Evaluation of in vitro antileishmanial efficacy of cyclosporin A and its non-immunosuppressive derivative, dihydrocyclosporin A.

BACKGROUND: New therapeutic drugs are urgently needed against visceral leishmaniasis because current drugs, such as pentavalent antimonials and miltefosine, produce severe side effects and development of resistance. Whether cyclosporine A (CsA) and its derivatives can be used as therapeutic drugs for visceral leishmaniasis has been controversial for many years. METHODS: In this study, we evaluated the efficacy of CsA and its derivative, dihydrocyclosporin A (DHCsA-d), against promastigotes and intracellular amastigotes of Leishmania donovani. Sodium stibogluconate (SSG) was used as a positive control. RESULTS: Our results showed that DHCsA-d was able to inhibit the proliferation of L. donovani promastigotes (IC50: 21.24 µM and 12.14 µM at 24 h and 48 h, respectively) and intracellular amastigotes (IC50: 5.23 µM and 4.84 µM at 24 and 48 h, respectively) in vitro, but CsA treatment increased the number of amastigotes in host cells. Both DHCsA-d and CsA caused several alterations in the morphology and ultrastructure of L. donovani, especially in the mitochondria. However, DHCsA-d showed high cytotoxicity towards cells of the mouse macrophage cell line RAW264.7, with CC50 values of 7.98 µM (24 h) and 6.65 µM (48 h). Moreover, DHCsA-d could increase IL-12, TNF-α and IFN-γ production and decrease the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. On the contrary, CsA decreased IL-12, TNF-α, and IFN-γ production and increased the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. The expression of L. donovani cyclophilin A (LdCyPA) in promastigotes and intracellular amastigotes and the expression of cyclophilin A (CyPA) in RAW 264.7 cells were found to be significantly downregulated in the CsA-treated group compared to those in the untreated group. However, no significant changes in LdCyPA and CyPA levels were found after DHCsA-d or SSG treatment. CONCLUSIONS: Our findings initially resolved the dispute regarding the efficacy of CsA and DHCsA-d for visceral leishmaniasis treatment. CsA showed no significant inhibitory effect on intracellular amastigotes. DHCsA-d significantly inhibited promastigotes and intracellular amastigotes, but it was highly cytotoxic. Therefore, CsA and DHCsA-d are not recommended as antileishmanial drugs.

Cinnamomum cassia exhibits antileishmanial activity against Leishmania donovani infection in vitro and in vivo.

BACKGROUND: There is a pressing need for drug discovery against visceral leishmaniasis, a life-threatening protozoal infection, as the available chemotherapy is antiquated and not bereft of side effects. Plants as alternate drug resources has rewarded mankind in the past and aimed in this direction, we investigated the antileishmanial potential of Cinnamomum cassia. METHODOLOGY: Dichloromethane, ethanolic and aqueous fractions of C. cassia bark, prepared by sequential extraction, were appraised for their anti-promastigote activity along with apoptosis-inducing potential. The most potent, C. cassia dichloromethane fraction (CBD) was evaluated for anti-amastigote efficacy in infected macrophages and nitric oxide (NO) production studied. The in vivo antileishmanial efficacy was assessed in L. donovani infected BALB/c mice and hamsters and various correlates of host protective immunity ascertained. Toxicity profile of CBD was investigated in vitro against peritoneal macrophages and in vivo via alterations in liver and kidney functions. The plant secondary metabolites present in CBD were identified by gas chromatography-mass spectroscopy (GC-MS). PRINCIPAL FINDINGS: CBD displayed significant anti-promastigote activity with 50% inhibitory concentration (IC50) of 33.6 µg ml-1 that was mediated via apoptosis. This was evidenced by mitochondrial membrane depolarization, increased proportion of cells in sub-G0-G1 phase, ROS production, PS externalization and DNA fragmentation (TUNEL assay). CBD also inhibited intracellular amastigote proliferation (IC50 14.06 µg ml-1) independent of NO production. The in vivo protection achieved was 80.91% (liver) and 82.92% (spleen) in mice and 75.61% (liver) and 78.93% (spleen) in hamsters indicating its profound therapeutic efficacy. CBD exhibited direct antileishmanial activity, as it did not specifically induce a T helper type (Th)-1-polarized mileu in cured hosts. This was evidenced by insignificant modulation of NO production, lymphoproliferation, DTH (delayed type hypersensitivity), serum IgG2a and IgG1 levels and production of Th2 cytokines (IL-4 and IL-10) along with restoration of pro-inflammatory Th1 cytokines (INF-γ, IL-12p70) to the normal range. CBD was devoid of any toxicity in vitro as well as in vivo. The chemical constituents, cinnamaldehyde and its derivatives present in CBD may have imparted the observed antileishmanial effect. CONCLUSIONS: Our study highlights the profound antileishmanial efficacy of C. cassia bark DCM fraction and merits its further exploration as a source of safe and effective antieishmanial compounds.

Knowledge, stigma, health seeking behaviour and its determinants among patients with post kalaazar dermal leishmaniasis, Bihar, India.

BACKGROUND: Lesishmaniasis is a neglected tropical disease endemic in Bihar, India. Inappropriate health seeking behaviour of post kala-azar dermal leishmaniasis (PKDL) patients may increase the disease duration, severity and transmissibility. Simultaneously, lack of knowledge and perceived stigma may also increase the length of delay in receiving treatment. This ultimately effects the kala-azar elimination program. METHODS: A cross sectional study was conducted in 120 confirmed PKDL patients, aged 18 years and older. Data related to knowledge and health seeking behaviour was collected by a pre-tested questionnaire. EMIC stigma scale was used for assessing the perceived stigma. Patients were personally interviewed after taking informed consent. Data analysis was done by using SPSS 16 software. RESULTS: The time between appearance of symptoms and first medical consultation (patient delay) ranged from 15 days to 5475 days (15 years) with a median of 285 days. The time between first medical consultations to onset of specific treatment (system delay) ranged from 2 to 5475 days with a median of 365 days. Many patients approached first to quacks (8.4%), homeopathic and ayurvedic practitioners (25.8%) upon recognition of symptoms. Majority of the patients (68.3%) had poor knowledge about PKDL and its vector. Type of skin lesions and gender had significant association with patient delay and system delay respectively (p<0.05). Distance to primary health centre (PHC) had significant association with patients delay as well as system delay (p<0.05). Patients with younger age, unmarried and polymorphic lesions had higher stigma (p<0.05). Patients with PKDL feel stigmatized in different areas. CONCLUSION: PKDL treatment delays were unacceptably high and patients had poor knowledge compounded with feelings of stigmatization. To reduce the delay, a system may be evolved to establish some sort of public-private collaboration, besides awareness programs should be tailored, and implemented for improving the patient education regarding the disease and its linkage with VL.

Protective inflammatory response against visceral leishmaniasis with potato tuber extract: A new approach of successful therapy.

The increasing number of drug resistance issue of Leishmania donovani strain to common drugs compels to develop new therapeutics against leishmaniasis with minimal toxicity. In this regard, bioactive phytocomponents may lead to the discovery of new medicines with appropriate efficiency. The important roles of Leishmania proteases in the virulence of Leishmania parasite make them very hopeful targets for the improvement of current remedial of leishmaniasis. As part of a hunt for new drugs, we have evaluated in vivo anti-leishmanial activity of serine protease inhibitor rich fraction (PTEx), isolated by sodium bisulfite extraction from potato tuber. The amastigote load of 25mg/kg body weight/day treated BALB/c mice showed 86.9% decrease in liver and 88.7% in case of spleen. This anti-leishmanial effect was also supported by PTEx induced immunomodulatory activity like acute formation of ROS and prolonged NO generation. The Th1/Th2 cytokine balance in splenocytes of PTEx treated animals was estimated and evaluated by ELISA assay as well as by mRNA expression using RT-PCR. Furthermore, significant survival rate (80%) was observed in PTEx treated hamsters. Thus, from the present observations we could accentuate the potential of PTEx to be employed as a new therapeutics from natural source against L. donovani. This might also provide a novel perception of natural serine protease inhibitor from potato tuber as an alternate approach for the treatment of visceral leishmaniasis.

The leishmanicidal activity of oleuropein is selectively regulated through inflammation- and oxidative stress-related genes.

BACKGROUND: Much research effort has been focused on investigating new compounds derived from low-cost sources, such as natural products, for treating leishmaniasis. Oleuropein derived from numerous plants, particularly from the olive tree, Olea europaea L. (Oleaceae), is a biophenol with many biological activities. Our previous findings showed that oleuropein exhibits leishmanicidal effects against three Leishmania spp. in vitro, and minimizes the parasite burden in L. donovani-infected BALB/c mice. The aim of the present study is to investigate the possible mechanism(s) that mediate this leishmanicidal activity. METHODS: We determined the efficacy of oleuropein in elevating ROS and NO production in L. donovani-infected J774A.1 macrophages and in explanted splenocytes and hepatocytes obtained from L. donovani-infected BALB/c mice. We also assessed the expression of genes that are related to inflammation, T-cell polarization and antioxidant defense, in splenocytes. Finally, we determined the ratios of specific IgG2a/IgG1 antibodies and DTH reactions in L. donovani-infected BALB/c mice treated with oleuropein. RESULTS: Oleuropein was able to elevate ROS production in both in vitro and in vivo models of visceral leishmaniasis and raised NO production in ex vivo cultures of splenocytes and hepatocytes. The extensive oxidative stress found in oleuropein-treated mice was obviated by the upregulation of the host's antioxidant enzyme (mGCLC) and the simultaneous downregulation of the corresponding enzyme of the parasite (LdGCLC). Moreover, oleuropein was able to mount a significant Th1 polarization characterized by the expression of immune genes (IL-12ß, IL-10, TGF-ß1, IFN-γ) and transcription factors (Tbx21 and GATA3). Moreover, this immunomodulatory effect was also correlated with an inhibitory effect on IL-1ß gene expression, rather than with the expression of IL-1α, IL-1rn and TNF-α. Furthermore, oleuropein-treated BALB/c mice mounted a delayed-type hypersensitivity (DTH) response and an elevated Leishmania-specific IgG2a/IgG1 ratio that clearly demonstrated an in vivo protective mechanism. CONCLUSION: The ability of Oleuropein to promote a Th1 type immune response in L. donovani-infected BALB/c mice points towards the candidacy of this bioactive compound as an immunomodulatory agent that may complement therapeutic approaches to leishmaniasis.

In vitro evaluation of traditionally used Surinamese medicinal plants for their potential anti-leishmanial efficacy.

ETHNOPHARMACOLOGICAL RELEVANCE: Plant-based preparations are extensively used in Surinamese folk medicine for treating leishmaniasis, but often without a scientific rationale. AIM OF THE STUDY: To evaluate 25 Surinamese medicinal plants for their potential efficacy against leishmaniasis. MATERIALS AND METHODS: Concentrated plant extracts were evaluated for their effect on the viability of L. (V.) guyanensis AMC, L. (L.) major NADIM5, and L. (L.) donovani GEDII promastigotes, as well as intracellular amastigotes of L. (L.) donovani BHU814 in infected THP-1 cells. Selectivity was assessed by cytotoxicity against THP-1 cells. RESULTS: The only plant extract that showed potentially meaningful anti-leishmanial activity was that from Solanum lycocarpum that displayed mean IC50 values of about 51, 61, and <16 µg/mL against L. (V) guyanensis, L. (L) major, and L. (L) donovani promastigotes, respectively; about 374 µg/mL against L. (L) donovani amastigotes; and >500 µg/mL against THP-1 cells. The Bryophyllum pinnatum, Inga alba, and Quassia amara extracts displayed moderate to high IC50 values against promastigotes (about 51 to >500 µg/mL) and/or amastigotes (about 224 to >500 µg/mL) but were relatively toxic to THP-1 cells (IC50 values <16 to about 42 µg/mL). The remaining plant extracts exhibited in many cases IC50 values close to, around, or above 500µg/mL against promastigotes, amastigotes, and THP-1 cells. CONCLUSIONS: The S. lycocarpum preparation may be useful against leishmaniasis and may have a good safety index, warranting further investigations into its active constituents and mechanism(s) of action.

Therapeutic efficacy of artemisinin-loaded nanoparticles in experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) is a fatal vector-borne parasitic syndrome attributable to the protozoa of the Leishmania donovani complex. The available chemotherapeutic options are not ideal due to their potential toxicity, high cost and prolonged treatment schedule. In the present study, we conjectured the use of nano drug delivery systems for plant-derived secondary metabolite; artemisinin as an alternative strategy for the treatment of experimental VL. Artemisinin-loaded poly lactic co-glycolic acid (ALPLGA) nanoparticles prepared were spherical in shape with a particle size of 220.0±15.0 nm, 29.2±2.0% drug loading and 69.0±3.3% encapsulation efficiency. ALPLGA nanoparticles administered at doses of 10 and 20mg/kg body weight showed superior antileishmanial efficacy compared with free artemisinin in BALB/c model of VL. There was a significant reduction in hepatosplenomegaly as well as in parasite load in the liver (85.0±5.4%) and spleen (82.0±2.4%) with ALPLGA nanoparticles treatment at 20mg/kg body weight compared to free artemisinin (70.3±0.6% in liver and 62.7±3.7% in spleen). In addition, ALPLGA nanoparticle treatment restored the defective host immune response in mice with established VL infection. The protection was associated with a Th1-biased immune response as evident from a positive delayed-type hypersensitivity reaction, escalated IgG2a levels, augmented lymphoproliferation and enhancement in proinflammatory cytokines (IFN-γ and IL-2) with significant suppression of Th2 cytokines (IL-10 and IL-4) after in vitro recall, compared to infected control and free artemisinin treatment. In conclusion, our results advocate superior efficacy of ALPLGA nanoparticles over free artemisinin, which was coupled with restoration of suppressed cell-mediated immunity in animal models of VL.

In vitro and in vivo evaluation of anti-leishmanial and immunomodulatory activity of Neem leaf extract in Leishmania donovani infection.

The toxicity and emergence of resistance to available chemical drugs against visceral leishmaniasis is evoking to explore herbal treatment. One such attempt with the Neem is being reported here. The current study is primarily focused to evaluate the anti-leishmanial effects of Neem leaf extracts. Among which, ethyl acetate fraction (EAF) alone was found to exhibit leishmanicidal effect validated through cytotoxicity assay and estimated its IC50 to be 52.4 µg/ml on the promastigote stage. Propidium iodide (PI) staining of dead cells substantiated the aforementioned activity. Carboxy fluorescein-diaceate succinimidyl ester (CFSE) staining of promastigotes has affirmed its anti-proliferation activity. The characteristic features such as DNA fragmentation, reduced mitochondrial membrane potential, increased sub G0/G1 phase parasites and increased reactive oxygen species (ROS) production in EAF treated promastigotes indicate the apoptosis like death. In addition, the reduced parasite burden both in vitro (viz. ~45% in human monocytic leukemia cell line (THP-1) and ~50% in peripheral blood mononuclear cells) and in vivo (spleen and liver) provides the evidence for its anti-leishmanial activity on amastigote stage. The increase of ROS levels in THP-1 and nitric oxide (NO) production from J774.1 cell line (mouse macrophages) upon EAF treatment was evidenced for oxidative killing of intracellular amastigotes. Active immunomodulatory activity at m-RNA level (viz. upregulation of Th1 cytokines, and downregulation of Th2 cytokines) both in vitro and in vivo was also shown to be exhibited by EAF. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of EAF revealed the presence of 14 compounds.

A cluster of four cases of cutaneous leishmaniasis by Leishmania donovani in Cyprus: a case series.

INTRODUCTION: Leishmaniasis is endemic in more than 95 countries and is the only tropical/subtropical vector-borne disease that has been endemic in Southern Europe for decades. To the best of our knowledge, this is the first case of cutaneous leishmaniasis by Leishmania donovani in a child and the first cluster with adult cases reported in Europe. CASE PRESENTATION: We describe a familial cluster of four cutaneous leishmaniasis cases among Greek Cypriots caused by L. donovani in a Paphos village, in Cyprus. A 6-year-old boy (Case number 1) had a persistent lesion in the left angle of his upper lip, a 60-year-old woman (Case number 2) presented with a 2 cm-diameter glabella lesion on her forehead, a 60-year-old man (Case number 3) developed a lesion on his moustache area and a 40-year-old woman (Case number 4) had a lesion on her neck. In Case number 3 the lesion was self-cured; the other cases recovered after surgical resection followed by liposomal amphotericin B (Case numbers 1 and 4) or thermotherapy and liposomal amphotericin B (Case number 2). CONCLUSIONS: This familial cluster of cutaneous leishmaniasis, due to the anthroponotic L. donovani, shows that the sand fly species responsible for transmitting this parasite species is found in the area around the three neighbouring houses involved. The factors favourable for the survival, spread and contact of the vector with people could be assessed in this area for the establishment of preventative measures to safeguard public health.

Apoptosis-like death in Leishmania donovani promastigotes induced by eugenol-rich oil of Syzygium aromaticum.

Leishmaniasis consists of a complex spectrum of infectious diseases with worldwide distribution of which visceral leishmaniasis or kala-azar caused by Leishmania donovani is the most devastating. In the absence of vaccines, chemotherapy remains the mainstay for the control of leishmaniasis. The drugs of choice are expensive and associated with multiple adverse side effects. Because of these limitations, the development of new antileishmanial compounds is imperative and plants offer prospects in this regard. The present work was conducted to study the antileishmanial potential of oil from Syzygium aromaticum flower buds (clove). The S. aromaticum oil was characterized by gas chromatography and GC-MS and eugenol as well as eugenyl acetate were found to be the most abundant compounds, composing 59.75 % and 29.24 %, respectively of the oil. Our findings have shown that eugenol-rich essential oil from S. aromaticum (EROSA) possesses significant activity against L. donovani, with 50 % inhibitory concentration of 21 ± 0.16 µg ml(-1) and 15.24 ± 0.14 µg ml(-1), respectively, against promastigotes and intracellular amastigotes. Alterations in cellular morphology and growth reversibility assay substantiated the leishmanicidal activity of EROSA. The leishmanicidal effect was mediated via apoptosis as confirmed by externalization of phosphatidylserine, DNA nicking by TdT-mediated dUTP nick-end labelling (TUNEL) assay, dyskinetoplastidy, cell cycle arrest at sub-G0-G1 phase, loss of mitochondrial membrane potential and reactive oxygen species generation. EROSA presented no adverse cytotoxic effects against murine macrophages even at 200 µg ml(-1). Our studies authenticate the promising antileishmanial activity of EROSA, which is mediated by programmed cell death, and, accordingly, EROSA may be a source of novel agents for the treatment of leishmaniasis.

Relapse after treatment with miltefosine for visceral leishmaniasis is associated with increased infectivity of the infecting Leishmania donovani strain.

UNLABELLED: Leishmania donovani is an intracellular protozoan parasite that causes leishmaniasis, which can range from a self-healing cutaneous disease to a fatal visceral disease depending on the infecting species. Miltefosine is currently the latest and only oral antileishmanial that came out of drug discovery pipelines in the past few decades, but recent reports indicate a significant decline in its efficacy against visceral leishmaniasis (also known as kala-azar) in the Indian subcontinent. This relapse rate of up to 20% within 12 months after treatment was shown not to be related to reinfection, drug quality, drug exposure, or drug-resistant parasites. We therefore aimed to assess other phenotypes of the parasite that may affect treatment outcome and found a significant association between the number of metacyclic parasites, parasite infectivity, and patient treatment outcome in the Indian subcontinent. Together with previous studies on resistance of L. donovani against pentavalent antimonials, these data suggest that the infectivity of the parasite, or related phenotypes, might be a more determinant factor for treatment failure in visceral leishmaniasis than drug susceptibility, warranting a reassessment of our current view on treatment failure and drug resistance in leishmaniasis and beyond. IMPORTANCE: The high miltefosine relapse rate poses a major challenge for the current Kala-Azar Elimination Program in the Indian subcontinent and other leishmaniasis control programs worldwide. This relapse rate could not be related to reinfection, drug-resistant parasites, or reduced treatment quality. Here we report that an increased infectivity of the parasite is associated with miltefosine relapse of visceral leishmaniasis (VL) patients. These results supplement those obtained with antimonial-resistant L. donovani where an increased infectivity was also observed. This challenges the current view of Leishmania drug susceptibility being the biggest parasitic factor that contributes to treatment failure in leishmaniasis. These selected more infectious parasites may pose an additional burden to leishmaniasis control programs, highlighting the importance of multifaceted control measures to achieve leishmaniasis elimination in the Indian subcontinent and other regions where leishmaniasis is endemic.

Extracts of Artemisia annua leaves and seeds mediate programmed cell death in Leishmania donovani.

Leishmaniasis is one of the major tropical parasitic diseases, and the condition ranges in severity from self-healing cutaneous lesions to fatal visceral manifestations. There is no vaccine available against visceral leishmaniasis (VL) (also known as kala-azar in India), and current antileishmanial drugs face major drawbacks, including drug resistance, variable efficacy, toxicity and parenteral administration. We report here that n-hexane fractions of Artemisia annua leaves (AAL) and seeds (AAS) possess significant antileishmanial activity against Leishmania donovani promastigotes, with GI(50) of 14.4 and 14.6 µg ml(-1), respectively, and the IC(50) against intracellular amastigotes was found to be 6.6 and 5.05 µg ml(-1), respectively. Changes in the morphology of promastigotes and growth reversibility analysis following treatment confirmed the leishmanicidal effect of the active fractions, which presented no cytotoxic effect on mammalian cells. The antileishmanial activity was mediated via apoptosis, as evidenced by externalization of phosphatidylserine, in situ labelling of DNA fragments by terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) and cell-cycle arrest at the sub-G(0)/G(1) phase. High-performance thin-layer chromatography (HPTLC) fingerprinting showed that the content of artemisinin in crude bioactive extracts (~1.4 µg per 100 µg n-hexane fraction) was too low to account for the observed antileishmanial activity. Characterization of the active constituents by GC-MS showed that α-amyrinyl acetate, ß-amyrine and derivatives of artemisinin were the major constituents in AAL and cetin, EINECS 211-126-2 and artemisinin derivatives in AAS. Our findings indicate the presence of antileishmanial compounds besides artemisinin in the n-hexane fractions of A. annua leaves and seeds.

Terpenoidal constituents of Eucalyptus loxophleba ssp. lissophloia.

CONTEXT: Eucalyptus has been a source of a number of biologically active compounds. The anti-leishmanial activity of terpenoids from Eucalyptus loxophleba (Benth.) ssp. lissophloia (Myrtaceae) has not yet been investigated. OBJECTIVE: Isolation of the terpenoidal constituents for evaluation of in vitro anti-leishmanial activity against the Leishmania donovani (Dd8 strain) promastigotes. MATERIALS AND METHODS: The chloroform-methanol (8:2) extract of dried leaves of Eucalyptus loxophleba was used to isolate terpenoidal constituents employing solvent partitioning, column chromatography and preparative high performance liquid chromatography and characterized from spectral data. The anti-leishmanial activity of the isolated compounds was tested in vitro using an Alamar blue assay against a culture of L. donovani (Dd8 strain) promastigotes. RESULTS: Two new naturally occurring triterpenes, named loxanic acid and 3-acetyl loxanic acid together with four known ursane triterpenoids and one bis-monoterpene glycoside, cuniloside B isolated from the leaves showed anti-leishmanial activity (IC(50) 133 to 235 µM) against the promastigotes of the tested strain. CONCLUSION: The terpenes isolated from the leaves of E. loxophleba showed moderate anti-leishmanial activity.

In vitro antileishmanial, antiplasmodial and cytotoxic activities of a new ventiloquinone and five known triterpenes from Parinari excelsa.

CONTEXT: Parinari excelsa Sabine (Chrysobalanaceae) is an indigenous tree from West and Eastern Africa. This tree is used in Ivory Coast as an antimalaria remedy. OBJECTIVE: The in vitro antiplasmodial and antileishmanial activities of the stem bark, the leaf and the major compounds from the stem bark were investigated. MATERIALS AND METHODS: The leaves and stem bark from P. excelsa were separately collected, air-dried and powdered. Two extracts (methylene chloride and methanol) were realized for both powders. Every extract was tested for its antiplasmodial and antileishmanial activities. Only the stem bark crude extracts were fractionated by column chromatography and their major components were analyzed by NMR, HRESIMS and IR methods. The compounds were tested for their antiplasmodial and antileishmanial activities. RESULTS: The comparison of the IC(50) values of the crude extracts were in this order: 3.41 (IC(50) of PeBMc) <4.10 (IC(50) of PeBMc) <4.42 (IC(50) of PeLMe) against P. falciparum and 5.19 (IC(50) of PeBMc) <12.32 (IC(50) of PeBMe) <19.33 (IC(50) of PeLMc) <32.37 (IC(50) of PeLMe) against L. donovani. The stem bark crude extracts were the most active against both parasites. Their fractionation leaded to a new ventiloquinone, five triterpenes and one chlorogenic acid. All these compounds were isolated for the first time from P. excelsa. High activities were observed with (3ß)-3-hydroxyolean-12-en-28-oic acid (IC(50) = 8.2 µM) and 3ß-hydroxyolean-5,12-dien-28-oic acid (IC(50) = 7.7 µM) against L. donovani. With the antiplasmodial activity, the best activity was observed with 16ß-hydroxylupane-1,20(29)-dien-3-one (IC(50) = 28.3 µM). DISCUSSION AND CONCLUSION: These findings demonstrated that the constituents of P. excelsa stem bark have in vitro antiplasmodial and antileishmanial activities.

Antileishmanial activity of cryptolepine analogues and apoptotic effects of 2,7-dibromocryptolepine against Leishmania donovani promastigotes.

Cryptolepine (5-methyl-10H-indolo [3, 2-b] quinoline), an indoloquinoline alkaloid (1) isolated from a medicinal plant traditionally used in Western Africa for treatment of malaria, has been shown to possess broad spectrum biological activity in addition to its antiplasmodial effect. Here, the antileishmanial properties of 11 synthetic derivatives of cryptolepine against Leishmania donovani parasites have been evaluated for the first time. 2,7-Dibromocryptolepine (8; IC50 0.5 ± 0.1 µM) was found to be the most active analogue against the promastigote form of a classical L. donovani strain (AG83) in comparison to the natural alkaloid, cryptolepine (1; IC50 1.6 ± 0.1 µM). Further, 8 was found to substantially inhibit the intracellular amastigote forms of two clinical isolates, one of them being an SbV-resistant strain of L. donovani. Moreover, the toxicity of 8 against normal mouse peritoneal macrophage cells was markedly lower than that of 1 (IC50 values: 9.0 ± 1.2 and 1.1 ± 0.3 µM, respectively), indicating 8 to be a prospective "lead" towards novel antileishmanial therapy. This was supported by studies on the mechanism of cytotoxicity induced by 8 in L. donovani promastigotes (AG83), which revealed the cytoplasmic and nuclear features of metazoan apoptosis. Light microscopic observation demonstrated a gradual decline in the motility, cell volume, and survival of the treated parasites with increasing incubation time. Flow cytometric analysis of phosphatidylserine externalization and distribution of cells in different phases of cell cycle confirmed the presence of a substantial percentage of cells in early apoptotic stage. Disruption of mitochondrial membrane integrity in terms of depolarization of membrane potential, and finally degradation of chromosomal DNA into oligonucleosomal fragments - the hallmark event of apoptosis - characterized the mode of cell death in L. donovani promastigotes.

Visceral leishmaniasis: experimental models for drug discovery.

Visceral leishmaniasis (VL) or kala-azar is a chronic protozoan infection in humans associated with significant global morbidity and mortality. The causative agent is a haemoflagellate protozoan Leishmania donovani, an obligate intracellular parasite that resides and multiplies within macrophages of the reticulo-endothelial system. Most of the existing anti-leishmanial drugs have serious side effects that limit their clinical application. As an alternate strategy, vaccination is also under experimental and clinical trials. The in vitro evaluation designed to facilitate rapid testing of a large number of drugs has been focussed on the promastigotes milt little attention on the clinically relevant parasite stage, amastigotes. Screening designed to closely reflect the situation in vivo is currently time consuming, laborious, and expensive, since it requires intracellular amastigotes and animal model. The ability to select transgenic Leishmania expressing reporter proteins, such as the green fluorescent proteins (GFP) or the luciferase opened up new possibilities for the development of drug screening models. Many experimental animal models like rodents, dogs and monkeys have been developed, each with specific features, but none accurately reproduces what happens in humans. Available in vitro and in vivo methodologies for antileishmanial drug screening and their respective advantages and disadvantages are reviewed.

Experimental chemotherapy with Allium sativum (Liliaceae) methanolic extract in rodents infected with Leishmania major and Leishmania donovani.

BACKGROUND & OBJECTIVES: Several plant products have been tested and found to possess antileishmanial activity. The present study was undertaken to establish whether methanolic extract of Allium sativum Linn has antileishmanial activity in comparison to standard drugs. METHODS: Methanolic extract of A. sativum bulbs was screened for in vitro and in vivo antileishmanial activity against Leishmania major strain (NLB 145) and L. donovani strain (NLB 065). Pentostam and Amphotericin B were used as standard drugs. BALB/c mice and golden hamsters (Mesocricetus auratus) were used in in vivo studies on L. major and L. donovani respectively. RESULTS: The extract exhibited very low cytotoxicity (IC50 >450 µg/ml) against Vero cells. The extract had significantly better (p <0.001) leishmanicidal activity against both species (IC50 34.22 µg/ml to L. major, 37.41 µg/ml to L. donovani) than Pentostam. However, the activity was significantly lower (p <0.001) than that of Amphotericin B against both the species. At a concentration of 250 µg/ml, the extract induced the production of 60 µM of nitric oxide, a ten-fold up-regulation in activated macrophages. The multiplication indices for L. major amastigotes treated in 100 µg/ml were significantly different (p <0.05). Treatment with the extract, daily for 28 days led to a significant reduction (p <0.05) in footpad swelling in BALB/c mice; similar activity noticed in the treatment with standard drugs. The Leishman-Donovan Units (LDU) for the extract treated animals were significantly higher (p <0.05) than those of standard drugs, but lower compared to the negative control. INTERPRETATION & CONCLUSION: Since the mechanism of action for the methanolic extract is apparently immunomodulatory, garlic compounds could be purified and tried as complementary medicine in the management of leishmaniases.

An unconventional form of actin in protozoan hemoflagellate, Leishmania.

Leishmania actin was cloned, overexpressed in baculovirus-insect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs.

In vitro activity of 10-deacetylbaccatin III against Leishmania donovani promastigotes and intracellular amastigotes.

Current treatments for leishmaniasis are unsatisfactory due to their route of administration, toxicity and expense but, most importantly, to the developed resistance of Leishmania to first-line drugs. Therefore, the identification of new effective targeted drugs is an urgent need. Since many studies have shown that medicinal plants contain compounds active against protozoa we have undertaken a study aiming to determine the antileishmanial activity of the taxoid 10-deacetylbaccatin III, isolated from dried needles and small branches of the European yew tree (Taxus baccata). Interestingly, 10-deacetylbaccatin III was found to selectively inhibit the growth of L. DONOVANI intracellular amastigotes within J774 murine macrophages in vitro at nanomolar concentrations with an IC(50) value of 70 nM. Concentrations of 10-deacetylbaccatin III as high as 5 microM did not affect J774 murine macrophages whereas 20 nM of taxol, used as a control, was toxic to macrophages. The compound also inhibited the growth of L. donovani promastigotes but at higher concentrations with a maximum level of inhibition of 35 %. Taxol inhibited promastigote growth at micromolar concentrations. Comparison of the effect of 10-deacetylbaccatin III to that of taxol on cell cycle progression and cellular morphology showed that their mechanisms of action are different. The 10-deacetylbaccatin III-treated promastigotes were slightly arrested in the G2/M phase whereas taxol-treated cells were blocked in the G2/M phase. In addition 10-deacetylbaccatin III treatment, contrary to taxol, did not affect cellular morphology.

Aloe vera leaf exudate induces a caspase-independent cell death in Leishmania donovani promastigotes.

Leishmaniasis constitutes a complex of diseases with clinical and epidemiological diversity and includes visceral leishmaniasis, a disease that is fatal when left untreated. In earlier studies, the authors reported that Aloe vera leaf exudate (AVL) is a potent antileishmanial agent effective in promastigotes of Leishmania braziliensis, Leishmania mexicana, Leishmania tropica, Leishmania major and Leishmania infantum and also in axenic amastigotes of Leishmania donovani. In the present study, it has been demonstrated that, in promastigotes of L. donovani (IC(50) = 110 microg ml(-1)), AVL mediates this leishmanicidal effect by triggering a programmed cell death. Incubation of promastigotes with AVL caused translocation of phosphatidylserine to the outer leaflet of the plasma membrane as measured by annexin V binding, which was accompanied by loss of mitochondrial membrane potential, release of cytochrome c into the cytosol and concomitant nuclear alterations that included chromatin condensation, deoxynucleotidyltransferase-mediated dUTP end labelling and DNA laddering. As this AVL-induced leishmanicidal effect could not be inhibited by protease inhibitors including Z-Val-Ala-dl-Asp (methoxy)-fluoromethylketone, a broad-spectrum caspase inhibitor, non-involvement of caspases and major proteases was suggested. Additionally, AVL treatment caused no increase in cytosolic Ca(2+) or generation of reactive oxygen species, indicating that although promastigote death was induced by an apoptotic-like mechanism similar to metazoan apoptosis, the pathways of induction and/or execution differed at the molecular level.