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1.
Leishmania donovani is an obligate intracellular protozoan parasite of macrophages; liver macrophages are, however, the only population of cells which express the resistant Lsh gene phenotype when these cells are infected in vitro. It was of interest to study in vitro the action of Con A-stimulated spleen cell lymphokines (LK) to protect or to cure liver macrophages from infection by L. donovani. Liver and peritoneal macrophages (PEC) from resistant (C57L/J) and susceptible (C57BL/6J) mice were infected in vitro with promastigotes before or after LK treatment; the percentage of infected macrophages was determined 4, 24, 48 and 72 h post-infection. Both macrophage populations were protected or cured by treatment with lymphokines; the cells of the resistant strain were protected or cured more effectively than those of the susceptible strain. The capacity for cure or for protection following LK treatment of liver and PEC macrophages was similar within each strain. Supernatants from the IL-2-produced MLA-144 cell line had no effect to protect or cure macrophages. This study indicates that the response of macrophages to the action of LK is also important in determining the susceptibility of mice to L. donovani; this model in vitro provides a good approximation of the response of macrophages to therapy.  相似文献   

2.
To determine the role of IFN-gamma in the activation of resident mouse peritoneal macrophages, crude macrophage-activating lymphokines were incubated with a monoclonal anti-murine IFN-gamma antibody. This treatment abolished the capacity of mitogen-induced lymphokines to enhance either H2O2 release or activity against the intracellular protozoa Toxoplasma gondii and Leishmania donovani. All macrophage-activating factor detected by these assays was also removed by passing the lymphokines over a Sepharose column to which the monoclonal anti-IFN-gamma antibody had been coupled. Therefore, pure murine rIFN-gamma was tested both in vitro and in vivo as a single activating agent. After 48 hr of pretreatment in vitro with 0.01 to 1 antiviral U/ml, macrophage H2O2-releasing capacity was enhanced an average of 6.4-fold; half-maximal stimulation was induced by 0.03 U/ml. Resident macrophages infected with T. gondii half-maximally inhibited parasite replication after 24 hr of preincubation with 0.14 U/ml of rIFN-gamma, and near complete inhibition was achieved by pretreatment with 100 U/ml. Half-maximal leishmanicidal activity was induced by 0.08 U/ml of rIFN-gamma, and 67 to 75% of intracellular L. donovani amastigotes were killed after macrophages were preincubated with 10 to 100 U/ml. Eighteen hours after parenteral injection of rIFN-gamma, peritoneal macrophages displayed a dose-dependent enhancement of H2O2-releasing capacity and antiprotozoal activity. Half-maximal enhancement required 85 to 250 U or rIFN-gamma given i.p. Peritoneal macrophages were also activated by rIFN-gamma injected i.v. and intramuscularly. These results suggest that, in the mouse model, IFN-gamma is likely to be a primary factor within mitogen-induced lymphokines responsible for activating macrophage oxidative metabolism and antiprotozoal activity, and indicate that rIFN-gamma is a potent activator of these effector functions both in vitro and in vivo. These findings provide a rationale for evaluating rIFN-gamma in the treatment of systemic intracellular infections, and indicate that murine models are appropriate for such studies.  相似文献   

3.
Leishmania donovani, the causative organism of human visceral leishmaniasis, invades host macrophages through its interaction with the cell surface molecules of target cells. The presence of a cell surface protein (Mr 34 kDa) having specific affinity toward hyaluronan (HA), a major extracellular matrix component, has been previously reported in macrophage cell lines. In order to identify the possible role of this HA-binding protein (HABP) in leishmaniasis, initially we demonstrated its overexpression in spleen, liver, macrophages, and serum of hamsters infected with L. donovani. We further observed higher levels of HABP in the macrophage cell line J774.G8 upon infection with L. donovani. Finally, we observed a significant increase in the level of HABP in the serum of patients with kala-azar. In order to understand its functional role in leishmaniasis, we report here a significant inhibition of cellular phosphorylation of HABP in hamster macrophages infected with L. donovani. Interestingly, the 34-kDa HABP was shown to bind with 2 proteins of promastigotes as well as amastigotes of L. donovani (with molecular masses of 55 kDa and 30 kDa respectively), suggesting a possible role for HABP in adhesion during the interaction of promastigotes and macrophages.  相似文献   

4.
BALB/cJ mice, which are homozygous for Lshs on chromosone 1, are genetically susceptible to Leishmania donovani (S3), but spontaneously reduce their parasite burdens late in the course of infection. Spleens from chronically infected mice (5 to 8 mo) were found to have T cells that responded to leishmanial Ag by proliferating and secreting immune IFN. An IFN-secreting T cell line derived from the spleen of a chronically infected mouse, after boosting with leishmanial Ag and treatment with Pentostam to kill residual parasites, was able to activate macrophages to kill amastigotes in vitro. Furthermore, after adoptive transfer of this cell line, naive BALB/cJ mice challenged with amastigotes demonstrated a 42-fold reduction in parasite burden compared to controls. Four of five clones derived from the protective T cell line secreted IFN and proliferated between days 7 and 14 after Ag stimulation. All clones were Ly1+2-, L3T4+. Another T cell line, which had Ly1+2-, L3T4+ phenotype, was derived from the lymph nodes of s.c. immunized, uninfected mice. It multipled but never produced IFN in response to leishmanial Ag and failed to protect macrophages against L. donovani infection in vitro or in vivo. This nonprotective T cell line and the fifth clone from the protective line, which also did not secrete IFN, proliferated between days 2 and 7 after Ag stimulation. In summary, leishmania-responsive helper/inducer T cells, which produced IFN but were slow to proliferate in response to Ag in vitro and which were able to activate macrophages to reduce amastigotes in vitro and in vivo, are present in chronically infected BALB/cJ mice and may mediate the decrease in parasite burden during chronic infection.  相似文献   

5.
After a subcutaneous injection of 100000 purified amastigotes of an isolate from a diffuse case of cutaneous leishmaniasis caused by the MHOM/BR/76/Ma-5 strain of Leishmania amazonensis, three inbred mouse strains developed a progressive nodular lesion, which evolved to an ulcerated lesion. Based on these data, mice of BALB/c, C57BL/6 or C57BL/10 could be classified as susceptible. The majority of mice developed metastases in the footpads, ear, tail, nose and oral mucosa. Amputation of the members related to the primary lesion was frequent. Experiments using the limiting dilution analysis showed that there was no correlation between lesion and parasite load. It has been demonstrated that these mouse strains could be considered excellent models for mucocutaneous leishmaniasis when infected with L. amazonensis. Metastatic lesions caused destruction of the nasal region with many parasitized macrophages under the epithelial surface of the nasal mucosa. Bone destruction occurred with an extensive inflammatory reaction presenting macrophages heavily parasitized by amastigotes. The parasites also spread to the periodontal ligament and other structures of the oral cavity, which could induce a severe inflammatory process. This study indicates that both nasal and oral lesions in mice infected by L. amazonensis were characterized by an inflammatory reaction with the presence of a high parasite load within macrophages.  相似文献   

6.
Resident peritoneal macrophages and macrophages elicited by injection of C3H/HeN mice with sterile inflammatory agents were exposed to amastigotes of Leishmania tropica in vitro and treated with lymphokines. Resident macrophages developed the capacity to kill intracellular parasites; microbicidal activity of activated resident cells ranged between 60 and 80%. In contrast, inflammatory macrophages responded poorly to lymphokines for intracellular killing of amastigotes; microbicidal activity of cells elicited with chronic inflammatory agents ranged between 0 and 45%. Defective intracellular killing of L. tropica by inflammatory macrophages was independent of the agent used to elicit the cells, but was clearly associated with the number of immature macrophages in the population. That intracellular killing capacity may reflect the presence of a killing mechanism in tissue-derived cells that is not yet developed in undifferentiated macrophages is supported by studies with peripheral blood monocytes: these cells were also incapable of eliminating intracellular amastigotes in the presence of potent activating factors. These observations on inflammatory macrophage interactions with amastigotes may provide important insights into the chronic nature of leishmanial disease.  相似文献   

7.
A series of H-2 and non-H-2 congenic resistant (CR) strains on a C57BL/10Sn background were infected with 10(7) amastigotes of Leishmania donovani. Non-H-2 congenic strains B10.LP-H-3b and B10.CE(30NX) and (B10.LP-H-3b x B10)F1 hybrids showed a very rapid decrease in liver-parasite burdens beyond day 21. Parasite counts for these strains at day 35 were significantly lower than for all other strains tested. The rapid decrease in parasite numbers, massive lymphocellular infiltration into the liver and strong delayed hypersensitivity reactions to parasite antigens in strains congenic for a portion of chromosome 2 indicated that acquired immunity to L. donovani was controlled by a dominant gene at or near the Ir-2 locus. In addition, B10.129(10M) mice, which differ from C57BL/10Sn at the H-11 locus, showed highly significant increases in parasite numbers at day 35. Other observations supporting the absence of acquired immunity in B10.129(10M) included negative delayed hypersensitivity tests to parasite antigens and the absence of lymphocellular infiltrate into the liver. Although the differences were not as pronounced, H-2 CR strains with H-2b, H-2a, and H-2k haplotypes also showed significantly greater decreases in parasite numbers by day 35 as compared to other H-2 CR strains.  相似文献   

8.
Natural resistance to the growth of Leishmania donovani in mice is controlled by a gene (Lsh) which is expressed, in an unknown fashion, in macrophages. Early net growth rate of the parasite is much higher in mice strains bearing the susceptible allele (Lshs) than in resistant (Lshr) mice. Intracellular events occurring in the Kupffer cells during this period have been studied at the ultrastructural level. It was found that the number of dividing amastigotes per thin section of infected cell was approximately 10-fold greater in susceptible (B10.A SgSn) than in resistant (A/J) strains of mice, both 7 and 14 days following infection. These findings support the hypothesis that high natural resistance to leishmaniasis (Lshr) is expressed as a microbistatic effect, exerted within the parasitized macrophage of the host.  相似文献   

9.
A. Hoerauf    Ch. Rascher    R. Bang    A. Pahl    W. Solbach    K. Brune    M. Röllinghoff  & H. Bang 《Molecular microbiology》1997,24(2):421-429
The antiparasitic effects of cyclosporin A were examined in leishmanial infection by analysing the role of CsA-binding proteins (cyclophilins) in the host–parasite interaction. We hypothesized that the leishmanicidal effects of CsA on Leishmania major infected macrophages might be mediated through a cyclophilin of either the parasite or the host cell. Two cyclophilins (20 and 22 kDa) were purified from L. major parasites and N-terminally sequenced. Although enzyme activity of these cyclophilins was inhibited by CsA, pretreatment of L. major parasites with CsA did not result in reduction of a subsequent macrophage infection, arguing against a role of L. major cyclophilins as infectivity potentiators. However, host-cell cyclophilin A (CypA) was found to be critically involved in the intracellular replication of L. major parasites in murine macrophages. An antisense oligonucleotide to murine CypA was constructed and added to cultures of peritoneal macrophages prior to infection with L. major parasites. This treatment strongly reduced the expression of CypA in macrophages and resulted in the inhibition of the intracellular replication of L. major amastigotes. These data indicate that interaction of amastigotes with host-cell cyclophilin is an important part of the intracellular replication machinery of L. major and define, for the first time, a direct involvement of a cyclophilin in the survival strategies of an intracellular parasite.  相似文献   

10.
Interleukin 1 (IL 1) is a principal mediator of the host immune response to microbial challenge. Accessory cells of the monocyte-macrophage series are a major source of this cytokine and are also chronically parasitized by protozoa of the genus Leishmania. This suggests that characterization of the macrophage IL 1 response to Leishmania would increase our understanding of the regulation of host immunity to these organisms. In the present study, the macrophage IL 1 response to Leishmania donovani was examined because infections with this organism have findings consistent with parasite-specific T cell unresponsiveness. Cytokine activity was measured either by direct stimulation or by co-stimulation of thymocytes. Conditioned media from BALB/c resident peritoneal macrophages infected with amastigotes of L. donovani contained no more IL 1 than did supernatant fluids of control cells. In contrast, supernatants from cells stimulated with lipopolysaccharide or heat-killed Listeria monocytogenes had significantly increased cytokine content. Resident cells infected with L. donovani for 4 hr before being stimulated with Listeria demonstrated a suppressed IL 1 response (approximately 40% of Listeria alone) to this secondary particulate stimulus. In contrast, the secondary response of leishmania-preinfected cells to lipopolysaccharide was not affected. To examine whether accessory cell nonresponsiveness to L. donovani (with respect to IL 1) was related to the state of macrophage activation, elicited peritoneal macrophages obtained by injection of proteose peptone were also studied. These cells responded to stimulation with lipopolysaccharide and fixed Staphylococcus aureus with increases in intracellular, membrane, and secreted cytokine activities. In contrast, L. donovani failed to induce any of these activities. This was found to be the case irrespective of whether amastigotes were alive or killed or opsonized with specific antibodies. Elicited cells preinfected with Leishmania responded normally to secondary stimulation with lipopolysaccharide, but not S. aureus (64% of Staphylococcus alone). In addition, attachment and penetration of L. donovani promastigotes and their subsequent conversion to amastigotes within macrophages failed to induce IL 1 synthesis. The findings of this study indicate that L. donovani has the ability to both evade and suppress the macrophage IL 1 response. Because this monokine provides an obligatory signal during macrophage-dependent T cell activation, evasion of signal transduction for IL 1 synthesis may be related to defects in cell-mediated immunity which occur during infections with this organism.  相似文献   

11.
Leishmania donovani causes human visceral leishmaniasis. The parasite infectious cycle comprises extracellular flagellated promastigotes that proliferate inside the insect vector, and intracellular nonmotile amastigotes that multiply within infected host cells. Using primary macrophages infected with virulent metacyclic promastigotes and high spatiotemporal resolution microscopy, we dissect the dynamics of the early infection process. We find that motile promastigotes enter macrophages in a polarized manner through their flagellar tip and are engulfed into host lysosomal compartments. Persistent intracellular flagellar activity leads to reorientation of the parasite flagellum toward the host cell periphery and results in oscillatory parasite movement. The latter is associated with local lysosomal exocytosis and host cell plasma membrane wounding. These findings implicate lysosome recruitment followed by lysosome exocytosis, consistent with parasite-driven host cell injury, as key cellular events in Leishmania host cell infection. This work highlights the role of promastigote polarity and motility during parasite entry.  相似文献   

12.
C3H/HeN and C3H/HeJ mice were infected ip with viable BCG, a macrophage-activating agent, and their peritoneal exudate macrophages exposed to Leishmania tropica amastigotes. Macrophages from BCG-infected C3H/HeN mice had both leishmanicidal activities described for lymphokine activation of C3H/HeN macrophages in vitro: increased resistance to L. tropica infection, followed by intracellular killing of the parasite. Macrophages from BCG-infected C3H/HeN mice were also activated to kill tumor cells in vitro. In contrast, macrophages from BCG-treated C3H/HeJ mice were not resistant to L. tropica infection, did not kill intracellular amastigotes over 72 hr in culture, and were not cytotoxic to tumor cells.  相似文献   

13.
Taxol, a mitotic spindle toxin, was found to selectively inhibit the proliferation of Leishmania donovani in vitro at nanomolar concentrations with an IC50 of 35 nM. Concentrations of taxol as high as 50 nM, however, did not affect J774A.1 murine macrophages. Taxol (30 nM) also inhibited amastigote multiplication within a J774A.1 macrophage cell line when used in a 10-day experiment. It resulted in the in vitro assembly of L. donovani microtubules in a dose-dependent manner. When promastigotes were exposed to different concentrations of taxol for 24 h, cells were largely blocked in the G2-M phase of the cell cycle and there was a marked reduction in the percentage of cells in the S phase. The selective nature of taxol action against the parasite and its effectiveness in controlling amastigote multiplication emphasise its use as a promising chemotherapeutic against kala-azar.  相似文献   

14.
Protozoan parasites of the genus Leishmania are important human pathogens that differentiate inside host macrophages into an amastigote life cycle stage. Although this stage causes the pathogenesis of leishmaniasis, only few proteins have been implicated in amastigote intracellular survival. Here we compare morphology, infectivity and protein expression of L. donovani LD1S grown in host free (axenic) culture, or exclusively propagated in infected hamsters, with the aim to reveal parasite traits absent in axenic but selected for in hamster-derived amastigotes through leishmanicidal host activities. Axenic and splenic amastigotes showed a striking difference in virulence and the ability to cause experimental hepato-splenomegaly in infected hamsters. 2D-DIGE analysis revealed statistically significant differences in abundance for 152 spots, with 14 spots showing fivefold or higher abundance in splenic amastigotes. Proteins identified by MS analysis include the anti-oxidant enzyme tryparedoxin peroxidase, and enzymes implicated in protein and amino acid metabolism. Analysis of parasite growth in vitro in minimal medium demonstrated increased survival of hamster-derived compared with axenic parasites under conditions that mimic the nutrient poor, cytotoxic phagolysosome. Thus, our comparative proteomics analysis sheds important new light on the biochemistry of bona fide amastigotes and informs on survival factors relevant for intracellular L. donovani infection.  相似文献   

15.
Eleven mouse strains were inoculated in footpads with amastigotes of Leishmania tropica and observed for 12 weeks. Liver and spleen impression smears from infected mice were examined for the presence of intracellular parasites. Four strains (BALB/cJ, C57L/J, NZW/N, and P/J) failed to heal the subcutaneous lesion and showed evidence of systemic infection; the remaining seven strains (A/J, C3H/HeJ, C3H/HeN, C3HeB/FeJ, C57BL/6J, C57BL/10J, and C57BL/10ScN) were each resistant to infection and resolved their lesions by Week 10. Macrophages from the four susceptible strains could not be activated to kill L. tropica amastigotes by treatment with soluble lymphocyte products in vitro. In contrast, macrophages from all seven resistant strains responded to lymphokine treatment and eliminated 80-90% of intracellular parasites. These results suggest that in vitro macrophage microbicidal activities predict the course of systemic leishmanial disease.  相似文献   

16.
The ability of BALB/c mice to resist reinfection with Leishmania donovani following chemotherapy was studied. BALB/c mice, infected with L. donovani, were treated on Days 7 and 8 postinfection with free, niosomal, or liposomal sodium stibogluconate. It was found that all three drug treatments caused a dramatic reduction in liver parasite burdens as measured on Days 6 and 29 post-treatment. On Day 6 postdrug treatment infection with L. donovani amastigotes, of mice from infected, drug-treated groups, along with age- and sex-matched uninfected controls, showed that at 23 days later, significantly fewer parasites were recovered from the livers of reinfected animals compared with controls given their first infection. Treatment of mice with sodium stibogluconate 6 days prior to a primary infection significantly reduced the number of parasites recovered 14 days later, especially using the carrier form of the drug. In vivo macrophage activity in the liver, as measured by the uptake of radiolabeled horseradish peroxidase immune complex, was significantly raised following stibogluconate treatment of infected but not uninfected mice. These results suggest that a state of resistance persists in the liver of infected mice following chemotherapy which may in part be due to local macrophage activation but also to an unsuspected persistance of the drug.  相似文献   

17.
Liposomes consisting of stearylamine (SA) and egg yolk phosphatidylcholine (PC) were studied for their cytotoxic activity against freshly transformed promastigotes and intracellular amastigotes of Leishmania donovani, the causative agent of visceral leishmaniasis. More than 99% of the parasites of strain AG83 were killed within 60 min by treatment with 22 mol% SA-PC liposomes (132 microg/ml total lipids). This was further confirmed by incubating the liposome-treated promastigotes at 22 C for 96 hr. The killing activity of the liposomes progressively decreased with lowering lipid concentration. However, weak cytotoxic activity was still detected at 6.6 microg/ml lipids. Leishmanicidal activity of the liposomes became stronger with increasing SA content but was reduced with the incorporation of cholesterol in the liposomes. A similar cytotoxic effect was observed on other Indian strains of L. donovani, for example PKDL and DD8, as well as on species such as Leishmania donovani S1, Leishmania donovani infantum, Leishmania tropica, Leishmania amazonensis, and Leishmania mexicana. However, freshly transformed promastigotes appeared to be more susceptible than the ones subcultured. The strong leishmanicidal activity of SA-PC liposomes was also demonstrated toward intracellular L. donovani amastigotes. The SA-bearing vesicles could effectively inhibit the growth and multiplication of the parasites within the macrophages. The cytolytic activity of these liposomes on leishmanial parasites and low toxicity on host macrophages may, thus, find application in the therapy of leishmaniasis.  相似文献   

18.
Leishmania donovani ADP-ribosylation factor-like protein 3A (LdARL-3A) is a small G protein isolated from the protozoan parasite L. donovani with no defined physiological function. Previously [Cuvillier, A., Redon, F., Antoine, J.-C., Chardin, P., DeVos, T., and Merlin, G. (2000) J Cell Sci 113: 2065-2074] we have shown that overexpression in L. amazonensis promastigotes of the mutated protein LdARL-3A-Q70L, which remains constitutively associated with GTP, leads to the disappearance of the flagellum but does not impair cell viability or growth. Here we report that parasites overexpressing LdARL-3A-Q70L can invade in vitro cultivated macrophages to the same extent as control cells, demonstrating that the flagellum is not necessary for attachment to or engulfment into macrophages. These infections are productive because amastigotes differentiate and multiply. However, aflagellated LdARL-3A-Q70L-overexpressing Leishmania promastigotes could not survive in experimentally infected Lutzomyia longipalpis insect vectors, in contrast to untransfected or native LdARL-3A-overexpressing cells. Overexpression of the native and mutated proteins did not modify in vitro procyclic to metacyclic lipophosphoglycan maturation or differentiation from procyclic to metacyclic promastigotes, nevertheless there is a block in transmission of Leishmania. Better understanding of LdARL-3A pathways, notably those regarding flagellum biogenesis, may lead to the future development of Leishmania-specific drugs, which may stop parasite transmission in nature without affecting other species.  相似文献   

19.
The antifungal and antileishmanial agent amphotericin B (AmB) was formulated in tripalmitin based nanosize lipid partices (emulsomes) for macrophage targeting for the treatment of visceral leishmaniasis (VL). Emulsomes were modified by coating them with macrophage-specific ligand (O-palmitoyl mannan, OPM). The antileishmanial activity of AmB (0.5 and 1?mg/kg) was investigated in-vivo against VL by the inhibition of parasitic load in the spleen of L. donovani infected hamsters after intraperitoneal injections of AmB-Doc (Mycol), plain emulsomes (TPEs) and OPM coated emulsomes (TPEs-OPM). The formulations were found to be less effective at the dose of 0.5?mg/kg. At the dose of 1?mg/kg, formulation TPEs-OPM eliminated intracellular amastigotes of L. donovani within splenic macrophages more efficiently (62.76?±?3.54 % parasite inhibition) than the formulation TPEs (42.68?±?2.36 % parasite inhibition) (P?相似文献   

20.
We examined in this work whether rTNF inhibits the capacity of Trypanosoma cruzi to multiply within murine macrophages or enhances the ability of the phagocytic host cells to destroy internalized parasites. We found that rTNF would not alter the fate of the trypanosomes within macrophages over a 48-h incubation period unless the latter cells were also treated with 1 ng/ml bacterial endotoxin (LPS). Treatment of macrophages with rTNF plus LPS, but not separate treatment with either rTNF or LPS, resulted in a significant decrease in the number of organisms per 100 macrophages with respect to values obtained with mock-treated macrophages. In addition, there was a significant reduction in the proportion of infected macrophages over the 48-h incubation period, indicating parasite clearance by the host cells. The combined effects of rTNF and LPS were seen when macrophages from CBA/J were used but not with LPS-insensitive macrophages from C3H/HeJ mice. Increased trypanosome killing by CBA/J macrophages treated with rTNF plus LPS was not seen when catalase was present in the culture medium, indicating a role for hydrogen peroxide in the cytotoxic effect. These results show that rTNF can affect the fate of T. cruzi within macrophages if LPS is present and point to destruction of internalized organisms rather than inhibition of parasite multiplication as the most likely explanation.  相似文献   

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