Leucine aminopeptidase and hatching of Schistosoma mansoni eggs

Leucine aminopeptidase (LAP) activity has been measured in extracts of eggs, miracidia, cercariae and adult worms of Schistosoma mansoni. Activity measured at pH 7.2 using L-leu-7-amino-4-trifluoro-methylcoumarin as substrate is 6- to 17-fold greater in eggs than in other life stages. LAP activity is also high in soluble egg antigen preparations and in hatching fluid. The release of LAP from eggs parallels hatching, and inhibitors of LAP also inhibit hatching. The possible role of LAP in the hatching process of S. mansoni eggs is discussed.     

Schistosoma mansoni: factors affecting hatching of eggs

The effect of light, oxygen tension, reducing conditions and thermal shock on egg hatching in Schistosoma mansoni were examined. Hatching was found to be unaffected by light or dark conditions or aerobic or anaerobic conditions. Cold shock from 15 to 120 sec was also ineffective in stimulating hatching. The reducing agents ascorbic acid and cysteine inhibited egg hatching. However, the oxidized forms of these compounds inhibited hatching as well, indicating that the reducing conditions they provided were not responsible for the inhibition.     

Effect of chemotherapy on the Schistosoma mansoni eggs

Praziquantel administered to mice with Schistosoma mansoni infection (50 cercarias/8 weeks) was observed to cause death of adult worms and disintegration of the eggs trapped within granulomas, sometimes with calcification, after the 4th day of treatment. Combined administration of oxamniquine/hycanthone to animals similarly infected, although quite effective in killing adult worms, did not interfere with the eggs in the tissue. The miracidium eclosion test was positive up to the 15th day after the curative treatment of these animals. Since praziquantel treatment causes a rapid destruction of eggs, possible serological and pathogenic effects are expected that may enable a faster reabsorption of granulomas by the host tissues than that produced by other equally effective drugs.     

Structural characterization of glycosphingolipids from the eggs of Schistosoma mansoni and Schistosoma japonicum

The granulomatous pathology in human intestinal schistosomiasisis induced primarily by the egg antigens of schistosome, a parasitictrematode. Glycolipids and glycoproteins were extracted fromthe eggs of the two major species which infect human, Schistosomamansoni and Schistosoma japonicum, for structural characterizationbased on highly sensitive mass spectrometric analysis coupledwith chemical derivatization. Here, we demonstrate that a seriesof uniquely multifucosylated glycosphingolipids constitute themajor egg glycolipids of S.mansoni but not of S.japonicum. TheS.mansoni glycosphingolipids were found to be extended by varyingnumbers of an unusual repeating unit,     

Size of hepatic granuloma produced by eggs of 2 geographic strains of Schistosoma mansoni, in mice

The authors have determined the mean diameter of granulomas in the liver of mice infected with cercariae from two different and well definite geographic strains of Schistosoma mansoni (LE, Belo Horizonte, MG, and SJ, S?o José dos Campos, SP). A total of 1,170 granulomas has been measured. Granulomas measured on the 60th day after infection showed larger size than the other ones measured on the 90th day. Modulation of the immunopathologic response was significantly more efficient for the LE strain, whereas the granulomas (with 60 and 90 days) related to SJ strain were significantly larger. Data suggested a higher pathogenicity for the SJ strain. It is speculated whether these findings could explain, in part, the occurrence of regional variations of the anatomo-clinical forms of schistosomiasis.     

Cysteinyl proteinases of Schistosoma mansoni eggs: purification and partial characterization

The egg stage of Schistosoma mansoni, a trematode blood fluke, is known to be responsible for an immunologically mediated granuloma formation. Proteolytic enzymes of S. mansoni eggs may be involved in the penetration of host tissue by eggs and/or may act as antigens to cause a humoral as well as a cell-mediated response leading to granuloma formation. Three acidic, thiol-dependent proteinases from the eggs of S. mansoni were isolated, and 2 major proteinases (I and II) were purified to homogeneity using chromatofocusing, AcA54 ultrogel chromatography, and thiopropyl-Sepharose 6B affinity chromatography. Proteinases I and II have molecular weights of 25,400 and 30,500, and isoelectric points of 6.0 and 5.6, respectively. These enzymes were found to be cathespin B-like cysteinyl proteinases based on similarities in molecular weight, isoelectric point, optimal assay pH, instability to neutral pH, substrate specificity, and inhibitor sensitivity. A monoclonal antibody, specific to S. mansoni egg proteinases was used in immunoblotting studies. Under native, but not under denaturing, conditions for gel electrophoresis, this monoclonal antibody reacted with egg proteinases. This antibody had previously been shown to recognize an antigen in the miracidial penetration glands of schistosome eggs.     

Isolation of a polysaccharide antigen from Schistosoma mansoni eggs.

A polysaccharide antigen was isolated from Schistosoma mansoni egg homogenates by the phenol procedure. The crude preparation (CPEA) contained at least two antigens. The more purified antigen (PEA) was isolated by sequential enzymatic treatment of CPEA with DNase, RNase, Pronase, and alpha-amylase. PEA was resistant to boiling, freezing and thawing, mild acid and alkali, and chloroform, but was destroyed with periodate. It gave a positive reaction with anthrone reagent. PEA was eluted in the wash fraction from a DEAE cellulose collumn and in the void volume of a Sephacryl 200 column. After immunoelectrophoresis and polyacrylamide electrophoresis there was little or no migration. Amino acid analysis failed to reveal ninhydrin-positive material in the a hydrolyzate of PEA. These resluts suggested that PEA is a neutral polysaccharide with a m.w. of more than 200,000 and contains no amino acids or hexosamine. Antibodies against PEA were detected in sera obtained from mice infected with S. mansoni. PEA is different from previously described antigens derived from schistosome eggs.     

Molecular characterization of an interleukin-4-inducing factor from Schistosoma mansoni eggs

The eggs of the parasitic trematode Schistosoma mansoni are powerful inducers of a T helper type 2 (Th2) immune response and immunoglobulin E (IgE) production. S. mansoni egg extract (SmEA) stimulates human basophils to rapidly release large amounts of interleukin (IL)-4, the key promoter of a Th2 response. Here we show purification and sequence of the IL-4-inducing principle of S. mansoni eggs (IPSE). Stimulation studies with human basophils using SmEA fractions and natural and recombinant IPSE as well as neutralization and immunodepletion studies using antibodies to recombinant IPSE demonstrate that IPSE is the bioactive principle in SmEA leading to activation of basophils and to expression of IL-4 and IL-13. Regarding the mechanism of action, blot analysis showed that IPSE is an IgE-binding factor, suggesting that it becomes effective via cross-linking receptor-bound IgE on basophils. Immunohistology revealed that IPSE is enriched in and secreted from the subshell area of the schistosome egg. We conclude from these data that IPSE may be an important parasite-derived component for skewing the immune response toward Th2.     

Partial purification of antigens from eggs of Schistosoma mansoni that elicit delayed hypersensitivity

Schistosoma mansoni egg antigens that elicit delayed hypersensitivity in appropriately sensitized guinea pigs were partially characterized by using ion exchange chromatography and preparative electrophoresis. At least three skin-reactive antigens were found, one of which was purified to homogeneity, as analyzed by polyacrylamide gel electrophoresis (PAGE). This antigen was not adsorbed to CM cellulose, migrated cathodal to guinea pig albumin on electrophoresis, and was adsorbed to DEAE cellulose. A second pool of antigenic activity was obtained by adsorption to CM cellulose and subsequent elution. DEAE cellulose chromatography and preparative electrophoresis of this pool indicated the presence of more than one antigen.     

Modulation of granulomatous hypersensitivity against Schistosoma mansoni eggs in mice vaccinated with culture-derived macrophages loaded with PIII

Granuloma modulation induced by antigen is an attractive model for vaccination studies of experimental schistosomiasis to test the effect of anti-pathology vaccine. We describe here an immunization procedure with culture derived macrophages-pulsed PIII, a known anionic antigen purified from S. mansoni adult worm, involved in the inhibition of granulomatous response to eggs. For our studies, peritoneal or spleen macrophages cultured over 15 days were loaded with PIII. Both macrophage sub-populations were capable to efficiently take up and subsequently present PIII to lymphocytes as evidenced by immunofluorescence assay. The vaccination of mice with intravenous injection of PIII-loaded macrophages potently induced antigen-specific immune response to S. mansoni antigens as determined by cell proliferation assay. This immunization procedure of mice caused significant decrease in hepatic granuloma formation and in vitro granuloma reaction to S. mansoni antigens coupled to polyacrylamide beads (PB-SEA, PB-SWAP or PB-PIII). Assessment of in vitro granuloma supernatant of spleen cells from PIII-loaded macrophages vaccinated mice revealed significant amounts of Th1-cytokines IFN-gamma and IL-2 compared to control cells. Collectively, our results indicate that culture derived-macrophages provided a valuable research tool to investigate aspects of immune response that promote modulation of granulomatous hypersensitivity to S. mansoni eggs in mice.     

Preservation of Schistosoma mansoni eggs and of infected livers as a source of eggs for the circumoval precipitin test

Eggs of Schistosoma mansoni and livers with numerous granulomata were preserved at -70 C in 1.7% salt solution plus an equal volume of water-soluble jelly. The eggs were suitable for circumoval tests after at least 1 yr of preservation and the livers supplied adequate eggs for the tests for at least 6 mo after preservation. This procedure may be useful where such storage facilities are available, and the COP test is used for diagnosis.     

The use of lyophilized Schistosoma mansoni eggs as antigenic particles in a radioimmunoassay

Lyophilized eggs of Schistosoma mansoni, when incubated briefly with serum from infected mice, bind antibodies, as made evident by subsequent binding of fluorescein labelled anti-IgG or 125I-labelled Protein A. On the basis of these findings, a radioimmunoassay was devised which employs whole lyophilized eggs (500 or 250 eggs/serum sample) as antigenic particles and 125I-labelled Protein A as a probe for antibody binding. Only 10 microliters of serum are required to obtain 90% of the maximal binding. Kinetic studies indicated that 70% of the maximal seropositivity develops in mice between five and six weeks after a light infection, reaches a maximum at eight weeks and fluctuates around a high plateau thereafter. Pre-incubation of the test serum with soluble egg antigen (SEA) considerably inhibits antibody binding to the eggs, suggesting that SEA-like antigens participate in the reaction.