Schistosoma mansoni:The Developmental Regulation and Immunolocalization of Antioxidant Enzymes

EXPERIMENTAL PARASITOLOGY ARTICLE NO. PR974150

86, 69–78 (1997)

Schistosoma mansoni: The Developmental Regulation and Immunolocalization of Antioxidant Enzymes Haiping Mei and Philip T. LoVerde Department of Microbiology, State University of New York, Buffalo, New York 14214, U.S.A. MEI, H., AND LOVERDE, P. T. 1996. Schistosoma mansoni: The developmental regulation and immunolocalization of antioxidant enzymes. Experimental Parasitology 86, 69–78. Antioxidant enzymes from S. mansoni, cytosolic Cu–Zn superoxide dismutase (CT-SOD), signal-peptide-containing SOD (SP-SOD), glutathione peroxidase (GPX), and glutathione transferase (GST) were compared for their relative levels of transcript expression throughout development in a semiquantitative reverse transcriptase-polymerase chain reaction assay. All of the antioxidant enzymes exhibited a similar pattern of developmental regulation. Adult worms have the highest level of specific mRNA compared with larval stages. GST shows the highest level of expression, being approximately 10-fold more abundant than CT-SOD and SP-SOD and 100-fold more abundant than GPX. This order of expression was nearly consistent for all the developmental stages studied. To localize the antioxidant enzymes, immunofluorescence staining was performed on 3-hr schistosomula and adult worms. GPX, SP-SOD, and CT-SOD were all found to be associated with the adult tegument and gut epithelium. SP-SOD was also associated with organelle and cell membranes of parenchymal cells and interestingly with the spines of adult worms. Schistosomula, on the other hand, showed little immunofluorescence. These studies further demonstrate the developmental regulation of antioxidant enzymes and localize them to the host–parasite interface, supporting the notion that they have a role in allowing adult worms to evade immune attack. © 1997 Academic Press INDEX DESCRIPTORS AND ABBREVIATIONS: Antioxidant enzymes; confocal microscopy; Schistosoma mansoni; cDNA, complementary deoxyribonucleic acid; CT-SOD, cytosolic Cu–Zn superoxide dismutase; SP-SOD, signal-peptide-containing Cu–Zn SOD; GST, glutathione S-transferase; GPX, glutathione peroxidase; mRNA, messenger ribonucleic acid; RT-PCR, reverse transcriptasepolymerase chain reaction.

parasite is most susceptible to immune elimination during the skin and lung stages of development and least susceptible as an adult parasite (Capron et al. 1987). Although current evidence suggests that immunity to schistosomes is multifactorial, a major component of the immune response is cell mediated, and, as part of that response, there is a release of toxic molecules such as reactive oxygen species (Butterworth 1984; Capron et al. 1987; Brophy and Pritchard 1992; Maizels et al. 1993). To protect against oxidant damage organisms possess antioxidant enzymes. In S. mansoni several antioxidant enzyme activities including Cu–Zn superoxide dismutase (SOD), glutathione peroxidase (GPX), cytochrome C peroxidase, glutathione S-transferase (GST), and glutathione reductase have been recognized (Mkoji et al. 1988a,b; Nare et al. 1990; Callahan et al. 1988; Brophy and Pritchard 1992; James 1994).

INTRODUCTION Schistosoma mansoni has a complex life cycle in which the free-living cercariae are able to penetrate the intact skin of humans in fresh water that contains them and transform into larvae, termed schistosomula. The schistosomula spend a few days in the skin, enter the venous circulation, migrate to the lungs (Days 5–7 postpenetration), and then move via the circulation to the hepato-portal circulation (>15 days). In the circulation of the liver, the worms develop into adults (Day 30), mate, and move to their final niche in the mesenteric circulation where they begin egg production (>35 days) (Clegg 1965). The host responds to infection with an aggressive immune response that limits challenge infection but has little or no effect on established adult worms (Smithers and Terry 1967; Capron et al. 1987). The schistosome 69

0014-4894/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.

70

MEI AND LOVERDE

However, catalase activity has not been detected in S. mansoni (Mkoji et al. 1988a). Two forms of Cu–Zn SOD, cytosolic SOD (CTSOD) and signal peptide-containing SOD (SPSOD) (Simurda et al. 1988; Cordeiro da Silva et al. 1992; Hong et al. 1992a,b; Mei et al. 1995a), GPX (Williams et al. 1991; Roche et al. 1994; Mei and LoVerde 1995b; Mei et al. 1996), and five isoforms of GST have been characterized (Balloul et al. 1987; O’Leary and Tracy 1988; Taylor et al. 1988; Henkle et al. 1990; Wright et al.1991; O’Leary et al. 1992; Walker et al. 1993). Enzyme activity for CT-SOD, GPX, and GST has been demonstrated to be developmentally regulated in that larval stages, which are the most susceptible to immune elimination, have the lowest specific activity whereas adult worms, which are the least susceptible to immune killing, have the highest specific activity (Mkoji et al. 1988a; Nare et al. 1990; Hong et al. 1992a; Mei et al. 1996; Roche et al. 1996). In this work, the relative abundance of antioxidant enzyme transcripts in S. mansoni were compared using a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR)

assay. Further, the distribution of selected antioxidant enzymes in adult schistosomes and 3-hr schistosomula was determined by immunofluorescence. MATERIALS AND METHODS Parasites. To maintain the life cycle, golden hamsters were exposed to cercariae of S. mansoni (NMRI strain) (Fletcher et al. 1981) produced in infected snails, Biomphalaria glabrata. Cercariae were transformed into schistosomula (Basch 1981) and the parasites were incubated in RPMI 1640 medium at 37°C for 3 hr. The portal veins and liver circulation of the infected hamsters were perfused to obtain 15-, 30-, and 42-day worms (Duvall and Dewitt 1967). Lung-stage worms were obtained by culturing chopped lung tissue from 7-day infected hamsters in RPMI 1640 medium for 3–5 hr and filtering the mixture through various sized meshed screens to collect the worms (Mangold and Knopf 1978). All parasites were used fresh or stored at −70°C. Quantitative RT-PCR to determine the mRNA levels of antioxidants (Murphy et al. 1993). RNA was extracted and quantitated from each developmental stage of S. mansoni according to previously published procedures (Chen et al. 1992). To optimize the amount of template for each RTPCR, a standard curve was established for each template. Five micrograms of total RNA from adult worms was reverse transcribed in a 50-ml mixture using random hexamers as primers. Varying dilutions of cDNA were used as templates to perform PCR using primer pairs of oligonucleotides representing CT-SOD, SP-SOD, GPX, GST, and a-tu-

TABLE I Oligonucleotides Used in Quantitative RT-PCR Gene

Oligonucleotides

CT-SOD-5 CT-SOD-3 CT-SOD-IN

ACG AGG ATG AAA GCT GTT TG-OH ACT GTT TCA CAG GGA GAA TAG G-OH TCT TGA GTA AAT TTG ACA ACA-OH

SP-SOD-5 SP-SOD-3 SP-SOD-IN

ATT AAA ATG ACA GTA TAT TCC-OH GTT CTA CTG CCT TCG TCT C-OH AGC CGG ATC AAA ATG TCT ACG-OH

GST-5a GST-3 GST-IN

ATG GCT GGC GAG CAT ATC-OH TCA GGA TAC TTG CCA GTT AG-OH TAT GAT CTG CGA ATC TCT G-OH

GPX-5 GPX-3 GPX-IN

ATG TCT TCA TCT CAC AAG TC-OH TTC GTC GAC TCA CTT CTT CTC CAA AAG C-OH CAC GAA CTT CTT AAT C-OH

a-Tubulin-5 a-Tubulin-3 a-Tubulin-IN

TGG AAC TTA TCG TCA ACT TTT CCA TCC-OH GAA GTG GAT ACG AGG ATA AGG TAC CAG-OH GGC GGT GGT ACT GGT TCT GGG TTC-OH

Note. 5,59 amplimer; 3,39 amplimer; IN, internal oligonucleotide. a For GST, the primers will anneal to isoforms GST-1 and GST-3.

S. mansoni

ANTIOXIDANT ENZYME EXPRESSION

bulin in a 50-ml mixture (Table 1). The primers were selected to produce PCR products around 500 bp in size for each gene. The PCR consisted of 23 cycles with each cycle consisting of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min. The PCR products were resolved on a 2% agarose gel and transferred onto a nitrocellulose membrane. Internal oligonucleotides end-labeled with g-32P were used as probes (Table 1). The blots were analyzed with a Betascope 601 (Betagen). For the semiquantitative RT-PCR 5 mg of total RNA from each developmental stage was reverse transcribed in a 50-ml mixture using random hexamers as primers. A 1:40 dilution of cDNA was used for PCR in a 50-ml mixture. Southern blot and Betascope analysis were performed as above. Immunofluorescence study of antioxidant enzymes in S. mansoni (Rekosh et al. 1988). Monoclonal antibodies for SP-SOD and GPX were available (Hong et al. 1992b; Mei et al. 1996). Rabbit anti-CT-SOD polyclonal serum was obtained by immunizing rabbits with 50 mg recombinant CT-SOD (Hong et al. 1992b) emulsified in Freund’s adjuvant, three times 3–4 weeks apart. The sera were affinitypurified over a CT-SOD-conjugated Sepharose column. The monoclonal antibodies and affinity-purified sera were used as primary antibody in immunolocalization experiments. Cryosections of adult worms and 3-hr schistosomula on glass slides (stored at −20°C) were soaked in PBS (pH 7.2) for 1–2 min and placed on top of wet 3MM filter paper in a sealed container. Anti-SP-SOD monoclonal antibody was diluted 1:10 to 1:100 in PBS. Anti-CT-SOD polyclonal antibody (affinity-purified) was diluted 1:10. Anti-GPX monoclonal antibodies from culture supernatants were diluted 1:2. Five hundred microliters of first antibody was added onto each slide and the slide was kept at room temperature for 45–60 min. The slide was washed in PBS. Fluoroscein isothiocyanate-conjugated secondary antibody was added onto the tissue section and incubated as above. The slide was washed in PBS buffer again. One drop of 70% glycerol was added to the section before a cover glass was applied. The slide was kept at 4°C in the dark. Immunofluorescence was observed using a Bio-Rad MRC-100 confocal microscope equipped with a krypton laser (Bio-Rad, Hercules, CA).

RESULTS A semiquantitative RT-PCR method (Murphy et al. 1993) was employed to determine the relative level of expression of various antioxidant enzyme transcripts. The method included RT-PCR and Southern hybridization with an internal oligonucleotide, which rendered high specificity and sensitivity to the detection system. a -Tubulin, a constitutively expressed structural protein in S. mansoni, was used as an internal control in the assay to standardize results (Duvaux-Miret et al. 1991). In Fig. 1, a-tu-

71

bulin levels kept a more or less steady level throughout all the stages assayed. CT-SOD, GPX, and GST all showed a similar pattern of regulated expression. The lowest levels were present in the egg, cercarial, and 3-hr schistosomula stages. The highest level existed in the 42-day adult worm. SP-SOD showed the same pattern of expression except that cercarial expression dropped slightly. A difference in the abundance of transcripts for each antioxidant enzyme at the same stage was demonstrated. Among the antioxidant enzymes, the level of expression from the highest to the lowest followed the order: GST, CTSOD, SP-SOD, and GPX. Interestingly, GST transcripts were 100-fold more abundant than GPX transcripts. Further, CT-SOD and SPSOD transcripts were 10 times more abundant than GPX transcripts. This order of expression level was nearly consistent in all the developmental stages examined. In order to define the distribution of the antioxidants in S. mansoni, immunolocalization of CT-SOD, SP-SOD, and GPX was performed on adult worms and 3-hr schistosomula using specific antibodies. CT-SOD is localized within the tegument and gut epithelium of the adult worm (Fig. 2). The subtegumental cytons appear to be responsible for the production of the protein for the tegument. SP-SOD is distributed within the tegument and gut epithelium (not shown) of the adult worm also. The spines on the ventral surface were distinctly stained as well. Parenchymal cells, especially cell and organellar membranes, were shown to contain SP-SOD (Fig. 2). GPX is associated with the tegument (Fig. 3) and gut epithelium (not shown). It seems that the gene is expressed in subtegumental cells and the GPX is transported to the tegument via cytoplasmic connections to the subtegumental cyton (Fig. 3). As expected GPX is localized in other cells such as muscle, where it likely performs a housekeeping function (Fig. 3). The controls which employed preimmunization sera or an irrelevant isotype-matched monoclonal antibody showed no fluorescence (Fig. 3C is representative of the results). For CT-SOD, SP-SOD, and GPX, the schis-

72

MEI AND LOVERDE

FIG. 1. RT-PCR determination of the mRNA levels of antioxidant enzymes. Equal amounts of cDNA from different developmental stages of the schistosome were used as templates to perform PCR. The PCR products were analyzed by Southern blot and results quantitated by Betascope. Each graph shows a plot of cpm versus developmental stage.

tosomula do not show strong staining. There is a lack of staining in the tegument (Fig. 4 is representative of the results). DISCUSSION Despite the fact that schistosomes engender a vigorous immune response, adult worms seem to be unaffected by this response and immunity is directed at the larval stages of a challenge infection (Smithers and Terry 1969; Capron et al. 1987). This has led to studies on mechanisms of immune evasion (Damian 1989; Pearce and Sher 1987; Maizels et al. 1993). Because a major mechanism of killing larval parasites occurs via cell-mediated processes and, depending on the experimental model, involves the release of toxic molecules by macrophages and/or eosinophils (Butterworth 1984; Capron et al. 1992; James 1992 for reviews), a hypothesis exists that states that parasite antioxidant enzymes play a role in protecting against the effects of resulting reactive oxygen species and thus represent one mechanism that allows adult worms

to persist (Callahan et al. 1988; Brophy and Pritchard 1992; Hong et al. 1992a; Mei et al. 1996, Roche et al. 1996). In both the RT-PCR and the immunofluorescence experiment, the adult worm, which is the least susceptible to host immune attack, showed the highest level of CT-SOD, SP-SOD, GPX, and GST antioxidant enzymes, while the most susceptible larval stages showed the lowest level of expression. In previous studies we (Hong et al. 1992a; Mei et al. 1996) and others (Roche et al. 1996) have shown that GPX and CT-SOD enzyme activity is developmentally regulated, with the highest level of activity occurring in the adult stage. Taken together these data support the notion that antioxidant enzymes play an important role in counteracting the immune attack from the host and thus protect the adult parasite. For antioxidant enzymes to play a crucial role in the survival of adult worms in the host, they need to be located at the host–parasite interface where the host cellular responses, including de-

S. mansoni

ANTIOXIDANT ENZYME EXPRESSION

73

FIG. 2. Localization by immunofluorescence of superoxide dismutases in adult worm cryosections. (A and B) Cross section of adult male reacted with rabbit anti-CT-SOD affinity-purified antibody diluted 1:10. The tegument (arrows) and intestinal epithelia (asterisk) exhibit intense fluorescence. (C and D) Cross section of adult male reacted with anti-SP-SOD monoclonal antibody diluted 1:100. The tegument (arrow) and spines (open arrow) exhibit fluorescence. In addition, cell and organellar membranes of parenchymal cells (boxed area) exhibit intense fluorescence. M, muscle layer.

granulation, respiratory burst, or release of nitric oxide, occur. The enrichment of antioxidant enzymes in the tegument as demonstrated in the present study may be an adaptive response of the schistosomes to protect themselves against the host cellular response. The reason that antioxidant enzymes are also concentrated in the gut epithelium may be because the intestinal lumen is filled with red and white blood cells, which may create an environment for the release or production of reactive oxygen species. Our previous studies suggested that SOD and GPX were associated with the tegument and the intestinal epithelium by demonstrating that the highest specific activity of each antioxidant enzyme in adult worms was in an NP-40 extract

(Hong et al. 1992a; Mei et al. 1996). However, our results are not in agreement with recent studies (Roche et al. 1996) showing that antibodies to a C-terminal peptide of GPX localized only to the vitelline cells of the female and only weakly to sections of the male worm. Perhaps the difference is due to the use of antibodies made to the entire molecule (this study) versus those made to a peptide (Roche et al. 1996). Interestingly, SP-SOD is also associated with cellular and organellar membranes as suggested by previous studies (Hong et al. 1993). However, why schistosomes have two forms of SOD (SP-SOD and CT-SOD) and why SP-SOD is associated with the spines and membranes are questions that remain unanswered.

74

MEI AND LOVERDE

FIG. 3. Localization by immunofluorescence of glutathione peroxidase in adult worm cryosections. (A) Cross section of adult male reacted with anti-GPX monoclonal antibody (culture supernatant). Inset shows subtegumental cyton with cytoplasmic connection (arrows) to tegument (asterisk). (B) As in A, except an oblique section of adult male through muscle (M) and tegument (asterisk). (C) Cross section of male worm reacted with preimmunization rabbit sera diluted 1:100. Similar results were obtained with nonschistosome monoclonal antibodies of the same isotype as anti-SP-SOD and anti-GPX monoclonal antibodies. Tegument (asterisk), muscle layer (M), subtegumental cyton and cytoplasmic connection to the tegument (arrows).

S. mansoni

ANTIOXIDANT ENZYME EXPRESSION

FIG. 4. Localization by immunofluorescence of glutathione peroxidase in 3-hr schistosomula. (A) Cross section of schistosomula reacted with anti-GPX monoclonal antibody. (B) Cross section of schistosomula reacted with irrelevant monoclonal antibody, isotype matched with the anti-GPX monoclonal antibody. Tegument (arrow); muscle layer (M).

75

76

MEI AND LOVERDE

The localization of GST in S. mansoni has been reported (Taylor et al. 1988; Holy et al. 1989; Porchet et al. 1994). GST isoenzymes 1, 2, and 3 are restricted to a subpopulation of parenchymal cells in males and females and the subectocytoplasmic core tissue of the dorsal tubercles of the male worm (Holy et al. 1989; Porchet et al. 1994). In addition GST immunoreactivity was identified in immature germinal cells of males and females and the ootype of the female worm (Porchet et al. 1994). Interestingly, GST does not localize to the tegument or the digestive tract of the adult parasite. This is in contrast to CT-SOD, SP-SOD, and GPX. However, this is consistent with data showing that GPX, and not GST, is the major antioxidant enzyme in adult worm NP-40 extract that acts to neutralize lipid hydroperoxide (Mei et al. 1996). The most obvious effect on the schistosome of free radicals is on lipid membranes, causing the release of lipid or phospholipid hydroperoxides. The lipid hydroperoxides will decompose further to secondary products such as toxic carbonyl species. As schistosomes do not contain catalase activity, the first line of enzymatic cellular defense against lipid peroxidation involves SOD and GPX, both of which are associated with the tegument. They act to suppress the initial free-radical chain reaction. The second line of defense against lipid peroxidation involves the detoxification of lipid hydroperoxides and reactive carbonyls by GST. It may well be that in the chain of events for detoxification of radicals, GST, which is primarily located in parencymal cells that connect to the dorsal tubercles by cytoplasmic processes (Holy et al. 1989), is not needed at the host–parasite interface and thus is not located there. Because the antioxidants SOD and GPX are indispensable parts of an integral protective system for the schistosomes, it is not surprising that they colocalize to the tegument and gut epithelium and demonstrate an almost parallel level of expression throughout development. For example, SOD and GPX have been shown to have a mutual protective effect (Hodgson and Fridovich 1975; Blum and Fridovich 1985). The distribution of antioxidant enzymes in

the outer covering (the tegument) and their apparent role in parasite survival suggest that they should be evaluated as experimental vaccine candidates, especially since they would be expected to target the adult parasite. In this regard GST, although not surface associated, has been identified as a vaccine candidate against the larval stages and adult female egg production (Balloul et al. 1987; Xu et al. 1991). ACKNOWLEDGMENTS We thank Dr. R. Summers, Head of the Imaging Core Facility, for assistance with Confocal Microscopy. This work was supported by NIAID Grant AI18867.

REFERENCES Balloul, J. M., Sondermeyer, P., Dreyer, D., Capron, M., Gryzch, J. M., Pierce, R. J., Carvallo, D., Lecocq, J. P., and Capron, A. 1987. Molecular cloning of a protective antigen against schistosomiasis. Nature, 326, 149–153. Basch, P. F. 1981. Cultivation of Schistosoma mansoni in vitro. I. Establishment of cultures from cercariae and development until pairing. Journal of Parasitology 67, 179– 185. Blum, J., and Fridovich, I. 1985. Inactivation of glutathione peroxidase by superoxide dismutase. Archives of Biochemistry and Biophysics 240, 500–508. Brophy, P. M., and Pritchard, D. I. 1992. Immunity to helminths: Ready to tip the biochemical balance? Parasitology Today 8, 419–422. Butterworth, A. E. 1984. Cell-mediated damage to helminths. Advances in Parasitology 23, 143–235. Callahan, H. L., Crouch, R. K., and James, E. R. 1988. Helminth anti-oxidant enzymes: A protective mechanism against host oxidants? Parasitology Today 4, 218–225. Capron, A., Dessaint, J. P., Capron, M., Ouma, J. H., and Butterworth, A. E. 1987. Immunity to schistosomes: Progress towards vaccine. Science, 238, 1065–1072. Capron, A., Dessaint, J. P., Capron, M., and Pierce, R. J. 1992. Schistosomiasis: From effector and regulation mechanisms in rodents to vaccine strategies in humans. Immunological Investigations 21, 409–422. Chen, L., Rekosh, D. M., and LoVerde, P. T. 1992. Schistosoma mansoni p48 eggshell protein gene: Characterization, developmentally regulated expression and comparison to the p14 eggshell protein gene. Molecular and Biochemical Parasitology 52, 39–52. Clegg, J. A. 1965. In vitro cultivation of Schistosoma mansoni. Experimental Parasitology 16, 133–147. Cordeiro da Silva, A., LePresle, T., Capron, A., and Pierce, R. J. 1992. Molecular cloning of a 16-kilodalton Cu/Zn superoxide dismutase from Schistosoma mansoni. Molecular and Biochemical Parasitology 52, 275–278. Damian, R. T. 1989. Molecular mimicry: Parasite evasion

S. mansoni

ANTIOXIDANT ENZYME EXPRESSION

and host defense. Current Topics in Microbiology and Immunology 145, 101–115. Duvall, R. H., and W. B. DeWitt. 1967. An improved perfusion technique for recovering adult schistosomes from laboratory animals. American Journal of Tropical Medicine and Hygiene 16, 483–486. Duvaux-Miret, O., Baratte, B., Dissous, C., and Capron, A. 1991. Molecular cloning and sequencing of the a-tubulin gene from Schistosoma mansoni. Molecular and Biochemical Parasitology 49, 337–340. Fletcher, M., LoVerde, P. T., and Woodruff, D. S. 1981. Genetic variation in Schistosoma mansoni: Enzyme polymorphisms in populations from Africa, Southwest Asia, South America, and the West Indies. American Journal of Tropical Medicine and Hygiene 30, 406–421. Henkle, K. J., Davern, K. M., Wright, M. D., Ramos, A. J., and Mitchell, G. F. 1990. Comparison of the cloned genes of the 26- and 28-kilodalton glutathione Stransferases of Schistosoma japonicum and Schistosoma mansoni. Molecular and Biochemical Parasitology 40, 23–34. Hodgson, E. K., and Fridovich, I. 1975. The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: Inactivation of the enzyme. Biochemistry 14, 5294–5299. Holy, J. M., O’Leary, K. A., Oaks, J. A., and Tracy, J. 1989. Immunocytochemical localization of the major glutathione S-transferases in adult Schistosoma mansoni. Journal of Parasitology 75, 181–190. Hong, Z., Kosman, D., Thakur, A., Rekosh, D., and LoVerde, P. T. 1992a. Identification and purification of a second form of Cu/Zn superoxide dismutase from Schistosoma mansoni. Infection and Immunity 60, 3641–3651. Hong, Z., LoVerde, P. T., Hammarskjold, M-L., and Rekosh, D. M. 1992b. Schistosoma mansoni: Cloning of a complementary DNA encoding a cytosolic superoxide dismutase and high-yield expression of the enymatically active gene product in Escherichia coli. Experimental Parasitology 75, 308–322. Hong, Z., LoVerde, P. T., Thakur, A., Hammarskjold, M-L., and Rekosh, D. 1993. Schistosoma mansoni: A Cu/Zn superoxide dismutase becomes glycosylated when expressed in mammalian cells and localizes to subtegumental region in adult schistosomes. Experimental Parasitology 76, 101–114. James, E. R. 1994. Superoxide dismutase. Parasitology Today 10, 481–484. James, S. L. 1992. Experimental models of immunization against schistosomes: Lessons for vaccine development. Immunological Investigations 21, 477–493. Maizels, R. M., Bundy, D. A., Selkirk, M. E., Smith, D. F., and Anderson, R. M. 1993. Immunological modulation and evasion by helminth parasites in human populations. Nature 365, 797–805. Mangold, B. L., and Knopf, P. M. 1978. The effect of assay conditions on the recovery of schistosomula from the

77

lungs of normal and resistant rats infected with Schistosoma mansoni. Journal of Parasitology 64, 813–821. Mei, H., Hirai, H., Tanaka, M., Hong, Z., Rekosh, D., and LoVerde, P. T. 1995. Schistosoma mansoni: Cloning and characterization of a gene encoding cytosolic Cu/Zn superoxide dismutase. Experimental Parasitology 80, 250– 259. Mei, H., and LoVerde, P. T. 1995b. Schistosoma mansoni: Cloning the gene encoding glutathione peroxidase. Experimental Parasitology 80, 319–322. Mei, H., Thakur, A., Schwartz, J., and LoVerde, P. T. 1996. Expression and characterization of glutathione peroxidase activity in the human blood fluke, Schistosoma mansoni. Infection and Immunity 64, 4299–4306. Mkoji, G. M., Smith, J. M., and Prichard, R. K. 1988a. Antioxidant systems in Schistosoma mansoni: Correlation between susceptibility to oxidant killing and the levels of scavengers of hydrogen peroxide and oxygen free radicals. International Journal for Parasitology 18, 661–666. Mkoji, G. M., Smith, J. M., and Prichard, R. K. 1988b. Antioxidant systems in Schistosoma mansoni: Evidence for their role in protection of the adult worms against oxidant killing. International Journal for Parasitology 18, 667– 673. Murphy, E., Hieny, S., Sher, A., and O’Garra, A. 1993. Detection of in vivo expression of interleukin-10 using a semi-quantitative polymerase chain reaction method in Schistosoma mansoni infected mice. Journal of Immunological Methods 162, 211–223. Nare, B., Smith, J. M., and Prichard, R. K. 1990. Schistosoma mansoni: Levels of antioxidants and resistance to oxidants increase during development. Experimental Parasitology 70, 389–397. O’Leary, K. A., Hathaway, K. M., and Tracy, J. W. 1988. Purification of three cytosolic glutathione S-transferases from adult Schistosoma mansoni. Archives of Biochemistry and Biophysics 264, 1–12. O’Leary, K. A., Hathaway, K. M., and Tracy, J. W. 1992. Schistosoma mansoni: Single-step purification and characterization of glutathione S-transferase isoenzyme 4. Experimental Parasitology 75, 47–55. Pearce, E. J., and Sher, A. 1987. Mechanisms of immune evasion in schistosomiasis. Contributions to Microbiology and Immunology 8, 219–232. Porchet, E., McNair, A., Caron, A., Kusnierz, J. P., Zemzoumi, K., and Capron, A. 1994. Tissue expression of the Schistosoma mansoni 28 kDa glutathione Stransferase. Parasitology 109, 565–572. Rekosh, D., Nygren, A., Flodby, P., Hammarskjold, M. L., and Wigzell, H. 1988. Coexpression of human immunodeficiency virus envelope proteins and tat from a single simina virus 40 late replacement vector. Proceedings of the National Academy of Sciences (USA) 85, 334–338. Roche, C., Williams, D. L., Khalife, J., LePresle, T., Capron, A., and Pierce, R. J. 1994. Cloning and characterization of the gene encoding Schistosoma mansoni glutathione peroxidase. Gene 138, 149–152.

78

MEI AND LOVERDE

Roche, C., J. L. Liu, T. LePresle, A. Capron, and R. J. Pierce. 1996. Tissue localization and stage-specific expression of the phospholipid hydroperoxide glutathione peroxidase of Schistosoma mansoni. Molecular and Biochemical Parasitology 75: 187–195. Simurda, M. C., van Keulen, H., Rekosh, D. M., and LoVerde, P. T. 1988. Schistosoma mansoni: Identification and analysis of an mRNA and a gene encoding superoxide dismutase (Cu/Zn). Experimental Parasitology 67, 73–84. Smithers, S. R., and Terry, R. J. 1969. Immunity in schistosomiasis. Annals of the New York Academy of Sciences 160, 826–840. Taylor, J. B., Vidal, A., Torpier, G., Meyer, D. J., Roitsch, C., Balloul, J. M., Southan, C., Sondermeyer, P., Pemble, S., Lecocq. J. P., Capron, A., and Ketter, B. 1988. The glutathione transferase activity and tissue distribution of a cloned Mr 28 K protective antigen of Schistosoma mansoni. EMBO Journal 7, 465–472. Walker, J., Crowley, P., Moreman, A. D., and Barrett, J.

1993. Biochemical properties of cloned glutathione Stransferases from Schistosoma mansoni and Schistosoma japonicum. Molecular and Biochemical Parasitology 61, 255–264. Williams, D. L., Pierce, R. J., Cookson, E., and Capron, A. 1991. Molecular cloning sequencing of glutathione peroxidase from Schistosoma mansoni. Molecular and Biochemical Parasitology 52, 127–130. Wright, M. D., Harrison, R. A., Melder, A. M., Newport, G. R., and Mitchell, G. F. 1991. Another 26-kilodalton glutathione S-transferase of Schistosoma mansoni. Molecular and Biochemical Parasitology 49, 177–180. Xu, C. B., Verwaerde, C., Gryzch, J. M., Fontaine, J., and Capron, A. 1991. A monoclonal antibody blocking the Schistosoma mansoni 28 kDa glutathione S-transferase activity reduces female worm fecundity and egg viability. European Journal of Immunology 21, 1801–1807. Received 25 September 1996; accepted with revision 6 January 1997