Cross-immunoreactivity between anti-potato apyrase antibodies and mammalian ATP diphosphohydrolases: potential use of the vegetal protein in experimental schistosomiasis

We have previously showed that Schistosoma mansoni ATP-diphosphohydrolase and Solanum tuberosum potato apyrase share epitopes and the vegetable protein has immunostimulatory properties. Here, it was verified the in situ cross-immunoreactivity between mice NTPDases and anti-potato apyrase antibodies produced in rabbits, using confocal microscopy. Liver samples were taken from Swiss Webster mouse 8 weeks after infection with S. mansoni cercariae, and anti-potato apyrase and TRITC-conjugated anti-rabbit IgG antibody were tested on cryostat sections. The results showed that S. mansoni egg ATP diphosphohydrolase isoforms, developed by anti-potato apyrase, are expressed in miracidial and egg structures, and not in granulomatous cells and hepatic structures (hepatocytes, bile ducts, and blood vessels). Therefore, purified potato apyrase when inoculated in rabbit generates polyclonal sera containing anti-apyrase antibodies that are capable of recognizing specifically S. mansoni ATP diphosphohydrolase epitopes, but not proteins from mammalian tissues, suggesting that autoantibodies are not induced during potato apyrase immunization. A phylogenetic tree obtained for the NTPDase family showed that potato apyrase had lower homology with mammalian NTPDases 1-4, 7, and 8. Further analysis of potato apyrase epitopes could implement their potential use in schistosomiasis experimental models.     

Mapping of the conserved antigenic domains shared between potato apyrase and parasite ATP diphosphohydrolases: potential application in human parasitic diseases

Evolutionary and closer structural relationships are demonstrated by phylogenetic analysis, peptide prediction and molecular modelling between Solanum tuberosum apyrase, Schistosoma mansoni SmATPase 2 and Leishmania braziliensis NDPase. Specific protein domains are suggested to be potentially involved in the immune response, and also seem to be conserved during host and parasite co-evolution. Significant IgG antibody reactivity was observed in sera from patients with American cutaneous leishmaniasis (ACL) and schistosomiasis using potato apyrase as antigen in ELISA. S. mansoni adult worm or egg, L. braziliensis promastigote (Lb) and Trypanosoma cruzi epimastigote (EPI) have ATP diphosphohydrolases, and antigenic preparations of them were evaluated. In ACL patients, IgG seropositivity was about 43% and 90% for Lb and potato apyrase, respectively, while IgM was lower (40%) or IgG (100%) seropositivity for both soluble egg (SEA) and adult worm (SWAP) antigens was higher than that found for potato apyrase (IgM=10%; IgG=39%). In Chagas disease, IgG seropositivity for EPI and potato apyrase was 97% and 17%, respectively, while the IgM was low (3%) for both antigens. The study of the conserved domains from both parasite proteins and potato apyrase could lead to the development of new drug targets or molecular markers.     

TGF-beta-associated enhanced susceptibility to leishmaniasis following intramuscular vaccination of mice with Leishmania amazonensis antigens

Leishmania amazonensis and Leishmania braziliensis are the main causal agents of anergic diffuse cutaneous leishmaniasis and hyperergic mucosal leishmaniasis in man, respectively. In this work we demonstrate that intramuscular vaccination of BALB/c mice with whole antigens of L. amazonensis (LaAg) but not L. braziliensis (LbAg) results in increased susceptibility to cutaneous leishmaniasis. LaAg vaccination resulted in an increased capacity of the draining lymph nodes to produce IL-10 and TGF-beta during antigen recall responses. In vitro cultivation with LaAg but not LbAg induced increased apoptosis of CD8+ T cells. Following infection with L. amazonensis, LaAg-vaccinated mice produced significantly more TGF-beta and a higher serum IgG1/IgG2a antibody ratio compared with LbAg-vaccinated and non-vaccinated animals. The association of TGF-beta with enhanced susceptibility to infection was confirmed in mice co-vaccinated with LaAg and neutralizing anti-TGF-beta antibodies. Upon parasite challenge, these animals developed much smaller lesion sizes and parasite burdens, comparable with non-vaccinated controls. The disease-promoting effect of LaAg vaccination is not a general event, as in contrast to BALB/c, the disease outcome in C57Bl/6 mice was unaltered. Together, these findings indicate that species-specific components of L. amazonensis activate overt TGF-beta production that predisposes more susceptible individuals to aggravated disease following vaccination.     

An antigenic domain within a catalytically active Leishmania infantum nucleoside triphosphate diphosphohydrolase (NTPDase 1) is a target of inhibitory antibodies

We identified a shared B domain within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detected in Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenized promastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, following SDS-PAGE and silver staining was identified as a single polypeptide of 50 kDa. By Western blots, it was recognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptide derived from the potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived from the Leishmania NTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantum NTPDase 1 and, also, its conserved B domain (r83–122). By immunoprecipitation assays and Western blots, immune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, the immune sera SB (44%) and SC or SD (87–99%) inhibited its activity, suggesting a direct effect on the B domain. By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respectively, confirming the B domain antigenicity.     

Leishmania amazonensis infection in nude mice.

Leishmania amazonensis is an intracellular protozoan parasite of macrophages. Cutaneous leishmaniasis in an immunocompetent host begins as papules or nodules followed by ulceration at the site of promastigote inoculation. In this study, the pathological changes of cutaneous leishmaniasis lesions in T cell deficient nude mice were examined. When infected with L. amazonensis promastigotes, nude mice developed non-ulcerative cutaneous nodules. By histological examination of cutaneous lesions, massive accumulation of vacuolated histiocytes containing amastigotes was observed in all the nude mice. Although infiltration of mononuclear and polymorphonuclear cells was seen in the lesions of immunocompetent mice, few such cells were observed in the lesions of nude mice. These results indicate the importance of T cells on the ulcer formation in cutaneous leishmaniasis.     

The 3A1-La monoclonal antibody reveals key features of Leishmania (L) amazonensis metacyclic promastigotes and inhibits procyclics attachment to the sand fly midgut

In this work, we characterise metacyclic promastigotes of Leishmania amazonensis, the causative agent of cutaneous and diffuse cutaneous leishmaniasis in the New World. To purify metacyclics from stationary culture by negative selection, we used the monoclonal antibody 3A1-La produced against procyclic promastigotes. The purified forms named 3A1-La(-) promastigotes, present key metacyclic characteristics: slender cell body and long flagella, ultrastructural features, resistance to complement lysis, high infectivity for macrophages and mice and reduced capacity for binding to the sand fly midgut. Moreover, the epitope recognised by 3A1-La is important for the promastigote attachment to the insect vector midgut epithelium. These results further characterise 3A1-La(-) promastigotes as metacyclic forms of L. amazonensis.     

The efficacy of 2-nitrovinylfuran derivatives against Leishmania in vitro and in vivo

Despite recent advances in the treatment of some forms of leishmaniasis, the available drugs are still far from ideal due to inefficacy, parasite resistance, toxicity and cost. The wide-spectrum antimicrobial activity of 2-nitrovinylfuran compounds has been described, as has their activity against Trichomonas vaginalis and other protozoa. Thus, the aim of this study was to test the antileishmanial activities of six 2-nitrovinylfurans in vitro and in a murine model of leishmaniasis. Minimum parasiticide concentration (MPC) and 50% inhibitory concentration (IC₅₀) values for these compounds against the promastigotes of Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis were determined, as were the efficacies of two selected compounds in an experimental model of cutaneous leishmaniasis (CL) caused by L. amazonensis in BALB/c mice. All of the compounds were active against the promastigotes of the three Leishmania species tested. IC₅₀ and MPC values were in the ranges of 0.8-4.7 µM and 1.7-32 µM, respectively. The compounds 2-bromo-5-(2-bromo-2-nitrovinyl)-furan (furvina) and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan (UC245) also reduced lesion growth in vivo at a magnitude comparable to or higher than that achieved by amphotericin B treatment. The results demonstrate the potential of this class of compounds as antileishmanial agents and support the clinical testing of Dermofural^(r) (a furvina-containing antifungal ointment) for the treatment of CL.     

Applications of recombinant Leishmania amazonensis expressing egfp or the beta-galactosidase gene for drug screening and histopathological analysis.

Leishmania amazonensis recombinants expressing the enhanced green fluorescent protein (egfp) gene or beta-galactosidase gene (lacZ) were constructed for drug screening and histopathological analysis. The egfp or lacZ in a leishmanial transfection vector, p6.5, was introduced into L. amazonensis promastigotes, and egfp or lacZ-carrying recombinant L. amazonensis, La/egfp and La/lacZ, respectively, were obtained. Expression of egfp or lacZ in both promastigotes and amastigotes could be clearly visualized by fluorescence microscopy or by light microscopy with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), respectively. Fluorescence signal and beta-galactosidase activity measured by a colorimetric reaction with chlorophenol red beta-D-galactopyranoside (CPRG) were well correlated to the numbers of these parasites. The inhibitory concentration (IC50) of a leishmanicidal drug, amphotericin B, in L. amazonensis promastigotes measured using La/egfp and La/lacZ was similar to that measured by conventional methods such as cell counting, thymidine incorporation and colorimetric assay. Furthermore, the fluorescence signal and absorbance of CPRG correlated well with the numbers of La/egfp and La/lacZ amastigotes in macrophages, respectively, suggesting La/egfp and La/lacZ can be a convenient and useful tool for drug screening not only in promastigotes, but also in amastigotes of L. amazonensis. La/lacZ collected from mouse tissues four weeks after the parasite infection were stained well with X-Gal. La/lacZ allowed parasite detection at high sensitivity in the tissues of infected mice and will be useful for following infections in macrophages in vivo. Thus, the marker-transfected Leishmania parasites constructed in this study will be useful for analyses of Leishmania parasites, especially at the intracellular stage.     

The role of IL-10 in promoting disease progression in leishmaniasis

To determine the role of IL-10 in cutaneous leishmaniasis, we examined lesion development following Leishmania major infection of genetically susceptible BALB/c mice lacking IL-10. Whereas normal BALB/c mice developed progressive nonhealing lesions with numerous parasites within them, IL-10(-/-) BALB/c mice controlled disease progression, and had relatively small lesions with 1000-fold fewer parasites within them by the fifth week of infection. We also examined a mechanism whereby Leishmania induced the production of IL-10 from macrophages. We show that surface IgG on Leishmania amastigotes allows them to ligate Fc gamma receptors on inflammatory macrophages to preferentially induce the production of high amounts of IL-10. The IL-10 produced by infected macrophages prevented macrophage activation and diminished their production of IL-12 and TNF-alpha. In vitro survival assays confirmed the importance of IL-10 in preventing parasite killing by activated macrophages. Pretreatment of monolayers with either rIL-10 or supernatants from amastigote-infected macrophages resulted in a dramatic enhancement in parasite intracellular survival. These studies indicate that amastigotes of Leishmania use an unusual and unexpected virulence factor, host IgG. This IgG allows amastigotes to exploit the antiinflammatory effects of Fc gamma R ligation to induce the production of IL-10, which renders macrophages refractory to the activating effects of IFN-gamma.     

Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydrolase isoform (SmATPDase 2) and immunolocalisation of this protein in the Schistosoma mansoni egg

A peptide (SmB2LJ; r175-194) that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation) or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenberg's envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs.     

Leishmania amazonensis: Biological and biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities

The presence of Leishmania amazonensis ecto-nucleoside triphosphate triphosphohydrolase activities was demonstrated using antibodies against different NTPDase members by Western blotting, flow cytometry, and immunoelectron microscopy analysis. Living promastigote cells sequentially hydrolyzed the ATP molecule generating ADP, AMP, and adenosine, indicating that this surface enzyme may play a role in the salvage of purines from the extracellular medium. The L. amazonensis ecto-NTPDase activities were insensitive to Triton X-100, but they were enhanced by divalent cations, such as Mg(2+). In addition, the ecto-NTPDase activities decreased with time for 96 h when promastigotes were grown in vitro. On the other hand, these activities increased considerably when measured in living amastigote forms. Furthermore, the treatment with adenosine, a mediator of several relevant biological phenomena, induced a decrease in the reactivity with anti-CD39 antibody, raised against mammalian E-NTPDase, probably because of down regulation in the L. amazonensis ecto-NTPDase expression. Also, adenosine and anti-NTPDase antibodies induced a significant diminishing in the interaction between promastigotes of L. amazonensis and mouse peritoneal macrophages.     

Axenic promastigote forms of Leishmania (Viannia) lainsoni as an alternative source for Leishmania antigen production

The present study demonstrates that axenic cultures of Leishmania (Viannia) lainsoni produce larger cell masses in NNN-LIT medium, as well as higher amounts of total proteins in cell extracts, than Leishmania (Leishmania) amazonensis. Antigenicity of L. (V.) lainsoni whole promastigotes is similar to that of L. (L.) amazonensis, as demonstrated by an indirect immunofluorescence diagnostic test using sera from human patients and dogs infected with visceral leishmaniasis. Infectivity of the L. (V.) lainsoni strain used in the present work was demonstrated by the detection by transmission-electron microscopy of tissue amastigotes in skin lesion samples from an experimentally infected hamster. Incubation of lesion fragments in NNN-LIT medium allowed us to obtain promastigote forms, which could be cultivated successfully in vitro. lsoenzyme analysis of such promastigotes confirmed the parasite strain as L. (V.) lainsoni, as compared to other Leishmania reference strains. Our data indicate that L. (V.) lainsoni is a useful alternative source for antigen production as well for use in assays that depend on large cell volumes of Leishmania spp. parasites.     

Protective immunity using recombinant human IL-12 and alum as adjuvants in a primate model of cutaneous leishmaniasis.

Protection from cutaneous leishmaniasis, a chronic ulcerating skin lesion affecting millions, has been achieved historically using live virulent preparations of the parasite. Killed or recombinant Ags that could be safer as vaccines generally require an adjuvant for induction of a strong Th1 response in murine models. Murine rIL-12 as an adjuvant with soluble Leishmania Ag has been shown to protect susceptible mice. We used 48 rhesus macaques to assess the safety, immunogenicity, and efficacy of a vaccine combining heat-killed Leishmania amazonensis with human rIL-12 (rhIL-12) and alum (aluminum hydroxide gel) as adjuvants. The single s.c. vaccination was found to be safe and immunogenic, although a small transient s.c. nodule developed at the site. Groups receiving rhIL-12 had an augmented in vitro Ag-specific IFN-gamma response after vaccination, as well as increased production of IgG. No increase in IL-4 or IL-10 was found in cell culture supernatants from either control or experimental groups. Delayed hypersensitivity reactions were not predictive of protection. Intradermal forehead challenge infection with 107 metacyclic L. amazonensis promastigotes at 4 wk demonstrated protective immunity in all 12 monkeys receiving 2 microgram rhIL-12 with alum and Ag. Partial efficacy was seen with lower doses of rhIL-12 and in groups lacking either adjuvant. Thus, a single dose vaccine with killed Ag using rhIL-12 and alum as adjuvants was safe and fully effective in this primate model of cutaneous leishmaniasis. This study extends the murine data to primates, and provides a basis for further human trials.     

Infection-induced respiratory burst in BALB/c macrophages kills Leishmania guyanensis amastigotes through apoptosis: possible involvement in resistance to cutaneous leishmaniasis

The immune mechanisms that underlie resistance and susceptibility to leishmaniasis are not completely understood for all species of Leishmania. It is becoming clear that the immune response, the parasite elimination by the host and, as a result, the outcome of the disease depend both on the host and on the species of the infecting Leishmania. Here, we analyzed the outcome of the infection of BALB/c mice with L. guyanensis in vivo and in vitro. We showed that BALB/c mice, which are a prototype of susceptible host for most species of Leishmania, dying from these infections, develop insignificant or no cutaneous lesions and eliminate the parasite when infected with promastigotes of L. guyanensis. In vitro, we found that thioglycollate-elicited BALB/c peritoneal macrophages, which are unable to eliminate L. amazonensis without previous activation with cytokines or lipopolysaccharide, can kill L. guyanensis amastigotes. This is the first report showing that infection of peritoneal macrophages with stationary phase promastigotes efficiently triggers innate microbicidal mechanisms that are effective in eliminating the amastigotes, without exogenous activation. We demonstrated that L. guyanensis amastigotes die inside the macrophages through an apoptotic process that is independent of nitric oxide and is mediated by reactive oxygen intermediates generated in the host cell during infection. This innate killing mechanism of macrophages may account for the resistance of BALB/c mice to infection by L. guyanensis.     

Trypanothione reductase activity is prominent in metacyclic promastigotes and axenic amastigotes of Leishmania amazonesis. Evaluation of its potential as a therapeutic target

The activity of trypanothione reductase in Leishmania amazonensis was evaluated and it was demonstrated that TR is expressed in the soluble fractions of infective promastigotes and amastigotes, while non-infective promastigotes expressed the enzyme at basal levels. This data allows an association of enzyme activity and the infective capacity of the parasite. We have also previously demonstrated that amidine compounds (N, N'-diphenyl-4-methoxy-benzamidine and pentamidine) were active against this parasite. Here, experiments concerning the effect of these compounds on TR activity, showed that both compounds significantly inhibited the enzyme. However, against glutathione reductase, only pentamidine showed a significant inhibitory action, suggesting an association with the toxic effects of this drug used in the clinic for the treatment of leishmaniasis.     

Antigenic extracts of Leishmania braziliensis and Leishmania amazonensis associated with saponin partially protects BALB/c mice against Leishmania chagasi infection by suppressing IL-10 and IL-4 production

This study evaluated two vaccine candidates for their effectiveness in protecting BALB/c mice against Leishmania chagasi infection. These immunogenic preparations were composed of Leishmania amazonensis or Leishmania braziliensis antigenic extracts in association with saponin adjuvant. Mice were given three subcutaneous doses of one of these vaccine candidates weekly for three weeks and four weeks later challenged with promastigotes of L. chagasi by intravenous injection. We observed that both vaccine candidates induced a significant reduction in the parasite load of the liver, while the L. amazonensis antigenic extract also stimulated a reduction in spleen parasite load. This protection was associated with a suppression of both interleukin (IL)-10 and IL-4 cytokines by spleen cells in response to L. chagasi antigen. No change was detected in the production of IFN-γ. Our data show that these immunogenic preparations reduce the type 2 immune response leading to the control of parasite replication.     

Species and stage specificity of Leishmania donovani antigens.

Monoclonal antibodies D2 and D13 were produced in mice using Leishmania donovani promastigote membrane fractions. To study the species and stage specificity of the antigens recognized by these antibodies, we examined amastigotes prepared in vitro and cultured promastigotes by indirect immunofluorescence with monoclonal antibodies D2 and D13. Monoclonal antibody D2 showed weak reactivity for 9 of 9 strains of L. donovani complex promastigotes and 8 of 9 amastigotes. In contrast, only 2 of 22 strains from other complexes yielded equivocal reactions. Monoclonal antibody D13, however, had much broader reactivity. D13 reacted with all the promastigotes and amastigotes of L. donovani complex isolates as well as with 10 of 22 promastigotes and 8 of 13 amastigotes from other complexes. The high degree of species specificity seen with monoclonal antibody D2 provides a rationale for further study of this antibody and its purified antigen for parasite identification and serodiagnosis of visceral leishmaniasis. The strong fluorescent signal noted with D13 and the presence of the D13 epitope on all L. donovani complex parasites supports studies on its role as an antigen in immunoprophylaxis of visceral leishmaniasis.     

The Abl and Arg kinases mediate distinct modes of phagocytosis and are required for maximal Leishmania infection

Leishmania, an obligate intracellular parasite, binds several receptors to trigger engulfment by phagocytes, leading to cutaneous or visceral disease. These receptors include complement receptor 3 (CR3), used by promastigotes, and the Fc receptor (FcR), used by amastigotes. The mechanisms mediating uptake are not well understood. Here we show that Abl family kinases mediate both phagocytosis and the uptake of Leishmania amazonensis by macrophages (Ms). Imatinib, an Abl/Arg kinase inhibitor, decreases opsonized polystyrene bead phagocytosis and Leishmania uptake. Interestingly, phagocytosis of IgG-coated beads is decreased in Arg-deficient Ms, while that of C3bi-coated beads is unaffected. Conversely, uptake of C3bi-coated beads is decreased in Abl-deficient Ms, but that of IgG-coated beads is unaffected. Consistent with these results, Abl-deficient Ms are inefficient at C3bi-opsonized promastigote uptake, and Arg-deficient Ms are defective in IgG1-opsonized amastigote uptake. Finally, genetic loss of Abl or Arg reduces infection severity in murine cutaneous leishmaniasis, and imatinib treatment results in smaller lesions with fewer parasites than in controls. Our studies are the first to demonstrate that efficient phagocytosis and maximal Leishmania infection require Abl family kinases. These results highlight Abl family kinase-mediated signaling pathways as potential therapeutic targets for leishmaniasis.