Carlos L. Zani

In vitro and in vivo experimental models for drug screening and development for Chagas disease

Chagas disease, a neglected illness, affects nearly 12-14 million people in endemic areas of Latin America. Although the occurrence of acute cases sharply has declined due to Southern Cone Initiative efforts to control vector transmission, there still remain serious challenges, including the maintenance of sustainable public policies for Chagas disease control and the urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox approximately 40 years ago, many natural and synthetic compounds have been assayed against Trypanosoma cruzi, yet only a few compounds have advanced to clinical trials. This reflects, at least in part, the lack of consensus regarding appropriate in vitro and in vivo screening protocols as well as the lack of biomarkers for treating parasitaemia. The development of more effective drugs requires (i) the identification and validation of parasite targets, (ii) compounds to be screened against the targets or the whole parasite and (iii) a panel of minimum standardised procedures to advance leading compounds to clinical trials. This third aim was the topic of the workshop entitled Experimental Models in Drug Screening and Development for Chagas Disease, held in Rio de Janeiro, Brazil, on the 25th and 26th of November 2008 by the Fiocruz Program for Research and Technological Development on Chagas Disease and Drugs for Neglected Diseases Initiative. During the meeting, the minimum steps, requirements and decision gates for the determination of the efficacy of novel drugs for T. cruzi control were evaluated by interdisciplinary experts and an in vitro and in vivo flowchart was designed to serve as a general and standardised protocol for screening potential drugs for the treatment of Chagas disease.

Screening of Asteraceae (Compositae) plant extracts for larvicidal activity against Aedes fluviatilis (Diptera: Culicidae)

Ethanol extracts of 83 plants species belonging to the Asteraceae (Compositae) family, collected in the State of Minas Gerais, Brazil, were tested for larvicidal activity against the mosquito Aedes fluviatilis--Diptera: Culicidae). The extract from Tagetes minuta was the most active with a LC90 of 1.5 mg/l and LC50 of 1.0 mg/l. This plant has been the object of several studies by other groups and its active components have already been identified as thiophene derivatives, a class of compounds present in many Asteraceae species. The extract of Eclipta paniculata was also significantly active, with a LC90 of 17.2 mg/l and LC50 of 3.3 mg/l and no previous studies on its larvicidal activity or chemical composition could be found in the literature. Extracts of Achryrocline satureoides, Gnaphalium spicatum, Senecio brasiliensis, Trixis vauthieri, Tagetes patula and Vernonia ammophila were less active, killing more than 50% of the larvae only at the higher dose tested (100 mg/l).

Trypanocidal activity and redox potential of heterocyclic- and 2-hydroxy-naphthoquinones

Abstract With the aim of understanding the influence of redox potentials on the trypanocidal activity, a series of quinones were tested in vitro with trypomastigotes of Trypanosoma cruzi and their first cathodic potentials (Epcl) measured by cyclic voltammetry. All quinones with Epcl > −0.72V were active, while most of those with Epcl

Synthesis and antimalarial activity of semicarbazone and thiosemicarbazone derivatives.

Seventeen semicarbazone and thiosemicarbazone derivatives were prepared and tested in vitro against a chloroquine resistant strain of Plasmodium falciparum (W2) to evaluate their antiplasmodial potential. Three thiosemicarbazones were found to be active against the parasite and non-toxic to human peripheral blood mononuclear cells (PBMC). Among these, compound 5b presented the lowest IC50 value against P. falciparum (7.2 microM) and was the least toxic in the PBMC proliferation assay (IC50=73.5 microM). It was selected for in vivo tests on mice infected with Plasmodium berghei (strain NK-65). The thiosemicarbazone 5b was able to reduce the parasitaemia by 61% at 20 mg/kg on day 7 after infection without any sign of toxicity to the animals. In comparison, the standard drug chloroquine at 15 mg/kg showed a reduction around 95%. These in vitro and in vivo results make 5b an interesting lead for further development.

Screening of Brazilian basidiomycetes for antimicrobial activity

A total of 103 isolates of basidiomycetes, representing 84 species from different Brazilian ecosystems, were evaluated for their antifungal and antibacterial activity in a panel of pathogenic and non-pathogenic microorganisms. Tissue plugs of the fruiting bodies were cultivated in liquid media and the whole culture extracted with ethyl acetate. Crude extracts from Agaricus cf. nigrecentulus, Agrocybe perfecta, Climacodon pulcherrimus, Gloeoporus thelephoroides, Hexagonia hydnoides, Irpex lacteus, Leucoagaricus cf. cinereus, Marasmius cf. bellus, Marasmius sp., Nothopanus hygrophanus, Oudemansiella canarii, Pycnoporus sanguineus, Phellinus sp., and Tyromyces duracinus presented significant activity against one or more of the target microorganisms. Eight isolates were active only against bacteria while three inhibited exclusively the growth of fungi. Two extracts presented wide antimicrobial spectrum and were active against both fungi and bacteria. Differences in the bioactivity of extracts obtained from isolates from the same species were observed.

Chemical communication in chagas disease vectors. source, identity, and potential function of volatiles released by the metasternal and Brindley's glands of Triatoma infestans adults

Compounds from the metasternal and Brindleys glands of the blood-sucking bug, Triatoma infestans, were identified by solid phase microextraction (SPME) and gas chromatography-mass spectrometry. Volatile compounds released by adult bugs during copulation or after mechanical disturbance were also characterized. Six compounds were identified and found consistently in all samples from metasternal glands. The most abundant were 3-pentanone, 2-methylbutanol, 3-pentanol, and an unidentified compound. The metasternal gland blends did not differ qualitatively between sexes. Compounds found in Brindleys glands were short chain acids, alcohols, esters, and a ketone with no qualitative differences between sexes. Isobutyric acid was the main component of this blend, and two new confirmed compounds were described as products of these glands: 2-butanone and 2-methylbutyric acid. 3-Pentanone was collected from the headspace over 33% of the copulating pairs of T. infestans. Volatiles found in the headspace of disturbed T. infestans adults included short-chain fatty acids, alcohols, esters, and ketones, with no qualitative differences between sexes. Both types of glands apparently discharge their contents after disturbance. However, most of the volatiles released by bugs after disturbance came from Brindleys glands. The locomotor activity of fourth instars increased significantly after stimulation with the odors emitted by disturbed adults, as compared with larvae stimulated by the odor of undisturbed adults or by clean air. We also studied the directional behavioral response of fifth instars to the disturbance scent in a locomotion compensator. Larvae exposed to volatiles released by disturbed adults walked away from the direction of the odor. The results suggest that this blend or part of it functions as an alarm pheromone for T. infestans. We suggest that the metasternal glands of this species are involved both in the sexual and the alarm contexts, and that the Brindleys glands probably have both alarm and defensive roles.

IL-4 and IL-13 regulate the induction of indoleamine 2,3-dioxygenase activity and the control ofToxoplasma gondii replication in human fibroblasts activated with IFN-γ

The ability of up‐regulatory [recombinant (r) IFN‐γ, rIFN‐β and rTNF‐α] and down‐regulatory (rIL‐4, rIL‐10 and rIL‐13) cytokines to control the expression of indoleamine 2,3‐dioxygenase (INDO) and anti‐Toxoplasma activity in the human fibrosarcoma cell line 2C4 was evaluated. Activation of fibroblasts with rIFN‐γ, rIFN‐β and rTNF‐α resulted in augmentation of INDO expression and activity leading to 40.0, 25.0 and 27.0 % inhibition of tachyzoite growth, respectively. An additive effect was observed when host cells were incubated with rIFN‐γ plus rTNF‐α. With regard to the down‐regulatory cytokines we observed that IL‐4 as well as IL‐13, but not IL‐10, induced significant inhibition of IFN‐γ‐induced control of parasite replication, INDO mRNA expression and tryptophan catabolism. Similarly, IL‐4 but not IL‐10 inhibited the cell surface expression of HLA‐DR and CD2 induced by IFN‐γ. Consistent with these findings we were able to detect by reverse transcription‐PCR the expression of mRNA for different chains of IL‐4 and IL‐13 receptors (IL‐4Rα, IL‐13Rα1 and IL‐13Rα2) but not for IL‐10 receptor in the 2C4 and other human lung fibroblast cell lines (LL24 and MRC5). Together our results indicate that IL‐4 and IL‐13, but not IL‐10, are implicated in the negative regulation of IFN‐γ‐induced anti‐Toxoplasma activity in human cells from fibroblast lineage.

Diversity and bioprospecting of fungal communities associated with endemic and cold-adapted macroalgae in Antarctica

We surveyed the distribution and diversity of fungi associated with eight macroalgae from Antarctica and their capability to produce bioactive compounds. The collections yielded 148 fungal isolates, which were identified using molecular methods as belonging to 21 genera and 50 taxa. The most frequent taxa were Geomyces species (sp.), Penicillium sp. and Metschnikowia australis. Seven fungal isolates associated with the endemic Antarctic macroalgae Monostroma hariotii (Chlorophyte) displayed high internal transcribed spacer sequences similarities with the psychrophilic pathogenic fungus Geomyces destructans. Thirty-three fungal singletons (66%) were identified, representing rare components of the fungal communities. The fungal communities displayed high diversity, richness and dominance indices; however, rarefaction curves indicated that not all of the fungal diversity present was recovered. Penicillium sp. UFMGCB 6034 and Penicillium sp. UFMGCB 6120, recovered from the endemic species Palmaria decipiens (Rhodophyte) and M. hariotii, respectively, yielded extracts with high and selective antifungal and/or trypanocidal activities, in which a preliminary spectral analysis using proton nuclear magnetic resonance spectroscopy indicated the presence of highly functionalised aromatic compounds. These results suggest that the endemic and cold-adapted macroalgae of Antarctica shelter a rich, diversity and complex fungal communities consisting of a few dominant indigenous or mesophilic cold-adapted species, and a large number of rare and/or endemic taxa, which may provide an interesting model of algal–fungal interactions under extreme conditions as well as a potential source of bioactive compounds.

Antifungal activity of extracts of some plants used in Brazilian traditional medicine against the pathogenic fungus Paracoccidioides brasiliensis

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by Paracoccidioides brasiliensis Almeida (Onygenales) that requires 1–2 years of treatment. In the absence of drug therapy, the disease is usually fatal, highlighting the need for the identification of safer, novel, and more effective antifungal compounds. With this need in mind, several plants employed in Brazilian traditional medicine were assayed on P. brasiliensis and murine macrophages. Extracts were prepared from 10 plant species: Inga spp. Mill. (Leguminosae), Schinus terebinthifolius Raddi (Anacardiaceae), Punica granatum L. (Punicaceae), Alternanthera brasiliana Kuntze (Amaranthaceae), Piper regnellii CDC. (Piperaceae), P. abutiloides Kunth (Piperaceae), Herissantia crispa L. Briz. (Malvaceae), Rubus urticaefolius Poir (Rosaceae), Rumex acetosa L. (Polygonaceae), and Baccharis dracunculifolia DC. (Asteraceae). Hexane fractions from hydroalcoholic extracts of Piper regnellii and Baccharis dracunculifolia were the most active against the fungus, displaying minimum inhibitory concentration (MIC) values of 7.8 μg/mL and 7.8–30 μg/mL, respectively. Additionally, neither of the extracts exhibited any apparent cytotoxic effects on murine macrophages at 20 μg/mL. Analyses of these fractions using gas chromatography-mass spectrometry (GC-MS) showed that the major components of B. dracunculifolia were ethyl hydrocinnamate (14.35%) and spathulenol (16.02%), while the major components of the hexane fraction of Piper regnellii were 1-methoxy-4-(1-propenyl) benzene (21.94%) and apiol (21.29%). The activities of these fractions against P. brasiliensis without evidence of cytotoxicity to macrophages justify their investigation as a potential source of new chemical agents for the treatment of PCM.

Antimicrobial activity of endophytic fungi associated with Orchidaceae in Brazil

The purpose of this study was to examine antimicrobial activity of endophytic fungi isolated from the leaves, stems, and roots of 54 species of Orchidaceae collected in a Brazilian tropical ecosystem. In total, 382 filamentous fungi and 13 yeast isolates were obtained and cultured to examine the production of crude extracts. Thirty-three percent of the isolates displayed antimicrobial activity against at least one target microorganism. The multivariate statistical analyses conducted indicate that the extracts of endophytic fungi isolated from leaves of terrestrial orchids in semideciduous forest were more active against Escherichia coli, whereas extracts of endophytic fungi from roots of rupicolous orchids collected in rock fields were more active against Candida krusei and Candida albicans. Among the fungi that were screened in the study, 22 isolates held their antimicrobial activities after replication and were therefore selected for assessment of the minimum inhibitory concentration (MIC), which ranged from 62.5 to 250 microg/mL and 7.8 to 250 microg/mL against bacteria and fungi, respectively. One isolate of Alternaria sp. and one isolate of Fusarium oxysporum presented the strongest antibacterial activity. Three Fusarium isolates, Epicoccum nigrum, and Sclerostagonospora opuntiae showed the greatest MIC values against the pathogenic yeasts. This study is the first survey investigating the bioactive potential of endophytic fungi associated with tropical Orchidaceae species present in Brazilian ecosystems.