Immucillins Impair Leishmania (L.) infantum chagasi and Leishmania (L.) amazonensis Multiplication In Vitro

Chemotherapy against visceral leishmaniasis is associated with high toxicity and drug resistance. Leishmania parasites are purine auxotrophs that obtain their purines from exogenous sources. Nucleoside hydrolases release purines from nucleosides and are drug targets for anti-leishmanial drugs, absent in mammal cells. We investigated the substrate specificity of the Leishmania (L.) donovani recombinant nucleoside hydrolase NH36 and the inhibitory effect of the immucillins IA (ImmA), DIA (DADMe-ImmA), DIH (DADMe-ImmH), SMIH (SerMe-ImmH), IH (ImmH), DIG (DADMe-ImmG), SMIG (SerMe-ImmG) and SMIA (SerME-ImmA) on its enzymatic activity. The inhibitory effects of immucillins on the in vitro multiplication of L. (L.) infantum chagasi and L. (L.) amazonensis promastigotes were determined using 0.05–500 μM and, when needed, 0.01–50 nM of each drug. The inhibition on multiplication of L. (L.) infantum chagasi intracellular amastigotes in vitro was assayed using 0.5, 1, 5 and 10 μM of IA, IH and SMIH. The NH36 shows specificity for inosine, guanosine, adenosine, uridine and cytidine with preference for adenosine and inosine. IA, IH, DIH, DIG, SMIH and SMIG immucillins inhibited L. (L.) infantum chagasi and L. (L.) amazonensis promastigote growth in vitro at nanomolar to micromolar concentrations. Promastigote replication was also inhibited in a chemically defined medium without a nucleoside source. Addition of adenosine decreases the immucillin toxicity. IA and IH inhibited the NH36 enzymatic activity (Ki = 0.080 μM for IA and 0.019 μM for IH). IA, IH and SMIH at 10 μM concentration, reduced the in vitro amastigote replication inside mice macrophages by 95% with no apparent effect on macrophage viability. Transmission electron microscopy revealed global alterations and swelling of L. (L.) infantum chagasi promastigotes after treatment with IA and IH while SMIH treatment determined intense cytoplasm vacuolization, enlarged vesicles and altered kinetoplasts. Our results suggest that IA, IH and SMIH may provide new chemotherapy agents for leishmaniasis.     

Interaction of Leishmania (L.) chagasi with the Vero cell line

The Vero cell line, a non-phagocytic cell, has supported the intracellular mechanism of Leishmania (L.) chagasi. This strain (MHOM/BR/501/MS00) was isolated from a human case of visceral leishmaniasis in Mato Grosso do Sul, Brazil and cultivated in Schneider's Drosophila medium with 20% of heat inactivated fetal calf serum. It was allowed to infect the Vero cells at a ratio of 10 to 20 promastigotes per cell. Within six hours of incubation, promastigote forms were found attached to Vero cells without any particular orientation. The number of amastigotes per cell increased during the incubation period. Results showed that promastigotes of L. (L.) chagasi could interact, transform to amastigote forms and multiply in non-phagocytic cells, demonstrating a new model to study the intracellular cycle of this protozoan.     

Oxidative stress of liver in hamsters infected with Leishmania (L.) chagasi

Oxidative stress as a mediator of hepatic tissue damage concurrent with Leishmania (L.) chagasi infection was investigated. Chemiluminescence in liver supernatant of hamsters infected with Leishmania (L.) chagasi showed a ratio of 1.53/ mg protein and 2.10/liver weight 90 days after infection when compared with the control. The malondialdehyde (MDA) levels also increased significantly both with and without addition of Fe3+/ascorbic acid in the reaction mixture, with a ratio of 2.12 and 1.55/mg protein or 2.91 and 2.12/liver weight, respectively. The parasite burden in the spleen, as a measure of infection severity, was 9.1+/-1.33 x 10(8) parasites/organ. On the 10th day of infection, the chemiluminescence also was significantly higher in infected hamsters than in the controls (ratio = 1.36/mg protein or 1.34/liver weight); however, the MDA levels were not different from those of controls. After 90 days of infection, significant correlations were observed between chemiluminescence and MDA concentration with and without the presence of Fe3+/ascorbic acid (r = 0.54, P = 0.0001; r = 0.56, P = 0.0001; respectively). The high infection/control ratio of both chemiluminescence and MDA concentration and the significant correlation between those events strongly indicate the occurrence of oxidative stress and lipid peroxidation as a mechanism of liver damage in cases of chronic infection by L. chagasi. The significant increase in chemiluminescence at 10 days of infection demonstrates that oxidative stress occurs very early, first consuming the antioxidants and then inducing lipid peroxidative damage later in the chronic stage of this disease.     

Leishmania (L.) infantum chagasi isolated from skin lesions of patients affected by non-ulcerated cutaneous leishmaniasis lead to visceral lesion in hamsters

In Central America, Leishmania (L.) infantum chagasi infection causes visceral leishmaniasis (VL) and non-ulcerated cutaneous leishmaniasis (NUCL). The aim of the present study was to evaluate the course of an experimental infection in hamsters caused by L. (L.) infantum chagasi isolated from patients affected by NUCL compared with a strain isolated from a patient with VL. Stationary phase parasites in culture were inoculated through subcutaneous and intraperitoneal routes in hamsters. Following the post-infection times, a histopathological study, parasite load and cytokine determination in skin from the cutaneous inoculation site and viscera were performed. Animals subcutaneously infected with the different strains did not develop macroscopic lesions at the inoculation site, and the histopathological changes in the dermis were very slight. Regarding the histopathological study of the viscera, we observed the portal mononuclear inflammatory infiltrate, the presence of nodules in the hepatic parenchyma and the proliferation of macrophages in the spleen, which increased over the infection course. Overall, the parasite load in the liver and spleen and in the total IgG titres in the sera of infected hamster showed an increase with the time of infection, regardless of the route of inoculation. Regarding cellular immunity, we did not observe an increase or decrease in pro- and anti-inflammatory cytokines compared to the healthy control, except for IL-10, which was evident in the infected animals. The data showed that strains isolated from NUCL cause visceral lesions in the hamsters regardless of the route of inoculation, and they were similar to parasites isolated from VL humans.     

Stage-specific pathways of Leishmania infantum chagasi entry and phagosome maturation in macrophages

The life stages of Leishmania spp. include the infectious promastigote and the replicative intracellular amastigote. Each stage is phagocytosed by macrophages during the parasite life cycle. We previously showed that caveolae, a subset of cholesterol-rich membrane lipid rafts, facilitate uptake and intracellular survival of virulent promastigotes by macrophages, at least in part, by delaying parasitophorous vacuole (PV)-lysosome fusion. We hypothesized that amastigotes and promastigotes would differ in their route of macrophage entry and mechanism of PV maturation. Indeed, transient disruption of macrophage lipid rafts decreased the entry of promastigotes, but not amastigotes, into macrophages (P<0.001). Promastigote-containing PVs were positive for caveolin-1, and co-localized transiently with EEA-1 and Rab5 at 5 minutes. Amastigote-generated PVs lacked caveolin-1 but retained Rab5 and EEA-1 for at least 30 minutes or 2 hours, respectively. Coinciding with their conversion into amastigotes, the number of promastigote PVs positive for LAMP-1 increased from 20% at 1 hour, to 46% by 24 hours, (P<0.001, Chi square). In contrast, more than 80% of amastigote-initiated PVs were LAMP-1+ at both 1 and 24 hours. Furthermore, lipid raft disruption increased LAMP-1 recruitment to promastigote, but not to amastigote-containing compartments. Overall, our data showed that promastigotes enter macrophages through cholesterol-rich domains like caveolae to delay fusion with lysosomes. In contrast, amastigotes enter through a non-caveolae pathway, and their PVs rapidly fuse with late endosomes but prolong their association with early endosome markers. These results suggest a model in which promastigotes and amastigotes use different mechanisms to enter macrophages, modulate the kinetics of phagosome maturation, and facilitate their intracellular survival.     

Experimental infection parameters in Galea spixii (Rodentia: Caviidae) with Leishmania infantum chagasi

In order to better understand the epidemiological transmission network of leishmaniasis, an endemic disease in Northeast Brazil, we investigated the susceptibility of Spix yellow-toothed cavies (Galea spixii) to the Leishmania infantum chagasi parasite. Nine cavies were experimentally infected, separated into three groups and monitored at 30, 90 and 180 days, respectively. Amastigotes were identified in the spleen slides of two cavies killed 180 days after infection. Antibodies against the L. i. chagasi were identified in one of the cavies. This demonstrates that G. spixii is in fact capable of maintaining a stable infection by L. i. chagasi without alterations in biochemical and hematological parameters of the host and without perceivable micro and macroscopic lesions.     

Use of a Recombinant Cysteine Proteinase from Leishmania (Leishmania) infantum chagasi for the Immunotherapy of Canine Visceral Leishmaniasis

Background

A recombinant cysteine proteinase from Leishmania (Leishmania) infantum chagasi (rLdccys1) was previously shown to induce protective immune responses against murine and canine visceral leishmaniasis. These findings encouraged us to use rLdccys1 in the immunotherapy of naturally infected dogs from Teresina, Piauí, a region of high incidence of visceral leishmaniasis in Brazil.

Methodology/Principal Findings

Thirty naturally infected mongrel dogs displaying clinical signs of visceral leishmaniasis were randomly divided in three groups: one group received three doses of rLdccys1 in combination with the adjuvant Propionibacterium acnes at one month interval between each dose; a second group received three doses of P. acnes alone; a third group received saline. The main findings were: 1) dogs that received rLdccys1 with P. acnes did not display increase of the following clinical signs: weight loss, alopecia, onychogryphosis, cachexia, anorexia, apathy, skin lesions, hyperkeratosis, ocular secretion, and enlarged lymph nodes; they also exhibited a significant reduction in the spleen parasite load in comparison to the control dogs; 2) rLdccys1-treated dogs exhibited a significant delayed type cutaneous hypersensitivity elicited by the recombinant antigen, as well as high IgG2 serum titers and low IgG1 serum titers; sera from rLdccys1-treated dogs also contained high IFN-γ and low IL-10 concentrations; 3) control dogs exhibited all of the clinical signs of visceral leishmaniasis and had low serum IgG2 and IFN-γ levels and high concentrations of IgG1 and IL-10; 4) all of the dogs treated with rLdccys1 were alive 12 months after treatment, whereas dogs which received either saline or P. acnes alone died within 3 to 7 months.

Conclusions/Significance

These findings illustrate the potential use of rLdccys1 as an additional tool for the immunotherapy of canine visceral leishmaniasis and support further studies designed to improve the efficacy of this recombinant antigen for the treatment of this neglected disease.     

Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB/c mice against Leishmania chagasi and Leishmania amazonensis challenge

Leishmania chagasi and Leishmania amazonensis are the etiologic agents of different clinical forms of human leishmaniasis in South America. In an attempt to select candidate antigens for a vaccine protecting against different Leishmania species, the efficacy of vaccination using Leishmania ribosomal proteins and saponin as adjuvant was examined in BALB/c mice against challenge infection with both parasite species. Mice vaccinated with parasite ribosomal proteins purified from Leishmania infantum plus saponin showed a specific production of IFN-γ, IL-12 and GM-CSF after in vitro stimulation with L. infantum ribosomal proteins. Vaccinated mice showed a reduction in the liver and spleen parasite burdens after L. chagasi infection. After L. amazonensis challenge, vaccinated mice showed a decrease of the dermal pathology and a reduction in the parasite loads in the footpad and spleen. In both models, protection was correlated to an IL-12-dependent production of IFN-γ by CD4⁺ and CD8⁺ T cells that activate macrophages for the synthesis of NO. In the protected mice a decrease in the parasite-mediated IL-4 and IL-10 responses was also observed. In mice challenged with L. amazonensis, lower levels of anti-parasite-specific antibodies were detected. Thus, Leishmania ribosomal proteins plus saponin fits the requirements to compose a pan-Leishmania vaccine.     

In vitro and experimental therapeutic studies of the calcium channel blocker bepridil: detection of viable Leishmania (L.) chagasi by real-time PCR

The need for novel and efficacious drugs against neglected parasitic diseases, such as Leishmaniasis and American Trypanosomiasis, is certainly apparent. In this work, we evaluated the in vitro potential of the calcium channel blocker bepridil against Leishmania spp. and Trypanosoma cruzi parasites and exploited an experimental assay using a hamster model with Leishmania (L.) chagasi, with a real-time PCR method for therapeutic evaluation. Bepridil was in vitro effective against promastigotes and intracellular amastigotes of L. (L.) chagasi, with 50% inhibitory concentration (IC₅₀) values of 3.81 and 21.55 μM, respectively. Leishmania (L.) amazonensis, L. (L.) major and L. (V.) braziliensis promastigotes and T. cruzi trypomastigotes were also susceptible to bepridil, with in vitro selectivity toward parasites and IC₅₀ values in the range of 3 to 7 μM. The mammalian cytotoxicity using LLC-MK2 cells resulted in an IC₅₀ value of 62.67 μM. However, bepridil showed lack of activity at 12 mg/kg in the experimental hamster model infected with L. (L.) chagasi parasites. However, the real-time PCR was a promising tool for the accurate and fast quantification of RNA of living parasites in the liver and spleen of infected hamsters after treatment, eliminating time-consuming light microscopy evaluations.     

Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae)

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37 masculineC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.     

Leishmania chagasi/infantum: further investigations on Leishmania tropisms in atypical cutaneous and visceral leishmaniasis foci in Central America

In Central America, apparently genetically identical Leishmania chagasi/infantum parasites cause cutaneous (CL) and visceral leishmaniasis (VL), the latter being more frequent in young children. The present study investigated if there were pathology-related differences in virulence between Honduran CL and VL strains using Mediterranean L. infantum strains as a reference. Macrophage infectivity and serum sensitivity, properties thought to be associated with virulence, were similar between CL and VL strains from both regions. Attention focused on the genome organisation of genes for two candidate virulence factors: Leishmania mitogen activated protein kinase (LMPK) and cysteine proteinase b (Cpb). Interestingly, the Mediterranean strains exhibited restriction enzyme polymorphisms associated with tropism for both LMPK and Cpb genes whereas no differences were observed for the Honduran strains. We also report relative genetic homogeneity of the Honduran strains as compared to the Mediterranean strains and discuss it in terms of the probable origin for the Central American L. chagasi/infantum.     

Generation of Luciferase-Expressing Leishmania infantum chagasi and Assessment of Miltefosine Efficacy in Infected Hamsters through Bioimaging

Background

The only oral drug available for the treatment of leishmaniasis is miltefosine, described and approved for visceral leishmaniasis in India. Miltefosine is under evaluation for the treatment of cutaneous leishmaniasis in the Americas although its efficacy for the treatment of human visceral leishmaniasis caused by Leishmania infantum chagasi has not been described. Drug efficacy for visceral leishmaniasis is ideally tested in hamsters, an experimental model that mimics human disease. Luciferase has been validated as a quantitative tool for the determination of parasite burden in experimental leishmaniasis. However, there are no reports of luciferase detection in the model of progressive visceral leishmaniasis in hamsters. Therefore, the aims of this study were to generate recombinant Leishmania infantum chagasi expressing the luciferase gene (Lc-LUC), characterize the biological properties of this transgenic line as compared with the wild-type parasites and evaluate miltefosine effectiveness in Lc-LUC infected hamsters.

Methodology/Principal Findings

A transgenic line containing a luciferase encoding gene integrated into the ribosomal DNA locus was obtained and shown to produce bioluminescence which correlated with the number of parasites. Lc-LUC growth curves and susceptibility to pentavalent antimony and miltefosine in vitro were indistinguishable from the wild-type parasites. The effectiveness of pentavalent antimony was evaluated in Lc-LUC infected hamsters through bioimaging and determination of Leishman Donovan Units. Both methods showed concordant results. Miltefosine was effective in the treatment of Lc-LUC-infected hamsters, as demonstrated by the reduction in parasite burden in a dose-dependent manner and by prolongation of animal survival.

Conclusions/Significance

Luciferase expressing parasites are a reliable alternative for parasite burden quantification in hamsters with advantages such as the possibility of estimating parasite load before drug treatment and therefore allowing distribution of animals in groups with equivalent mean parasite burden. Miltefosine was effective in vivo in an L. infantum chagasi experimental model of infection.     

In vitro and in vivo activity of meglumine antimoniate produced at Farmanguinhos-Fiocruz, Brazil, against Leishmania (Leishmania) amazonensis, L (L.) chagasi and L (Viannia) braziliensis

The leishmanicidal activity of four batches of meglumine antimoniate, produced in Farmanguinhos-Fiocruz, Brazil (TAMs), was assessed and compared to Glucantime(R)-Aventis Pharma Ltda. Using the amastigote-like in vitro model, the active concentrations of Sb v varied from 10microg/ml to 300 microg/ml for L. (L.) chagasi and from 50microg/ml to 300microg/ml for L. (L.) amazonensis, with no statistically significant differences among the four batches of TAMs and Glucantime(R). The inhibitory concentrations (IC50) determined by the amastigote-infected macrophage model for TAM01/03 and Glucantime(R) were, respectively: 26.3microg/ml and 127.6microg/ml for L. chagasi, 15.4microg /ml and 22.9microg/ml for L. amazonensis, and 12.1microg/ml and 24.2microg/ml for L. (V.) braziliensis. The activities of the four batches of TAMs were confirmed in an in vivo model by assessing, during eight weeks skin lesions caused by L. braziliensis in hamster that were treated with 20mg Sb v/Kg/day for 30 consecutive days. The meglumine antimoniate produced by Farmanguinhos was as effective as the reference drug, Glucantime(R)-Aventis, against three species of Leishmania that are of medical importance in Brazil.     

Evaluation of an in vitro and in vivo model for experimental infection with Leishmania (Viannia) braziliensis and L. (V.) peruviana

Leishmania (Viannia) braziliensis and L. (V.) peruviana are two parasite species characterized by a very different pathogenicity in humans despite a high genetic similarity. We hypothesized previously that L. (V.) peruviana would descend from L. (V.) braziliensis and would have acquired its 'peruviana' character during the southward colonization and adaptation of the transmission cycle in the Peruvian Andes. In order to have a first appreciation of the differences in virulence between both species, we evaluated an in vitro and in vivo model for experimental infection. A procedure was adapted to enrich culture forms in infective stages and the purified metacyclics were used to infect macrophage cell lines and golden hamsters. The models were tested with 2 representative strains of L. (V.) braziliensis from cutaneous and mucosal origin respectively and 2 representative strains of L. (V.) peruviana from Northern and Southern Peru respectively. Our models were reproducible and sensitive enough to detect phenotypic differences among strains. We showed in vitro as well as in vivo that the L. (V.) braziliensis was more infective than L. (V.) peruviana. Furthermore, we found that in vitro infectivity patterns of the 4 strains analysed, were in agreement with the geographical structuring of parasite populations demonstrated in our previous studies. Further work is needed to confirm our results with more strains of different geographical origin and their specific clinical outcome. However, our data open new perspectives for understanding the process of speciation in Leishmania and its implications in terms of pathogenicity.     

Optimization of the 503 antigen induction strategy of Leishmania infantum chagasi expressed in Escherichia coli M15

Abstract

Leishmaniosis is a complex of diseases that can be fatal, if not given proper attention. Despite its relevance in the public health system, there is no vaccine capable of preventing the disease in humans so far and its treatment is expensive and aggressive to human health. The present study aims to optimize the induction parameters of the 503 Leishmania i. chagasi antigen expressed in recombinant Escherichia coli M15. The induction at different cell densities was evaluated in order to analyze the influence of the induction time on the yield of the protein of interest. In this segment, lactose and isopropyl-β-d-thiogalactopyranoside (IPTG) were used as inducer molecules, using various concentrations: 0.1?g/L, 1.0?g/L, and 10?g/L for lactose and 20?μM, 100?μM, 500?μM, and 1000?μM for IPTG. The results presented that the concentration of IPTG that obtained the higher antigen levels was that of 100?μM (0.087?g/L), a 10-fold lower concentration than was being previously used in this type of system and for lactose, it was 1?g/L (0.016?g/L). Thus, the induction with 100?μM allowed obtaining the antigen with a concentration 5.6 times higher than the lactose induction maximum concentration.     

Vaccination with L. infantum chagasi Nucleosomal Histones Confers Protection against New World Cutaneous Leishmaniasis Caused by Leishmania braziliensis

Background

Nucleosomal histones are intracellular proteins that are highly conserved among Leishmania species. After parasite destruction or spontaneous lysis, exposure to these proteins elicits a strong host immune response. In the present study, we analyzed the protective capability of Leishmania infantum chagasi nucleosomal histones against L. braziliensis infection using different immunization strategies.

Methodology/Principal Findings

BALB/c mice were immunized with either a plasmid DNA cocktail (DNA) containing four Leishmania nucleosomal histones or with the DNA cocktail followed by the corresponding recombinant proteins plus CpG (DNA/Protein). Mice were later challenged with L. braziliensis, in the presence of sand fly saliva. Lesion development, parasite load and the cellular immune response were analyzed five weeks after challenge. Immunization with either DNA alone or with DNA/Protein was able to inhibit lesion development. This finding was highlighted by the absence of infected macrophages in tissue sections. Further, parasite load at the infection site and in the draining lymph nodes was also significantly lower in vaccinated animals. This outcome was associated with increased expression of IFN-γ and down regulation of IL-4 at the infection site.

Conclusion

The data presented here demonstrate the potential use of L. infantum chagasi nucleosomal histones as targets for the development of vaccines against infection with L. braziliensis, as shown by the significant inhibition of disease development following a live challenge.