Effect of protein kinase inhibitors on the growth, morphology, and infectivity of Leishmania promastigotes

The effect of two protein kinase inhibitors, staurosporine and H-7, on the growth, morphology and infectivity of Leishmania major and Leishmania amazonensis promastigotes was examined. Incubation with H-7 (600 μM) for up to one hour had no effect on parasite growth, morphology or infectivity. Staurosporine, however, was cytotoxic for promastigotes and incubation for 1, 5 or 15 minutes with 10 μM inhibitor killed 19, 34 and 59 %, respectively, of the parasites. Longer incubations, up to one hour, at this concentration did not increase parasite killing. However, treatment with 25 μM staurosporine for one hour was highly toxic, only 4 % of the promastigotes surviving after 72 h. Lower concentrations of staurosporine, 0.25 and 2.5 μM, had only minor effects on parasite growth. Incubation of either L. major or L. amazonensis with staurosporine (10 μM for 10 minutes) caused marked morphological changes in the size and appearance of the flagellar pocket, and/or cytoplasm of the viable parasites. Treated parasites were still capable of infecting mouse peritoneal macrophages and causing disease in BALB/c mice, though the treated parasites were less virulent than control promastigotes. These results indicate that staurosporine, while inhibiting promastigote growth, does not prevent differentiation to amastigotes and amastigote replication. Received: 26 June 1996 / Accepted: 20 August 1996     

Morphological alterations and growth inhibition of Leishmania (L.)amazonensis promastigotes exposed to zidovudine (AZT)

Leishmania parasites cause a worldwide public health disease and its treatment is still based on pentavalent antimonials which present financial and toxicologic limitations. Some nucleosidic derivatives have demonstrated anti-leishmanial properties and this study aims to evaluate the in vitro morphologic alterations and growth inhibition of Leishmania (L.) amazonensis promastigotes exposed to zidovudine at several concentrations. The citotoxicity of zidovudine (AZT) to macrophages was determined by an MTT assay. After which the promastigotes were exposed to concentrations of AZT, ranging from 1 to 50 μM. The evaluation of survival and morphometry alterations were performed in two distinct phases of in vitro growth, on the third and sixth days, representing the logarithmic and stationary phases, respectively. Slides with the promastigotes were photographed and analyzed using Image J. A significant reduction of parasite number in the logarithmic phase of in vitro growth was observed when the parasites were submitted to 20, 30, 40, and 50 μM of AZT. Morphometric alterations were observed such as an increase in width of the body, cytoplasmic granulations and vacuolizations. These data indicate the toxicity of AZT which prevents the parasite's multiplication, indicating a promising use of AZT as an anti-leishmania drug.     

Involvement of the macrophage mannose-6-phosphate receptor in the recognition of Leishmania mexicana amazonensis

Significant differences were found in the ability of resident mouse peritoneal macrophages to ingest amastigote and promastigote forms of Leishmania mexicana amazonensis. Differences in the association index of the parasites to the macrophages were also found between infective and non-infective promastigotes. Evidence was obtained suggesting that the macrophage receptor, which recognizes mannose-6-phosphate-containing units found in lysosomal enzymes, is involved in the association with the macrophage of promastigotes, but not of amastigotes. Addition of mannose-6-phosphate, its structural analogue fructose-1-phosphate, Hansenula holstii phosphomannan or the mannose-6-phosphate-containing lysosomal enzyme -D-mannosidase to the interaction medium, markedly inhibits the association of the parasites with macrophages.     

The effect of phospholipase A2 from <Emphasis Type="Italic">Crotalus durissus collilineatus</Emphasis> on <Emphasis Type="Italic">Leishmania (Leishmania) amazonensis</Emphasis> infection

In this study, the effect of phospholipase A2 (PLA2) derived from Crotalus durissus collilineatus was evaluated in vitro and in vivo on experimental cutaneous leishmaniasis. The promastigote and amastigote forms treated with PLA2 presented increased growth rate. In vivo studies showed that PLA2-treated Leishmania (Leishmania) amazonensis promastigotes increased the size of lesions in BALB/c mice, and histopathological analysis showed numerous necrotic regions presenting a higher density of polymorphonuclear, mononuclear, and amastigote cells. Additionally, infected macrophages treated with PLA2 were able to generate prostaglandin E2 (PGE2). Cytokine quantification showed that the supernatant from infected macrophages presented moderate and high amounts of IL-2 and IL-10, respectively. However, in PLA2-treated infected macrophages, suppression of IL-2 levels occurred, but not of IL-10 levels. Observation also revealed that both the supernatant and lysate of L. (L.) amazonensis promastigotes exhibited PLA2 activity, which, in the presence of dexamethasone, showed no reduction in their activities; while glucocorticoid maintained the ability of promastigote forms to infect macrophages, which presented values similar to controls. In conclusion, the results indicate that PLA2 may be a progression factor for cutaneous leishmaniasis, since the PLA2 effect suppressed IL-2 levels and generated PGE2, an inflammatory lipid mediator.     

In vitro activity of the essential oil of <Emphasis Type="Italic">Cymbopogon citratus</Emphasis> and its major component (citral) on <Emphasis Type="Italic">Leishmania amazonensis</Emphasis>

Leishmaniasis causes considerable mortality throughout the world, affecting more than 12 million people. Cymbopogon citratus (DC) Stapf, Family Poaceae, is a widely used herb in tropical countries and is also known as a source of ethnomedicines. In this study, the inhibitory effect and the morphological and ultrastructural alterations on Leishmania amazonensis by the essential oil (EO) of C. citratus and its main constituent, citral, were evaluated. The results showed that the antiproliferative activity of EO on promastigotes and axenic amastigotes, and intracellular amastigote forms of L. amazonensis was significantly better than citral, and indicated a dose-dependent effect. Neither compound showed a cytotoxic effect on macrophage strain J774G8. The promastigote forms of L. amazonensis underwent remarkable morphological and ultrastructural alterations compared with untreated cultures. These alterations were visible by light, scanning, and transmission electron microscopy of promastigotes treated with EO and citral at concentrations corresponding to the IC₅₀ (1.7 and 8.0 μg/ml) and IC₉₀ (3.2 and 25 μg/ml), respectively, after 72 h of incubation. This study revealed that citral-rich essential oil from C. citratus has promising antileishmanial properties, and is a good candidate for further research to develop a new anti-protozoan drug.     

The effect of verapamil on in vitro susceptibility of promastigote and amastigote stages of <Emphasis Type="Italic">Leishmania tropica</Emphasis> to meglumine antimoniate

Pentavalent antimonials are the standard treatment for cutaneous leishmaniasis (CL) with low efficacy and resistance is emerging. CL is increased significantly in respect to incidence rate and expanding to new foci. In the present study, the effect of verapamil on in vitro susceptibility of promastigote and amastigote stages of Leishmania tropica to meglumine antimoniate (MA, Glucantime) was evaluated using colorimetric assay (MTT) and in a macrophage model, respectively. Verapamil, as a calcium channel blocker, affects drug uptake by preventing of drug efflux from the cells. In promastigote form, several concentrations of MA with or without verapamil showed significant decrease (P < 0.05) in optical density. The overall mean IC₅₀ value with combination of MA plus verapamil (IC50 = 116.03 μg/ml) was significantly less than MA (IC50 = 225.14 μg/ml) alone (P < 0.05) for promastigote stage. Similarly, the amastigote stage was more susceptible to treatment with MA plus verapamil to that of MA alone (P < 0.05). Analysis of overall effect of different concentrations of MA alone, compared with combination of MA plus verapamil by mean infection rate of amastigotes in each macrophage showed a significant difference (P < 0.05).These findings indicated some degree of synergistic effects between MA and verapamil on in vitro susceptibility of L. tropica to MA. Further works are required to evaluate this synergistic effect on animal model or volunteer human subjects.     

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.     

Antileishmanial activity of crude extract and coumarin from <Emphasis Type="Italic">Calophyllum brasiliense</Emphasis> leaves against <Emphasis Type="Italic">Leishmania amazonensis</Emphasis>

Infections by protozoans of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials, which show renal and cardiac toxicity. As part of a search for new drugs against leishmaniasis, we evaluated the in vitro leishmanicidal activity of the (−) mammea A/BB. The compound (−) mammea A/BB is a coumarin-type mammea purified from a dichloromethane crude extract of leaves of Calophyllum brasiliense Cambess (Clusiaceae). The isolated compound was characterized using spectral analyses by UV, infrared, nuclear magnetic resonance of ¹H, ¹³C, distortionless enhancement by polarization transfer, correlation spectroscopy, heteronuclear multiple bond correlation, and heteronuclear multiple quantum coherence. The compound (−) mammea A/BB showed significant activity against promastigote and amastigote forms of L. amazonensis, with IC₅₀ (50% inhibition concentration of cell growth) at a concentration of 3.0 and 0.88 μg/ml and IC₉₀ (90% inhibition concentration of cell growth) of 5.0 and 2.3 μg/ml, respectively. The coumarin (−) mammea A/BB showed no cytotoxicity against J774G8 macrophages in culture, when it was tested at high concentrations that inhibited promastigote forms. Electron microscopy studies revealed considerable ultrastructural changes when promastigote forms of L. amazonensis were treated with 3.0 μg/ml of the coumarin (−) mammea A/BB for 72 h. We observed significant changes such as mitochondrial swelling with concentric membranes in the mitochondrial matrix and intense exocytic activity in the region of the flagellar pocket. Other alterations included the appearance of binucleate cells and multiple cytoplasmic vacuolization. These results showed that (−) mammea A/BB is a potent growth inhibitor of L. amazonensis and caused important changes in the parasite’s ultrastructure. This study provided new perspectives on the development of novel drugs with leishmanicidal activity obtained from natural products.     

<Emphasis Type="Italic">Leishmania major</Emphasis> protein disulfide isomerase as a drug target

Leishmaniasis is a major health problem worldwide and tools available for their control are limited. Effective vaccines are still lacking, drugs are toxic and expensive, and parasites develop resistance to chemotherapy. In this context, new antimicrobials are urgently needed to control the disease in both human and animal. Here, we report the enzymatic and functional characterization of a Leishmania virulence factor, Leishmania major Protein disulfide isomerase (LmPDI) that could constitute a potential drug target. LmPDI possesses domain structure organization similar to other PDI family members (a, a′, b, b′ and c domains), and it displays the three enzymatic and functional activities specific of PDI family members: isomerase, reductase and chaperone. These results suggest that LmPDI plays a key role in assisting Leishmania protein folding via its capacity to catalyze formation, breakage, and rearrangement of disulfide bonds in nascent polypeptides. Moreover, Bacitracin, a reductase activity inhibitor, and Ribostamycin, a chaperone activity inhibitor, were tested in LmPDI enzymatic assays and versus Leishmania promastigote in vitro cultures and Leishmania amastigote multiplication inside infected THP-1-derived macrophages. Bacitracin inhibited both isomerase and reductase activities, while Ribostamycin had no effect on the chaperone activity. Interestingly, Bacitracin blocked in vitro promastigote growth as well as amastigote multiplication inside macrophages with EC₅₀ values of 39 μM. These results suggest that LmPDI may constitute an interesting target for the development of new anti-Leishmania drugs.     

Anti-leishmania activity of semi-purified fraction of <Emphasis Type="Italic">Jacaranda puberula</Emphasis> leaves

The crude methanolic extract from leaves of Jacaranda puberula showed activity against Leishmania (Leishmania) amazonensis. The extract presented active against promastigote forms with an inhibitory concentration 50% (IC₅₀) value of 88.0 μg/ml, but only moderated activity against amastigote forms; however in higher concentrations the extract showed cytotoxic effects. The bio-guided chromatographic fractionation the crude methanolic extract against amastigotes yielded a fraction with an IC₅₀ value of 14.0 μg/ml (without cytotoxic activity) in relation to the crude extract (IC₅₀ value, 359.0 μg/ml). These data indicate that J. puberula leaves contain active compounds, which should be further investigated for the development of new potential drugs against cutaneous leishmaniasis.     

Megasome biogenesis in Leishmania amazonensis : a morphometric and cytochemical study

Megasomes are large lysosomes found in the amastigote stage of Leishmania species belonging to the mexicana complex. The biogenesis of megasomes was investigated by transmission electron microscopy during the transformation of promastigotes into the amastigote form of L. amazonensis maintained in axenic cultures. Mainly small vacuoles with low electron density were found in the promastigote and early intermediate forms. Morphometrical analysis showed an increase in the volume density of these structures during the transformation process. Cysteine proteinase was localized in this structure by immunocytochemical assay. Membrane-bounded structures filled with electron-dense material were also found in significant amounts from the 2nd day on. These structures were relatively abundant, both in axenic and lesion-derived amastigotes, but not in stable long-term axenic amastigote culture. A three-dimensional reconstruction of lesion-derived amastigotes and axenic amastigotes of L. amazonensis demonstrated that megasomes comprise almost 5% of the total cell volume. In addition, the development of other organelles was examined during the transformation process. Received: 18 May 2000 / Accepted: 31 August 2000     

Leishmania amazonensis Amastigotes Trigger Neutrophil Activation but Resist Neutrophil Microbicidal Mechanisms

Neutrophils are the first cells to infiltrate to the site of Leishmania promastigote infection, and these cells help to reduce parasite burden shortly after infection is initiated. Several clinical reports indicate that neutrophil recruitment is sustained over the course of leishmaniasis, and amastigote-laden neutrophils have been isolated from chronically infected patients and experimentally infected animals. The goal of this study was to compare how thioglycolate-elicited murine neutrophils respond to L. amazonensis metacyclic promastigotes and amastigotes derived from axenic cultures or from the lesions of infected mice. Neutrophils efficiently internalized both amastigote and promastigote forms of the parasite, and phagocytosis was enhanced in lipopolysaccharide (LPS)-activated neutrophils or when parasites were opsonized in serum from infected mice. Parasite uptake resulted in neutrophil activation, oxidative burst, and accelerated neutrophil death. While promastigotes triggered the release of tumor necrosis factor alpha (TNF-α), uptake of amastigotes preferentially resulted in the secretion of interleukin-10 (IL-10) from neutrophils. Finally, the majority of promastigotes were killed by neutrophils, while axenic culture- and lesion-derived amastigotes were highly resistant to neutrophil microbicidal mechanisms. This study indicates that neutrophils exhibit distinct responses to promastigote and amastigote infection. Our findings have important implications for determining the impact of sustained neutrophil recruitment and amastigote-neutrophil interactions during the late phase of cutaneous leishmaniasis.     

Alterations in membrane fluidity, lipid metabolism, mitochondrial activity, and lipophosphoglycan expression in pentamidine-resistant Leishmania

Cytoplasmic and mitochondrial membranes were studied in wild-type promastigotes of Leishmania donovani and L. amazonensis treated with pentamidine and in the parasites resistant to this drug. Analyses by flow cytometry showed membrane fluidification in resistant cells and a modification of the lipidic metabolism in pentamidine-treated cells and in resistant clones as compared with wild-type ones. Pentamidine decreased the amounts of membrane polar lipids, i.e., phospholipids, and neutral lipidic droplets in the cytoplasm. Perturbation of lipid constituents did not induce modifications of membrane acid phosphatase and protease activities. Lipophosphoglycan, the major cell-surface glycoconjugate, was no longer detected by Western blotting in resistant cells. The use of rhodamine 123 showed a decrease in transmembrane mitochondrial potential in wild-type cells treated with pentamidine and in resistant parasites. In conclusion, cytoplasmic and mitochondrial membranes and lipidic metabolism are altered in pentamidine-resistant parasites. Received: 15 May 1997 / Accepted: 16 July 1997     

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     

Antiparasitic activity of biochanin A,an isolated isoflavone from fruits of <Emphasis Type="Italic">Cassia fistula</Emphasis> (Leguminosae)

The fractionation through bioguided antileishmanial activity of the dichloromethane extract of Cassia fistula fruits (Leguminosae) led to the isolation of the active isoflavone biochanin A, identified by spectroscopic methods. This compound showed 50% effective concentration (EC₅₀) value of 18.96 μg/mL against promastigotes of Leishmania (L.) chagasi. The cytotoxicity of this substance against peritoneal macrophages resulted in an EC₅₀ value of 42.58 μg/mL. Additionally, biochanin A presented an anti-Trypanosoma-cruzi activity, resulting in an EC₅₀ value of 18.32 μg/mL and a 2.4-fold more effectiveness than benznidazole. These results contribute with novel antiprotozoal compounds for future drug design studies.     

Antileishmanial activity of imidothiocarbamates and imidoselenocarbamates

In the present study, a family of 15 imidothio- and imidoselenocarbamates (1–15) analogs have been synthesized and screened for their in vitro antileishmanial potential against Leishmania infantum promastigotes. The six most active ones (2, 4, 7, 13, 14, and 15) were also tested in an axenic amastigote model. In order to establish their selectivity indexes (SI) the cytotoxic effect of each compound was also assayed against Jurkat and THP-1 cell lines. Compounds 2 and 4, both with a pyridine moiety, showed a moderate antileishmanial activity with an IC₅₀ value of 4.68 ± 0.46 and 3.03 ± 0.24 μM, respectively, in the amastigote model. The activity was compared with that of standard drugs, edelfosine (IC₅₀ = 0.82 ± 0.13 μM) and miltefosine (IC₅₀ = 2.84 ± 0.10 μM). Related to selectivity, the SI of both compounds are similar to those of the standard drugs when compared against the THP-1 cell line. Moreover, compound 4 was able to reduce the number of amastigote-infected THP-1 cells to 40% of that observed in untreated controls after a 96-h period of treatment. These derivatives thus represent two new leads for further studies aimed at establishing their mechanism of action.     

GC-MS analysis and antileishmanial activities of two Turkish propolis types

Propolis is a honeybee product with a very complex chemical composition and various pharmacological properties. This study was aimed to investigate antileishmanial activities of “Bursa” and “Hatay” propolis samples against Leishmania infantum and Leishmania tropica strains. Propolis samples were analysed with the gas chromatography-mass spectrometry technique. Promastigotes were incubated in Roswell Park Memorial Institute culture medium in the absence and presence of several concentrations (50, 100, 250, 500, 750, and 1,000 μg/mL) of each propolis sample. The viability and cell morphology of promastigotes in each concentration were examined after 24, 48, 72, and 96 h of incubation. The growth of leishmania parasites was significantly suppressed in the presence of 500, 750, and 1,000 μg/mL of Hatay propolis. Bursa propolis was found to be efficient in inhibiting the growth of leishmania promastigotes in culture media at these concentrations, 250, 500, 750, and 1,000 μg/mL. Thus, the in vitro results showed that the Hatay and Bursa propolis samples decreased significantly the proliferation of L. infantum and L. tropica parasites (p < 0.001); however, Bursa propolis was found to be more effective than Hatay propolis against leishmania promastigotes. These two natural products may be useful agents in the prevention of leishmanial infections.     

In vitro and in vivo antileishmanial efficacy of nitazoxanide against Leishmania donovani

Control of Leishmania infection relies primarily on chemotherapy, and the current drug available for treating leishmaniasis is limited. Nitazoxanide (NTZ) is a broad spectrum antiparasitic agent with activity against protozoa, nematodes, cestodes, and trematodes. In the present study, the in vitro antileishmanial efficacy of NTZ was evaluated by incubation of Leishmania donovani promastigotes with NTZ, indicating that NTZ can affect the ultrastructure of parasite promastigote and efficiently inhibit the parasite growth. Moreover, 200 μg/ml NTZ inhibited >90% of promastigotes growth, showing similar activity of the reference drug amphotericin B (P > 0.05). Therapeutic efficacy of NTZ against L. donovani-infected BALB/c mice demonstrated that oral NTZ produced a significant reduction of parasite burden in spleen and liver from L. donovani-infected mice, compared with the untreated mice (P < 0.05). These results indicated NTZ may be a novel therapeutic drug for leishmaniasis.     

Monoclonal antibodies recognizing determinants specific for the promastigote stage of Leishmania mexicana

Two monoclonal antibodies (IX-IF9-D8 and IX-5H9-CI) produced to a membrane enriched fraction of Leishmania mexicana amazonensis promastigotes have been demonstrated to be specific for the promastigote (insect) form and not the amastigote (mammalian host) form of the parasite. The antigens recognized by these monoclonal antibodies are not found on amastigotes isolated from infected animals or on amastigotes isolated from a macrophage cell line J774 infected initially with promastigotes. The antigens are not re-expressed by amastigotes cultured at 34°C; however, amastigotes cultured at 24°C that have begun transformation into promastigotes do express these antigens. The level of expression of these antigens in cultures of amastigotes undergoing transformation into promastigotes, increases with time from 16 to 36 h and appears to correlate with the percentage of promastigotes. Two protein molecules with apparent molecular weights of 40 000 and 92 000 have been identified by radioimmune precipitation as associated with L. mexicana promastigote stage specific determinants.