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The majority of cells and microorganisms have a nonspherical shape and complex structure that challenge the interpretation of their spectral features. To address this issue, two approximations to the core-shell Mie theory were proposed. These included the approximation of light extinction by an ellipsoid with representation of the extinction by an equivalent sphere and representation of the extinction by a population of ellipsoidal particles with those of two weighted particle orientations. These hypotheses were first tested through numerical interpretation of the theoretical extinction spectra of prolate nucleated ellipsoids mimicking biological cells generated with anomalous diffraction approximation used as a reference method. Theoretical cases of fixed and random particle orientations demonstrated excellent capabilities of the proposed approach to retrieve the size, shape, and composition parameters of the model particles. Second, the UV-visible spectra of Leishmania species, promastigotes, elongated cells with prominent nuclei, were interpreted. The retrieved estimates of the protozoa size, shape, nucleus size, and nucleotide composition were in agreement with the corresponding microscopy estimates and literature values. Both theoretical tests and experimental results illustrated that the proposed approach can be successfully applied to estimate the structural and compositional parameters of cells from spectroscopic measurements.

Infection with Leishmania spp. causes a disease with multifaceted clinical manifestations in humans. The treatment for leishmaniasis is dependent on a limited range of drugs. Here we investigated the antileishmanial activity of eupomatenoid-5, a neolignan isolated from leaves of Piper regnellii var. pallescens. We showed that eupomatenoid-5 had a dose-dependent activity during 72h of treatment, exhibiting IC(50) of 9.0microg/mL and 13.0microg/mL for promastigote and axenic amastigote forms, respectively, and IC(50) of 5.0microg/mL for intracellular amastigote forms of Leishmania amazonensis. When L. amazonensis was treated with eupomatenoid-5, it underwent considerable ultrastructural alterations, as shown by transmission electron microscopy. Among the alterations was the appearance of intense exocytic activity in the region of the flagellar pocket, myelin-like figures, and vacuoles in the cytoplasm of parasites treated with 9.0microg/mL. Cells treated with 25.0microg/mL showed a very large structure, apparently an extension of the endoplasmic reticulum. Also, mitochondrial swelling was detected at this concentration, indicating damage and significant change in this organelle. A cytotoxicity assay showed that the action of the isolated compound is more specific for protozoa and it is not toxic to macrophages. Our studies indicated that eupomatenoid-5 might be a potential new drug for the treatment of leishmaniasis, because this compound displays interesting antileishmanial activity in vitro against promastigote, axenic amastigote, and intracellular amastigote forms of L. amazonensis.

The current treatment for leishmaniasis is unsatisfactory due to toxic side effects, high cost, and problems with drug resistance. Various approaches have been used to identify novel drug candidates to treat Leishmania sp. parasites including the use of re-purposed drugs. Furazolidone is a nitrofuran derivative with antiprotozoal and antibacterial activity and is used for the treatment of giardiasis. In the present work, we determined the in vitro antileishmanial activity of furazolidone and its ability to induce ultrastructural alterations of parasites. Promastigotes of Leishmania (L.) chagasi, Leishmania (V.) braziliensis, Leishmania (L.) major, and Leishmania (L.) amazonensis were highly susceptible to furazolidone, with IC(50) values ranging between 0.47 and 0.73 microg/mL. Furazolidone was also very effective against L. chagasi intracellular amastigotes, and despite mammalian cytotoxicity, the selectivity index was 8.0 in human monocytes. The drug also had limited toxicity in mice erythrocytes. Furazolidone demonstrated specific activity against Leishmania, a potential consequence of the lack of macrophage nitric oxide activation. As determined by electron transmission microscopy, drug treatment induced severe damage to the parasite mitochondria and nucleus. This older oral drug is an effective agent for the treatment of L. (L.) chagasi in vitro and is a novel candidate for further experimental studies.

The interactions between the Leishmania parasite and its mammalian host cells are strongly regulated dynamic processes that take place at the molecular, cellular and organ level. Several different interaction models have been developed to take advantage of the development of imaging technologies. Here, we first review how conventional imaging techniques have been applied to fixed Leishmania-loaded tissue and cell samples. Then, we show how transgenic Leishmania expressing fluorescent or bioluminescent reporters allowed characterization of their tissue and cell host niches. Most notably, the use of whole-body imaging or intravital microscopy techniques has allowed accurate real-time monitoring of parasites in their environment. Finally, we discuss how innovative imaging technologies will allow further refinement of our understanding of the interplay between Leishmania and its hosts.

It was found that in 50% of clinically diagnosed cases of cutaneous leishmaniasis in Damascus, microscopic examination failed to detect the presence of the amastigote form of the parasite inside the macrophages or in the extracellular media, i.e. 50% were false negative. However, in over 1000 photographs from 32 slides of 16 cases, the presence of microorganisms was noted in the extracellular fluid that did not match any of the known blood cells or components but did match the promastigote form as seen by general and electron microscopy. Since the promastigote form is not generally thought to be present in humans, this finding needs further investigation.

The present study analyses complement resistance, cell surface carbohydrates expression, lipidic composition and morphology in vivo and in vitro, of Leishmania (Viannia) shawi, a parasite identified in the Amazon region, Pará state, in 1989. We demonstrated that promastigotes in the stationary (STAT) growth phase are more resistant to complement lysis than in the logarithmic (LOG) growth phase. Ultrastructural analyses and imidazol technique showed accumulation of lipids in STAT growth phase promastigotes, which was confirmed by biochemical approach. Light and electron microscopy of skin lesion in hamster footpads caused by promastigotes in STAT growth phase, 90 days post inoculation, showed amastigotes inside of macrophage and free in the tissue surrounded by collagen fibers as well as extensive inflammatory reaction with tissue destruction. We also demonstrated, using lectins by agglutination assays and flow cytometry, the presence of fucose, mannose and/or glucose carbohydrate residues on the surface of LOG and STAT promastigotes. The results constitute the first characterization essay combining biochemical and morphological approaches dedicated to LOG and STAT growth phase promastigotes of L. (V) shawi contributing for a better knowledge of this poorly studied species of the New World.

Leishmaniasis is a human parasitic disease caused by infection by the protozoan Leishmania spp. Chemotherapy is currently the only treatment available, but its efficacy is increasingly challenged by the rising incidence of resistance and the frequent severe side effects associated with first-line drugs. Thus the development of leads with distinct mechanisms of action is urgently needed. A strategy often used for this purpose consists of assaying for leishmanicidal activity drugs formerly developed for other applications, such as amphotericin B (antifungal) or miltefosine (antitumor), among others, to profit from previous pharmacological and toxicological studies. Kahalalide F (KF) is a tumoricidal cyclic depsipeptide currently under phase II clinical trials for several types of cancer and psoriasis. Its mechanism of action has not been fully elucidated. Here we report the leishmanicidal activity of KF and its synthetic analogues at a micromolar range of concentrations. Its lethality is strongly linked to the alteration of the plasma membrane (PM) of the parasite based on (i) a rapid depolarization of the PM and uptake of the vital dye SYTOX Green upon its addition; (ii) evidence of severe morphological damage to the membrane of the parasite, as shown by transmission electron microscopy; and (iii) a rapid drop in the intracellular ATP levels, which correlates significantly with the leishmanicidal activity for active analogues, some of them with significant improvement of their therapeutic index with respect to the parental molecule. In addition to the basic knowledge obtained, this class of lethal mechanism is considerably less prone to the induction of resistance than classical drugs. All together, these observations foster further studies for the optimization of KF and its analogues as new anti-Leishmania leads with a new mode of action.

Neutrophils are short-lived leukocytes that die by apoptosis, necrosis, and NETosis. Upon death by NETosis, neutrophils release fibrous traps of DNA, histones, and granule proteins named neutrophil extracellular traps (NETs), which can kill bacteria and fungi. Inoculation of the protozoan Leishmania into the mammalian skin causes local inflammation with neutrophil recruitment. Here, we investigated the release of NETs by human neutrophils upon their interaction with Leishmania parasites and NETs' ability to kill this protozoan. The NET constituents DNA, elastase, and histones were detected in traps associated to promastigotes by immunofluorescence. Electron microscopy revealed that Leishmania was ensnared by NETs released by neutrophils. Moreover, Leishmania and its surface lipophosphoglycan induced NET release by neutrophils in a parasite number- and dose-dependent manner. Disruption of NETs by DNase treatment during Leishmania-neutrophil interaction increased parasite survival, evidencing NETs' leishmanicidal effect. Leishmania killing was also elicited by NET-rich supernatants from phorbol 12-myristate 13-acetate-activated neutrophils. Immunoneutralization of histone during Leishmania-neutrophil interaction partially reverted Leishmania killing, and purified histone killed the parasites. Meshes composed of DNA and elastase were evidenced in biopsies of human cutaneous leishmaniasis. NET is an innate response that might contribute to diminish parasite burden in the Leishmania inoculation site.

Endocytosis is essential for eukaryotic cell survival and has been well characterized in mammal and yeast cells. Among protozoa it is also important for evading from host immune defenses and to support intense proliferation characteristic of some life cycle stages. Here we focused on the contribution of morphological and cytochemical studies to the understanding of endocytosis in Trichomonas, Giardia, Entamoeba, Plasmodium, and trypanosomatids, mainly Trypanosoma cruzi, and also Trypanosoma brucei and Leishmania.

Anti-proliferative effects are described for newly synthesised copper (II) complexes of two triazolo-pyrimidine derivatives (1,2,4-triazolo-[1,5-a]pyrimidine, tp, and 5,7-dimethyl 1,2,4-triazolo-[1,5-a]pyrimidine, dmtp) against to Trypanosoma cruzi and Leishmania (Viannia) peruviana. Of the compounds assayed, those that presented the ligand tp and auxiliary ligand 1,10-phenanthroline (C24b, C49) were most highly active against to T. cruzi with IC(50) within the range of the reference drug benznidazole. These compounds, together with C35 were the most effective against L. (V.) peruviana with an IC(50) greater than that presented by reference drugs (Pentostam and Glucantim). These compounds were not toxic to the host cell. IC(25) diminished the infection capacity and severely reduced the multiplication of intracellular forms of T. cruzi, and L. (V.) peruviana. In the case of T. Cruzi, the transformation to trypomastigote was seriously depressed. Copper (II) complexes C24b, C49 and C35, acted on the energy metabolism of the parasites at the level of the NAD(+)/NADH balance and at the level of the organelle membranes, causing degradation and cell death.

Cysteine proteinases have been implicated in many aspects of protozoan parasite pathogenesis. These hydrolases are normally found as zymogens, and some classes in trypanosomatids possess a long C-terminal extension (CTE), for which no function has been assigned. In this paper we hypothesize that the CTE domain of Lpcys2, the abundant lysosomal cysteine proteinase of Leishmania pifanoi amastigotes, is involved in host cell infection. Confirming previous reports that this peptide is highly immunogenic in Trypanosoma cruzi, we detected antibodies against CTE in sera of leishmaniasis patients. We produced a polyclonal antibody specific to Lpcys2 CTE and determined that this antibody was capable of recognizing both L. pifanoi and Leishmania amazonensis cysteine proteinases. Using this antibody, we detected a predominant localization of Lpcys2 CTE in the lysosome and flagellar pocket of cultured axenic amastigotes of both parasite species; however, its location was shifted towards the surface of the parasites during macrophage infection. We examined the role of Lpcys2 CTE in macrophage infection and found a significant reduction in the percentage of infected cells when macrophages were infected with L. pifanoi and L. amazonensis in the presence of anti-CTE antibody. This study suggests a role for leishmanial cysteine proteinases CTE at early stages of infection.

Polyamines are important regulators of growth and differentiation in a variety of cells, including parasitic protozoa. Promastigotes of Leishmania species have high levels of putrescine and spermidine and their growth can be inhibited by polyamine biosynthesis antagonists. The putrescine analogue 1,4-diamino-2-butanone (DAB) is microbicidal against Tritrichomonas foetus and Trypanosoma cruzi, so we tested its effects on Leishmania amazonensis proliferation, viability, organization, putrescine transport and synthesis as well as in vitro infectivity. DAB impaired promastigote proliferation dose-dependently (IC(50) 144 microM) and the parasite putrescine concentration was reduced by nearly 50 %. This analogue markedly inhibited both ornithine decarboxylase activity and [H(3)]putrescine uptake by promastigotes. Pre-treatment with DAB for 24 h led to compensatory enhancement of putrescine uptake, indicating an adaptive mechanism in DAB-treated parasites. Remarkably, DAB caused mitochondrial damage, assessed by transmission electron microscopy, and 3 h treatment with 1 mM DAB enhanced lipid peroxidation, whereas incubation with 10 mM DAB or for 24 h resulted in decreased peroxidation levels in the parasites. This effect was probably due to the loss of mitochondrial function, demonstrated by the diminished reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), not observed in macrophages. Murine macrophages infected with L. amazonensis amastigotes treated with DAB had parasite loads significantly (P<0.05) lower than controls, presumably due to interference with putrescine uptake and/or synthesis. These results suggest that putrescine may be involved in leishmanial survival, possibly by maintaining the parasite's mitochondrial function. The use of analogues to interfere with polyamine/diamine synthesis and transport may shed light on its function in intracellular parasite survival and lead to identification of new targets for leishmaniasis chemotherapy.

The eukaryotic exosome is a complex of at least 11 proteins that is required for various 3'-5' exoribonucleolytic RNA processing and degradation reactions. The minimal core consists of 6 RNase PH and 3 S1 domain subunits; various additional proteins may be associated. We describe here the purification of native exosome from Leishmania tarentolae. The yield is sufficient for structural studies of the native exosome. Electron microscopy and image reconstruction of negatively stained preparations revealed the expected six-membered ring structure at 35 A resolution. An additional density suggested that RRP6 and its partner EAP3 (equivalent to Rrp47) might be located at the top of the exosome and at the side of the hexameric ring. No exonuclease or polyadenylation activity was detected in the exosome preparations.

Field emission scanning electron microscopy (FE-SEM) provides a range of strategies for investigating the structural organization of biological systems, varying from isolated macromolecules to tissue organization and whole organisms. This review covers some of the results so far obtained using FE-SEM observation and various protocols of sample fixation to analyze the structural organization of parasitic protozoa and their interaction with host cells. The employment of FE-SEM can be broadened through the use of gold-labeled molecules or tracers, gradual extraction by detergents, and cleavage techniques. These analyses provide significant contributions to the characterization of these organisms concerning ultrastructure, cytoskeleton, motility and intracellular behavior.

This article provides a summary and discussion of properties of Leishmania amazonensis-loaded mouse macrophages. It illustrates how high-throughput analysis is expected to contribute to deciphering features displayed by macrophages when they are subverted as host cells for replicating Leishmania amastigotes. Firstly, we discuss features of mouse mononuclear phagocytes in steady-state conditions, including the phagocytosis of apoptotic cells. Secondly, we discuss results from ongoing investigations aimed at characterizing transcriptional signatures displayed by BALB/c mouse bone marrow-derived macrophages housing replicating L. amazonensis amastigotes. After a brief presentation on the feasibility of high-throughput microscopy relying on our robust culture system, we share some perspectives on the perpetuation of L. amazonensis in their hosts. Within this latter context, a novel question is formulated and its relevance is discussed: do the Leishmania amastigotes that persist within the mammalian dermis reach a non-replicating developmental stage? If so, is this developmental stage the only one displaying the features required for further development as promastigotes within the sand fly gut lumen?

Genetically encoded cationic anti-microbial peptides (AMPs) are essential components of the ancient and non-specific innate immune system, which is the principal defence mechanism of all species of life, with the primary role to kill infectious microorganisms. Amphibian skin is one of the richest natural sources of such molecules, which are produced by holocrine-type dermal glands and released upon stimulation. This review highlights the attractive and unique structural/functional properties of temporins and bombinins H, two families of short and mildly cationic peptides, isolated from the skin of frogs belonging to Rana and Bombina genera, respectively. Beside improving our knowledge on the role of AMPs in the regulation of the innate immunity, the biological significance of the existence of multiple forms of a prototypic peptide sequence within the same organism and the implication of short peptides in the endotoxin neutralization, these two classes of AMPs can be also considered as valid candidates for the design of novel anti-infective and anti-sepsis drugs.

Mitogen-activated protein kinases are well-known mediators of signal transduction of higher eukaryotes regulating important processes like proliferation, differentiation, stress response and apoptosis. In Leishmania, the typical three-tiered module of MAP kinase signal transduction pathways is present. However, typical activators like cell surface receptors and substrates such as RNA polymerase II transcription factors are missing. Here, I describe the set of 15 putative mitogen-activated protein kinases encoded in the Leishmania genome and discuss their potential function.

Infections by protozoans of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials, which show renal and cardiac toxicity. As part of a search for new drugs against leishmaniasis, we evaluated the in vitro leishmanicidal activity of the (-) mammea A/BB. The compound (-) mammea A/BB is a coumarin-type mammea purified from a dichloromethane crude extract of leaves of Calophyllum brasiliense Cambess (Clusiaceae). The isolated compound was characterized using spectral analyses by UV, infrared, nuclear magnetic resonance of (1)H, (13)C, distortionless enhancement by polarization transfer, correlation spectroscopy, heteronuclear multiple bond correlation, and heteronuclear multiple quantum coherence. The compound (-) mammea A/BB showed significant activity against promastigote and amastigote forms of L. amazonensis, with IC(50) (50% inhibition concentration of cell growth) at a concentration of 3.0 and 0.88 mug/ml and IC(90) (90% inhibition concentration of cell growth) of 5.0 and 2.3 microg/ml, respectively. The coumarin (-) mammea A/BB showed no cytotoxicity against J774G8 macrophages in culture, when it was tested at high concentrations that inhibited promastigote forms. Electron microscopy studies revealed considerable ultrastructural changes when promastigote forms of L. amazonensis were treated with 3.0 microg/ml of the coumarin (-) mammea A/BB for 72 h. We observed significant changes such as mitochondrial swelling with concentric membranes in the mitochondrial matrix and intense exocytic activity in the region of the flagellar pocket. Other alterations included the appearance of binucleate cells and multiple cytoplasmic vacuolization. These results showed that (-) mammea A/BB is a potent growth inhibitor of L. amazonensis and caused important changes in the parasite's ultrastructure. This study provided new perspectives on the development of novel drugs with leishmanicidal activity obtained from natural products.

Leishmaniasis is an important disease in widely dispersed regions of the world. In South America, visceral leishmaniasis (VL) is mainly caused by Leishmania chagasi. The morbidity associated with the infection is high, and death may occur in some untreated patients. Treatment has been based upon pentavalent antimonial drugs for more than half a century and problems, including development of resistance to antimonials and lack of efficacy against VL/HIV co-infections, have emphasized the need for new drugs. Squalene synthase (SQS) is an essential enzyme for the biosynthesis of protozoal sterol molecules. In this work, nineteen synthetic quinuclidines, potentially inhibitors of SQS, were tested against promastigote forms of L. chagasi and the IC50 values of the compounds were determined. The most active compounds had IC50 values of around 30 nM and induced complete growth arrest and cell lysis at sub-micromolar concentrations. We analyzed the morphological structure of the parasites treated with these compounds by transmission electron microscopy of thin sections. Treated parasites showed significant ultrastructural changes, which varied from discrete alterations to total destruction of the cells, depending on the drug concentration and the time of incubation. One important change observed was a typical swelling of the unique and highly branched mitochondrion, where the inner membrane lost its organization. There was an increase in the number of autophagosomal structures. Changes in the organization of the nuclear chromatin and alterations in the flagellar pocket and flagellar membrane were also observed.

Leishmania (Viannia) lainsoni was first described in 1987 in the state of Pará, in the Brazilian Amazon region. The initial characterization of this parasite was performed based only in light microscopy techniques. Posterior studies with this Leishmania species, which focused on biochemistry and molecular assays, showed its divergent position in the Leishmania genus. In this study, we characterize the ultrastructure of culture promastigote forms throughout the growth curve. Our results demonstrate a time-dependent accumulation of electron-dense deposit in the acidocalcisome matrix. We have also analyzed, by transmission electron microscopy, the chronic experimental skin lesion induced in hamster. The experimental infection assay showed adhesion of the intracellular parasites to the parasitophorous vacuole membrane and the occurrence of free vacuoles in the lesion site containing amastigote forms (the amastigote forms morphometrical data were summarized). Our morphological evidences suggest a possible alternative surviving mechanism for L. (Viannia) lainsoni in chronic lesion site.

A novel type of ribonucleoprotein (RNP) complex has been described from the kinetoplast-mitochondria of Leishmania tarentolae. The complex, termed the 45S SSU*, contains the 9S small subunit rRNA but does not contain the 12S large subunit rRNA. This complex is the most stable and abundant mitochondrial RNP complex present in Leishmania. As shown by tandem mass spectrometry, the complex contains at least 39 polypeptides with a combined molecular mass of almost 2.1 MDa. These components include several homologs of small subunit ribosomal proteins (S5, S6, S8, S9, S11, S15, S16, S17, S18, MRPS29); however, most of the polypeptides present are unique. Only a few of them show recognizable motifs, such as protein-protein (coiled-coil, Rhodanese) or protein-RNA (pentatricopeptide repeat) interaction domains. A cryo-electron microscopy examination of the 45S SSU* fraction reveals that 27% of particles represent SSU homodimers arranged in a head-to-tail orientation, while the majority of particles are clearly different and show an asymmetric bilobed morphology. Multiple classes of two-dimensional averages were derived for the asymmetrical particles, probably reflecting random orientations of the particles and difficulties in correlating these views with the known projections of ribosomal complexes. One class of the two-dimensional averages shows a SSU moiety attached to a protein mass or masses in a monosome-like appearance. The combined mass spectrometry and electron microscopy data thus indicate that the majority 45S SSU* particles represents a heterodimeric complex in which the SSU of the Leishmania mitochondrial ribosome is associated with an additional protein mass. The biological role of these particles is not known.

Experimental chronic (45-day-old) skin lesion in hamster hind foot induced by Leishmania (Viannia) lainsoni infection showed the presence of promastigote forms in the tissue, inside parasitophorous vacuoles, as assessed by transmission electron microscopy. Experimental in vitro interaction (24 and 48 h) between Leishmania (V.) lainsoni and J774-G8 macrophage cells also demonstrated the same profile. This morphological aspect is unusual, since in this parasite genus only amastigote forms have been described as the resistant and obligate intracellular forms.

Photodynamic therapy (PDT) is emerging as a potential therapeutic modality in the clinical management of cutaneous leishmaniasis (CL). In order to establish a rationale for effective PDT of CL, we investigated the impact of the molecular charge and structure of photosensitizers on the parasitic phototoxic response. Two photosensitizers from the benzophenoxazine family that bear an overall cationic charge and two anionic porphyrinoid molecules were evaluated. The photodynamic activity of the photosensitizers decreases in the following order: EtNBSe > EtNBS > BpD > PpIX. The studies suggest that compared to hydrophobic anionic photosensitizers, the hydrophilic cationic benzophenoxazine analogs provide high effectiveness of PDT possibly due to (1) their strong attraction to the negatively charged parasitic membrane, (2) their hydrophilicity, (3) their high singlet oxygen quantum yield, and (4) their efficacy in targeting intracellular organelles.

A 3D pharmacophore was generated to describe the antileishmanial activity of dinitroaniline sulfonamides by CATALYST 3D-QSAR methodology, and this pharmacophore was used to search the Maybridge database. Two compounds identified in this search, BTB 06237 and BTB 06256, were highly active with IC(50) values against L. donovani amastigotes of 0.5 +/- 0.2 and 2.3 +/- 0.8 microM, respectively. BTB 06237 also reduced parasite burdens in L. mexicana-infected J774 macrophages at low micromolar concentrations. Unlike the dinitroaniline sulfonamides, the active compounds did not display antimitotic effects against Leishmania. Transmission electron microscopy showed that the single parasite mitochondrion becomes dilated following incubation with BTB 06237, and fluorescence microscopy demonstrated that this organelle fragments into intensely staining spheres when treated with a mitochondrion-specific dye. The mitochondrial membrane potential was also dissipated in BTB 06237-treated parasites. These results indicate that BTB 06237 is an intriguing antileishmanial lead compound that likely interferes with mitochondrial function.

Leishmaniasis is a group of diseases with a large spectrum of clinical manifestations caused by protozoans of the genus Leishmania. Here we demonstrate the leishmanicidal activity of the essential oil of Ocimum gratissimum as well as its main constituent, eugenol. The eugenol-rich essential oil of O. gratissimum progressively inhibited Leishmania amazonensis growth at concentrations ranging from 100 to 1000 microg/ml. The IC50 (sub-inhibitory concentration) of the essential oil for promastigotes and amastigotes were respectively 135 and 100 microg/ml and the IC50 of eugenol was 80 microg/ml for promastigote forms. L. amazonensis exposed to essential oil at concentrations corresponding to IC50 for promastigotes and for amastigotes underwent considerable ultrastructural alterations, as shown by transmission electron microscopy. Two or more nuclei or flagella were observed in 31% and 23.3% of treated amastigote and promastigote forms, respectively, suggesting interference in cell division. Considerable mitochondrial swelling was observed in essential oil-treated promastigotes and amastigotes, which had the inner mitochondrial membrane altered, with a significant increase in the number of cristae; in some amastigotes the mitochondrial matrix became less electron-dense. The minimum inhibitory concentration for both promastigotes and amastigotes was 150 microg/ml. Pretreatment of mouse peritoneal macrophages with 100 and 150 microg/ml essential oil reduced the indices of association between promastigotes and the macrophages, followed by increased in nitric oxide production by the infected macrophages. The essential oil showed no cytototoxic effects against mammalian cells. This set of results suggests that O. gratissimum essential oil and its compounds could be used as sources for new antileishmanial drugs.

Whole-cell and soluble extracts of Leishmania promastigotes have both been used as skin test antigens and have also been tested as vaccine candidates. However, the differences in antigenicity between soluble and particulate Leishmania fractions are not known. We evaluated in vitro responses of PBMC from 30 American tegumentary leishmaniasis (ATL) patients and seven noninfected donors to different antigen preparations from Leishmania promastigotes, namely Leishmania amazonensis and L. braziliensis whole-cell extracts, as well as soluble and particulate fractions of L. amazonensis. All Leishmania antigen preparations stimulated significantly higher proliferation and interferon (IFN)-gamma production (but not interleukin (IL)-10 production) in PBMC from the leishmaniasis patients than in cells from the control subjects. The L. braziliensis whole-cell extract stimulated significantly higher cell proliferation and IFN-gamma production than the L. amazonensis whole-cell extract in the group of patients but not in the control group. This result can be explained by the fact that the patients were infected with L. braziliensis. Again in the group of patients, the PBMC proliferative responses as well as the levels of IFN-gamma and IL-10 stimulated by L. amazonensis whole-cell extract were significantly greater than those elicited by the L. amazonensis soluble fraction but were not significantly different from those elicited by the L. amazonensis particulate fraction. We found a higher antigenicity of the particulate fraction as compared to the soluble fraction, what suggests that the antigens present in the particulate fraction account for most of the antigenicity of whole-cell Leishmania promastigote antigen extracts.

To study the role of Natural Killer (NK) cells in Leishmania infection, peritoneal macrophages from BALB/c mice were infected with Leishmania (Leishmania) amazonensis promastigotes and incubated with interleukin-2 (IL-2)-activated NK (A-NK) cells at different ratios of A-NK cells to infected macrophages (5:1, 1:1, 0.2:1). The A-NK cells were added either together with the parasites (0-h group) or 24 h later (24-h group). Morphological studies of the cultures revealed predominance of parasitic debris within macrophages that were in close contact with A-NK cells and the decrease in parasite recovery was directly proportional to the A-NK cell concentration used. Interferon-gamma (IFN-gamma) and IL-12 were detected in the supernatant at levels proportional to the A-NK cell concentration used. No significant difference was observed between the groups with respect to NO levels in the culture supernatant. When A-NK cells were added directly to the L. (L.) amazonensis promastigote cultures, the parasite recovery decreased proportional to the number of A-NK cells added. In vivo studies demonstrated smaller lesion sizes in animals inoculated with both parasites and A-NK cells compared with parasites alone. Histopathology of the skin lesions from animals receiving A-NK cells together with the parasites showed moderate parasitism and a nodular inflammatory infiltrate formed by mononuclear cells and a few vacuolized macrophages. In contrast, animals inoculated only with the parasites showed a highly parasitized dermis with infiltration of intensely vacuolized macrophages. These results demonstrate the role of A-NK cells in parasite lysis and in resistance of macrophages to L. (L.) amazonensis in the early phase of infection.

The presence of actin in Leishmania has recently been demonstrated, but the functional form of this protein (filamentous actin) has not yet been identified. We report here that the putative coronin homologue identified in the Leishmania genome is invariably associated with the filament-like structures of actin in Leishmania promastigotes. The occurrence of filamentous structures is significantly increased upon overexpression of Leishmania coronin as its GFP fusion product in Leishmania cells. However, expression of Leishmania actin or coronin alone in mammalian cells does not result in formation of any filament-like structures of Leishmania actin or association of Leishmania coronin with mammalian filamentous actin, but coexpression of both the proteins in these cells leads to formation of filamentous structures containing Leishmania actin and coronin. The high specificity of Leishmania coronin for Leishmania actin could be attributed to its unique structure as it differs from other coronins not only in the unique region but also in the actin-binding site and leucine zipper motif. These results taken together indicate that Leishmania contains a novel form of coronin which colocalizes with actin in filament-like structures in these cells.

The present study demonstrates that axenic cultures of Leishmania (Viannia) lainsoni produce larger cell masses in NNN-LIT medium, as well as higher amounts of total proteins in cell extracts, than Leishmania (Leishmania) amazonensis. Antigenicity of L. (V.) lainsoni whole promastigotes is similar to that of L. (L.) amazonensis, as demonstrated by an indirect immunofluorescence diagnostic test using sera from human patients and dogs infected with visceral leishmaniasis. Infectivity of the L. (V.) lainsoni strain used in the present work was demonstrated by the detection by transmission-electron microscopy of tissue amastigotes in skin lesion samples from an experimentally infected hamster. Incubation of lesion fragments in NNN-LIT medium allowed us to obtain promastigote forms, which could be cultivated successfully in vitro. lsoenzyme analysis of such promastigotes confirmed the parasite strain as L. (V.) lainsoni, as compared to other Leishmania reference strains. Our data indicate that L. (V.) lainsoni is a useful alternative source for antigen production as well for use in assays that depend on large cell volumes of Leishmania spp. parasites.

Characterization of infective metacyclic promastigotes of Leishmania spp can be an essential step in several experimental protocols. Metacyclic forms of all Leishmania species display a typical morphology with short, narrow cell body, and an elongated flagellum. This feature suggests that metacyclics can be distinguished from procyclic forms by non-fluorimetric flow cytometric parameters thus enabling the follow-up of their appearance and acquisition of specific properties, during metacyclogenesis in in vitro cultures. Here we describe the flow cytometric parameters of stage-specific promastigotes of Leishmania major, Leishmania donovani, Leishmania amazonensis, and Leishmania braziliensis. Our findings were validated by optical microscopy morphology and specific procyclic labeling with FITC-peanut agglutinin. Furthermore, we show that parasite's distribution in the plot during differentiation in culture is not species specific and that the parasites displaying low forward-angle light scatter (FSC(low)) are three times more infective than the FSC(high) ones. The method here described can be applied to the identification of metacyclics of different Leishmania spp within the whole stationary population.

The antileishmanial efficacy of four novel quinoline derivatives was determined in vitro against Leishmania chagasi, using extracellular and intracellular parasite models. When tested against L. chagasi-infected macrophages, compound 3b demonstrated 8.3-fold greater activity than did the standard pentavalent antimony. No significant activity was found for compounds 3a, 4a, and 4b. The antilesihmanial effect of compound 3b was independent of host cell activation, as demonstrated by nitric oxide production. Ultrastructural studies of promastigotes treated with compound 3b showed mainly enlarged mitochondria, with matrix swelling and reduction in the number of cristae. Synthetic analogues based on the quinoline ring structure, already an established template for antiparasitic drugs, could provide further useful compounds.

In the present study, we evaluated the effects of hyperbaric oxygen (HBO) exposure in both Leishmania amazonensis life stages (promastigotes and amastigotes) and on macrophage cultures infected with the parasite. HBO treatment protocols, which can be tolerated by humans and animals, induced irreversible metabolic damage and affected parasite morphology, growth and ability to transform. The observation that the antioxidant N-acetylcysteine (NAC) prevents some of these deleterious effects indicated an involvement of oxidative stress during parasite HBO exposure. In addition, HBO exposed L. amazonensis-infected macrophage cultures showed reduction of the percentage of infected cells and of the number of intracellular parasites per cell. Thus, the demonstration that HBO, a therapy used in the management of different diseases, is toxic for both L. amazonensis life stages and can alter macrophage susceptibility to the infection encourages further studies of this therapy in animal models of Leishmania infection.

Leishmaniasis encompasses a wide range of infections caused by the human parasitic protozoan species belonging to the Leishmania genus. It appears frequently as an opportunistic disease, especially in virus-infected immunodepressed people. Similarly to other pathogens, parasites became resistant to most of the first-line drugs. Therefore, there is an urgent need to develop antiparasitic agents with new modes of action. Gene-encoded antimicrobial peptides are promising candidates, but so far only a few of them have shown anti-protozoa activities. Here we found that temporins A and B, 13-amino acid antimicrobial peptides secreted from the skin of the European red frog Rana temporaria, display anti-Leishmania activity at micromolar concentrations, with no cytolytic activity against human erythrocytes. To the best of our knowledge, temporins represent the shortest natural peptides having the highest leishmanicidal activity and the lowest number of positively charged amino acids (a single lysine/arginine) and maintain biological function in serum. Their lethal mechanism involves plasma membrane permeation based on the following data. (i) They induce a rapid collapse of the plasma membrane potential. (ii) They induce the influx of the vital dye SYTOX Green. (iii) They reduce intracellular ATP levels. (iv) They severely damage the membrane of the parasite, as shown by transmission electron microscopy. Besides giving us basic important information, the unique properties of temporins, as well as their membranolytic effect, which should make it difficult for the pathogen to develop resistance, suggest them as potential candidates for the future design of antiparasitic drugs with a new mode of action.

In trypanosomatids (Trypanosoma and Leishmania), protozoa responsible for serious diseases of mankind in tropical and subtropical countries, core carbohydrate metabolism including glycolysis is compartmentalized in peculiar peroxisomes called glycosomes. Proper biogenesis of these organelles and the correct sequestering of glycolytic enzymes are essential to these parasites. Biogenesis of glycosomes in trypanosomatids and that of peroxisomes in other eukaryotes, including the human host, occur via homologous processes involving proteins called peroxins, which exert their function through multiple, transient interactions with each other. Decreased expression of peroxins leads to death of trypanosomes. Peroxins show only a low level of sequence conservation. Therefore, it seems feasible to design compounds that will prevent interactions of proteins involved in biogenesis of trypanosomatid glycosomes without interfering with peroxisome formation in the human host cells. Such compounds would be suitable as lead drugs against trypanosomatid-borne diseases.

Objectives: Analysis of the effect of edelfosine, ilmofosine and miltefosine on Leishmania amazonensis and of potential targets of these lysophospholipid analogues., Methods: Quantification and ultrastructural analysis of the effect of lysophospholipid analogues on promastigote forms and on infected peritoneal macrophages, and flow cytometry analysis of treated promastigotes labelled with propidium iodide and rhodamine 123 (Rh123)., Results: The lysophospholipid analogues presented potent antiproliferative activity with IC50/3 days of 1.9-3.4 microM for promastigotes and 4.2-9.0 microM for intracellular amastigotes. Treatment with these analogues in Schneider medium for 1 day led to a dose-dependent decrease in Rh123 fluorescence, an effect more accentuated in edelfosine-treated parasites, suggesting interference with the potential of the mitochondrial membrane. In both forms of L. amazonensis, edelfosine induced extensive mitochondrial damage, multinucleation and, in promastigotes, also led to plasma membrane alterations, formation of autophagic structures and membranous arrangements inside the flagellar pocket., Conclusions: The alkylglycerophosphocholines edelfosine and ilmofosine were more active than the alkylphosphocholine miltefosine against promastigotes and intracellular amastigotes of L. amazonensis, and ultrastructural and flow cytometry data indicate the mitochondrion as a target of edelfosine.

This is the first report of cutaneous leishmaniasis in kangaroos where infection was acquired within Australia. The diagnosis is based on the clinical criteria used for humans, the lesion histopathology, the detection and isolation of parasites from the lesions, and the analysis of the small subunit ribosomal RNA genes using the polymerase chain reaction. Despite a clear indication that the parasites belong to the genus Leishmania, no assignation to a known Leishmania species could be made using these or other less conserved genetic loci such as the non-transcribed spacer of the mini-exon repeat. As is the case in humans, some but not all animals harbouring lesions had antibodies to the isolated parasites or to several other Leishmania species. The isolated parasites displayed two well characterised Leishmania glycoconjugates, the lipophosphoglycan and proteophosphoglycan. They were infectious for mouse macrophages in vitro and established long-term infection at 33 degrees C but not at 37 degrees C. Our findings raise the possibility of transmission to humans, which may be unrecognised and suggest the possibility that imported species of Leishmania could become endemic in Australia.

To study the occurrence and subcellular distribution of actin in trypanosomatid parasites, we have cloned and overexpressed Leishmania donovani actin gene in bacteria, purified the protein, and employed the affinity purified rabbit polyclonal anti-recombinant actin antibodies as a probe to study the organisation and subcellular distribution of actin in Leishmania cells. The Leishmania actin did not cross react with antimammalian actin antibodies but was readily recognized by the anti-Leishmania actin antibodies in both the promastigote and amastigote forms of the parasite. About 10(6) copies per cell of this protein (M(r) 42.05 kDa) were present in the Leishmania promastigote. Unlike other eukaryotic actins, the oligomeric forms of Leishmania actin were not stained by phalloidin nor were dissociated by actin filament-disrupting agents, like Latrunculin B and Cytochalasin D. Analysis of the primary structure of this protein revealed that these unusual characteristics may be related to the presence of highly diverged amino acids in the DNase I-binding loop (amino acids 40-50) and the hydrophobic plug (amino acids 262-272) regions of Leishmania actin. The subcellular distribution of actin was studied in the Leishmania promastigotes by employing immunoelectron and immunofluorescence microscopies. This protein was present not only in the flagella, flagellar pocket, nucleus and the kinetoplast but it was also localized on the nuclear, vacuolar and cytoplasmic face of the plasma membranes. Further, the plasma membrane-associated actin was colocalised with subpellicular microtubules, while most of the actin present in the kinetoplast colocalised with the k-DNA network. These results clearly indicate that Leishmania contains a novel form of actin which may structurally and functionally differ from other eukaryotic actins. The functional significance of these observations is discussed.

Trypanosomatid parasites are able to use their flagella for attachment to cuticular surfaces within their arthropod hosts. In this study the attachment mechanism of Leishmania promastigotes was investigated using a new and quantifiable in vitro assay system. The results showed that hemidesmosomal flagellar attachment to three different plastic substrates occurred (Melinex, Polyvinyl, Thermanox). Attachment density was increased by scratching the surface of the substrate or by coating with the hydrocarbons n-octacosane and paraffin. Variation in attachment density was observed, depending on the culture medium and the parasite isolate used. All four species examined, L. braziliensis, L. donovani, L. major and L. mexicana, were capable of flagellar attachment in vitro. Collectively, these data indicate that flagellar attachment is mediated by a non-specific hydrophobic interaction in Leishmania species.

3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is a key enzyme in the sterol biosynthesis pathway, but its subcellular distribution in the Trypanosomatidae family is somewhat controversial. Trypanosoma cruzi and Leishmania HMGRs are closely related in their catalytic domains to bacterial and eukaryotic enzymes described but lack an amino-terminal domain responsible for the attachment to the endoplasmic reticulum. In the present study, digitonin-titration experiments together with immunoelectron microscopy were used to establish the intracellular localization of HMGR in these pathogens. Results obtained with wild-type cells and transfectants overexpressing the enzyme established that HMGR in both T. cruzi and Leishmania major is localized primarily in the mitochondrion and that elimination of the mitochondrial targeting sequence in Leishmania leads to protein accumulation in the cytosolic compartment. Furthermore, T. cruzi HMGR is efficiently targeted to the mitochondrion in yeast cells. Thus, when the gene encoding T. cruzi HMGR was expressed in a hmg1 hmg2 mutant of Saccharomyces cerevisiae, the mevalonate auxotrophy of mutant cells was relieved, and immunoelectron analysis showed that the parasite enzyme exhibits a mitochondrial localization, suggesting a conservation between the targeting signals of both organisms.

The present work showed the presence of a megasome in Leishmania (Leishmania) chagasi amastigotes. Transmission electron microscopy analysis of ultrathin serial sections and three-dimensional reconstruction allowed visualization of large structures in amastigote forms of L. (L.) chagasi and a multivesicular tubule-lysosome structure in metacyclic promastigotes. Morphometric data showed that the relative volume occupied by the megasome and the multivesicular tubule (MVT)-lysosome structures was about 5% and 3.2%, respectively, in amastigotes and promastigotes of L. (L.) chagasi. Further characterization of the megasome in L. (L.) chagasi amastigotes was carried out by immunolabeling of cysteine proteinase, whereas the lysosomal content of amastigotes and promastigotes was confirmed by arylsulfatase cytochemistry.

This paper describes the synthesis of some novel azasterols based on (20R,22xi)-5alpha-pregnan-20-(piperidin-2-yl)-3beta,20-diol. These compounds are potential inhibitors of the enzyme sterol 24-methyltransferase (24-SMT), which is a vital enzyme in the biosynthesis of ergosterol and related 24-alkyl sterols. Structure-activity studies were undertaken to understand the important features for activity against the enzyme, with the aim of increasing activity and selectivity. The compounds were evaluated for inhibition of recombinant Leishmania major 24-SMT and the effect of compounds on sterol composition and parasite proliferation. Essentially, compounds which showed good activity against the recombinant enzyme had a significant effect on the sterol composition and growth of parasites. The activity of compounds was found to be related to the basicity and stereochemical location of the nitrogen. Also, presence of an unprotected 3beta-OH seemed to be important for activity. However, some azasterols which were not good inhibitors of 24-SMT also showed antiproliferative activity, suggesting that there may be other modes of actions of these compounds.

This study investigated the mechanism of action and efficacy of CO2 laser rays and hypertonic sodium chloride (NaCl) with different concentrations in treatment of cutaneous leishmaniasis (CL) as assessed clinically, parasitologically, histopathologically by light & transmission electron microscopy (TEM) and immunologically by RT-PCR for gene expression of interleukin-13 (IL-13). Eighty mice were divided into four groups. The first was non infected control group (n=16) and the second was infected and served as non-treated control (n=16). The third (n=32) and fourth groups (n=16) were subjected to NaCl injection and CO2 laser respectively. Results showed that clinical healing by CO2 laser were nearly similar to normal appearance, but differed according to concentration of NaCl, as confirmed by ultrastructure and immunohistopathologic features of the host cells and surrounding skin tissue. IL-13 mRNA was significantly decreased after treatment denoting that Th2 cytokine (IL-13) is important for the development of strategies to prevent the induction of pathologic processes. It is concluded that CO2 laser then 7% NaCl are good modalities for CL treatment and is recommended wherever possible.

A stable 100-kD complex from mitochondria of Leishmania tarentolae containing two RNA-binding proteins, Ltp26 and Ltp28, was identified by cross-linking to unpaired 4-thiouridine nucleotides in a partially duplex RNA substrate. The genes were cloned and expressed and the complex was reconstituted from recombinant proteins in the absence of RNA or additional factors. The Ltp26 and Ltp28 proteins are homologs of gBP27 and gBP29 from Crithidia fasciculata and gBP25 and gBP21 from Trypanosoma brucei, respectively. The purified Ltp26/Ltp28 complex, the individual recombinant proteins, and the reconstituted complex are each capable of catalyzing the annealing of complementary RNAs, as was previously shown for gBP21 from T. brucei. A high-molecular-weight RNP complex consisting of the Ltp26/Ltp28 complex and several 55-60-kD proteins together with guide RNA could be purified from mitochondrial extract of L. tarentolae transfected with Ltp28-TAP. This complex also interacted in a less stable manner with the RNA ligase-containing L-complex and with the 3' TUTase. The Ltp26/Ltp28 RNP complex is a candidate for catalyzing the annealing of guide RNA and pre-edited mRNA in the initial step of RNA editing.

Parasitic protozoa comprise a large number of species, some of which are agents of important diseases. They are also of interest from the point of view of cell biology since they contain special organelles and structures. This review analyses our present knowledge of (1). the glycosomes, found in members of the Kinetoplastida order, (2). the hydrogenosomes found in some anaerobic protozoa, especially in trichomonads, (3). the acidocalcisomes, recently described in several protozoa, and (4). structures and organelles participating in the endocytic pathway in trypanosomatids.

This work describes a simple method to yield large amounts of Leishmania amastigote-like forms in axenic cultures using promastigotes as the starting population. The method described induced extracellular amastigote transformation of Leishmania amazonensis (97%), Leishmania braziliensis (98%) and Leishmania chagasi (90%). The rounded parasites obtained in axenic cultures were morphologically similar, even at the ultrastructural level, to intracellular amastigotes. Moreover, the axenic amastigotes remained viable as measured by their ability to revert back to promastigotes and to infect BALB/c mice. L. amazonensis and L. braziliensis promastigotes and axenic amastigotes differed in terms of their Western blot profiles. A 46 kDa protein was recognized by specific antibodies only in axenic and lesion-derived L. amazonensis amastigotes and not in promastigotes.

The endocytic pathway of Leishmania parasites has recently come under intense research focus through the development of several markers for various compartments of this pathway. Through these studies a novel multivesicular tubule lysosome has been discovered in promastigote-stage parasites. This organelle has a highly dynamic role during parasite growth and differentiation. This review discusses recent insights into the Leishmania lysosome with respect to its organisation within the endocytic pathway, stage-specific functions, and biogenesis.

Unicellular organisms, such as the protozoan parasite Leishmania, can be stimulated to show some morphological and biochemical features characteristic of mammalian apoptosis. This study demonstrates that under a variety of stress conditions such as serum deprivation, heat shock and nitric oxide, cell death can be induced leading to genomic DNA fragmentation into oligonucleosomes. DNA fragmentation was observed, without induction, in the infectious stages of the parasite, and correlated with the presence of internucleosomal nuclease activity, visualisation of 45 to 59 kDa nucleases and detection of TUNEL-positive nuclei. DNA fragmentation was not dependent on active effector downstream caspases nor on the lysosomal cathepsin L-like enzymes CPA and CPB. These data are consistent with the presence of a caspase-independent cell death mechanism in Leishmania, induced by stress and differentiation that differs significantly from metazoa.

A synthetic lipid A analog (ONO-4007) exhibits antileishmanial activity by activating Leishmania-infected macrophages in experimental leishmaniasis. In the present in vitro study, ONO-4007 at concentrations between 0.01 and 1.00 mg/mL markedly inhibited the proliferation of Leishmania major and L. amazonensis promastigotes. Ultrastructurally, L. major-infected macrophages showed degenerated intracellular amastigotes after exposure to ONO-4007. Leishmania-infected macrophages treated with ONO-4007 showed poorly developed parasitophorous vacuoles. High levels of tumor necrosis factor-alpha were induced by ONO-4007 in Leishmania-infected macrophages. In this in vivo study, L. amazonensis-infected BALB/c mice were treated with a dose of 30 mg/kg of ONO-4007 by perilesional and peritoneal injections. The skin lesion size was assessed before treatment with ONO-4007 and at eight weeks after injection. The lesion size was significantly suppressed in mice perilesionally injected with ONO-4007 (P < 0.01) compared with the controls. The data from our present in vitro and in vivo studies indicate that ONO-4007 has an antileishmanial effect.

Ajoene [(E,Z)-4,5,9 trithiadodeca 1,6,11 triene 9-oxide], the major bioactive compound derived from garlic, shows a potent trypanolytic and antimicotic activity. In this paper we evaluate its effect on Leishmania mexicana(Lm:MHOM/VE/80/NR), L eishmania mexicana venezuelensis (Lmv: MHOM/VE/80/H16), L eishmania mexicana amazonensis (Lma: M112, IFLA/BR/67/PH8) and L eishmania donovani chagasi (Ldch: MHOM/BR/74/PP75). Ajoene showed a potent leishmanicidal activity in vitro against all species studied. Concentrations higher than 0.3 microM led to total inhibition of growth, and 10 microM induced 100% lysis of Leishmaniaafter 96 h of incubation in a chemically defined culture medium. The 50% inhibitory concentration (IC(50)) for lysis, for all species, was about to 2 microM. The effect was dose-dependent and a threefold increase in concentration (30 microM) produced 100% lysis of cultured forms after 72 h. Ultrastructural studies showed a time- and dose-dependent morphological alteration of the mitochondrial membrane and nuclear envelope, as well as the formation of large autophagic vacuoles.