Roles of CR3 and mannose receptors in the attachment and ingestion of Leishmania donovani by human mononuclear phagocytes.

Leishmania donovani is an obligate intracellular parasite of mammalian macrophages. Two macrophage receptors, the mannose-fucose receptor (MFR) and the receptor for complement component C3bi, CR3, were examined for their roles in the attachment and ingestion of L. donovani by human monocyte-derived macrophages. Two monoclonal antibodies which bind to the human CR3, anti-Mo1 and anti-Mac-1, inhibited both attachment and ingestion of L. donovani promastigotes after preincubation with human monocyte-derived macrophages; attachment was inhibited by 40 and 62% by anti-Mo1 and anti-Mac-1, respectively, and ingestion was inhibited by 34 and 51% by anti-Mo1 and anti-Mac-1, respectively. The interaction between promastigotes and CR3 may not have involved the C3bi-binding site on CR3, however, because a monoclonal antibody which exhibits specificity for this site, OKM10, inhibited promastigote attachment by only 18%. In contrast, OKM1, which is believed to react with the alternate lectinlike binding site on CR3, inhibited ingestion by 65%. MFR activity was inhibited using the soluble MFR ligands, mannan and mannosylated bovine serum albumin, which also inhibited promastigote attachment by 40 and 37%, respectively. The simultaneous inhibition of both CR3 (by anti-Mac-1) and the MFR (by either mannan or mannosylated bovine serum albumin) resulted in a greater decrease in promastigote attachment than inhibition of either receptor alone. Additionally, the reduction of MFR activity by allowing macrophages to adhere to a mannan-coated surface followed by the addition of anti-CR3 antibodies resulted in an 81% inhibition of promastigote ingestion, a greater decrease than was obtained by manipulation of either receptor alone. The results suggest that the MFR and CR3 independently participate in the attachment and ingestion of L. donovani promastigotes by human macrophages.     

IFN-γ is directly Cytostatic to the Extracellular Form (Promastigote) of Leishmania donovani

We investigated the effect of various human cytokines on the in vitro growth of Leishmania donovani (L. donovani ) promastigotes. rhIFN-α, rhIFN-β, rhIL-1, rhIL-2, rhIL-3, rhIL-4, rhIL-6 and rhM-CSF had no effect. By contrast. rhIFN-γ was directly growth inhibitory to L. donovani promastigotes in a dose-dependent manner. Anti-IFN-γ antibody, but neither anti-IFN-α nor an ti-IFN-β antibody, abolished this promastigote growth Inhibitory effect of rhIFN-γ. L. donovani promastigotes were not lysed by rhIFN-γ as determined by ⁵¹Cr-release assay. These data indicate that rhIFN-γ is cytostatic. not cytotoxic, to L. donovani promastigotes.     

Amastigote stage-specific monoclonal antibodies against Leishmania major.

Monoclonal antibodies were produced against gamma-irradiated amastigotes of Leishmania major. Five antibodies (T16 through T20) were selected which reacted in enzyme-linked immunoassays with the intracellular stage of the parasite. These antibodies did not react with promastigotes of L. major or Leishmania donovani. One of the monoclonal antibodies (T16) reacted with amastigotes of Leishmania mexicana amazonensis and L. donovani. Western blotting (immunoblotting) and immunoprecipitation of [35S]methionine-labeled amastigotes demonstrates that T16 reacted with multiple L. major amastigote components between 12 and 180 kilodaltons. Antibody T20 was shown to recognize a low-molecular-mass doublet (less than 26 kilodaltons) in both [14C]leucine- and [35S]methionine-labeled amastigotes. A protein of less than 180 kilodaltons was also weakly recognized by T17, T19, and T20 in metabolically labeled amastigotes. This protein reacted strongly with T16. The reactive antigens could be identified on the surface of amastigotes isolated from the lesions of infected mice and on newly transformed amastigotes within 24 h after the infection of mouse peritoneal macrophages by promastigotes. These monoclonal antibodies should prove useful for the diagnosis of L. major in human tissue biopsies.     

Interaction of Leishmania donovani promastigotes with human monocyte-derived macrophages: parasite entry, intracellular survival, and multiplication.

Leishmania donovani promastigotes were incubated with human monocyte-derived macrophages in vitro to assess the role of macrophages in the early stage of visceral leishmaniasis. Adherent mononuclear cells, obtained from nonimmune human donors, were cultivated on glass cover slips for 5 days and then incubated with axenically grown promastigotes in the presence of heat-inactivated autologous serum. Promastigotes attached to macrophages with either their flagellar or aflagellar ends, and macrophage pseudopodia formed around them. Intracellular parasites were identified within phagocytic vacuoles by electron microscopy, and the parasites assumed a form similar to that of amastigotes obtained from infected hamster spleens. Initially, 67 +/- 5% of the macrophages were infected with a mean of 4.2 +/- 0.7 parasites per infected cell. After 6 days of incubation, 79 +/- 7% of the macrophages were infected with 15.9 +/- 3.2 parasites per infected cell. The total number of parasites per monolayer increased from 4.8 +/- 0.8 x 10(5) to 1.8 +/- 0.4 x 10(6) (P less than 0.05). Dividing parasites were identified in macrophage vacuoles by electron microscopy. Human monocyte-derived macrophage vacuoles by electron microscopy. Human monocyte-derived macrophages can phagocytize promastigotes, allow the conversion of promastigotes to an amastigote-like state, and support intracellular multiplication.     

The immunochemical structure and surface arrangement of Leishmania donovani lipophosphoglycan determined using monoclonal antibodies

Using intact Leishmania donovani promastigotes or purified L. donovani lipophosphoglycan (LPG) as immunogens, we have derived four LPG-specific monoclonal antibodies (MAbs). Two of these MAbs recognize an epitope consisting of the repeating phosphorylated galactose beta-1,4-mannose disaccharide portion of the molecule and cross-reacted with LPG from Leishmania major. These MAbs bound to the surface of living promastigotes of both species. The two other MAbs bound to the phosphosaccharide core structure of LPG and did not bind to the surface of living parasites, presumably due to masking of the core region. Experiments using all four MAbs with an LPG-deficient promastigote mutant indicated that both the repeat epitope and phosphosaccharide core were present in these cells, suggesting that incomplete assembly was responsible for the absence of intact LPG.     

RNA interference reveals a role for TLR2 and TLR3 in the recognition of Leishmania donovani promastigotes by interferon-gamma-primed macrophages

Leishmania donovani promastigotes evade the induction of a proinflammatory response during their invasion of naive macrophages. However, their entry into IFN-gamma-primed macrophages is accompanied by the secretion of nitric oxide (NO) and proinflammatory cytokines. In the present study, we addressed the hypothesis that priming with IFN-gamma induces the expression of a receptor that enables mouse macrophages to recognize L. donovani promastigotes. We observed that in IFN-gamma-primed macrophages, L. donovani promastigotes stimulated Interleukin-1 receptor-associated kinase-1 (IRAK-1) activity. We next showed that Toll-like receptor (TLR)3 is barely detectable in naive macrophages but is expressed in IFN-gamma-treated macrophages. Silencing of TLR3, TLR2, IRAK-1 and myeloid differentiation factor 88 (MyD88) expression by RNA interference revealed that both TLR are involved in the secretion of NO and TNF-alpha induced by L. donovani promastigotes. Using L. donovani mutants, we showed that TLR2-mediated responses are dependent on Galbeta1,4Manalpha-PO(4)-containing phosphoglycans, whereas TLR3-mediated responses are independent of these glycoconjugates. Furthermore, our data indicate a participation of TLR2 and TLR3 in the phagocytosis of L. donovani promastigotes and a role for TLR3 in the leishmanicidal activity of the IFN-gamma-primed macrophages. Collectively, our data are consistent with a model where recognition of L. donovani promastigotes depends on the macrophage activation status and requires the expression of TLR3.     

Susceptibility of 3H-thymidine-prelabelled Leishmania donovani amastigotes and promastigotes to the cytotoxic activity of peritoneal, splenic and liver macrophages

C57BL/6 macrophage populations from spleen and liver, the main organs for the manifestation of visceral leishmaniasis, were investigated for their ability to perform spontaneous phagocytosis-associated killing of 3H-thymidine (3H-TdR)-prelabelled L. donovani amastigotes and promastigotes. The results showed that organ macrophages from spleen and liver killed L. donovani amastigotes and promastigotes spontaneously with high efficiency. This consistent finding was first detectable at 2-3 h, and the reaction was completed at 12 h. This type of killing was strongly enhanced when spleen and liver macrophages were activated. This phagocytosis-associated killing mechanism may contribute, to a large extent, in maintaining the infection under control in vivo, by drastically reducing the amount of parasites that is required to establish intracellular parasitism. To be able to assay phagocytosis-associated destruction of both promastigotes and amastigotes, a reproducible system for the production in vitro of Leishmania donovani amastigotes by the macrophage cell-line J774 was developed. The DNA of the Leishmania amastigotes was labelled with 3H-TdR with high efficiency. The spontaneous label release of prelabelled L. donovani amastigotes was comparable to that of prelabelled promastigotes over an assay period of 24 h.     

The kinetoplastid membrane protein 11 of Leishmania donovani and African trypanosomes is a potent stimulator of T-lymphocyte proliferation.

Kinetoplastid membrane protein 11 (KMP-11) from Leishmania donovani is an abundant 11-kDa surface membrane glycoprotein. Lymph node cells from mice of six different H-2 haplotypes immunized with KMP-11 or with L. donovani promastigotes were stimulated to proliferate in vitro KMP-11. Primed purified T cells required antigen presentation since they were not stimulated unless KMP-11-pulsed or L. donovani-infected macrophages were added. Promastigotes of a wide variety of Leishmania species and procyclic forms of African trypanosomes stimulated proliferation of KMP-11-primed or L. donovani promastigote-primed lymph node cells. All of the Leishmania promastigotes and African trypanosomes tested contained an 11-kDa protein, as detected by immunoblotting with KMP-11-specific monoclonal antibodies. The widespread distribution of the 11-kDa (KMP-11) molecules and their ability to stimulate strong T-lymphocyte proliferation in a non-H-restricted fashion suggest that they may be important molecules for induction of cell-mediated immune responses.     

Purification and enzymatic activity of an NADH-fumarate reductase and other mitochondrial activities of Leishmania parasites

A 65 kD membrane-associated NADH-fumarate reductase subunit, which has a molecular weight similar to that of one of the enzyme subunits from bacteria, was purified from Leishmania donovani promastigotes. NADH-fumarate reductase and other mitochondrial enzymatic activities of L. major and L. donovani promastigotes and amastigotes were investigated. The presence of NADH-fumarate reductase was demonstrated in digitonin-permeabilized L. major promastigotes and mitochondria of L. major and L. donovani promastigotes and amastigotes. The activity of solubilized NADH-fumarate reductase was measured in L. major and L. donovani promastigotes. Succinate exhibited a clear concentration-dependent inhibitory effect on fumarate reductase, whereas fumarate also exhibited a clear concentration-dependent inhibitory effect on succinate dehydrogenase. The data indicate that fumarate reductase is an obligatory component of the respiratory chain of the parasite. Since the enzyme is an important component in the intermediate metabolism in the Leishmania parasite and is absent in mammalian cells, it could be a potential target for antileishmanial drugs.     

Autoantibodies to crude human leucocyte interferon (IFN), native human IFN, recombinant human IFN-alpha 2b and human IFN-gamma in healthy blood donors.

Recently, naturally occurring antibodies to IFN-alpha were discovered in a few systemic lupus erythematosus (SLE) and cancer patients; however, in most patients monitored for anti-IFN antibodies before treatment, no antibodies were found. In an attempt to explain the 'IFN-blocking effect' that we observed in all serum samples we investigated 200 sera from healthy blood donors. We isolated the globulin fraction, and used rabbit anti-human IgG and IgM columns, protein A columns and T-gel affinity chromatography to isolate human IgG and IgM. All sample fractions were tested in a biological IFN neutralization assay by means of a sensitive MTT-assay. We found that normal human serum contained autoantibodies to crude human leucocyte IFN, native human fibroblast IFN, recombinant human leucocyte IFN-alpha 2b and recombinant human IFN-gamma, and that these naturally occurring antibodies were biologically active immunoglobulins of IgG and IgM type. These anti-IFN antibodies were also present in purified human normal immunoglobulin pools. We conclude that all humans have naturally occurring anti-interferon antibodies in their serum, and it is a tempting theory that human cytokines and lymphokines are, at least partly, regulated by immunoglobulins.     

Purification and characterization of a membrane-bound acid phosphatase of Leishmania mexicana.

As defined by the reaction with monoclonal antibodies, Leishmania mexicana promastigotes contain two acid phosphatases which together comprise about 90% of the cellular activity. A first enzyme recognized by monoclonal antibody AP4 is largely membrane-bound. The protein has an apparent molecular weight of 70,000-72,000, carries about seven N-linked glycan chains and is present in approximately 16,000 copies per cell. The protein is also expressed in the amastigote stage. A second enzyme reactive with monoclonal antibody AP3, that also recognizes lipophosphoglycan and a secreted acid phosphatase, is mainly found in the soluble fraction of promastigote lysates. It is suggested that this enzyme is the precursor of the secreted protein. The N-terminal sequences of the phosphatase recognized by AP4 and the secreted enzyme are similar but not identical. AP4 does not cross-react with phosphatase activity of Leishmania major or Leishmania donovani promastigotes, while AP3 recognizes part of the cellular and all of the secreted phosphatase activity of L. donovani promastigotes but not that of L. major which does not release an acid phosphatase into the culture medium.     

Nitric oxide induced expression of stress proteins in virulent and avirulent promastigotes of Leishmania donovani

Intracellular survival and replication of Leishmania donovani inside macrophage is essential for establishment of the disease. Cytokines play an important role in this process through activation or inhibition of macrophage antimicrobial activity. Nitric oxide (NO) has been demonstrated to be the principal effector molecule mediating intracellular killing of Leishmania. We have examined the effect of NO and various other cytokines on stress protein synthesis by promastigotes of L. donovani virulent and avirulent strains. Virulent promastigotes exposed to NO showed appreciable increase in relative synthesis of HSPs 83, 70 and 65. The overexpression of HSPs on exposure of parasite to NO was observed to be more pronounced at 37 degrees C than at 24 degrees C. In contrast, the avirulent promastigotes responded by an increase in relative synthesis of HSP70 alone at 37 degrees C. Furthermore, treatment of promastigotes of L. donovani with gammaIFN, TGF-beta or IL-4 did not significantly alter the stress proteins expression. The overexpression of HSPs in promastigotes of L. donovani in response to sublethal doses of NO suggests that HSPs may be playing a protective role for parasite survival in the mammalian host. This is further supported by the observation that a significantly higher induction of HSPs is seen in the virulent as compared to the avirulent strain of L. donovani.     

Leishmania donovani promastigotes evade the activation of mitogen-activated protein kinases p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase-1/2 during infection of naive macrophages

The protozoan parasite Leishmania fails to activate naive macrophages for proinflammatory cytokines production, and selectively impairs signal transduction pathways in infected macrophages. Because mitogen-activated protein kinases (MAPK)- and NF-kappaB-dependent signaling pathways regulate proinflammatory cytokines release, we investigated their activation in mouse bone marrow-derived macrophages (BMM) exposed to Leishmania donovani promastigotes. In naive BMM, the parasite failed to induce the phosphorylation of p38 MAPK, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK)1/2, as well as the degradation of IkappaB-alpha. The use of L. donovani mutants defective in the biosynthesis of lipophosphoglycan revealed that evasion of ERK1/2 activation requires surface expression of the repeating unit moiety of this virulence determinant. In IFN-gamma-primed BMM, L. donovani promastigotes strongly induced the phosphorylation of p38 MAPK and ERK1/2, and the use of selective inhibitors for ERK (PD98059) and p38 MAPK (SB203580) revealed that both kinases are required for L. donovani-induced TNF-alpha but not NO(2)(-) release. Collectively, these data suggest that both p38 MAPK and ERK1/2 pathways participate in some Leishmania-induced responses in IFN-gamma-primed BMM. The ability of L. donovani promastigotes to avoid MAPK and NF-kappaB activation in naive macrophages may be part of the strategy evolved by this parasite to evade innate immune responses.     

抗杜氏利什曼原虫犬分离株单克隆抗体的研制及用于病犬血清循环抗原的检测

本文报道用杜氏利什曼原虫大分离株前鞭毛体膜抗原免疫BALB/c小鼠的脾细胞与Sp2/0瘤细胞融合,得到多株分泌抗体较高且稳定的细胞株。进一步筛选出McAb C11-G10-A4可用于检测病犬血清循环抗原。用McAb-AST检测采自甘肃省黑热病流行区的30份犬血清,阳性率30％。与骨髓涂片查病原体的总符合率为86.7％,阳性符合率达100％。69份非流行区对照大血清检测结果,仅1份出现阳性反应,可见本试验的敏感性较高,特异性较强(98.55％)。     

Adjuvanticity and protective immunity elicited by Leishmania donovani antigens encapsulated in positively charged liposomes.

In the search for a leishmaniasis vaccine, extensive studies of cutaneous leishmaniasis have been carried out. Investigations in this regard with the visceral form are limited. As an initial step in the identification of the protective molecules, leishmanial antigens extracted from the membranes of Leishmania donovani promastigotes, alone or in association with liposomes, were evaluated for their immunogenicity and ability to elicit a protective immune response against challenge infection. Intraperitoneal immunization of hamsters and BALB/c mice with the leishmanial antigens conferred protection against infection with the virulent promastigotes. Encapsulation in positively charged liposomes significantly enhanced the protective efficacy of these antigens. The splenic parasite burden of hamsters was reduced by 97% after 6 months of infection. BALB/c mice exhibited 87 and 81.3% protection in the liver and spleen, respectively, after 4 months of infection. These protected animals elicited profound delayed-type hypersensitivity and increased levels of Leishmania-specific immunoglobulin G (IgG) antibodies. Protection in mice also coincided with elevated levels of IgM and IgA antibodies, which decreased with disease progression in the control-infected animals. Although both IgG1 and IgG2a antibodies were present in the sera of infected mice, IgG1 appeared to be the predominant isotype, suggesting a preferential induction of the Th2 type of immune response over that of Th1. Effective stimulation of all the IgG isotypes, particularly IgG2a, after immunization with liposome encapsulated antigens seems to be responsible for the significant levels of resistance against the disease. Taken together, these data indicate a potential for the liposomal antigens as a vaccine which could trigger both humoral and cell-mediated immune responses.     

Inhibition of Leishmania donovani promastigote internalization into murine macrophages by chemically defined parasite glycoconjugate ligands.

Leishmania donovani, the agent of human visceral leishmaniasis, is an intracellular parasite that must be recognized and internalized by host macrophages to complete its biological cycle. In a search for possible ligands for macrophage surface receptors, glycoconjugates were obtained from Leishmania promastigotes by aqueous, phenol-aqueous, and alkaline extraction. A fucose-mannose glycoproteic ligand, a lipopeptidephosphoglycan, and a phosphate mannogalactan ligand were purified from promastigotes and analyzed for their chemical contents, with special attention to their glycidic moieties. Sugars that were identified as components of these glycoconjugates were tested for their capacity to inhibit promastigote internalization by BALB/c peritoneal macrophages in vitro. Neutral hexoses showed little inhibitory activity; fucose, charged monosaccharides, and a mannose polymer showed the highest activity. Two of the glycoconjugates (fucose-mannose glycoproteic ligand and phosphate mannogalactan ligand) purified from promastigotes were potent inhibitors of internalization, 75% inhibition being obtained at concentrations of 6 to 10 micrograms/ml. The simultaneous presence of both ligands in low concentrations yielded an increase in inhibitory activity above that found for each ligand alone, indicating that promastigotes may use at least two receptor sites for penetration into macrophages. These ligands are specific inhibitors of L. donovani promastigote phagocytosis, since 10 micrograms of each ligand per ml interfered neither with internalization of yeast cells nor with phagocytosis of Leishmania adleri promastigotes.     

Methylglyoxal-catabolizing enzymes of Leishmania donovani promastigotes

Methylglyoxal is a toxic metabolite with growth inhibitory properties against Leishmania donovani promastigotes. We have shown in the present study that both log and stationary phase promastigotes of L. donovani can catabolize methylglyoxal to D-lactate as the major end product. The specific activity of methylglyoxal reductase was found to be the highest of all the catabolic enzymes. In contrast, the anabolic pathway for methylglyoxal could not be detected. Moreover, when control promastigotes or promastigotes in which the glycolytic pathway was inhibited were incubated with glucose, glycerol or dihydroxyacetone phosphate as energy source, neither methylglyoxal nor D-lactate could be detected.     

Subspecies-specific surface antigens of promastigotes of the Leishmania donovani complex.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of proteins and externally exposed labeled surface constituents were analyzed in promastigotes of three etiological agents of kala azar (Leishmania donovani, HS70 strain from India; L. chagasi, Imperatriz strain from Brazil; L. infantum, ITMPA K263 strain from Morocco and MO strain from France). Coomassie blue-stained gels showed similar protein patterns for L. donovani and L. chagasi and a more distinct one for L. infantum. Surface radioiodination with two different methods, lactoperoxidase and IODO-GEN, gave identical autoradiographic patterns for each parasite. Four major labeled proteins with apparent Mr values of 65,000, 60,000, 50,000, and 26,000 were detected in both L. chagasi and L. donovani. However, the radioiodinated polypeptide pattern of L. infantum only showed two major bands with an apparent Mr of 62,000 and a doublet of 26,000 to 23,000. Immunoprecipitation of detergent extracts of labeled promastigote subspecies with immune sera from rabbits immunized with either L. chagasi or L. infantum and from patients and mice infected with these two parasites, as well as with a monoclonal antibody against the surface of L. donovani promastigotes, demonstrated that the surface antigenic expression of L. infantum is different from that noticed in the two other subspecies, which are similar. Immunofluorescence experiments with some of these antibodies confirmed these results. The present findings should be considered in taxonomic and immunological studies in visceral leishmaniasis.     

Leishmania donovani requires functional Cdc42 and Rac1 to prevent phagosomal maturation

Leishmania donovani promastigotes survive inside macrophage phagosomes by inhibiting phagosomal maturation. The main surface glycoconjugate on promastigotes, lipophosphoglycan (LPG), is crucial for survival and mediates the formation of a protective shell of F-actin around the phagosome. Previous studies have demonstrated that this effect involves inhibition of protein kinase C alpha. The present study shows that functional Cdc42 and Rac1 are required for the formation of F-actin around L. donovani phagosomes. Moreover, we present data showing that phagosomes containing LPG-defective L. donovani, which is unable to induce F-actin accumulation, display both elevated levels of periphagosomal F-actin and impaired phagosomal maturation in macrophages with permanently active forms of Cdc42 and Rac1. We conclude that L. donovani engages Cdc42 and Rac1 to build up a protective coat of F-actin around its phagosome to prevent phagosomal maturation.     

Solid-phase radioimmunoassay of IgA, IgG, and IgM antibodies to human rotavirus.

A solid-phase radioimmunoassay (RIA) has been developed for the detection of human rotavirus-specific IgA, IgG, and IgM antibodies. Nebraska calf diarrhea virus grown in LLC-MK2 cell cultures in the presence of trypsin was directly adsorbed onto polystyrene balls, and antibodies that attached to the virus-coated balls were detected by subsequent binding of 125I-labeled antibodies specific to human alpha, gamma or mu chains of human Iga, IgG, or IgM immunoglobulins. A total of 116 serum specimens from 58 adult patients were tested. Binding ratios between the positive and the negative serum varied between 5 and 15, occasionally being 20 or more in the IgA and IgG assays, but rarely exceeding 3 in the IgM assay. The RIA was found to be more sensitive in detecting antibodies to rotavirus than the complement fixation (CF) test, the RIA titers obtained being 50--100 times as high as the CF titers. The method described offers a possibility of evaluating the immune response to human rotavirus and of detecting recent infection.