Leishmania and the pathogenesis of HIV infection

Acquired immunodeficiency syndrome (AIDS) and leishmaniasis overlap in several parts of the world, and microorganisms responsible for these human diseases infect and replicate within the macrophage. Therefore, the opportunity that the pathogenesis of the human immunodeficiency virus (HIV) and Leishmania infections could be modulated within dually infected individual is optimized. The most prominent clinical feature of AIDS is the development of debilitating secondary infections induced by several opportunistic microorganisms, including protozoa. Michel Tremblay, Martin Olivier and Richard Bernier here focus on the recently reported information on the putative cofactor role that the intracellular pathogen of the genus Leishmania may play in the pathogenesis of HIV infection.     

Antigen-stimulated lymphokines from patients with cutaneous leishmaniasis induce monocyte killing of Leishmania major intracellular amastigotes

The specific immune response of patients with cutaneous leishmaniasis including the ability of their lymphokines to enhance the monocytes' leishmaniacidal activity was studied. In 16 patients with cutaneous leishmaniasis, their concanavalin A-induced lymphocyte proliferative responses, interferon-gamma and interleukin 2 activities and the ability of their concanavalin A-induced lymphokines to kill monocyte intracellular amastigotes were not different from normal controls. Antigen-stimulated lymphocyte cultures showed that 13 of 13 patients had an increased lymphocyte proliferative response; 11 of 16 produced interleukin 2 and 12 of 13 produced interferon-gamma; in addition, 10 of 11 of these antigen-induced supernatants increased the monocytes' killing of Leishmania major amastigotes. Antibody levels to parasite membrane antigens determined by radioimmunoassay showed that 8 of 13 titers were greater than 10 and 4 of 13 titers were 2 to 10 times higher than control. Our findings demonstrate that patients with cutaneous leishmaniasis elicit a specific immune response to L. major antigens and part of this response is the production of lymphokine capable of promoting monocyte killing of intracellular amastigotes.     

Sensitivity of Leishmania braziliensis promastigotes to meglumine antimoniate (glucantime) is higher than that of other Leishmania species and correlates with response to therapy in American tegumentary leishmaniasis

The first line drugs for the treatment of leishmaniasis are antimonial derivatives. Poor clinical response may be credited to factors linked to the host, the drug, or the parasite. We determined the sensitivity of Leishmania sp. promastigotes and amastigotes by counting parasites exposed to increasing concentrations of meglumine antimoniate (Glucantime). Leishmania braziliensis promastigotes were significantly more sensitive than those belonging to other species. The sensitivity of L. braziliensis isolates from patients with unfavorable clinical outcome, such as therapeutic failure or relapse, was significantly lower than those from patients who had clinical cure. Poor clinical response to therapy (therapeutic failure or relapse) was also associated with inadequate antimonial therapy. We also found a significant and positive correlation between promastigotes and intracellular amastigotes with regard to their in vitro susceptibilities to meglumine antimoniate. Our data provide evidence for an association between the sensitivity of promastigotes to antimonials in vitro and clinical response to therapy in American tegumentary leishmaniasis. The high sensitivity of the local L. braziliensis to meglumine antimoniate in vitro provides an explanation for the good clinical response of cutaneous leishmaniasis in the municipality of Rio de Janeiro, Brazil, even when low-dose regimens are employed.     

The diverse and dynamic nature of Leishmania parasitophorous vacuoles studied by multidimensional imaging

An important area in the cell biology of intracellular parasitism is the customization of parasitophorous vacuoles (PVs) by prokaryotic or eukaryotic intracellular microorganisms. We were curious to compare PV biogenesis in primary mouse bone marrow-derived macrophages exposed to carefully prepared amastigotes of either Leishmania major or L. amazonensis. While tight-fitting PVs are housing one or two L. major amastigotes, giant PVs are housing many L. amazonensis amastigotes. In this study, using multidimensional imaging of live cells, we compare and characterize the PV biogenesis/remodeling of macrophages i) hosting amastigotes of either L. major or L. amazonensis and ii) loaded with Lysotracker, a lysosomotropic fluorescent probe. Three dynamic features of Leishmania amastigote-hosting PVs are documented: they range from i) entry of Lysotracker transients within tight-fitting, fission-prone L. major amastigote-housing PVs; ii) the decrease in the number of macrophage acidic vesicles during the L. major PV fission or L. amazonensis PV enlargement; to iii) the L. amazonensis PV remodeling after homotypic fusion. The high content information of multidimensional images allowed the updating of our understanding of the Leishmania species-specific differences in PV biogenesis/remodeling and could be useful for the study of other intracellular microorganisms.     

Transgenic, fluorescent Leishmania mexicana allow direct analysis of the proteome of intracellular amastigotes

Investigating the proteome of intracellular pathogens is often hampered by inadequate methodologies to purify the pathogen free of host cell material. This has also precluded direct proteome analysis of the intracellular, amastigote form of Leishmania spp., protozoan parasites that cause a spectrum of diseases that affect some 12 million patients worldwide. Here a method is presented that combines classic, isopycnic density centrifugation with fluorescent particle sorting for purification by exploiting transgenic, fluorescent parasites to allow direct proteome analysis of the purified organisms. By this approach the proteome of intracellular Leishmania mexicana amastigotes was compared with that of extracellular promastigotes that are transmitted by insect vectors. In total, 509 different proteins were identified by mass spectrometry and database search. This number corresponds to approximately 6% of gene products predicted from the reference genome of Leishmania major. Intracellular amastigotes synthesized significantly more proteins with basic pI and showed a greater abundance of enzymes of fatty acid catabolism, which may reflect their living in acidic habitats and metabolic adaptation to nutrient availability, respectively. Bioinformatics analyses of the genes corresponding to the protein data sets produced clear evidence for skewed codon usage and translational bias in these organisms. Moreover analysis of the subset of genes whose products were more abundant in amastigotes revealed characteristic sequence motifs in 3'-untranslated regions that have been linked to translational control elements. This suggests that proteome data sets may be used to identify regulatory elements in mRNAs. Last but not least, at 6% coverage the proteome identified all vaccine antigens tested to date. Thus, the present data set provides a valuable resource for selection of candidate vaccine antigens.     

Cutaneous leishmaniasis infection in Balb/c mice using a Leishmania tropica strain isolated from Turkey.

Leishmania tropica, which is endemic in Turkey, is the causative agent of cutaneous leishmaniasis. Leishmania tropica promastigotes (2 x 10(7)) isolated from a patient with dermal leishmaniasis and reproduced in NNN medium were inoculated subcutaneously into the footpads of 10 Balb/c mice. Cutaneous leishmaniasis developed on the footpads of 4 mice approximately 45 days later. Leishmania tropica amastigotes were observed in smear slides and then cultivated in NNN medium. Balb/c mice are a suitable laboratory model for this isolate of L. tropica and thus a source of amastigotes for studies on the immunology, chemotherapy, and pathogenicity of cutaneous leishmaniasis.     

Concurrent cutaneous,visceral and ocular leishmaniasis caused by Leishmania (Viannia) braziliensis in a kidney transplant patient

Although cases of leishmaniasis co-infection have been described in acquired immunodeficiency syndrome patients as well as those who have undergone organ transplants, to our knowledge, the present report is the first documented case of simultaneous cutaneous, visceral and ocular leishmaniasis due to Leishmania (Viannia) braziliensis in a transplant patient. The patient had been using immunosuppressive drugs since receiving a transplanted kidney. The first clinical signs of leishmaniasis included fever, thoracic pain, hepatosplenomegaly, leucopenia and anemia. The cutaneous disease was revealed by the presence of amastigotes in the skin biopsy. After three months, the patient presented fever with conjunctive hyperemia, intense ocular pain and low visual acuity. Parasites isolated from iliac crest, aqueous humor and vitreous body were examined using a range of molecular techniques. The same strain of L. (V.) braziliensis was responsible for the different clinical manifestations. The immunosuppressive drugs probably contributed to the dissemination of Leishmania.     

Leishmanicidal activity of stearylamine-bearing liposomes in vitro

Liposomes consisting of stearylamine (SA) and egg yolk phosphatidylcholine (PC) were studied for their cytotoxic activity against freshly transformed promastigotes and intracellular amastigotes of Leishmania donovani, the causative agent of visceral leishmaniasis. More than 99% of the parasites of strain AG83 were killed within 60 min by treatment with 22 mol% SA-PC liposomes (132 microg/ml total lipids). This was further confirmed by incubating the liposome-treated promastigotes at 22 C for 96 hr. The killing activity of the liposomes progressively decreased with lowering lipid concentration. However, weak cytotoxic activity was still detected at 6.6 microg/ml lipids. Leishmanicidal activity of the liposomes became stronger with increasing SA content but was reduced with the incorporation of cholesterol in the liposomes. A similar cytotoxic effect was observed on other Indian strains of L. donovani, for example PKDL and DD8, as well as on species such as Leishmania donovani S1, Leishmania donovani infantum, Leishmania tropica, Leishmania amazonensis, and Leishmania mexicana. However, freshly transformed promastigotes appeared to be more susceptible than the ones subcultured. The strong leishmanicidal activity of SA-PC liposomes was also demonstrated toward intracellular L. donovani amastigotes. The SA-bearing vesicles could effectively inhibit the growth and multiplication of the parasites within the macrophages. The cytolytic activity of these liposomes on leishmanial parasites and low toxicity on host macrophages may, thus, find application in the therapy of leishmaniasis.     

Biphenylquinuclidines as inhibitors of squalene synthase and growth of parasitic protozoa

In this paper we describe the preparation of some biphenylquinuclidine derivatives and their evaluation as inhibitors of squalene synthase in order to explore their potential in the treatment of the parasitic diseases leishmaniasis and Chagas disease. The compounds were screened against recombinant Leishmania major squalene synthase and against Leishmania mexicana promastigotes, Leishmania donovani intracellular amastigotes and Trypanosoma cruzi intracellular amastigotes. Compounds that inhibited the enzyme, also reduced the levels of steroids and caused growth inhibition of L. mexicana promastigotes. However there was a lower correlation between inhibition of the enzyme and growth inhibition of the intracellular parasites, possibly due to delivery problems. Some compounds also showed growth inhibition of T. brucei rhodesiense trypomastigotes, although in this case alternative modes of action other than inhibition of SQS are probably involved.     

Identification and characterization of T cell-stimulating antigens from Leishmania by CD4 T cell expression cloning

Persistent immunity against Leishmania: infections in humans is mediated predominantly by CD4(+) T cells of the Th1 phenotype. Herein we report the expression cloning of eight Leishmania: Ags using parasite-specific T cell lines derived from an immune donor. The Ags identified by this technique include the flagellar proteins alpha- and beta-tubulin, histone H2b, ribosomal protein S4, malate dehydrogenase, and elongation factor 2, as well as two novel parasite proteins. None of these proteins have been previously reported as T cell-stimulating Ags from Leishmania: beta-tubulin-specific T cell clones generated against Leishmania: major amastigotes responded to Leishmania:-infected macrophages and dendritic cells. IFN-gamma enzyme-linked immunospot analysis demonstrated the presence of T cells specific for several of these Ags in PBMC from self-healing cutaneous leishmaniasis patients infected with either Leishmania: tropica or L. major. The responses elicited by Leishmania: histone H2b were particularly striking in terms of frequency of histone-specific T cells in PBMC (1 T cell of 6000 PBMC) as well as the percentage of responding donors (86%, 6 of 7). Ags identified by T cells from immune donors might constitute potential vaccine candidates for leishmaniasis.     

The surface membrane of Leishmania mexicana mexicana: comparison of amastigote and promastigote using freeze-fracture cytochemistry

The freeze fracture replica technique has been used to compare the plasma membranes of amastigote and promastigote stages of Leishmania mexicana mexicana with respect to intramembranous particle (integral protein) distribution and to beta-hydroxysterols content as revealed by the distribution of lesions induced by the polyene antibiotic filipin. Intramembranous particle (IMP) density was greater in promastigote than in amastigote plasma membranes. Intramembranous particles were more abundant in the protoplasmic face (PF) than in the exoplasmic face (EF) of promastigotes, but this situation was found to be reversed in amastigotes. Filipin-induced lesions in glutaraldehyde-fixed parasites indicated higher levels of beta-hydroxysterols in the amastigote than in the promastigote plasma membrane, and in the promastigote flagellar membrane than in the body membrane. Amphotericin B (a related polyene antibiotic used in chemotherapy of leishmaniasis) induced IMP aggregation in the PF of unfixed amastigotes but did not appear to influence sterol distribution as demonstrated by freeze-fracture of subsequently-fixed and filipin-treated organisms.     

Specificity of the rapid rK39 antigen-based immunochromatographic test Kalazar Detect(r) in patients with cutaneous leishmaniasis in Brazil

The aim of this study was to evaluate the specificity of a rapid immunochromatographic test that was developed to detect antibodies against the rK39 antigen for the diagnosis of visceral leishmaniasis (VL). This evaluation was performed using sera from patients with a confirmed diagnosis of active cutaneous leishmaniasis. The sera from 272 patients with a confirmed diagnosis of localised cutaneous leishmaniasis (CL) who resided in an area endemic for Leishmania braziliensis in Brazil were obtained before the initiation of antileishmanial treatment. Kalazar Detect^(r)(InBios, Seattle, WA) recombinant K39 antigen-based immunochromatographic strips were used according to the manufacturer''s instructions. The test results were evaluated independently by two examiners in sequential order. The positive controls for the test included five serum samples from five patients with parasitologically confirmed diagnosis of VL caused by Leishmania infantum in Brazil. Overall, 100% of the samples obtained from patients with CL were negative, confirming the absence of a serological cross-reaction for individuals with cutaneous disease when these patients were evaluated using the rapid test. The lack of a cross-reaction in patients who were infected by parasites of the same genus highlights the specificity of the rK39 antigen for the diagnosis of VL in areas with the sympatric circulation of L. braziliensis and L. infantum.     

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world''s most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly¹. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited ²;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance ³. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.In vitro promastigotes ⁴ and axenic amastigotes assays⁵ are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).     

Antileishmanial activities and mechanisms of action of indole-based azoles

Two 3-(alpha-azolylbenzyl)indoles were evaluated against Leishmania amastigotes. Both compounds proved to be very active against intracellular and axenic amastigotes. The IC50 values of the imidazole derivative, PM17, and the triazole analogue, PM19, against L. mexicana axenic amastigotes, were 4.4 +/- 0.1 and 6.4 +/- 0.1 microM, respectively. Against intracellular amastigotes, PM17 produced a 66% decrease of leishmanial burden at 1 microM and PM19 had an IC50 of 1.3 microM. In a Balb/c mice model of L. major leishmaniasis, administration of PM17 led to a clear-cut parasite burden reduction: 98.9% in the spleen, 79.0% in the liver and 49.9% in the popliteal node draining the cutaneous lesion. As anticipated, it was brought to the fore that PM17 decreases ergosterol biosynthesis leading to membrane fungal cell alterations. Moreover it was proved that this imidazole antifungal agent induces a parasite burden-correlated decrease in interleukine-4 production both in the splenocyte and the popliteal node of the mouse.     

Diagnostic value of rK39 dipstick in zoonotic visceral leishmaniasis in Turkey

K39 is a repetitive immunodominant epitope in a kinesin-related protein expressed predominantly in the amastigotes of visceral Leishmania spp. Enzyme immunoassays of patient's sera with recombinant K39 (rK39) proved to be highly specific and sensitive for diagnosis of active visceral leishmaniasis (VL, kala-azar). The same assays in dipstick format were also found effective for diagnosis of both human VL (HVL) and canine VL (CanVL) in most endemic areas of these diseases. Fifty-eight human patients and 22 dogs, clinically suspected of kala-azar, were screened with rK39 dipstick in comparison with the conventional methods of diagnosis, i.e., microscopic examinations of bone marrow and lymph node aspirates and immunofluorescent antibody tests (IFAT), respectively. Sixteen patients and 12 dogs were found to be rK39 dipstick positive. The results were corroborated with those of parasitological examinations, except 1, rK39-positive but smear-negative, case in each group. IFAT of the 2 discordant cases gave positive results. The rK39 dipstick is thus reliable for diagnosis of both HVL and canVL cases in Turkey.     

Structure-activity relationship of antileishmanials neolignan analogues

Twenty-two synthetic analogues of neolignans comprising beta-ketoethers and beta-ketosulfides were obtained from condensation reactions among beta-bromoketones and phenols or thiophenols, respectively, in basic solutions, and assayed in vitro for activity against intracellular Leishmania amazonensis and Leishmania donovani amastigotes, the causative agents of cutaneous and visceral leishmaniasis. The highest selective activity was found for compounds with sulfur bridges, whereas beta-ketosulphoxides and beta-ketosulphones had significantly less growth inhibitory activity. Compounds 2-[(4-chlorophenyl)thio]propan-1-one and 1-(3,4-dimethoxy)-2-[(4-methylphenyl)thio]propan-1-one were the most potent, inhibiting the growth parasite species by over 90% at microgram/mL, but only compound 1-(3,4-dimethoxy)-2-[(4-methylphenyl)thio]propan-1-one was selectively toxic to the parasites.     

Detection of Leishmania (Viannia) DNA in blood from patients with American cutaneous leishmaniasis

The aim of the present study was to investigate the presence of Leishmania (Viannia) subgenus DNA in peripheral blood from patients with cutaneous lesions due to American cutaneous leishmaniasis. The buffy coats from 68 blood samples were analyzed by polymerase chain reaction using the MP1L/MP3H primers. The parasite DNA was detected in 2 (3.4%) out of 59 patients who had amastigotes present in samples taken from lesions. The presence of Leishmania (Viannia) DNA in the blood of these patients indicates hematogeneous parasite dissemination.     

Leishmania infantum Amastigotes Trigger a Subpopulation of Human B Cells with an Immunoregulatory Phenotype

Visceral leishmaniasis is caused by the protozoan parasites Leishmania infantum and Leishmania donovani. This infection is characterized by an uncontrolled parasitization of internal organs which, when left untreated, leads to death. Disease progression is linked with the type of immune response generated and a strong correlation was found between disease progression and serum levels of the immunosuppressive cytokine IL-10. Other studies have suggested a role for B cells in the pathology of this parasitic infection and the recent identification of a B-cell population in humans with regulatory functions, which secretes large amounts of IL-10 following activation, have sparked our interest in the context of visceral leishmaniasis. We report here that incubation of human B cells with Leishmania infantum amastigotes resulted in upregulation of multiple cell surface activation markers and a dose-dependent secretion of IL-10. Conditioned media from B cells incubated with Leishmania infantum amastigotes were shown to strongly inhibit CD4⁺ T-cell activation, proliferation and function (i.e. as monitored by TNF and IFNγ secretion). Blockade of IL-10 activity using a soluble IL-10 receptor restored only partially TNF and IFNγ production to control levels. The parasite-mediated IL-10 secretion was shown to rely on the activity of Syk, phosphatidylinositol-3 kinase and p38, as well as to require intracellular calcium mobilization. Cell sorting experiments allowed us to identify the IL-10-secreting B-cell subset (i.e. CD19⁺CD24⁺CD27^-). In summary, exposure of human B cells to Leishmania infantum amastigotes triggers B cells with regulatory activities mediated in part by IL-10, which could favor parasite dissemination in the organism.     

Fitness of Leishmania donovani Parasites Resistant to Drug Combinations

Drug resistance represents one of the main problems for the use of chemotherapy to treat leishmaniasis. Additionally, it could provide some advantages to Leishmania parasites, such as a higher capacity to survive in stress conditions. In this work, in mixed populations of Leishmania donovani parasites, we have analyzed whether experimentally resistant lines to one or two combined anti-leishmanial drugs better support the stress conditions than a susceptible line expressing luciferase (Luc line). In the absence of stress, none of the Leishmania lines showed growth advantage relative to the other when mixed at a 1:1 parasite ratio. However, when promastigotes from resistant lines and the Luc line were mixed and exposed to different stresses, we observed that the resistant lines are more tolerant of different stress conditions: nutrient starvation and heat shock-pH stress. Further to this, we observed that intracellular amastigotes from resistant lines present a higher capacity to survive inside the macrophages than those of the control line. These results suggest that resistant parasites acquire an overall fitness increase and that resistance to drug combinations presents significant differences in their fitness capacity versus single-drug resistant parasites, particularly in intracellular amastigotes. These results contribute to the assessment of the possible impact of drug resistance on leishmaniasis control programs.