Evaluation of Leishmanization Using Iranian Lizard Leishmania Mixed With CpG-ODN as a Candidate Vaccine Against Experimental Murine Leishmaniasis.

Background and Objectives: The live non-pathogenic Leishmania tarantolae has recently provided a promising approach as an effective vaccine candidate against experimental leishmaniasis (ILL). Here, we evaluated the immunoprotective potential of the live Iranian Lizard Leishmania mixed with CpG adjuvant against L. major infection in BALB/c mice. Methods: Four groups of female BALB/c mice were included in the study. The first and second groups received PBS and CpG, respectively. The immunized groups received 2 × 105 ILL promastigotes and the CpG-mixed ILL (ILL+CpG). Injections were performed subcutaneously in the right footpad. Three weeks later, all mice were challenged with 2 × 105 metacyclic promastigotes of Leishmania majorEGFP ; inoculation was done in the left footpad. The measurement of footpad swelling and in vivo fluorescent imaging were used to evaluate disease progress during infection course. Eight weeks after challenge, all mice were sacrificed and the cytokines levels (IFN-γ, IL-4, and IL-10) and sera antibodies concentrations (IgG2a and IgG1) using ELISA assay, nitric oxide production using Griess assay, and arginase activity in cultured splenocytes, were measured. In addition, direct fluorescent microscopy analysis and qPCR assay were used to quantify the splenic parasite burden. Result: The results showed that mice immunized with ILL+CpG were protected against the development of the dermal lesion. Moreover, they showed a significant reduction in the parasite load, in comparison to the control groups. The observed protection was associated with higher production of IFN-γ, as well as a reduction in IL-4 level. Additionally, the results demonstrated that arginase activity was decreased in ILL+CpG group compared to other groups. Conclusion: Immunization using ILL+CpG induces a protective immunity; indicating that ILL with an appropriate adjuvant would be a suitable choice for vaccination against leishmaniasis.

A Phospholipase-A Activity in Soluble Leishmania Antigens Causes Instability of Liposomes.

AIM: This study aimed to investigate the existence of phospholipase-A (PLA) activity in Soluble L. major Antigens (SLA) because of no reports for it so far. Liposomes were used as sensors to evaluate PLA activity. OBJECTIVES: Liposomal SLA consisting of Egg Phosphatidylcholine (EPC) or Sphingomyelin (SM) were prepared by two different methods in different pH or temperatures and characterized by Dynamic Light Scattering (DLS) and Thin Layer Chromatography (TLC). METHODS: Lipid hydrolysis led to the disruption of EPC liposomal SLA in both methods but the Film Method (FM) produced more stable liposomes than the Detergent Removal Method (DRM). RESULT: The preparation of EPC liposomal SLA at pH 6 via FM protected liposomes from hydrolysis to some extent for a short time. EPC liposomes but not SM liposomes were disrupted in the presence of SLA. CONCLUSION: Therefore, a phospholipid without ester bond such as SM should be utilized in liposome formulations containing PLA as an encapsulating protein.

Immunogenicity of HLA-DR1 and HLA-A2 peptides derived from Leishmania major Gp63 in golden hamsters.

AIMS: This study aimed to evaluate the toxicity and humoral and cellular immune response of three heterologous vaccines against Leishmania infantum, yet containing synthetic peptides from Leishmania major in the experimental model in hamsters. METHODS AND RESULTS: Through bioinformatics analyses, two Leishmania major Gp63 peptides were predicted and selected for vaccine formulations. Hamsters were divided into four groups, with each group receiving doses of three vaccine formulations containing HLA-DR1 or HLA-A2 peptides plus MontanideTM or both associated with the adjuvant. The animals received three vaccine doses and were evaluated for toxicity after each dose, in addition to being analysed for the production of antibodies and lymphoproliferation on day 211 after the last vaccine dose. Peptides predicted in association with oily adjuvant induced a humoral response and strong lymphoproliferation to Leishmania infantum antigen-specific stimulation.

Olive (Olea europaea) leaf extract alters the cytokine profile of Leishmania major-infected macrophages: New insight into the underlying mechanism.

This study aimed to identify the effects of olive leaf extract (OLE) on IFNγ, TNFα, TGFß and nitric oxide (NO) resulted from macrophages infected with Leishmania major (L. major) amastigotes in the culture medium. High-performance liquid chromatography (HPLC) was used to analyse the level of Oleuropein in plant extract. To evaluate the immunomodulatory effects of OLE, the isolated BALB/c mice peritoneal macrophages were infected with L. major promastigotes and treated with 6.25, 12.5 and 25 µg/mL concentrations of OLE. To assess the cytokines, supernatants of cell cultures were harvested after 12, 24 and 48 hours. Cytokine production was evaluated by ELISA. Nitrite accumulation in the culture medium was assessed using the Griess reaction. The level of Oleuropein in the extract was 18.45% by HPLC. According to results, the production of IFNγ and TNFα was significantly increased when the infected and/or not infected macrophages with L. major promastigotes were affected by different concentrations of OLE. Conversely, the production of TGFß was significantly decreased under the same conditions. Furthermore, the colorimetric determination of NO accumulation in the culture medium indicated that OLE has no effect on NO production. The study corroborates the immunomodulatory effects of OLE on L. major-infected macrophages.

Caffeic acid combined with autoclaved Leishmania major boosted the protection of infected BALB/c mice by enhancing IgG2 production, IFN-γ/TGF-ß and iNO synthase/arginase1 ratios, and the death of infected phagocytes.

BACKGROUND: Immunization with killed Leishmania promastigotes without adjuvant was considered as safe, but gave variable rates of protection. Taking advantage of the immuno-modulatory effect of caffeic acid (CA), a natural polyphenolic antioxidant, we investigated its potentiating effect in autoclaved Leishmania major (ALM)-induced protection of Leishmania major-infected BALB/c. METHODS: First, BALB/c mice were sensitized for 6 weeks either with CA, or ALM alone or combined with caffeic acid (ALM-CA) or Freund's adjuvant (ALM-FA), and subsequently infected with L. major promastigotes. Second, to test the curative effect, CA was given daily for 5 weeks to susceptible mice, starting on week 4 post-infection. Sera, footpads and lymph nodes (LNs) were collected at week 9 post-infection and submitted to biochemical or histological analyses. RESULTS: Compared to the respective controls, our results showed that CA directly healed footpad lesions and reduced the hallmarks of cutaneous leishmaniasis including oxidative inflammation, parasite load, and phagocytes influx and infestation. In sensitized mice, the protection enhanced gradually from ALM-FA, CA, ALM to ALM-CA in parallel to decreased seric IgGt levels. In contrast to ALM-FA, the combined effect of ALM and CA increased specific isotype IgG2, and decreased IL-17 and MCP-1, and phagocyte influx, as attested by the concomitant reduction in myeloperoxidase (MPO) and α-naphthyl acetate esterase (ANAE) activities. ALM-CA shifted IFN-γ/TGF-ß and iNO synthase/arginase1 (iNOS/Arg1) balances in a Th1 immune response that control efficiently cutaneous lesions and LNs hypertrophy, and reactivate the death of infected phagocytes. CONCLUSIONS: Therefore, CA combined with ALM synergizes with L. major antigens for priming innate cells, through early polarization to optimal Th1 response that leads to IFN-γ and iNOS-dependent leishmanicidal activity of neutrophils and macrophages.

The Immunology of a Healing Response in Cutaneous Leishmaniasis Treated with Localized Heat or Systemic Antimonial Therapy.

BACKGROUND: The effectiveness of systemic antimonial (sodium stibogluconate, Pentostam, SSG) treatment versus local heat therapy (Thermomed) for cutaneous leishmaniasis was studied previously and showed similar healing rates. We hypothesized that different curative immune responses might develop with systemic and local treatment modalities. METHODS: We studied the peripheral blood immune cells in a cohort of 54 cutaneous Leishmania major subjects treated with SSG or TM. Multiparameter flow cytometry, lymphoproliferative assays and cytokine production were analyzed in order to investigate the differences in the immune responses of subjects before, on and after treatment. RESULTS: Healing cutaneous leishmaniasis lead to a significant decline in circulating T cells and NKT-like cells, accompanied by an expansion in NK cells, regardless of treatment modality. Functional changes involved decreased antigen specific CD4+ T cell proliferation (hyporesponsiveness) seen with CD8+ T cell depletion. Moreover, the healing (or healed) state was characterized by fewer circulating regulatory T cells, reduced IFN-γ production and an overall contraction in polyfunctional CD4+ T cells. CONCLUSION: Healing from cutaneous Leishmaniasis is a dynamic process that alters circulating lymphocyte populations and subsets of T, NK and NKT-like cells. Immunology of healing, through local or systemic treatments, culminated in similar changes in frequency, quality, and antigen specific responsiveness with immunomodulation possibly via a CD8+ T cell dependent mechanism. Understanding the evolving immunologic changes during healing of human leishmaniasis informs protective immune mechanisms.

T cell lipid peroxidation induces ferroptosis and prevents immunity to infection.

The selenoenzyme glutathione peroxidase 4 (Gpx4) is a major scavenger of phospholipid hydroperoxides. Although Gpx4 represents a key component of the reactive oxygen species-scavenging network, its relevance in the immune system is yet to be defined. Here, we investigated the importance of Gpx4 for physiological T cell responses by using T cell-specific Gpx4-deficient mice. Our results revealed that, despite normal thymic T cell development, CD8(+) T cells from T(ΔGpx4/ΔGpx4) mice had an intrinsic defect in maintaining homeostatic balance in the periphery. Moreover, both antigen-specific CD8(+) and CD4(+) T cells lacking Gpx4 failed to expand and to protect from acute lymphocytic choriomeningitis virus and Leishmania major parasite infections, which were rescued with diet supplementation of high dosage of vitamin E. Notably, depletion of the Gpx4 gene in the memory phase of viral infection did not affect T cell recall responses upon secondary infection. Ex vivo, Gpx4-deficient T cells rapidly accumulated membrane lipid peroxides and concomitantly underwent cell death driven by ferroptosis but not necroptosis. These studies unveil an essential role of Gpx4 for T cell immunity.

Alginate microspheres encapsulated with autoclaved Leishmania major (ALM) and CpG-ODN induced partial protection and enhanced immune response against murine model of leishmaniasis.

A suitable adjuvant and delivery system are needed to enhance efficacy of vaccines against leishmaniasis. In this study, alginate microspheres as an antigen delivery system and CpG-ODN as an immunoadjuvant were used to enhance immune response and induce protection against an experimental autoclaved Leishmania major (ALM) vaccine. Alginate microspheres were prepared by an emulsification technique and the characteristics of the preparation such as size, encapsulation efficiency and release profile of encapsulates were studied. Mean diameter of microspheres was determined using SEM (Scanning Electron Microscopy) and particle size analyzer. The encapsulation efficiency was determined using Lowry protein assay method. The integrity of ALM antigens was assessed using SDS-PAGE. Mean diameter of microspheres was 1.8±1.0µm. BALB/c mice were immunized three times in 3-weeks intervals with ALM+CpG-ODN loaded microspheres [(ALM+CpG)(ALG)], ALM encapsulated alginate microspheres [(ALM)(ALG)], (ALM)(ALG)+CpG, ALM+CpG, ALM alone or PBS. The intensity of infection induced by L. major challenge was assessed by measuring size of footpad swelling. The strongest protection was observed in group of mice immunized with (ALM+CpG)(ALG). The groups of mice received (ALM+CpG)(ALG), (ALM)(ALG)+CpG, (ALM)(ALG) and ALM+CpG were also showed a significantly (P<0.05) smaller footpad swelling compared with the group that received either ALM alone or PBS. The mice immunized with (ALM+CpG)(ALG) or ALM+CpG showed the significantly (P<0.05) highest IgG2a/IgG1 ratio. The IFN-γ level was significantly (P<0.0001) highest in group of mice immunized with either (ALM)(ALG)+CpG or ALM+CpG. It is concluded that alginate microspheres and CpG-ODN adjuvant when are used simultaneously induced protection and enhanced immune response against ALM antigen.

Immunomodulatory properties of borage (Echium amoenum) on BALB/c mice infected with Leishmania major.

Leishmaniasis is caused by parasitic protozoa transmitted by the bite of a female sand fly and is currently endemic in 88 countries. BALB/c mice are highly susceptible to the infection with the parasite Leishmania major, and this susceptibility has been attributed, in part, to the expansion of Th2 cells, production of their cytokines, and downregulation of Th1 cytokine, interferon gamma (IFN-γ). In this report, we used both aqueous and alcoholic extracts of Iranian borage (Echium amoenum Fisch & C.A. Mey) for treatment of L. major infection in BALB/c mice. We found that both extracts had immunomodulatory properties and increased the level of IFN-γ and lowered the parasite burden in the proximal lymph nodes and prevented the necrosis of the footpad as compared with the untreated infected mice. These results may provide a basis for further studies directed toward the use of the Iranian borage against L. major infection.

Comparative evaluation of two vaccine candidates against experimental leishmaniasis due to Leishmania major infection in four inbred mouse strains.

Experimental leishmaniasis in BALB/c and C57BL/6 mice are the most investigated murine models that were used for the preclinical evaluation of Leishmania vaccine candidates. We have previously described two new inbred mouse strains named PWK and MAI issued from feral founders that also support the development of experimental leishmaniasis due to L. major. In this study, we sought to determine whether different mouse inbred strains generate concordant or discordant results when used to evaluate the potential of Leishmania proteins to protect against experimental leishmaniasis. To this end, two Leishmania proteins, namely, LACK (for Leishmania homolog of receptor for activated C kinase) and LmPDI (for L. major protein disulfide isomerase) were compared for their capacity to protect against experimental leishmaniasis in PWK, MAI, BALB/c, and C57BL/6 inbred mouse strains. Our data show that the capacity of Leishmania proteins to confer protection depends on the mouse strain used, stressing the important role played by the genetic background in shaping the immune response against the pathogen. These results may have important implications for the preclinical evaluation of candidate Leishmania vaccines: rather than using a single mouse strain, a panel of different inbred strains of various genetic backgrounds should be tested in parallel. The antigen that confers protection in the larger range of inbred strains may have better chances to be also protective in outbred human populations and should be selected for clinical trials.

[Immuno-modulating effects of eukaryotic expressing vectors of IL-12 and GM-CSF associated to DNA-based vaccination against experimental cutaneous leishmaniasis in BALB/c mouse]. / Effets immuno-modulateurs des vecteurs d'expressions eucaryotes de l'il-12 et du GM-CSF en association avec la vaccination à base d'ADN contre la leishmaniose cutanée expérimentale chez la souris BALB/c.

Different works of DNA based vaccination against leishmaniasis highlight the complexity of the induced immune responses to fight against the disease. In this work, we exploited the capacity of IL-12 and GMC-SF to activate immune cell mediators and effectors to induce a Th1 response, more capable of clearing the parasite. To generate these immunomodulating activities, we associated eukaryotic expressing vectors of murine IL-12 and GMC-SF to several DNA based vaccine candidates encoding to several L. (L.) major antigens, in the BALB/c mouse. When mice were challenged with a high parasitic load in the hind footpad, no additional protective effect could be generated. However, when the challenge was carried out in the inner face of the ear with a small parasitic load, the association of plasmids encoding to IL-12 and GMC-SF to DNA based vaccination, the protective effects were increased.

Nitric oxide synthase and cytokines gene expression analyses in Leishmania-infected RAW 264.7 cells treated with an extract of Pelargonium sidoides (Eps 7630).

A modern aqueous-ethanolic formulation of the roots of Pelargonium sidoides (Eps 7630), elaborated from the traditional herbal medicine used in areas of southern Africa, is effectively employed for the treatment of ENT and respiratory tract infections in modern phytotherapy. Previous studies have demonstrated antibacterial and immunomodulatory activities. To gain insight into the mode of action at the molecular level, gene expression analyses for the inducible nitric oxide synthase and the cytokines interleukin (IL)-1, IL-12, IL-18, tumour necrosis factor (TNF)-alpha, interferon (IFN)-alpha, and IFN-gamma, were performed using reverse transcription-polymerase chain reaction (RT-PCR). The experiments were carried out in parallel in non-infected and in Leishmania major-infected RAW 264.7 cells and the expression profiles were compared with those mediated by IFN-gamma+LPS. Eps 7630 induced low mRNA levels in non-infected cells, and it considerably up-regulated the transcript expressions in parasitised cells. Interestingly, and in contrast to activation by IFN-gamma+LPS, Eps 7630 also stimulated infected cells to produce IFN-gamma mRNA. A similar expression profile was observed for the methanol-insoluble fraction (MIF) of Eps 7630 and gallic acid, a trace constituent of the extract, while the methanol-soluble fraction and umckalin, an exclusive and representative member of the occurring coumarins, proved to be devoid of any remarkable gene-inducing capabilities. The present results provide not only convincing support for the improvement of immune functions as previously demonstrated in functional bioassays, but also evidence for activation at the transcriptional level and suggest that the underlying inducing principle is located in the MIF.

Immune response and protection assay of recombinant major surface glycoprotein of Leishmania (rgp63) reconstituted with liposomes in BALB/c mice.

In this study the ability of recombinant gp63 entrapped in liposomes to induce immune response and protection against L. major infection in susceptible BALB/c mice was studied. Liposomes containing rgp63 (Lip-rgp63) were prepared from egg lecithin and cholesterol using detergent solubilization method. Immunization of BALB/c mice with rgp63 alone conferred a partial protection while entrapment of rgp63 in liposomes significantly increased the rate of protection (P<0.05). The parasite burden of spleen in mice challenged with L. major was significantly (p<0.001) lower in group of mice immunized with rgp63 alone or Lip-rgp63, however, the least parasite burden was seen in Lip-rgp63 group. Both rgp63 alone and Lip-rgp63 elicited significant delayed-type hypersensitivity (DTH) response compared to controls (p<0.01), however, the DTH response of PBS-rgp63 was less than the Lip-rgp63. Titration of anti-Leishmania IgG isotypes (IgG2a/IgG1) showed a preferential Th1 type of immune response only in mice immunized with Lip-rgp63. The results indicate that liposomes might be used as a suitable immunoadjuvant for development of Leishmania vaccine.

Transmission blocking vaccine studies in leishmaniasis: II. Effect of immunisation using Leishmania major derived 63 kilodalton glycoprotein, lipophosphoglycan and whole parasite antigens on the course of L. major infection in BALB/c mice.

BACKGROUND: Safe, effective and inexpensive vaccines may be the most practical tool for control of any form of leishmaniasis. Leishmaniasis produces a state of pre-immunition which is the underlying mechanism for prolonged immunity to re-infection. Low doses of parasites has been shown to be able to induce protection in mice. It is not known, however, how immune sera from a susceptible host immunised with Leishmania-derived antigens when taken in by the sandfly affects the development and the subsequent transmission of the parasite to naive hosts. OBJECTIVE: To monitor the course of disease in BALB/c mice following challenge using L. major infected P. duboscqi which had previously fed on immunised mice. METHODS: BALB/c mice were immunised adequately with Leishmania major-derived antigens namely, crude whole parasite (WPA), recombinant 63 kilodalton glycoprotein (rgp63), lipophosphoglycan (LPG) and a cocktail composed of rgp63 plus LPG antigens. Laboratory reared Phlebotomus duboscqi sandflies, the natural vector for L. major were later allowed to feed on immunised animals, interrupted and allowed to continue feeding on infected animals for an equal amount of time until they became fully engorged. The sandflies were maintained on apples as a carbohydrate source in an insectary maintained at a temperature of 25 degrees C and 80% relative humidity. On the seventh day these sandflies were used to infect naive BALB/c mice and the course of infection followed for a period of at least three months. RESULTS: Mice infected using sandflies which had previously fed on WPA or rgp63-immunized mice showed disease exacerbation as the infection progressed, whereas those infected using sandflies which had previously fed on LPG-immunised mice had the least lesion sizes compared to control mice infected using sandflies which had fed on saline immunised mice (p < 0.05). CONCLUSIONS: Results from this study indicate that the course of L. major infection in BALB/c mice was dependent on the infective dose of parasites transmitted by the sandflies. Results from this study suggests that sub-infective doses of the parasite from sandflies previously fed on animals immunised with Leishmania-derived antigens needs to be evaluated for their potential in vaccine development against Leishmania infections.

Transmission blocking vaccine studies in leishmaniasis: I. Lipophosphoglycan is a promising transmission blocking vaccine molecule against cutaneous leishmaniasis.

BACKGROUND: New strategies for control of leishmaniasis is needed as chemotherapy using antimonial drugs is prolonged, expensive, associated with side effects and relapses. Vector control has limitations and a vaccine which may be the best approach is not available. OBJECTIVES: To assess the level of inhibition of promastigote development and gut morphology in infected Phlebotomus duboscqi sandflies fed on different groups of BALB/c mice immunised with rgp63, lipophosglycan (LPG) or their cocktail and whole parasite antigens prepared from L. major culture-derived promastigotes. METHODS: BALB/c mice were immunised adequately with Leishmania major-derived antigens namely, crude whole parasite (WPA), recombinant 63 kilodalton glycoprotein (rgp63), LPG and a cocktail composed of rgp63 plus LPG antigens. Laboratory reared Phlebotomus duboscqi sandflies, the natural vector for L. major were later allowed to feed on immunised animals, interrupted and allowed to continue feeding on infected animals for an equal amount of time until they became fully engorged. The sandflies were maintained on apples as a carbohydrate source in an insectary maintained at a temperature of 25 degrees C and 80% relative humidity. Some of the sandflies were dissected on days 2, 4 and 6 after feeding and observed using the light and the transmission electron microscopy for any changes in their gut morphology. The remaining sandflies were all dissected on the sixth day post-feeding and examined for procyclics, nectomonads, haptomonads and metacyclic promastigote forms of Leishmania. RESULTS: Sandflies which had previously fed on WPA, LPG plus rgp63 cocktail and LPG-immunised mice showed the lowest infection rates compared to control sandflies fed on saline immunised mice (p < 0.05). A significant number of procyclic promastigotes, the first developmental form of the parasite in culture as well as in the sandfly was observed in sandflies which fed on LPG-immunised mice (p < 0.05). The dominant parasite form in sandflies which fed on rgp63 or LPG-immunised mice was the nectomonad form but very few of the infective metacyclic forms (p < 0.05). Control sandflies fed on saline immunised or infected mice alone displayed a normal pattern of parasite development up to the metacyclic stage. Studies showed that two possible mechanisms through which immune sera from immunised mice may cause inhibition of parasite development is by exflagellation of nectomonad forms and degeneration of the sandfly midgut epithelium as revealed by light and electron microscopy studies respectively. CONCLUSIONS: This study has shown that immune-mediated transmission blocking may be applied to Leishmania infections. Based on observation of the procyclic promastigotes, the dominance of the nectomonad forms, low infectivity rates in sandflies fed on LPG-immunised mice, we concluded that LPG stands out to be a promising transmission blocking vaccine candidate in leishmaniasis.

Long-term protection of mice against Leishmania major with a synthetic peptide vaccine.

A single synthetic T cell epitope (PT3), obtained from the histidine zinc-binding region of the metalloprotease gp63, was employed in a vaccine trial using two virulent strains of L. major. When a single subcutaneous injection of PT3 was delivered with the Thl stimulating adjuvant poloxamer 407, BALB/c mice were protected for at least 10 months against the disease. Vaccinated mice were largely free of lesions on termination of the experiment. Protection was similar for both L. major MRHP/SU/59 Neals and L. major WHOM/IR/-/173 strains which manifest different disease sequelae. Thus, these data provide evidence that a single subcutaneous injection of a single synthetic T cell epitope is sufficient to provide long-lasting protection against two highly virulent strains of L. major in BALB/c mice.

The Leishmania promastigote surface antigen 2 complex is differentially expressed during the parasite life cycle.

The promastigote surface antigen 2 (PSA-2) complex comprises a family of antigenically similar polypeptides of M(r) 96,000, 80,000 and 50,000, anchored to the membrane with glycosylphosphatidylinositol. Although PSA-2 was initially detected only in promastigotes, Northern blot analysis indicated that mRNA transcripts are also present in amastigotes. Unlike the situation in promastigotes, where at least four major transcripts (2.6-5.3 kb) were detected, only one major (2.6 kb) and two minor transcripts were present in amastigotes. A cDNA clone encoding a member of the PSA-2 family expressed in amastigotes was isolated using DNA probes. The predicted protein sequence of M(r) 40,000 is distinct from promastigote sequences, but shows significant similarity to previously described members of the family from L major and L amazonensis. Antibodies to the carboxyl terminal sequence conserved in all L major PSA-2 studied to date, as well as antibodies affinity purified on the amastigote cDNA-derived polypeptide recognized a major M(r) 50,000 amastigote polypeptide. Immuno-electron microscopy localized both promastigote and amastigote PSA-2 to the cell surface. The expression of PSA-2 polypeptides during the transformation of amastigotes into promastigotes was ordered in a time-dependent manner, with the promastigote M(r) 80000 polypeptide appearing first, followed by the M(r) 96000 polypeptide. In contrast to the glycosylphosphatidylinositol anchor of promastigote PSA-2, which could be hydrolysed by phosphatidylinositol-specific phospholipase C, the amastigote form was resistant to this enzyme.