Leishmania mexicana: a cytochemical and quantitative study of lysosomal enzymes in infected rat bone marrow-derived macrophages

The cellular localization and activity of the lysosomal enzymes acid phosphatase, trimetaphosphatase, and arylsulfatase were studied in rat bone marrow-derived macrophages infected with Leishmania mexicana amazonensis amastigotes. The specific activity of acid phosphatase normalized for protein content was similar in normal macrophages and in isolated amastigotes, whereas the latter were markedly deficient in trimetaphosphatase and arylsulfatase activities. It is thus likely that trimetaphosphatase and arylsulfatase activities detected in infected macrophages were of host cell origin. The activities of the three enzymes, assayed biochemically, varied independently in the infected macrophages. While arylsulfatase activity was unchanged after infection, the activity of acid phosphatase increased by 19, 40, and 94% at 6, 24, and 48 hr, respectively. Trimetaphosphatase activity rose only slightly during the first 24 hr after infection but increased by 74% at 48 hr. The rise in acid phosphatase activity could be accounted for only partially by multiplication of the amastigotes. Thus, as for trimetaphosphatase, these results suggest enhanced macrophage synthesis of acid phosphatase and/or reduced enzyme degradation by the infected macrophages. The reduction in host cell lysosomes previously described (Ryter et al. 1983; Barbieri et al. 1985) was confirmed but appearance of lysosomal enzyme activity in the parasitophorous vacuole is documented in the present report. Thus, Leishmania do not seem to reduce the amount and the activity of host lysosomal enzymes.     

Leishmania (L.) infantum chagasi isolated from skin lesions of patients affected by non-ulcerated cutaneous leishmaniasis lead to visceral lesion in hamsters

In Central America, Leishmania (L.) infantum chagasi infection causes visceral leishmaniasis (VL) and non-ulcerated cutaneous leishmaniasis (NUCL). The aim of the present study was to evaluate the course of an experimental infection in hamsters caused by L. (L.) infantum chagasi isolated from patients affected by NUCL compared with a strain isolated from a patient with VL. Stationary phase parasites in culture were inoculated through subcutaneous and intraperitoneal routes in hamsters. Following the post-infection times, a histopathological study, parasite load and cytokine determination in skin from the cutaneous inoculation site and viscera were performed. Animals subcutaneously infected with the different strains did not develop macroscopic lesions at the inoculation site, and the histopathological changes in the dermis were very slight. Regarding the histopathological study of the viscera, we observed the portal mononuclear inflammatory infiltrate, the presence of nodules in the hepatic parenchyma and the proliferation of macrophages in the spleen, which increased over the infection course. Overall, the parasite load in the liver and spleen and in the total IgG titres in the sera of infected hamster showed an increase with the time of infection, regardless of the route of inoculation. Regarding cellular immunity, we did not observe an increase or decrease in pro- and anti-inflammatory cytokines compared to the healthy control, except for IL-10, which was evident in the infected animals. The data showed that strains isolated from NUCL cause visceral lesions in the hamsters regardless of the route of inoculation, and they were similar to parasites isolated from VL humans.     

Leishmania donovani: Acquired resistance to visceral leishmaniasis in the golden hamster

The ability to acquire resistance to visceral leishmaniasis was studied in the golden hamster. Hamsters were infected subcutaneously with Leishmania donovani and challenged 6 weeks later by an intracardial route of inoculation. Parasitization in previously infected and control hamsters after challenge was followed by spleen and liver impression smears. Hamsters receiving a previous subcutaneous infection showed significantly lower numbers of visceral parasites after challenge than control animals. Differences in parasitization between the two groups were detectable as early as 2 days after challenge. Promastigotes and both hamster or cotton rat infected spleen tissue were effective in inducing acquired resistance to infection. The golden hamster is discussed as a model for the study of immunity to kala-azar.     

Fusion of host cell secondary lysosomes with the parasitophorous vacuoles of Leishmania mexicana-infected macrophages.

Secondary lysosomes of cultured mouse peritoneal macrophages were labeled with the electron-dense colloid saccharated iron oxide; the identity of the labeled structures was checked by the Gomori reaction for acid phosphatase. Amastigotes of Leishmania mexicana mexicana derived from mouse lesions were used to infect these macrophages in vitro. In electron micrographs of thin sections of infected macrophages the labeled secondary lysosomes were seen fused with the parasitophorous vacuoles without preventing subsequent multiplication of the parasites. A similar fusion probably occurs in vivo, and may provide a pathway through which not only nutrients but also drugs and host antibodies could reach the intracellular parasite.     

Fusion of Host Cell Secondary Lysosomes with the Parasitophorous Vacuoles of Leishmania mexicana-inlected Macrophages

SYNOPSIS. Secondary lysosomes of cultured mouse peritoneal macrophages were labeled with the electron-dense colloid saccharated iron oxide; the identity of the labeled structures was checked by the Gomori reaction for acid phosphatase. Amastigotes of Leishmania mexicana mexicana derived from mouse lesions were used to infect these macrophages in vitro. In electron micrographs of thin sections of infected macrophages the labeled secondary lysosomes were seen fused with the parasitophorous vacuoles without preventing subsequent multiplication of the parasites. A similar fusion probably occurs in vivo , and may provide a pathway through which not only nutrients but also drugs and host antibodies could reach the intracellular parasite.     

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 with Leishmania 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.     

Effects of long-term in vitro cultivation on Leishmania donovani promastigotes

Promastigotes of Leishmania donovani that had been subcultured in modified Tobie's medium for 2 to 3 years showed decreased infectivity and lack of virulence for hamsters and mice compared to newly transformed promastigotes. Amastigotes derived from these long-term promastigote cultures decreased in number rapidly in hamsters, but only slightly in mice, over a 48-day period. In cultured mouse and hamster macrophages infected in vitro, amastigotes derived from long-term cultures rapidly decreased to low numbers, which persisted. The same pattern was seen in macrophages treated with catalase, an inhibitor of the oxygen-dependent killing mechanism of the macrophage. Promastigotes from long-term cultures also differed from virulent first-passage promastigotes in size, growth patterns in Tobie's medium, and in the quantities of some of their antigens.     

Leishmania-induced inhibition of macrophage antigen presentation analyzed at the single-cell level

A number of studies have previously examined the capacity of intracellular Leishmania parasites to modulate the capacity of macrophages to process and present Ags to MHC class II-restricted CD4(+) T cells. However, the bulk culture approaches used for assessing T cell activation make interpretation of some of these studies difficult. To gain a more precise understanding of the interaction between Leishmania-infected macrophages and effector T cells, we have analyzed various parameters of T cell activation in individual macrophage-T cell conjugates. Leishmania-infected macrophages efficiently stimulate Ag-independent as well as Ag-dependent, TCR-mediated capping of cortical F-actin in DO.11 T cells. However, infected macrophages are less efficient at promoting the sustained TCR signaling necessary for reorientation of the T cell microtubule organizing center and for IFN-gamma production. A reduced ability to activate these T cell responses was not due to altered levels of surface-expressed MHC class II-peptide complexes. This study represents the first direct single-cell analysis of the impact of intracellular infection on the interaction of macrophages with T cells and serves to emphasize the subtle influence Leishmania has on APC function.     

Suppression of macrophage lysosomal enzymes after Leishmania donovani infection

In order to have an insight into the role of host lysosomal enzymes in the intracellular survival of Leishmania parasites, the activities of beta-galactosidase, alpha-mannosidase, and N-acetyl-beta-D-glucosaminidase were studied in peritoneal macrophages of hamsters infected with L. donovani. There was a significant decrease of all three lysosomal enzymes after infection. Heat-killed or formalin-treated parasites failed to inhibit the enzymes, instead a slight stimulation was observed. Purified excreted factor from promastigotes had no effect on the enzymes except beta-galactosidase which was inhibited up to 20%. Inhibition of enzymes was not due to increased secretion after infection. The absence of induction of any endogenous macrophage inhibitor was confirmed by mixed experiments. The levels of 5'-nucleotidase and lactate dehydrogenase remained unchanged after infection. Thus, the inhibition of lysosomal enzymes appears to be the effect of infection process and reflects to actua decrease rather than increased secretion or the action of any inhibitors present in Leishmania promastigotes.     

Experimental transmission of Leishmania tropica to hamsters and mice by the bite of Phlebotomus sergenti

Phlebotomus sergenti is a natural vector of Leishmania tropica. However, the ability of P. sergenti to transmit L. tropica by bite has not been proven experimentally yet. We have transmitted L. tropica to golden hamsters and BALB/c mice by the bite of P. sergenti. Sand flies and Leishmania both originated from an anthroponotic cutaneous leishmaniasis focus in Urfa, Turkey. P. sergenti females from a laboratory colony were infected by feeding on lesions of needle-inoculated hamsters or mice. Gravid females were allowed to refeed on uninfected hosts 9-15 d after the infective feeding. At the second feeding, some infected females took a full blood meal, while others only a partial one; some females failed to feed at all. The ability of infected females to take a blood meal did not correlate with the parasite transmissibility. In four BALB/c mice, lesions developed after 1-6 months. In two albino hamsters (Mesocricetus auratus), lesions developed 1 month after the infective feeding, and Leishmania could be reisolated from these sites. Another hamster did not develop a lesion; however, the feeding site and the adjacent ear were PCR positive 1 year after infective feeding. Our results show that dissemination to other parts of host body occurs in L. tropica after sand fly bite. Experimental transmission of the parasite confirms that P. sergenti is a natural vector of L. tropica.     

Down-regulation of MARCKS-related protein (MRP) in macrophages infected with Leishmania.

Leishmania, a protozoan parasite of macrophages, has been shown to interfere with host cell signal transduction pathways including protein kinase C (PKC)-dependent signaling. Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP, MacMARCKS) are PKC substrates in diverse cell types. MARCKS and MRP are thought to regulate the actin network and thereby participate in cellular responses involving cytoskeletal rearrangement. Because MRP is a major PKC substrate in macrophages, we examined its expression in response to infection by Leishmania. Activation of murine macrophages by cytokines increased MRP expression as determined by Western blot analysis. Infection with Leishmania promastigotes at the time of activation or up to 48 h postactivation strongly decreased MRP levels. Leishmania-dependent MRP depletion was confirmed by [3H]myristate labeling and by immunofluorescence microscopy. All species or strains of Leishmania parasites tested, including lipophosphoglycan-deficient Leishmania major L119, decreased MRP levels. MRP depletion was not obtained with other phagocytic stimuli including zymosan, latex beads, or heat-killed Streptococcus mitis, a Gram-positive bacterium. Experiments with [3H]myristate labeled proteins revealed the appearance of lower molecular weight fragments in Leishmania-infected cells suggesting that MRP depletion may be due to proteolytic degradation.     

Leishmania mexicana: acid phosphatase ultrastructural cytochemistry of infected mouse macrophage cultures treated with phenazine methosulfate

Bone marrow-derived mouse macrophage cultures infected with Leishmania mexicana amazonensis amastigotes were given a 2-hr pulse with 10 microM phenazine methosulfate (PMS), a cationic electron carrier which destroys the intracellular parasites. Cultures were fixed at different times after the PMS pulse and processed for the detection of acid phosphatase (AcP) activity at the electron microscopic level. Only a small proportion of nontreated, infected macrophages stained for AcP. In contrast, 2 to 6 hr after exposure to PMS, many infected cells displayed AcP-positive lysosomes and parasitophorous vacuoles. This increased AcP reactivity paralleled the reduction in the percentage of morphologically intact parasites. In addition, qualitative observations indicated that while nontreated infected cells contained only few recognizable lysosomes, the lysosomal complement noticeably increased a few hours after exposure to PMS. Most intact intracellular amastigotes were not stained, but damaged parasites were often positive for AcP. Twenty hours after the PMS pulse, the percentage of AcP-positive macrophages dropped to the levels initially present in noninfected cultures and all of the parasites were destroyed. Exposure of noninfected macrophages to PMS did not affect their AcP reactivity.     

Experimental American leishmaniasis and Chagas' disease in the Brazilian squirrel monkey: effect of dual infection on antibodies to parasite antigens.

Adult, laboratory-bred squirrel monkeys (Saimiri sciureus) were infected with either Leishmania braziliensis braziliensis or L. b. panamensis and, 42 weeks later, they were challenge-infected with Trypanosoma cruzi. Another group of monkeys was infected with T. cruzi and challenged with L. b. braziliensis after 42 weeks. Immunoblotting was used to examine parasite antigens bound by antibodies in plasma obtained from the monkeys during the course of primary and challenge infections. During primary infections Leishmania-infected monkeys produced antibodies which bound to a number of Leishmania antigens, most notably a Leishmania antigen of 72 kDa, which were not recognized by antibodies produced by the monkeys given a primary infection of T. cruzi. These Leishmania-induced antibodies were no longer detectable 42 weeks after primary infections. However, when the Leishmania-infected monkeys were challenged with T. cruzi they once again produced antibodies capable of binding numerous Leishmania antigens, including the antigen of 72 kDa, which had not been recognized by antibodies produced by the monkeys with primary T. cruzi infections. A similar phenomenon was observed in T. cruzi-infected animals following Leishmania challenge.     

Apoptosis of Leishmania (Leishmania) chagasi amastigotes in hamsters infected with visceral leishmaniasis

Apoptosis in amastigotes from hamsters infected with visceral leishmaniasis was absent 30-day post-infection but appeared 90-day post-infection in the liver and spleen, as analysed using the TUNEL method. Necrosis was not present in these tissues and the nuclei of macrophages harbouring apoptotic amastigotes were preserved. Amastigote DNA fragmentation was demonstrated using agarose gel electrophoresis. DNA fragmentation was evident 90-day post-infection, coinciding with the occurrence of apoptosis of amastigotes in the tissues. Apoptosis of Leishmania amastigotes in vivo may constitute a mechanism that regulates growth of the parasite population during infection.     

Chemical and physical characterization of a proline-rich polypeptide from sheep colostrum.

Lysosome formation was induced in cells of the renal medulla by feeding rats on a K+-deficient diet. The role of the endoplasmic reticulum in the production of acid phosphatase, a typical lysosomal enzyme, was examined. Lysosomal and microsomal fractions were prepared for study by differential centrifugation of homogenates of renal papilla and inner stripe of red medulla. Acid phosphatase activity in the microsomal fraction was distinguished from the activity in the lysosomal fraction in normal tissue by differences in pH optima, tartrate inhibition, distribution of multiple forms after polyacrylamide-gel electrophoresis and detergent-sensitivity. During progressive K+ depletion, acid phosphatase activity in both microsomal and lysosomal fractions of the tissue increased 3-fold. In the lysosomes, K+ depletion was associated with the appearance of a new band of acid phosphatase. The neuraminidase-sensitivity of this band on polyacrylamide-gel electrophoresis indicated that the enzyme protein had been modified by the addition of sialic acid residues. K+ depletion also altered the lysosomal enzyme so that thiol compounds were able to stimulate its activity.     

Inhibition of human seminal fluid and Leishmania donovani phosphatases by molybdate heteropolyanions

Inhibition of a tartrate-resistant acid phosphatase (ACP) from Leishmania donovani and the tartrate-sensitive ACP from human seminal fluid (prostatic ACP) was examined using a series of 13 molybdate-containing heteropolyanions. The heteropolyanions were divided into four groups based on the number of molybdenum atoms they contain: Group I, Mo4; Group II, Mo6-8; Group III, Mo12; Group IV, Mo18. Two of the four groups, those consisting of compounds that contain either an Mo4 unit or an Mo18 unit with a heteroatom in the central cavity, were potent inhibitors and exhibited the highest degree of selectivity against the leishmanial and seminal fluid ACPs. The inhibition of prostatic ACP by complex E2 could be completely reversed by dialysis. Little inhibition of the acid phosphatase, beta-glucuronidase, or alpha-mannosidase from human spleen was observed with complexes B' and E2. For the seminal fluid phosphatase, the Ki values obtained with arsenate and vanadate depended markedly on pH, suggesting that, unlike most other phosphatases, the conformation of the inhibitor binding site on human seminal fluid ACP is pH-dependent. Results of competition experiments performed with various inhibitor pairs indicated that complex D2 binds to the active site of prostatic ACP while complex M binds at some site on the enzyme that affects the active site. Binding of complex M also modifies the affinity of the enzyme for other inhibitors such as vanadate. The potency of several heteropolyanion complexes and their selective inhibition of pathophysiologically significant acid phosphatases indicate that these compounds may have value as tools for study of the structure and function of this class of enzyme and perhaps in the therapy of human disease.     

Metastatic capability of Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis in golden hamsters

The pattern and kinetics of internal dissemination and frequency of cutaneous metastatic lesions resulting from experimental infection of golden hamsters with Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis were examined. Nineteen strains were evaluated: 16 L. (V.) panamensis isolated from patients and 3 L. (V.) guyanensis, 2 isolated from human cases and 1 WHO reference strain originating from a sandfly vector. Lymphatic dissemination occurred within 3 mo and was observed for 16 of 16 (100%) of L. (V.) panamensis and 3 of 3 (100%) of L. (V.) guyanensis. Parasites were cultured infrequently from liver and spleen: 3 of 125 (2%) L. (V.) panamensis and 1 of 22 (5%) L. (V.) guyanensis. Decreased frequency of isolation from the inoculation site and draining lymph nodes over time was accompanied by increased frequency of isolation from distant lymph nodes. Dilution of triturated tissue samples resulted in an increased efficiency of parasite culture. Both primary lesions and secondary cutaneous metastatic lesions were more severe in hamsters infected with L. (V.) guyanensis than with L. (V.) panamensis. Cutaneous metastatic lesions were produced more frequently by L. (V.) guyanensis, 24 of 46 hamsters (52%), than by L. (V.) panamensis, 28 of 252 hamsters (11%). Individual Leishmania strains displayed distinctive propensities to produce cutaneous metastases, manifested as a reproducible phenotype. Metastatic pathogenicity was independent of the inoculum dose, supporting the dissociation of infectivity and pathogenicity.     

Enhancement of macrophage IL-1 production by Leishmania major infection in vitro and its inhibition by IFN-gamma

Peritoneal cells from highly susceptible BALB/c mice were infected with Leishmania major and cultured for various times in vitro. The culture supernatants contained significant levels of IL-1 which were consistently higher than those in the cell cultures stimulated with an optimal concentration of LPS. This finding extends to a macrophage cell line, P388D1, and peritoneal exudate cells stimulated with starch in vivo. However, the level of IL-1 produced was significantly reduced when the cells were preincubated with a lymphokine preparation (supernatant of Con A-stimulated rat spleen cells). The level of IL-1 produced seems to be directly correlated with the degree of parasitization of the macrophages. A similar and dose-dependent reduction in IL-1 production by infected macrophages could also be obtained when the cells were preincubated with IFN-gamma. This finding is in direct contrast to that of visceral leishmaniasis in which peritoneal macrophages from BALB/c mice infected with Leishmania donovani not only fail to produce IL-1 but also lose the capacity to produce IL-1. This apparent discrepancy is discussed in terms of a possible difference in the induction of cell-mediated immunity between the two leishmanial diseases.