Schistosoma mansoni: activity responses in vitro to praziquantel.

The effects of praziquantel, a novel antischistosomal compound, on the activity of adult Schistosoma mansoni (Liverpool strain) in vitro were investigated. Worm activity was modified at all concentrations of praziquantel. High concentrations (above 0.5 microgram/ml) produced rapid paralysis, whilst low concentrations of praziquantel stimulated worm activity. It is concluded that such activity modification could lead to worm displacement in vivo.     

Anthelmintic activity of triclabendazole

Triclabendazole was tested in vitro and in vivo against a range of helminths. Although in vitro activity was found against Hymenolepis diminuta (0.5 microgram/ml), Fasciola hepatica (2.5 micrograms/ml), Taenia crassiceps and Schistosoma mansoni (50 micrograms/ml), in vivo activity was only found against F. hepatica, a single oral dose of 40 mg/kg killing 99% of adult flukes in the rat. This spectrum of activity suggests a mechanism of action unlike that of other benzimidazole anthelmintics.     

Activation of complement by Schistosoma mansoni schistosomula: killing of parasites by the alternative pathway and requirement of IgG for classical pathway activation.

Living Schistosoma mansoni schistosomula incubated with normal chicken, guinea pig, human, and monkey sera were killed after 4 hr contact at 37 degrees C. The following data indicate that this action is dependent on the activation of the alternative complement pathway (AP): a) the inactivity of RB, RD, and zymosan-treated serum against schistosomula; b) the partial activity of RD restored in FD; c) the full effect of the C4-deficient guinea pig, C2-deficient human, and the agammaglobulinemic human sera; d) the consumption of both the AP and FB after the incubation of NHS with schistosomula; e) the detection of C3d breakdown product during the contact of the C2-deficient human serum with these young parasites. Killing by serum was decreased as the immature schistosomes developed and was completely absent against 4-day-old lung schistosomula (LS). In other experiments, it was demonstrated that schistosomula, in the presence of IgG, were able to initiate complement activation also through the classical pathway (CP). However, the CP does not appear to play a role in the schistosomulicidal activity of complement. The in vivo relevance of these observations is considered.     

Synthesis and activity of new triphenylphosphonium derivatives of betulin and betulinic acid against Schistosoma mansoni in vitro and in vivo

We studied the antischistosomal activity of betulin, betulinic acid and its 9 triphenylphosphonium derivatives characterized by a covalently linkage of the hydrophobic fragment of triterpenoid at C(2)- or C(30)-position with the triphenylphosphonium moiety via a hydrocarbon bridge. The triphenylphosphonium salts showed in vitro antischistosomal activity against newly transformed schistosomula (NTS) and adult worms of Schistosoma mansoni at low micromolar concentrations. In contrast betulin and betulinic acid were inactive against NTS and adult S. mansoni. Of the 9 triphenylphosphonium derivatives tested, the allyl salts 10 (IC₅₀ of 0.76 μg/mL) and 11 (IC₅₀ of 0.64 μg/mL) demonstrated the highest antischistosomal activity against adult S. mansoni. Low worm burden reductions of 22% were observed in vivo for these two compounds. In conclusion, triphenylphosphonium derivatives were obtained from available natural betulin by simple transformations, rendering it practical and useful for large scale application. However, further structural modifications are necessary to translate the promising antischistosomal in vitro activities into in vivo.     

Biological effect of interferon-gamma during the course of experimental infection of rat by Schistosoma mansoni

We demonstrate here that a second mechanism of platelet activation dependent on lymphokine could also take place in the expression of platelet cytotoxicity against Schistosoma mansoni in vitro. Indeed, IgE, as previously described, but also IFN-gamma, present in the sera of infected rats, together induce platelets from normal rats into cytotoxic effectors for the parasitic larvae. This second mechanism appears also effective in vivo since the passive transfer of normal platelets treated by recombinant IFN-gamma (rIFN-gamma) and the administration of rIFN-gamma to rats conferred a protective immunity to S. mansoni.     

Evidence for the mode of antischistosomal action of hycanthone

Evidence is presented which supports the hypothesis that the mode of action, or a slight variant thereof, suggested by Hartman and Hulbert (11) to account for the mutagenic effects of hycanthone (HC) is the mechanism whereby HC exerts its antischistosomal activity. HC is metabolically activated to a reactive ester which, upon dissociation, alkylates DNA. If resistant schistosomes are unaffected because they cannot convert HC to a reactive ester they should be killed upon direct exposure to an appropriately esterified drug. Hycanthone N-methylcarbamate (HNMC) was synthesized and shown to bind to DNA and also alkylate 4-(p-nitrobenzyl)-pyridine. When tested with schistosomes kept in vitro, HNMC caused an irreversible inhibition of 3H-uridine incorporation not only in sensitive S. mansoni (as HC does) but also in HC-resistant and immature S. mansoni worms and S. japonicum worms which are only transiently inhibited by HC. After in vitro contact with HNMC for 1 h both sensitive and resistant schistosomes died in three weeks if either kept in culture or re-transplanted into the host animal. Mice infected with HC-resistant schistosomes showed a drastic worm reduction after in vivo HNMC administration.     

Lack of correlation between schistosomicidal and anticholinergic properties of hycanthone and related drugs

Visual observation of the motor activity of Schistosoma mansoni kept in vitro showed an increase of activity in the presence of hycanthone (HC). In addition, HC caused a delay in the paralytic effects of carbachol. Similar results were observed in the presence of oxamniquine (OXA). The same pattern of motor activity, however, was shown by HC-resistant worms, by Schistosoma japonicum, and by worms exposed to drug precursors (lucanthone and UK-3883), which are not schistosomicidal in vitro. Other analogs with in vitro killing activity (IA-4 and IA-4 N-oxide) showed minimal anticholinergic effects. The anticholinergic effects of HC and OXA were quickly reversible in vitro and in vivo, whereas their antischistosomal effects are irreversible and delayed. Incubation of schistosomes with high concentrations of carbachol or with anticholinergic drugs failed to compete with the schistosomicidal effects of HC. These results are viewed as contradictory to the hypothesis that HC kills schistosomes by blocking their acetylcholine receptors.     

Sex- and stage-related sensitivity of Schistosoma mansoni to in vivo and in vitro praziquantel treatment

The efficacy of praziquantel against a Puerto Rican strain of Schistosoma mansoni was assessed using both in vivo and in vitro approach. The drug effective dose (50%) in the infected mouse model was about 30 times higher when determined against 28-day-old infections than against 7-week-old parasites. Single-sex female infections were also largely refractory to treatment and single-sex male infections moderately refractory, in comparison with bisexual infections. The in vitro approach consisted of overnight exposure of parasite cultures to various drug concentrations, followed by several days of culture in drug-free medium. In vitro results confirmed in vivo data and allowed for the observation of schistosome morphological phenomena after praziquantel exposure. Early worm contraction was observed in all cases, even after exposure to sub-lethal concentrations of praziquantel or upon exposure of the largely refractory 28-day-old schistosomes. In these instances, however, worms resumed movements and normal shape upon drug removal and were able to survive. The inference of these observations on the clinical use of praziquantel and on its mechanism of action is discussed.     

Schistosoma mansoni: Antischistosomal activity of the four optical isomers and the two racemates of mefloquine on schistosomula and adult worms in vitro and in vivo

Recent studies have shown that mefloquine (MQ) reveals interesting antischistosomal properties. We examined the antischistosomal activities of the erythro and threo isomers and racemates of MQ on newly transformed schistosomula (NTS) and adult Schistosoma mansoni in vitro and in mice harbouring adult S. mansoni. The in vitro effects in the presence and absence of haemin were monitored by means of microcalorimetry, scanning electron microscopy and phenotypic evaluation. Incubation of NTS with the erythro derivatives at concentrations of 3 μg/ml and above resulted in convulsions, granularity, decrease in heat flow, and death while NTS incubated with the threo derivatives were only affected at high concentrations (100 μg/ml). Extensive tegumental alterations, decrease in metabolic activity, viability, and death were observed when adult schistosomes had been exposed to 10 μg/ml of the erythro compounds. Moderate tegumental and viability changes but reduced heat production rates were observed with the threo derivatives at 10 μg/ml. In the presence of haemin, all MQ derivatives showed pronounced antischistosomal properties against adult S. mansoni in vitro. In vivo, MQ derivatives achieved statistically significant total and female worm burden reductions ranging between 65.4% and 100%. The highest total worm burden reductions of 93.4% and 90.2% were observed following treatment with the erythro and threo racemates, respectively. In conclusion, the optical isomers and racemates of MQ show only moderate stereoselectivity, in particular in vivo. Our results may enhance our understanding of the mechanism of action and therapeutic profile of MQ derivates on schistosomes.     

Praziquantel, a new board-spectrum antischistosomal agent.

Praziquantel, (2-cyclohexylcarbonyl)-1,2,3,6,7,11b-hexa-hydro-2H pyrazino[2,1a]isoquinolin-4-one, belongs to a new series of antischistosomal compounds. The results of a detailed study of the efficacy of praziquantel on Schistosoma mansoni in mice, Mastomys and Syrian hamsters are described. Praziquantel is effective after oral and all parenteral routes of administration tested. The amount of praziquantel required to achieve parasite reductions of at least 95% depends on the host species and on the routes and schedules of administration. Total doses range from 200--1,000 mg/kg in mice and from 100--500 mg/kg for Mastomys and hamsters. In all three species, splitting of the total dose into 3 or more fractional doses given within 1 day approximately doubles the efficacy over that achieved after a single oral administration of the same total dose. A single subcutaneous dose is only slightly more effective, whilst a single intramuscular injection in olive oil is about twice as effective as a single oral administration. Praziquantel is very effective against the invading stages and slightly less against schistosomules up to an age of 7 days. It is less effective against 2- to 4-week-old juveniles, but is effective again against 5-week-old and older schistosomes. Praziquantel is equally effective against both sexes of S. mansoni. It is less effective against unpaired and therefore juvenile female worms, but fully effective against single male worms. The efficacy of praziquantel on S. mansoni in mice is not influenced by the strain or the sex of the host, the worm burden or the age of the infection. Considering all data available, praziquantel promises to be a very potent antischistosomal drug.     

Parasitological and biochemical parameters in Schistosoma mansoni-infected mice treated with methanol extract from the plants Chenopodium ambrosioides, Conyza dioscorides and Sesbania sesban

This study aims to detect the antischistosomal properties of the plants' Chenopodium ambrosioides, Conyza dioscorides and Sesbania sesban methanol extract against Schistosoma mansoni in infected mice, including determination of total protein and albumin levels and the activities of alanine and aspartate transaminases (AlT, AsT) and acid and alkaline phosphatases (AcP and AkP) enzymes in the serum of infected treated mice. Male Swiss albino mice were infected with S. mansoni and orally treated with methanol extract of the plants C. ambrosioides (1250 mg/kg/day), C. dioscorides and S. sesban (1000 mg/kg/day from each) for 2 consecutive days 7 weeks post infection (PI). In addition, treatment of mice with the tested dose of each plant extract was successively done (i.e. the 1st extract followed by the 2nd and 3rd one with an hour interval). Parasitological and biochemical parameters were assessed. Nine weeks PI, the reduction rates of worm load/mouse treated with either C. dioscorides (1000 mg/kg), C. ambrosioides (1250 mg/kg) or S. sesban (1000 mg/kg) were 40.9%, 53.7% and 54.4%, respectively. Successive treatment raised the reduction rates of worm load/mouse to 66.3% and the ova/g tissue in liver to 76.9%. Moreover, serum total protein and albumin levels and activities of AlT, Ast, AcP and AkP enzymes of infected treated mice were improved in comparison with those of infected untreated ones. It is concluded that administration of C. dioscorides, C. ambrosioides and S. sesban methanol extract to infected mice exhibited a moderate antischistosomal effect. Successive treatment improved the antischistosomal properties of these plant species, hence ameliorated the liver functions of treated mice that may suggest degenerations of liver granulomas and regenerative changes.     

Development of chiral praziquantel analogues as potential drug candidates with activity to juvenile Schistosoma japonicum

A series of chiral praziquantel analogues were synthesized and evaluated against Schistosoma japonicum both in vitro and in vivo. All compounds exhibited low to considerable good activity in vivo. Remarkably, worm reduction rate of R-3 was 60.0% at a single oral dose of 200 mg/kg against juvenile stage of Schistosoma japonicum. The target compounds displayed in vivo antischistosomal activity against both Schistosoma japonicum and Schistosoma mansoni. Furthermore, all R-isomers displayed stronger antischistosomal activity than S-isomers in vivo, indicating R-isomers were the active enantiomers, while S-isomers were less active ones. This structure activity relationship (SAR) could have important implications in further drug development for schistosomiasis.     

Synthesis of new praziquantel analogues: potential candidates for the treatment of schistosomiasis

An efficient synthesis of antischistosomal drug praziquantel and analogues was achieved and the synthetic route designed was to afford structurally diverse analogues for better structure-activity relationship understanding. Total of nineteen PZQ analogues with structural variations at amide, piperazine and aromatic moieties have been synthesized and fully characterized. Among all the new analogues tested for antischistosomal activity, one dimethoxy tetrahydroisoquinoline analogue and two tetrahydro-β-carboline analogues exhibited moderate activity against adult Schistosoma mansoni. Tetrahydro-β-carboline analogues showed moderate activity whereas the presence of p-trifluoromethylbenzoyl and p-toluenesulphonyl moieties resulted in complete suppression of antischistosomal activity.     

Interleukin-6 is the main mediator of the interaction between monocytes and platelets in the killing of Schistosoma mansoni

We demonstrate the existence of a cooperation between monocytes and platelets for the killing of Schistosoma mansoni. Indeed, supernatants obtained after a 24 hr adherence of normal human monocytes were able to induce, in a dose dependent manner, the cytotoxicity of normal human platelets towards the young larvae of S.mansoni in vitro. The physicochemical analysis of the supernatants showed that a factor exhibiting a pl of 4.8-4.9 was responsible of this effect, suggesting a role of IL-6, detected in the supernatants, in this induction. This was confirmed by the neutralization of the cytotoxic effect by a polyclonal serum against IL6 whereas polyclonal sera against IL-1 beta or TNF-alpha, the other cytokines present in the supernatants, did not modify the cytotoxicity observed. Finally human recombinant IL-6 induces the platelet cytotoxic function, demonstrating a direct effect of IL6 on blood platelets.     

Subclasses of rat IgG active in the killing of schistosomula of Schistosoma mansoni in vitro and in vivo

Schistosomula of Schistosoma mansoni are known to be killed in vitro by complement and IgG (lethal antibody). To investigate whether this mechanism reflects the in vivo situation, we isolated IgG subclasses from sera of infected rats and assayed their ability to promote the complement-mediated killing of schistosomula in vitro as well as to protect normal recipients from a challenge infection. We found that a serum fraction containing only IgG2a + IgG2b has lethal activity to schistosomula in vitro, whereas a fraction containing only IgG1 + IgG2c fails to kill schistosomula in the presence of complement. The assay of protective activity has shown that the same fraction containing the lethal activity (IgG2a + IgG2b) was able to reduce the number of schistosomula recovered from lungs. These results provide evidence of the participation of IgG2a and/or IgG2b, but not IgG1 or IgG2c, in protective immunity to S. mansoni in rats, possibly through a complement-mediated mechanism.     

Oxamniquine, praziquantel and lovastatin association in the experimental Schistosomiasis mansoni

The activity of lovastatin associated with oxamniquine or praziquantel against schistosomiasis mansoni was evaluated in mice infected with Schistosoma mansoni. Forty days after infection, mice were treated with lovastatin, 400 mg/kg for five consecutive days by oral route, and on the last day of this sequence with 50 mg/kg oxamniquine or with 200 mg/kg praziquantel, both by oral route, single dose. Fifteen days later, the animals were perfused in parallel with an untreated control group. Studies were carried out in vitro, using lovastatin in culture medium containing S. mansoni worms proceeding from experimentally infected mice. In the in vivo trials, the association of lovastatin with oxamniquine or praziquantel did not show any additive action, but there were oogram changes when lovastatin was associated with oxamniquine. In vitro lovastatin was able to interrupt the maturation of S. mansoni eggs, which remained at the 1st or 2nd stages, depending on the dose used. The total number of morphologically dead eggs found in culture of worms exposed to 2 microg/ml or 4 microg/ml concentrations of lovastatin was significantly higher than the number of viable eggs. Using the probe Hoescht 33258 it was observed that 70% of the eggs considered morphologically viable in the treated groups (against 16% in the control group) were labeled, indicating that the majority of the viable eggs had membrane permeability increased due to lovastatin action.     

Schistosoma mansoni: schistosomulicidal activity of macrophages isolated from liver granulomas of infected mice

Mice infected with Schistosoma mansoni develop T cell-mediated granulomatous reactions around disseminated parasite eggs. In this study, granuloma-derived leucocytes have been examined for schistosomulicidal capacity by the use of in vitro cytotoxicity assays. Adherent macrophages, that were shown by electron microscopy to exhibit no gross morphological abnormalities, were unable to mediate significant mortality in the absence of serum factors. When cocultured with immune serum and complement, however, these cells killed +/- 26% of the larvae at a cell:target ratio of 5000:1. In contrast, granuloma-derived cell populations that were enriched for eosinophils (50-70% eosinophil content) showed only minimal cytotoxic potential. This may be related to observed structural changes in the eosinophil lysosomal granules, or perhaps to blocking of the cell-surface receptors by immune complexes. It is concluded that granuloma macrophages, activated by egg antigen-sensitised T lymphocytes, may serve as effector cells in immunity to schistosomules.     

Effect of artemether administered alone or in combination with praziquantel to mice infected with Plasmodium berghei or Schistosoma mansoni or both

The artemisinins have become key drugs for the treatment and control of malaria, particularly within artemisinin-based combination therapies. Since the artemisinins also exhibit antischistosomal properties, their use in areas where malaria and schistosomiasis are co-endemic may have an effect on both diseases and co-infection might alter drug efficacy. We assessed the antimalarial and antischistosomal efficacies of artemether in mice infected with Plasmodium berghei or Schistosoma mansoni or both parasites concurrently. Three oral doses of 400 mg/kg artemether at 14-day intervals reduced total and female S. mansoni worm burdens by 98.7-100%, regardless of a concurrent P. berghei infection. When four daily doses of 55 mg/kg artemether were administered, which is a standard treatment schedule to cure P. berghei-infected mice, significantly lower total and female S. mansoni worm burden reductions were observed (73.1-89.2%). Artemether, administered at both of the above-mentioned treatment schemes, showed excellent antimalarial efficacy with no indications of delayed clearance of P. berghei or recrudescence, also in mice co-infected with S. mansoni. Co-infection with P. berghei had no effect on S. mansoni worm burden reductions following artemether-praziquantel combinations. Our findings point to the need for epidemiological studies in areas where malaria and schistosomiasis co-exist and where artemisinin-based combination therapies are introduced, since artemisinin-based combination therapies as part of a malaria control package may have ancillary benefits against schistosomiasis.     

T-cell-mediated regression of "spontaneous" and of Epstein-Barr virus-induced B-cell transformation in vitro: studies with cyclosporin A

The regression of Epstein-Barr (EB) virus-transformed B-cell outgrowth which is seen in experimentally-infected cultures of blood mononuclear (UM) cells from healthy seropositive donors can be abolished in medium containing the T-cell-suppressive agent cyclosporin A (CSA) at concentrations of 0.05 microgram/ml and above. CSA mediates its effect within the first 4 days post-infection of the UM cells and this prevents subsequent in vitro generation of the EB virus-specific cytotoxic-T-cell response which normally brings about regression. Regression can be fully restored by supplementing the CSA-treated culture with interleukin 2 (IL-2)-containing culture supernatants or indeed with purified IL-2 itself, suggesting that CSA mediates its effect in this system through inhibiting the endogenous production of IL-2 which is required to amplify the virus-specific cytotoxic response. "Spontaneous transformation" to EB virus genome-positive lymphoblastoid cell lines in noninfected cultures of UM cells from healthy seropositive donors, though rare in normal medium, is enhanced to such a degree in the presence of CSA that, for many donors, the phenomenon becomes titratable against input cell dose across the 2.0 X 10(6)-2.5 X 10(5) cells/culture range. Cell mixing experiments suggest that the spontaneously transformed cell lines which arise with such efficiency under these conditions do so not by direct in vitro outgrowth of progenitor cells transformed by the virus in vivo, but by a two-step mechanism involving virus release and secondary infection in vitro.     

Schistosoma mansoni: In vitro schistosomicidal activity and tegumental alterations induced by piplartine on schistosomula

Schistosomiasis is one of the most important parasitic infections in humans that occur in many tropical and subtropical countries. Currently, the control of schistosomiasis rests with a single drug, praziquantel, which is effective against adult worms but not the larval stages. Recent studies have shown that piplartine, an amide isolated from plants of the genus Piper (Piperaceae), reveals interesting antischistosomal properties against Schistosoma mansoni adult worms. Here, we report the in vitro antischistosomal activity of piplartine on S. mansoni schistosomula of different ages (3h old and 1, 3, 5, and 7days old), and examine alterations on the tegumental surface of worms by means of confocal laser scanning microscopy. Piplartine at a concentration of 7.5μM caused the death of all schistosomula within 120h. The lethal effect occurred in a dose-dependent manner and was also dependent on the age of the parasite. Microscopy observation revealed extensive tegumental destruction, including blebbing, granularity, and a shorter body length. This report provides the first evidence that piplartine is able to kill schistosomula of different ages and reinforce that piplartine is a promising compound that could be used for the development of new schistosomicidal agent.