Identification of exposed components on the surface of adult Schistosoma mansoni by lactoperoxidase-catalysed iodination

Adult schistosomes have been labelled with ¹²⁵I using the lactoperoxidase-catalysed technique modified to cause minimal worm damage. After surface membrane removal and characterization, at least 13 labelled proteins were identified together with a large amount of labelled glycolipids, free fatty acids and phospholipids, especially phosphatidyl ethanolamine. Cationised ferritin has been used to stimulate surface membrane turnover of iodinated worms and the shedding of covalently bound ¹²⁵I-counts used as an index of turnover. Finally worms have been iodinated before and after stimulation of membrane turnover in chemically defined media and the patterns of labelled proteins were compared.     

Changes in glucose metabolism and cyanide sensitivity in Schistosoma mansoni during development

Schistosoma mansoni was studied by biochemical and electrophysiological techniques to follow the physiological changes occurring during transformation in the mammalian host. Volume conducted electrical potentials and measurement of CO₂, evolution indicate that 3 h post-transformational schistosomula are highly sensitive to cyanide. By 24 h after transformation, evolution of CO₂ under control conditions is reducted by 77% from 3 h levels, while lactate excretion rises by 84%. Cyanide does not affect the frequency or magnitude of endogenous electrical transients, but does eliminate 83% of the already reduced levels of CO₂ evolved in 24 schistosomula. Electrophysiological analyses indicate that the timecourse of metabolic changes in skin- and mechanically transformed schistosomula are similar, and incubation of schistosomula in 200 μg ml^?1 puromycin does not alter the onset of cyanide insensitivity. The adult parasite evolves a low level of CO₂ which is reduced by 88% in the presence of 1 mM cyanide. No significant Pasteur effect is detected, however, and endogenous electrical activity as well as mechanical responses of the adult musculature are unaffected by cyanide exposure. Our results indicate that schistosomula continue to rely on cyanide-sensitive respiratory components for at least 3 h after transformation; by 24 h, however, the parasites are metabolically similar to the adult stage, i.e., they depend on lactate fermentation for most of their energy requirements.     

Surface antigens of Schistosoma mansoni

The outer tegument membrane of 18 h artificially prepared schistosomula of Schistosoma mansoni was labelled using the non-permeant diazonium salt of [¹²⁵I]iodosulphanilic acid. Eight iodinated surface proteins were identified by SDS-polyacrylamide gel electrophoresis. Three of these proteins, one of which is glycosylated, can be precipitated by immune serum.     

Isolation and characterization of surface antigens from Schistosoma mansoni schistosomula

Surface antigens of Schistosoma mansoni schistosomula were isolated using antibodies produced in rat and human schistosomiasis. Three immunoreactive surface proteins of 40 000, 37 000 and 32 000 daltons were identified by SDS—PAGE analysis of immune complexes formed by incubation of a detergent extract of surface labelled schistosomula with infected rat sera. Surface antigens of similar molecular weight were also isolated when using sera of patients with schistosomiasis. Binding of schistosomula surface antigens to specific antibodies was substantially inhibited by components released by adult worms. The results suggest that these schistosomula surface antigens could be involved in the immune response against challenge infection but their protective role in immunity still remains to be determined.     

Antigenic properties of Schistosoma mansoni aminopeptidases: Evolution during the development in mammalian hosts

The involvement in the immunity to schistosomiasis of an aminopeptidase activity of schistosomula, 20-day-old and adult worms of Schistosoma mansoni has been studied. This activity, hydrolysing leucine-p-nitroanilide and alanine-p-nitroanilide at pH 7.4 was immunoprecipitated by various infected rat and human sera. These antigenic enzymes were expressed at day 28 after infection in the rat and seemed to be specific for the Schistosoma species. Several aminopeptidase activities were found after analysis by isoelectric focusing of the adult extract but only the pI 8.3 peak was antigenic. Three antigenic peaks were demonstrated after AcA 34 Ultrogel filtration. The biological relevance of these antigenic enzymes in schistosomiasis is discussed.     

Adenylate cyclase in adults and cercariae of Schistosoma mansoni

Adenylate cyclase of adult Schistosoma mansoni is activated by serotonin (5-hydroxytryptamine). Serotonin activation is markedly enhanced by GTP. The poorly hydrolyzed analogs of GTP, guanylyl imidophosphate (Gpp(NH)p) and guanosine 5′-(3-O-thio)triphosphate activate the schistosome cyclase in the absence of serotonin. The activating effect of these nucleotides is increased in the presence of serotonin. Female adult schistosomes have only 40% as much NaF-stimulated activity as do the male parasites. In addition, the serotonin stimulation of adenylate cyclase in the females is only 20% as much as the males. Maximal activation of adenylate cyclase with NaF reveals that the adult parasites have tenfold higher activity than the cercariae. Cercarial adenylate cyclase shows very poor response to serotonin in the presence of GTP. Serotonin caused more significant activation of cercarial adenylate cyclase in the presence of guanylyl imidophosphate.     

Schistosoma mansoni: Modulation of schistosomular lipid composition by serum

Human serum and foetal calf serum have been compared in terms of their ability to modify the biochemical and immunological properties of the schistosomular surface. Artificially transformed schistosomula were incubated in the presence of serum for 24 h and then radioiodinated using the chloramine T method. With this method only lipids are labelled. Foetal calf serum produces a net loss of lipids from the schistosomula, particularly of mono- and diglycerides. Human serum however, promotes not only a loss of mono- and diglycerides, but also a substantial uptake of cholesterol and triglycerides. Schistosomula recovered from the lungs of mice could also be labelled and contained besides triglycerides, an increased amount of cholesterol esters. The modulation of surface lipids in worms cultured with human serum correlates with the observation that such schistosomula develop significantly greater protection against cosinophil-mediated cytotoxicity in vitro than do individuals incubated with foetal calf serum. On the other hand, schistosomula cultured in the presence of either human serum or foetal calf serum develop the same degree of protection against complement-dependent lethal antibody; this result indicates that resistance against complement-mediated damage may be independent of the uptake of cholesterol and/or triglycerides, and might involve only limited alterations in the surface configuration of the schistosomulum.     

Major phospholipids and phosphatidylcholine synthesis in adult Schistosoma mansoni

The major phospholipids present in the phospholipid extract of Schistosoma mansoni were phosphatidylcholine (28%), phosphatidylethanolamine (25%), phosphatidylserine (15%) and phosphatidylglycerol (8%). The synthesis of phosphatidylcholine in S. mansoni adults occurred by the choline to phosphatidylcholine or Kennedy pathway. Incorporation of CDPcholine and choline into the phosphatidylcholine of worm slices appeared linear over time with no demonstrable sex differences in choline incorporation. A slight difference in the incorporation of CDPcholine by separate sexes was evident. Methylation of phosphatidylethanolamine to phosphatidylcholine could not be demonstrated.     

The effect of praziquantel on calcium in Hymenolepis diminuta

Praziquantel (PZ) at concentrations down to 5 × 10^?8 M induced a rapid contraction of Hymenolepis diminuta musculature. This effect was accompanied by a strong inhibition of ⁴⁵Ca²⁺ incorporation which showed some dependence on Ca²⁺ concentration. Ca²⁺ efflux experiments showed that PZ markedly stimulated the release of Ca²⁺ from tapeworms preloaded with ⁴⁵Ca²⁺, with the effluxed Ca²⁺ being derived from a small fast pool and a larger slow pool. This stimulatory effect appeared, like PZ-induced muscle contraction, to be independent of external Ca²⁺. By carrying out ⁴⁵Ca²⁺ exchange experiments under near equilibrium conditions and atomic absorption spectroscopy it could be demonstrated that PZ resulted in a net excretion of endogenous Ca²⁺. In PZ-induced contracted worms adenylate nucleotide levels and the adenylate energy charge were not significantly different from those of untreated control worms. Also, PZ had no effect on Ca²⁺-stimulated ATPase activity of the tapeworm's tegumental brush border. Nor did the drug alter the activities of Ca²⁺-ATPases in whole homogenates of worms or mitochondria, microsomal or soluble fractions. Although the mechanism of PZ-induced changes in Ca²⁺ transport was not elucidated, it is suggested that the sustained release of endogenous Ca²⁺ may affect the sequence of excitation-contraction coupling and that such interference may cause the observed massive contraction of the tapeworm's musculature.     

Pharmacodynamics of pentoxifylline and/or praziquantel in murine schistosomiasis mansoni

Pentoxifylline (PTX) was proved to exert both anti-inflammatory and anti-fibrotic effects, and was used therapeutically in this experimental model to investigate its role alone or with praziquantel (PZQ) in Schistosoma mansoni-infected mice, and to explore its impact on the tissue expression of transforming growth factor-beta1 (TGF-beta1). S. mansoni-infected mice were divided into seven groups: Control untreated (I), treated with curative dose of PZQ, 500 mg/kg/day for 2 consecutive days (II), or subcurative dose, 100 mg/kg/day for 2 consecutive days (III), treated with PTX (10 mg/kg/day for 5 days/wk) alone for 4 weeks (IV) or in addition to subcurative dose of PZQ (V), and treated with PTX alone for 8 weeks (VI) or in addition to subcurative dose of PZQ (VII). All animals were killed 10 weeks post infection. Parasitological assessment of worm burden, tissue egg load and oogram pattern was carried out. The degree of granulomatous fibrosis and eosinophilic cell population was quantified in Sirius-red-stained sections and tissue transforming growth factor beta-1 expression was estimated immunohistochemically. Serum ALAT and GGT, as well as hepatic content of reduced GSH, were measured. The results revealed the highest percent of worm reduction and dead ova in groups (II) and (VII) accompanied by significant diminution in granulomatous parameters, collagen content and TGF-beta1 tissue expression. Moreover, treatments with PTX and/or PZQ ameliorated the liver functions. In conclusion, prolonged treatment with PTX has a potent anti-fibrogenic role especially when used in the early stages of infection, with limited toxic effects on schistosome worms and eggs. Thus, PTX can be used as an adjuvant therapeutic tool with anti-helminthic drugs in the treatment of human schistosomiasis.     

Uptake of galactose by the epithelial syncytium of Schistosoma mansoni

A steady-state compartmental analytical solution to radiolabeled galactose fluxes into and out of S. mansoni using a three compartment model is presented. Experiments included phlorizin inhibition, Na⁺-free incubations and inhibition experiments with glucose and 3-O-methylglucose. The inward rate constants exceeded the outward exchange rates by a factor of 1.55 in females and 1.85 in males. Phlorizin, Na⁺-free conditions and the other hexoses reduced the inward exchange rates such that net secretion of galactose would be favored since the inward to outward exchange rates were less than unity. In the Na⁺-free incubations, the outward exchange rate was increased but not in the other experiments. The steady-state K_t values were increased in Na⁺-free incubations and during inhibitions by glucose and 3-O-methylglucose, but not in response to phlorizin. The size of the exchangeable tissue pool of galactose was decreased in a predictable manner when the exchange rates across the surface epithelial syncytium were reduced by glucose, 3-O-methylglucose, phlorizin and Na⁺-free incubations. The results were generally consistent with previous findings of stereospecific Na⁺-coupled uptake of galactose by S. mansoni.     

Immunological and parasitological parameters in Schistosoma mansoni-infected mice treated with crude extract from the leaves of Mentha x piperita L.

Schistosomiasis is a chronic disease caused by an intravascular trematode of the genus Schistosoma. Praziquantel is the drug used for treatment of schistosomiasis; nevertheless failure of treatment has been reported. Consequently, the identification of new effective schistosomicidal compounds is essential to ensure the effective control of schistosomiasis in the future. In this work we investigated the immunomodulatory and antiparasitic effects of the crude leaves extract of Mentha x piperita L. (peppermint) on murine Schistosomiasis mansoni. Female Balb/c mice were infected each with 50 S. mansoni cercariae and divided into three experimental groups: (I) untreated; (II) treated daily with M. x piperita L. (100 mg/kg) and III) treated on 1/42/43 days post-infection with Praziquantel (500 mg/kg). Another group with uninfected and untreated mice was used as a control. Subsequently, seven weeks post-infection, S. mansoni eggs were counted in the feces, liver and intestine. Worms were recovered by perfusion of the hepatic portal system and counted. Sera levels of IL-10, IL-5, IL-13, IFN-γ, IgG1, IgE and IgG2a were assayed by ELISA. Animals treated with a daily dose of M. x piperita L. showed increased sera levels of IL-10, IFN-γ, IgG2a and IgE. Besides, M. x piperita L. treatment promoted reduction in parasite burden by 35.2% and significant decrease in egg counts in the feces and intestine.     

Use of recombinant calreticulin and cercarial transformation fluid (CTF) in the serodiagnosis of Schistosoma mansoni

Schistosomiasis is traditionally diagnosed by microscopic detection of ova in stool samples, but this method is labour intensive and its sensitivity is limited by low and variable egg secretion in many patients. An alternative is an ELISA using Schistosoma mansoni soluble egg antigen (SEA) to detect anti-schistosome antibody in patient samples. SEA is a good diagnostic marker in non-endemic regions but is of limited value in endemic regions, mainly because of its high cost and limited specificity. Here we assess seven novel antigens for the detection of S. mansoni antibody in an endemic region (the Northern Nile Delta). Using recombinant S. mansoni calreticulin (CRT) and fragments thereof, anti-CRT antibodies were detected in the majority of 97 patients sera. The diagnostic value of some of these antigens was, however, limited by the presence of cross-reacting antibody in the healthy controls, even those recruited in non-endemic areas. Cercarial transformation fluid (CTF), a supernatant that contains soluble material released by the cercariae upon transformation to the schistosomula, is cheaper and easier to produce than SEA. An ELISA using CTF as the detection antigen had a sensitivity of 89.7% and an estimated specificity of 100% when used in non-endemic regions, matching the performance of the established SEA ELISA. CTF was substantially more specific than SEA for diagnosis in the endemic region, and less susceptible than SEA to cross-reacting antibody in the sera of controls with other protozoan and metazoan infections.     

The incorporation and utilization of radiolabelled lipids by adult Schistosoma mansoni in vitro

Live adult Schistosoma mansoni, maintained in vitro were able to absorb and utilize radiolabelled arachidonic acid, linolenic acid, 3-sn-phosphatidylcholine, tripalmitylgylcerol and cholesterol in the culture medium. Differential centrifugation demonstrated that all these lipids were incorporated into the parasite's various membrane structures. Analysis by thin-layer chromatography of the labelled compounds which resulted from incubation with the labelled lipids revealed that arachidonic acid, linolenic acid and fatty acids of tripalmitylglyceron and 3-sn-phosphatidylcholine was present largely as triacylglycerol with smaller amounts of labelled diacylglycerol, phospholipids and fatty acids. The labelled polar head group of 3-sn-phosphatidylcholine was cleaved from the molecule during incorporation, which suggested that hydrolysis of complex lipids is an integral part of the absorption mechanism. Cholesterol was not apparently altered during incorporation or further metabolized. It was also observed that arachidonic acid was incorporated more readily than the other lipids, however, no prostaglandin biosynthesis was detected.     

Preparation and partial characterisation of a multilamellar body fraction from Schistosoma mansoni

Multilaminate vesicles were purified, from homogenised whole Schistosoma mansoni adults, by differential density centrifugation on sucrose gradients, following methods previously employed for isolating multilamellar bodies (MLB) from mammalian lung. Morphometric analysis, on electron micrographs, of the pelleted fraction revealed that the pellet contained at least 56% MLB (by volume of solid material).An apparent projection core was described in both fixed MLB from our fraction and in unfixed MLB from a freeze thaw preparation of whole worms. The phospholipid-protein ratio of the MLB fraction was 1.6:1. The major phospholipid classes were separated and identified by quantitative, two dimensional, thin layer chromatography on silica gel. Phosphatidylcholine was the predominant phospholipid in the MLB fraction, comprising 57% of the total phospholipids. Phosphatidic acid phosphatase, previously reported from lung MLB but not schistosomes, was detected in the fraction. The high activity of this enzyme suggests a more active role for schistosome MLB than that of a mere reservoir of preformed membrane precursors.     

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

Two monoclonal antibodies of the IgE class (54.10) and of the IgG₁ 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 mcIgG₁ 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.     

Molecular characterization of mitochondrial DNA provides evidence for the recent introduction of Schistosoma mansoni into America

Mitochondrial DNA was analyzed from 6 strains of Schistosoma mansoni with 12 restriction enzymes, corresponding to 40 restriction sites or about 1.5% of the coding region. An extensive length polymorphism among strains of S. mansoni was found, with size ranging from 16 500 bp to 24 900 bp. Five restriction sites among 40 were polymorphic; phylogenetic analysis using parsimony criteria supports the idea that American strains were introduced very recently from Africa. Two large fragments (2800 and 5400 bp), representing half of the coding region were cloned. The location of some genes was determined using heterospecific multigenes probes and suggests that this platyhelminth genome differs from all other known mtDNA gene organization.     

Transcriptional profiling of the oesophageal gland region of male worms of Schistosoma mansoni

The intestinal tract of schistosomes opens at the mouth and leads into the foregut or oesophageal region that is lined with syncytium continuous with the apical cytoplasm of the tegument. The oesophagus is surrounded by a specialised gland, the oesophageal gland. This gland releases materials into the lumen of the oesophagus and the region is thought to initiate the lysis of erythrocytes and neutralisation of immune effectors of the host. The oesophageal region is present in the early invasive schistosomulum, a stage potentially targetable by anti-schistosome vaccines. We used a 44k oligonucleotide microarray to identify highly up-regulated genes in microdissected frozen sections of the oesophageal gland of male worms of S. mansoni. We show that 122 genes were up-regulated 2-fold or higher in the oesophageal gland compared with a whole male worm tissue control. The enriched genes included several associated with lipid metabolism and transmembrane transport as well as some micro-exon genes. Since the oesophageal gland is important in the initiation of digestion and the fact that it develops early after invasion of the mammalian host, further study of selected highly up-regulated functionally important genes in this tissue may reveal new anti-schistosome intervention targets for schistosomiasis control.     

Identification of a sialic acid containing glycocalyx on the surface of Schistosoma mansoni

Ruthenium red fixation of adult Schistosoma mansoni revealed the existence of a negatively charged layer external to the outer bilayer, which was morphologically similar to the glycocalyx of other cell types. Regional and sexual differences were found in the extent and organisation of the surface coat, which can be correlated with interfacial free energy, adhesiveness and protection from immune effectors. Neuraminidase treatment confirmed the presence of surface sialic acid. Mechanical or skin penetrated schistosomula, maintained in vitro for 24 h were found not to have a glycocalyx and this may relate to their increased susceptibility to immune killing. Lung stage schistosomula however, did bind ruthenium red to their surface.     

Down-regulation of specific antigen-driven cytokine production in a population with endemic Schistosoma japonicum infection

Schistosome antigen-driven cytokine responses and antischistosome antibody levels of residents of a Schistosoma japonicum endemic island in Poyang Lake, Jiangxi Province were studied before and 45 days after treatment with praziquantel. IL-4, IL-5, IL-10 and INF-gamma were all detected in the supernatants of whole-blood cultures after stimulation with schistosome soluble egg antigen (SEA) and soluble worm antigen preparation (SWAP). The percentages of subjects producing detectable amounts of each cytokine assayed were higher in the group who were negative by stool examination at the start of the study than in those who were initially stool positive. After praziquantel treatment the percentages of subjects producing both type I and type II cytokines increased. This suggests that the production of both types of cytokine was down-regulated in the presence of live, egg-laying S. japonicum adult worms but that this was reversible by treatment. In contrast, the antibody studies showed higher levels of SWAP and SEA-specific antibodies (IgE, total IgG, IgG4, IgM) in subjects who were originally stool-positive than in those who were stool-negative. After treatment specific IgE responses were elevated, but total IgG and IgG4 anti-SEA and IgM anti-SWAP antibody levels all fell significantly.