In vitro and in vivo activity of meglumine antimoniate produced at Farmanguinhos-Fiocruz, Brazil, against Leishmania (Leishmania) amazonensis, L (L.) chagasi and L (Viannia) braziliensis

The leishmanicidal activity of four batches of meglumine antimoniate, produced in Farmanguinhos-Fiocruz, Brazil (TAMs), was assessed and compared to Glucantime®-Aventis Pharma Ltda. Using the amastigote-like in vitro model, the active concentrations of Sb v varied from 10µg/ml to 300 µg/ml for L. (L.) chagasi and from 50µg/ml to 300µg/ml for L. (L.) amazonensis, with no statistically significant differences among the four batches of TAMs and Glucantime®. The inhibitory concentrations (IC50) determined by the amastigote-infected macrophage model for TAM01/03 and Glucantime® were, respectively: 26.3µg/ml and 127.6µg/ml for L. chagasi, 15.4µg /ml and 22.9µg/ml for L. amazonensis, and 12.1µg/ml and 24.2µg/ml for L. (V.) braziliensis. The activities of the four batches of TAMs were confirmed in an in vivo model by assessing, during eight weeks skin lesions caused by L. braziliensis in hamster that were treated with 20mg Sb v/Kg/day for 30 consecutive days. The meglumine antimoniate produced by Farmanguinhos was as effective as the reference drug, Glucantime®-Aventis, against three species of Leishmania that are of medical importance in Brazil.

Cloning, expression and characterisation of an HtrA-like serine protease produced in vivo by Mycobacterium leprae

Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40°C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.

Experimental chemotherapy for Chagas disease: 15 years of research contributions from in vivo and in vitro studies

Chagas disease, which is caused by the intracellular parasite Trypanosoma cruzi, is a neglected illness with 12-14 million reported cases in endemic geographic regions of Latin America. While the disease still represents an important public health problem in these affected areas, the available therapy, which was introduced more than four decades ago, is far from ideal due to its substantial toxicity, its limited effects on different parasite stocks, and its poor activity during the chronic phase of the disease. For the past 15 years, our group, in collaboration with research groups focused on medicinal chemistry, has been working on experimental chemotherapies for Chagas disease, investigating the biological activity, toxicity, selectivity and cellular targets of different classes of compounds on T. cruzi. In this report, we present an overview of these in vitro and in vivo studies, focusing on the most promising classes of compounds with the aim of contributing to the current knowledge of the treatment of Chagas disease and aiding in the development of a new arsenal of candidates with anti-T. cruzi efficacy.

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.

In vitro and in vivo effects of Enterococcus faecalis CECT7121 on Toxocara canis

The aim of the present paper was to evaluate the larvicidal effect of Enterococcus faecalis CECT7121 (Ef7121) on the Toxocara canis cycle both in vitro and in vivo. For the in vitro experiments, T. canis larvae were incubated with the supernatants of Ef7121 (EI) and mutant Ef7121 (EIm), in a pre-culture of Ef7121 (EII) and in a fresh culture with Ef7121 (EIII) and the Ef7121 mutant strain (EIIIm). The viability of the larvae was calculated after a 48 h incubation. A significant reduction of the viability of T. canis larvae was observed in EI, EII and EIII. A decrease of this inhibitory effect was observed in EIm and EIIIm (p = 0.008). In the in vivo experiments, mice were orally inoculated with three doses of Ef7121. To study the probiotic persistence in the intestine, the animals were sacrificed every four days and their intestines were dissected. The initial average bacterial levels were 9.7 x 10(4) for Ef7121 (colony forming units/g). At the end of the assay the levels were 1.46 x 10(4). No bacterial translocation was detected in mesenteric lymphatic nodules and spleen. Ef7121 interference with the biological cycle was evaluated in mice challenged with T. canis. The interference was significant when the mice were challenged with probiotic and T. canis simultaneously (p = 0.001), but it was not significant when the challenge was performed 15 days after administration of the bacterial inoculum (p = 0.06). In conclusion, Ef7121 possessed in vitro and in vivo larvicidal activity.

The in vivo antimalarial activity of methylene blue combined with pyrimethamine, chloroquine and quinine

The effectiveness of methylene blue (MB) combined with pyrimethamine (PYR), chloroquine (CQ) or quinine (Q) was examined in a classical four-day suppressive test against a causative agent of rodent malaria, Plasmodium berghei. A marked potentiation was observed when MB was administered at a non-curative dose of 15 mg/kg/day in combination with PYR (0.19 mg/kg/day) or Q (25 mg/kg/day). No synergy was found between MB (15 mg/Kg) and CQ (0.75 mg/Kg). Our results suggest that the combination of MB with PYR or Q may improve the efficacy of these currently used antimalarial drugs.

In vitro and in vivo antimalarial activity and cytotoxicity of extracts, fractions and a substance isolated from the Amazonian plant Tachia grandiflora (Gentianaceae)

Tachia sp. are used as antimalarials in the Amazon Region and in vivo antimalarial activity of a Tachia sp. has been previously reported. Tachia grandiflora Maguire and Weaver is an Amazonian antimalarial plant and herein its cytotoxicity and antimalarial activity were investigated. Spectral analysis of the tetraoxygenated xanthone decussatin and the iridoid aglyone amplexine isolated, respectively, from the chloroform fractions of root methanol and leaf ethanol extracts was performed. In vitro inhibition of the growth of Plasmodium falciparum Welch was evaluated using optical microscopy on blood smears. Crude extracts of leaves and roots were inactive in vitro. However, chloroform fractions of the root and leaf extracts [half-maximal inhibitory concentration (IC50) = 10.5 and 35.8 µg/mL, respectively] and amplexine (IC50= 7.1 µg/mL) were active in vitro. Extracts and fractions were not toxic to type MRC-5 human fibroblasts (IC50> 50 µg/mL). Water extracts of the roots of T. grandiflora administered by mouth were the most active extracts in the Peters 4-day suppression test in Plasmodium berghei-infected mice. At 500 mg/kg/day, these extracts exhibited 45-59% inhibition five to seven days after infection. T. grandiflora infusions, fractions and isolated substance have potential as antimalarials.

In vivo assessment of antiretroviral therapy-associated side effects

Antiretroviral therapy has been associated with side effects, either from the drug itself or in conjunction with the effects of human immunodeficiency virus infection. Here, we evaluated the side effects of the protease inhibitor (PI) indinavir in hamsters consuming a normal or high-fat diet. Indinavir treatment increased the hamster death rate and resulted in an increase in triglyceride, cholesterol and glucose serum levels and a reduction in anti-oxLDL auto-antibodies. The treatment led to histopathological alterations of the kidney and the heart. These results suggest that hamsters are an interesting model for the study of the side effects of antiretroviral drugs, such as PIs.

The efficacy of 2-nitrovinylfuran derivatives againstLeishmania in vitro and in vivo

Despite recent advances in the treatment of some forms of leishmaniasis, the available drugs are still far from ideal due to inefficacy, parasite resistance, toxicity and cost. The wide-spectrum antimicrobial activity of 2-nitrovinylfuran compounds has been described, as has their activity against Trichomonas vaginalis and other protozoa. Thus, the aim of this study was to test the antileishmanial activities of six 2-nitrovinylfurans in vitro and in a murine model of leishmaniasis. Minimum parasiticide concentration (MPC) and 50% inhibitory concentration (IC50) values for these compounds against the promastigotes of Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis were determined, as were the efficacies of two selected compounds in an experimental model of cutaneous leishmaniasis (CL) caused by L. amazonensis in BALB/c mice. All of the compounds were active against the promastigotes of the three Leishmania species tested. IC50 and MPC values were in the ranges of 0.8-4.7 µM and 1.7-32 µM, respectively. The compounds 2-bromo-5-(2-bromo-2-nitrovinyl)-furan (furvina) and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan (UC245) also reduced lesion growth in vivo at a magnitude comparable to or higher than that achieved by amphotericin B treatment. The results demonstrate the potential of this class of compounds as antileishmanial agents and support the clinical testing of Dermofural(r) (a furvina-containing antifungal ointment) for the treatment of CL.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellowperoba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium bergheiin mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.

In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii

The polar hydroethanolic extract from Selaginella sellowii(SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.

Efeito da disponibilidade de hospedeiro e de alimento nas características biológicas de Trichogramma galloi Zucchi (Hymenoptera, Trichogrammatidae)

Características biológicas de Trichogramma galloi Zucchi, 1988 foram avaliadas em laboratório onde esses parasitóides foram criados com ovos de Diatraea saccharalis (Lepidoptera, Pyralidae), com ou sem mel, e expostos a ovos do hospedeiro após 0, 6, 12, 24, 36, 48, 60, 72 e 84 horas da emergência. As taxas de parasitismo e de viabilidade mostraram-se elevadas para indivíduos que receberam alimento. A razão sexual não foi influenciada pelo alimento. O número de indivíduos por ovo somente mostrou diferença para aqueles adultos que não receberam alimento e permaneceram 6 horas sem ovos do hospedeiro. Conferindo o efeito da disponibilidade de ovos, somente a razão sexual, com ou sem mel, não mostrou diferenças. Os resultados mostram que T. galloi necessita de um suprimento de carboidratos e que o tempo pode influenciar a capacidade reprodutiva.

A comunidade de abelhas (Hymenoptera, Apidae s. l.) em uma área restrita de campo natural no Parque Estadual de Vila Velha, Paraná: diversidade, fenologia e fontes florais de alimento

Coletas sistemáticas de abelhas em uma área restrita no Parque Estadual de Vila Velha, Paraná, no período de outubro de 2002 a outubro de 2003, resultaram em 1552 espécimes pertencentes a 181 espécies. Estas espécies estão distribuídas em 58 gêneros, 24 tribos e 5 subfamílias. As plantas visitadas correspondem a 113 espécies, em 72 gêneros e 38 famílias. Megachile com 20 espécies foi o gênero mais rico e Ceratina o gênero mais abundante dentre os gêneros nativos. Apis mellifera foi a espécie mais coletada, correspondendo a 28% do total de indivíduos, e Bombus atratus foi a espécie mais abundante dentre as abelhas nativas. A riqueza e a equitabilidade nos meses foram variáveis, sendo março o mais rico e novembro o de maior equitabilidade. Apesar de tradicionalmente considerados parte das estepes sulinas, os campos de Vila Velha apresentam uma fauna de abelhas contendo várias espécies típicas de cerrado. O igual número de espécies entre as subfamílias Apinae e Halictinae também apontam para uma peculiaridade de sua fauna. Listas de abelhas e plantas coletadas são apresentadas em anexo.

Efeitos da temperatura e do alimento no desenvolvimento de Dysdercus maurus Distant (Hemiptera, Pyrrhocoridae)

Dysdercus maurus Distant, 1901 (Hemiptera, Pyrrhocoridae) é uma importante praga de Gossypium spp. (algodoeiro), Citrus Sinensis Osbeck (Rutaceae) (laranjeira) e Citrus reticulata (Rutaceae) (tangerineira), além de sementes de Chorisia speciosa St. Hil. (paineira). Este trabalho objetivou avaliar os efeitos da temperatura e do alimento no desenvolvimento de D. maurus. Foram realizados oito tratamentos, seis em que os percevejos foram alimentados com sementes de paineira e mantidos a 15, 18, 20, 25 e 30 ± 1ºC, UR 80 ± 3% e fotofase de 12 h ou em condições ambientais de laboratório (23,5 ± 2,6ºC, UR 73,3 ± 9,9 %), e dois em que foram alimentados com sementes de algodão variedade IAC-22 e mantidos a 25 e 30ºC. Em todos os tratamentos foram observados cinco estágios imaturos. O aumento da temperatura proporcionou diminuição do tempo de desenvolvimento. A temperatura de 15ºC foi letal para ovos e ninfas de D. maurus. A menor mortalidade de ninfas ocorreu quando os percevejos foram alimentados com sementes de algodão a 25ºC (24,07%). A menor temperatura base (Tb) foi obtida para o 1º ínstar (11,54ºC) e a maior para o 2º ínstar (15,33ºC). As fêmeas de D. maurus necessitam de maior quantidade de graus-dias (329,93 graus-dias) que os machos (300,49 graus-dias) para atingir o estádio adulto.

Efeito de diferentes fontes de carboidrato e da privação de alimento sobre aspectos biológicos de Telenomus remus Nixon (Hymenoptera, Scelionidae)

O efeito de cinco fontes de carboidratos, da água e da ausência de alimentação foi avaliado sobre aspectos biológicos de T. remus. Os tratamentos constituíram-se de: (a) fêmeas não alimentadas, e alimentadas com (b) água destilada; (c) mel puro; (d) xarope de glicose (Karo®); (e) solução de glicose 1M; (f) solução de frutose 1M; (g) solução de sacarose 1M; (h) solução de glicose+frutose+sacarose 1M; (i) solução de glicose+frutose+sacarose 3M. Os alimentos foram disponibilizados durante todo o experimento. Posturas de Sodoptera frugiperda com aproximadamente 100 ovos (< 24h) foram ofertadas às fêmeas de T. remus. O parasitismo, a razão sexual e a longevidade foram avaliados. No aspecto parasitismo, as fêmeas alimentadas com mel e soluções de glicose, frutose e sacarose mostraram maior eficiência, não diferindo significativamente entre si, enquanto que as fêmeas não alimentadas e alimentadas somente com água apresentaram níveis mais baixos de parasitismo. Já as fêmeas alimentadas com xarope de glicose (Karo®), solução de glicose+frutose+sacarose 1M e 3M apresentaram parasitismo mais baixo quando comparados com os outros tratamentos, não diferindo significativamente entre si. Quanto à razão sexual, os tratamentos não apresentaram diferenças significativas, mantendo-se em torno de 0,57. No que se refere à longevidade, os insetos mantiveram-se vivos em média 5 dias quando alimentados com qualquer um dos carboidratos, já aqueles alimentados com água ou não alimentados sobreviveram apenas por 1 dia. Logo, conclui-se que o mel, glicose, frutose e sacarose mostram-se mais adequados para a alimentação de fêmeas de T. remus.