Leishmania infantum: stage-specific activity of pentavalent antimony related with the assay conditions

The determination of the intrinsic sensitivity of Leishmania strains to pentavalent antimonials in clinical trials, before treatment is begun, is essential in order to avoid failures and to allow alternative drugs to be chosen. A comparative study of SbV activity on promastigotes, axenic amastigote-like cells, and intracellular amastigotes of Leishmania infantum, when administered in the form of meglumine antimoniate and free, in hydrochloric solution, was performed. Results indicate that the conditions under which the promastigotes were cultured affect the IC(50) obtained, although results were homogeneous when the products were assayed on axenic-like and intracellular amastigotes. The IC(50) obtained for SbV in the form of meglumine antimoniate or in hydrochloric solution on promastigotes cultured in Schneider's medium depends on the growth rate of the culture and therefore could be regulated by modifying the fetal calf serum concentration in the medium. The pH of the culture medium strongly affected the activity of meglumine antimoniate but not that of the SbV hydrochloric solution on promastigotes cultured in Schneider's medium. This influence of pH was observed to a much lesser extent when promastigotes were cultured on M199 or RPMI media. In homogeneous culture conditions, which included the regulation of the promastigote growth rate through the heat-inactivated fetal calf serum concentration in the medium and the dilution of the meglumine antimoniate with Schneider's medium at pH 6.5, the activity of SbV, free or in the form of meglumine antimoniate, was the same in promastigotes, intracellular amastigotes, and axenic amastigote-like cells.     

Sensitivity to Glucantime of Leishmania viannia isolated from patients prior to treatment

In vitro sensitivity to pentavalent antimony (SbV) as meglumine antimoniate (Glucantime) of Leishmania of the Viannia subgenus isolated prior to treatment from patients with uncomplicated cutaneous leishmaniasis was evaluated for intracellular amastigotes in the U-937 human monocytic cell line and log phase promastigotes. The 50% effective dose (ED50) of pharmaceutical and additive-free formulations of Glucantime were determined based on the kinetics of the response of Leishmania Viannia to SbV in vitro. ED50 to SbV was inversely related to time of exposure to drug. The potency of the additive-free formulation of Glucantime was significantly greater than that of the pharmaceutical formulation, irrespective of the parasite form. In vitro sensitivity to SbV ranged from < 5.3 micrograms/ml to > 170.0 micrograms/ml. Under the conditions used, 11 (39%) of 28 strains were sensitive to clinically achievable serum concentrations of SbV. No correlation was observed between the total amount of SbV required for healing of lesions and the in vitro response to the pharmaceutical formulation of Glucantime. In contrast, a significant correlation (P = 0.001) was observed between clinical response and the in vitro sensitivity of promastigotes to the additive-free formulation of Glucantime. The greater potency of the additive-free formulation of Glucantime, the correlation of in vitro sensitivity of promastigotes to this formulation and the clinical response to treatment, and the effect of time of exposure to SbV demonstrate the importance of assay conditions on the outcome and interpretation of in vitro evaluation of drug sensitivity.     

Leishmania infantum infection rates in Phlebotomus perniciosus fed on naturally infected dogs under antimonial treatment

Dogs naturally infected with Leishmania infantum Nicolle were treated with three courses of meglumine antimoniate. Changes were observed in the dogs' clinical signs, antibody titres and in infection rates of Phlebotomus perniciosus Newstead fed on the dogs. A large reduction in the sandfly infection rate was observed for 4-5 months after the first treatment. The use of antimonial drugs is advocated for the control of canine leishmaniasis and to reduce risks of L.infantum transmission.     

Responses of Leishmania (Viannia) braziliensis cutaneous infection to N-methylglucamine antimoniate in the rhesus monkey (Macaca mulatta) model

The antileishmanial efficacy of the reference drug N-methylglucamine antimoniate (Glucantime) was evaluated in groups of rhesus monkeys with acute and chronic Leishmania (Viannia) braziliensis cutaneous infection. The therapeutic responses in experimental animals to either a low dose (5 mg/kg body wt/day for 28 days) or a routine dose (20 mg/kg/day for 28 days) of pentavalent antimony were similar to those reported in the human disease. Primates were cured of their lesions after treatment, but with cryptic parasitism and/or relapse. The rhesus model of L. (V.) braziliensis cutaneous leishmaniasis therefore provides an additional resource for preclinical trials with newer drugs.     

Leishmania mexicana: enzyme activities of amastigotes and promastigotes and their inhibition by antimonials and arsenicals

A major difference between the metabolism of Leishmania species amastigotes and cultured promastigotes was found in the area of CO2 fixation and phosphoenolpyruvate metabolism. Malate dehydrogenase (EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) were at much higher activities in amastigotes than promastigotes of both L. m. mexicana and L. donovani, whereas the reverse was true of pyruvate kinase (EC 2.7.1.40). Pyruvate carboxylase (EC 6.4.1.1) and malic enzyme (carboxylating) (EC 1.1.1.40) could not be detected in L. m. mexicana amastigotes. Promastigotes of L. m. mexicana had a high NAD-linked glutamate dehydrogenase activity in comparison to amastigotes, whereas NADP-linked glutamate dehydrogenase activity was detected only in amastigotes. Leishmania m. mexicana culture promastigotes were killed in vitro by the trivalent antimonial Triostam (LD50, 20 micrograms/ml) and the trivalent arsenical melarsen oxide (LD50, 20 micrograms/ml), but they were unaffected by Pentostam. Neither antimonial drug significantly inhibited leishmanial hexokinase (EC 2.7.1.2), phosphofructokinase (EC 2.7.1.11), pyruvate kinase, malate dehydrogenase or phosphoenolpyruvate carboxykinase, whereas melarsen oxide was a potent inhibitor of all the enzymes tested except phosphoenolpyruvate carboxykinase.     

Inhibition of Leishmania donovani promastigote DNA topoisomerase I and human monocyte DNA topoisomerases I and II by antimonial drugs and classical antitopoisomerase agents

We have compared the inhibitor sensitivities of DNA topoisomerase I (TOPI) from Leishmania donovani promastigotes and TOPs I and II of human monocytes using pentavalent and trivalent antimonials (SbV, SbIII) and classical TOP inhibitors. Bis-benzimidazoles (Hoechst-33258 and -33342) were potent inhibitors of both parasite and human TOPI, but Hoechst-33342 was markedly less cytotoxic to promastigotes than to monocytes in vitro. Leishmania donovani was also considerably less sensitive than monocytes to camptothecin, both at enzyme and cellular levels. Sodium stibogluconate (SSG) was the only antimonial to inhibit TOPI, exhibiting a significant (P < 0.05) 3-fold greater potency against the L. donovani enzyme but showed low cytotoxicities against intact promastigotes. The SbV meglumine antimoniate failed to inhibit TOPI and showed negligible cytotoxicities, whereas SbIII drugs were lethal to parasites and monocytes yet poor inhibitors of TOPI. Monocyte TOPII was inhibited by bis-benzimidazoles and insensitive to antimonials and camptothecin. The disparity between the high leishmanicidal activity and low anti-TOPI potency of SbIII indicates that in vivo targeting of L. donovani TOPI by the reductive pathway of antimonial activation is improbable. Nevertheless, the potent direct inhibition of TOPI by SSG and the differential interactions of camptothecin with L. donovani and human TOPI support the possibility of developing parasite-specific derivatives.     

Development of Luciferase Expressing Leishmania donovani Axenic Amastigotes as Primary Model for In Vitro Screening of Antileishmanial Compounds

The development of new therapeutic leads against leishmaniasis relies primarily on screening of a large number of compounds on multiplication of clinically irrelevant transgenic promastigotes. The advent of the successful in vitro culture of axenic amastigotes allows the development of transgenic axenic amastigotes as a primary screen which can test compounds in a high throughput mode like promastigotes, still representative of the clinically relevant mammalian amastigotes stage. The present study reports the development of luciferase-tagged axenic amastigotes of Leishmania donovani, the causative agent of Indian Kala-azar, for in vitro drug screening. Luciferase expressing promastigotes were transformed to axenic amastigotes at a low pH and high temperature without the loss of luciferase expression. As compared to transgenic promastigotes, the luciferase expressing axenic amastigotes exhibited more sensitivity to antileishmanial drugs, particularly to pentavalent antimony (~2.8-fold) and also to the test compounds. Hence, the developed luciferase expressing axenic amastigotes make an ideal choice for high throughput drug screening for antileishmanial compounds.     

A proteogenomic approach to map the proteome of an unsequenced pathogen - Leishmania donovani

Visceral leishmaniasis or kala azar is the most severe form of leishmaniasis and is caused by the protozoan parasite Leishmania donovani. There is no published report on L. donovani genome sequence available till date, although the genome sequences of three related Leishmania species are already available. Thus, we took a proteogenomic approach to identify proteins from two different life stages of L. donovani. From our analysis of the promastigote (insect) and amastigote (human) stages of L. donovani, we identified a total of 22,322 unique peptides from a homology-based search against proteins from three Leishmania species. These peptides were assigned to 3711 proteins in L. infantum, 3287 proteins in L. major, and 2433 proteins in L. braziliensis. Of the 3711 L. donovani proteins that were identified, the expression of 1387 proteins was detectable in both life stages of the parasite, while 901 and 1423 proteins were identified only in promastigotes and amastigotes life stages, respectively. In addition, we also identified 13 N-terminally and one C-terminally extended proteins based on the proteomic data search against the six-frame translated genome of the three related Leishmania species. Here, we report results from proteomic profiling of L. donovani, an organism with an unsequenced genome.     

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.     

The surface membrane of Leishmania. I. The effects of lectins on different stages of Leishmania braziliensis.

The infective stages of Leishmania braziliensis, amastigotes and promastigotes subcultured a limited number of times, were agglutinated by Ricinus communis agglutinin and Concanavalin A. These results suggest that terminal ligands similar or identical with alpha-D mannose, alpha-D glucose (specific receptors for Con A), and alpha-D galactose (specific receptor for RCA) are present in the surface membrane of L. braziliensis. Noninfective promastigotes from the same stock, but subcultured approximately 500 times, were not agglutinated by RCA suggesting either the absence of the alpha-D galactose groups in the surface membrane or their presence in a very reduced number. Agglutination with soybean agglutinin, wheat germ agglutinin, or phytohemagglutinin P was not observed in any of the L. braziliensis forms tested. The difference in polysaccharide residues on the surface membrane of L. braziliensis may be related to the different pathogenic properties of the cell.     

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.     

Infection-induced respiratory burst in BALB/c macrophages kills Leishmania guyanensis amastigotes through apoptosis: possible involvement in resistance to cutaneous leishmaniasis

The immune mechanisms that underlie resistance and susceptibility to leishmaniasis are not completely understood for all species of Leishmania. It is becoming clear that the immune response, the parasite elimination by the host and, as a result, the outcome of the disease depend both on the host and on the species of the infecting Leishmania. Here, we analyzed the outcome of the infection of BALB/c mice with L. guyanensis in vivo and in vitro. We showed that BALB/c mice, which are a prototype of susceptible host for most species of Leishmania, dying from these infections, develop insignificant or no cutaneous lesions and eliminate the parasite when infected with promastigotes of L. guyanensis. In vitro, we found that thioglycollate-elicited BALB/c peritoneal macrophages, which are unable to eliminate L. amazonensis without previous activation with cytokines or lipopolysaccharide, can kill L. guyanensis amastigotes. This is the first report showing that infection of peritoneal macrophages with stationary phase promastigotes efficiently triggers innate microbicidal mechanisms that are effective in eliminating the amastigotes, without exogenous activation. We demonstrated that L. guyanensis amastigotes die inside the macrophages through an apoptotic process that is independent of nitric oxide and is mediated by reactive oxygen intermediates generated in the host cell during infection. This innate killing mechanism of macrophages may account for the resistance of BALB/c mice to infection by L. guyanensis.     

Development of Leishmania (Viannia) panamensis lesions and relationship of numbers of amastigotes to lesion area on antimony-treated and untreated hamsters

Young adult (60-70-g) male golden hamsters (Mesocricetus auratus) each were injected intradermally at the dorsal base of the tail with 15 x 10(6) promastigotes of Leishmania (Viannia) panamensis (MHOM/PA/83/WR539), and progression and regression of subsequent lesions were evaluated for up to 17 wk postinfection (PI) as to area, weight, and number of amastigotes within lesions in untreated hamsters and in hamsters treated with meglumine antimoniate (Glucantime). In untreated hamsters total area of lesion, weight, and numbers of amastigotes generally increased rapidly and concomitantly up to 3-4 wk PI. Amastigote numbers tended to decrease from 4 to 11 wk PI and subsequently the numbers of amastigotes within the lesions decreased rapidly, whereas relatively little change occurred in the area and weight of the lesions. Meglumine antimoniate treatment of cutaneous hamster lesions resulted in marked concomitant decrease in size of the lesions and numbers of amastigotes within the lesions examined 1 wk after treatment. Measurement of the area of cutaneous leishmanial lesions thus would appear to be a valid method of evaluating the efficacy of promising compounds against L. panamensis in hamsters when measurements are taken 3-5 wk after experimental infection and reflects the number of amastigotes present in the lesion.     

A marmoset model for some New World dermatotropic leishmaniases

Callithrix penicillata is a good model for dermatotropic strains of some New World Leishmania species (L. mexicana mexicana, L. m. amazonensis, and L. braziliensis guyanensis). Both amastigotes and promastigotes readily infect these primates, with a relative short period of incubation. The developed lesions parallel those found in man, and despite the self healing presented by some marmosets, cell and serum immunological responses are presented. Skin alterations are confined to the site of inoculum, and visceralization is very rarely found. A good response to standard antimonial treatment makes the model useful for preclinical experiments with promising compounds.     

Leishmania (Viannia) braziliensis metacyclic promastigotes purified using Bauhinia purpurea lectin are complement resistant and highly infective for macrophages in vitro and hamsters in vivo

In this study we characterised metacyclogenesis in axenic culture of Leishmania (Viannia) braziliensis, the causative agent of mucocutaneous leishmaniasis in the New World. Metacyclogenesis of other species of Leishmania has been shown by morphological changes as well as molecular modifications in the lipophosphoglycan, the major cell surface glycoconjugate of the promastigotes. In order to obtain metacyclic forms of L. braziliensis we tested a panel of different lectins. Our results showed that Bauhinia purpurea lectin facilitated the purification of metacyclic promastigotes from stationary-phase culture by negative selection. The B. purpurea non-agglutinated promastigotes had a slender short cell body and long flagella, typical of metacyclic morphology. The ultrastructural analysis showed that B. purpurea non-agglutinated promastigotes have a dense and thicker glycocalyx. They are resistant to complement lysis, and highly infective for macrophage in vitro and hamsters in vivo. Contrary to procyclic promastigotes, B. purpurea non-agglutinated forms were poorly recognised by sand fly gut epithelial cells. These results suggest that the B. purpurea non-agglutinated promastigotes are the metacyclic forms of L. braziliensis.     

In vitro evaluation of new terpenoid derivatives against Leishmania infantum and Leishmania braziliensis

The activity of five (1-5) abietane phenol derivatives against Leishmania infantum and Leishmania braziliensis was studied using promastigotes and axenic and intracellular amastigotes. Infectivity and cytotoxicity tests were performed with J774.2 macrophage cells using Glucantime as a reference drug. The mechanisms of action were analysed by performing metabolite excretion and transmission electron microscopy ultrastructural studies. Compounds 1-5 were more active and less toxic than Glucantime. The infection rates and mean number of parasites per cell observed in amastigote experiments showed that derivatives 2, 4 and 5 were the most effective against both L. infantum and L. braziliensis. The ultrastructural changes observed in the treated promastigote forms confirmed that the greatest cell damage was caused by the most active compound (4). Only compound 5 caused changes in the nature and amounts of catabolites excreted by the parasites, as measured by 1H nuclear magnetic resonance spectroscopy. All of the assayed compounds were active against the two Leishmania species in vitro and were less toxic in mammalian cells than the reference drug.     

Fusion between Leishmania amazonensis and Leishmania major parasitophorous vacuoles: live imaging of coinfected macrophages

Protozoan parasites of the genus Leishmania alternate between flagellated, elongated extracellular promastigotes found in insect vectors, and round-shaped amastigotes enclosed in phagolysosome-like Parasitophorous Vacuoles (PVs) of infected mammalian host cells. Leishmania amazonensis amastigotes occupy large PVs which may contain many parasites; in contrast, single amastigotes of Leishmania major lodge in small, tight PVs, which undergo fission as parasites divide. To determine if PVs of these Leishmania species can fuse with each other, mouse macrophages in culture were infected with non-fluorescent L. amazonensis amastigotes and, 48 h later, superinfected with fluorescent L. major amastigotes or promastigotes. Fusion was investigated by time-lapse image acquisition of living cells and inferred from the colocalization of parasites of the two species in the same PVs. Survival, multiplication and differentiation of parasites that did or did not share the same vacuoles were also investigated. Fusion of PVs containing L. amazonensis and L. major amastigotes was not found. However, PVs containing L. major promastigotes did fuse with pre-established L. amazonensis PVs. In these chimeric vacuoles, L. major promastigotes remained motile and multiplied, but did not differentiate into amastigotes. In contrast, in doubly infected cells, within their own, unfused PVs metacyclic-enriched L. major promastigotes, but not log phase promastigotes--which were destroyed--differentiated into proliferating amastigotes. The results indicate that PVs, presumably customized by L. major amastigotes or promastigotes, differ in their ability to fuse with L. amazonensis PVs. Additionally, a species-specific PV was required for L. major destruction or differentiation--a requirement for which mechanisms remain unknown. The observations reported in this paper should be useful in further studies of the interactions between PVs to different species of Leishmania parasites, and of the mechanisms involved in the recognition and fusion of PVs.     

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.     

Cutaneous leishmaniasis infection in Balb/c mice using a Leishmania tropica strain isolated from Turkey.

Leishmania tropica, which is endemic in Turkey, is the causative agent of cutaneous leishmaniasis. Leishmania tropica promastigotes (2 x 10(7)) isolated from a patient with dermal leishmaniasis and reproduced in NNN medium were inoculated subcutaneously into the footpads of 10 Balb/c mice. Cutaneous leishmaniasis developed on the footpads of 4 mice approximately 45 days later. Leishmania tropica amastigotes were observed in smear slides and then cultivated in NNN medium. Balb/c mice are a suitable laboratory model for this isolate of L. tropica and thus a source of amastigotes for studies on the immunology, chemotherapy, and pathogenicity of cutaneous leishmaniasis.