Glycosphingolipid antigens of Leishmania (Leishmania) amazonensis amastigotes identified by use of a monoclonal antibody.

Monoclonal antibodies directed against Leishmania (Leishmania) amazonensis amastigotes were produced. One monoclonal antibody (1C3) selected by indirect immunofluorescence reacted with both amastigotes and promastigotes of L. (L.) amazonensis. Glycolipid extraction from L. (L.) amazonensis amastigotes and separation by high-performance thin-layer chromatography followed by immunoblotting demonstrated that 1C3 reacts with two glycosphingolipids which migrate chromatographically similarly to ceramide-N-acetylneuraminic acid (GM1) and ceramide-N-tetrose-di-acetylneuraminic acid (GD1a). The antibody did not react with glycosphingolipids from L. (L.) amazonensis promastigotes. Immunoprecipitation of 125I- and 35S-methionine-labeled promastigotes demonstrated that 1C3 recognizes gp63 from L. (L.) amazonensis promastigotes. Biosynthetic incorporation of labeled lipids by L. (L.) amazonensis amastigotes indicated that the glycosphingolipids reactive with 1C3 contain oleic acid in their structures. Surface labeling with galactose oxidase and sodium boro[3H]hydride indicated that galactose is present in 1C3-reactive antigens, strongly suggesting that these glycosphingolipids are localized on the surface of L. (L.) amazonensis amastigotes. Inhibition experiments of macrophage infection implicated the 1C3-reactive glycosphingolipids from L. (L.) amazonensis amastigotes in Leishmania invasion. The role of gp63 in promastigote-macrophage attachment was also demonstrated by inhibition experiments performed with 1C3, consistent with data from the literature.     

A radioiodinated peptidyl diazomethane detects similar cysteine proteinases in amastigotes and promastigotes ofLeishmania (L.) mexicana andL. (L.) amazonensis

Cysteine proteinase activities were examined in lesion amastigotes as well as in stationary-phase promastigotes ofLeishmania (L.) mexicana andLeishmania (L.) amazonensis isolates. Enzyme detection in gelatin gels revealed that amastigotes of threeL. (L.) mexicana isolates (M379, IOC-0561, and IP) shared similar proteinases, including the multiple low-molecular-weight (25–35 kDa) cysteine proteinases. High cysteine proteinase activity was also observed inL. (L.) amazonensis amastigotes, but the banding profile was different in two of the isolates examined. Promastigotes displayed fewer low-molecular-weight proteinase bands, and these were much less intense as compared with those of lesion amastigotes. Independently of theLeishmania isolates and developmental stages examined, incubation of the parasites for 2 h with 0.2 M radioiodinatedN-benzyloxycarbonyl-tyrosyl-alanyl diazomethane (Z-Tyr[¹²⁵I]-AlaCHN₂) markedly and selectively labeled bands comigrating with the 28- and 31-kDa cysteine proteinases. Under reducing conditions, labeling was associated with four similar polypeptides (29–34 kDa), which were also detected when incubation with Z-Tyr[¹²⁵I]-AlaCHN₂ was carried out after cell lysis. Labeling was completely abolished if lysates were first incubated with 20 M E-64 and then exposed to the¹²⁵I-tagged inhibitor, thus confirming the specificity of the compound toward cysteine proteinases.     

Comparative analysis of the nitric oxide production by<Emphasis Type="Italic"> Leishmania</Emphasis> sp.

The present report explores a comparative analysis of nitric oxide (NO^·) production by three different species of Leishmania (L. amazonensis, L. braziliensis and L. chagasi). Among these species, L. braziliensis produced the highest amount of NO^·, measured in the supernatants of promastigotes cultures as nitrite, a stable by-product derived from NO^·. We have previously described the expression of a constitutive nitric oxide synthase (cNOS) in L. amazonensis promastigotes and axenic amastigotes. Comparing those results with the present work, using immunofluorescence assay, it was shown that both L. braziliensis and L. chagasi also express a cNOS. Immunostaining experiments showed that promastigotes from early passages of these species in culture had a strong immunoreactivity against anti-cNOS and anti-endothelial cell NOS, in comparison with the same parasite cultured for long time, suggesting a correlation between the NO^· production and the presence of metacyclic forms prominent in those newly isolated parasites. These data corroborate findings of a higher NO^· production by those parasites, following the growth curve. The relationship between the two NO^·-generating systems in the parasite and in their host cell warrants further investigation. The presence of cNOS raises the possibility of a similar type of cross-talk or down-regulation between the NO^· signaling systems in host cells and the lower eukaryotic-like Leishmania sp.     

Further evidences on a new diagnostic approach for monitoring human Leishmania (L.) infantum chagasi infection in Amazonian Brazil

This was a prospective study carried out during a period over 2 years (May/2006–September/2008) with a cohort of 1,099 individuals of both genders, aged 1 year old and older, from an endemic area of American visceral leishmaniasis (AVL) in Pará state, Brazil. The object was to analyze the prevalence and incidence of human Leishmania (L.) infantum chagasi infection as well as the dynamics evolution of its clinical-immunological profiles prior identified: (1) asymptomatic infection (AI); (2) symptomatic infection (SI = AVL); (3) sub-clinical oligosymptomatic infection (SOI); (4) sub-clinical resistant infection (SRI) and; (5) indeterminate initial infection (III). The infection diagnosis was performed by using both the indirect fluorescent antibody test and leishmanin skin test with amastigotes and promastigotes antigens of L. (L.) i. chagasi, respectively. A total of 187 cases of infection were recorded in the prevalence (17%), 117 in the final incidence (6.9%), and 304 in the accumulated prevalence (26.7%), which provided the following distribution into the clinical-immunological profiles: AI, 51.6%; III, 22.4%; SRI, 20.1%; SOI, 4.3%; and SI (=AVL), 1.6%. The major finding regarding the dynamics evolution of infection was concerned to III profile, from which the cases of infection evolved to either the resistant profiles, SRI (21 cases, 30.8%) and AI (30 cases, 44.1%), or the susceptible SI (=AVL; 1 case, 1.5%); the latter 16 cases remained as III till the end of the study. These results provided the conclusion that this diagnostic approach may be useful for monitoring human L. (L.) i. chagasi infection in endemic area and preventing the high morbidity of severe AVL cases.     

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

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

Serine protease activities in Leishmania (Leishmania) chagasi promastigotes

The present work reports the isolation, biochemical characterization, and subcellular location of serine proteases from aqueous, detergent soluble, and culture supernatant of Leishmania chagasi promastigote extracts, respectively, LCSII, LCSI, and LCSIII. The active enzyme molecular masses of LCSII were about 105, 66, and 60 kDa; of LCSI, 60 and 58 kDa; and of LCSIII, approximately 76 and 68 kDa. Optimal pH for the enzymes was 7.0 for LCSI and LCSIII and 8.5 for LCSII, and the optimal temperature for all enzymes was 37°C, using α-N-ρ-tosyl-l-arginine methyl ester as substrate. Assay of thermal stability indicated that LCSIII is the more stable enzyme. Hemoglobin, bovine serum albumin, and ovalbumin were hydrolyzed by LCSII and LCSI but not by LCSIII. Inhibition studies suggested that enzymes belong to the serine protease class modulated by divalent cations. Rabbit antiserum against 56-kDa serine protease of Leishmania amazonensis identified proteins in all extracts of L. chagasi. Furthermore, immunocytochemistry demonstrated that serine proteases are located in flagellar pocket region and cytoplasmic vesicles of L. chagasi promastigotes. These findings indicate that L. chagasi serine proteases differ from L. amazonensis proteases and all known flagellate proteases, but display some similarities with serine proteases from other Leishmania species, suggesting a conservation of this enzymatic activity in the genus.     

Eosinophil chemotactic activity in Leishmania amazonensis promastigotes

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

Putrescine and spermidine transport in Leishmania

The transport of putrescine and spermidine into Leishmnania donovani promastigotes and Leishmania mexicana promastigotes and amastigotes has been characterised. Polyamine transport was shown to be saturable and temperature-sensitive for both developmental stages of Leishmania. Transport was pH-dependent with pH optima of 7.4 and 5.5 for promastigotes and amastigotes, respectively. The uptake process was independent of extracellular Na+, but inhibited by protonophores and H+-ATPase inhibitors. Kinetic analyses of polyamine transport showed that Km and Vmax differed between promastigotes of the two species and between promastigotes and amastigotes of L. mexicana. Inhibition data suggest that putrescine and spermidine use different transporters. The aromatic diamidine pentamidine, the drug of choice for treatment of antimonial-resistant cases of leishmaniasis, inhibited both putrescine and spermidine transport non-competitively.     

Role of beta-D-galactofuranose in Leishmania major macrophage invasion

The role of glycosylinositol phospholipid 1 (GIPL-1) of Leishmania (Leishmania) major in the interaction of promastigotes and amastigotes with macrophages was analyzed. Monoclonal antibody MEST-1, which recognizes glycolipids containing terminal galactofuranose (Galf) residues (E. Suzuki, M. S. Toledo, H. K. Takahashi, and A. H. Straus, Glycobiology 7:463-468, 1997), was used to detect GIPL-1 in Leishmania by indirect immunofluorescence and to analyze its role in macrophage infectivity. L. major promastigotes showed intense fluorescence with MEST-1, and GIPL-1 was detected in both amastigote and promastigote forms by high-performance thin-layer chromatography immunostaining by using MEST-1. Delipidation of L. major promastigotes with isopropanol-hexane-water eliminated the MEST-1 reactivity, confirming that only GIPL-1 is recognized in either amastigotes or promastigotes of this species. The biological role of GIPL-1 in the ability of L. major to invade macrophages was studied by using either Fab fragments of MEST-1 or methylglycosides. Preincubation of parasites with Fab fragments reduced macrophage infectivity in about 80% of the promastigotes and 30% of the amastigotes. Preincubation of peritoneal macrophages with p-nitrophenyl-beta-galactofuranoside (10 mM) led to significant ( approximately 80%) inhibition of promastigote infectivity. These data suggest that a putative new receptor recognizing beta-D-Galf is associated with L. major macrophage infectivity and that GIPL-1 containing a terminal Galf residue is involved in the L. major-macrophage interaction.     

A simple and reproducible method to obtain large numbers of axenic amastigotes of different<Emphasis Type="Italic"> Leishmania</Emphasis> species

This work describes a simple method to yield large amounts of Leishmania amastigote-like forms in axenic cultures using promastigotes as the starting population. The method described induced extracellular amastigote transformation of Leishmania amazonensis (97%), Leishmania braziliensis (98%) and Leishmania chagasi (90%). The rounded parasites obtained in axenic cultures were morphologically similar, even at the ultrastructural level, to intracellular amastigotes. Moreover, the axenic amastigotes remained viable as measured by their ability to revert back to promastigotes and to infect BALB/c mice. L. amazonensis and L. braziliensis promastigotes and axenic amastigotes differed in terms of their Western blot profiles. A 46 kDa protein was recognized by specific antibodies only in axenic and lesion-derived L. amazonensis amastigotes and not in promastigotes.     

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

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

Effect of trypsin and 2-mercaptoethanol on the exposure of sugar residues on the surface ofLeishmania donovani chagasi

Using transmission electron microscopy, goldlabeled lectins, morphometry and enzyme-linked lectin assay, we could show that treatment of promastigotes ofLeishmania donovani chagasi with trypsin did not interfere with the binding of lectins (concanavalin A, peanut agglutinin, wheat germ agglutinin andRicinus communis agglutinin) to the parasite surface. These observations are in agreement with results we previously obtained using a biochemical approach. Treatment of fixed promastigotes with 2-mercaptoethanol induced a significant increase in the density of concanavalin A (Con A) receptors on the surface ofL. d. chagasi in relation to the control. We suggest that this increase is due to the unfolding of one or more surface glycoproteins after cleavage of disulfide bonds between cystein residues in adjacent protein loops, exposing second-order Con A receptors that are otherwise hidden in the protein quaternary structure.     

Development ofLeishmania chagasi (Kinetoplastida: Trypanosomatidae) in the second blood-meal of its vectorLutzomyia longipalpis (Diptera: Psychodidae)

Light and electron microscopy investigations were carried out to compare the development ofLeishmania chagasi inLutzomyia longipalpis females that took a second blood-meal and others that took a single blood-meal. The establishment of the parasite in the foregut and the thoracic midgut of the vector was not severely affected by the intake of the second blood-meal. At 3 days after the intake of the second blood-meal, a rapid increase in the proportion of metacyclic promastigotes detected in the midgut and proboscis of the vector was noticed. No similar increase in the proportion of metacyclic promastigotes observed in the females that took a single blood-meal was noticeable. The results indicate a higher probability of transmission of the parasite to the vertebrate host at the third bite of the vector than at the second bite.     

Susceptibility of 3H-thymidine-prelabelled Leishmania donovani amastigotes and promastigotes to the cytotoxic activity of peritoneal, splenic and liver macrophages

C57BL/6 macrophage populations from spleen and liver, the main organs for the manifestation of visceral leishmaniasis, were investigated for their ability to perform spontaneous phagocytosis-associated killing of 3H-thymidine (3H-TdR)-prelabelled L. donovani amastigotes and promastigotes. The results showed that organ macrophages from spleen and liver killed L. donovani amastigotes and promastigotes spontaneously with high efficiency. This consistent finding was first detectable at 2-3 h, and the reaction was completed at 12 h. This type of killing was strongly enhanced when spleen and liver macrophages were activated. This phagocytosis-associated killing mechanism may contribute, to a large extent, in maintaining the infection under control in vivo, by drastically reducing the amount of parasites that is required to establish intracellular parasitism. To be able to assay phagocytosis-associated destruction of both promastigotes and amastigotes, a reproducible system for the production in vitro of Leishmania donovani amastigotes by the macrophage cell-line J774 was developed. The DNA of the Leishmania amastigotes was labelled with 3H-TdR with high efficiency. The spontaneous label release of prelabelled L. donovani amastigotes was comparable to that of prelabelled promastigotes over an assay period of 24 h.     

Amastigote stage-specific monoclonal antibodies against Leishmania major.

Monoclonal antibodies were produced against gamma-irradiated amastigotes of Leishmania major. Five antibodies (T16 through T20) were selected which reacted in enzyme-linked immunoassays with the intracellular stage of the parasite. These antibodies did not react with promastigotes of L. major or Leishmania donovani. One of the monoclonal antibodies (T16) reacted with amastigotes of Leishmania mexicana amazonensis and L. donovani. Western blotting (immunoblotting) and immunoprecipitation of [35S]methionine-labeled amastigotes demonstrates that T16 reacted with multiple L. major amastigote components between 12 and 180 kilodaltons. Antibody T20 was shown to recognize a low-molecular-mass doublet (less than 26 kilodaltons) in both [14C]leucine- and [35S]methionine-labeled amastigotes. A protein of less than 180 kilodaltons was also weakly recognized by T17, T19, and T20 in metabolically labeled amastigotes. This protein reacted strongly with T16. The reactive antigens could be identified on the surface of amastigotes isolated from the lesions of infected mice and on newly transformed amastigotes within 24 h after the infection of mouse peritoneal macrophages by promastigotes. These monoclonal antibodies should prove useful for the diagnosis of L. major in human tissue biopsies.     

Partial anaerobiosis induces infectivity of Leishmania infantum promastigotes

Leishmania infantum stationary-phase promastigotes could acquire infectivity via preincubation in a partially anaerobic medium (95% air/5% CO₂) for 16 h before the infection, whereas promastigotes were efficiently destroyed when no CO₂ was present. Incubation of L. infantum promastigotes with additional glucose (20 and 50 mM) greatly increased infection parameters in the absence of CO₂; this is consistent with a “reverse Pasteur effect.” Results showed that culture at 33 °C permitted survival and amastigote multiplication (a nearly 10-fold increase in amastigotes as compared with those observed in 37 °C cultures). This finding was obtained with the two strains of L. infantum tested (Doba and PB75). Received: 28 November 1998 / Accepted: 17 December 1998     

Valeriana wallichii root extracts and fractions with activity against Leishmania spp

Leishmanial diseases, posing a public health problem worldwide, are caused by Leishmania parasites with a dimorphic life cycle alternating between the promastigote and amastigote forms. Promastigotes transmitted by the vector are transformed into amastigotes residing in the host tissue macrophages. Presently, new antiparasitic agents are needed against Leishmania donovani and Leishmania major, the respective organisms causing visceral and cutaneous leishmaniasis, since the available treatments are unsatisfactory due to toxicity, high cost, and emerging drug resistance. Over the years, traditional medicinal flora throughout the world enriched the modern pharmacopeia. Hence, roots of 'Indian Valerian' (Valeriana wallichii DC) were studied for its antileishmanial activity for the first time. The methanol and chloroform extracts showed activity against L. donovani promastigotes and both promastigotes and amastigotes of L. major. The most active fraction, F3, obtained from the chloroform extract, showed IC(50) at ～ 3-7 μg/ml against both the promastigotes and 0.3 μg/ml against L. major amastigotes. On investigation of the mechanism of cytotoxicity in L. donovani promastigotes, the 'hall-mark' events of morphological degeneration, DNA fragmentation, externalization of phosphatidyl serine, and mitochondrial membrane depolarization indicated that F3 could induce apoptotic death in leishmanial cells. Therefore, the present study revealed a novel and unconventional property of V. wallichii root as a prospective source of effective antileishmanial agents.     

Fast high yield of pure Leishmania (Leishmania) infantum axenic amastigotes and their infectivity to mouse macrophages

Leishmania (L.) infantum (syn. Leishmania chagasi) is a dimorphic protozoan parasite that lives in promastigote and amastigote form in its sandfly vector and mammalian hosts, respectively. Here, we describe an in vitro culture system for the generation of a pure population of L. infantum axenic amastigotes after only 4 days incubation in culture medium supplemented with fetal calf serum, human urine, l-glutamine, and HEPES at 37oC (pH 5.5). Ultrastrutural analysis and infection assays in two macrophage populations (Kupffer cells (KUP) and peritoneal macrophages (PM)) infected with axenic amastigotes demonstrated that they maintained morphological and biochemical (A2 expression) features and a similar infection pattern to tissue-derived L. infantum amastigotes. The susceptibility of the macrophage lines to axenic or tissue-derived amastigotes and promastigotes was investigated. We found a completely different susceptibility profile for KUP and PM. Liver macrophages, both KUP and immigrant macrophages, are intimately involved in the response to L. infantum infection; this difference in susceptibility is probably related to their capacity to eliminate these parasites. Our in vitro system was thus able to generate axenic amastigotes that resemble tissue-derived amastigotes both in morphology and infectivity pattern; this will help in further investigation of the biological characteristics of the host–parasite relationship as well as the process of pathogenesis.     

Leishmania mexicana pifanoi: in vivo and in vitro interactions between amastigotes and macrophages

Macrophages of the cell line J774 were used in a comparative study of virulence involving amastigote stages ofLeishmania mexicana pifanoi isolated from macrophages (AMA-M) of the aforementioned cell line, amastigote forms grown in the UM-54 cell-free medium (AMA-C), and promastigote stages. The macrophage cultures were inoculated with AMA-M and AMA-C at the culture cell to parasite ratios of 13, 15, and 110. The macrophages were exposed to either kind of amastigotes for 24, 48, and 72 h. At the end of each of these periods, and for each dilution, the percentages of macrophages harboring the parasites within their cytoplasm and the mean numbers of intracellular parasites/macrophage were estimated on the basis of examination of 200 phagocytes. When either AMA-M or AMA-C were employed, after 24 h, the percentages of infected macrophages were, respectively, 84.5%, 89.0%, and 94.5% for the three aforementioned dilutions, the majority of the phagocytes containing 1–5 parasites. After 48- and 72-h exposures, the macrophages harbored 6–11 and 11–20 amastigotes/cell, respectively. Evidently intracellular multiplication of the amastigotes has taken place. In contrast to the results obtained with the amastigote forms, after inoculations of the macrophage cultures with promastigotes at the dilutions previously used for amastigotes, only 48–78 phagocytes were found to contain intracellular stages within their cytoplasm. Many macrophages were parasite-free, especially when exposed to fewer promastigotes. Experiments in which 5×10⁶ promastigotes, AMA-M, or AMA-C were inoculated into the footpads of hamsters yielded the following results with regard to terminal footpad volumes: 1.57, 3.31, and 3.32 cm³, respectively. Evidently both kinds of amastigotes had equal virulence for hamsters; however, the promastigote stages were much less virulent for these experimental hosts.This report is part of a dissertation submitted to the Graduate School, University of Massachusetts at Amherst, in partial fulfillment of the requirements for the Doctor of Philosophy degree in Zoology