Search

Current treatments for cutaneous and visceral leishmaniasis are toxic, expensive, difficult to administer, and limited in efficacy and availability. Disulfiram has primarily been used to treat alcoholism. More recently, it has shown some efficacy as therapy against protozoan pathogens and certain cancers, suggesting a wide range of biological activities. We used an ex vivo system to screen several thiuram disulfide compounds for antileishmanial activity. We found five compounds (compound identifier [CID] 7188, 5455, 95876, 12892, and 3117 [disulfiram]) with anti- Leishmania activity at nanomolar concentrations. We further evaluated these compounds with the addition of divalent metal salts based on studies that indicated these salts could potentiate the action of disulfiram. In addition, clinical studies suggested that zinc has some efficacy in treating cutaneous leishmaniasis. Several divalent metal salts were evaluated at 1 μM, which is lower than the normal levels of copper and zinc in plasma of healthy individuals. The leishmanicidal activity of disulfiram and CID 7188 were enhanced by several divalent metal salts at 1 μM. The in vitro therapeutic index (IVTI) of disulfiram and CID 7188 increased 12- and 2.3-fold, respectively, against L. major when combined with ZnCl 2 . The combination of disulfiram with ZnSO 4 resulted in a 1.8-fold increase in IVTI against L. donovani . This novel combination of thiuram disulfides and divalent metal ions salts could have application as topical and/or oral therapies for treatment of cutaneous and visceral leishmaniasis.

Leishmaniasis is a vector-borne zoonotic infection affecting people in tropical and subtropical regions of the world. Current treatments for cutaneous leishmaniasis are difficult to administer, toxic, expensive, and limited in effectiveness and availability. Here we describe the development and application of a medium-throughput screening approach to identify new drug candidates for cutaneous leishmaniasis using an e x vivo l ymph node e xplant c ulture (ELEC) derived from the draining lymph nodes of Leishmania major -infected mice. The ELEC supported intracellular amastigote proliferation and contained lymph node cell populations (and their secreted products) that enabled the testing of compounds within a system that mimicked the immunopathological environment of the infected host, which is known to profoundly influence parasite replication, killing, and drug efficacy. The activity of known antileishmanial drugs in the ELEC system was similar to the activity measured in peritoneal macrophages infected in vitro with L. major . Using the ELEC system, we screened a collection of 334 compounds, some of which we had demonstrated previously to be active against L. donovani , and identified 119 hits, 85% of which were confirmed to be active by determination of the 50% effective concentration (EC 50 ). We found 24 compounds (7%) that had an i n v itro t herapeutic i ndex (IVTI; 50% cytotoxic/effective concentration [CC 50 ]/EC 50 ) > 100; 19 of the compounds had an EC 50 below 1 μM. According to PubChem searchs, 17 of those compounds had not previously been reported to be active against Leishmania . We expect that this novel method will help to accelerate discovery of new drug candidates for treatment of cutaneous leishmaniasis.

Malnutrition is thought to contribute to more than one-third of all childhood deaths via increased susceptibility to infection. Malnutrition is a significant risk factor for the development of visceral leishmaniasis, which results from skin inoculation of the intracellular protozoan Leishmania donovani . We previously established a murine model of childhood malnutrition and found that malnutrition decreased the lymph node barrier function and increased the early dissemination of L. donovani . In the present study, we found reduced numbers of resident dendritic cells (conventional and monocyte derived) but not migratory dermal dendritic cells in the skin-draining lymph nodes of L. donovani -infected malnourished mice. Expression of chemokines and their receptors involved in trafficking of dendritic cells and their progenitors to the lymph nodes was dysregulated. C-C chemokine receptor type 2 (CCR2) and its ligands (CCL2 and CCL7) were reduced in the lymph nodes of infected malnourished mice, as were CCR2-bearing monocytes/macrophages and monocyte-derived dendritic cells. However, CCR7 and its ligands (CCL19 and CCL21) were increased in the lymph node and CCR7 was increased in lymph node macrophages and dendritic cells. CCR2-deficient mice recapitulated the profound reduction in the number of resident (but not migratory dermal) dendritic cells in the lymph node but showed no alteration in the expression of CCL19 and CCL21. Collectively, these results suggest that the malnutrition-related reduction in the lymph node barrier to dissemination of L. donovani is related to insufficient numbers of lymph node-resident but not migratory dermal dendritic cells. This is likely driven by the altered activity of the CCR2 and CCR7 chemoattractant pathways.

The acquisition of immunity following subclinical or resolved infection with the intracellular parasiteLeishmania donovanisuggests that vaccination could prevent visceral leishmaniasis (VL). The LACK (Leishmaniahomolog of receptors for activated C kinase) antigen is of interest as a vaccine candidate for the leishmaniases because of its immunopathogenic role in murineL. majorinfection. Immunization of mice with a truncated (24-kDa) version of the 36-kDa LACK antigen, delivered in either protein or DNA form, was found previously to protect against cutaneousL. majorinfection by redirecting the early T-cell response away from a pathogenic interleukin-4 (IL-4) response and toward a protective Th1 response. The amino acid sequence of theLeishmaniap36(LACK) antigen is highly conserved, but the efficacy of this vaccine antigen in preventing disease caused by strains other thanL. majorhas not been determined. We investigated the efficacy of a p36(LACK) DNA vaccine against VL because of the serious nature of this form of leishmaniasis and because it was unclear whether the LACK vaccine would be effective in a model where there was not a dominant pathogenic IL-4 response. We demonstrate here that although the LACK DNA vaccine induced a robust parasite-specific Th1 immune response (IFN-γ but not IL-4 production) and primed for an in vivo T-cell response to inoculated parasites, it did not induce protection against cutaneous or systemicL. donovanichallenge. Coadministration of IL-12 DNA with the vaccine did not enhance the strong vaccine-induced Th1 response or augment a protective effect.

Current therapy for leishmaniasis is unsatisfactory. Efficacious and safe oral therapy would be ideal. We examined the efficacy of SCH 56592, an investigational triazole antifungal agent, against cutaneous infection with Leishmania amazonensis and visceral infection with Leishmania donovani in BALB/c mice. Mice were infected in the ear pinna and tail with L. amazonensis promastigotes and were treated with oral SCH 56592 or intraperitoneal amphotericin B for 21 days. At doses of 60 and 30 mg/kg/day, SCH 56592 was highly efficacious in treating cutaneous disease, and at a dose of 60 mg/kg/day, it was superior to amphotericin B at a dose of 1 mg/kg/day. The means of tail lesion sizes were 0.32 ± 0.12, 0.11 ± 0.06, 0.17 ± 0.07, and 0.19 ± 0.08 mm for controls, SCH 56592 at 60 and 30 mg/kg/day, and amphotericin B recipients, respectively ( P = 0.0003, 0.005, and 0.01, respectively). Parasite burden in draining lymph nodes confirmed these efficacy findings. In visceral leishmaniasis due to L. donovani infection, mice treated with SCH 56592 showed a 0.5- to 1-log-unit reduction in parasite burdens in the liver and the spleen compared to untreated mice. Amphotericin B at 1 mg/kg/day was superior to SCH 56592 in the treatment of visceral infection, with a 2-log-unit reduction in parasite burdens in both the liver and spleen. These studies indicate very good activity of SCH 56592 against cutaneous leishmaniasis due to L. amazonensis infection and, to a lesser degree, against visceral leishmaniasis due to L. donovani infection in susceptible BALB/c mice.

Current therapy for leishmaniasis is unsatisfactory because parenteral antimonial salts and pentamidine are associated with significant toxicity and failure rates. We examined the efficacy of KY62, a new, water-soluble, polyene antifungal, against cutaneous infection with Leishmania amazonensis and against visceral infection with Leishmania donovani in susceptible BALB/c mice. Mice were infected with L. amazonensis promastigotes in the ear pinna and in the tail and were treated with KY62 or amphotericin B. The cutaneous lesions showed a remarkable response to therapy with KY62 at a dose of 30 mg per kg of body weight per day. At this dose, the efficacy of KY62 was equivalent to or better than that of amphotericin B at 1 to 5 mg/kg/day. Mice infected intravenously with 10 7 L. donovani promastigotes and treated with KY62 showed a 4-log reduction in the parasite burden in the liver and spleen compared to untreated mice. These studies indicate potent activity of KY62 against experimental cutaneous leishmaniasis caused by L. amazoniensis and against experimental visceral leishmaniasis caused by L. donovani .

The Syrian golden hamster ( Mesocricetus auratus ) is uniquely susceptible to a variety of intracellular pathogens and is an excellent model for a number of human infectious diseases. The molecular basis for this high level of susceptibility is unknown, and immunological studies related to this model have been limited by the lack of available reagents. In this report we describe the cloning and sequence analysis of portions of the Syrian hamster interleukin 2 (IL-2), IL-4, gamma interferon (IFN-γ), tumor necrosis factor alpha, IL-10, IL-12p40, and transforming growth factor β cDNAs. In addition, we examined the cytokine response to infection with the intracellular protozoan Leishmania donovani in this animal model. Sequence analysis of the hamster cytokines revealed 69 to 93% homology with the corresponding mouse, rat, and human nucleotide sequences and 48 to 100% homology with the deduced amino acid sequences. The hamster IFN-γ, compared with the mouse and rat homologs, had an additional 17 amino acids at the C terminus that could decrease the biological activity of this molecule and thus contribute to the extreme susceptibility of this animal to intracellular pathogens. The splenic expression of these genes in response to infection with L. donovani , the cause of visceral leishmaniasis (VL), was determined by Northern blotting. VL in the hamster is a progressive, lethal disease which very closely mimics active human disease. In this model there was pronounced expression of the Th1 cytokine mRNAs, with transcripts being detected as early as 1 week postinfection. Basal expression of IL-4 in uninfected hamsters was prominent but did not increase in response to infection with L. donovani . IL-12 transcript expression was detected at low levels in infected animals and paralleled the expression of IFN-γ. Expression of IL-10, a potent macrophage deactivator, increased throughout the course of infection and could contribute to the progressive nature of this infection. These initial studies are the first to examine the molecular immunopathogenesis of a hamster model of VL infection and indicate that progressive disease in this model of VL is not associated with early polarization of the splenic cellular immune response toward a Th2 phenotype and away from a Th1 phenotype.

Infection with the protozoan Leishmania donovani can cause serious visceral disease or subclinical infection in humans. To better understand the pathogenesis of this dichotomy, we have investigated the host cellular immune response to cutaneous or visceral infection in a murine model. Mice infected in the skin developed no detectable visceral parasitism, whereas intravenous inoculation resulted in hepatosplenomegaly and an increasing visceral parasite burden. Spleen cells from mice with locally controlled cutaneous infection showed strong parasite-specific proliferative and gamma interferon (IFN-γ) responses, but spleen cells from systemically infected mice were unresponsive to parasite antigens. The in situ expression of IFN-γ, interleukin-4 (IL-4), IL-10, IL-12, and inducible nitric oxide synthase (iNOS) mRNAs was determined in the spleen, draining lymph node (LN), and cutaneous site of inoculation. There was considerably greater expression of IFN-γ and IL-12 p40 mRNAs in the LN draining a locally controlled cutaneous infection than in the spleen following systemic infection. Similarly, there was a high level of IFN-γ production by LN cells following subcutaneous infection but no IFN-γ production by spleen cells following systemic infection. Splenic IL-4 expression was transiently increased early after systemic infection, but splenic IL-10 transcripts increased throughout the course of visceral infection. IL-4 and IL-10 mRNAs were also increased in the LN following cutaneous infection. iNOS mRNA was detected earlier in the LN draining a cutaneous site of infection compared to the spleen following systemic challenge. Thus, locally controlled cutaneous infection was associated with antigen-specific spleen cell responsiveness and markedly increased levels of IFN-γ, IL-12, and iNOS mRNA in the draining LN. Progressive splenic parasitism was associated with an early IL-4 response, markedly increased IL-10 but minimal IL-12 expression, and delayed expression of iNOS.

The nature of the host cellular immune response largely determines the expression of disease following infection with the intracellular protozoans Leishmania spp. In experimental animals control and resolution of infection are mediated by gamma interferon and tumor necrosis factor alpha (TNF-alpha), whereas disease progression is associated with the production of interleukin 4 (IL-4), IL-5, IL-10, and transforming growth factor beta (TGF-beta). We have analyzed the profile of cytokine gene expression directly in the lesions of 13 patients with localized cutaneous leishmaniasis due to Leishmania mexicana. All but one patient had a single lesion, and the time of evolution ranged from 8 days to 18 months. Cytokine gene expression was quantitated by reverse transcriptase PCR and interpolation from a standard curve. Gamma interferon, TNF-alpha, IL-1 alpha, IL-6, IL-10, and TGF-beta gene expression was present in all samples. IL-3 and IL-4 gene expression was barely detectable in 1 and 3 of 13 samples, respectively. IL-2 and IL-5 mRNAs were not found. A significant increase in the expression of IL-1 alpha, TNF-alpha, IL-10, and TGF-beta was observed in late lesions (> or = 4 months) compared with that in early lesions (< or = 2 months). Because of their inhibitory effects on macrophage function, the expression of IL-10 and TGF-beta may play a role in the immunopathogenesis of chronic cutaneous leishmaniasis.

In regions where leishmaniasis is endemic, clinical disease is usually reported more frequently among males than females. This difference could be due to disparate risks of exposure of males and females, but gender-related differences in the host response to infection may also play a role. Experimental studies of the influence of gender onLeishmaniainfection have not included parasites of the subgenusViannia, which is the most common cause of cutaneous leishmaniasis in the Americas. Mice are not readily susceptible to infection byLeishmania(Viannia) spp., but cutaneous infection of hamsters withL.(V.)panamensisorL.(V.)guyanensisresulted in chronic lesions typical of the human disease caused by these parasites. Strikingly, infection of male hamsters resulted in significantly greater lesion size and severity, an increased rate of dissemination to distant cutaneous sites, and a greater parasite burden in the draining lymph node than infection in female animals. Two lines of evidence indicated this gender-related difference in disease evolution was determined at least in part by the sex hormone status of the animal. First, prepubertal male animals had smaller and/or less severe cutaneous lesions than adult male animals. Second, infection of testosterone-treated female animals resulted in significantly larger lesions than in untreated female animals. The increased severity of disease in male compared to female animals was associated with significantly greater intralesional expression of interleukin-4 (IL-4) (P= 0.04), IL-10 (P= 0.04), and transforming growth factor β (TGF-β) (P< 0.001), cytokines known to promote disease in experimental leishmaniasis. There was a direct correlation between the expression of TGF-β mRNA and lesion size (Spearman's correlation coefficient = 0.873;P< 0.001). These findings demonstrate an inherent risk of increased disease severity in male animals, which is associated with a more permissive immune response.

Visceral leishmaniasis caused by the intracellular parasiteLeishmania donovaniis a significant public health problem in many regions of the world. Because of its large genome and complex biology, developing a vaccine for this pathogen has proved to be a challenging task and, to date, protective recombinant vaccine candidates have not been identified. To tackle this difficult problem, we adopted a reductionist approach with the intention of identifying cDNA sequences in anL. donovaniamastigote cDNA library that collectively or singly conferred protection against parasite challenge in a murine model of visceral leishmaniasis. We immunized BALB/c mice with plasmid DNA isolated and pooled from 15 cDNA sublibraries (∼2,000 cDNAs/sublibrary). Following systemic challenge withL. donovani, mice immunized with 6 of these 15 sublibraries showed a significantly reduced (35- to 1,000-fold) hepatic parasite burden. Because of the complexity and magnitude of the sequential fractionation-immunization-challenge approach, we restricted our attention to the two sublibraries that conferred the greatest in vivo protection. From one of these two sublibraries, we identified several groups of cDNAs that afforded protection, including a set of nine novel cDNAs and, surprisingly, a group of five cDNAs that encodedL. donovanihistone proteins. At each fractionation step, the cDNA sublibraries or the smaller DNA fractions that afforded in vivo protection against the parasite also induced in vitro parasite-specific T helper 1 immune responses. Our studies demonstrate that immunization with sequential fractions of a cDNA library is a powerful strategy for identifying anti-infective vaccine candidates.

Immunity in human leishmaniasis is mediated by sensitized T lymphocytes; however, the antigens involved in eliciting this immunity have not been defined. We describe the generation of human T-lymphocyte clones derived from two patients with healed leishmaniasis. By use of one- and two-dimensional cellular immunoblotting techniques, we directly identified the parasite antigens recognized by these clones. To our knowledge, these are the first leishmanial antigens identified to which CD4+, gamma interferon-producing T cells from immune individuals have been shown to respond, the strategy may be of general use for the identification of antigens involved in immunity in this disease.