania major: parasite interactions suggesting sexuality

In five experiments, Leishmania (Leishmania) major (MRHO/SU/59/P-strain) grew poorly when seeded in FYTS medium supplemented with 15 per cent fetal calf serum, but presented several peculiar pairs of promastigotes diametrically opposed and attached at their posterior ends (5.8-13.5 per cent). As seen in Giemsa-stained smears, a ring-like line and/or an enlargement, generally occured at the parasite junction. A close proximity of nuclei, which sometimes were difficult to distinguish from each other, was also observed at this junction. Several of these pairs appeared to be composed of fused cells in which the nuclei could be apparently fused, as shown by fluorescence microscopy to detect ß-tubulin and DNA, and by scanning electron microscopy. Under other culture conditions these pairs were absent or occurred at very low rates (0.2-2.2 per cent). Such pairs differ markedly from longitudinally dividing cells and resemble those described in two other Leishmania species, as well as in Herpetomonas megaseliae and Phytomonas davidi, suggesting steps of a putative sexual process.