Mucin-like peptides from Echinococcus granulosus induce antitumor activity.

There is substantial evidence suggesting that certain parasites can have antitumor properties. We evaluated mucin peptides derived from the helminth Echinococcus granulosus (denominated Egmuc) as potential inducers of antitumor activity. We present data showing that Egmuc peptides were capable of inducing an increase of activated NK cells in the spleen of immunized mice, a fact that was correlated with the capacity of splenocytes to mediate killing of tumor cells. We demonstrated that Egmuc peptides enhance LPS-induced maturation of dendritic cells in vitro by increasing the production of IL-12p40p70 and IL-6 and that Egmuc-treated DCs may activate NK cells, as judged by an increased expression of CD69. This evidence may contribute to the design of tumor vaccines and open new horizons in the use of parasite-derived molecules in the fight against cancer.

Intraspecies variation in Trypanosoma cruzi GPI-mucins: biological activities and differential expression of α-galactosyl residues.

The glycosylphosphatidylinositol (GPI)-anchored mucins of Trypanosoma cruzi trypomastigotes play an important immunomodulatory role during the course of Chagas disease. Here, some biological activities of tGPI-mucins from four T. cruzi isolates, including benznidazole-susceptible (BZS-Y), benznidazole-resistant (BZR-Y), CL, and Colombiana, were evaluated. GPI-mucins were able to differentially trigger the production of interleukin-12 and nitric oxide in BALB/c macrophages and modulate LLC-MK2 cell invasion. The significance of these variations was assessed after analysis of the terminal α-galactosyl residues. Enzymatic treatment with α-galactosidase indicated a differential expression of O-linked α-galactosyl residues among the strains, with higher expression of this sugar in BZS-Y and BZR-Y T. cruzi populations followed by Colombiana and CL. Unweighted pair group method analysis of the carbohydrate anchor profile and biological parameters allowed the clustering of two groups. One group includes Y and CL strains (T. cruzi II and VI), and the other group is represented by Colombiana strain (T. cruzi I).

Glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi trypomastigotes induce in vivo leukocyte recruitment dependent on MCP-1 production by IFN-gamma-primed-macrophages.

Glycosylphosphatidylinositol-anchored mucin-like glycoproteins from Trypanosoma cruzi trypomastigotes (tGPI-mucins) activate macrophages in vitro to produce proinflammatory cytokines, chemokines, and nitric oxide. These effects of tGPI-mucins may be important in the ensuing immune response to T. cruzi. Here, we have sought evidence for a role of tGPI-mucins in mediating leukocyte recruitment in vivo. tGPI-mucins are highly effective in promoting cell recruitment in the pleural cavity of mice primed with IFN-gamma-inducing agents but not in naïve mice. Maximal recruitment was observed at a dose between 250 and 1250 ng tGPI-mucins. There was a significant elevation in the levels of MCP-1 in the pleural cavity of primed animals injected with tGPI-mucins, and in vivo neutralization of MCP-1 abolished leukocyte recruitment. Pretreatment with anti-MIP-1alpha or anti-RANTES had no effect on the recruitment induced by tGPI-mucins. MCP-1 immunoreactivity was detected in pleural macrophages, and macrophages produced MCP-1 in vitro, especially after priming with IFN-gamma. Finally, tGPI-mucins induced significant leukocyte recruitment in primed C3H/HeJ but not in TLR2-deficient mice. Together, our results suggest that T. cruzi-derived GPI-mucins in conjunction with IFN-gamma may drive tissue chemokine production and inflammation and bear a significant role in the pathogenesis of Chagas disease.

Separation of Galfbeta1-->XGlcNAc and Galpbeta1-->XGlcNAc (X = 3, 4, and 6) as the alditols by high-pH anion-exchange chromatography and thin-layer chromatography: characterization of mucins from Trypanosoma cruzi.

The O-linked N-acetylglucosamine oligosaccharides in the mucins of Trypanosoma cruzi may contain galactofuranose or galactopyranose, depending on the strain, one of the components being the disaccharide. Since galactofuranose is a site for antibody recognition, it is desirable to have a sensitive method for the detection of the galactofuranosyl structures. In this paper, we present procedures for the separation of Galfbeta1-->XGlcNAc and Galpbeta1-->XGlcNAc (X = 3, 4, and 6) as the corresponding alditols by high-pH anion-exchange chromatography with pulse amperometric detection. All the isomeric disaccharides could be resolved on a CarboPac PA-10 column, the galactofuranose-containing disaccharides being more retained in the column. GlcNAcol and Galfbeta1-->4(Galpbeta1-->6)GlcNAcol could be analyzed in the same run. The compounds could also be separated by thin-layer chromatography on silica gel 60, a convenient method for analysis of the radiolabeled alditols obtained by reductive beta-elimination in the presence of NaB(3)H(4). Both methods were applied for the analysis of the O-linked sugars in the mucins of T. cruzi CL 14 and revealed that they contained only N-acetylglucosamine and the disaccharide Galpbeta1-->4GlcNAc.

Removal of sialic acid from mucin-like surface molecules of Trypanosoma cruzi metacyclic trypomastigotes enhances parasite-host cell interaction.

The 35/50 kDa mucin-like surface glycoprotein (gp35/50) of Trypanosoma cruzi metacyclic trypomastigotes has been implicated in mammalian cell invasion. In this study we investigated whether the sialyl residues of gp35/50 are required for interaction of parasites with target cells. After treatment with bacterial neuraminidase, the metacyclic forms (G strain) remained reactive with the monoclonal antibody (mAb) 10D8 but lost their reactivity with mAb 3C9, that recognizes sialic acid-containing epitopes on gp35/50, and entered HeLa cells in significantly higher numbers as compared to untreated controls. Resialylation of gp35/50, by incubation of parasites with T. cruzi trans-sialidase and sialyl lactose, restored the reactivity with mAb 3C9 as well as the affinity for sialic acid specific lectin. Accordingly, the rate of invasion of resialylated parasites was reduced to levels similar to those observed before desialylation. Purified G strain gp35/50, desialylated by neuraminidase treatment, bound to HeLa cells more than its sialylated counterpart. The Ca2+ signaling activity, which has been associated with cell invasion, was also determined by measuring the cytosolic Ca2+ concentration ([Ca2+]i), in HeLa cells upon interaction with sonicated extracts from untreated or neuraminidase-treated parasites, or with purified gp35/50 in its sialylated or desialylated form. Consistent with the results of cell invasion assay, the desialylated parasite preparations, as well as the sialic acid free gp35/50, induced an average elevation in [Ca2+]i significantly higher than that triggered by untreated controls. None of these effects, namely the increase in infectivity and Ca2+ signaling activity, was observed with neuraminidase-treated CL strain metacyclic trypomastigotes, which express a variant form of sialic acid gp35/50 molecule that is not recognized by mAb 10D8 and apparently is not involved in target cell invasion.

Glycoconjugates isolated from Trypanosoma cruzi but not from Leishmania species membranes trigger nitric oxide synthesis as well as microbicidal activity in IFN-gamma-primed macrophages.

In the present study, we investigated the role of glycosylphosphatidylinositol-anchored mucin-like glycoproteins (GPI-mucins) from Trypanosoma cruzi trypomastigotes in triggering the synthesis of nitric oxide as well as the microbicidal activity in murine macrophages. Our results show that GPI-mucins isolated from trypomastigote membranes are potent inducers of nitric oxide synthesis by IFN-gamma-primed macrophages, even at concentrations as low as 10 ng/ml. Our data also indicate the important role of glycosylphosphatidylinositol anchors from GPI-mucins as the second signal responsible for induction of nitric oxide synthesis by macrophages. To further investigate the role of these parasite molecules in inducing parasiticidal function, we cultured macrophages in the presence or absence of trypomastigote GPI-mucins and/or IFN-gamma and then infected these cells with either Leishmania spp. or T. cruzi. IFN-gamma was sufficient to induce microbial activity in macrophages infected with T. cruzi trypomastigotes. In contrast, killing of different species of Leishmania was further enhanced when macrophages exposed to IFN-gamma were also costimulated with trypomastigote-derived GPI-mucins. Our results also indicate that different glycolipids obtained from Leishmania major or Leishmania donovani (i.e., lipophosphoglycans or glycoinositolphospholipids) were unable to potentiate nitric oxide synthesis and/or microbicidal activity displayed by IFN-gamma-primed macrophages.