Schistosoma Mansoni Larvae Research Articles

Molluscicidal and Larvicidal Potency of N-Heterocylic Analogs against Biomophalaria alexandrina Snails and Schistosoma mansoni Larval Stages.

This work describes the synthesis of quinoline-based N--heterocyclic arenes and their biological evaluation as molluscicides against adult Biomophalaria alexandrina snails as well as larvicides against Schistosoma mansoni larvae (miracidia and cercariae). Molecular docking studies were demonstrated to investigate their affinity for cysteine protease protein as an interesting target for antiparasitics. Compound AEAN showed the best docking results followed by APAN in comparison to the co-crystallized ligand D1R reflected by their binding affinities and RMSD values. The egg production, hatchability of B. alexandrina snails and ultrastructural topography of S. mansoni cercariae using SEM were assessed. Biological evaluations (hatchability and egg-laying capacity) revealed that the quinoline hydrochloride salt CAAQ was the most effective compound against adult B. alexandrina snails, whereas the indolo-quinoline derivative APAN had the most efficiency against miracidia, and the acridinyl derivative AEAA was the most effective against cercariae and caused 100% mortality. CAAQ and AEAA were found to modulate the biological responses of B. alexandrina snails with/without S. mansoni infection and larval stages that will affect S. mansoni infection. AEAA caused deleterious morphological effects on cercariae. CAAQ caused inhibition in the number of eggs/snail/week and reduced reproductive rate to 43.8% in all the experimental groups. CAAQ and AEAA can be recommended as an effective molluscicide of plant origin for the control program of schistosomiasis.

Evolutionary epidemiology of schistosomiasis: linking parasite genetics with disease phenotype in humans

Here we assess the role of parasite genetic variation in host disease phenotype in human schistosomiasis by implementing concepts and techniques from environmental association analysis in evolutionary epidemiology. Schistosomiasis is a tropical disease that affects more than 200 million people worldwide and is caused by parasitic flatworms belonging to the genus Schistosoma. While the role of host genetics has been extensively studied and demonstrated, nothing is yet known on the contribution of parasite genetic variation to host disease phenotype in human schistosomiasis. In this study microsatellite genotypes of 1561 Schistosoma mansoni larvae collected from 44 human hosts in Senegal were linked to host characteristics such as age, gender, infection intensity, liver and bladder morbidity by means of multivariate regression methods (on each parasite locus separately). This revealed a highly significant association between allelic variation at the parasite locus L46951 and host infection intensity and bladder morbidity. Locus L46951 is located in the 3′ untranslated region of the cGMP-dependent protein kinase gene that is expressed in reproductive organs of adult schistosome worms and appears to be linked to egg production. This putative link between parasite genetic variation and schistosomiasis disease phenotype sets the stage for further functional research.

In vivo assay of IgE activities on the expulsion of intestinal adult worms

A physiological role of immunoglobulin E (IgE) is to promote parasitic helminth expulsion. This assertion is largely based on a series of studies carried out by Capron's laboratory. They observed that IgE is an essential component of protective immunity against Schistosoma mansoni larvae both in vitro and in vivo. Then, another group reported that IgE-deficient mice show higher worm burdens than wild-type (WT) mice when mice are infected with Trichinella spiralis. Although these studies indicate anti-helminth activities of IgE targeted on larvae forms, they do not prove the fighting effects of IgE on adult worms. In contrast, a recent study demonstrates an expelling activity of IgE for adult worms through an adoptive transfer of immune serum-derived IgE into Strongyloides venezuelensis-infected mice. Here, I describe how IgE is purified from S. venezuelensis-immune sera and is transferred into infected mice to examine its effect on worm expulsion. This method will be used to advance our understanding the mechanism of S. venezuelensis expulsion and explore S. venezuelensis antigens recognized by IgE. Moreover, adoptive transfers of IgE purified from immune sera will be applicable to other helminth infection models to investigate physiological roles of IgE.

Schistosoma mansoni Larvae Do Not Expand or Activate Foxp3+ Regulatory T Cells during Their Migratory Phase.

Foxp3+ regulatory T (Treg) cells play a key role in suppression of immune responses during parasitic helminth infection, both by controlling damaging immunopathology and by inhibiting protective immunity. During the patent phase of Schistosoma mansoni infection, Foxp3+ Treg cells are activated and suppress egg-elicited Th2 responses, but little is known of their induction and role during the early prepatent larval stage of infection. We quantified Foxp3+ Treg cell responses during the first 3 weeks of murine S. mansoni infection in C57BL/6 mice, a time when larval parasites migrate from the skin and transit the lungs en route to the hepatic and mesenteric vasculature. In contrast to other helminth infections, S. mansoni did not elicit a Foxp3+ Treg cell response during this early phase of infection. We found that the numbers and proportions of Foxp3+ Treg cells remained unchanged in the lungs, draining lymph nodes, and spleens of infected mice. There was no increase in the activation status of Foxp3+ Treg cells upon infection as assessed by their expression of CD25, Foxp3, and Helios. Furthermore, infection failed to induce Foxp3+ Treg cells to produce the suppressive cytokine interleukin 10 (IL-10). Instead, only CD4+ Foxp3− IL-4+ Th2 cells showed increased IL-10 production upon infection. These data indicate that Foxp3+ Treg cells do not play a prominent role in regulating immunity to S. mansoni larvae and that the character of the initial immune response invoked by S. mansoni parasites contrasts with the responses to other parasitic helminth infections that promote rapid Foxp3+ Treg cell responses.

Efficiency of Citrus limon extract on biological and molecular activities of Biomphalaria alexandrina snails

Molluscicides have gained a great attention in recent years and proved to be a good way of snailcontrol. C. limon was tested as a molluscicide against adult Biomophalaria alexandrina snailsand a larvicide against Schistosoma mansoni larvae (miracidia and cercariae). Lethalconcentrations, LC50 and LC90 values were detected for both tests (744.17 and 1821.25 ppm,respectively). B. alexandrina snails, non-infected and infected with S. mansoni were exposed toCitrus limon peel extract concentration of 150 ppm for 4 weeks compared to non expoed with C.limon and/or infected controls. The meracidea, cercariae, egg production, protein electrophoresisand DNA fragmentation analysis and organ histology of B. alexandrina were assessed. C. limonexposure stimulated the growth rate, increased the number of eggs/snail/week (59.31 ± 5.71),inhibited reproductive rate by 20.6 %, hatchability rate (57.7 %) and stimulation of cercarialproduction. Histological investigation of C. limon extract exhibited necrosis and deformation ofhermaphrodite and digestive glands of infected and non-infected B. alexandrina snails. C. limonexposure led to increase in total protein intensity of the hermaphrodite-digestive glands in bothexposed/exposed-infected snails. RNA intensity was increased in C. limon exposed andexposed-infected snails during the experimental period. Necrotic DNA form was a characteristicof C. limon and/or infection.

In vitro screening of selected medicinal plants against Schistosoma mansoni larvae

Schistosomiasis, caused by various members of the trematode flatworms (Schistosoma species) is the second most important parasitic disease next to malaria. Resistance and low susceptibility towards praziquantel, the only available schistosomicidal drug, urge the search for new drugs. This study aimed at assessing the in vitro schistosomicidal effects of several medicinal plants traditionally used for the treatment of schistosomiasis or other helminths [1]. The crude MeOH extracts of selected plants Artemisia absinthium L. and A. abrotanum L. (aerial parts) Phytolacca dodecandra L. (roots), Curcuma longa L. (roots), Zingiber officinale Roscoe (roots), Punica granatum L. (peels, fruits and the aqueous extract of the fruits) and Citrus reticulata Blanco (peels) were tested against juvenile worms (schistosomulae) of S. mansoni by using the standard visual larval assay. All crude extracts, except P. granatum and C. reticulata exhibited significant antischistosomal effect. The highest activity was displayed by C. longa (IC50 4.06µg/ml), followed by A. abrotanum and Z. officinalis extracts with IC50 values of 11.1µg/ml and 11.73µg/ml, respectively. In the next step, the crude MeOH extracts were subjected to a liquid-liquid partitioning scheme between aqueous MeOH, hexane and CHCl3 and the subextracts were retested in the same assay. Generally, the lipohilic subextracts retained the initial schistosomicidal potential, whereas the aq. MeOH subextracts were mostly inactive at 100µg/ml concentration. The current study highlights the potential of plants against Schistosoma infections and confirms their use in traditional medicine. It also warrants phytochemical studies on the active plants to identify their active principles.

In the absence of IL-12, the induction of Th1-mediated protective immunity by the attenuated schistosome vaccine is impaired, revealing an alternative pathway with Th2-type characteristics.

Vaccination of mice with irradiated Schistosoma mansoni larvae confers high levels of immunity which is mediated by Th1-type lymphocytes. To investigate a possible role for IL-12 in the induction of protection, we have compared the immune response of IL-12 p40-deficient (KO) mice and their C57BL/6 (WT) counterparts following vaccination. Cultured lymph node cells from KO mice had markedly altered cytokine profiles with significantly decreased production of IFN-gamma increased IL-4. Correspondingly, KO mice had enhanced levels of IgE. After challenge, cells recovered from the lungs of KO mice secreted abundant IL-4 and IL-5 but little IFN-gamma, while flow cytometric and histological analysis of lung cell populations recorded a very high proportion of eosinophils. The levels of protection in KO mice were substantially lower than in their WT counterparts, demonstrating the importance of IL-12 and Th1-mediated immune responses. This conclusion is reinforced by the administration of rIL-12 to KO mice immediately after vaccination which led to increased IFN-gamma and the restoration of protective immunity. Nevertheless, the data also indicated that the limited levels of protection induced in KO mice occur via an IL-12-independent pathway, possibly mediated by Th2 cells.

Inhibition of Cysteine Proteinase from Schistosoma mansoni Larvae by a-Macroglobulin from the Plasma of Biomphalaria glabrata

The hemolymph of Biomphalaria glabrata, a molluscan host of Schistosoma mansoni, contains an alpha-macroglobulin proteinase inhibitor (alphaM). In this study we have demonstrated that this host molecule inhibits a cysteine proteinase produced by larval S. mansoni. Inhibition by alphaM involves conformational changes through proteolytic cleavage by the proteinase, thus the enzyme must be active for interactions to occur. A specific cysteine proteinase inhibitor (E64) was used to block the interaction between parasite cysteine proteinase and host alphaM during an in vitro parasite killing assay. Increased sporocyst mortality was not observed in hemolymph from susceptible strains of B. glabrata when E64 was included, nor was there decreased killing in similarly treated hemolymph from a resistant strain. This suggests that the inhibition of this parasite proteinase by host alphaM is not involved in processes determining either resistance or susceptibility to this trematode.

Phagocytosis of latex beads by Biomphalaria glabrata hemocytes is modulated in a strain-specific manner by adsorbed plasma components

Circulating phagocytes of the mollusc Biomphalaria glabrata help determine the fate of invading Schistosoma mansoni larvae. In mediating cytotoxicity against this trematode parasite, phagocytic hemocytes may interact with unidentified plasma components. In this study, positively and negatively charged latex beads were coated with plasma for use in in vitro phagocytosis assays. Hemocytes from S. mansoni-susceptible and -resistant strains of B. glabrata were tested for their ability to internalize beads coated with homologous and heterologous plasma components. Electrophoretic analysis showed that differences in the plasma polypeptides adsorbed to latex beads were charge-related, but no consistent strain-specific differences were detected. Hemocytes from a susceptible strain phagocytosed negatively charged latex pretreated in plasma form a resistant strain of snail at a higher rate than beads treated in plasma from the susceptible strain. Pretreatment of susceptible hemocytes with resistant plasma reduced subsequent phagocytosis of beads coated with resistant plasma, consistent with blocking of receptors on the susceptible hemocytes with resistant strahl plasma components. Exogenous lectins detected no differences in the carbohydrates on beads coated with plasma from either snail strahl. However, strain differences in endogenous lectins are suggested by differential binding of neoglycoproteins to plasmacoated beads. The plasma components) responsible for the differential phagocytosis of beads treated in the two plasma strains by hemocytes from the susceptible strahl of B. glabrata remain(s) to be identified.

High-affinity IgE receptor on eosinophils is involved in defence against parasites

Parasitic infections are often associated with eosinophilia and high levels of immunoglobulin E (IgE). This observation has led to speculation that eosinophils and IgE may act together in the immune response against parasites. In support of this hypothesis, IgE and eosinophils participate in cytotoxic reactions directed against Schistosoma mansoni larvae in vitro. Furthermore, epidemiological studies have shown an inverse correlation between levels of specific IgE and rates of infection with Schistosoma. The low-affinity IgE receptor (Fc epsilon RII/CD23) was first incriminated in eosinophil activation. The fact that the high-affinity IgE receptor (Fc epsilon RI) is not only expressed on mast cells and basophils but also on Langerhans cells led us to investigate the presence of Fc epsilon RI on eosinophils. Here we show that Fc epsilon RI is expressed on eosinophils from hypereosinophilic patients, is involved in eosinophil degranulation, and participates in eosinophil-mediated cytotoxicity against S. mansoni. Our results indicate that Fc epsilon RI may play a major part in immune defence against parasites.

Separate induction of human blood platelet aggregation or cytotoxicity by different concentrations of PAF-acether and thrombin.

Using decreasing concentrations of PAF-acether or thrombin, it was possible to observe on human platelets, first, aggregation, classically associated to activation, then , below a threshold, cytotoxicity towards Schistosoma mansoni larvae, proposed here as stimulation. These two activities appeared as distinct and antithetic. However, their induction might be the consequence of triggering of the same receptors with different intensity, since PAF-induced, but not thrombin-induced, cytotoxicity could be inhibited with specific PAF-antagonists BN 52021 and BN 52024 also known to inhibit PAF-induced aggregation. These results give credit to the hypothesis that haemostatic and cytotoxic properties of platelets are two distinct functions of these blood elements.

Plasmapheresis in a patient with severe asthma associated with auto‐antibodies to platelets

Although the pathogenesis of aspirin-sensitive asthma remains to be specified, it is known that in the presence of acetylsalicylic acid or non-steroidal anti-inflammatory drugs, platelets from aspirin-sensitive asthmatics have been described as generating cytocidal mediators that killed parasite targets such as Schistosoma mansoni larvae. Here we report, in a patient with corticosteroid-dependent asthma associated with aspirin sensitivity, the presence of circulating IgE antibodies against 55 kD and 68 kD platelet antigens. In addition, the serum from this patient was shown to contain a factor able to trigger the release of cytocidal mediators from his platelets as well as from normal individual platelets. This platelet stimulatory activity was presumably supported by IgE antibodies or immune complexes. After informed consent the patient was submitted to plasma exchanges. Plasma removal induced clinical improvement, anti-platelet antibody decrease, and the reduction of the platelet stimulatory activity. All clinical symptoms disappeared within 2 weeks. The disease remained quiescent for 2 months, and daily requirements for prednisone (20-5 mg), and beta-agonist (10-16 to 0-1 inhalations) could be kept at a low level follow-up. The plasma exchanges were delayed by 3 mg kg-1 azathioprine with the maintenance of clinical improvement. A relapse occurred after the arrest of immunosuppressive therapy with the reappearance of both asthma attacks and anti-platelet antibodies, as well as the increase of the platelet stimulatory activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphocyte‐mediated inhibition of platelet cytotoxic functions during Hymenoptera venom desensitization: characterization of a suppressive lymphokine

Recently, it has been shown that platelets, through a receptor for the Fc fragment of IgE, could be specially triggered by venom allergens in hypersensitivity to hymenoptera, generating cytocidal mediators toward Schistosoma mansoni larvae, and oxygen metabolites measured by chemiluminescence. After rush immunotherapy, a depressed platelet response was demonstrated to be associated with the production of lymphokine(s). Here we report the characterization of a factor present in supernatants of antigen-stimulated T cells from patients after hymenoptera venom desensitization which is able to inhibit platelet cytotoxic functions in a dose-dependent manner. The optimal inhibition was observed with supernatants obtained after T lymphocyte stimulated with 10(-5) micrograms venom allergen/ml. Once specifically produced the platelet-suppressive effect of lymphocyte supernatants was not antigen specific. The producing T cell subpopulation was identified as CD8+. This lymphokine had an approximate molecular mass of 25 kDa and a pI of 4.8. It was heat and acid stable and sensitive to trypsin and proteinase K but not to neuraminidase. This platelet inhibitory activity was absorbed by platelet membrane suggesting its binding to a receptor. These properties were very similar to a previously described platelet activity suppressive lymphokine, suggesting the participation of this lymphokine in the mechanisms of rush desensitization.

Enhancement of human eosinophil cytotoxicity and leukotriene synthesis by biosynthetic (recombinant) granulocyte-macrophage colony-stimulating factor.

Abstract Culture medium conditioned by activated human T lymphocytes enhances the in vitro cytotoxicity of purified human eosinophils toward Schistosoma mansoni larvae, suggesting the existence of a mechanism for T lymphocyte regulation of eosinophil function. Here we show that purified biosynthetic (recombinant) human T lymphocyte granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced markedly two eosinophil functions: cytotoxicity toward schistosomula by a mean of 676%, and calcium ionophore A23187-induced generation of leukotriene C4 (LTC4) by a mean of 135%. Augmentation of each eosinophil function by GM-CSF was time- and dose-dependent, with a dose-response relationship at concentrations between 1 and 20 pM. Tumor necrosis factor (TNF) enhanced eosinophil cytotoxicity with slower kinetics, a different dose-dependence relationship, and to a lower maximum, as compared with GM-CSF. There was no detectable effect of TNF on calcium ionophore A23187-induced generation of LTC4. The effect of GM-CSF on arachidonic acid metabolism to LTC4 reached a plateau with 60 min of incubation before stimulation with ionophore, and was characterized by an initial augmentation of the intracellular level of LTC4 and a subsequent increment in extracellular LTC4. Thus, GM-CSF can serve as a mediator for T lymphocyte regulation of functions of mature eosinophils. It is also the first defined macromolecule known to enhance metabolism of membrane-derived arachidonic acid via the 5-lipoxygenase pathway.

Identification of Schistosoma mansoni antigens by means of biologically active monoclonal antibodies

Two monoclonal antibodies of the IgE class (54.10) and of the IgG 1 class (27.21), that were shown previously to possess biological activity against Schistosoma mansoni larvae, were used for identification of surface antigens of the cercariae and schistosomula. This was performed by immunoprecipitation, immunoaffinity chromatography and immunoblotting. The epitope reactive with 27.21 mcIgG 1 is present on four polypeptides (60, 50, 27 and 19 kDa) derived from the parasite. The 60 kDa is specific to cercariae, whereas the 50 kDa is a glycoprotein shared both by cercariae and schistosomula. The antigen reactive with the 54.10 mcIgE was isolated by affinity chromatography on 54.10 column, and contained three major peptides of 125, 94 and 30 kDa. The 125 and 94 kDa band probably originate from the same protein, since they yield almost identical peptide maps. The isolated antigen retained its biological activity as demonstrated in the basophils degranulation assay.

The respiratory burst is not required for killing of intracellular and extracellular parasites by a lymphokine‐activated macrophage cell line

The macrophage cell line, IC-21, was found to be incapable of producing the oxygen products associated with the respiratory burst. However, IC-21 cells were activated by lymphokine (LK) to kill intracellular (Leishmania donovani amastigotes) and extracellular (Schistosoma mansoni larvae) parasites, as well as tumor cells. In each case, the cytotoxicity exhibited by activated IC-21 cells and activated peritoneal macrophages was indistinguishable. However, nonactivated IC-21 cells were unable to kill L. donovani log-growth phase promastigotes, while nonactivated peritoneal macrophages destroyed greater than 90% of the initial infection. These results indicate that amastigotes and schistosome larvae are susceptible to killing by nonoxidative cytotoxic mechanism induced by lymphokine activation but, on the other hand, support the concept that the killing of log-growth phase promastigotes by nonactivated cells is dependent upon the respiratory burst. We propose that the IC-21 cell line may be a useful model for studying nonoxidative killing functions of activated macrophages.

A new function for platelets: IgE-dependent killing of schistosomes.

Several killing mechanisms against schistosomes have been described in vitro, involving cellular and humoral factors. Neutrophils, eosinophils--with an accessory role for mast cells--monocytes and macrophages have been shown to exhibit cytotoxic properties against Schistosoma mansoni larvae, in association with antibodies of various isotypes or with complement (reviewed in ref. 1). Lymphocyte participation in effector functions is mediated mainly through lymphokines inducing cytotoxic macrophages, and, in certain cases, directly by T cells. The experiments reported here show that platelets, taken from rats after specific periods of infection with S. mansoni, were able to kill schistosomula, and that normal human or rat platelets acquired toxic properties towards the same target in the presence of serum from infected individuals. The humoral factor involved in this process was shown to be IgE, and evidence was obtained of a Fc receptor for IgE on human and rat platelets. The passive transfer of immune platelets to normal rats conferred a high degree of protection towards a challenge infection by the parasite.

Protein synthesis during cercaria-schistosomulum transformation and early development of the Schistosoma mansoni larvae

1. 1. Protein synthesis during early development of Schistosoma mansoni larva was investigated in vitro and assessed by labelled amino-acid incorporation. 2. 2. The rate of protein synthesis was low at the first 6 hr, increasing gradually as the time elapsed. 3. 3. The rate of 14C-leucine incorporation was strongly dependent on the enrichment of the incubation media with an amino-acid mixture. 4. 4. The protein synthesis was not affected by the addition of 100 and 200 μg/ml of cycloheximide and chloroamphenicol to the incubation media. On the other hand, puromycin (500 μg/ml) and actinomycin (0.65–1.30 μg/ml) strongly inhibited protein synthesis. 5. 5. Proteins of tegument incorporated 2–3 times more isotope than those of the larval body.