Activity of Brazilian and Bulgarian propolis against different species of Leishmania

Extracts of propolis samples collected in Brazil and Bulgaria were assayed against four Leishmania species--Leishmania amazonensis, L. braziliensis, L. chagasi from the New World, and L. major from the Old World--associated to different clinical forms of leishmaniasis. The composition of the extracts has been previously characterized by high temperature high resolution gas chromatography coupled to mass spectrometry. Considering the chemical differences among the extracts and the behavior of the parasites, it was observed significant differences in the leishmanicidal activities with IC50/1 day values in the range of 2.8 to 229.3 microg/ml . An overall analysis showed that for all the species evaluated, Bulgarian extracts were more active than the ethanol Brazilian extract. As the assayed propolis extracts have their chemical composition determined it merits further investigation the effect of individual components or their combinations on each Leishmania species.     

Production and characterization of species-specific monoclonal antibodies against Leishmania donovani for immunodiagnosis

Sixteen species-specific monoclonal antibodies were produced against membranes of Leishmania donovani. These antibodies only reacted with determinants present on L. donovani. No cross-reactions were found with any other species of Leishmania or with membranes of Trypanosoma cruzi. An extensive analysis of the binding specificities of selected antibodies was carried out by using whole promastigote homogenates as antigen. Monoclonal antibodies D-1, D-2, D-3, and D-4 correctly identified all 44 L. donovani stocks from a cross-panel of 84 New and Old World Leishmania stocks. Antibodies D-1 and D-2 were also useful for species classification by immunofluorescence. No cross-reactions were observed with any other Leishmania species examined. Based on either Western blot and/or radioimmunoprecipitation analyses, five distinct groups of molecules associated with L. donovani-specific antigenic determinants were identified. These molecules range in m.w. from 18 to 84 kilodaltons. The antigenic molecules recognized by antibodies D-2, D-10, and D-13 are also recognized by antibodies present in sera from patients with visceral leishmaniasis (kala-azar). Kala-azar sera obtained from cases in both the Old and New World specifically compete with these monoclonal antibodies for the appropriate antigenic determinants in Western blot analysis. These monoclonal antibodies and/or the purified protein antigens may be useful in the development of a serologic assay for the clinical diagnosis of visceral leishmaniasis caused by L. donovani and in epidemiologic studies of leishmaniasis.     

Analysis of ribosomal DNA internal transcribed spacer sequences of the Leishmania donovani complex

To understand phylogenetic relationships of species and strains within the Leishmania donovani complex, we have analyzed the ribosomal DNA internal transcribed spacer (ITS) sequences of 27 Leishmania infantum, 2 Leishmania chagasi, 18 L. donovani and 5 Leishmania archibaldi strains of different zymodemes and geographical origin. Eight ITS sequence types were found. All detected sequence variation within ITS1 and ITS2 was based on 12 polymorphic microsatellites. The L. infantum strains from the Mediterranean region, China and L. chagasi from the New World formed a phylogenetic group well separated from the second main group including all strains from East Africa and India. Within the latter group three distinct phylogenetic subgroups could be differentiated: (1) L. donovani (Sudan/Ethiopia, China) + L. archibaldi (Sudan), (2) L. donovani (Sudan/Ethiopia) + L. infantum (Sudan) + L. archibaldi (Sudan/Ethiopia), and (3) L. donovani (Kenya, India). These groups are not consistent with previous species definitions based on isoenzyme analyses, e.g. L. infantum is polyphyletic and L. archibaldi is not supported as a distinct species. Two groups of Indian strains could be differentiated, one of which has an identical sequence type to the strains from Kenya. Three main lineages of strains can thus be differentiated in East Africa: two quite distantly related groups of strains from Sudan/Ethiopia, and a third group including all strains from Kenya, which is more closely related to part of the Indian strains than to any of the Sudanese/Ethiopian groups. The ITS sequence analysis presented here supports the need for revision of the taxonomy of the L. donovani complex.     

Anti-leishmanial and anti-trypanosomal activities of 1,4-dihydropyridines: In vitro evaluation and structure-activity relationship study

Leishmaniasis and Chagas' disease constitute a relevant health and socio-economic problem in Latin America, Africa, and Asia. The therapeutic interventions rely on inefficient and highly toxic drugs with systemic side effects in patients. Considering the multiple biological activities of the calcium channel blockers and the high versatility of 1,4-dihydropyridines, eight clinically used 1,4-dihydropyridines (azelnidipine, amlodipine, cilnidipine, lercanidipine, nicardipine, nifedipine, nimodipine and nitrendipine) were in vitro tested against Leishmania and Trypanosoma cruzi parasites, and their cytotoxicity was tested against mammalian cells. In addition, a QSAR study was performed in order to delineate further structural requirements for the anti-protozoan activity and to predict the biological potency of 1,4-dihydropyridines. The tested compounds were effective against Leishmania (L.) amazonensis, Leishmania (V.)braziliensis, Leishmania (L.) chagasi, and Leishmania (L.) major promastigotes, L. (L.) chagasi intracellular amastigotes and T. cruzi trypomastigotes with 50% inhibitory concentration (IC(50)) values in the range of 2.6-181μM. The QSAR provided useful information about the structural features of the anti-protozoan activities, including diphenylpropyl and diphenylmethylazetidin groups at position 4 of the 1,4-dihydropyridine ring, allowing the prediction of two novel potential anti-protozoan analogs.     

Experimental infection of canine peritoneal macrophages with visceral and dermotropic Leishmania strains.

A study was carried out using macrophages cultured from the peritoneal exudate of dogs infected in vitro with three species of Leishmania: L. (L.) chagasi, L. (Viannia) braziliensis and L. (L.) amazonensis with the aim of investigating the growth kinetics and infectivity of these species in the host cell. Results were expressed as the percentage of macrophages infected measured at 24 hr intervals over six days in RPMI - 1640 culture medium at a temperature of 34-35 degrees C. The findings open the possibility of using canine peritoneal cells as a model for the screening of leishmanicide drugs and to study the pathogenesis of these species.     

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.     

In vitro antileishmanial and antitrypanosomal activities of flavanones from Baccharis retusa DC. (Asteraceae)

Leishmaniasis and Chagas' are parasitic protozoan diseases that affect the poorest population in the world, causing a high mortality and morbidity. As a result of highly toxic and long-term treatments, novel, safe and more efficacious drugs are essential. In this work, the CH(2)Cl(2) phase from MeOH extract from the leaves of Baccharis retusa DC. (Asteraceae) was fractioned to afford two flavonoids: naringenin (1) and sakuranetin (2). These compounds were in vitro tested against Leishmania spp. promastigotes and amastigotes and Trypanosoma cruzi trypomastigotes and amastigotes. Compound 2 presented activity against Leishmania (L.) amazonensis, Leishmania (V.) braziliensis, Leishmania (L.) major, and Leishmania (L.) chagasi with IC(50) values in the range between 43 and 52 μg/mL and against T. cruzi trypomastigotes (IC(50)=20.17 μg/mL). Despite of the chemical similarity, compound 1 did not show antiparasitic activity. Additionally, compound 2 was subjected to a methylation procedure to give sakuranetin-4'-methyl ether (3), which resulted in an inactive compound against both Leishmania spp. and T. cruzi. The obtained results indicated that the presence of one hydroxyl group at C-4' associated to one methoxyl group at C-7 is important to the antiparasitic activity. Further drug design studies aiming derivatives could be a promising tool for the development of new therapeutic agents for Leishmaniasis and Chagas' disease.     

Inhibition of macrophage invasion by monoclonal antibodies specific to Leishmania (Viannia) braziliensis promastigotes and characterisation of their antigens

Monoclonal antibodies that specifically recognise Leishmania (Viannia) braziliensis promastigotes were produced and termed SST-2, SST-3 and SST-4. SST-2 recognises a conformational epitope present in a 24-28 kDa doublet and in a 72 kDa component, as verified by Western blotting. Indirect immunofluorescence showed that the antigen recognised by SST-2 is distributed homogeneously on the parasite surface. SST-3 recognises a flagellar glycoprotein of approximately 180 kDa. The reactivity of this mAb was abolished by sodium m-periodate treatment, indicating that SST-3 reacts with a carbohydrate epitope of the 180 kDa antigen. SST-4 recognises a conformational epitope of a 98 kDa antigen. SST-2, SST-3 and SST-4 were specific to L. (V.) braziliensis promastigote forms. Indirect immunofluorescence did not show reactivity of SST-2 or SST-3 with amastigotes of L. (V.) braziliensis, or with promastigotes of Leishmania (Viannia) panamensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) naiffi, Leishmania (Viannia) lainsoni, Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) major, or Leishmania (Leishmania) chagasi. We also evaluated the involvement of SST-2, SST-3 and SST-4 antigens in parasite-macrophage interaction. Fab fragments of SST-3 and SST-4 significantly inhibited the infectivity of L. (V.) braziliensis promastigotes to mouse peritoneal macrophages.     

Inhibition by Dications of in vitro growth of Leishmania major and Leishmania tropica: causative agents of old world cutaneous leishmaniasis

Old World cutaneous leishmaniasis is caused by infection with Leishmania major and Leishmania tropica. Pentamidine and related dications exhibit broad spectrum antiprotozoal activity. Based on the previously reported efficacy of these compounds against related organisms, 18 structural analogs of pentamidine were evaluated for in vitro antileishmanial activity, using pentamidine as the standard reference drug for comparison. Furan analogs and reversed amidine compounds were examined for activity against L. major and L. tropica promastigotes. The most active compounds against both Leishmania species were in the reversed amidine series. DB745 and DB746 exhibited the highest activity against L. major and DB745 was the most active compound against L. tropica. Both of these compounds exhibited 50% inhibitory concentrations (IC50) below 1 nM for L. major. Ten reversed amidines were also tested for their ability to inhibit growth in an axenic amastigote model. Nine of 10 reversed amidine analogs were active at concentrations below 1 nM. These results justify further study of dicationic compounds as potential new agents for treating cutaneous leishmaniasis.     

Reproductive strategies and population structure in Leishmania: substantial amount of sex in Leishmania Viannia guyanensis

Leishmania species of the subgenus Viannia and especially Leishmania Viannia guyanensis are responsible for a large proportion of New World leishmaniasis cases. Since a recent publication on Leishmania Viannia braziliensis, the debate on the mode of reproduction of Leishmania parasites has been reopened. A predominant endogamic reproductive mode (mating with relatives), together with strong Wahlund effects (sampling of strains from heterogeneous subpopulations), was indeed evidenced. To determine whether this hypothesis can be generalized to other Leishmania Viannia species, we performed a population genetic study on 153 human strains of L. (V.) guyanensis from French Guiana based on 12 microsatellite loci. The results revealed important homozygosity and very modest linkage disequilibrium, which is in agreement with a high level of sexual recombination and substantial endogamy. These results also revealed a significant isolation by distance with relatively small neighbourhoods and hence substantial viscosity of Leishmania populations in French Guiana. These results are of epidemiological relevance and suggest a major role for natural hosts and/or vectors in parasite strain diffusion across the country as compared to human hosts.     

Identification of New World Leishmania species from Peru by biochemical techniques and multiplex PCR assay

We have characterized diverse strains or species of Leishmania isolated in humans that are currently circulating throughout Peru, by means of isoenzymatic characterization, kDNA analysis by restriction enzymes, and multiplex PCR assay. The cluster analysis gave five groups. Cluster 1 includes L. (L.) donovani together with the isolates LP4 and LP7, forming the donovani complex. Thus, this complex corresponds to the New World visceral form, L. (L.) chagasi. Cluster 2 is formed by the isolates LP1-LP3, LP6, LP10, LP9, and LP11, phylogenetically intermediate between Cluster 1 and Cluster 3, or they can be treated as hybrids. Cluster 3 is divided into two subgroups: one formed by L. (V.) peruviana, together with the isolates LP14 and LP5, and the second one formed by L. (V.) brazilensis and the isolate LP8. These two subgroups form part of the brazilensis complex. The three strains of L. (L.) infantum [L. (L.) infantum I and II and la LSI] make up Cluster 4. In Cluster 5, we include the three Mexican strains (LM1-LM3) forming one subgroup while we would place L. (L.) amazonensis in another subgroup. These two subgroups would comprise the complex mexicana.     

Antileishmanial activity of polycyclic derivatives

33 polycyclic derivatives have been studied and tested on Leishmania donovani and L. major promastigotes. Their antileishmanial activity was assessed in vitro and an assay of their cytotoxicity was realized on human myelomonocytic cell line. The reference molecules used in the assays were amphotericin B and pentamidine. Among the compounds tested, 29 possess an antileishmanial activity; 25 of those were more active against L. donovani than amphotericin B, and nine were as effective as amphotericin B against L. major. Many synthesized derivatives were more active against L. donovani than against L. major. The cytotoxicity studies have shown that among the thirty-three derivatives tested, 12 molecules have an IC50 towards THP-1 cells about equal than that reference drugs, the 21 other derivatives are much less toxic. A 3D QSAR study was undertaken and has permitted to predict activity against L. donovani and L. major and to highlight critical area to optimize activity against the two species.     

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.     

Synthesis and anti-leishmanial activity of 1-aryl-β-carboline derivatives against Leishmania donovani

β-carbolines from various natural and synthetic sources have been known to show diverse biological activities. As a part of our current ongoing project to search for potent natural product-derived anti-leishmanial compounds, we have synthesized a series of substituted 1-aryl-β-carboline derivatives. A total of 22 compounds were synthesized and tested in vitro against Leishmania donovani, out of which 6 compounds (4, 5, 10, 11, 19 and 22) showed notably more activity than the standard miltefosine (IC(50) 12.07±0.82 μM), with compound 4 being the most potent (IC(50) 2.16±0.26 μM).     

Direct Comparison of the Efficacy and Safety of Oral Treatments with Oleylphosphocholine (OlPC) and Miltefosine in a Mouse Model of L. major Cutaneous Leishmaniasis

Background

Cutaneous leishmaniasis (CL) represents a range of skin diseases caused by infection with Leishmania parasites and associated with tissue inflammation and skin ulceration. CL is clinically widespread in both the Old and New World but lacks treatments that are well tolerated, effective and inexpensive. Oleylphosphocholine (OlPC) is a new orally bioavailable drug of the alkylphosphocholine family with potent antileishmanial activity against a broad range of Leishmania species/strains.

Methodology/principal findings

The potential of OlPC against Old World CL was evaluated in a mouse model of Leishmania (L.) major infection in BALB/c mice. Initial dose-response experiments showed that an oral daily dose of 40 mg/kg of OlPC was needed to impact time to cure and lesion sizes. This dose was then used to directly compare the efficacy of OlPC to the efficacy of the antileishmanial drugs miltefosine (40 mg/kg/day), fluconazole (160 mg/kg/day) and amphotericin B (25 mg/kg/day). OlPC, miltefosine and fluconazole were given orally for 21 days while amphotericin B was administered intraperitoneally for 10 days. Ulcer sizes and animal weights were followed up on a weekly basis and parasitemia was determined by means of a real-time in vivo imaging system which detects luminescence emitted from luciferase-expressing infecting L. major parasites. Amphotericin B and OlPC showed excellent efficacy against L. major lesions in terms of reduction of parasitic loads and by inducing complete healing of established lesions. In contrast, treatment with miltefosine did not significantly affect parasitemia and lesion sizes, while fluconazole was completely ineffective at the dose regimen tested.

Conclusions/Significance

Given the data showing the outstanding efficacy and tolerability of OlPC, our results suggest that OlPC is a promising new drug candidate to improve and simplify current clinical management of L. major CL.     

Brazilian flora extracts as source of novel antileishmanial and antifungal compounds

Natural products have long been providing important drug leads for infectious diseases. Leishmaniasis is a protozoan parasitic disease found mainly in developing countries, and it has toxic therapies with few alternatives. Fungal infections have been the main cause of death in immunocompromised patients and new drugs are urgently needed. In this work, a total of 16 plant species belonging to 11 families, selected on an ethnopharmacological basis, were analyzed in vitro against Leishmania (L.) chagasi, Leishmania (L.) amazonensis, Candida krusei, and C. parapsilosis. Of these plant species, seven showed antifungal activity against C. krusei, five showed antileishmanial activity against L. chagasi and four against L. amazonensis, among them species of genus Plectranthus. Our findings confirm the traditional therapeutic use of these plants in the treatment of infectious and inflammatory disorders and also offer insights into the isolation of active and novel drug prototypes, especially those used against neglected diseases as Leishmaniasis.     

Effect of HIV protease inhibitors on New World Leishmania

The incidence of HIV/Leishmania co-infection decreases after antiretroviral drug therapy; therefore, the in vitro and in vivo activity of three antiretroviral drugs against Leishmania (Viannia) braziliensis and L. (L.) amazonensis was evaluated. Different concentrations of indinavir (IDV), atazanavir (ATV), and ritonavir (RTV) were added to promastigote cultures, and the 50% inhibitory concentration (IC50) was determined. IDV and RTV were also evaluated against intracellular amastigotes, and the Infection Index determined. BALB/c mice, infected with L. (L.) amazonensis in the left footpad, were treated orally with IDV and RTV for 30days, and monitored by measuring the footpad thickness and parasite load of regional lymph nodes and spleen. For promastigotes, IDV exhibited an IC50 value of 100μM against L.(L.) amazonensis. The RTV IC50 for L. (L.) amazonensis and L. (V.) braziliensis were 40 and 2.3μM, respectively, and the ATV IC50 for L. (V.) braziliensis was 266μM. For intracellular amastigotes, IDV (25, 50, and 100μM) significantly decreased the Infection Index of L. (L.) amazonensis (56.8%, 47.9%, and 65.0%) and L. (V.) braziliensis (37.8%, 48.7%, and 43.2%). RTV (12.5, 25, and 50μM) decreased the infection index of L. (L.) amazonensis by 26.3%, 42.4%, and 44.0%, and that of L. (V.) braziliensis by 27.6%, 37.3%, and 39.2%. Antiretroviral-treated mice had a significant reduction in footpad thickness after the third week of IDV and after the fifth week of RTV treatment. However, there was no reduction in parasite load. These results suggest that IDV and RTV have anti-Leishmania activity, but only in higher concentrations.     

Monoclonal antibodies that react with Leishmania (Viannia) naiffi

Monoclonal antibodies were raised against Leishmania (Viannia) naiffi, which recently has been characterized as a new species. BALB/c mice were immunized with membrane-enriched fractions of a mixture of L. (V.) naiffi isolates. Subsequent fusion of immunized splenocytes with NS-1 myeloma cells resulted in the production of 5 Mabs (N1-N5). Screening by ELISA and indirect immunofluorescence against an extensive cross-panel of Leishmania strains revealed that N3 was species-specific and could thus be used to identify L. (V.) naiffi. N2 and N5 reacted only with strains of L. (V.) naiffi and Leishmania (Viannia) braziliensis, and therefore could be used in conjunction with N3 to identify L. (V.) braziliensis parasites. These species-specific Mabs will therefore be useful additions to the panel of antibodies already available for the identification of Leishmania species. N1 and N4 were found to recognize a small, glycosylated molecule present on all L. (V.) naiffi and L. (V.) braziliensis isolates that is related to the GIPL family of membrane glycophospholipids previously described for Leishmania (Leishmania) major.     

Protective immunity against the protozoan Leishmania chagasi is induced by subclinical cutaneous infection with virulent but not avirulent organisms

Protective immunity against Leishmania major is provided by s.c. immunization with a low dose of L. major promastigotes or with dihydrofolate-thymidylate synthase gene locus (DHFR-TS) gene knockout L. major organisms. Whether these vaccine strategies will protect against infection with other Leishmania species that elicit distinct immune responses and clinical syndromes is not known. Therefore, we investigated protective immunity to Leishmania chagasi, a cause of visceral leishmaniasis. In contrast to L. major, a high dose s.c. inoculum of L. chagasi promastigotes was required to elicit protective immunity. Splenocytes from mice immunized with a high dose produced significantly greater amounts of IFN-gamma and lower TGF-beta than mice immunized with a low dose of promastigotes. The development of protective immunity did not require the presence of NK cells. Protection was not afforded by s.c. immunization with either attenuated L. chagasi or with L. major promastigotes, and s.c. L. chagasi did not protect against infection with L. major. Subcutaneous immunization with DHFR-TS gene knockouts derived from L. chagasi, L. donovani, or L. major did not protect against L. chagasi infection. We conclude that s.c. inoculation of high doses of live L. chagasi causes a subclinical infection that elicits protective immune responses in susceptible mice. However, L. chagasi that have been attenuated either by long-term passage or during the raising of recombinant gene knockout organisms do not elicit protective immunity, either because they fail to establish a subclinical infection or because they no longer express critical antigenic epitopes.