Cercarial Extracts Research Articles

Comparison of cysteine peptidase activities inTrichobilharzia regentiandSchistosoma mansonicercariae

Cercariae of the bird schistosome Trichobilharzia regenti and of the human schistosome Schistosoma mansoni employ proteases to invade the skin of their definitive hosts. To investigate whether a similar proteolytic mechanism is used by both species, cercarial extracts of T. regenti and S. mansoni were biochemically characterized, with the primary focus on cysteine peptidases. A similar pattern of cysteine peptidase activities was detected by zymography of cercarial extracts and their chromatographic fractions from T. regenti and S. mansoni. The greatest peptidase activity was recorded in both species against the fluorogenic peptide substrate Z-Phe-Arg-AMC, commonly used to detect cathepsins B and L, and was markedly inhibited (> 96%) by Z-Phe-Ala-CHN2 at pH 4.5. Cysteine peptidases of 33 kDa and 33-34 kDa were identified in extracts of T. regenti and S. mansoni cercariae employing a biotinylated Clan CA cysteine peptidase-specific inhibitor (DCG-04). Finally, cercarial extracts from both T. regenti and S. mansoni were able to degrade native substrates present in skin (collagen II and IV, keratin) at physiological pH suggesting that cysteine peptidases are important in the pentration of host skin.

Schistosoma mansoni proteases Sm31 (cathepsin B) and Sm32 (legumain) are expressed in the cecum and protonephridia of cercariae.

Adult Schistosoma mansoni parasites live in the bloodstream of their vertebrate hosts where they consume red blood cells. Hemoglobin, released from the ingested red blood cells, is degraded by a variety of parasite proteases, including Sm31 (cathepsin B) and Sm32 (schistosome legumain). In this study the localization pattern of the Sm31 and Sm32 enzymes in cercariae (the infectious life cycle stage) was examined. Antibodies generated against recombinant Sm31 and Sm32 recognize their respective proteins in Western blots of soluble parasite extracts. Highest levels are seen in adult female extracts, whereas the level of both proteins is below detection in cercarial extracts. However, in fixed, whole cercariae, both proteins are seen in the cecum and protonephridia. In the cecum, the staining pattern has a granular appearance, suggesting that the proteins are packaged in vesicles. In the protonephridial system, Sm31 and Sm32 are detected in all 8 flame cells in the cercarial body and in both flame cells in the cercarial tail. The distribution of the 2 proteins differs in the flame cells. Examination of immunostained cercariae using laser scanning confocal microscopy shows that whereas Sm31 is located in the tubule cell, Sm32 is found in both the tubule cell and its adjoining cap cell. These findings suggest that the proteins are involved in the proposed excretory and osmoregulatory roles of flame cells.

Cleavage of Human IgE Mediated by Schistosoma mansoni

Background: IgE-mediated mechanisms are important in protection against helminth parasites. However, schistosomes are long-lived in mammalian hosts, presumably as a result of immune evasion strategies. We sought evidence for one such strategy, namely specific cleavage of host IgE. Methods: Human IgE, IgA and IgG were incubated with extracts from cercarial and schistosomular stages of Schistosoma mansoni or with schistosomular culture supernatants. The resulting products were analysed by Western blotting with Ig-specific antibodies. Numerous protease inhibitors were assessed for ability to inhibit the observed cleavage of IgE by the extracts. Partial purification of the IgE-proteolytic activity from cercarial extract was achieved by gel filtration. To test IgE function, we compared the abilities of untreated and schistosomular-treated IgE to mediate rosette formation through interaction with Fcε receptors. Results: Cercarial and schistosomular extracts were found to cleave human, mouse and rat IgE but not human IgA1, IgA2 or IgG1. Schistosomular culture supernatants displayed similar proteolytic activity towards IgE. Immunoblotting suggested that cleavage occurred close to the Cε2/Cε3 domain interface of the IgE heavy chain. PMSF and elastatinal inhibited cleavage, suggesting that the protease involved is an elastase-like serine protease, particularly since porcine pancreatic elastase also cleaved IgE to give similar-sized products. Further, the chloromethyl ketone derivatized peptide MeO-Suc-Ala-Ala-Pro-Leu- CMK, known to specifically inhibit the schistosome elastase, prevented IgE cleavage. Cleavage of human IgE rendered the antibody molecule unable to interact with U937 cells expressing FcεRII. Conclusions: An elastase-like protease in S. mansoni is able to render IgE non-functional.

Schistosome extracts with heat shock factor activity revealed by the gel shift assay.

To understand the regulated expression of stage-specific genes of schistosomes, it is necessary to identify regulatory DNA elements and DNA-binding proteins that control the level of gene transcription. Here we describe the preparation of Schistosoma mansoni extracts with active transcription factors detected by the electrophoretic mobility shift assay (EMSA). We analysed the hsp70 system of S. mansoni because the promoters of the hsp70 gene contain two heat shock elements (HSE) that differ from the consensus sequence (CnnGAAnnTTCnnG) at one (HSEI) or three (HSEII) positions, and it is known that transcriptional activation of hsp70 genes is mediated by interaction of HSE with the heat shock factor (HSP). Analyses of parasite extracts from different developmental stages demonstrate the presence of putative HSF that correlates with the pattern of hsp70 mRNA expression (cercariae-, schistosomula+, adult worms+). Cercarial extracts did not show binding of 32P-labelled HSEI or HSEII. Extracts of schistosomula and of adult worms kept at 37 or 42 degrees C showed binding of HSEI but not HSEII. The specificity of HSEI-HSF complex formation was ascertained by inhibition experiments. The EMSA experiments and structural features of the hsp70 promoter indicate that HSEI is the major DNA element responsible for transcriptional activation of the hsp70 gene, while the HSEII may be a redundant sequence with minor (if any) regulatory function. The extracts reported here can be used to study transcriptional control of other schistosome genes.

Anti-nuclear antibodies associated with schistosomiasis and anti-schistosomal antibodies associated with SLE.

Various autoantibodies to nuclear antigens were detected by ELISA in pooled sera of 15 ICR mice 9 weeks after infection with schistosoma. The binding of this serum with acute infection to cercarial extract was inhibited by prior incubation of the sera with DNA, polynucleotides and cercarial extract. Sera from 9 BALB/c mice with experimentally induced lupus, reacted with cercarial extract. No binding was noted with normal mouse sera (NMS). Similarly, sera of SLE patients (N = 113) reacted with the parasite extracts in a higher incidence (14.2%) compared to NHS (3.7%) (P < 0.05). Immunoblot analysis showed common features to SLE sera reacting with two antigenic "groups". The first are antigens that also were recognized by sera of mice infected with schistosomiasis (60, 85, and 94 kDa cercarial proteins). The second group entailed proteins that bound with SLE sera only (10-18 kDa, 29 kDa and 52 kDa). It can be concluded that antinuclear autoantibodies can be found in sera of acute infected mice with schistosomiasis, while SLE sera may react with 3 schistosomal extracts in a higher incidence than NHS (P < 0.05). These results suggest an autoimmune response in schistosomiasis. It is possible that a parasite infection (as bilharzia) may be one of the "trigger" mechanisms for SLE in a subject with the suitable immunogenetic and hormonal background.

Identification of free and conjugated ecdysteroids in cercariae of the schistosomeTrichobilharzia ocellata

Extracts of cercariae of the avian schistosome Trichobilharzia ocellata were analysed for the presence of ecdysteroids by radioimmunoassay, high-performance liquid chromatography monitoring fractions by radioimmunoassay, and gas chromatography/mass spectrometry (selected ion monitoring). Both free ecdysteroids and polar conjugated ecdysteroids were detected in the cercarial extracts. The free ecdysteroid fraction, as well as the hydrolysed polar conjugated ecdysteroid fraction, contained both ecdysone and 20-hydroxyecdysone in approximately equal amounts. The amount of ecdysteroids detected is comparable to those found in other platyhelminths. A possible role for the ecdysteroids in the development of the parasite and/or the interactions between the parasite and its intermediate host, the freshwater snail Lymnaea stagnalis, is discussed.

Schistosoma mansoni: Stage-specific expression of muscle-specific genes

It was previously shown that an antigen preparation termed 9B obtained from Schistosoma mansoni cercarial extracts partially (34%) protects mice from challenge infection with cercariae (R. Tarrab-Hazdai et al., J. Immunol. 135, 2772, (1985)). To characterize some of the proteins which comprise this preparation, rabbit antibodies to the 9B antigen preparation were used to screen cDNA libraries of cercariae and adult worms. We isolated and sequenced cDNA clones encoding three proteins: calcium-binding protein, paramyosin, and myosin. The calcium-binding protein was previously shown to be expressed in cercariae but not in sporocysts or adult worms (D. Ram et al., Mol. Biochem. Parasitol. 34, 167, (1989)). Northern blots showed the presence of paramyosin and myosin mRNAs in sporocysts and adult worms but not in cercariae. Antibodies to paramyosin detected the protein in sporocysts and adult worms as well as in cercariae. These findings explain, in part, the protective activity of the 9B antigen preparation against challenge infection.

Schistosoma mansoni: Immunogenic glycoproteins of the cercarial glycocalyx

Immunochemical studies at the level of the light and electron microscope showed that a monoclonal antibody, 128 C3 3 , was directed to an epitope in the glycocalyx of Schistosoma mansoni cercariae. Immunoprecipitation of surface labeled cercarial extracts with this monoclonal antibody demonstrated that the glycocalyx is composed of at least five components, including a very large molecular size polypeptide and polypeptides of 220, 180, 170, and 15 kDa. After transformation of cercariae to schistosomula, these polypeptides were shed from the surface and were therefore no longer accessible to surface labeling. Monoclonal antibody 128 C3 3 was also reactive with a 38 kDa polypeptide from schistosomula; this polypeptide was weakly expressed on the surface of cercariae. Analysis of immunoprecipitates of radioiodinated protein extracts of cercariae, newly transformed schistosomula, and 36 hr in vitro cultured schistosomula showed that the 180 and 170 kDa polypeptides continued to be expressed within the organism following transformation, but were not accessible to surface labeling. Lectin binding studies revealed differences in the oligosaccharide composition of the six polypeptides. With the exception of the 15 kDa antigen, all the polypeptides reactive with 128 C3 3 were highly immunogenic in infected mice and humans.

Anaphylaxis with Schistosoma mansoni extracts in normal and infected mice

Methods generally utilized for studies on anaphylaxis to protein antigens such as determination of histamine release to the blood, hemoconcentration, histamine release from peritoneal mast cells and passive cutaneous anaphylaxis (PCA) were used to investigate some aspects of the anaphylaxis to parasite antigens in Schistosoma mansoni infected mice. The release of histamine to the blood and significant rates of hemoconcentration were induced by intravenous injection of schistosomula or cercarial extracts into 10-13 weeks infected mice. Cercarial, schistosomula, worm tegument and soluble egg antigens were able to trigger histamine release from peritoneal mast cells from chronically infected mice. In spite of the PCA reaction beeing detected within 2 hours of sensitization (IgG1antibodies) in 6 of 8 tested sera from chronically infected mice, no detectable reactions were obtained after 48 hours sensitization (IgE antibodies). Although IgE was not detected in the circulation, by the PCA technique, the results indicate that the infected mice contained IgE antibodies bound to their mast cells.

Schistosoma mansoni: Eicosanoid production by cercariae

Cercariae of Schistosoma mansoni are stimulated to penetrate skin by certain free fatty acids. The cercariae have an active arachidonate cascade, presumably using host skin essential fatty acids as cascade precursors. Exposing cercariae to 3.3 m M linoleate for 1, 10, and 60 min resulted in production of a wide variety of eicosanoids. Using high-performance liquid chromatography, eicosanoids coeluting with prostaglandin E 2, D 2, and A 2, leukotriene B 4, and 5-hydroxyeicosatetraenoic acid standards were identified, as well as unidentified peak positions. Radioimmunoassay confirmed the presence of immunoreactive prostaglandin E 1, and E 2, and 5- and 15-hydroxyeicosatetraenoic acids in cercarial extracts. No eicosanoid production occurred when cercariae were exposed to 3.3 m M oleate and 1 or 330 μ M linoleate. Both high-performance liquid chromatography and radioimmunoassay data indicated that cercariae regulate the production of eicosanoids through time. It is postulated that arachidonate metabolism and subsequent eicosanoid production are required for successful cercarial penetration.

Strain differences in lymphocyte responses and in vitro suppressor cell induction between Schistosoma mansoni-infected C57BL/6 and CBA mice.

Splenic lymphocyte blastogenic responses to mitogens and antigens were compared in Schistosoma mansoni-infected C57BL/6 and CBA mice. At 1 and 2 weeks of infection, elevated responses to phytohemagglutinin (PHA) were noted in C57BL/6, but not in CBA cultures. During the same period, elevated responses to lipopolysaccharide were observed in cultures from CBA, but not from C57BL/6 mice. Responses to both mitogens were depressed in both strains later in infection. The magnitude of antigen-specific responses was greater in CBA than in C57BL/6 cultures. A cercarial extract (CE) induced significant suppressor cell activity in normal C57BL/6 cell cultures as assessed by inhibition of syngeneic cell responsiveness to PHA. CE did not induce suppressive activity in CBA cultures. CE-induced suppression was unaffected by adherent cell removal, but was sensitive to anti-Thy 1.2 plus complement treatment. CE also directly inhibited PHA-stimulated C57BL/6 cell cultures, but enhanced cultures of CBA mice. The significance of these findings is discussed in relation to the relative strain differences seen in antigen-specific lymphocyte proliferation and the levels of protection induced after immunization with irradiated cercariae.

Enzymes in Schistosoma intercalatum and the relative status of the Lower Guinea and Zaire strains of the parasite

Seven enzyme systems [phosphoglucomutases (PGM), glucose phosphate isomerases (GPI), glucose-6-phosphate dehydrogenases (G6PD), malate dehydrogenases (MDH), laetate dehydrogenases (LDH), acid phosphatases (AcP) and hexokinases] in extracts of adult worms from two isolates of Schistosoma intercalatum, one from Zaire and one from Cameroun, were compared by isoelectric focusing. Two systems (GPI and PGM) were also compared in extracts of cercariae. Distinctive differences between the strains were found in the LDH system and even more marked differences in the G6PD and PGM systems (the latter were apparent in both adult worm and cercarial extracts). These observations are discussed in conjunction with existing evidence on the results of intermediate host infection experiments and of experimental hybridization between the two strains of S. intercalatum. In turn these aspects are discussed in the light of what is known about other species of African Schistosoma. It is concluded that any definite decision on the relative status of the two strains of S. intercalatum is still premature.

Comparison of proteolytic enzymes from cercarial extract and secretion

1. 1. Both cercarial extract and secretion were fractionated in casein polyacrylamide gel producing 6–7 and 3–4 bands respectively. 2. 2. Most of the proteolytic activity was concentrated in the fast-moving band. 3. 3. The main proteolytic activity from secretion was easily purified by molecular sieve chromatography. 4. 4. The proteolytic and esterolytic activities are eluted in the same peak.

Resistance to infection with Schistosoma mansoni after immunization with worm extracts or live cercariae: role of cytotoxic antibody in mice and guinea pigs.

Abstract : Mice and guinea pigs were immunized with adult and cercarial extracts prepared in different ways. Most animals responded by producing moderate to high levels of cytotoxic antibody; but no correlation with resistance to infection could be detected. Initial experiments with a 3M KC1 extract and with cercarial exoantigen produced partial resistance; several attempts to reproduce these results tailed. Mice immunized either by live or attenuated cercariae, while developing very low levels of cytotoxic antibody, were highly resistant to challenge infection. These results are discussed in terms of a failure to induce a cooperative immune mechanism required for the action of cytotoxic antibody. It is suggested that homocytotropic antibodies may play a gatekeeper role by initiating a reaction that promotes the transloction of serum cytotoxic antibodies and cells into the tissue surrounding the migrating schistosomules.

Schistosoma mansoni: Calcium Content of Cercariae and Its Effects on Protease Activity In vitro

The localization and quantitation of calcium in the protease-containing preacetabular glands of Schistosoma mansoni cercariae were determined by electron probe analysis and atomic absorption spectroscopy. Levels of calcium in these glands appear to exceed 8 to 10 M. In vitro, these high levels of calcium inhibit the cercarial proteases reversibly. It is suggested that calcium functions to control protease activity in situ in these organisms. The infective stage of the human blood fluke, Schistosoma mansoni, infects its host by penetrating the skin. The penetration process is thought to be mediated by proteolytic enzymes present in the preacetabular glands of this organism (Lewert and Lee, 1956; Stirewalt and Fregeau, 1966; Gazzinelli et al., 1966; Dresden and Asch, 1972). Moreover, it has been demonstrated that these glands contain large amounts of calcium (Stirewalt, 1959; Lewert et al., 1966). Lewert and Lee (1956) originally reported that these proteases are stimulated at low concentrations of these cations and we have confirmed this observation (Dresden and Edlin, 1974). Recently we determined that the proteolytic enzymes in extracts of this organism are inhibited by high concentrations of calcium and magnesium (Dresden and Edlin, 1974). We suggested that calcium might play a significant role in controlling the activity of these proteases by acting as an inhibitor of protease activity in situ. The results described in this report provide qualitative and quantitative evidence on the levels of calcium in these glands and its effects on protease activity. MATERIALS AND METHODS Infected snails, Biomphalaria glabrata (Puerto Rican strain), were allowed to shed their cercariae at room temperature in beakers containing dechlorinated tap water or 1 mM Tris-HCl, pH 7.8. Using a depression slide and a dissecting microscope, individual cercariae were transferred by disposable pipette. For electron probe analysis single cercariae were dried on silica discs at room temperature as rapidly as possible, aided by withdrawal of water with a micropipette. For calcium analysis, individual cercariae (50 Received for publication 10 October 1974. t 200) were counted and transferred to appropriate tubes. Spectrophotometric analysis of calcium was by the method of Williams and Wilson (1961). CaCl2 and CaCO3 (0.5 to 100 ,ug) were used as standards. Atomic absorption spectroscopy for calcium was determined following extraction of 100 to 200 cercariae in 5% lanthanum chloride-1% HC1 at 100 C for 10 min. Calcium contributed by sources other than the cercariae was corrected for by using uninfected snails. Protease activity in cercarial extracts was measured using Azocoll substrate as described previously (Dresden and Edlin, 1974). RESULTS AND DISCUSSION The results of the electron probe X-ray analysis and scanning electron microscopy are shown in Figure la-c. These figures represent a single cercaria examined by scanning electron microscopy (Fig. la) and by X-ray probe analysis for calcium (Fig. lb) and phosphorus (Fig. lc). A number of individual cercariae were examined by these combined techniques. The results demonstrate dramatically a concentration of calcium in the region of the preacetabular glands, and a fairly uniform distribution of phosphorus throughout the body of the cercariae. Using the tail region as a control, scanning X-ray analysis was carried out for 20to 40-sec intervals at 25 kv with a 0.185-,uamp sample current. These experiments demonstrated that calcium is at least 1,000 to 2,000 times more abundant in the preacetabular gland region than in the tail region. Magnesium levels in the gland region were not significantly elevated over those in the tail region. While these experiments established the localization of calcium, it would have been necessary to determine the thickness and composition of the cercariae in order to determine

Nicarbazin in schistosome infections: I. Antibody formation in mice and hamsters infected with Schistosoma mansoni

The immunoprecipitin response of mice infected with Schistosoma mansoni and treated with nicarbazin, an egg suppressive agent, is significantly lower than in untreated-infected mice. The precipitin response to cercarial extract is virtually abolished in treated mice indicating common antigenic determinants with eggs of the same species. Haemagglutinins to adult worms are also significantly diminished in treated mice. Finally, circumoval precipitins are absent in treated mice when the drug is given continuously to infected mice in order to prevent egg laying by the female adult parasite. The results suggest that a significant portion of the precipitating antibody produced in schistosome infections reactive with cercariae and adult worms, as well as eggs, is probably a secondary antibody response due to common antigenic determinants found in eggs.

Proteolytic enzymes in extracts of Schistosoma mansoni Cercariae

The enzymatic mechanisms by which cercariae of Schistosoma mansoni invade mammalian skin have been investigated. Cercarial extracts prepared by sonication were assayed for activity against a variety of substrates including purified components of connective tissues. These extracts contain activity against gelatin, casein, Azocoll and elastin, but do not hydrolyze undenatured soluble or fibrous collagen. Cercarial extracts are very active against a chondromucoprotein complex from cartilage but do not hydrolyze the major mucopolysaccharide component of this complex. Sephadex G-150 chromatography of cercarial extracts revealed the existence of multiple peaks of proteolytic activity. It is suggested that carcarial proteases exert their main activity against the protein backbone of the protein-polysaccharide present in mammalian connective tissues.

Immunopathology of Dermal Reactions Induced by Schistosoma Mansoni Cercariae and Cercarial Extract *

Abstract Mice infected with Schistosoma mansoni and normal mice were exposed to viable S. mansoni cercariae, and the exposed areas were biopsied at ½, 1, 2, 4, 8, 16, 24, 36, 48, and 120 hours. The initial histologic response (2–8 hours after challenge) of previously infected animals was characterized by edema and a progressive, predominantly polymorphonuclear infiltrate. By 16 and 24 hours, the schistosomules were the foci of intense mixed cell infiltrates, and by 36 and 48 hours, dense mononuclear infiltrates were evident. Normal mice showed only edema and slight polymorphonuclear infiltrates, and by 16 hours most of the schistosomules had migrated through the dermis. Intradermal challenge of infected and uninfected mice with a crude saline cercarial extract produced reactions similar to those described for the two groups after cercarial penetration. Normal mice were also challenged by cercarial exposure or injection of cercarial extract after passive transfer of either lymphoid cells or serum from infected or normal mice. Sera from infected mice passively immunized normal mice, which then responded with an early (5-hour) polymorphonuclear infiltrate against cercariae. Lymphoid cells from infected mice passively immunized normal mice which then developed a late (30-hour) mononuclear infiltrate against either schistosomules or cercarial extract. It is concluded that mice develop both humoral and cell-mediated immune responses against the cercarial stage of S. mansoni.

Evidence of transplacental passage of specific antibody in schistosomiasis mansoni in man.

Transplacental passage of specific antibodies from mother to neonatal infant was demonstrated in Schistosoma mansoni infections in man. This passage of antibody was demonstrated by the circumoval precipitin method, the Ouchterlony double-diffusion method with adult worm and cercarial extracts of S. mansoni, and by the cercarial slide-flocculation method. Immunoglobulin levels between mothers and their infants were compared by simple radial immunodiffusion. Levels of immunoglobulin G of mothers and their infants were similar; however, no immunoglobulin A was detected in the serum from the neonatal infants, and only three of 16 infants had measurable amounts of immunoglobulin M by the methods used. We believe this to be presumptive evidence that the transplacental passage of specific antibodies from mothers to their neonatal infants in S. mansoni infections is due to serum IgG.

Schistosoma mansoni: Generation of anaphylatoxin by cercarial extracts

Extracts from Schistosoma mansoni cercariae generate in plasma or serum an activity which contracts the isolated guinea pig ileum. This contraction is inhibited by mepiramine (an anti-histaminic drug). The activity formed by cercarial extracts produces cross-desensitization with the classical anaphylatoxin generated by agar, and induces mast cell degranulation in guinea pig mesentery as well as enhancement of vascular permeability in guinea pig skin. The generation of this anaphylatoxin was blocked by the previous heating of the serum at 56 °C for 30 minutes and by the presence in mixture of 1 × 10 −2 M Na 33HEDTA. The possible role of this pharmacological active material in the process of skin penetration by cercariae is discussed.