Antileishmanial phenylpropanoids from Alpinia galanga (Linn.) Willd

Hexane, chloroform and ethyl acetate extracts (100 microg/ml) of Alpinia galanga rhizomes exhibited significant activity in vitro against promastigotes of L. donovani. Twelve compounds namely, methyleugenol (1), p-coumaryl diacetate (2), 1'-acetoxychavicol acetate (3), 1'-acetoxyeugenol acetate (4), trans-p-acetoxycinnamyl alcohol (5), trans-3,4-dimethoxycinnamyl alcohol (6), p-hydroxybenzaldehyde (7), p-hydroxycinnamaldehyde (8), trans-p-coumaryl alcohol (9), galangin (10), trans-p-coumaric acid (11) and galanganol B (12) were isolated from these extracts. Of these, compounds 2, 3, 4 and 5 were found most active in vitro against promastigotes of L. donovani with IC50 values of 39.3, 32.9, 18.9 and 79.9 microM respectively. This is the first report of antileishmanial activity of the extracts and isolated constituents of A. galanga.     

Arylanthranilodinitriles: a new biaryl class of antileishmanial agents

A series of anthranilodinitrile-based biaryls were synthesized and evaluated in vitro against extracellular promastigotes and intracellular amastigotes of Leishmania donovani. Among various screened compounds, a biaryl with trifluoromethyl group 5f showed 83% inhibition against promastigotes and 70% inhibition against amastigotes of L. donovani at 8 and 20microg/mL concentrations, respectively.     

Antileishmanial and trypanocidal activity of Brazilian Cerrado plants

The side effects and the emerging resistance to the available drugs against leishmaniasis and trypanosomiasis led to the urgent need for new therapeutic agents against these diseases. Thirty one extracts of thirteen medicinal plants from the Brazilian Cerrado were therefore evaluated in vitro for their antiprotozoal activity against promastigotes of Leishmania donovani, and amastigotes of Trypanosoma cruzi. Among the selected plants, Casearia sylvestris var. lingua was the most active against both L. donovani and T. cruzi. Fifteen extracts were active against promastigotes of L. donovani with concentrations inhibiting 50% of parasite growth (IC50) between 0.1-10 microg/ml, particularly those of Annona crassiflora (Annonaceae), Himatanthus obovatus (Apocynaceae), Guarea kunthiana (Meliaceae), Cupania vernalis (Sapindaceae), and Serjania lethalis (Sapindaceae). With regard to amastigotes of T. cruzi, extracts of A. crassiflora, Duguetia furfuracea (Annonaceae), and C. sylvestris var. lingua were active with IC50 values between 0.3-10 microg/ml. Bioassay fractionations of the more active extracts are under progress to identify the active antiparasite compounds.     

Anti-parasite activity of nucleoside analogues: the metabolism of carbocyclic inosine in promastigotes of Leishmania tropica and Leishmania donovani and its activity against amastigotes of Leishmania donovani in vitro

Carbocyclic inosine is a potent inhibitor for the growth of the promastigote form of Leishmania tropica and Leishmania donovani. In culture, the EC50 values of carbocyclic inosine are 8.3 X 10(-8) and 1.3 X 10(-7) M for the promastigotes of L. tropica and L. donovani, respectively. On the other hand, it is less toxic towards mouse mammary tumor FM3A cells: the EC50 value is 2.7 X 10(-4)M. Carbocyclic inosine is metabolized by Leishmania promastigotes to give carbocyclic adenosine-5'-triphosphate(aristeromycin-5'-triphosphate) and carbocyclic guanosine-5'-triphosphate. This metabolic conversion provides a mechanism for the parasite-selective toxicity of carbocyclic inosine. Carbocyclic inosine was found to be active against L. donovani amastigotes in an in vivo-like cultivation in vitro.     

Synthesis and in vitro antileishmanial activity of 5-substituted-2'-deoxyuridine derivatives

We report herein the synthesis and the in vitro antileishmanial evaluation of 5-substituted-2'-deoxyuridine nucleosides. The most active compound against Leishmania donovani promastigotes was Thia-dU (3a) with an IC50 =3 microM. This compound exhibited the same activity as zidovudine (3'-azido-2'-deoxythymidine) used as nucleoside reference compound. Considering the cytotoxicity of synthetic compounds on peritoneal murine macrophages, the most toxic compound was MeThio-dU (3d) with a MTC at 10 microM. Only Methia-dU (3b) was active against intramacrophagic amastigotes with an IC50 =6.5 microM. This latter can now be evaluated in vivo, for further investigations through structure-based drug design.     

Developmental regulation of proline transport in Leishmania donovani

Leishmania donovani are the causative agents of kala azar in humans. These organisms cycle between the proline-rich environment of the sand fly vector (extracellular promastigotes) and the sugar-rich condition in the mammalian host (intracellular amastigotes). Parasites have adapted to these extreme changes in proline concentrations: promastigotes utilize proline as a carbon source, whereas amastigotes utilize sugars and fatty acids. Previous studies have suggested that promastigotes and amastigotes express distinct proline transporters. However, the information available on these transporters is limited. In this work, proline transport was investigated in axenic L. donovani cultures. Three transport systems were identified: cation-dependent and -independent proline transporters in promastigotes (systems A and B, respectively) and a single cation-independent transporter in amastigotes (system C). Systems A and C have broad specificity to almost all amino acids and obtain optimum activity at acidic pH ranges (pH 6 and 5, respectively). System B is more specific to proline, as it is inhibited by only five amino acids. Temperature response analyses indicated that the transporters of both promastigotes and amastigotes perform best at 37 degrees C. The activity of system A during parasite differentiation was assessed. The transport activity of system A disappeared 3 days after promastigotes were induced to differentiate into amastigotes. In these cells, elevated temperature and acidic pH each suppressed the activity of system A. When amastigotes were induced to differentiate back into promastigotes, system A resumed its activity 24 h after differentiation was initiated. In conclusion, L. donovani obtain proline transport systems that are stage specific, regulated by both pH and temperature. This paper constitutes the first investigation of amino acid transport in axenic L. donovani.     

Leishmanicidal activity of stearylamine-bearing liposomes in vitro

Liposomes consisting of stearylamine (SA) and egg yolk phosphatidylcholine (PC) were studied for their cytotoxic activity against freshly transformed promastigotes and intracellular amastigotes of Leishmania donovani, the causative agent of visceral leishmaniasis. More than 99% of the parasites of strain AG83 were killed within 60 min by treatment with 22 mol% SA-PC liposomes (132 microg/ml total lipids). This was further confirmed by incubating the liposome-treated promastigotes at 22 C for 96 hr. The killing activity of the liposomes progressively decreased with lowering lipid concentration. However, weak cytotoxic activity was still detected at 6.6 microg/ml lipids. Leishmanicidal activity of the liposomes became stronger with increasing SA content but was reduced with the incorporation of cholesterol in the liposomes. A similar cytotoxic effect was observed on other Indian strains of L. donovani, for example PKDL and DD8, as well as on species such as Leishmania donovani S1, Leishmania donovani infantum, Leishmania tropica, Leishmania amazonensis, and Leishmania mexicana. However, freshly transformed promastigotes appeared to be more susceptible than the ones subcultured. The strong leishmanicidal activity of SA-PC liposomes was also demonstrated toward intracellular L. donovani amastigotes. The SA-bearing vesicles could effectively inhibit the growth and multiplication of the parasites within the macrophages. The cytolytic activity of these liposomes on leishmanial parasites and low toxicity on host macrophages may, thus, find application in the therapy of leishmaniasis.     

Enzymes of carbohydrate metabolism in Leishmania donovani amastigotes

A method for the isolation of Leishmania donovani amastigotes from infected hamster spleen and liver tissues is described. Over 85% of the isolated amastigotes were viable as judged by acridine orange-ethidium bromide staining and in vitro transformation to the promastigote form. A comprehensive survey of the enzymes of carbohydrate metabolism in L. donovani amastigotes and promastigotes was conducted. Amastigotes and promastigotes possess all of the enzymes of the Embden-Meyerhof pathway, hexose monophosphate shunt, and tricarboxylic acid cycle. Cell-free extracts of both forms demonstrate an active glutamate dehydrogenase, thus linking activity which permits entry of pyruvate into the tricarboxylic acid cycle. Both forms demonstrate an active glutamate dehydrogenase, thus linking amino acid metabolism with carbohydrate metabolism. Pyruvate carboxylase, the enzyme responsible for replenishment of C4 acids by heterotrophic CO2 fixation into pyruvate, was also demonstrable in the tissue and insect forms. In general, activities of promastigote enzymes are higher than the amastigote enzymes. Differences between the vertebrate (amastigote) and invertebrate (promastigote) forms in their potential to utilize carbohydrates as substrates would appear to be quantitative rather than qualitative.     

Species and stage specificity of Leishmania donovani antigens.

Monoclonal antibodies D2 and D13 were produced in mice using Leishmania donovani promastigote membrane fractions. To study the species and stage specificity of the antigens recognized by these antibodies, we examined amastigotes prepared in vitro and cultured promastigotes by indirect immunofluorescence with monoclonal antibodies D2 and D13. Monoclonal antibody D2 showed weak reactivity for 9 of 9 strains of L. donovani complex promastigotes and 8 of 9 amastigotes. In contrast, only 2 of 22 strains from other complexes yielded equivocal reactions. Monoclonal antibody D13, however, had much broader reactivity. D13 reacted with all the promastigotes and amastigotes of L. donovani complex isolates as well as with 10 of 22 promastigotes and 8 of 13 amastigotes from other complexes. The high degree of species specificity seen with monoclonal antibody D2 provides a rationale for further study of this antibody and its purified antigen for parasite identification and serodiagnosis of visceral leishmaniasis. The strong fluorescent signal noted with D13 and the presence of the D13 epitope on all L. donovani complex parasites supports studies on its role as an antigen in immunoprophylaxis of visceral leishmaniasis.     

Leishmania donovani: long-term culture of axenic amastigotes at 37 degrees C

L. donovani promastigotes were subjected to heat treatment yielding an axenic amastigote stage which was long-term cultured at 37 degrees C. No differences were observed between the growth rates of axenic amastigotes and promastigotes. Flow cytometry-derived DNA histograms of axenic amastigotes and promastigotes were typical of exponentially growing cell populations. Moreover, axenic amastigotes were metabolically active as evidenced by the release of an immunoprecipitable extracellular acid phosphatase (SAcP) into their culture supernatant. Cell transformation was confirmed by transmission electronmicroscopic examination of thin sections and extended by fracture-flip survey which allowed differentiation of cell membranes. The ultrastructure and nanoanatomy of axenic amastigotes was identical to that of intracellular amastigotes. The production of large amounts of heat-shock axenic amastigotes suitable for biochemical and biological studies of differentiation in Leishmania donovani may have important implications in the development of prevention and/or treatment strategies.     

Infectivity and virulence of Leishmania donovani promastigotes: a role for media,source, and strain of parasite

Transformation of promastigotes of Leishmania donovani strain AG83 from amastigotes derived from an infected animal was studied in three media, Schneider's Drosophila medium (SDM), Medium 199 (M199), and biphasic M199 (B-M199) with 10% fetal bovine serum. The media, SDM and B-M199, both supported a more efficient transformation of promastigotes in comparison with M199. Infectivity studies in hamsters and BALB/c mice showed that promastigotes isolated in B-M199 were several folds more infective than those obtained from M199. Comparison of the infectivity and virulence of promastigotes of AG83, with a recent isolate of kala-azar, SL94, harvested under similar conditions, revealed greater infectivity of SL94 for both macrophages and animal models. The present study demonstrates that the medium used for the conversion of amastigotes to promastigotes plays a major role in determining the infectivity of the freshly transformed L. donovani promastigotes in hamsters and BALB/c mice. The source and the strain of the parasite also influence the outcome of L. donovani infection.     

In vitro and in vivo leishmanicidal activity of Dysoxylum binectariferum and its fractions against Leishmania donovani

The leishmanicidal effect of crude ethanolic extract of stem bark of Dysoxylum binectariferum and its fractions has been investigated against Leishmania donovani, the causative agent of visceral leishmaniasis. Ethanolic extract was lethal to promastigotes as well as amastigote forms in macrophage system at the concentration of 100 microg/ml. Chloroform fraction significantly inhibited promastigote multiplication and was also active against amastigotes in infected J774A.1 macrophages at 100 microg/ml. Hexane fraction was moderately active and the other fractions were inactive against both the forms. When tested in vivo in hamsters, ethanolic extract was toxic at 500 mg/kg whereas exhibited marginal activity (67.7+/-5.3%) at 250 mg/kg x 5, p.o. on day 7 post treatment (p.t.) which increases slightly (69+/-4.7) by day 30 p.t. Chloroform and n-hexane fractions exhibited 64.3+/-4% and 47.8+/-4.6% parasite inhibition at the dose of 100 mg/kg x 5 p.o., respectively. The pure compound, rohitukine, obtained from chloroform fraction showed weaker in vitro activity and was ineffective in infected hamsters. The lead potential of this plant need further investigations.     

Over-expression of Leishmania major MAP kinases reveals stage-specific induction of phosphotransferase activity

During the infectious cycle, protozoan parasites undergo various developmental transitions and switch virulence factors in response to extracellular signals in insect vectors and human hosts. Despite the importance of environmental sensing in parasite pathogenicity, little is known about the pathways that transduce extracellular signals into stage-specific gene expression. Here, we used a transgenic approach to gain insight into localisation and activity of three green fluorescence protein (GFP)-tagged Leishmania major mitogen-activated protein kinases, LmaMPK4, 7 and 10. The GFP-LmaMPKs in both L. major and Leishmania donovani transgenic lines showed predominant cytoplasmic localisation and the over-expression had no effect on promastigote morphology, growth and the ability to differentiate into stationary-phase metacyclics for L. major and axenic amastigotes for L. donovani. We isolated the GFP-tagged MPKs from parasite extracts and tested their phosphotransferase activity across various culture conditions. For all three GFP-LmaMPKs, kinase activity was low or absent in promastigote extracts but significantly increased in L. major promastigotes after exposure to pH 5.5 and 34 degrees C, and in axenic L. donovani amastigotes. Enhanced activity correlated with increased GFP-LmaMPK phosphorylation as judged by phospho-specific fluorescent staining of the immuno-precipitated kinases. We could extend these findings to the endogenous LmaMPK10, which accumulated in the phospho-protein fraction of axenic amastigotes but not promastigotes, and thus follows the stage-specific phosphorylation profile of episomally expressed GFP-LmaMPK10. These results provide evidence for the functional conservation of Leishmania MAP kinases in parasite environmental sensing and underscore the potential of transgenic approaches to gain insight into signaling events during the Leishmania life cycle.     

Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics

In this report, we describe an in vitro culture system for the generation and propagation of axenic amastigotes from the well characterised 1S-CL2D line of Leishmania donovani. Fine structure analyses of these in vitro-grown amastigotes demonstrated that they possessed morphological features characteristic of L. donovani tissue-derived amastigotes. Further, these axenic amastigotes (LdAxAm) were shown to synthesise and release a secretory acid phosphatase isoform similar to that produced by intracellular amastigotes. Such LdAxAm also expressed surface membrane 3'-nucleotidase enzyme activity similar to that of tissue-derived amastigotes. Moreover, LdAxAm, in contrast to promastigotes, expressed significant levels of the amastigote-specific A2 proteins. In addition, LdAxAm, derived from long term cultures of Ld 1S-CL2D promastigotes, had significant infectivity for both human macrophages in vitro and for hamsters in vivo. Thus, the in vitro culture system described herein provides a useful tool for the generation of large quantities of uniform populations of axenic amastigotes of the L. donovani 1S-CL2D line. The availability of such material should greatly facilitate studies concerning the cell and molecular biology of this parasite developmental stage.     

Synthesis of chalcone analogues with increased antileishmanial activity

Eighteen analogues of an active natural chalcone were synthesized using xanthoxyline and some derivatives, and these analogues were tested for selective activity against both promastigotes and intracellular amastigotes of Leishmania amazonensis in vitro. Three analogues (10, 12, and 19) containing nitro, fluorine or bromine groups, respectively, displayed increased selective activity against the parasites as compared with the natural chalcone. The nitrosylated chalcone 10 was also tested intralesionally in infected mice and was found to be as effective as Pentostan reference drug at a dose 100 times higher than that of the chalcone in controlling both the lesion growth and the parasite burden.     

Synthesis of marine alkaloid: 8,9-dihydrocoscinamide B and its analogues as Novel class of antileishmanial agents

A series of marine alkaloid 8,9-dihydrocoscinamide B, its analogues and indolylglyoxylamide derivatives have been synthesized and screened for their in vitro antileishmanial activity profile in promastigote and amastigote models. Compounds 7 and 10 have shown 99-100% inhibition against promastigotes and 97-98% inhibition against amastigotes at a concentration of 10 microg/ml.     

Identification of amastigote-specific antigens of Leishmania donovani using kala-azar patient sera

Antigenic characterization of the soluble fraction of axenic amastigotes of Leishmania donovani ( strain Dd8, causative agent of Indian kala-azar) and their comparison with promastigotes is reported. The axenic amastigotes were assessed for their immunological status employing anti-A2 monoclonal antibody which is extremely specific for L. donovani amastigotes. SDS-PAGE of 35[S] methionine labeled proteins of the two parasite stages exhibited few stage specific and some conserved antigens in both the stages. An increased synthesis of heat shock proteins was observed in axenic amastigotes. Western blot experiments employing sera of kala azar positive patients identified immunodominent antigens of 116,83,26 and 12 kDa in axenic amastigotes which were not present in promastigotes. These amastigote stage specific antigens may have immense potential in immunodiagnosis and prophylaxis of kala-azar.     

Differential survival of Leishmania donovani amastigotes in human monocytes

Leishmania donovani is an important intracellular protozoal pathogen of man; it is found solely within macrophages in its amastigote stage in humans, and exists in its extracellular, flagellated promastigote stage in the sandfly, its arthropod vector. To determine if either stage of L. donovani was capable of surviving within monocytes--the oxidatively active precursors of tissue macrophages--interactions of the parasite with human monocytes were studied in vitro. Amastigotes and promastigotes were ingested to a comparable degree by monocytes; whereas 79% of promastigotes were killed within 48 hr, however, amastigotes survived and multiplied threefold over 5 days. Promastigotes, which have been shown to be sensitive to hydrogen peroxide-peroxidase-halide microbicidal mechanisms, elicited a phagocytic oxidative burst that was 49% of the response to serum-opsonized zymosan, as assessed by luminol-enhanced chemiluminescence. NBT was reduced to formazan in 71% of monocytes exposed to promastigotes. The death of promastigotes within monocytes could be attributed at least in part to oxidative microbicidal mechanisms because there was no significant decrease in the number of cell-associated parasites in monocytes from donors with chronic granulomatous disease of childhood. In contrast to promastigotes, amastigotes survived within monocytes, despite eliciting an oxidative response that was 27% of the response produced by serum-opsonized zymosan; this response was not significantly different from that produced by promastigotes. In a phagocyte-free system, amastigotes were found to be sevenfold more resistant than were promastigotes to the lethal effects of hydrogen peroxide. The survival of L. donovani in human monocytes is thus dependent on the parasite stage; promastigotes are ingested, they elicit an oxidative burst, and the majority are killed by oxidative microbicidal mechanisms, whereas amastigotes are ingested and survive to parasitize human monocytes successfully, despite eliciting a phagocytic oxidative burst.     

Characterization of the elongating alpha-D-mannosyl phosphate transferase from three species of Leishmania using synthetic acceptor substrate analogues

Leishmania express lipophosphoglycans and proteophosphoglycans that contain Galbeta1-4Manalpha1-P phosphosaccharide repeat structures assembled by the sequential addition of Manalpha1-P and betaGal. The synthetic acceptor substrate Galbeta1-4Manalpha1-P-decenyl and a series of analogues were used to probe Leishmania alpha-D-mannosyl phosphate transferase activity. We show that the activity detected with Galbeta1-4Manalpha1-P-decenyl is the elongating alpha-D-mannosyl phosphate transferase associated with lipophosphoglycan biosynthesis (eMPT(LPG)). Differences in the apparent K(m) values for the donor and acceptor substrates were found using L. major, L. mexicana, and L. donovani promastigote membranes, but total activity correlated with the number of lipophosphoglycan repeats. Further comparisons showed that lesion-derived L. mexicana amastigotes, that do not express lipophosphoglycan, lack eMPT(LPG) and that nondividing L. major metacyclic promastigotes contain 5-fold less eMPT(LPG) activity than dividing procyclic promastigotes. The fine specificity of promastigote eMPT(LPG) activity was determined using 24 synthetic analogues of Galbeta1-4Manalpha1-P-decenyl. The three species gave similar results: the negative charge of the phosphodiester and the C-6 hydroxyl of the alphaMan residue are essential for substrate recognition, the latter most likely acting as a hydrogen bond acceptor. The C-6' hydroxyl of the betaGal residue is required for substrate recognition as well as for catalysis. The rate of Manalpha1-P transfer declines with increasing acceptor substrate chain length. The presence of a monosaccharide substituent at the C-3 position of the terminal betaGal residue abrogates Man-P transfer, showing that chain elongation must precede side chain modification during lipophosphoglycan biosynthesis. In contrast, substitution of the penultimate phosphosaccharide repeat does not abrogate transfer but is slightly stimulatory in L. mexicana and inhibitory in L. major.