Identification of cytoplasmic soluble antigens related to the major surface antigens ofLeishmania braziliensis braziliensis andL. donovani chagasi

This study describes the identification of aqueous-soluble antigens inLeishmania promastigotes immunologically and biochemically closely related to the major surface antigen. Proteins from surface-iodinatedL. braziliensis braziliensis andL. donovani chagasi promastigotes, extracted and separated by partitioning in the detergent Triton X-114, were analyzed. Immunoblotting of the extracted proteins, using homologous antisera, showed recognition of a 72-kDa labeled, amphiphilic antigen ofL. b. braziliensis and a 65-kDa surface antigen ofL. d. chagasi. The respective homologous sera also recognized non-labeled hydrophilic antigens, similar in their apparent molecular weights to the major surface antigens. The amphiphilic and hydrophilic antigens of each species were found to share common antigenic determinants, inasmuch as monospecific antibodies that recognized the amphiphilic protein reacted with the hydrophilic antigen. Structural homology was also obtained in the peptide-digestion profiles of the amphiphilic and the respective hydrophilic major antigens. Zymogram assay showed that both amphibilic and hydrophilic fractions displayed proteolytic activity that could be directly attributed to the majorL. b. braziliensis andL. d. chagasi antigens. The hydrophilic antigens found in this study are probably not hydrolytic products of the surface antigens and occur in large quantities in the promastigote cytosol.     

Eosinophil chemotactic activity in Leishmania amazonensis promastigotes

 Tissue eosinophilia was observed in the subcutaneous tissue of mice shortly after their inoculation not only with living but also with lysed promastigotes of Leishmania amazonensis. Intraperitoneal inoculation of lysed promastigotes from five different Leishmania species (L. donovani, L. chagasi, L. tropica, L. amazonensis, and L. braziliensis) induced eosinophil accumulation in the mouse peritoneum. This eosinophil infiltration was also detected in C5-deficient AKR mice, indicating complement independent eosinophil chemotaxis by the parasite. The induced eosinophils were hypodense, suggesting activation of the cells. Finally, we demonstrated in vitro eosinophil chemotactic activity in the promastigote lysates using purified eosinophils and blind well chambers. These results suggest the presence of an eosinophil chemotactic factor in Leishmania, a protozoan parasite. Received: 6 November 1995 / Accepted: 31 January 1996     

Furazolidone is a selective in vitro candidate against Leishmania (L.) chagasi: an ultrastructural study

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 IC₅₀ values ranging between 0.47 and 0.73 μg/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.     

Antileishmanial activity and ultrastructural alterations of Leishmania (L.) chagasi treated with the calcium channel blocker nimodipine

In a search for novel antileishmanial drugs, we investigated the activity of the calcium channel blocker nimodipine against Leishmania spp. and explored the ultrastructural damages of parasites induced by nimodipine after a short period of incubation. Nimodipine was highly effective against promastigotes and intracellular amastigotes of Leishmania (L.) chagasi, with 50% inhibitory concentration values of 81.2 and 21.5 μM, respectively. Nimodipine was about fourfold more effective than the standard pentavalent antimony against amastigotes and showed a Selectivity Index of 4.4 considering its mammalian cells toxicity. Leishmania (L.) amazonensis and Leishmania (L.) major promastigotes were also susceptible to nimodipine in a range concentration between 31 and 128 μM. Ultrastructural studies of L. (L.) chagasi revealed intense mitochondria damage and plasma membrane blebbing, resulting in a leishmanicidal effect as demonstrated by the lack of mitochondrial oxidative metabolism. The amastigote-killing effect suggests other mechanism than macrophage activation, as no upregulation of nitric oxide was seen. This calcium channel blocker is an effective in vitro antileishmanial compound and if adequately studied could be used as a novel drug candidate or as a novel drug lead compound for drug design studies against leishmaniasis.     

Immune response induced by New World<Emphasis Type="Italic"> Leishmania</Emphasis> species in C57BL/6 mice

In the present study, C57BL/6 mice were inoculated with metacyclic Leishmania amazonensis or L. braziliensis promastigotes. While these animals were capable of controlling the infection by L. braziliensis, they developed chronic lesions with elevated numbers of parasites when infected by L. amazonensis. The differences in parasite control were associated with a decreased production of IFN- and TNF by lymph node cells from L. amazonensis-infected mice. Furthermore, these animals presented decreased spleen cell proliferation and activation of germinal centers. In addition, we compared the ability of these parasites to hydrolyze extracellular ATP and AMP. While the ATPase activity of both parasite species was similar, L. amazonensis promastigotes presented higher AMP hydrolytic activity. This increased activity may lead to an increased production of adenosine, which has been shown to present anti-inflammatory activity and may thus be involved in the establishment of the immunosuppression observed in mice infected by L. amazonensis.     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

Salivary gland homogenates from wild‐caught sand flies Lutzomyia flaviscutellata and Lutzomyia (Psychodopygus) complexus showed inhibitory effects on Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis infection in BALB/c mice

During the natural transmission of Leishmania parasites, the infected sand fly female regurgitates promastigotes into the host's skin together with its saliva. It has been reported that vector saliva contains immunomodulatory molecules that facilitate the establishment of infection. Thus, the main objective of this study was to evaluate the specificity of Lutzomyia (Lu.) flaviscutellata and Lu. (Psychodopygus) complexus salivas on the infectivity of Leishmania (L.) (Leishmania) amazonensis and L. (Viannia) braziliensis, respectively. BALB/c mice were inoculated into the skin of hind footpad with L. (L.) amazonensis and L. (V.) braziliensis promastigotes in the absence or presence of Lu. flaviscutellata and Lu. (P.) complexus salivary gland homogenates (SGHs). The evolution of the infection was evaluated by lesion size, histopathological analysis and determination of the parasite load in the skin biopsies collected from the site of infection at 4 and 8 weeks PI. The lesion size and the parasite load of both groups of mice infected in the presence of SGHs were smaller than the control groups. The histopathological features showed that the inflammatory reaction was less prominent in the groups of mice infected in the presence of both SGHs when compared to the control group. The results showed that the presence of SGHs of Lu. flaviscutellata and Lu. (P.) complexus led to induction of processes that were disadvantageous to parasite establishment during infection by L. (L.) amazonensis and L. (V.) braziliensis. An inhibitory effect on Leishmania infection could be observed in both groups inoculated with SGHs, especially when the SGH from Lu. (P.) complexus was used.     

Detection ofLeishmania parasites by DNA in situ hybridization with non-radioactive probes

In situ hybridization techniques develop rapidly into diagnostic tools of considerable value for detection of viruses and bacteria. Here we report the application of this technique for the detection ofLeishmania parasites. Biotin-labelled total promastigote DNA was hybridized to culturedLeishmania parasites and to blood and impression smears of infected mice. In promastigotes kinetoplasts were strongly stained, nuclei somewhat more diffuse. In amastigotes both nuclear and kinetoplast DNA hybridized strongly. Amastigotes were easily detected in tissue of infected mice by their stable configuration of kinetoplast and nuclei. Cross-hybridization was observed betweenLeishmania donovani andL. tropica, but not between these two andL. braziliensis orTrypanosoma cruzi. A minor aspecific staining of host cell nuclei in the smears did not interfere with the detectability of the parasites.     

PCR for identification of species causing American cutaneous leishmaniasis

Parasites of the complexes Leishmania (Leishmania) mexicana, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) chagasi coexist within the same endemic areas of the American Continent. They produce similar clinical manifestations, yet not all respond well to treatment with anti-leishmania drugs. Thus, high specificity and sensitivity are needed to improve diagnosis and treatment. We developed a highly specific and sensitive polymerase chain reaction based diagnostic method that permits the identification of parasites belonging to the genus Leishmania and the differentiation between parasites belonging to the L. (L.) mexicana and L. (V.) braziliensis complexes and the identification of species of the L. (L.) mexicana complex, such as L. (L.) mexicana, Leishmania (L.) amazonensis, and Leishmania (L.) venezuelensis. This PCR permits the specific identification of Leishmania species in tissues of patients with different clinical forms of leishmaniasis. Its high sensitivity and specificity allow a precise diagnosis in lesions of patients that harbor few parasites, where the microscopic evaluation is unreliable. Additionally, this PCR could be a valuable tool for the identification of Leishmania species in mammalian reservoirs and sand fly vectors present in the American Continent. This work was supported by the CONACyT 47256-M and DGAPA IN221806-3     

Nitric oxide production by Leishmania-infected macrophages and modulation by prostaglandin E2

Nitric oxide (NO), produced by the nitric oxide synthase (iNOS) enzyme, is the most-important molecule responsible for the killing of Leishmania parasites by macrophages. In previous work we have demonstrated that, after activation with recombinant human interferon-γ and/or bacterial lipopolysaccharide, human macrophages infected with Leishmania infantum are able to produce nitric oxide and to express nitric oxide synthase. The arachidonate derivative prostaglandin E₂ has been shown to modulate various macrophage activities, and in particular nitric oxide production, sometimes with opposite effects, related to experimental conditions. In this work we have evaluated nitric oxide release and parasite killing by peripheral blood-derived L. infantum-infected human macrophages in vitro stimulated with lipopolysaccharide and simultaneously treated with prostaglandin E₂. Experiments were also performed in the presence of the nitric oxide synthase inhibitor l-N ^G monomethylarginine (l-NMMA) and of the cyclooxygenase inhibitor indomethacin. Nitric oxide release in supernatants of macrophage cultures was measured by the Griess reaction for nitrites. Parasite killing was microscopically evaluated by fluorescent dyes. Results demonstrated that macrophages stimulated with lipopolysaccharide and treated with prostaglandin E₂ exhibited increased nitric oxide producation and parasite killing, which were significantly reduced by either l-NMMA or indomethacin. In indomethacin-treated macrophages, nitric oxide production and leishmanicidal ability were partially restored by the addition of exogenous prostaglandin E₂. Taken together, these results indicate that prostaglandin E₂ may be involved in nitric oxide production, and possibly in the host-protective immune response against Leishmania. Moreover, the demonstration of a stimulatory role of prostaglandin E₂ on nitric oxide production induced by intracellular pathogens in humans is interesting in the light of a possible pharmacological regulation of nitric oxide by modulation of prostaglandin E₂ synthesis. Received: 27 March 2001 / Accepted: 24 August 2001     

Selection and phenotype characterization of potassium antimony tartrate-resistant populations of four New World Leishmania species

In the present study, we selected in vitro populations of Leishmania Viannia guyanensis, L.V. braziliensis, L. Leishmania amazonensis and L.L. infantum chagasi that were resistant to potassium antimony tartrate (SbIII). The resistance index of these populations varied from 4- to 20-fold higher than that of their wild-type counterparts. To evaluate the stability of the resistance phenotype, these four resistant populations were passaged 37 to 47 times in a culture medium without SbIII. No change was observed in the resistance indexes of L.V. guyanensis (19-fold) and L.L. infantum chagasi (4-fold). In contrast, a decrease in the resistance index was observed for L.V. braziliensis (from 20- to 10-fold) and L.L. amazonensis (from 6- to 3-fold). None of the antimony-resistant populations exhibited cross-resistance to amphotericin B and miltefosine. However, the resistant populations of L.V. braziliensis, L.L. amazonensis and L.L. infantum chagasi were also resistant to paromomycin. A drastic reduction was observed in the infectivity in mice for the resistant L.V. guyanensis, L.L. amazonensis and L.V. braziliensis populations. The SbIII-resistant phenotype of L.V. braziliensis was stable after one passage in mice. Although the protocol of induction was the same, the SbIII-resistant populations showed different degrees of tolerance, stability, infectivity in mice and cross-resistance to antileishmanial drugs, depending on the Leishmania species.     

Killed but Metabolically Active Leishmania infantum as a Novel Whole-Cell Vaccine for Visceral Leishmaniasis

There are currently no effective vaccines for visceral leishmaniasis, the second most deadly parasitic infection in the world. Here, we describe a novel whole-cell vaccine approach using Leishmania infantum chagasi promastigotes treated with the psoralen compound amotosalen (S-59) and low doses of UV A radiation. This treatment generates permanent, covalent DNA cross-links within parasites and results in Leishmania organisms termed killed but metabolically active (KBMA). In this report, we characterize the in vitro growth characteristics of both KBMA L. major and KBMA L. infantum chagasi. Concentrations of S-59 that generate optimally attenuated parasites were identified. Like live L. infantum chagasi, KBMA L. infantum chagasi parasites were able to initially enter liver cells in vivo after intravenous infection. However, whereas live L. infantum chagasi infection leads to hepatosplenomegaly in mice after 6 months, KBMA L. infantum chagasi parasites were undetectable in the organs of mice at this time point. In vitro, KBMA L. infantum chagasi retained the ability to enter macrophages and induce nitric oxide production. These characteristics of KBMA L. infantum chagasi correlated with the ability to prophylactically protect mice via subcutaneous vaccination at levels similar to vaccination with live, virulent organisms. Splenocytes from mice vaccinated with either live L. infantum chagasi or KBMA L. infantum chagasi displayed similar cytokine patterns in vitro. These results suggest that KBMA technology is a potentially safe and effective novel vaccine strategy against the intracellular protozoan L. infantum chagasi. This approach may represent a new method for whole-cell vaccination against other complex intracellular pathogens.     

In vitro leishmanicidal activity of Tityus discrepans scorpion venom

Leishmania parasites are sensitive to peptides with antimicrobial and ion-channel inhibitory activity. Because scorpion venoms are rich sources of such peptides, the leishmanicidal effect of Tityus discrepans venom was investigated. A negative correlation between cell growth and venom concentration was observed for venom-treated cultures of Leishmania (L.) mexicana mexicana promastigotes; 50% growth inhibition was obtained at 0.4 μg/ml. Light microscopy showed rounded, highly vacuolated L. (L.) m. mexicana cells with impaired flagellar motion after 15 min of incubation at 35 μg/ml. Ultrastructural studies confirmed an intense cytoplasm vacuolation and also enlargement of the flagellar pocket. Survival rates for New World Leishmania promastigotes (75% venom effective concentration, μg/ml) obtained after acute (1 h) venom toxicity tests were: L. (L.) m. mexicana (2.3), Leishmania (V.) braziliensis (11.3), and Leishmania (L.) chagasi (56.2). Heat (90°C) treatment of venom and fraction TdII abolished most of their leishmanicidal effect. Acute toxicity assays performed with Sephadex G-50 fractions indicated that leishmanicidal activity is associated with the venom lowest molecular mass components (2.8–7.4 kDa), as determined by MALDI-TOF mass spectrometry.     

Murine epidermal Langerhans cells do not express inducible nitric oxide synthase

In Leishmania-infected macrophages (MΦ), the formation of reactive nitrogen intermediates by the inducible isoform of nitric oxide synthase (iNOS) is critical for the killing of the intracellular parasites. We have recently shown that, in addition to MΦ, epidermal Langerhans cells (LC) can phagocytose Leishmania major, but they do not allow parasite replication. Therefore, we analyzed whether LC and MΦ display the same leishmanicidal effector mechanism. Unlike MΦ, stimulation of unselected epidermal cells with interferon-γ/lipopoly-saccharide did not lead to the release of nitric oxide (NO), and inhibition of NO production had no effect on the rate of infection of LC. iNOS mRNA was clearly detectable in MΦ as well as unselected epidermal cells (the majority of which consists of keratinocytes) after stimulation with different cytokines. In contrast, pure LC obtained by single-cell picking from cytokine-activated or L. major-infected epidermal cells did not express iNOS mRNA. Addition of the NO donor S-nitroso-N-acetylpenicillamine to already-infected LC did not alter their rate of infection, indicating that LC do not utilize exogenous NO for the control of intracellular Leishmania. These results suggest that in the L. major-infected skin, activated MΦ and keratinocytes, but not LC have the ability to express iNOS activity. Therefore, an as yet unidentified, NO-independent mechanism appears to be responsible for the control of parasite replication in LC.     

Organization of H locus conserved repeats in <Emphasis Type="Italic">Leishmania (Viannia) braziliensis</Emphasis> correlates with lack of gene amplification and drug resistance

Resistance to antimonials is a major problem when treating visceral leishmaniasis in India and has already been described for New World parasites. Clinical response to meglumine antimoniate in patients infected with parasites of the Viannia sub-genus can be widely variable, suggesting the presence of mechanisms of drug resistance. In this work, we have compared L. major and L. braziliensis mutants selected in different drugs. The cross-resistance profiles of some cell lines resembled those of mutants bearing H locus amplicons. However, amplified episomal molecules were exclusively detected in L. major mutants. The analysis of the L. braziliensis H region revealed a strong conservation of gene synteny. The typical intergenic repeats that are believed to mediate the amplification of the H locus in species of the Leishmania sub-genus are partially conserved in the Viannia species. The conservation of these non-coding elements in equivalent positions in both species is indicative of their relevance within this locus. The absence of amplicons in L. braziliensis suggests that this species may not favour extra-chromosomal gene amplification as a source of phenotypic heterogeneity and fitness maintenance in changing environments.     

Intramuscular immunization with p36(LACK) DNA vaccine induces IFN-γ production but does not protect BALB/c mice against Leishmania chagasi intravenous challenge

Acute visceral leishmaniasis is a progressive disease caused by Leishmania chagasi in South America. The acquisition of immunity following infection suggests that vaccination is a feasible approach to protect against this disease. Since Leishmania homologue of receptors for activated C kinase (LACK) antigen is of particular interest as a vaccine candidate because of the prominent role it plays in the pathogenesis of experimental Leishmania major infection, we evaluated the potential of a p36(LACK) DNA vaccine in protecting BALB/c mice challenged with L. chagasi. In this study, mice received intramuscular (i.m.) or subcutaneous (s.c.) doses of LACK DNA vaccine. We evaluated the production of vaccine-induced cytokines and whether this immunization was able to reduce parasite load in liver and spleen. We detected a significant production of interferon gamma by splenocytes from i.m. vaccinated mice in response to L. chagasi antigen and to rLACK protein. However, we did not observe a reduction in parasite load neither in liver nor in the spleen of vaccinated animals. The lack of protection observed may be explained by a significant production of IL-10 induced by the vaccine.     

Distinct Roles for MyD88 and Toll-Like Receptor 2 during Leishmania braziliensis Infection in Mice

We have previously reported that Leishmania braziliensis infection can activate murine dendritic cells (DCs) and upregulate signaling pathways that are essential for the initiation of innate immunity. However, it remains unclear whether Toll-like receptors (TLRs) are involved in L. braziliensis-mediated DC activation. To address this issue, we generated bone marrow-derived DCs from MyD88^−/− and TLR2^−/− mice and examined their responsiveness to parasite infection. While wild-type DCs were efficiently activated to produce cytokines and prime naïve CD4⁺ T cells, L. braziliensis-infected MyD88^−/− DCs exhibited less activation and decreased production of interleukin-12 (IL-12) p40. Furthermore, MyD88^−/− mice were more susceptible to infection in that they developed larger and prolonged lesions compared to those in control mice. In sharp contrast, the lack of TLR2 resulted in an enhanced DC activation and increased IL-12 p40 production after infection. As such, L. braziliensis-infected TLR2^−/− DCs were more competent in priming naïve CD4⁺ T cells in vitro than were their controls, findings which correlated with an increased gamma interferon production in vivo and enhanced resistance to infection. Our results suggest that while MyD88 is indispensable for the generation of protective immunity to L. braziliensis, TLR2 seems to have a regulatory role during infection.Leishmaniasis is a vector-borne disease that has a great socioeconomic impact in many tropical and neotropical countries (40). Leishmania parasites multiply as flagellated promastigotes in the midguts of sand flies and are transmitted to the vertebrate host via the bites of parasite-carrying female flies (3, 22). The insult at the bite site initiates a strong neutrophil influx and parasite capture by these cells (38). Interestingly, some of the captured parasites remain viable, and these infected neutrophils actually facilitate the silent entry of parasites into macrophages (Mφs) (29), where parasites survive and replicate as intracellular amastigotes (3). The magnitude and nature of inflammatory responses at the infection site and the profile of subsequent T-cell responses determine the outcome of the infection. In South America, Leishmania braziliensis infection causes cutaneous leishmaniasis in most cases and mucocutanous leishmaniasis in some individuals. The latter is a severe and disfiguring form of the disease. At present, it remains unclear why the infection is controlled in some individuals but progressive in others (40).Dendritic cell (DC)-pathogen interactions are initiated by interaction between receptors on DCs and pathogen-associated molecular patterns, including lipopolysaccharide (LPS), glycolipids, and nucleic acids. Signals through Toll-like receptors (TLRs) can induce DC maturation and the production of proinflammatory cytokines (20, 39), thereby bridging the innate and adaptive immune responses (9). Upon ligand binding, downstream signaling of all TLRs (with the exception of TLR3) uses the adaptor protein MyD88 (32). Gene knockout studies in mice have suggested that TLR signaling is essential for the immune responses against Leishmania parasites (52). For example, MyD88 and TLR4 contribute to the control of Leishmania major infection in C57BL/6 mice (27, 33). TLR9 is involved in NK cell activation in animal models of visceral (Leishmania donovani) and cutaneous (L. major and L. braziliensis) leishmaniasis (30, 45), while TLR2 and TLR3 are required for the intracellular killing of L. donovani in gamma interferon (IFN-γ)-primed Mφs (15). Leishmania lypophosphoglycan (LPG), an abundant molecule in the surfaces of promastigotes, not only is a virulence factor for some Leishmania species (e.g., L. major and L. donovani) (49) but also acts as a ligand for TLR2-mediated signaling (5). However, different species of Leishmania display relatively high variations (biochemical modifications) in LPG molecules (7). In the case of L. braziliensis, the procyclic form of the parasite lacks side chain sugar substitutions on its LPG, whereas the metacyclic form appears to contain decreased amounts of LPG compared to other Leishmania species (47). On the DC surface, TLR2 is present as preexisting heterodimers of TLR2/1 and/or TLR2/6, recognizing triacylated and diacylated lipoproteins, respectively (51). TLR2 has been shown to be important for NK cell activation in vitro by purified L. major LPG (5); however, the functional roles of TLR2 remain largely unclear during both parasite-DC interactions and the course of Leishmania infection in vivo.Most inbred strains of mice are genetically resistant to L. braziliensis infection, due to the capacity of mice to establish a strong Th1 response (43). This self control of infection is accompanied by the selective expansion of IFN-γ-producing CD4⁺ T cells, which induce nitric oxide production in infected Mφs to promote parasite killing (3, 12). We have previously revealed that several key molecules in the innate immunity pathways (e.g., STAT1, STAT3, and ISG15) were upregulated in L. braziliensis-infected DCs and that such DCs were highly efficient in priming CD4⁺ T cells in vitro and in vivo (53). However, it remains unclear whether DC-Leishmania cell interactions in the absence of MyD88 and TLR2 impact T-cell functions and in vivo containment of infection. In the present study, we generated bone marrow-derived DCs (BMDCs) from MyD88^−/− and TLR2^−/− mice and examined their responsiveness to L. braziliensis infection. We found that infected MyD88^−/− DCs showed low levels of cell activation and interleukin-12 (IL-12) p40 production, which correlated with increased susceptibilities of these mice to L. braziliensis infection and decreased expansion of IFN-γ-producing and IL-17-producing CD4⁺ T cells during the course of infection. Given that most TLR pathways share MyD88 and that TLR2 is involved in LPG recognition, we then examined the role of TLR2 in L. braziliensis recognition. Contrary to MyD88^−/− DCs, the lack of TLR2 enhanced DC activation, IL-12 p40 production, and T-cell priming in vitro. Consequently, TLR2^−/− mice were more resistant to infection than were the control mice, a finding that was associated with enhanced IFN-γ production in the draining lymph nodes (dLN). Collectively, our results show that while MyD88 is critical for L. braziliensis recognition in vitro and in vivo, TLR2 appears to have a regulatory role in modulating immune responses to the parasite.     

A new experimental culture medium for cultivation of Leishmania amazonensis: its efficacy for the continuous in vitro growth and differentiation of infective promastigote forms

Parasites from the genus Leishmania cause a variety of disease states in humans and other mammals in tropical and subtropical regions, which include cutaneous, mucocutaneous and visceral leishmaniasis. The elaboration of a culture medium for the in vitro cultivation of Leishmania spp., which promotes the growth and differentiation of the parasites, is an important tool for diagnosis, biochemical, biological and immunological studies in the genus. Herein, we have reported the development of a rapid, inexpensive and reliable monophasic culture medium. The novel medium, designated PBHIL, promoted an excellent parasite growth, generating high quantities of promastigotes with long-term viability, and was able to induce cellular differentiation of L. amazonensis promastigotes to the amastigote-like forms (93%). Additionally, we reported the influence of this novel medium on the biochemical characteristics of L. amazonensis and on the interaction of this parasite parasites with mammalian macrophages.     

Curcumin overcomes the inhibitory effect of nitric oxide on Leishmania

Upon Leishmania infection, macrophages are activated to produce nitrogen and oxygen radicals simultaneously. It is well established that the infected host cells rely on nitric oxide (NO) as the major weapon against the intracellular parasite. In India where leishmaniasis is endemic, the spice turmeric is used prolifically in food and for insect bites. Curcumin, the active principle of turmeric, is a scavenger of NO. This report shows that curcumin protects promastigotes and amastigotes of the visceral species, Leishmania donovani, and promastigotes of the cutaneous species, L. major, against the actions of S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and DETANONOate, which release NO, 3-morpholino-sydnonimine hydrochloride (SIN-1), which releases NO and superoxide, and peroxynitrite, which is formed from the reaction of NO with superoxide. Thus, curcumin, as an antioxidant, is capable of blocking the action of both NO and NO congeners on the Leishmania parasite.