Biosynthesis of anti-leishmanial natural products in callus cultures of Artemisia scoparia.

Clinically, available synthetic chemotherapeutics in the treatment for leishmaniasis are associated with serious complications, such as toxicity and emergence of resistance. Natural products from plants can provide better remedies against the Leishmania parasite and can possibly minimize the associated side effects. In this study, various extracts of the callus cultures of Artimisia scoparia established in response to different plant growth regulators (PGRs) were evaluated for their anti-leishmanial effects against Leishmania tropica promastigotes, followed by an investigation of the possible mechanism of action through reactive apoptosis assay using fluorescent microscopy. Amongst the different callus extracts, higher anti-leishmanial activity (IC50:19.13 µg/mL) was observed in the callus raised in-vitro in the presence of 6-Benzylaminopurine (BA) plus 2,4-Dichlorophenoxyacetic Acid (2,4-D) at the concentration of 1.5 mg/L, each. Further, the results of apoptosis assay showed a large number of early-stage apoptotic (EA) and late-stage apoptotic (LA) cells in the Leishmania under the effect of callus extract grown in-vitro at BA plus 2,4-D. For the determination of the potent natural products in the callus extracts responsible for the anti-leishmanial activity, extracts were subjected to Gas chromatography-mass spectrometry (GC-MS) for the metabolite analysis. Nonetheless, higher levels of the metabolites, such as nerolidol (22%), pelletierine (18%), aspidin (15%) and ascaridole (11%) were detected in the callus grown in vitro at BA plus 2,4-D (1.5 mg/L, each). This protocol determines a novel method of production of anti-leishmanial natural products through callus cultures of A. scoparia, a medicinal plant.

[Infecting glial cells with antimony resistant Leishmania tropica: A new ex-vivo model]. / Glia hücrelerinin antimona dirençli Leishmania tropica ile enfekte edilmesi: Yeni bir ex-vivo modeli.

Leishmaniasis is a vector-borne zoonotic disease that shows different clinical features like cutaneous, mucocutaneous, visceral and viscerotropic forms. The protocols used in the treatment of leishmaniasis are toxic and have many limitations during administration. One of the limitations of treatment is the resistance against the protocols in practice. There is also a need to define new treatment options especially for resistant patients. Ex-vivo models using primary cell cultures may be a good source for evaluating new drug options in patients with antimony resistance, in addition to in-vitro and in-vivo studies. In this study, it was aimed to define a new ex-vivo culture model to evaluate treatment options in patients with cutaneous leishmaniasis who did not respond to treatment. In our experimental model of ex-vivo infection, Leishmania tropica promastigotes isolated from a case previously diagnosed with cutaneous leishmaniasis were used. The primary astroglial cell culture used for the ex-vivo model was prepared from 2-3 days old neonatal Sprague Dawley rat brains under sterile conditions by the modification McCarthy's method. The astroglia cells, which reached sufficient density, were infected with antimony resistant L.tropica promastigotes. After 24 hours of incubation, the supernatant on the cells were collected, the cell culture plate was dried at room temperature, then fixed with methyl alcohol and stained with Giemsa to search for L.tropica amastigotes. Amastigotes were intensely observed in glia cells in primary cell cultures infected with L.tropica promastigotes. No promastigotes were seen on Giemsa stained preparations of the precipitates prepared from the bottom sediment after the centrifugation of the liquid medium removed from the infected plates. In this study, promastigotes from a cutaneous leishmaniasis patient unable to respond to pentavalent antimony therapy were shown to infect rat glia cells and converted to amastigote form. This amastigote glial cell model, as far as we know, is the first model in the literature produced by L.tropica. The occurrence of L.tropica amastigote forms in glia cells may be indicative of the ability of Leishmania species to infect the central nervous system. The central nervous system may be an area for the Leishmania amastigotes to escape from the immune system in cases of leishmaniasis without a treatment response. Our study is important because it is the first study to show the infection of glia cells with L.tropica amastigotes.

The associations of Leishmania major and Leishmania tropica aspects by focusing their morphological and molecular features on clinical appearances in Khuzestan Province, Iran.

Cutaneous leishmaniasis has various phenotypic aspects consisting of polymorphic amastigotes with different genetic ranges. Samples were collected from suspected patients of Khuzestan province. Prepared smears were stained, scaled, and measured using ocular micrometer. The Cyt b, ITS-rDNA, and microsatellite genes of Leishmania were amplified and Leishmania species were identified by molecular analyses. Of 150 examined suspected patients, 102 were identified to Leishmania species (90 L. major, nine L. tropica, and three unidentified). The amastigotes of 90 L. major had regular and different irregular shapes within three clinical lesions with no and/or low genetic diversity. Three haplotypes of Cyt b of L. major were found but no variation was observed using ITS-rDNA gene. Interesting findings were that all nine L. tropica had regular amastigote shapes with more genetic variations, also a patient which had coinfection of L. major, L. tropica, and Crithidia. At least two L. major and L. tropica were identified in suspected patients of the regions. Different irregular amastigotes' shapes of L. major can be explained by various reservoir hosts and vectors. In contrast, more molecular variations in L. tropica could be justified by genetic characters. Unidentified Leishmania could be mixed pathogens or nonpathogens with mammals' Leishmania or Crithidia.

Miltefosine-induced apoptotic cell death on Leishmania major and L. tropica strains.

The aim of this study was to assess the cytotoxic effects of various concentrations of miltefosine on Leishmania major (MRHO/IR/75/ER) and L. tropica (MHOM/IR/02/Mash10) promastigotes and to observe the programmed cell death features. The colorimetric MTT assay was used to find L. major and L. tropica viability and the obtained results were expressed as 50% inhibitory concentration (IC50). Also, 50% effective doses (ED50) for L. major and L. tropica amastigotes were also determined. Annexin-V FLUOS staining was performed to study the cell death properties of miltefosine using FACS analysis. Qualitative analysis of the total genomic DNA fragmentation was performed by agarose gel electrophoresis. Furthermore, to observe changes in cell morphology, promastigotes were examined using light microscopy. In both strains of L. major and L. tropica, miltefosine induced dose-dependent death with features of apoptosis, including cell shrinkage, DNA laddering, and externalization of phosphatidylserine. The IC50 was achieved at 22 µM and 11 µM for L. major and L. tropica after 48 hr of incubation, respectively. ED50 of L. major and L. tropica amastigotes were 5.7 µM and 4.2 µM, respectively. Our results indicate that miltefosine induces apoptosis of the causative agent of cutaneous leishmaniasis in a dose-dependent manner. Interestingly, L. major did not display any apoptotic changes when it was exposed to miltefosine in concentrations sufficient to kill L. tropica.

Leishmania tropica: the effect of darkness and light on biological activities in vitro.

Leishmania parasites can be exposed to effects of light in their vectors and hosts, at various periods. However, there is no information about the effects of light on Leishmania parasites. The aim of this study is to investigate the effects of light on various cell parameters of Leishmania tropica, in vitro. All experiments were conducted on L. tropica promastigotes and amastigote-macrophage cultures, using flow cytometric analysis, MTT and phenol-sulfuric acid assay, DAPI and Giemsa. The results showed that the morphology of parasites has changed; the cell cycle has been affected and this caused parasites to remain at G0/G1 phase. Furthermore the proliferation, infectivity, glucose consumption and mitochondrial dehydrogenase activities of parasites were decreased. Thus, for the first time, in this study, the effects of light on biological activities of Leishmania parasites were shown. These new information about parasites' biology, would be very important to investigate the effects of light on the parasites in infected vectors and hosts.

GC-MS analysis and antileishmanial activities of two Turkish propolis types.

Propolis is a honeybee product with a very complex chemical composition and various pharmacological properties. This study was aimed to investigate antileishmanial activities of "Bursa" and "Hatay" propolis samples against Leishmania infantum and Leishmania tropica strains. Propolis samples were analysed with the gas chromatography-mass spectrometry technique. Promastigotes were incubated in Roswell Park Memorial Institute culture medium in the absence and presence of several concentrations (50, 100, 250, 500, 750, and 1,000 µg/mL) of each propolis sample. The viability and cell morphology of promastigotes in each concentration were examined after 24, 48, 72, and 96 h of incubation. The growth of leishmania parasites was significantly suppressed in the presence of 500, 750, and 1,000 µg/mL of Hatay propolis. Bursa propolis was found to be efficient in inhibiting the growth of leishmania promastigotes in culture media at these concentrations, 250, 500, 750, and 1,000 µg/mL. Thus, the in vitro results showed that the Hatay and Bursa propolis samples decreased significantly the proliferation of L. infantum and L. tropica parasites (p < 0.001); however, Bursa propolis was found to be more effective than Hatay propolis against leishmania promastigotes. These two natural products may be useful agents in the prevention of leishmanial infections.

Miltefosine-Induced Apoptotic Cell Death on Leishmania major and L. tropica Strains

The aim of this study was to assess the cytotoxic effects of various concentrations of miltefosine on Leishmania major (MRHO/IR/75/ER) and L. tropica (MHOM/IR/02/Mash10) promastigotes and to observe the programmed cell death features. The colorimetric MTT assay was used to find L. major and L. tropica viability and the obtained results were expressed as 50% inhibitory concentration (IC50). Also, 50% effective doses (ED50) for L. major and L. tropica amastigotes were also determined. Annexin-V FLUOS staining was performed to study the cell death properties of miltefosine using FACS analysis. Qualitative analysis of the total genomic DNA fragmentation was performed by agarose gel electrophoresis. Furthermore, to observe changes in cell morphology, promastigotes were examined using light microscopy. In both strains of L. major and L. tropica, miltefosine induced dose-dependent death with features of apoptosis, including cell shrinkage, DNA laddering, and externalization of phosphatidylserine. The IC50 was achieved at 22 microM and 11 microM for L. major and L. tropica after 48 hr of incubation, respectively. ED50 of L. major and L. tropica amastigotes were 5.7 microM and 4.2 microM, respectively. Our results indicate that miltefosine induces apoptosis of the causative agent of cutaneous leishmaniasis in a dose-dependent manner. Interestingly, L. major did not display any apoptotic changes when it was exposed to miltefosine in concentrations sufficient to kill L. tropica.

In vitro antileishmanial activity of Adana propolis samples on Leishmania tropica: a preliminary study.

Propolis (bee glue) is a natural resinous hive product, collected from various plant sources. It has attracted much attention as a useful substance applied in medicine due to its pharmacological activities. It was aimed to investigate the in vitro effects of an ethanolic extract of Adana propolis samples on the growth of Leishmania tropica. Parasite cells were treated with five concentrations (25, 50, 100, 50, 500, and 750 microg/ml) of the propolis. The number of promastigotes in each concentration was calculated using a hemocytometer slide at 24, 48, and 72 h after being harvested. In the experiments, it was determined that the concentrations up to 100 mug/ml of the propolis did not exhibit antileishmanial activity against the parasites cells. At these concentrations, there was no changes in terms of morphologically. In addition, there was no statistically significant difference in terms of cell count between control and these three groups (p > 0.05). However, in culture media containing the propolis samples at 250, 500, and 750-microg/ml concentrations, statistically significant differences in cell counts were observed, as compared to the control group (p < 0.05). Our results demonstrate that ethanolic extracts of Adana propolis samples reduce the proliferation of L. tropica parasites significantly.

A bifunctional tRNA import receptor from Leishmania mitochondria.

In kinetoplastid protozoa, import of cytosolic tRNAs into mitochondria occurs through tRNAs interacting with membrane-bound proteins, the identities of which are unknown. The inner membrane RNA import complex of Leishmania tropica contains multiple proteins and is active for import in vitro. RIC1, the largest subunit of this complex, is structurally homologous to the conserved alpha subunit of F1 ATP synthase. The RIC1 gene complemented an atpA mutation in Escherichia coli. Antisense-mediated knockdown of RIC1/F1alpha in Leishmania resulted in depletion of several mitochondrial tRNAs belonging to distinct subsets (types I and II) that interact cooperatively or antagonistically within the import complex. The knockdown-induced defect in import of type I tRNAs was rectified in a reconstituted system by purified RIC1/F1alpha alone, but recovery of type II tRNA import additionally required a type I tRNA. RIC1/F1alpha formed stable complexes with type I, but not type II, tRNAs through the cooperation of its nucleotide binding and C-terminal domains. Thus, RIC1/F1alpha is a type I tRNA import receptor. As expected of a bifunctional protein, RIC1/F1alpha is shared by both the import complex and by respiratory complex V. Alternative use of ancient respiratory proteins may have been an important step in the evolution of tRNA import.

gamma-Tubulin in Leishmania: cell cycle-dependent changes in subcellular localization and heterogeneity of its isoforms.

A panel of six anti-peptide antibodies recognizing epitopes in different regions of the gamma-tubulin molecule was used for the characterization and localization of gamma-tubulin during cell cycle in Leishmania promastigotes. Immunofluorescence microscopy revealed the presence of gamma-tubulin in the basal bodies, posterior pole of the cell, and in the flagellum. Furthermore, the antibodies showed punctuate staining in the subpellicular microtubule. This complex localization pattern was observed in both interphase and dividing cells, where staining of posterior poles and the subpellicular corset was more prominent. In posterior poles, gamma-tubulin co-distributed with the 210-kDa microtubule-interacting protein and the 57-kDa protein immunodetected with anti-vimentin antibody. Immunogold electron microscopy on thin sections of isolated flagella showed that gamma-tubulin was associated with the paraflagellar rod (PFR) that runs adjacent to the axonemal microtubules. Under different extraction conditions, gamma-tubulin in Leishmania was found only in insoluble cytoskeletal fractions, in contrast to tubulin dimers that were both in soluble and cytoskeletal pool. Two-dimensional electrophoresis revealed multiple charge variants of gamma-tubulin. Posttranslational modifications of Leishmania gamma-tubulin might therefore have an important role in the regulation of microtubule nucleation and interaction with other proteins. The complex pattern of gamma-tubulin localization and its properties indicate that gamma-tubulin in Leishmania might have other function(s) besides microtubule nucleation.

Leishmania major: differential regulation of the surface metalloprotease in amastigote and promastigote stages.

During its life cycle, the protozoan parasite Leishmania major alternates from an intracellular amastigote form in the mammalian host to a flagellated promastigote form in the insect vector. The expression of the surface metalloprotease (PSP) during differentiation in vitro was investigated by Western and Northern blots, by immunoprecipitation of cells metabolically labeled with [35S]methionine or labeled at the surface with radioactive iodine, and by quantification of the proteolytic activity in substrate-containing polyacrylamide gels. We report that the surface metalloprotease is down-regulated at both the mRNA and the protein level in amastigotes, where it represents less than 1% of the equivalent proteolytic activity detected in promastigotes. A significant amount of mRNA is detected 4 hr after the onset of differentiation. The expression of the protease begins at that time and reaches steady state 8 hr later. The synthesis of PSP precedes the complete morphological differentiation to the promastigote stage and the appearance of the lipophosphoglycan, another major promastigote surface component. In contrast to PSP, a family of mercaptoethanol-activated proteases present in the amastigote exists only at a reduced level in the promastigote. The confinement of the surface metalloprotease to the insect stage of the parasite suggests that it has no physiological function in the parasitism maintenance of mammalian host macrophages.

Real-time quantitative elemental analysis and mapping: microchemical imaging in cell physiology.

Recent advances in widely available microcomputers have made the acquisition and processing of digital quantitative X-ray maps of one to several cells readily feasible. Here we describe a system which uses a graphics-based microcomputer to acquire spectrally filtered X-ray elemental image maps that are fitted to standards, to display the image in real time, and to correct the post-acquisition image map with regard to specimen drift. Both high-resolution quantitative energy-dispersive X-ray images of freeze-dried cyrosections and low-dose quantitative bright-field images of frozen-hydrated sections can be acquired to obtain element and water content from the same intracellular regions. The software programs developed, together with the associated hardware, also allow static probe acquisition of data from selected cell regions with spectral processing and quantification performed on-line in real time. In addition, the unified design of the software program provides for off-line processing and analysing by several investigators at microcomputers remote from the microscope. The overall experimental strategy employs computer-aided imaging, combined with static probes, as an essential interactive tool of investigation for biological analysis. This type of microchemical microscopy facilitates studies in cell physiology and pathophysiology which focus on mechanisms of ionic (elemental) compartmentation, i.e. structure-function correlation at cellular and subcellular levels; it allows investigation of intracellular concentration gradients, of the heterogeneity of cell responses to stimuli, of certain fast physiological events in vivo at ultrastructural resolution, and of events occurring with low incidence or involving cell-to-cell interactions.

Changes in the shape of Leishmania major promastigotes in response to hexoses, proline, and hypo-osmotic stress.

Leishmania major promastigotes in late-log phase are generally long and slender, and remain so during a 1 h incubation in buffer without exogenous substrate. When glucose, 2-deoxyglucose, fructose, mannose, or proline are added, the cells become shorter and more rounded. The shape change in response to glucose is complete within 20 min and is reversible upon incubating the cells without substrate. Galactose, 3-O-methylglucose, 6-deoxyglucose, sucrose, maltose, ribose, glycerol, alanine, glutamate or aspartate do not cause the shape change. Decreasing the osmolarity of the medium causes a rounding of the cells similar to that observed in the presence of glucose, and increasing the osmolarity inhibits the shape change in response to glucose. Inhibitors of glucose transport and 2nd messenger analogs do not affect the shape change.

[Cell fusion in Leishmania (Kinetoplastida, Trypanosomatidae)]. / Fusion cellulaire chez les Leishmania (Kinetoplastida, Trypanosomatidae).

Cell fusion in Leishmania infantum and L. tropica was recorded by means of a camera and a video system. Promastigotes were obtained from cultures in both cases. Fusion began with attachment of the posterior extremities of two ovoid flagellates. Complete fusion, with disappearance of adjacent cell membranes took about five minutes. In parallel, the flagella appear larger and shorter. These observations support the hypothesis of genetic exchanges suggested by analysis of DNA and enzymatic proteins.

Amastigote stage-specific monoclonal antibodies against Leishmania major.

Monoclonal antibodies were produced against gamma-irradiated amastigotes of Leishmania major. Five antibodies (T16 through T20) were selected which reacted in enzyme-linked immunoassays with the intracellular stage of the parasite. These antibodies did not react with promastigotes of L. major or Leishmania donovani. One of the monoclonal antibodies (T16) reacted with amastigotes of Leishmania mexicana amazonensis and L. donovani. Western blotting (immunoblotting) and immunoprecipitation of [35S]methionine-labeled amastigotes demonstrates that T16 reacted with multiple L. major amastigote components between 12 and 180 kilodaltons. Antibody T20 was shown to recognize a low-molecular-mass doublet (less than 26 kilodaltons) in both [14C]leucine- and [35S]methionine-labeled amastigotes. A protein of less than 180 kilodaltons was also weakly recognized by T17, T19, and T20 in metabolically labeled amastigotes. This protein reacted strongly with T16. The reactive antigens could be identified on the surface of amastigotes isolated from the lesions of infected mice and on newly transformed amastigotes within 24 h after the infection of mouse peritoneal macrophages by promastigotes. These monoclonal antibodies should prove useful for the diagnosis of L. major in human tissue biopsies.

Development of sandfly forms of Leishmania major in sucrose solutions.

Stages of Leishmania developing in the vector include different morphs that are exposed first to ingested blood and then to sugar meals. This study sought to determine whether stages occurring in the latter medium could be induced by culturing in sugar-based media. In sucrose solutions, L. major continued to divide and multiplied by 38-46%. Paramastigotes and aflagellates are forms present in late stages of Leishmania infection in Phlebotomus papatasi. They constituted 79% of the forms in sucrose medium, but a maximum of 15% in NNN. Rate and degree of transformation varied as a function of the stage of growth of the NNN starter culture. Motility was lost in sucrose media but was retained in a mixture of sucrose and Ringer's solution. In the latter mixture, the parasites exhibited transformation as well as attachment to the substrate and morphological changes of the flagellum similar to those occurring in the sandfly vector. Parasites from sucrose medium and from P. papatasi reacted similarly, whereas those from NNN reacted differently to a monoclonal antibody. It is suggested that transformation of L. major in sucrose media resembles this process in the vector.

Leishmania promastigotes are recognized by the macrophage receptor for advanced glycosylation endproducts.

In this paper we demonstrate the involvement of the macrophage receptor for advanced glycosylation endproducts (AGE) in the phagocytosis of Leishmania major promastigotes. Blocking of this receptor with the ligand, AGE-BSA, leads to a 50% decrease in phagocytosis relative to controls, and a comparable decrease in the respiratory burst. The inhibition of phagocytosis by AGE-BSA was specific to leishmania. The binding of zymosan or C3bi-RBC and the phagocytosis of IgG-RBC or latex beads was not affected by the presence of AGE-BSA. Blocking of both the AGE receptor and CR3 decreases leishmania binding by nearly 90%, and reduces the respiratory burst by 80%, indicating that the two receptors account for the bulk of L. tropica promastigote recognition and uptake by the macrophage.