Gérzia M. C. Machado

Antileishmanial activity of lapachol analogues

The antileishmanial activity of lapachol, isolapachol, and dihydrolapachol, along with soluble derivatives (potassium salt) and acetate was obtained. All the compounds were assayed against metacyclic promastigotes of two different species of Leishmania associated to tegumentar leishmaniasis: L. amazonensis and L. braziliensis. All compounds presented significant activity, being isolapachol acetate the most active against promastigotes, with IC50/24h = 1.6 +/- 0.0 microg/ml and 3.4 +/- 0.5 microg/ml for, respectively, L. amazonensis and L. braziliensis. This compound was also assayed in vivo against L. amazonensis and showed to be active. Its toxicity in vitro was also established, and at concentration similar to the IC50, no toxicity was evidenced. In all experiments, pentamidine isethionate was used as a reference drug. The present results reinforce the potential use of substituted hydroxyquinones and derivatives as promising antileishmanial drugs and suggest a continuing study within this class of compounds.

Anti-leishmanial activity of alkaloidal extract from Aspidosperma ramiflorum

Infections due to protozoa of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials (SbV), which present renal and cardiac toxicity. Besides, the precise chemical structure and mechanism of action of these drugs are unknown up to date. In order to find new drugs against leishmaniasis, we have been studying extracts of Brazilian trees. In the present study, we have evaluated the effectiveness of an alkaloid extract of Aspidosperma ramiflorum Muell. Arg. (Apocynaceae), against the extracellular forms promastigotes of L. (L.) amazonensis and L. (V.) braziliensis. The alkaloid extract of A. ramiflorum was much more effective against L. (L.) amazonensis (LD50 < 47 microg/ml) than L. (V.) braziliensis. Based on these in vitro results against L. (L.) amazonensis new studies should be made to find the compounds with anti-leishmanial activity.

Trypanosoma cruzi in the opossum Didelphis marsupialis: Parasitological and serological follow-up of the acute infection

The opossum Didelphis marsupialis is known to be among the most important wild reservoirs of Trypanosoma cruzi and one in which the trypanosome may go through both the usual vertebrate intracellular cycle in its tissues and an extracellular cycle in the lumen of its scent glands. The species is highly resistant to heavy inocula and, depending on the parasite strain, experimental infections may be permanent or self limited. Aiming to understand the mechanisms involved in this parasite-host interaction we made a study of the acute phase of infection with different T. cruzi strains. Strains F, G-49 and G-327 produced durable infections with relatively high parasitemia and invasion of the scent glands, while equivalent inocula of the Y strain resulted in scanty parasitemia of short duration, no invasion of the SG, and no evidence of persistent parasitism. A smaller inoculum of G-49 produced only subpatent though persistent parasitemia and no invasion of the scent glands. The humoral immune response was less marked in the Y group; among the other groups IgM and IgG antibodies increased to high levels, higher in the G-49 group. The increase in IgG coincided with a drop of parasitemia to subpatent levels. Two opossums inoculated directly in the scent glands with culture forms of the Y strain had a short-lived subpatent parasitemia, but the parasites remained in the glands and serum Ig antibodies reached high levels. Immunoblot analysis showed that the sera of the inoculated opossums recognized few T. cruzi antigens (more in the F strain) in comparison with those of mice. However, with the only exception of those subcutaneously inoculated with the Y strain and including two naturally infected specimens, all the opossums sera recognized a 90-kDa peptide in all T. cruzi strains. Our results confirm that opossums are able to selectively eliminate some strains of T. cruzi and indicate that the mechanism involved in this selection is probably not related to the humoral immune response. In infections by strains that are able to establish a permanent foothold in opossum tissues, there are indications that IgG antibodies participate in the control of the parasite population of the acute phase but are unable to prevent the chronic phase. It was once more demonstrated that the opossum infected scent glands function as diffusion chambers for parasite antigens but that, on the other hand, the parasites are here protected against the mechanisms developed by the host to control their population.

Nitric oxide biosynthesis by Leishmania amazonensis promastigotes containing a high percentage of metacyclic forms

Due to the diversity of its physiological and pathophysiological functions and general ubiquity, the study of nitric oxide (NO) has become of great interest. In this work, it was demonstrated that Leishmania amazonensis promastigotes produces NO, a free radical synthesized from l-arginine by nitric oxide synthase (NOS). A soluble NOS was purified from L. amazonensis promastigotes by affinity chromatography (2′, 5′-ADP-agarose) and on SDS-PAGE the enzyme migrates as a single protein band of 116.2 (±6) kDa. Furthermore, the presence of a constitutive NOS was detected through indirect immunofluorescence using anti-cNOS and in NADPH consumption assays. The present work show that NO production, detected as nitrite in culture supernatant, is prominent in promastigotes preparations with high number of metacyclic forms, suggesting an association with the differentiation and the infectivity of the parasite.

Chemical and Biological Analyses of the Essential Oils and Main Constituents of Piper Species

The essential oils obtained from leaves of Piper duckei and Piper demeraranum by hydrodistillation were analyzed by gas chromatography-mass spectrometry. The main constituents found in P. demeraranum oil were limonene (19.3%) and β-elemene (33.1%) and in P. duckei oil the major components found were germacrene D (14.7%) and trans-caryophyllene (27.1%). P. demeraranum and P. duckei oils exhibited biological activity, with IC50 values between 15 to 76 μg mL−1 against two Leishmania species, P. duckei oil being the most active. The cytotoxicity of the essential oils on mice peritoneal macrophage cells was insignificant, compared with the toxicity of pentamidine. The main mono- and sesquiterpene, limonene (IC50 = 278 μM) and caryophyllene (IC50 = 96 μM), were tested against the strains of Leishmania amazonensis, and the IC50 values of these compounds were lower than those found for the essential oils of the Piper species. The HET-CAM test was used to evaluate the irritation potential of these oils as topical products, showing that these oils can be used as auxiliary medication in cases of cutaneous leishmaniasis, with less side effects and lower costs.

Synthesis and activity of novel tetrazole compounds and their pyrazole-4-carbonitrile precursors against Leishmania spp

A new series of 5-(1-aryl-3-methyl-1H-pyrazol-4-yl)-1H-tetrazole derivatives (4a-m) and their precursor 1-aryl-3-methyl-1H-pyrazole-4-carbonitriles (3a-m) were synthesized and evaluated as antileishmanials against Leishmania braziliensis and Leishmania amazonensis promastigotes in vitro. In parallel, the cytotoxicity of these compounds was evaluated on the RAW 264.7 cell line. The results showed that among the assayed compounds the substituted 3-chlorophenyl (4a) (IC50/24h=15±0.14 μM) and 3,4-dichlorophenyl tetrazoles (4d) (IC50/24h=26±0.09 μM) were the most potent against L. braziliensis promastigotes, as compared the reference drug pentamidine, which presented IC50=13±0.04 μM. In addition, 4a and 4d derivatives were less cytotoxic than pentamidine. However, these tetrazole derivatives (4) and pyrazole-4-carbonitriles precursors (3) differ against each of the tested species and were more effective against L.braziliensis than on L. amazonensis.

Chemical and biological evaluation of essential oils with economic value from Lauraceae species

This work compares the chemical composition of the essential oils from the leaves of Licaria canella collected in two different seasons. The results of this investigation were compared with the leaf essential oils of other species of the Lauraceae family, Aniba canelilla, collected at the same time. Both essential oils were analyzed by GC-FID and GC-MS. The results demonstrated a larger predominance of benzenoids, being the main constituent benzyl benzoate for L. canella and 1-nitro-2-phenylethane for A. canelilla. The comparison of the biological activities showed that L. canella (IC50 19 µg mL-1) was more active against Leishmania amazonensis strains and less cytotoxic in macrophage cultures than A. canelilla (IC50 40 µg mL-1). On the other hand, the L. canella oil displayed a higher cytotoxicity against Artemia salina with a lethal concentration (LC50), equal to 5.25 µg mL-1.

Description of Leishmania (Leishmania) forattinii sp. n., a new parasite infecting opossums and rodents in Brazil

A new parasite species of Leishmania is described, L. (Leishmania) forattinii sp. n., which was isolated from a pooled triturate of liver and spleen of a opossum (Didelphis marsupialis aurita) and from skin samples from a rodent (Proechmys iheringi denigratus), captured in primary forest on the Atlantic Cost of Brazil. Our results on the basis of biological and molecular criteria indicate that this taxonomically distinct parasite ias a new species of the L. mexicana complex, but closely related to L. (L.) aristidesi Laison & shaw, 1979, as revelated by phenetic and phylogenetic numerical analyses of the enzyme data. L. forattinii was clearly distinguishable from other Leishmania species of the genus usisng enzyme electrophoresis, monoclonal antibodies, molecular karyotypes, analysis of restriction enzyme digestion patterns of kinetoplast DNA (kDNA), as well as the use of kDNA hybridization procedures.

A New Enzymatic Variant of Leishmania (Leishmania) forattinii Isolated from Proechimys iheringi (Rodentia, Echimydae) in Espírirto Santo, Brazil

Taxonomic studies of leishmanial isolates fromthe New World indicate tremendous diversitywithin this genus (E Cupolillo et al. 1994 Am JTrop Med Hyg 50: 296-311). A number of newLeishmania species from sylvan areas of theNeotropics have been described recently. Some ofthose parasites are associated with disease in hu-mans; others appear to be restricted to lower or-ders of mammals, such as rodents and edentates(G Grimaldi et al. 1989 Am J Trop Med Hyg 41:687-725). However, some of the latter parasitesmay yet be shown capable of causing human dis-ease, particularly in persons with altered cellularimmune responses.During a survey of potential reservoir hosts ofLeishmania done in the State of Espirito Santo, Bra-zil (1982-1985), three leishmanial parasites (des-ignated with stock codes MPRO/BR/82/RV203,MPRO/BR/82/RV228, and MPRO/BR/85/RV260)were isolated from skin lesion samples taken fromspiny-rats, Proechimys iheringi (Rodentia,Echimydae), captured in a secondary forest locatedin the municipality of Viana (A Falqueto et al. 1985Mem Inst Oswaldo Cruz 80: 497). In studies us-ing an indirect radioimmune binding assay (RIA)and a large panel of monoclonal antibodies (Mabs)derived for selected species of Leishmania , the iso-lates were characterized as members of the L.mexicana complex. These strains were differentfrom those species infective to humans, in that theyreacted with either the L.(L.) amazonensis or L.(L.) mexicana specific MAbs. Furthermore, thiswas also the case with cloned organisms from twostocks showing that they were not mixed popula-tions of Leishmania (G Grimaldi et al. 1987 Am JTrop Med Hyg 36: 270-287). This group of dis-tinct parasites presented, however, similarserodeme patterns in relation to the new recentlydescribed species of the Leishmania subgenus, L.(L.) forattinii (E Yoshida et al. 1993 Mem InstOswaldo Cruz 88: 397-406).The latter parasite was isolated from sylvaticreservoir hosts (Didelphis marsupialis aurita andP. iheringi denigratus ) captured on regions endemicfor American cutaneous leishmaniasis in Brazil,respectively in the municipalities of Conchas, SaoPaulo (E Yoshida et al. 1979 Rev Inst Med Trop SPaulo 21: 110-113) and Tres Bracos, Bahia (ABarreto et al. 1985 Rev Soc Bras Med Trop 18:243-246). The general morphology and the growthcharacteristics in vitro of the new species were simi-lar to those of other L. mexicana complex para-sites. Here we have further studied the Espirito Santoisolates using additional techniques for the char-acterization of Leishmania . The molecular proce-dures used for typing the strains (isoenzyme elec-trophoresis, blot enzyme binding assay usingMAbs, restriction endonuclease fragment patternsof k-DNA, and molecular karyotype analysis) havebeen described in detail in previous publications(Yoshida et al. 1979, 1983 loc. cit. , Cupolillo et al.1994 loc. cit., A Franco et al. 1997 Mem InstOswaldo Cruz 92: 63-68).Results of electrophoretic analysis were basedon the following enzymatic loci, namely: glucose-6-phosphate dehydrogenase (G6PDH), malate de-hydrogenase (MDH), isocitrate dehydrogenase(IDHNADP), 6-phosphogluconate dehydrogenase(6PGDH), nucleotidase (NH1 and NH2), glucosephosphate isomerase (GPI), phosphoglucomutase(PGM), proline dipeptidase (PEPD), leucine pep-tidase (PEP2), malic enzyme (ME), mannose phos-phate isomerase (MPI), and aconitate hydratase(ACON). The enzyme profiles of the selected iso-lated signature MPRO/BR/82/RV228, which rep-resents the poorly defined group of parasites from

Minicircle kDNA microheterogeneity in Endotrypanum indicate diversity within this genus

A comparison of kDNA restriction-endonuclease fragment patterns from strains representing selected Endotrypanum zymodemes was done by schizodeme analysis. As the degree of heterogeneity within mini-circles varied among species or strains of Endotrypanum, the fingerprint obtained with each of the restriction enzymes was unique for each of these parasites. The data have revealed that this trypanosomatid genus is much more complex than it was originally thought to be.