Localization and activity of various lysosomal proteases in Leishmania amazonensis-infected macrophages.

In mammalian hosts, Leishmania amastigotes are obligatory intracellular parasites of macrophages and multiply within parasitophorous vacuoles of phagolysosomal origin. To understand how they escape the harmful strategies developed by macrophages to kill ingested microorganisms, it is important to obtain information on the functional state of parasitophorous vacuole. For this purpose, we studied the intracellular distribution and activity of host lysosomal proteases in rat bone marrow-derived macrophages infected with Leishmania amazonensis amastigotes. Localization of cathepsins B, H, L, and D was investigated by using specific immunoglobulins. In uninfected macrophages, these enzymes were located in perinuclear granules (most of them were probably secondary lysosomes) which, after infection, disappeared progressively. In infected macrophages, cathepsins were detected mainly in the parasitophorous vacuoles, suggesting that the missing secondary lysosomes had fused with these organelles. Biochemical assays of various proteases (cathepsins B, H, and D and dipeptidyl peptidases I and II) showed that infection was accompanied by a progressive increase of all activities tested, except that of dipeptidyl peptidase II, which remained constant. No more than 1 to 10% of these activities could be attributed to amastigotes. These data indicate that (i) Leishmania infection is followed by an increased synthesis and/or a reduced catabolism of host lysosomal proteases, and (ii) amastigotes grow in a compartment rich in apparently fully active proteases. Unexpectedly, it was found that infected and uninfected macrophages degraded endocytosed proteins similarly. The lack of correlation in infected macrophages between increase of protease activities and catabolism of exogenous proteins could be linked to the huge increase in volume of the lysosomal compartment.     

In vitro light and electron microscope studies on different virulent promastigotes ofLeishmania donovani in hamster peritoneal macrophages

Summary Hamster peritoneal macrophages were infected with arivulent and virulent promastigotes of aL. donovani strain using various ratios (11; 110) of parasites and peritoneal cells. Light microscope studies have shown that there was a significant difference in the number of parasites taken up by phagocytic cells between the macrophage cultures infected with avirulent and virulent promastigotes at 4 h as well as during the following 14 days of infection. In both virulent groups the number of amastigotes were sharply increased. However, the surviving parasites were eliminated continuously when the macrophage cultures were infected with avirulent parasites. Electron microscope examinations of the different infected macrophage cultures did not show any difference in the localization of the surviving parasites. At one and 24 h post-infection, parasites have been observed in typical parasitophorous vacuoles. However, by day 4, 7, and 14 post-infections, the majority of intact parasites were surrounded by a four-laminar membrane without a space between parasite and vacuole membrane. Besides, some amastigotes were seen in large parasitophorous vacuoles. It seemed as if some of these amastigotes were trying to leave the parasitophorous vacuoles. In all cases acid phosphatase could be demonstrated in the parasitophorous vacoules and around the parasites indicating that the lysosomes of the host cell have been fused with the parasitophorous vacuole. It is indicated that the virulentLeishmania parasites are more resistant to the digestive system of the macrophages.     

Antigen presentation by Leishmania mexicana-infected macrophages: activation of helper T cells by a model parasite antigen secreted into the parasitophorous vacuole or expressed on the amastigote surface

Leishmania are protozoan parasites which invade mammalian macrophages and multiply as amastigotes in phagolysosomes (parasitophorous vacuoles). Using L. mexicana and bone marrow-derived macrophages (BMM), the question is addressed whether infected BMM induced to express major histocompatibility complex class II molecules can present defined antigens to specific T helper type 1 cells. As a model antigen, a membrane-bound acid phosphatase (MAP), a minor protein associated with intracellular vesicles in amastigotes, was either overexpressed at the surface of the parasites or overexpressed in a soluble form leading to antigen secretion into the parasitophorous vacuole. Presentation of MAP epitopes by these three types of amastigotes was then compared for macrophages containing live parasites or amastigotes inactivated by drug treatment. It is shown that surface-exposed and secreted MAP can be efficiently presented to T cells by macrophages harboring live amastigotes. Therefore, the parasitophorous vacuole communicates by vesicular membrane traffic with the plasmalemma of the host cell. The intracellular MAP of wild-type cells or the abundant lysosomal cysteine proteinases are not or only inefficiently presented, respectively. After killing of the parasites, abundant proteins such as overexpressed MAP and the cysteine proteinases efficiently stimulate T cells, while wild-type MAP levels are not effective. We conclude that intracellular proteins of intact amastigotes are not available for presentation, while after parasite inactivation, presentation depends on antigen abundance and possibly stability. The cell biological and possible immunological consequences of these results are discussed.     

Parasitophorous vacuoles of Leishmania amazonensis-infected macrophages maintain an acidic pH.

Leishmania amastigotes are intracellular protozoan parasites of mononuclear phagocytes which reside within parasitophorous vacuoles of phagolysosomal origin. The pH of these compartments was studied with the aim of elucidating strategies used by these microorganisms to evade the microbicidal mechanisms of their host cells. For this purpose, rat bone marrow-derived macrophages were infected with L. amazonensis amastigotes. Intracellular acidic compartments were localized by using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine as a probe. This indicator, which can be detected by light microscopy by using immunocytochemical methods, mainly accumulated in perinuclear lysosomes of uninfected cells, whereas in infected cells, it was essentially localized in parasitophorous vacuoles, which thus appeared acidified. Phagolysosomal pH was estimated quantitatively in living cells loaded with the pH-sensitive endocytic tracer fluoresceinated dextran. After a 15- to 20-h exposure, the tracer was mainly detected in perinuclear lysosomes and parasitophorous vacuoles of uninfected and infected macrophages, respectively. Fluorescence intensities were determined from digitized video images of single cells after processing and automatic subtraction of background. We found statistically different mean pH values of 5.17 to 5.48 for lysosomes and 4.74 to 5.26 for parasitophorous vacuoles. As for lysosomes of monensin-treated cells, the pH gradient of parasitophorous vacuoles collapsed after monensin was added. This very likely indicates that these vacuoles maintain an acidic internal pH by an active process. These results show that L. amazonensis amastigotes are acidophilic and opportunistic organisms and suggest that these intracellular parasites have evolved means for survival under these harsh conditions and have acquired plasma membrane components compatible with the environment.     

Liposomal chemotherapy in visceral leishmaniasis: An ultrastructural study of an intracellular pathway

The intracellular fate of liposomes administered intracardially was examined in the liver and spleen of hamsters experimentally infected withLeishmania donovani. Separate groups of animals were treated with liposomes containing either an antileishmanial agent, a colloidal gold marker, or saline. Ultrastructural examinations of lysosomal interactions with the parasitophorous vacuole and with phagocytized liposomes were made. Lysosomes readily fused with the parasitophorous vacuoles but appeared to have little effect on the parasite, possibly due to the production of enzyme inhibitors. Liposomes rapidly became localized in lysosomes subsequent to endocytosis by macrophages. Morphologic evidence suggested that secondary lysosomes containing liposomal residues then fused with the parasitophorous vacuole. Aspects of one possible pathway are discussed which may account for the greatly enhanced effectiveness of liposomal chemotherapy for experimental visceral leishmaniasis.     

Localization and activity of different lysosomal proteases in rat macrophages infected by Leishmania amazonensis

Leishmania are protozoans of the trypanosomatidae family that cause human infections. The amastigote form of Leishmania is an obligate intracellular parasite of mononuclear phagocytes that multiplies within parasitophorous vacuoles (pv) of phagolysosomal origin. To investigate the strategies which allow Leishmania to withstand these potentially cytotoxic conditions, the distribution and activities of various lysosomal peptidases in rat macrophages infected or uninfected with Leishmania amazonensis amastigotes were studied. Specific immunoglobulins against cathepsins (cat.) B, H, L and D were used to localize these endopeptidases by immunocytochemistry. Results showed that most or even all of the secondary lysosomes in the host cell fuse with parasite-filled phagosomes, leading to translocation of the proteases in the parasitophorous vacuoles. A further study consisted in assays of five protease activities: dipeptidylpeptidases (DPP) I and II (exopeptidases), cat. B, cat. H and cat. D. Infection of macrophages was followed by a gradual increase in all these protease activities except for DPP II. These increases were apparently not related to parasite protease activities. It seems that infection by Leishmania amazonensis is followed by increased synthesis and/or reduced catabolism of host cell lysosomal proteases or alternatively by inactivation of endogenous inhibitors. Amastigote infectivity may be related, at least in part, to the development of mechanisms that allow the parasite to withstand unfavorable environmental conditions.     

Multiplication of Leishmania in human macrophages in vitro.

To facilitate in vitro studies of the immunology of human leishmaniasis, we developed a method of growing pathogenic Leishmania in human monocyte-derived macrophages. After 6 days of incubation, adherent mononuclear cells were infected with Leishmania donovani amastigotes obtained from infected hamster spleen cells or with L. tropica amastigotes obtained from infected BALB/c tissue mouse footpad. Forty-eight percent of the macrophages were initially infected, with a mean of 3.0 amastigotes per infected macrophage. After 6 days of incubation, 59% of macrophages were infected and contained 8.8 amastigotes per infected macrophage, representing 2.9-fold multiplication. Electron microscopy revealed the presence of dividing parasites within phagolysosomes. These observations indicate that Leishmania survive and multiply within human monocyte-derived macrophages despite fusion of secondary lysosomes with the parasitophorous vacuole.     

Receptor-mediated entry of peroxidases into the parasitophorous vacuoles of macrophages infected with Leishmania Mexicana amazonensis

Leishmania amastigotes, obligatory parasites of macrophages, lodge and multiply within long-lived phagolysosomelike "parasitophorous vacuoles" (PV). The glycoprotein horseradish peroxidase (HRP) was shown, by light and electron microscopic cytochemistry, to enter the PVs of rat in vitro-derived bone marrow macrophages infected with Leishmania mexicana amazonensis. Uptake was obtained both in preinfected macrophages incubated with HRP and in macrophages pulsed with HRP, infected, and further incubated in ligand-free medium. Peroxidase positive and negative PVs could coexist in the same macrophages. Infected macrophages commonly displayed fewer labeled secondary lysosomes than noninfected cells. Lactoperoxidase (LP) was also shown, by light microscopy, to enter the PVs of rat macrophages. Uptake of HRP and of LP was blocked by mannan, supporting the mannose receptor mediated recognition of these ligands. Transfer of HRP to PVs was much less efficient in resident mouse peritoneal macrophages, even at 50 X higher ligand concentrations. Such macrophages expressed negligible mannose receptor function. The efficient mannan-inhibitable uptake of HRP by rat marrow macrophages was confirmed biochemically. Bulk HRP uptake in infected and noninfected cultures was found to be similar. Peroxidases should be useful in further studies of endocytosis by Leishmania-infected macrophages and in the development of lysosomotropic macrophage-targeted drug carriers.     

Localization of major histocompatibility complex class II molecules in phagolysosomes of murine macrophages infected with Leishmania amazonensis

Leishmania-infected macrophages are potential antigen-presenting cells for CD4+ T lymphocytes, which recognize parasite antigens bound to major histocompatibility complex class II molecules (Ia). However, the intracellular sites where Ia and antigens may interact are far from clear, since parasites grow within the modified lysosomal compartment of the host cell, whereas Ia molecules seem to be targeted to endosomes. To address this question, the expression and fate of Ia molecules were studied by immunocytochemistry in Leishmania amazonensis-infected murine macrophages stimulated with gamma interferon. In uninfected macrophages, Ia molecules were localized on the plasma membrane and in perinuclear vesicles, but they underwent a dramatic redistribution after infection, since most of the intracellular staining was then associated with the periphery of the parasitophorous vacuoles (p.v.) and quite often polarized towards amastigote-binding sites. The Ii invariant chain, which is transiently associated with Ia during their intracellular transport, although well expressed in infected macrophages, apparently did not reach the p.v. Similar findings were observed with macrophages from mice either resistant or highly susceptible to Leishmania infection. In order to determine the origin of p.v.-associated Ia, the fate of plasma membrane, endosomal, and lysosomal markers, detected with specific antibodies, was determined after infection. At 48 h after infection, p.v. was found to exhibit a membrane composition typical of mature lysosomes. Overall, these data suggest that (i) Ia located in p.v. originate from secondary lysosomes involved in the biogenesis of this compartment or circulate in several endocytic organelles, including lysosomes and (ii) p.v. could play a role in antigen processing and presentation. Alternatively, the presence of high amounts of Ia in p.v. could be due to a Leishmania-induced mechanism by means of which this organism may evade the immune response.     

Infection of murine peritoneal macrophages withToxoplasma gondii exposed to ultraviolet light

Exposure ofToxoplasma trophozoites to ultraviolet light (UV; 2,539 Å) remarkably inhibited intracellular multiplication of the toxoplasmas within cultured mouse peritoneal macrophages. These toxoplasmas possessed the ability to induce normal parasitophorous vacuoles (PV) and underwent gradual degeneration in the PV without participation of host-cell lysosomes. Apparently, the basic conformation of the PV, i.e., the association of mitochondria, rough endoplasmic reticulum, and microtubules of the host cell and the presence of microvillous infoldings, was maintained as seen under the electron microscope even after the toxoplasmas had died within the PV. Even PV, in which debris of the toxoplasmas could be observed, did not show the sign of fusion with ferritin-labeled secondary lysosomes.     

Intravital luminescence-serologic study of intracellular processes during phagocytosis of brucellas by macrophages

The ingestion of a heterologous brucellar luminescent antiserum by cultures of macrophages from intact and immune guinea pigs and its accumulation in the cytoplasmic granules (secondary lysosomes) and its entry into phagocytic vacuoles (phagosomes) were studied. The accumulation of antiserum in the phagosomes was judged from the appearance of specific luminescence of the phagocytosed brucellas. Accumulation of luminescent antigen-antibody complex in the cytoplasmic granules also was investigated at various time intervals after phagocytosis of brucellas treated with brucellar luminescent serum. No difference was found between macrophages from intact and immune animals as regards the character of pinocytosis of the luminescent sera. Differences between macrophages were found in the rate of passage of the antisera from the lysosomes into the phagosomes and the rate of formation of luminescent granules after phagocytosis of brucellas treated with luminescent antiserum.     

Isolation of the intracellular stage of Trypanosoma cruzi and its interaction with mouse macrophages in vitro.

Interaction between Trypanosoma cruzi spheromastigotes (amastigotes) and mouse macrophages was studied. The spheromastigotes, isolated from the spleens of infected mice, were incorporated and digested by the macrophages. By use of horseradish peroxidase labeling of the macrophage lysosomes we showed that fusion of lysosomes with phagocytic vacuoles containing T. cruzi occurred. Parasites showing alterations indicative of digestion were seen inside the phagocytic vacuoles. Our results suggest that intracellular sphermastigotes of T. cruzi, isolated from the spleens of infected mice, and not able to induce a protective infection in mouse macrophages maintained in vitro.     

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     

Freeze-fracture and cytochemistry study of the interaction between Leishmania mexicana amazonensis and macrophages

The process of interaction between macrophages and promastigote and amastigote forms of Leishmania mexicana amazonensis was analyzed using freeze fracture and cytochemistry. The promastigotes inside endocytic vacuoles of macrophages presented an altered distribution of intramembranous particles and a wavy aspect of the plasma membrane. However, amastigotes did not show such alterations. The membrane alterations are probably caused by intracellular cell lysis of the promastigotes by the macrophages. An accumulation of intramembranous particles was seen in the plasma membrane of amastigote forms in the area of adhesion to the macrophages. The parasitophorous vacuole membrane had intramembranous particles randomly distributed. The enzyme activity of Mg++-ATPase, 5'-nucleotidase and NAD(P)H-oxidase was cytochemically detected, at the ultrastructural level, in normal mouse peritoneal macrophages and in macrophages infected with Leishmania mexicana amazonensis. Mg++-ATPase and 5'-nucleotidase are uniformly distributed throughout the macrophage's plasma membrane but were not detected in the membrane lining endocytic vacuoles containing ingested parasites (parasitophorous vacuole). NAD(P)H-oxidase activity was seen in those portions of the macrophage's plasma membrane which enter in direct contact with parasites and also in association with the membrane of the parasitophorous vacuole. The amount of reaction product, indicative of NAD(P)H-oxidase activity, was larger in macrophages which interacted with the promastigote than in those which interacted with the amastigote form of L. mexicana amazonensis. Concanavalin A binding sites and anionic sites of the macrophage's surface, labeled before the interaction, are not interiorized together with the parasites, however, are observed in endocytic vacuoles which do not contain parasites.     

Biogenesis of Leishmania major-harboring vacuoles in murine dendritic cells

In mammalian hosts, Leishmania sp. parasites are obligatory intracellular organisms that invade macrophages and dendritic cells (DC), where they reside in endocytic organelles termed parasitophorous vacuoles (PV). Most of the present knowledge of the characteristics of PV harboring Leishmania sp. is derived from studies with infected macrophages. Since DC play a key role in host resistance to leishmaniasis, there is a need to understand the properties and biogenesis of PV in Leishmania sp.-infected DC. Therefore, we determined the acquisition of endosomal and lysosomal molecules by Leishmania major-containing compartments in DC at different maturation stages, using fluorescence labeling and confocal microscopy. The results show that newly formed phagosomes in DC rapidly develop into late endosomal compartments. However, the small GTPase Rab7, which regulates late fusion processes, was found only in PV of mature bone marrow-derived DC (BMDC); it was absent in immature BMDC, suggesting an arrest of their PV biogenesis at the stage of late endosomes. Indeed, fusion assays with endocytic tracers demonstrated that the fusion activity of L. major-harboring PV toward lysosomes is higher in mature BMDC than in immature BMDC. The inhibition of PV-lysosome fusion in DC is dependent upon the viability and life cycle stage of the parasite, because live promastigotes blocked the fusion almost completely, whereas killed organisms and amastigotes induced a considerable level of fusion activity. The differences in the fusion competences of immature and mature DC may be relevant for their distinct functional activities in the uptake, transport, and presentation of parasite antigens.     

Polyketide synthase (PKS) reduces fusion of Legionella pneumophila-containing vacuoles with lysosomes and contributes to bacterial competitiveness during infection

L. pneumophila-containing vacuoles (LCVs) exclude endocytic and lysosomal markers in human macrophages and protozoa. We screened a L. pneumophila mini-Tn10 transposon library for mutants, which fail to inhibit the fusion of LCVs with lysosomes by loading of the lysosomal compartment with colloidal iron dextran, mechanical lysis of infected host cells, and magnetic isolation of LCVs that have fused with lysosomes. In silico analysis of the mutated genes, D. discoideum plaque assays and infection assays in protozoa and U937 macrophage-like cells identified well established as well as novel putative L. pneumophila virulence factors. Promising candidates were further analyzed for their co-localization with lysosomes in host cells using fluorescence microscopy. This approach corroborated that the O-methyltransferase, PilY1, TPR-containing protein and polyketide synthase (PKS) of L. pneumophila interfere with lysosomal degradation. Competitive infections in protozoa and macrophages revealed that the identified PKS contributes to the biological fitness of pneumophila strains and may explain their prevalence in the epidemiology of Legionnaires’ disease.     

Interaction ofTrypanosoma cruzi with macrophages in vitro: Dissociation of the attachment and internalization phases by low temperature and cytochalasin B

Chicken macrophages, obtained by cultivation of blood monocytes, were infected with epimastigote and bloodstream trypomastigote forms ofTrypanosoma cruzi strain Y. The percentage of macrophages containing parasites within parasitophorous vacuoles and of flagellates attached to cell surfaces was determined. By incubation of the macrophages at 4°C or in the presence of cytochalasin B it was possible to dissociate the attachment from the internalization phases in the process of infection of macrophages. Both treatments had a marked effect on the internalization of epimastigote and trypomastigote forms. Cytochalasin B treatment and placement of the macrophages at 4° C before infection inhibited this process by about 99 and 96%, respectively. These results suggest that endocytosis is the principal mechanism of internalization ofT. cruzi by macrophages. They show also that epimastigote and trypomastigote forms ofT. cruzi have a different rate of adhesion to the macrophage surface.     

Subverted transferrin trafficking in Leishmania- infected macrophages

The intracellular fate of human transferrin (HTf) in macrophages infected by Leishmania was investigated. Binding of HTf-gold complexes at 4 °C was competitively inhibited by native holoHTf but not by apoHTf. Infected and uninfected macrophages displayed rather distinct HTf trafficking. Pulse-chase experiments using uninfected macrophages loaded with 15-nm gold-conjugated bovine serum albumin (BSA) and then incubated with 5-nm gold-conjugated HTf revealed a remarkable segregation of these tracers in distinct compartments. Nevertheless, Leishmania-infected macrophages presented extensive particle colocalization at both 60 min and 18 h. Light and electron microscopy immunolabeling indicated that HTf was delivered to the parasitophorous vacuole, formed patches on the amastigote surface, and was endocytosed via the flagellar pocket. Double-staining assays showed the colocalization of biotinylated HTf and its receptor in association with the parasitophorous vacuole. To approach the Tf-binding sites of amastigotes we performed HTf-fluorescein isothiocyanate (FITC) assays. Staining was diffuse at 4 °C and punctate at 35 °C, and only the former was sensitive to ethidium bromide, indicating an eventual temperature-dependent endocytic process. Within parasites, HTf was found in cysteine-proteinase-rich structures, suggesting that the protein can be endocytosed by intracellular amastigotes and sorted to the parasite endosomal-lysosomal compartments rather than being recycled. The treatment of infected macrophages with holoHTf, but not apoHTf, promoted the parasite's intracellular survival. These results suggest that Leishmania amastigotes can exploit and subvert the host-cell endocytic system and indicate the role of Tf-carried iron in the outcome of leishmanial infection. Received: 20 May 1998 / Accepted: 18 June 1998     

CHÉDIAK-HIGASHI SYNDROME: REPORT OF A CASE WITH AUTOPSY AND ELECTRON MICROSCOPIC STUDIES*

A case of a 6-month-old boy with Chédiak-Higashi syndrome was studied by autopsy and electron microscopy. Electron microscopy of leukocytes from peripheral blood and bone marrow indicated that the abnormal intracytoplasmic granules or vacuoles identifiable in light microscopy fell into 3 categories; (1) giant lysosomes; (2) autophagic vacuoles; and (3) phagocytized cells. These observations may support the recent concept that Chédiak-Higashi syndrome is a possible lysosomal disease. Autopsy revealed various tissues of the patient infiltrated by histiocytic reticulum cells and lymphocytes, and unusual findings including an infiltration of these cells in and around the splenic trabecular veins and brain vessels as well as multifocal collections of the histiocytic cells in the brain. The histological feature of these infiltrates may represent a reactive type of histiocytic reticulosis or lympho-histiocytosis, probably secondary to some metabolic disorder to which lysosomal dysfunctions may play an important role.     

Antigen presentation by Leishmania mexicana-infected macrophages: Activation of helper T cells specific for amastigote cysteine proteinases requires intracellular killing of the parasites

Leishmania mexicana amastigotes proliferate in the phagolysosomes of mammalian macrophages. The parasites abundantly synthesize lysosomal cysteine proteinases, which are encoded by the lmcpb gene family. One of these genes was overexpressed in Escherichia coli, and the purified recombinant protein was used as an antigen to induce and establish a T helper 1 (T_h1) cell line. The T cells recognize epitopes shared by the native cysteine proteinases and the recombinant protein. Infected bone marrow-derived macrophages induced to express major histocompatibility complex class II molecules by interferon (IFN)-γ do not affect parasite viability. These macrophages fail to stimulate the proliferation of the T cell line. In contrast, strong T cell stimulation is observed after the parasites are killed by treatment with L-leucine methylester, or after activation of macrophages by IFN-γ and tumor necrosis factor-α. It is concluded that infected macrophages efficiently present this lysosomal Leishmania antigen once the parasites are inactivated and degraded. This observation may be of considerable relevance for the outcome of Leishmania infections provided that it can be extended to other parasite antigens.