A short-term method to evaluate anti-leishmania drugs by inhibition of stage differentiation in Leishmania mexicana using flow cytometry.

Leishmaniasis is a vector-borne neglected tropical disease caused by the Leishmania spp. Parasite. The disease is transmitted to humans and animals by the bite of infected female sandflies during the ingestion of bloodmeal. Because current drug treatments induce toxicity and parasite resistance, there is an urgent need to evaluate new drugs. Most therapeutics target the differentiation of promastigotes to amastigotes, which is necessary to maintain Leishmania infection. However, in vitro assays are laborious, time-consuming, and depend on the experience of the technician. In this study, we aimed to establish a short-term method to assess the differentiation status of Leishmania mexicana (L. mexicana) using flow cytometry. Here, we showed that flow cytometry provides a rapid means to quantify parasite differentiation in cell culture as reliably as light microscopy. Interestingly, we found using flow cytometry that miltefosine reduced promastigote-to-amastigote differentiation of L. mexicana. We conclude that flow cytometry provides a means to rapidly assay the efficacy of small molecules or natural compounds as potential anti-leishmanials.

Involvement of Akt and the antiapoptotic protein Bcl-xL in the inhibition of apoptosis of dendritic cells by Leishmania mexicana.

The intracellular parasite Leishmania mexicana inhibits camptothecin (CPT)-induced apoptosis of monocyte-derived dendritic cells (moDC) through the down-regulation of p38 and JNK phosphorylation, while the kinase Akt is maintained active for 24 h. In addition, the infection of moDC with L. mexicana promastigotes increases the protein presence of the antiapoptotic protein Bcl-xL. In the present work, we aimed to investigate the role of Akt in the inhibition of apoptosis of moDC by L. mexicana and in the modulation of the expression of the antiapoptotic proteins Bcl-2, Mcl-1 and Bcl-xL. moDC were infected with L. mexicana metacyclic promastigotes and treated with CPT, an Akt inhibitor, or both and the mitochondrial outer membrane permeabilization (MOMP) and protein presence of active caspase 3, Bcl-2, Mcl-1 and Bcl-xL were evaluated. Our results show that the specific inhibition of Akt reverts the apoptosis protective effect exerted by L. mexicana on moDC reflected by a reduction in MOMP, caspase 3 activation, and upregulation of Bcl-xL. Interestingly, we also found that the infection of moDC with L. mexicana promastigotes induces a decrease in Bcl-2 along with an isoform change of Mcl-1, this independently to Akt activity. We demonstrated that Akt is deeply involved in the inhibition of apoptosis of moDC by L. mexicana.

Amentoflavone isolated from Selaginella sellowii Hieron induces mitochondrial dysfunction in Leishmania amazonensis promastigotes.

Leishmaniasis chemotherapy is a bottleneck in disease treatment. Although available, chemotherapy is limited, toxic, painful, and does not lead to parasite clearance, with parasite resistance also being reported. Therefore, new therapeutic options are being investigated, such as plant-derived anti-parasitic compounds. Amentoflavone is the most common biflavonoid in the Selaginella genus, and its antileishmanial activity has already been described on Leishmania amazonensis intracellular amastigotes but its direct action on the parasite is controversial. In this work we demonstrate that amentoflavone is active on L. amazonensis promastigotes (IC50 = 28.5 ± 2.0 µM) and amastigotes. Transmission electron microscopy of amentoflavone-treated promastigotes showed myelin-like figures, autophagosomes as well as enlarged mitochondria. Treated parasites also presented multiple lipid droplets and altered basal body organization. Similarly, intracellular amastigotes presented swollen mitochondria, membrane fragments in the lumen of the flagellar pocket as well as autophagic vacuoles. Flow cytometric analysis after TMRE staining showed that amentoflavone strongly decreased mitochondrial membrane potential. In silico analysis shows that amentoflavone physic-chemical, drug-likeness and bioavailability characteristics suggest it might be suitable for oral administration. We concluded that amentoflavone presents a direct effect on L. amazonensis parasites, causing mitochondrial dysfunction and parasite killing. Therefore, all results point for the potential of amentoflavone as a promising candidate for conducting advanced studies for the development of drugs against leishmaniasis.

Benzimidazole Derivatives as New and Selective Inhibitors of Arginase from Leishmania mexicana with Biological Activity against Promastigotes and Amastigotes.

Leishmaniasis is a disease caused by parasites of the Leishmania genus that affects 98 countries worldwide, 2 million of new cases occur each year and more than 350 million people are at risk. The use of the actual treatments is limited due to toxicity concerns and the apparition of resistance strains. Therefore, there is an urgent necessity to find new drugs for the treatment of this disease. In this context, enzymes from the polyamine biosynthesis pathway, such as arginase, have been considered a good target. In the present work, a chemical library of benzimidazole derivatives was studied performing computational, enzyme kinetics, biological activity, and cytotoxic effect characterization, as well as in silico ADME-Tox predictions, to find new inhibitors for arginase from Leishmania mexicana (LmARG). The results show that the two most potent inhibitors (compounds 1 and 2) have an I50 values of 52 µM and 82 µM, respectively. Moreover, assays with human arginase 1 (HsARG) show that both compounds are selective for LmARG. According to molecular dynamics simulation studies these inhibitors interact with important residues for enzyme catalysis. Biological activity assays demonstrate that both compounds have activity against promastigote and amastigote, and low cytotoxic effect in murine macrophages. Finally, in silico prediction of their ADME-Tox properties suggest that these inhibitors support the characteristics to be considered drug candidates. Altogether, the results reported in our study suggest that the benzimidazole derivatives are an excellent starting point for design new drugs against leishmanisis.

Micro-CT visualization of a promastigote secretory gel (PSG) and parasite plug in the digestive tract of the sand fly Lutzomyia longipalpis infected with Leishmania mexicana.

Leishmaniasis is a debilitating disease of the tropics, subtropics and southern Europe caused by Leishmania parasites that are transmitted during blood feeding by phlebotomine sand flies (Diptera: Psychodidae). Using non-invasive micro-computed tomography, we were able to visualize the impact of the laboratory model infection of Lutzomyia longipalpis with Leishmania mexicana and its response to a second blood meal. For the first time we were able to show in 3D the plug of promastigote secretory gel (PSG) and parasites in the distended midgut of whole infected sand flies and measure its volume in relation to that of the midgut. We were also able to measure the degree of opening of the stomodeal valve and demonstrate the extension of the PSG and parasites into the pharynx. Although our pilot study could only examine a few flies, it supports the hypothesis that a second, non-infected, blood meal enhances parasite transmission as we showed that the thoracic PSG-parasite plug in infected flies after a second blood meal was, on average, more than twice the volume of the plug in infected flies that did not have a second blood meal.

High-speed, three-dimensional imaging reveals chemotactic behaviour specific to human-infective Leishmania parasites.

Cellular motility is an ancient eukaryotic trait, ubiquitous across phyla with roles in predator avoidance, resource access, and competition. Flagellar motility is seen in various parasitic protozoans, and morphological changes in flagella during the parasite life cycle have been observed. We studied the impact of these changes on motility across life cycle stages, and how such changes might serve to facilitate human infection. We used holographic microscopy to image swimming cells of different Leishmania mexicana life cycle stages in three dimensions. We find that the human-infective (metacyclic promastigote) forms display 'run and tumble' behaviour in the absence of stimulus, reminiscent of bacterial motion, and that they specifically modify swimming direction and speed to target host immune cells in response to a macrophage-derived stimulus. Non-infective (procyclic promastigote) cells swim more slowly, along meandering helical paths. These findings demonstrate adaptation of swimming phenotype and chemotaxis towards human cells.

The kinesin of the flagellum attachment zone in Leishmania is required for cell morphogenesis, cell division and virulence in the mammalian host.

Leishmania parasites possess a unique and complex cytoskeletal structure termed flagellum attachment zone (FAZ) connecting the base of the flagellum to one side of the flagellar pocket (FP), an invagination of the cell body membrane and the sole site for endocytosis and exocytosis. This structure is involved in FP architecture and cell morphogenesis, but its precise role and molecular composition remain enigmatic. Here, we characterized Leishmania FAZ7, the only known FAZ protein containing a kinesin motor domain, and part of a clade of trypanosomatid-specific kinesins with unknown functions. The two paralogs of FAZ7, FAZ7A and FAZ7B, display different localizations and functions. FAZ7A localizes at the basal body, while FAZ7B localizes at the distal part of the FP, where the FAZ structure is present in Leishmania. While null mutants of FAZ7A displayed normal growth rates, the deletion of FAZ7B impaired cell growth in both promastigotes and amastigotes of Leishmania. The kinesin activity is crucial for its function. Deletion of FAZ7B resulted in altered cell division, cell morphogenesis (including flagellum length), and FP structure and function. Furthermore, knocking out FAZ7B induced a mis-localization of two of the FAZ proteins, and disrupted the molecular organization of the FP collar, affecting the localization of its components. Loss of the kinesin FAZ7B has important consequences in the insect vector and mammalian host by reducing proliferation in the sand fly and pathogenicity in mice. Our findings reveal the pivotal role of the only FAZ kinesin as part of the factors important for a successful life cycle of Leishmania.

Divergent Cytochrome c Maturation System in Kinetoplastid Protists.

In eukaryotes, heme attachment through two thioether bonds to mitochondrial cytochromes c and c1 is catalyzed by either multisubunit cytochrome c maturation system I or holocytochrome c synthetase (HCCS). The former was inherited from the alphaproteobacterial progenitor of mitochondria; the latter is a eukaryotic innovation for which prokaryotic ancestry is not evident. HCCS provides one of a few exemplars of de novo protein innovation in eukaryotes, but structure-function insight of HCCS is limited. Uniquely, euglenozoan protists, which include medically relevant kinetoplastids Trypanosoma and Leishmania parasites, attach heme to mitochondrial c-type cytochromes by a single thioether linkage. Yet the mechanism is unknown, as genes encoding proteins with detectable similarity to any proteins involved in cytochrome c maturation in other taxa are absent. Here, a bioinformatics search for proteins conserved in all hemoprotein-containing kinetoplastids identified kinetoplastid cytochrome c synthetase (KCCS), which we reveal as essential and mitochondrial and catalyzes heme attachment to trypanosome cytochrome c KCCS has no sequence identity to other proteins, apart from a slight resemblance within four short motifs suggesting relatedness to HCCS. Thus, KCCS provides a novel resource for studying eukaryotic cytochrome c maturation, possibly with wider relevance, since mutations in human HCCS leads to disease. Moreover, many examples of mitochondrial biochemistry are different in euglenozoans compared to many other eukaryotes; identification of KCCS thus provides another exemplar of extreme, unusual mitochondrial biochemistry in an evolutionarily divergent group of protists.IMPORTANCE Cytochromes c are essential proteins for respiratory and photosynthetic electron transfer. They are posttranslationally modified by covalent attachment of a heme cofactor. Kinetoplastids include important tropical disease-causing parasites; many aspects of their biology differ from other organisms, including their mammalian or plant hosts. Uniquely, kinetoplastids produce cytochromes c with a type of heme attachment not seen elsewhere in nature and were the only cytochrome c-bearing taxa without evidence of protein machinery to attach heme to the apocytochrome. Using bioinformatics, biochemistry, and molecular genetics, we report how kinetoplastids make their cytochromes c Unexpectedly, they use a highly diverged version of an enzyme used for heme-protein attachment in many eukaryotes. Mutations in the human enzyme lead to genetic disease. Identification of kinetoplastid cytochrome c synthetase, thus, solves an evolutionary unknown, provides a possible target for antiparasite drug development, and an unanticipated resource for studying the mechanistic basis of a human genetic disease.

Effect of Prophylactic Vaccination with the Membrane-Bound Acid Phosphatase Gene of Leishmania mexicana in the Murine Model of Localized Cutaneous Leishmaniasis.

Leishmaniasis is a disease caused by an intracellular protozoan parasite of the genus Leishmania. Current treatments for leishmaniasis are long, toxic, and expensive and are not available in some endemic regions. Attempts to develop an effective vaccine are feasible, but no vaccine is in active clinical use. In this study, the LmxMBA gene of Leishmania mexicana was selected as a possible vaccine candidate using the reverse vaccinology approach, and the prophylactic effect generated by DNA vaccination with this gene in a murine model of cutaneous leishmaniasis was evaluated. The results showed that prophylactic vaccination with pVAX1::LmxMBA significantly reduced the size of the lesion and the parasitic load on the footpad, compared to the control groups. At a histological level, a smaller number of parasites were evident in the dermis, as well as the absence of connective tissue damage. Mice immunized with plasmid pVAX1::LmxMBA induced immunity characterized by an increase in the IgG2a/IgG1 > 1 ratio and a higher rate of lymphocyte proliferation. In this study, immunization with the plasmid promoted an improvement in the macroscopic and microscopic clinical manifestations of the experimental infection by L. mexicana, with a T helper 1 response characterized by an IgG2a/IgG1 > 1 ratio and high lymphoproliferative response. These findings support immunization with the plasmid pVAX1::LmxMBA as a preventive strategy against cutaneous infection of L. mexicana.

Chemical inhibition of ß-glucocerebrosidase does not affect phagocytosis and early containment of Leishmania by murine macrophages.

Gaucher disease is a lysosomal storage disease in which a genetic deficiency in ß-glucocerebrosidase leads to the accumulation of glycosphingolipids in lysosomes. Macrophages are amongst the cells most severely affected in Gaucher disease patients. One phenotype associated with Gaucher macrophages is the impaired capacity to fight bacterial infections. Here, we investigate whether inhibition of ß-glucocerebrosidase activity affects the capacity of macrophages to phagocytose and act on the early containment of human pathogens of the genus Leishmania. Towards our aim, we performed in vitro infection assays on macrophages derived from the bone marrow of C57BL/6 mice. To mimic Gaucher disease, macrophages were incubated with the ß-glucocerebrosidase inhibitor, conduritol B epoxide (CBE), prior to contact with Leishmania. This treatment guaranteed that ß-glucocerebrosidase was fully inhibited during the contact of macrophages with Leishmania, its enzymatic activity being progressively recovered along the 48 h that followed removal of the inhibitor. Infections were performed with L. amazonensis, L. infantum, or L. major, so as to explore potential species-specific responses in the context of ß-glucocerebrosidase inactivation. Parameters of infection, recorded immediately after phagocytosis, as well as 24 and 48 h later, revealed no noticeable differences in the infection parameters of CBE-treated macrophages relative to non-treated controls. We conclude that blocking ß-glucocerebrosidase activity during contact with Leishmania does not interfere with the phagocytic capacity of macrophages and the early onset of leishmanicidal responses.

Key role of the macrophage microtubule network in the intracellular lifestyle of Leishmania amazonensis.

We conducted a study to decipher the mechanism of the formation of the large communal Leishmania amazonensis-containing parasitophorous vacuole (PV) and found that the macrophage microtubule (MT) network dynamically orchestrates the intracellular lifestyle of this intracellular parasite. Physical disassembly of the MT network of macrophage-like RAW 264.7 cells or silencing of the dynein gene, encoding the MT-associated molecular motor that powers MT-dependent vacuolar movement, by siRNA resulted in most of the infected cells hosting only tight parasite-containing phagosome-like vacuoles randomly distributed throughout the cytoplasm, each insulating a single parasite. Only a minority of the infected cells hosted both isolated parasite-containing phagosome-like vacuoles and a small communal PV, insulating a maximum of two to three parasites. The tight parasite-containing phagosome-like vacuoles never matured, whereas the small PVs only matured to a small degree, shown by the absence or faint acquisition of host-cell endolysosomal characteristics. As a consequence, the parasites were unable to successfully complete promastigote-to-amastigote differentiation and died, regardless of the type of insulation.

Differential Regulation of l-Arginine Metabolism through Arginase 1 during Infection with Leishmania mexicana Isolates Obtained from Patients with Localized and Diffuse Cutaneous Leishmaniasis.

l-Arginine metabolism through arginase 1 (Arg-1) and inducible nitric oxide synthase (NOS2) constitutes a fundamental axis for the resolution or progression of leishmaniasis. Infection with Leishmania mexicana can cause two distinct clinical manifestations: localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). In this work, we analyzed in an in vivo model the capacity of two L. mexicana isolates, one obtained from a patient with LCL and the other from a patient with DCL, to regulate the metabolism of l-arginine through Arg-1 and NOS2. Susceptible BALB/c mice were infected with L. mexicana isolates from both clinical manifestations, and the evolution of the infection as well as protein presence and activity of Arg-1 and NOS2 were evaluated. The lesions of mice infected with the DCL isolate were bigger, had higher parasite loads, and showed greater protein presence and enzymatic activity of Arg-1 than the lesions of mice infected with the LCL isolate. In contrast, NOS2 protein synthesis was poorly or not induced in the lesions of mice infected with the LCL or DCL isolate. The immunochemistry analysis of the lesions allowed the identification of highly parasitized macrophages positive for Arg-1, while no staining for NOS2 was found. In addition, we observed in lesions of patients with DCL macrophages with higher parasite loads and stronger Arg-1 staining than those in lesions of patients with LCL. Our results suggest that L. mexicana isolates obtained from patients with LCL or DCL exhibit different virulence or pathogenicity degrees and differentially regulate l-arginine metabolism through Arg-1.

Cytokine regulation of female-macrophage resistance to Leishmania mexicana parasites. Role of IL-12p70.

In previous studies, carried out in humans, we showed that females are resistant to Leishmania mexicana infection. We also showed that 17ß-estradiol (E2) induces killing of parasites inside of murine macrophages. In this work, we compared, for the first time, L mexicana survival inside of male (male BMDM) and female (female BMDM) bone marrow-derived macrophages (BMDM) treated in vitro with E2 or dihydrotestosterone (DHT). We also compared their levels of nitric oxide (NO), interleukin (IL)-6, IL-10, IL-12p70 and tumour necrosis factor (TNF-α). We found that female BMDM are a lot less susceptible to infection as compared with male BMDM. 17ß-estradiol induced killing of most parasites inside of female BMDM. Dihydrotestosterone, on the other hand, induced some parasite killing inside of some infected male BMDM. Interleukin-6 levels were higher in female BMDM treated with either hormone. Neither TNF-α nor IL-10 levels showed significant differences compared with sham controls. Interestingly IL-12p70 was more abundantly produced by sham female BMDM as compared with sham male BMDM. Only female BMDM treated with E2 trigger a robust IL-12p70 production, but it was significantly reduced in male BMDM. This suggests IL-12p70 is an important factor in female-macrophage resistance to L mexicana parasites.

Metabolomic Profile of BALB/c Macrophages Infected with Leishmania amazonensis: Deciphering L-Arginine Metabolism.

BACKGROUND: Leishmaniases are neglected tropical diseases that are caused by Leishmania, being endemic worldwide. L-arginine is an essential amino acid that is required for polyamines production on mammal cells. During Leishmania infection of macrophages, L-arginine is used by host and parasite arginase to produce polyamines, leading to parasite survival; or, by nitric oxide synthase 2 to produce nitric oxide leading to parasite killing. Here, we determined the metabolomic profile of BALB/c macrophages that were infected with L. amazonensis wild type or with L. amazonensis arginase knockout, correlating the regulation of L-arginine metabolism from both host and parasite. METHODS: The metabolites of infected macrophages were analyzed by capillary electrophoresis coupled with mass spectrometry (CE-MS). The metabolic fingerprints analysis provided the dual profile from the host and parasite. RESULTS: We observed increased levels of proline, glutamic acid, glutamine, L-arginine, ornithine, and putrescine in infected-L. amazonensis wild type macrophages, which indicated that this infection induces the polyamine production. Despite this, we observed reduced levels of ornithine, proline, and trypanothione in infected-L. amazonensis arginase knockout macrophages, indicating that this infection reduces the polyamine production. CONCLUSIONS: The metabolome fingerprint indicated that Leishmania infection alters the L-arginine/polyamines/trypanothione metabolism inside the host cell and the parasite arginase impacts on L-arginine metabolism and polyamine production, defining the infection fate.

A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation.

Adaptation to starvation is integral to the Leishmania life cycle. The parasite can survive prolonged periods of nutrient deprivation both in vitro and in vivo. The identification of parasite proteins synthesised during starvation is key to unravelling the underlying molecular mechanisms facilitating adaptation to these conditions. Additionally, as stress adaptation mechanisms in Leishmania are linked to virulence as well as infectivity, profiling of the complete repertoire of Newly Synthesised Proteins (NSPs) under starvation is important for drug target discovery. However, differential identification and quantitation of low abundance, starvation-specific NSPs from the larger background of the pre-existing parasite proteome has proven difficult, as this demands a highly selective and sensitive methodology. Herein we introduce an integrated chemical proteomics method in L. mexicana promastigotes that involves a powerful combination of the BONCAT technique and iTRAQ quantitative proteomics Mass Spectrometry (MS), which enabled temporally resolved quantitative profiling of de novo protein synthesis in the starving parasite. Uniquely, this approach integrates the high specificity of the BONCAT technique for the NSPs, with the high sensitivity and multiplexed quantitation capability of the iTRAQ proteomics MS. Proof-of-concept experiments identified over 250 starvation-responsive NSPs in the parasite. Our results show a starvation-specific increased relative abundance of several translation regulating and stress-responsive proteins in the parasite. GO analysis of the identified NSPs for Biological Process revealed translation (enrichment P value 2.47e-35) and peptide biosynthetic process (enrichment P value 4.84e-35) as extremely significantly enriched terms indicating the high specificity of the NSP towards regulation of protein synthesis. We believe that this approach will find widespread use in the study of the developmental stages of Leishmania species and in the broader field of protozoan biology.

Diffuse cutaneous leishmaniasis with oral involvement in a patient of Northern Mexico.

Diffuse cutaneous leishmaniasis is a rare chronic infectious disease, associated with Leishmania mexicana and L. amazonensis, presenting as multiple non-ulcerative painless nodules, with a tendency to relapse soon after treatment. We report a case of a 56-year-old Mexican woman exhibiting nodular lesions, plaques, crusts and scars involving the whole body. A solitary nodule was present at the junction between hard and soft palates. Diffuse cutaneous leishmaniasis is a disfiguring disease resulting in severe scarring if untreated.

Development of Leishmania mexicana in Lutzomyia longipalpis in the absence of sugar feeding.

The leishmaniases are caused by Leishmania parasites and transmitted through the bites of phlebotomine sand flies. During parasite development inside the vector's midgut, promastigotes move towards the stomodeal valve, a mechanism that is crucial for transmission. It has been reported that the sugar meal acquired by sand flies during feeding between bloodmeals is essential for the development and migration of parasites. We demonstrated that the distribution of Leishmania mexicana parasites was affected by the sugar meals obtained by the sand flies. Promastigote migration towards the cardia region seems to be only partially based on the stimuli provided by sugar molecules. In the absence of sugars, significant amounts of parasites developed in the hindgut. In addition, sugar meals were important for the survival of sand flies, especially during blood digestion, presumably supporting their energy requirements.

Macrophage priming is dispensable for NLRP3 inflammasome activation and restriction of Leishmania amazonensis replication.

The NLRP3 inflammasome is activated in response to multiple stimuli and triggers activation of caspase-1 (CASP1), IL-1ß production, and inflammation. NLRP3 activation requires two signals. The first leads to transcriptional regulation of specific genes related to inflammation, and the second is triggered when pathogens, toxins, or specific compounds damage cellular membranes and/or trigger the production of reactive oxygen species (ROS). Here, we assess the requirement of the first signal (priming) for the activation of the NLRP3 inflammasome in bone marrow-derived macrophages (BMDMs) infected with Leishmania amazonensis. We found that BMDMs express the inflammasome components NLRP3, ASC, and CASP1 at sufficient levels to enable the assembly and activation of NLRP3 inflammasome in response to infection. Therefore, priming was not required for the formation of ASC specks, CASP1 activation (measured by fluorescent dye FAM-YVAD), and restriction of L. amazonensis replication via the NLRP3 inflammasome. By contrast, BMDM priming was required for CASP1 cleavage (p20) and IL-1ß secretion, because priming triggers robust up-regulation of pro-IL-1ß and CASP11 that are important for efficient processing of CASP1 and IL-1ß. Taken together, our data shed light into the cellular and molecular processes involved in activation of the NLRP3 in macrophages by Leishmania, a process that is important for the outcome of Leishmaniasis.

Effect of Two Different Isolates of Leishmania mexicana in the Production of Cytokines and Phagocytosis by Murine Dendritic Cells.

Species of the genus Leishmania are the causal agents of leishmaniasis, a disease with diametrically different clinical manifestations that have been attributed to the species and host immune response. Some Leishmania species, including Leishmania mexicana, are capable of causing both localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). Therefore, it is possible that intraspecific differences may exist that contribute to the development of distinct clinical forms. Dendritic cells (DC) are important host cells of Leishmania spp. parasites, and cytokine production and phagocytosis upon infection with the parasite are significant for the outcome of the disease. In the present study we analyzed the production of IL-12, TNF-α, and IL-10 by DC infected with L. mexicana amastigotes isolated from a patient with LCL (amastigote = Lac) and from a patient with DCL (amastigote = Diact) by murine DC. Furthermore, we compared the frequency of phagocytosis of L. mexicana amastigotes of each isolate by fluorescence and optical microscopy and by flow cytometry. We show that the infection of DC with Diact amastigotes elicited the secretion of IL-10, TNF-α, and IL-12 by DC to a major extent as compared to the infection with Lac amastigotes. On the other hand, Lac and Diact amastigotes were similarly phagocytosed by DC, but interestingly there were more vacuoles in DC infected with Diact amastigotes. Our results suggest that isolates from a same species of Leishmania, such as L. mexicana, with different degrees of virulence according to the clinical manifestation they cause, differ in their capacity to elicit cytokine production and form vacuoles in DC.

Automated identification of flagella from videomicroscopy via the medial axis transform.

Ubiquitous in eukaryotic organisms, the flagellum is a well-studied organelle that is well-known to be responsible for motility in a variety of organisms. Commonly necessitated in their study is the capability to image and subsequently track the movement of one or more flagella using videomicroscopy, requiring digital isolation and location of the flagellum within a sequence of frames. Such a process in general currently requires some researcher input, providing some manual estimate or reliance on an experiment-specific heuristic to correctly identify and track the motion of a flagellum. Here we present a fully-automated method of flagellum identification from videomicroscopy based on the fact that the flagella are of approximately constant width when viewed by microscopy. We demonstrate the effectiveness of the algorithm by application to captured videomicroscopy of Leishmania mexicana, a parasitic monoflagellate of the family Trypanosomatidae. ImageJ Macros for flagellar identification are provided, and high accuracy and remarkable throughput are achieved via this unsupervised method, obtaining results comparable in quality to previous studies of closely-related species but achieved without the need for precursory measurements or the development of a specialised heuristic, enabling in general the automated generation of digitised kinematic descriptions of flagellar beating from videomicroscopy.