Leishmania (L.) amazonensis LaLRR17 increases parasite entry in macrophage by a mechanism dependent on GRP78.

Leishmaniases affect 12 million people worldwide. They are caused by Leishmania spp., protozoan parasites transmitted to mammals by female phlebotomine flies. During the life cycle, promastigote forms of the parasite live in the gut of infected sandflies and convert into amastigotes inside the vertebrate macrophages. The parasite evades macrophage's microbicidal responses due to virulence factors that affect parasite phagocytosis, survival and/or proliferation. The interaction between Leishmania and macrophage molecules is essential to phagocytosis and parasite survival. Proteins containing leucine-rich repeats (LRRs) are common in several organisms, and these motifs are usually involved in protein­protein interactions. We have identified the LRR17 gene, which encodes a protein with 6 LRR domains, in the genomes of several Leishmania species. We show here that promastigotes of Leishmania (L.) amazonensis overexpressing LaLRR17 are more infective in vitro. We produced recombinant LaLRR17 protein and identified macrophage 78 kDa glucose-regulated protein (GRP78) as a ligand for LaLRR17 employing affinity chromatography followed by mass spectrometry. We showed that GRP78 binds to LaLRR17 and that its blocking precludes the increase of infection conferred by LaLRR17. Our results are the first to report LRR17 gene and protein, and we hope they stimulate further studies on how this protein increases phagocytosis of Leishmania.

Assessing nystatin cream treatment efficacy against Leishmania (L.) amazonensis infection in BALB/c model.

The current scenario for cutaneous leishmaniasis treatment includes the use of first and second-choice drugs, both therapeutic strategies presenting several adverse effects and being related to an increment of treatment-refractory parasite strains. These facts encourage the search for new treatment approaches, including repositioning drugs, such as nystatin. Although in vitro assays show that this polyene macrolide compound has leishmanicidal activity, no in vivo evidence for a similar activity has been shown so far for the commercial nystatin cream formulation. This work assessed the effects of nystatin cream (25,000 IU/g) administered on mice in an amount to completely cover the paw surface of BALB/c mice infected with Leishmania (L.) amazonensis once a day, until a total of up to 20 doses. The data presented herein points to unequivocal evidence that treatment with this formulation causes a statistically significant reduction of swelling/edema in mice paws when compared to animal groups not submitted to this treatment regimen after the fourth week of infection: lesion sizes at the sixth (p = 0.0159), seventh (p = 0.0079) and eighth (p = 0.0079) week. Furthermore, swelling/edema reduction relates to a decrease in parasite load in the footpad (∼48%) and in draining lymph nodes (∼68%) at eight weeks post-infection. This is the first report of the effectiveness of nystatin cream used as a topical treatment in BALB/c model for cutaneous leishmaniasis.

Unsaturated lipids modulating the interaction of the antileishmanial isolinderanolide E with models of cellular membranes.

The present work evaluated the antiprotozoal activity of isolinderanolide E, isolated from the Brazilian plant Nectandra oppositifolia, against promastigote forms of Leishmania (Leishmania) amazonensis. The compound exhibited an EC50 value of 20.3 µM, similar to the positive control miltefosine (IC50 of 19.4 µM), and reduced toxicity to macrophages (CC50 > 200 µM). Based on these results, Langmuir monolayers of two unsaturated lipids: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), were employed as a model of mammalian and parasite membranes, respectively, to study the interaction of isolinderanolide E at a molecular level. The films were characterized with tensiometry (surface pressure-area isotherms and surface pressure-time curves), infrared spectroscopy, and Brewster angle microscopy (BAM). This compound changed the profile of the isotherms leading to fluid DOPC and DOPE monolayers, which were not able to attain rigid states even with compression. Infrared spectroscopy showed that the bioactive compound decreases the trans/gauche ratio conformers related to the molecular conformational disorder. BAM showed the formation of specific aggregates upon drug incorporation. In conclusion, isolinderanolide E changes the thermodynamic, mechanical, structural, and morphological characteristics of the monolayer of these unsaturated lipids, which may be essential to understand the action at the molecular level bioactives in biointerfaces.

Galectin-3 plays a protective role in Leishmania (Leishmania) amazonensis infection.

Leishmania (L.) amazonensis is one of the species responsible for the development of cutaneous leishmaniasis in South America. After entering the vertebrate host, L. (L.) amazonensis invades mainly neutrophils, macrophages and dendritic cells. Studies have shown that gal-3 acts as a pattern recognition receptor. However, the role of this protein in the context of L. (L.) amazonensis infection remains unclear. Here, we investigated the impact of gal-3 expression on experimental infection by L. (L.) amazonensis. Our data showed that gal-3 plays a role in controlling parasite invasion, replication and the formation of endocytic vesicles. Moreover, mice with gal-3 deficiency showed an exacerbated inflammatory response. Taken together, our data shed light to a critical role of gal-3 in the host response to infection by L. (L.) amazonensis.

Tetrahydrophthalazinone Inhibitor of Phosphodiesterase with In Vitro Activity against Intracellular Trypanosomatids.

The phosphodiesterase inhibitor tetrahydrophthalazinone NPD-008 was explored by phenotypic in vitro screening, target validation, and ultrastructural approaches against Trypanosoma cruzi NPD-008 displayed activity against different forms and strains of T. cruzi (50% effective concentration [EC50], 6.6 to 39.5 µM). NPD-008 increased cAMP levels of T. cruzi and its combination with benznidazole gave synergistic interaction. It was also moderately active against intracellular amastigotes of Leishmania amazonensis and Leishmania infantum, confirming a potential activity profile as an antitrypanosomatid drug candidate.

Eosinophils increase macrophage ability to control intracellular Leishmania amazonensis infection via PGD2 paracrine activity in vitro.

Clinical and experimental studies have described eosinophil infiltration in Leishmania amazonensis infection sites, positioning eosinophils strategically adjacent to the protozoan-infected macrophages in cutaneous leishmaniasis. Here, by co-culturing mouse eosinophils with L. amazonensis-infected macrophages, we studied the impact of eosinophils on macrophage ability to regulate intracellular L. amazonensis infection. Eosinophils prevented the increase in amastigote numbers within macrophages by a mechanism dependent on a paracrine activity mediated by eosinophil-derived prostaglandin (PG) D2 acting on DP2 receptors. Exogenous PGD2 mimicked eosinophil-mediated effect on managing L. amazonensis intracellular infection by macrophages and therefore may function as a complementary tool for therapeutic intervention in L. amazonensis-driven cutaneous leishmaniasis.

Assessing the composition of the plasma membrane of Leishmania (Leishmania) infantum and L. (L.) amazonensis using label-free proteomics.

Protozoan parasites of the genus Leishmania are causative agents of leishmaniasis, a wide range of diseases affecting 12 million people worldwide. The species L. infantum and L. amazonensis are etiologic agents of visceral and cutaneous leishmaniasis, respectively. Most proteome analyses of Leishmania have been carried out on whole-cell extracts, but such an approach tends to underrepresent membrane-associated proteins due to their high hydrophobicity and low solubility. Considering the relevance of this category of proteins in virulence, invasiveness and the host-parasite interface, this study applied label-free proteomics to assess the plasma membrane sub-proteome of L. infantum and L. amazonensis. The number of proteins identified in L. infantum and L. amazonensis promastigotes was 1168 and 1455, respectively. After rigorous data processing and mining, 157 proteins were classified as putative plasma membrane-associated proteins, of which 56 proteins were detected in both species, six proteins were detected only in L. infantum and 39 proteins were exclusive to L. amazonensis. The quantitative analysis revealed that two proteins were more abundant in L. infantum, including the glucose transporter 2, and five proteins were more abundant in L. amazonensis. The identified proteins associated with distinct processes and functions. In this regard, proteins of L. infantum were linked to metabolic processes whereas L. amazonensis proteins were involved in signal transduction. Moreover, transmembrane transport was a significant process among the group of proteins detected in both species and members of the superfamily of ABC transporters were highly represented. Interestingly, some proteins of this family were solely detected in L. amazonensis, such as ABCA9. GP63, a well-known virulence factor, was the only GPI-anchored protein identified in the membrane preparations of both species. Finally, we found several proteins with uncharacterized functions, including differentially abundant ones, highlighting a gap in the study of Leishmania proteins. Proteins characterization could provide a better biological understanding of these parasites and deliver new possibilities regarding the discovery of therapeutic targets, drug resistance and vaccine candidates.

Efficacy of Topical Treatment with (-)-Epigallocatechin Gallate, A Green Tea Catechin, in Mice with Cutaneous Leishmaniasis.

The treatment of leishmaniasis includes pentavalent antimony drugs but, because of the side effects, toxicity and cases of treatment failure or resistance, the search of new antileishmanial compounds are necessary. The aims of this study were to evaluate and compare the in vitro antileishmanial activity of four green tea catechins, and to assess the efficacy of topical (-)-epigallocatechin gallate in a cutaneous leishmaniasis model. The antileishmanial activity of green tea catechins was evaluated against intracellular amastigotes, and cytotoxicity was performed with human monocytic cell line. BALB/c mice were infected in the ear dermis with Leishmania (Leishmania) amazonensis and treated with topical 15% (-)-epigallocatechin gallate, intraperitoneal Glucantime, and control group. The efficacy of treatments was evaluated by quantifying the parasite burden and by measuring the lesions size. (-)-Epigallocatechin gallate and (-)-epigallocatechin were the most active compounds with IC50 values <59.6 µg/mL and with a selectivity index >1. Topical treatment with (-)-epigallocatechin gallate decreased significantly both lesion size and parasite burden (80.4% inhibition) compared to control group (p < 0.05), and moreover (-)-epigallocatechin gallate showed a similar efficacy to Glucantime (85.1% inhibition), the reference drug for leishmaniasis treatment.

Contribution of spinal cord glial cells to L. amazonensis experimental infection-induced pain in BALB/c mice.

BACKGROUND: The cellular and molecular pathophysiological mecha\nisms of pain processing in neglected parasitic infections such as leishmaniasis remain unknown. The present study evaluated the participation of spinal cord glial cells in the pathophysiology of pain induced by Leishmania amazonensis infection in BALB/c mice. METHODS: Mice received intra-plantar (i.pl.) injection of L. amazonensis (1 × 105) and hyperalgesia, and paw edema were evaluated bilaterally for 40 days. The levels of TNF-α and IL-1ß, MPO activity, and histopathology were assessed on the 40th day. ATF3 mRNA expression was assessed in DRG cells at the 30th day post-infection. Blood TNF-α and IL-1ß levels and systemic parasite burden were evaluated 5-40 days after the infection. At the 30th day post-infection L. amazonensis, the effects of intrathecal (i.t.) treatments with neutralizing antibody anti-CX3CL1, etanercept (soluble TNFR2 receptor), and interleukin-1 receptor antagonist (IL-1ra) on infection-induced hyperalgesia and paw edema were assessed. In another set of experiments, we performed a time course analysis of spinal cord GFAP and Iba-1 (astrocytes and microglia markers, respectively) and used confocal immunofluorescence and Western blot to confirm the expression at the protein level. Selective astrocyte (α-aminoadipate) and microglia (minocycline) inhibitors were injected i.t. to determine the contribution of these cells to hyperalgesia and paw edema. The effects of i.t. treatments with glial and NFκB (PDTC) inhibitors on spinal glial activation, TNF-α, IL-1ß, CX3CR1 and CX3CL1 mRNA expression, and NFκB activation were also evaluated. Finally, the contribution of TNF-α and IL-1ß to CX3CL1 mRNA expression was investigated. RESULTS: L. amazonensis infection induced chronic mechanical and thermal hyperalgesia and paw edema in the infected paw. Mechanical hyperalgesia was also observed in the contralateral paw. TNF-α, IL-1ß, MPO activity, and epidermal/dermal thickness increased in the infected paw, which confirmed the peripheral inflammation at the primary foci of this infection. ATF3 mRNA expression at the ipsilateral DRG of the infected paw was unaltered 30 days post-infection. TNF-α and IL-1ß blood levels were not changed over the time course of disease, and parasitism increased in a time-dependent manner in the ipsilateral draining lymph node. Treatments targeting CX3CL1, TNF-α, and IL-1ß inhibited L. amazonensis-induced ongoing mechanical and thermal hyperalgesia, but not paw edema. A time course of GFAP, Iba-1, and CX3CR1 mRNA expression indicated spinal activation of astrocytes and microglia, which was confirmed at the GFAP and Iba-1 protein level at the peak of mRNA expression (30th day). Selective astrocyte and microglia inhibition diminished infection-induced ipsilateral mechanical hyperalgesia and thermal hyperalgesia, and contralateral mechanical hyperalgesia, but not ipsilateral paw edema. Targeting astrocytes, microglia and NFκB diminished L. amazonensis-induced GFAP, Iba-1, TNF-α, IL-1ß, CX3CR1 and CX3CL1 mRNA expression, and NFκB activation in the spinal cord at the peak of spinal cord glial cells activation. CX3CL1 mRNA expression was also detected in the ipsilateral DRG of infected mice at the 30th day post-infection, and the i.t. injection of TNF-α or IL-1ß in naïve animals induced CX3CL1 mRNA expression in the spinal cord and ipsilateral DRG. CONCLUSIONS: L. amazonensis skin infection produces chronic pain by central mechanisms involving spinal cord astrocytes and microglia-related production of cytokines and chemokines, and NFκB activation contributes to L. amazonensis infection-induced hyperalgesia and neuroinflammation.

Nitric oxide-loaded chitosan nanoparticles as an innovative antileishmanial platform.

Leishmaniasis is a neglected tropical disease that demands for new therapeutic strategies due to adverse side effects and resistance development promoted by current drugs. Nitric oxide (NO)-donors show potential to kill Leishmania spp. but their use is limited because of their instability. In this work, we synthesize, characterize, and encapsulate S-nitroso-mercaptosuccinic acid into chitosan nanoparticles (NONPs) and investigate their activity on promastigotes and intracellular amastigotes of Leishmania (Leishmania) amazonensis. Cytotoxicity on macrophages was also evaluated. We verified that NONPs reduced both forms of the parasite in a single treatment. We also noticed reduction of parasitophorous vacuoles as an evidence of inhibition of parasite growth and resolution of infection. No substantial cytotoxicity was detected on macrophages. NONPs were able to provide a sustained parasite killing for both L. (L.) amazonensis infective stages with no toxicity on macrophages, representing a promising nanoplatform for cutaneous leishmaniasis.

Efficacy of lapachol on treatment of cutaneous and visceral leishmaniasis.

Leishmaniasis is one of the most important neglected diseases worldwide. It is a life-threatening disease and causes significant morbidity, long-term disability, and early death. Treatment involves disease control or use of intervention measures, although the currently used drugs require long-lasting therapy, and display toxicity and reduced efficacy. The use of natural products isolated from plants, such as lapachol, an abundant naphthoquinone naturally occurring in South American Handroanthus species (Tabebuia, Bignoniaceae), is a promising option for the treatment of leishmaniasis. In this study, we investigated the leishmanicidal activity of lapachol in vitro and in vivo against Leishmania infantum and L. amazonensis, causative agents of visceral and cutaneous leishmaniasis, respectively. Low cytotoxicity in HepG2 cells (3405.8 ±â€¯261.33 µM), good anti-Leishmania activity, and favorable selectivity indexes (SI) against promastigotes of both L. amazonensis (IC50 = 79.84 ±â€¯9.10 µM, SI = 42.65) and L. infantum (IC50 = 135.79 ±â€¯33.04 µM, SI = 25.08) were observed. Furthermore, anti-Leishmania activity assays performed on intracellular amastigotes showed good activity for lapachol (IC50 = 191.95 µM for L. amazonensis and 171.26 µM for L. infantum). Flow cytometric analysis demonstrated that the cytotoxic effect of lapachol in Leishmania promastigotes was caused by apoptosis-like death. Interestingly, the in vitro leishmanicidal effect of lapachol was confirmed in vivo in murine models of visceral and cutaneous leishmaniasis, as lapachol (25 mg/kg oral route for 24 h over 10 days) was able to significantly reduce the parasitic load in skin lesions, liver, and spleen, similar to amphotericin B, the reference drug. These results reinforce the therapeutic potential of lapachol, which warrants further investigations as an anti-leishmaniasis therapeutic.

Insights into the tracking of the cysteine proteinase B COOH-terminal polypeptide of Leishmania (Leishmania) amazonensis by surface plasmon resonance.

Leishmania (Leishmania) amazonensis has adaptive mechanisms to the host environment that are guided by its proteinases, including cysteine proteinase B (CPB), and primarily its COOH-terminal region (Cyspep). This work aimed to track the fate of Cyspep by surface plasmon resonance (SPR) of promastigotes and amastigotes to gain a greater understanding of the adaptation of this parasite in both hosts. This strategy consisted of antibody immobilization on a COOH1 surface, followed by interaction with parasite proteins and epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64). Pro-CPB and Cyspep were detected using specific polyclonal antibodies against a recombinant Cyspep in both parasite forms. The parasitic supernatants from amastigotes and promastigotes exhibited higher anti-Cyspep recognition compared with that in the subcellular fractions. As the supernatant of the promastigote cultures exhibited resonance unit values indicative of an effective with to E-64, this result was assumed to be Pro-CPB detection. Finally, after using three sequential SPR assay steps, we propose that amastigotes and promastigotes release Cyspep into the extracellular environment, but only promastigotes release this polypeptide as Pro-CPB.

The role of the P2X7 receptor in murine cutaneous leishmaniasis: aspects of inflammation and parasite control.

Leishmania amazonensis is the etiological agent of diffuse cutaneous leishmaniasis. The immunopathology of leishmaniasis caused by L. amazonensis infection is dependent on the pathogenic role of effector CD4+ T cells. Purinergic signalling has been implicated in resistance to infection by different intracellular parasites. In this study, we evaluated the role of the P2X7 receptor in modulating the immune response and susceptibility to infection by L. amazonensis. We found that P2X7-deficient mice are more susceptible to L. amazonensis infection than wild-type (WT) mice. P2X7 deletion resulted in increased lesion size and parasite load. Our histological analysis showed an increase in cell infiltration in infected footpads of P2X7-deficient mice. Analysis of the cytokine profile in footpad homogenates showed increased levels of IFN-γ and decreased TGF-ß production in P2X7-deficient mice, suggesting an exaggerated pro-inflammatory response. In addition, we observed that CD4+ and CD8+ T cells from infected P2X7-deficient mice exhibit a higher proliferative capacity than infected WT mice. These data suggest that P2X7 receptor plays a key role in parasite control by regulating T effector cells and inflammation during L. amazonensis infection.

Crotoxin stimulates an M1 activation profile in murine macrophages during Leishmania amazonensis infection.

American tegumentary leishmaniasis is caused by different species of Leishmania. This protozoan employs several mechanisms to subvert the microbicidal activity of macrophages and, given the limited efficacy of current therapies, the development of alternative treatments is essential. Animal venoms are known to exhibit a variety of pharmacological activities, including antiparasitic effects. Crotoxin (CTX) is the main component of Crotalus durissus terrificus venom, and it has several biological effects. Nevertheless, there is no report of CTX activity during macrophage - Leishmania interactions. Thus, the main objective of this study was to evaluate whether CTX has a role in macrophage M1 polarization during Leishmania infection murine macrophages, Leishmania amazonensis promastigotes and L. amazonensis-infected macrophages were challenged with CTX. MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] toxicity assays were performed on murine macrophages, and no damage was observed in these cells. Promastigotes, however, were affected by treatment with CTX (IC50 = 22·86 µg mL-1) as were intracellular amastigotes. Macrophages treated with CTX also demonstrated increased reactive oxygen species production. After they were infected with Leishmania, macrophages exhibited an increase in nitric oxide production that converged into an M1 activation profile, as suggested by their elevated production of the cytokines interleukin-6 and tumour necrosis factor-α and changes in their morphology. CTX was able to reverse the L. amazonensis-mediated inhibition of macrophage immune responses and is capable of polarizing macrophages to the M1 profile, which is associated with a better prognosis for cutaneous leishmaniasis treatment.

Studies in vitro on infectivity and sensitivity to antileishmanial drugs in New World Leishmania species transfected with the green fluorescent protein [pIR3(-)-eGFP].

Current chemotherapeutic agents for leishmaniasis have several disadvantages interfering with the effective treatment and therefore more and better antileishmanial drugs are needed. Discovery of candidates for leishmaniasis treatment requires not only accurate and precise methodologies but also well-known biological system to measure infectivity of parasites and antileishmanial activity of the new compounds. Significant variation in the in vitro and in vivo infectivity and sensitivity to established and experimental drugs in Leishmania strains are reported. This work reports the in vitro biological behavior and antileishmanial drugs sensitivity of different green fluorescent protein transfectant Leishmanias strains. The in vitro growth kinetic and infectivity to U937 cells vary slightly in the Leishmania transfectant strains in comparison with their correspondant wild-type. However, the insertion of the pIR3(-)-eGFP may affect the sensitivity of the parasites to meglumine antimoniate (MA) and miltefosine but not to amphotericin B (AMB) and pentamidine isethionate. In consequence, AMB or pentamidine isethionate but not MA or miltefosine should be used as antileishmanial control drugs during in vitro assays of antileishmanial activity. Furthermore, is recommended to test compounds against more than one Leishmania strain in order to verify that the antileihmanial activity of these compound is similar among species.

Evaluation on the leishmanicidal activity of 2-N,N'-dialkylamino-1,4-naphthoquinone derivatives.

Parasites of the Leishmania genus are the causative agents of leishmaniasis in humans, a disease that affects more than 12 million people worldwide. In this study was evaluated in vitro leishmanicidal activity of 2-N,N'-dialkylamino-1,4-naphthoquinone derivatives, covering a series of fourteen 2-N-morpholino-, 2-N-thiomorpholino, 2-N-piperidino, 2-N-(N4-methyl)-piperazino naphthoquinones (1a-n) derived from nor-lapachol and lawsone, belong to some other di-alkyaminoderivatives. At the cytotoxicity assay on peritoneal macrophages, the compounds possessing larger alkyl groups and N-methyl-piperazino moiety (1d, 1h, 1i and 1k), showed toxic effects similar to the standard drug used pentamidine. However, the other compounds of the series showed no deleterious effect on the host cell. Meanwhile, these cytotoxic derivatives (1d, 1h and 1i) had pronounced leishmanicidal activity against L. amazonensis promastigotes, and treatments with six other compounds (1d, 1e, 1f, 1h, 1k and 1n) had significant effect leishmanicidal against L. chagasi promastigotes. In the assay against L. chagasi amastigotes, eight compounds (1a, 1b, 1c, 1d, 1h, 1i, 1k and 1m) showed significant activity. Moreover, the compounds (1a, 1b, 1c, and 1m) showed effect against amastigotes of L. chagasi and not being toxic to the host cell. These data show the derivatives as promising substances for research leishmanicidal activity.

Distinct courses of infection with Leishmania (L.) amazonensis are observed in BALB/c, BALB/c nude and C57BL/6 mice.

Leishmania (L.) amazonensis [L. (L.) amazonensis] is widely distributed in Brazil and its symptomatic infections usually lead to few localized lesions and sometimes to diffuse cutaneous form, with nodules throughout the body, anergy to parasite antigens and poor therapeutic response. The variability of these manifestations draws attention to the need for studies on the pathophysiology of infection by this species. In this study, we analysed the course and immunological aspects of L. (L.) amazonensis infection in BALB/c and C57BL/6 strains, both susceptible, but displaying different clinical courses, and athymic BALB/c nude, to illustrate the role of T cell dependent responses. We analysed footpad thickness and parasite burden by in vivo imaging. Furthermore, we evaluated the cellular profile and cytokine production in lymph nodes and the inflammatory infiltrates of lesions. Nude mice showed delayed lesion development and less inflammatory cells in lesions, but higher parasite burden than BALB/c and C57BL/6. BALB/c and C57BL/6 mice had similar parasite burdens, lesion sizes and infiltrates until 6 weeks after infection, and after that C57BL/6 mice controlled the infection. Small differences in parasite numbers were observed in C57BL/6 macrophages in vitro, indicating that in vivo milieu accounts for most differences in infection. We believe our results shed light on the role of host immune system in the course of L. (L.) amazonensis infection by comparing three mouse strains that differ in parasitaemia and inflammatory cells.

Performance of a real time PCR for leishmaniasis diagnosis using a L. (L.) infantum hypothetical protein as target in canine samples.

Visceral leishmaniasis represents an important public health issue in different parts of the world, requiring that measures be put in place to control the spread of the disease worldwide. The canine leishmaniasis diagnosis is not easy based on clinical signs, since dogs may not develop the infection with recognizable signs. Thus, the laboratorial diagnosis is essential to ascertain the incidence and prevalence of canine leishmaniasis especially in areas with major control efforts. Although, the diagnosis can be performed by the use of different approaches, the molecular methods such as PCR have become an indispensable tool for leishmaniases diagnosis. A TaqMan assay for real-time PCR (Linj31-qPCR) was developed to determine the parasite occurrence in clinical cases of leishmaniasis. The assay targets an L. (L.) infantum hypothetical protein region. The specificity of the assay was verified by using Leishmania World Health Organization reference strains including parasites belonging to subgenus L. (Leishmania), subgenus L. (Viannia), other Leishmania species and Trypanosoma cruzi. The sensitivity was verified by using isolates of L. (L.) amazonensis and L. (L.) infantum. The usefulness of the assay for diagnosis was ascertained by testing 277 samples from dogs in regions endemic for visceral and/or cutaneous leishmaniasis and from regions in which leishmaniasis was not endemic in São Paulo State, Brazil. Diagnosis of canine visceral leishmaniasis (CVL) was determined on these animals by conventional PCR and three serological tests. The dog samples were divided into four groups. I, dogs with CVL (n = 101); II, dogs with other diseases and without CVL (n = 97); III, dogs with American cutaneous leishmaniasis (n = 7), and, IV, dogs without CVL (n = 72) from areas where leishmaniasis was not endemic as control group. Results indicated that Linj31-qPCR was able to identify parasites belonging to subgenus L. (Leishmania) with no cross-amplification with other parasite subgenera. The Linj31-qPCR detected Leishmania parasites DNA in 98% of samples from Group I. In conclusion this methodology can be used as routine diagnostic tools to detect parasites from subgenus Leishmania.

Leishmanicidal Activity of (+)-Phyllanthidine and the Phytochemical Profile of Margaritaria nobilis (Phyllanthaceae).

The effects of the Securinega alkaloid (+)-phyllanthidine on Leishmania (L.) amazonensis and the first chemical investigation of Margaritaria nobilis L.f. (Phyllanthaceae) are described. Treating the parasites with this alkaloid caused a dose-dependent reduction in promastigote growth of 67.68% (IC50 82.37 µg/mL or 353 µM) and in amastigote growth of 83.96% (IC50 49.11 µg/mL or 210 µM), together with ultrastructural alterations in the promastigotes. No cytotoxic effect was detected in mammalian cells (CC50 1727.48 µg/mL or CC50 5268 µM). Classical chromatographic techniques and spectral methods led to the isolation and identification of betulinic acid, kaempferol, corilagin, gallic acid and its methyl ester, besides (+)-phyllanthidine from M. nobilis leaves and stems. Margaritaria nobilis is another source of the small group of Securinega alkaloids, together with other Phyllanthaceae (Euphorbiaceae s.l.) species. The low toxicity to macrophages and the effects against promastigotes and amastigotes are suggestive that (+)-phyllanthidine could be a promising antileishmanial agent for treating cutaneous leishmaniasis.

Modulation of inflammation response to murine cutaneous Leishmaniasis by homeopathic medicines: Antimonium crudum 30cH.

BACKGROUND: Leishmaniasis is a zoonotic disease caused by protozoan parasites of the mononuclear phagocytic system. The modulation activity of these cells can interfere in the host/parasite relationship and influences the prognosis. METHODS: We evaluated the effects of the homeopathic preparation Antimonium crudum 30cH on experimental infection induced by Leishmania (L.) amazonensis. Male Balb/c mice were inoculated with 2 × 10(6)Leishmania (L.) amazonensis promastigotes into the footpad and, after 48 h (acute phase) or 60 days (chronic phase), cell population of lymphocytes and phagocytes present in the peritoneal washing fluid and spleen were analyzed by flow cytometry and histopathology, with histometry of the subcutaneous primary lesion, local lymph node and spleen. Immunohistochemistry was performed to quantify CD3 (T lymphocyte), CD45RA (B lymphocyte) and CD11b (phagocytes) positive cells. RESULTS: In treated mice, during the acute phase, there was significant increase of the macroscopic lesion, associated to inflammatory edema, as well increase in the number of free amastigotes and B lymphocytes inside the lesion. Increase of B lymphocytes (predominantly B-2 cells) was also seen in the local lymph node, spleen and peritoneum. In the chronic phase, the inflammatory process in the infection focus was reduced, with reduced phagocyte migration and peritoneal increase of B-1a cells (precursors of B-2 immunoglobulin producers cells) and T CD8+ cells. CONCLUSION: The treatment of mice with Antimonium crudum 30cH induced a predominantly B cell pattern of immune response in Leishmania (L.) amazonensis experimental infection, alongside the increase of free amastigote forms number in the infection site. The clinical significance of this study is discussed, further studies are suggested.