Description and ultrastructure of Leishmania zuckermani n. sp. amastigotes detected within the erythrocytes of the South African gecko Pachydactylus turneri Gray, 1864.

In erythrocytes recovered from blood of geckoes of the species Pachydactylus turneri collected in Gauteng Province, Republic of South Africa, leishmania zuckemani n. sp. were detected. Giemsa stained erythrocytes contained amastigotes, either single or numerous, in loose assemblies or in a compact rounded oggregates which may condense to become a round basophilic bodies with a central hollow. This new species of Leishmania differs from all previously described species in being almost exclusively parasitic in circulating erythrocytes. Three to seven amastigotes lodged all within one, or divided between several parasitophorous vacuoles were detected at the EM level. The amastigotes demonstrated essentially all the cytological components characteristic of leishmania species known to parasitize mammals. A point which emphasizes an already suggested close affiliation between mammalian and lizard Leishmania.     

Fusion between Leishmania amazonensis and Leishmania major parasitophorous vacuoles: live imaging of coinfected macrophages

Protozoan parasites of the genus Leishmania alternate between flagellated, elongated extracellular promastigotes found in insect vectors, and round-shaped amastigotes enclosed in phagolysosome-like Parasitophorous Vacuoles (PVs) of infected mammalian host cells. Leishmania amazonensis amastigotes occupy large PVs which may contain many parasites; in contrast, single amastigotes of Leishmania major lodge in small, tight PVs, which undergo fission as parasites divide. To determine if PVs of these Leishmania species can fuse with each other, mouse macrophages in culture were infected with non-fluorescent L. amazonensis amastigotes and, 48 h later, superinfected with fluorescent L. major amastigotes or promastigotes. Fusion was investigated by time-lapse image acquisition of living cells and inferred from the colocalization of parasites of the two species in the same PVs. Survival, multiplication and differentiation of parasites that did or did not share the same vacuoles were also investigated. Fusion of PVs containing L. amazonensis and L. major amastigotes was not found. However, PVs containing L. major promastigotes did fuse with pre-established L. amazonensis PVs. In these chimeric vacuoles, L. major promastigotes remained motile and multiplied, but did not differentiate into amastigotes. In contrast, in doubly infected cells, within their own, unfused PVs metacyclic-enriched L. major promastigotes, but not log phase promastigotes--which were destroyed--differentiated into proliferating amastigotes. The results indicate that PVs, presumably customized by L. major amastigotes or promastigotes, differ in their ability to fuse with L. amazonensis PVs. Additionally, a species-specific PV was required for L. major destruction or differentiation--a requirement for which mechanisms remain unknown. The observations reported in this paper should be useful in further studies of the interactions between PVs to different species of Leishmania parasites, and of the mechanisms involved in the recognition and fusion of PVs.     

Importance of lymphokines in the control of multiplication and dispersion of Leishmania donovani within liver macrophages of resistant and susceptible mice

Leishmania donovani is an obligate intracellular parasite of mammalian macrophages. The immunosuppressant cyclosporin A (CsA), which inhibits the production of interleukin (IL)-1, IL-2, and interferon-gamma, increased infections 3-fold without affecting expression of the Lsh gene. The objective of this study was to determine how activation of macrophages by lymphokines affects the multiplication and propagation of the parasite within liver macrophages. Susceptible C57BL/6J and resistant C57L/J mice were treated with 200 mg/kg CsA and then infected intravenously with 10(7) amastigotes. Two weeks later macrophages were collected from the liver by perfusion, plated on coverslips, and incubated for 4, 24, and 48 hr. The percentage of infected macrophages and the number of amastigotes/100 cells were determined after staining the cells with Giemsa's stain. The number of infected macrophages and amastigotes per macrophage was significantly greater in animals of both strains that had been treated with CsA. This study demonstrated clearly that lymphokines or other soluble mediators produced by T cells act, in part, to control infection by L. donovani by minimizing both multiplication within macrophages and their dispersion.     

Leishmania major: excreted factor, calcium ions, and the survival of amastigotes

Mouse macrophages infected with amastigotes of Leishmania major contain about 40% more intracellular exchangeable calcium than control macrophages. Similar elevation of intracellular exchangeable calcium was observed in macrophages engulfing red blood cells coated with purified excreted factor from L. major. The rate of cytolysis of red blood cells coated with excreted factor was significantly lower than that of uncoated controls. Excreted factor strongly binds calcium; thus, the possible role of a microenvironment rich in calcium bound to excreted factor within the phagolysosome in protecting the amastigotes may be considered.     

Control of infection with Leishmania major in susceptible BALB/c mice lacking the common gamma-chain for FcR is associated with reduced production of IL-10 and TGF-beta by parasitized cells

Previous studies have shown that the in vitro ligation of FcgammaRs with IgG-opsonized Leishmania amastigotes promotes IL-10 production by macrophages. In addition, infection of either BALB/c mice lacking the common gamma-chain of Fc receptors (FcgammaR(-/-)) or mice genetically altered to lack circulating Ab (J(H)D) with Leishmania pifanoi results in reduced and delayed lesion development and a deficit in the recruitment of inflammatory cells into infected lesions. We show in this study that FcgammaR(-/-) mice can control infection with Leishmania major and totally resolve cutaneous lesions. The ability to eventually control infection is not associated with a reduction in lesion inflammation or a reduction in the ability of Leishmania to parasitize cells through week 6 of infection. The immune response in healing FcgammaR(-/-) mice is associated with a reduction in numbers of cells producing Th2-type cytokines, including IL-4 and IL-10, but not an increase in numbers of IFN-gamma-producing cells characteristic of a dominant Th1-type response. Instead, we observe a reduction in levels of IL-10 and TGF-beta within infected lesions, including reduced levels of these cytokines within parasitized macrophages. Together, these results suggest that uptake of opsonized parasites via FcgammaRs may be a strong in vivo stimulus for the production of anti-inflammatory cytokines that play a role in susceptibility to infection.     

Leishmanicidal activity of Yucatecan medicinal plants on Leishmania species responsible for cutaneous leishmaniasis

The leishmanicidal activity of 15 extracts and 4 pure metabolites obtained from Urechites andrieuxii, Colubrina greggii, Dorstenia contrajerva, and Tridax procumbens was evaluated using the newly developed MTS ({3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay, optimized for promastigotes of Leishmania major, Leishmania tropica, and Leishmania aethiopica, as well as for L. aethiopica axenic amastigotes. The assay was then used for calculating the percentage of viable stationary phase parasites after a 24-hr treatment with each plant extract or pure metabolite. The 3 most active samples, 2 from C. greggii (NCG-5C and DCG-3A) and 1 from T. procumbens (TPZ-2A), showed LD50 values of 62.4, 7.2, and 18.5 microg/ml, respectively, on stationary promastigotes, and of 94.2, 27.1, and 95.2 microg/ml, on amastigotes of L. aethiopica. Moreover, TPZ-2A and DCG-3A significantly reduced the percentage of infected monocyte-derived macrophages (THP-I). The percentage of infected cells decreased from 69.9% +/- 2.5% to 20.8% +/- 2% when the cells were treated with the DCG-3A fraction and to 14.9% +/- 0.5% when treated with TPZ-2A, without significantly decreasing the number of human cells. These findings indicate the presence of potentially bioactive metabolites in the roots of C. greggii and in T. procumbens and reflect the importance of pursuing the bioassay-guided purification of these metabolites.     

The ultrastructure of the parasitophorous vacuole formed by Leishmania major

Protozoan parasites of Leishmania spp. invade macrophages as promastigotes and differentiate into replicative amastigotes within parasitophorous vacuoles. Infection of inbred strains of mice with Leishmania major is a well-studied model of the mammalian immune response to Leishmania species, but the ultrastructure and biochemical properties of the parasitophorous vacuole occupied by this parasite have been best characterized for other species of Leishmania. We examined the parasitophorous vacuole occupied by L. major in lymph nodes of infected mice and in bone marrow-derived macrophages infected in vitro. At all time points after infection, single L. major amastigotes were wrapped tightly by host membrane, suggesting that amastigotes segregate into separate vacuoles during replication. This small, individual vacuole contrasts sharply with the large, communal vacuoles occupied by Leishmania amazonensis. An extensive survey of the literature revealed that the single vacuoles occupied by L. major are characteristic of those formed by Old World species of Leishmania, while New World species of Leishmania form large vacuoles occupied by many amastigotes.     

Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi

The origin of Trypanosoma cruzi slender and broad forms found in the circulation of the mammalian host has remained obscure and, unlike what has been proposed for African trypanosomes, no precise form-function relationship has been ascribed to them. We show here that parasites circulating in the blood of infected animals display a high degree of polymorphism. Around 10% of the forms found circulating in mice during the acute phase of infection were amastigotes, and the other 90% included slender and broad trypomastigotes and intermediate forms between amastigotes and trypomastigotes. Slender trypomastigotes, from blood or cell culture, undergo extracellularly morphological rearrangements in which the parasites become gradually broader and transform into amastigotes. By scanning electron microscopy a progressive internalization of the flagellum and reorganization of the cell shape in a helical fashion were observed in parasites undergoing transformation. After 48 hr of extracellular incubation the parasite population consisted exclusively of amastigotes with a short protruding flagellum. The morphological changes were associated with the expression of different surface antigens defined by monoclonal antibodies: the trypomastigote-specific antigens Ssp-1 (a 100-120-150-Mr glycoprotein), Ssp-2 (a 70-Mr glycoprotein), Ssp-3 (undefined), and Ssp-4, an amastigote-specific surface antigen. Ssp-4 was also detected on intracellular amastigotes (in vitro and in vivo). We conclude that trypomastigotes are programmed to develop into amastigotes whether or not they enter cells, and that the differentiation can occur in the blood of the vertebrate host. These findings raise some questions regarding conventional views on the life cycle of T. cruzi.     

Monoclonal antibodies specific for the amastigote stage of Leishmania pifanoi. I. Characterization of antigens associated with stage- and species-specific determinants

Eight mAb were produced against membrane-enriched preparations of Leishmania pifanoi amastigotes either grown in axenic culture (P-1 through P-6) or isolated from macrophage cell culture (P-7 and P-8). Two mAb produced against promastigote membranes (P-9 and P-10) were found to be specific against this stage. Antibodies P-1 through P-8 on analysis by radioimmune binding only reacted with determinants present on amastigotes. mAb P-2, P-4, and P-8 also reacted with Leishmania amazonensis amastigotes but not promastigotes. No cross-reactions were found on any other species of Leishmania or with membranes of Trypanosoma cruzi epimastigotes or amastigotes. An indirect immunofluorescence assay using mAb P-1 through P-8 confirmed the stage specificity and binding to L. pifanoi axenically grown amastigotes, amastigotes within infected hamster tissue, and amastigotes within J774.1 macrophages. When Western blot analysis of amastigote membranes was conducted, one distinct group of molecules associated with L. pifanoi-specific determinants was identified. mAb P-1, P-3, P-5, P-7, and P-8 bound to molecules Mr 43 and 34 kDa. Promastigote-specific mAb P-9 recognized a diffuse pattern from 88 to greater than 200 kDa, and mAb P-10 localized a second class of proteins with Mr53 kDa. On immunoprecipitation of solubilized [35S]methionine-labeled amastigotes, mAb P-2 recognized a doublet of Mr 35 and 33 kDa and another doublet at Mr 17.5 and 13.5 kDa. mAb P-4 and P-7 each precipitated a band at Mr 34 kDa. These studies indicate that antigenically the axenically cultured amastigote is closely related to macrophage-derived amastigote. These mAb and/or purified protein Ag may be useful in studying stage differentiation, monitoring transformation, and for further taxonomic, epidemiologic, and immunologic studies of New World leishmaniasis.     

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.     

Leishmania amazonensis: humoral response to amastigote excreted-antigens in murine leishmaniasis

With the purpose of studying the antigenic role that factors excreted by Leishmania amastigotes might have during murine infection, immunoblots were carried out with sera from C57BL/6 and BALB/c mice infected with two strains of Leishmania (L.) amazonensis, NR and IFLA/BR. Both strains differ widely in virulence in BALB/c mice. BALB/c but not C57BL/6 sera recognized several excretion products. The excreted antigens showed a strong response towards IgG1 and IgG2a isotypes whilst they reacted only weakly against IgG2b and IgG3. A low-molecular weight antigen (about 20 kDa) excreted by both Leishmania strains was strongly recognized by IgG1 from BALB/c mice sera infected with IFLA/BR, the most virulent strain. Sera from NR infected mice were incapable of recognizing this antigen in spite of its presence in NR excreted products. The results indicate that the humoral immune response to excreted antigens of amastigotes depends on both the host genetic background and the parasite strain.     

Applications of recombinant Leishmania amazonensis expressing egfp or the beta-galactosidase gene for drug screening and histopathological analysis.

Leishmania amazonensis recombinants expressing the enhanced green fluorescent protein (egfp) gene or beta-galactosidase gene (lacZ) were constructed for drug screening and histopathological analysis. The egfp or lacZ in a leishmanial transfection vector, p6.5, was introduced into L. amazonensis promastigotes, and egfp or lacZ-carrying recombinant L. amazonensis, La/egfp and La/lacZ, respectively, were obtained. Expression of egfp or lacZ in both promastigotes and amastigotes could be clearly visualized by fluorescence microscopy or by light microscopy with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), respectively. Fluorescence signal and beta-galactosidase activity measured by a colorimetric reaction with chlorophenol red beta-D-galactopyranoside (CPRG) were well correlated to the numbers of these parasites. The inhibitory concentration (IC50) of a leishmanicidal drug, amphotericin B, in L. amazonensis promastigotes measured using La/egfp and La/lacZ was similar to that measured by conventional methods such as cell counting, thymidine incorporation and colorimetric assay. Furthermore, the fluorescence signal and absorbance of CPRG correlated well with the numbers of La/egfp and La/lacZ amastigotes in macrophages, respectively, suggesting La/egfp and La/lacZ can be a convenient and useful tool for drug screening not only in promastigotes, but also in amastigotes of L. amazonensis. La/lacZ collected from mouse tissues four weeks after the parasite infection were stained well with X-Gal. La/lacZ allowed parasite detection at high sensitivity in the tissues of infected mice and will be useful for following infections in macrophages in vivo. Thus, the marker-transfected Leishmania parasites constructed in this study will be useful for analyses of Leishmania parasites, especially at the intracellular stage.     

Selective killing of Leishmania infected mouse macrophages by 6-methylpurine 2′-deoxyriboside

6-methylpurine 2′-deoxyriboside killed mouse macrophages infected with amastigotes of $\text{Leishmania donovani}$ and $\text{Leishmania mexicana}$, but did not affect the growth of non-parasitized cells. $\text{Leishmania}$ extracts cleaved the non-toxic 6-methylpurine 2′-deoxyriboside to 6-methylpurine, a potent adenine antimetabolite for mammalian cells. By eliminating macrophages latently infected with $\text{Leishmania donovani}$ amastigotes, 6-methylpurine 2′-deoxyriboside could augment the effects of leishmanicidal agents $\text{in vivo}$.     

Interleukin-12 augments a Th1-type immune response manifested as lymphocyte proliferation and interferon gamma production in Leishmania infantum-infected dogs

The dog is the major reservoir for human visceral leishmaniasis caused by Leishmania infantum. Interleukin-12 is considered to have an essential role in the development of both innate and adaptive immunity to Leishmania spp. and other intracellular pathogens. This study focused on the influence of IL-12 in experimental and natural canine visceral leishmaniasis. Responses of peripheral blood mononuclear cells to IL-12, interleukin-10 and Leishmania soluble antigen were evaluated in L. infantum experimentally infected oligosymptomatic beagles, uninfected beagles, naturally infected polysymptomatic dogs, and their matched uninfected controls. Leishmania soluble antigen induced strong peripheral blood mononuclear cells proliferation both in experimentally infected dogs (median stimulation index [SI]=15.01), and in naturally infected dogs (SI=8.86), but not by cells from the control groups. IL-12 addition further enhanced cell proliferation in naturally (SI=14.95), but not in experimentally infected animals. Peripheral blood mononuclear cells from experimentally infected dogs were able to produce significant amounts of IFN-gamma (3.39 ng/ml) upon LSA stimulation, but no such production was detected in cells from naturally infected or control animals. Interestingly, addition of IL-12 reversed the inhibitory effect of LSA on IFN-gamma production by cells from polysymptomatic naturally infected dogs and the uninfected beagles (4.84 and 7.45 ng/ml, respectively), and further increased IFN-gamma production by peripheral blood mononuclear cells from experimentally infected oligosymptomatic dogs (29.28 ng/ml). IFN-gamma mRNA expression correlated well with IFN-gamma production. Addition of IL-10 to Leishmania soluble antigen stimulated peripheral blood mononuclear cells inhibited proliferation and IFN-gamma production in experimentally infected dogs. Thus, the ability of IL-12 to augment IFN-gamma production by peripheral blood mononuclear cells from dogs with experimental or natural symptomatic canine visceral leishmaniasis makes it a good candidate for cytokine therapy in dogs that are refractory to current therapy.     

Parasite accessory cell interactions in murine leishmaniasis. I. Evasion and stimulus-dependent suppression of the macrophage interleukin 1 response by Leishmania donovani

Interleukin 1 (IL 1) is a principal mediator of the host immune response to microbial challenge. Accessory cells of the monocyte-macrophage series are a major source of this cytokine and are also chronically parasitized by protozoa of the genus Leishmania. This suggests that characterization of the macrophage IL 1 response to Leishmania would increase our understanding of the regulation of host immunity to these organisms. In the present study, the macrophage IL 1 response to Leishmania donovani was examined because infections with this organism have findings consistent with parasite-specific T cell unresponsiveness. Cytokine activity was measured either by direct stimulation or by co-stimulation of thymocytes. Conditioned media from BALB/c resident peritoneal macrophages infected with amastigotes of L. donovani contained no more IL 1 than did supernatant fluids of control cells. In contrast, supernatants from cells stimulated with lipopolysaccharide or heat-killed Listeria monocytogenes had significantly increased cytokine content. Resident cells infected with L. donovani for 4 hr before being stimulated with Listeria demonstrated a suppressed IL 1 response (approximately 40% of Listeria alone) to this secondary particulate stimulus. In contrast, the secondary response of leishmania-preinfected cells to lipopolysaccharide was not affected. To examine whether accessory cell nonresponsiveness to L. donovani (with respect to IL 1) was related to the state of macrophage activation, elicited peritoneal macrophages obtained by injection of proteose peptone were also studied. These cells responded to stimulation with lipopolysaccharide and fixed Staphylococcus aureus with increases in intracellular, membrane, and secreted cytokine activities. In contrast, L. donovani failed to induce any of these activities. This was found to be the case irrespective of whether amastigotes were alive or killed or opsonized with specific antibodies. Elicited cells preinfected with Leishmania responded normally to secondary stimulation with lipopolysaccharide, but not S. aureus (64% of Staphylococcus alone). In addition, attachment and penetration of L. donovani promastigotes and their subsequent conversion to amastigotes within macrophages failed to induce IL 1 synthesis. The findings of this study indicate that L. donovani has the ability to both evade and suppress the macrophage IL 1 response. Because this monokine provides an obligatory signal during macrophage-dependent T cell activation, evasion of signal transduction for IL 1 synthesis may be related to defects in cell-mediated immunity which occur during infections with this organism.     

Leishmania mexicana: comparative fine structure of amastigotes and promastigotes in vitro and in vivo

Amastigotes of Leishmania mexicana pifanoi were cultivated by serial transfers in cell-free medium UM-54 at 33 and 35 C. Electron microscopy was used to analyze the structural relationships among promastigotes, axenically cultured amastigotes, and amastigotes in footpads of infected hamsters. These studies revealed very close structural similarities between culture and hamster derived amastigotes. However, both of these amastigotes differed from the promastigotes in the following aspects. The flagellum of promastigotes contained a paraxial rod originating at the axosome level within the flagellar pocket, whereas the flagellum of amastigotes lacks this structure. The flagellar pocket of promastigotes was usually small whereas amastigotes had a distended reservoir. Subpellicular microtubules of promastigotes terminated at the posterior end, whereas those of amastigotes ended subterminally. Membrane bounded vesicles were present only in amastigotes. These results along with the biologic and antigenic comparisons indicate that amastigotes obtained from axenic cultures are related very closely to amastigotes from infected hamster footpads and that their relationship to promastigotes is far more distant.     

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).