Killing of Leishmania tropica amastigotes by factors in normal human serum

Amastigotes of Leishmania tropica and L. donovani were incubated with fresh or heat-inactivated normal human serum. Viability was estimated by amastigote conversion to promastigote forms and by the ability of serum-treated amastigotes to infect human monocytes. L. tropica, a parasite that causes local skin infection, was killed by fresh but not by heat-inactivated serum. The serum cytotoxic effect on L. tropica was inhibited by EDTA but not by Mg-EGTA. C2-deficient serum killed normally; C6-deficient serum was ineffective. These data indicate that L. tropica is killed by the complement membrane attack complex, in a sequence of reactions initiated by components of the alternate pathway. In contrast, L. donovani, a parasite that causes systemic visceral leishmaniasis, was 10-fold less susceptible to the cytotoxic effects of normal serum. Thus, a profound difference exists in the susceptibility of amastigotes of two species of Leishmania to a defense mechanism present in human serum. Serum complement factors may play an important role in limiting L. tropica to the skin. The resistance of L. donovani to such factors may be the primary reason for its ability to escape from the site of inoculation and cause catastrophic, disseminated disease.     

Characterisation of Bangladeshi Leishmania isolated from kala-azar patients by isoenzyme electrophoresis

To identify the prevalent Leishmania species in Bangladesh, a total of nine patients aged 4-35 years, were studied; six (66.7%) of them were below 20 years of age. All the patients were clinically diagnosed to have visceral leishmaniasis; their haematological profile was in accordance with leishmaniasis and all were improved after treatment with sodium stibogluconate. All the aspirated materials (eight bone marrows and one splenic aspirate) yielded growth of Leishmania parasite in NNN media; Leishman-Donovan bodies were found in seven (77.8%) of them in a Giemsa stained smear. Aldehyde test (AT) was positive in all the nine cases examined, whereas, complement fixation test (CFT) was positive in seven (77.8%) and indirect fluorescent antibody test (IFAT) in eight (88.9%) cases. In this study, five of the nine isolates from kala-azar patients were characterised by isoenzyme analysis comparing with five WHO reference strains viz., Leishmania (Leishmania) donovani (DD8), L.(L.) donovani (HU3), L.(L.) infantum (IPT-1), L.(L.) tropica (K-27) and L.(L.) major (5-ASKH) using cellulose acetate electrophoresis. By analysing 11 soluble isoenzymes it was found that all five WHO reference strains had distinct electrophoretic mobility of the isoenzymes studied. No interspecies difference was observed amongst the five isolates from kala-azar patients examined and their isoenzyme profiles were consistent with WHO reference strain of L.(L.) donovani (DD8) but different from L.(L.) donovani (HU3).     

Characterization of isolates and clones of leishmania by analysis of kinetoplast DNA

The genetic characterization of pathogenic isolates of Leishmania was attempted by analysis of the molecular properties of kinetoplast DNA (kDNA) minicircles. Unit minicircle size is not conserved during speciation of Leishmania since the minicircles of strains and clones of L t major are smaller (700 bp) than those found in certain strains of L mexicana ssp (820 bp), L donovani (850 bp) or L t tropica (900 bp). Schizodeme analysis of minicircles reveals a high degree of sequence divergence in kDNA of Leishmania with the degree of microheterogeneity varying between species. This sequence divergence allows the discrimination of species, strains, and clones of Leishmania into schizodemes. Southern blot hybridization experiments reveal that at high stringency overall minicircle sequence homology is conserved among clones and strains of one species (L t major) but not between different species. This property of minicircle DNA permits the use of kDNA probes as a species-specific diagnostic test for the identification of unknown Leishmania isolates. The properties of kDNA from an L t tropica strain LRC-L32 (a “recidiva” organism) are so diverged from those of L t major strains as to support the classification [22,23] of L t tropica and L t major as separate species of Leishmania rather than subspecies of L tropica.     

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.     

Screening and evaluation of antiparasitic and in vitro anticancer activities of Panamanian endophytic fungi

Many compounds produced by fungi have relevant pharmaceutical applications. The purpose of this study was to collect and isolate endophytic fungi from different regions of Panama and then to test their potential therapeutic activities against Leishmania donovani, Plasmodium falciparum, and Trypanosoma cruzi as well as their anticancer activities in MCF-7 cells. Of the 25 fungal isolates obtained, ten of them had good anti-parasitic potential, showing selective activity against L. donovani; four had significant anti-malarial activity; and three inhibited the growth of T. cruzi. Anticancer activity was demonstrated in four isolates. Of the active isolates, Edenia sp. strain F0755, Xylaria sp. strain F1220, Aspergillus sp. strain F1544, Mycoleptodiscus sp. strain F0194, Phomopsis sp. strain F1566, Pycnoporus sp. strain F0305, and Diaporthe sp. strain F1647 showed the most promise based on their selective bioactivity and lack of toxicity in the assays.     

Carbocyclic inosine as a potent anti-leishmanial agent: the metabolism and selective cytotoxic effects of carbocyclic inosine in promastigotes of Leishmania tropica and Leishmania donovani

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.     

Antileishmanial activity, cytotoxicity and QSAR analysis of synthetic dihydrobenzofuran lignans and related benzofurans

A series of synthetic dihydrobenzofuran lignans and related benzofurans were evaluated for their cytotoxicity in a screening panel consisting of various human tumour cell lines, and for their antiprotozoal activity against L. donovani (axenic amastigotes), chloroquine resistant Plasmodium falciparum (strain K1), Trypanosoma brucei rhodesiense and T. cruzi, and for cytotoxicity on L6 cells. No promising cytotoxicities against human tumour cell lines were observed for newly synthesised compounds, but the dimerisation product of some lipophylic esters of caffeic acid, such as compound 2g, showed a high activity against chloroquine-resistant P. falciparum (strain K1) (IC50 0.43 microg/mL) and L. donovani (axenic amastigotes) (IC50 0.12 microg/mL), which was confirmed in an infected macrophage assay (IC50 0.19 microg/mL). QSAR models for the cytotoxic and antileishmanial activity were generated using Quasar receptor surface modelling.     

Comparison of the protein kinase and acid phosphatase activities of five species of Leishmania

Promastigotes from log phase and stationary phase cultures of Leishmania donovani, L. braziliensis panamensis, L. tropica, L. major, and L. mexicana amazonensis were analyzed for their content of protein kinase and acid phosphatase activities. Cell surface, histone-specific protein kinase activity was 1.3- to 2.8-fold higher in stationary phase cells of all species except for L. tropica in which the activities of stationary and log phase cells were equal; L. mexicana amazonensis had the highest histone-specific protein kinase activity and L. donovani the lowest. When viable, motile promastigotes of all five species were incubated for 10 min with [gamma-32P]ATP and Mg2+ (10 mM) in the absence of exogenous histone acceptor; about one dozen proteins were phosphorylated in each case. Both log phase and stationary phase promastigotes of all five species extensively phosphorylated a 50-kDa protein that had the mobility of tubulin. Incubation of pure calf brain tubulin with [gamma-32P]ATP and purified L. donovani protein kinase resulted in extensive phosphorylation of the former. Highly infective metacyclic forms (PNA-) of L. major, isolated from a stationary culture using the peanut agglutinin (PNA), contained eight times more histone-specific protein kinase activity than noninfective log phase cells (PNA+). The PNA- and PNA+ forms of L. major both phosphorylated a 50-kDa protein when incubated with [gamma-32P]ATP and magnesium or manganese ions (10 mM); the 50-kDa protein was precipitated by anti-tubulin rabbit antibodies. Extracts of all five species contained large amounts of acid phosphatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The activity of diguanidino and 'reversed' diamidino 2,5-diarylfurans versus Trypanosoma cruzi and Leishmania donovani

The in vitro activity of 20 dicationic molecules containing either diguanidino or reversed amidine cationic groups were evaluated versus Trypanosoma cruzi and Leishmania donovani. The most active compounds were in the reversed amidine series and six exhibited IC(50) values of less than 1 micro mol versus T. cruzi and five gave similar values versus 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.     

Analyses of surface membrane carbohydrates in parasitic flagellates of the order kinetoplastida using lectins.

Crithidia fasciculata, Leishmania donovani, Leishmania major, Leishmania mexicana amazonensis, Leishmania tropica, Leishmania tarentolae, Trypanosoma sp. from Formosan bats (Tb), Trypanosoma lewisi, Trypanosoma musculi, and different strains of Trypanosoma cruzi (Tc) were cultivated at 27 degrees C in a liquid culture medium. Flagellates harvested from log phase culture were analyzed for their lectin agglutinating characteristics with concanavalin A (Con A), Peanut agglutinin, Ricinus communis agglutinin 120, soybean agglutinin (SBA), Ulex europeus agglutinin (UEA) and wheat germ agglutinin (WGA). Results indicated that all these flagellates might have D-galactose and methyl- alpha-D-manopyranoside on their surface. The presence of L-Fucose, which complexes specifically with UEA, could not be demonstrated on the surface of these flagellates. Results from quantitative comparison of surface molecules of Tb and the Tulahuen strain of Tc suggested that Tb may have more WGA-binding molecules while Tc may have more ConA-binding molecules. Pretreatment of the flagellates with 0.05% trypsin at 37 degrees C for 30 minutes caused some reduction of agglutination titers. Cell agglutination with lectins was completely inhibited or reversed in the presence of the specific lectin-binding monosaccharides.     

Human mixed infections of Leishmania spp. and Leishmania-Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme

Parasites belonging to Leishmania braziliensis, Leishmania donovani, Leishmania mexicana complexes and Trypanosoma cruzi (clones 20 and 39) were searched in blood, lesions and strains collected from 28 patients with active cutaneous leishmaniasis and one patient with visceral leishmaniasis. PCR-hybridization with specific probes of Leishmania complexes (L. braziliensis, L. donovani and L. mexicana) and T. cruzi clones was applied to the different DNA samples. Over 29 patients, 8 (27.6%) presented a mixed infection Leishmania complex species, 17 (58.6%) a mixed infection Leishmania-T. cruzi, and 4 (13.8%) a multi Leishmania-T. cruzi infection. Several patients were infected by the two Bolivian major clones 20 and 39 of T. cruzi (44.8%). The L. braziliensis complex was more frequently detected in lesions than in blood and a reverse result was observed for L. mexicana complex. The polymerase chain reaction-hybridization design offers new arguments supporting the idea of an underestimated rate of visceral leishmanisis in Bolivia. Parasites were isolated by culture from the blood of two patients and lesions of 10 patients. The UPGMA (unweighted pair-group method with arithmetic averages) dendrogram computed from Jaccard's distances obtained from 11 isoenzyme loci data confirmed the presence of the three Leishmania complexes and undoubtedly identified human infections by L. (V.) braziliensis, L. (L.) chagasi and L. (L.) mexicana species. Additional evidence of parasite mixtures was visualized through mixed isoenzyme profiles, L. (V.) braziliensis-L. (L.) mexicana and Leishmania spp.-T. cruzi.The epidemiological profile in the studied area appeared more complex than currently known. This is the first report of parasitological evidence of Bolivian patients with trypanosomatidae multi infections and consequences on the diseases' control and patient treatments are discussed.     

Utilization of leucine and acetate as carbon sources for sterol and fatty acid biosynthesis by Old and New World Leishmania species, Endotrypanum monterogeii and Trypanosoma cruzi.

The relative roles of acetate and leucine in the provision of a carbon source for fatty acid and sterol biosynthesis in several trypanosomatid species were investigated using 14C- and 13C-labelled acetate, glucose and leucine as substrates. Promastigotes of Leishmania species synthesized a large proportion of their sterol from leucine. L. major (LV39), L. amazonensis and L. mexicana were the most efficient utilizers of leucine, producing at least 70-77% of their sterol from leucine; L. braziliensis, L. donovani and L. tropica apparently produced less sterol from leucine (23-36%) and L. major (LV561), L. adleri and L. panamamensis were intermediate, utilizing leucine to provide 51-58% of their sterol. In all the cases the balance of the sterol produced was apparently synthesized from carbon arising from acetate. The related trypanosomatid Endotrypanum monterogeii also produced a large amount (77%) of its sterol from leucine rather than acetate. By contrast Trypanosoma cruzi elaborated only 8% of its sterol from leucine and used acetate far more effectively than the Leishmania species for sterol biosynthesis. The fatty acid moieties of the triacylglycerols and phospholipids were produced from acetate. Leucine was also incorporated into the fatty acids to varying extents in the different organisms showing that leucine can also be metabolized in trypanosomatids to generate acetyl-CoA.     

Hexose and Glycerol Absorption by Some Trypanosomatidae

SYNOPSIS. Culture forms of 9 species of Trypanosomatidae were studied. Of these, Crithidia fasciculata, Leishmania brasiliensis, L. donovani, L. tropica, Trypanosoma conorhini, T. cruzi and T. rangeli consumed significantly less glycerol than glucose, both when the 2 substrates were offered singly or simultaneously. On the other hand, T. gambiense and T. rhodesiense consumed as much glycerol as glucose when the 2 substrates were given separately. When both substrates were offered simultaneously, more glycerol than glucose was consumed, but the sum of glucose + glycerol carbon taken up approximated closely that taken from glucose alone. This held for species consuming only little glycerol, indicating that in all cases some mutual inhibition took place. Atttempts to adapt T. cruzi to glycerol consumption were unsuccessful. When T. gambiense and T. rhosiense were cultivated in the presence of glycerol, their normrapid glycerol consumption remained also unchanged but, curiothey consumed more glucose than before "adaptation." Iodoacetami and NaF strongly inhibited glucose and glycerol absorption of 7 gambiense ; phloridzin, deoxygalactose and deoxyglucose gave nonificant inhibition. KCN slightly stimulated glucose absorption, weakly inhibited glycerol absorption. Omission of sodium from medium was without effect, while lack of CO₂ markedly inhiglucose and glycerol uptake. Galactose was not a satisfactory sub strate for T. gambiense and T. rhodesiense , nor were deoxygalactos and deoxyglucose. These 3 hexoses were consumed in small amonly and did not allow maintenance of motility.     

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.     

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.     

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.     

Kinetoplastid flagellates: surface-reactive carbohydrates detected by fluorescein-conjugated lectins

Membrane-associated carbohydrate residues of 3 isolates of Leishmania derived from etiological agents of visceral leishmaniasis (VL), postkala-azar dermal leishmaniasis (PKDL), and cutaneous leishmaniasis (CL), as well as 2 other nonpathogenic insect gut kinetoplastid flagellates, Bodo sp. and Herpetomonas sp., were characterized with the aid of 8 fluorescein-conjugated lectins. Four lectins, concanavalin A, Dolichos biflorus, phytohemagglutinin P, Ricinus communis agglutinin, bound to all kinetoplastid flagellates at different concentrations. All Leishmania promastigotes showed reactions with Ulex agglutinin. Although these lectins were bound to all kinetoplastids, the site and intensity of binding was different. All skin-dwelling Leishmania parasites, viz., Leishmania donovani of PKDL and Leishmania tropica of CL showed unique selectivity toward peanut agglutinin (PNA), soybean agglutinin, and wheatgerm agglutinin (WGA). More interestingly, Herpetomonas showed positive fluorescence with PNA and WGA, whereas Bodo was negative. The results demonstrated that no lectin could distinguish between the pathogenic and nonpathogenic status of kinetoplastid flagellates. Moreover, the antigenic (carbohydrate) profiles of Herpetomonas corresponded more closely to those of L. tropica, whereas Bodo shared some common lectin receptors with L. donovani of VL.     

Haemoflagellates: commercially available liquid media for rapid cultivation.

The successful cultivation of a variety of haemoflagellates in three different liquid media is reported. These media include medium 199, Grace's insect tissue-culture medium and Schneider's drosophila medium, each in combination with 30% (v/v) foetal calf serum. These media were used to cultivate Old and New World species of visceral and cutaneous human Leishmania, as well as Leishmania species isolated from sandflies, rodents, and reptiles. Four strains of Trypanosoma cruzi, an isolate of T. R-angeli and and an isolate of T. lewisi have also been cultivated in these media. One or more of these media have been used to cultivate 121 strains of haemoflagellates, including at least 14 different species (11 Leishmania and 3 Trypanosoma) and many geographic isolates or strains. The Leishmania include L. braziliensis, L. peruviana, L. mexicana, L. tropica, L. donovani, L. chagasi, L. enriettii, L. hertigi, L. hoogstraali, L. adleri, and L. agamae. Using the Schneider's based medium, we have obtained primary isolates of both cutaneous and visceral Leishmania of man and of experimentally infected laboratory rodents and canines. Freeze-dried preparations of the Schneider's based medium that were reconsituted with distilled water after 24 months of storage at ambient temperature have proven to be suitable cultivation media. This feature makes the media valuable field tools. The various species of human Leishmania cultivated in these media have in our experience demonstrated no differences in growth rate, viability after liquid nitrogen preservation, or infectivity for laboratory animals and tissue-culture cells compared with promastigotes derived from blood-agar cultivation.     

Monoclonal antibodies specific for Leishmania tropica. I. Characterization of antigens associated with stage- and species-specific determinants

Four monoclonal antibodies (T-1 through T-4), which were produced to membrane-enriched preparations of Leishmania tropica major promastigotes, reacted specifically with the members of L. tropica complex. The antibodies T-1 and T-4 react exclusively with L. t. major and L. t. minor. The remaining two monoclonal antibodies bind, in addition, to L. t. aethiopica and weakly to L. mexicana amazonensis. No significant cross-reactivity was observed with L. donovani, L. braziliensis braziliensis, and Trypanosoma cruzi. Antibodies T-1, T-2, and T-4 were found to be specific for the promastigote stage (insect) of L. tropica. Antibody T-3 reacts with both the amastigote and promastigote stages of the parasite. All of the monoclonal antibodies react with cell surface components on intact promastigotes. The protein antigens containing the species-specific determinants recognized by each of the four antibodies were identified by radioimmunoprecipitation of solubilized 125I-labeled L. t. major promastigotes. A single 50 kilodalton protein is recognized by clone T-4. T-1 recognized two high m.w. proteins (100 and 200 kilodaltons). These two antigens plus an additional protein of lower m.w. (70 kilodaltons) are also immunoprecipitated by the antibodies T-2 and T-3, demonstrating that species-specific determinants are present on several different cell surface proteins of L. t. major.