Proc. Nati. Acad. Sci. USA Vol. 89, pp. 778-781, January 1992 Immunology

Immunosuppression in the definitive and intermediate hosts of the human parasite Schistosoma mansoni by release of immunoactive neuropeptides (neuroimmunomodulation/conformational changes/neutral endopeptidase/parasite neuropeptides/host-parasite interactions)

ODILE DUVAUX-MIRET*, GEORGE B. STEFANOt, ERIC M. SMITHS, COLETTE Dissous*,

AND ANDRE

CAPRON*

*Centre d'Immunologie et de Biologie Parasitaire, Institut National de la Sante et de la Recherche M6dicale Unit6 167, Centre National de la Recherche Scientifique 624, Institut Pasteur, 59019 Lille Cedex, France; tMultidisciplinary Center for the Study of Aging, State University of New York, Old Westbury, NY 11568; and tDepartments of Microbiology and Psychiatry and Behavioral Sciences, University of Texas Medical Branch, Galveston, TX 77550

Communicated by Berta Scharrer, October 14, 1991

ABSTRACT Evidence supporting the concept that the parasitic trematode Schistosoma mansoni may escape immune reactions from its vertebrate (man) or invertebrate (the freshwater snail Biomphalaria glabrata) hosts by using signal molecules it has in common with these hosts was obtained by the following experiments. The presence of immunoactive proopiomelanocortin (POMC)-derived peptides [corticotropin (ACTH), 13-endorphin] in, and their release from, S. mansoni was demonstrated. Coincubation of adult worms with human polymorphonuclear leukocytes or B. glabrata immunocytes led to the appearance of a-melanotropin (MSH) in the medium. The conclusion that this a-MSH resulted from conversion ofthe parasite ACTH by neutral endopeptidase 24.11 (NEP) present on these cells was supported by the fact that the a-MSH level in the medium was markedly reduced by addition of the specific NEP inhibitor phosphoramidon. This interpretation is substantiated by the fact that no conversion was observed in comparable tests with human monocytes, which exhibit no NEP activity. a-MSH has the capacity to inactivate formerly active immunocytes not only from the definitive host (man, hamster) but also from the intermediate host (B. glabrata), as determined by microscopic computer-assisted examination of conformational changes. POMC-derived peptides have been detected in B. glabrata hemolymph 2, 10, and 24 days after infection by S. mansoni miracidia. Immunocytes from infected snails were found to be inactivated, and this inactivation was prevented by antibodies directed against ACTH and a-MSH. The immunoactive P-endorphin released from S. mansoni does not appear to be subject to enzymatic conversion. Since it is active at lower concentrations, it may be used for distant signalng.

nuclear cells (PMN) (2, 3) as well as the migration of invertebrate immunocytes (4). Recently, it also was found that ACTH can exert the same immunosuppressive cellular effects through conversion into a-MSH by means of neutral endopeptidase 24.11 (NEP or "enkephalinase"), since the enzyme is present on the surface of both mammalian PMN and invertebrate immunocytes (5, 6). The aim of the present study was to test our hypothesis that S. mansoni may take advantage of these immunoactive phylogenetically conserved molecules to interfere with the normal immune response of the host. More precisely, the role of the parasite POMC-derived peptides in immunosuppression in the definitive host (man) and in the intermediate host (the freshwater snail Biomphalaria glabrata) has been examined.

MATERIALS AND METHODS Parasites and Hosts. A Puerto Rican strain of S. mansoni was maintained in the laboratory in the hamster Mesocricetus auratus and in the intermediate host B. glabrata. Adult worms were collected by portal perfusion of 40-day-infected hamsters and were washed thoroughly with minimal essential medium (Eagle's), containing penicillin at 100 units/ml and streptomycin at 50 ,ug/ml (MEM-PS) at 37°C. For RIAs, the worms were sedimented, drained, and lyophilized. Incubations of worms were carried out at 37°C in MEM-PS in a 5% CO2 atmosphere for 2 hr at a concentration of 50 pairs of worms (male plus female) per ml in culture plates. Coincubation with human PMN was performed under the same conditions in the presence of 3 x 106 human PMN per ml, prepared from healthy donors (7), in Linbro microwell tissue culture plates (Flow Laboratories). The enzyme inhibitors bestatin and phosphoramidon were added as indicated at a final concentration of 0.1 mM. B. glabrata hemolymph and hemocytes were withdrawn through the snail shell with a microsyringe (26 gauge needle /2 inch long) in the pericardium from either healthy or infected specimens. Hemocytes were pelleted, if necessary, by 500 x g centrifugation and resuspended in phosphatebuffered saline. Hemolymph supernatant, after a 15-min centrifugation at 10,000 x g, was collected and pooled from 10-15 age-matched snails. Hamster PMN were prepared as described elsewhere (7). Structural Analysis of Immunocytes. Changes in conformation of hamster and human PMN or B. glabrata hemocytes based on measurements of cell area and cell perimeter were determined by the use of American Innovision (San Diego)

Thus far the scarcity of information on the mechanisms enabling the survival of Schistosoma mansoni in its definitive and intermediate hosts has stood in the way of the eradication of schistosomiasis, a major human parasitic disease. In this report we provide evidence indicating that the interaction between parasite and host involves the dispatch by the parasite of the same signal molecules as those found in the host into the immediate vicinity of the infestation, thus interfering with the host immune function. We have recently demonstrated the presence of proopiomelanocortin (POMC)-derived peptides [immunoactive corticotropin (adrenocorticotropic hormone, ACTH), a-melanotropin (melanocyte-stimulating hormone, a-MSH), and ,B-endorphin] in all stages of the life cycle of S. mansoni (1). This finding is highly significant because a-MSH has been shown to counteract the inflammatory action of interleukin 1 and tumor necrosis factor by inhibiting human polymorpho-

Abbreviations: POMC, proopiomelanocortin; PMN, polymorphonuclear leukocytes; ACTH, corticotropin (adrenocorticotropic hormone); MSH, melanotropin (melanocyte-stimulating hormone); NEP, neutral endopeptidase 24.11.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

778

Immunology: Duvaux-Miret et al. form-factor calculations as described in detail elsewhere (8). Briefly, the form factor is equal to Ac/AT = [LT/Lc]2, where AT is the area of a circle with the same perimeter as that of the given cell and LT is the perimeter of a circle with the same area as that of the given cell; Ac and Lc represent the actual area and perimeter of the cell. The computer-assisted image and video analysis system were the same as previously described (9). The immunocytes were prepared and treated as noted in detail elsewhere (5, 9). In the cardiac aspiration of immunocytes from infected molluscs the cells were mixed on the slide with saline and the antibodies (anti-ACTH and anti-MSH) were diluted to 1:100. Incubations under the in vitro conditions were for 40 min, based on previous experiments (6, 10). Statistical analysis was carried out by means of Student's t test. The mean value from 10 separate trials, each trial representing a single cell measurement, was averaged with three additional mean values similarly obtained to provide the value for each point on the various graphs. RIA. All samples were lyophilized to dryness prior to RIA and examined as previously noted in detail (11). Briefly, ACTH, MSH, and f-endorphin levels were determined by use of commercially available kits (Incstar, Stillwater, MN). The sensitivity of the ACTH and MSH assay was approximately 15 pg/ml, and that for P3-endorphin was 5 pg/ml. Using peptide standards, we found less than 0.1% crossreactivity for the inappropriate peptide in the assays. Lyophilized hemolymph from infected snails or incubation medium of worms was assayed according to the kits' instructions. All three assays are based on the experimental sample binding to specific antibodies adsorbed to a test tube, thereby inhibiting the binding of 1251-labeled peptide standards. Each assay needed 50-100 jA of sample. Bound radiolabeled tracer was quantitated with a y counter. Values in pg/ml were extrapolated from the standard curve, based on the percent inhibition of binding of radiolabeled tracer. Materials. Synthetic peptides (ACTH, a-MSH, 13-MSH, ,3-endorphin) were purchased from Peninsula Laboratories. Bestatin and phosphoramidon were purchased from Sigma. Antibodies used to reverse hemocyte activation were obtained from Incstar.

RESULTS Definitive Host. ACTH-like and P-endorphin-like substances were detected in the incubation medium of adult worms incubated in MEM-PS and assayed for POMCderived peptides (Table 1, Experiment A). Interestingly, their level in the medium was relatively higher than that found in the parasites, suggesting that the amount found in the medium Table 1. RIA of ACTH, a-MSH, and 3-endorphin in S. mansoni adult worms and incubation medium a-MSH ACTH 13-Endorphin Sample Experiment A, peptide conc. in pmol/g wet wt 0.2% ± 0.07 1.840 ± 0.11 0.222 ± 0.12 Parasites ND 0.740 ± 0.13 Medium 1.265 ± 0.32 Experiment B, peptide conc. in fmol/ml 2.1 ± 0.9 29.7 ± 0.0 PMN ND 2.0 ± 0.7 5.0 ± 1.3 22.0 ± 0.0 PMN + phos. 1.3 ± 0.1 4.1 ± 0.1 36.0 ± 5.1 PMN + bestatin Experiment A: Adult worms (1-1.5 g wet weight) were incubated in MEM-PS at 370C for 2 hr in a 5% CO2 atmosphere (50 couples per ml). Peptides were assayed either in the worms prior to incubation or in the incubation medium after incubation. Experiment B: Fifty pairs ofadult worms were incubated in the presence of 3 x 106 human PMN in 1 ml of MEM-PS with no additions or with phosphoramidon (phos.) or bestatin at 0.1 mM. Incubation medium was then assayed for the considered peptides after lyophilization. All values are expressed as mean ± SEM. ND, not detectable.

Proc. Natl. Acad. Sci. USA 89 (1992)

779

resulted from de novo synthesis and release from the adult worm. Furthermore, under these conditions a-MSH was detected only within the adult worms, not in the incubation medium. This may be due to the absence of release of this compound. Since Smith et al. (6) demonstrated that NEP may be involved in the conversion of ACTH to an a-MSH-like molecule, the parasite ACTH-like molecule was tested for its potential to be converted to the antiinflammatory peptide a-MSH by coincubating adult worms (for 2 hr) with human PMN, which contain NEP on their surface (5) (Table 1, Experiment B). This time was chosen on the basis of results of the study by Smith et al. (6). Under these conditions, the ACTH-like material was no longer present and immunoactive a-MSH was detected in the incubation medium. The role of NEP in this conversion was ascertained by use of the specific NEP inhibitor phosphoramidon, which significantly reduced the amount of immunoactive a-MSH in the medium. By contrast, bestatin, an aminopeptidase inhibitor that does not affect NEP activity, did not modify the levels of ACTH in the medium. These results strongly suggest that human PMN have the ability to convert the parasite immunoactive ACTH to immunoactive a-MSH. This was further substantiated by performing the same incubation with B. glabrata immunocytes, which also contain NEP on their surface. This experiment gave basically the same results (not shown). On the contrary, when adult worms were coincubated with human monocytes, which are devoid of NEP, no a-MSH was found (data not shown). The amount of the parasite immunoactive ACTH was increased in the presence of phosphoramidon, since the worm continued to release it and it was not degraded. Bestatin also increased the amount of ACTH-like material, an observation that remains unexplained; it may be indicative of a multienzymatic pathway (6). It should also be noted that immunoactive /3-endorphin levels were not affected by the peptidase inhibitors used during immunocyte incubations, indicating that this peptide appears to be resistant to proteolytic attack by membrane-bound peptidases. Cells from both mammalian hosts (human and M. auratus) as well as from the intermediate mollusc host B. glabrata were tested for their ability to respond to a- and (3-MSH (Fig. 1). The degree of activation of the immunocytes was quantified by calculation of the form-factor, which is inversely related to their perimeter. The lower the form-factor, the more ameboid the cell shape (9). In this assay, the cells responded to a- or B-MSH by an increase in form-factor. The sensitivity of the vertebrate PMN was higher than that of the mollusc immunocytes (effective concentration 10-9 and 10-7 M, respectively), as had already been described for human PMN and Mytilus edulis immunocytes (4, 6). Within an hour, the presence of ACTH in the immunocyte incubation medium did not affect the cells' activation, since conversion requires more than 1 hr, as noted elsewhere (6). Intermediate Host. For the determination of the activity of these peptides in the intermediate host, ACTH, a-MSH, and ,3-endorphin were assayed in the hemolymph of infected B. glabrata at different developmental stages of the sporocysts and cercaria (Table 2). All peptides were detected in infected snails, whereas none were found in noninfected controls. High levels of a-MSH were detected approximately 30 hr after infection (day 2), corresponding to the formation of the mother sporocyst, which escapes encapsulation (12). At day 10, when all daughter sporocysts have completed their migration into the digestive gland (12), all three peptides were present in the hemolymph. Finally, at day 24, when mature cercariae are liberated into the water, only immunoactive ACTH could be detected in the hemolymph. Peptide concentrations in hemolymph ranged between 10-12 and 10-10 M, an order of magnitude lower than that effective in immunosuppression. We believe this reflects a higher concentra-

780

Immunology: Duvaux-Miret et al.

Proc. Natl. Acad. Sci. USA 89 (1992)

A 0.800

\P)t A '>0.01 nosuppression of the host(s) has been demonstrated also in the case of protozoan and to some extent of helminthic 0.600 l A infections (17). This can be achieved by evading the host immune response by molecular mimicry, phyletic conver* or other mechanisms (18, 19). Some parasite-derived gence, 0.400 A ** molecules have been shown to interfere directly with the host metabolic and/or immune activities for protozoans (20), cestodes (21), nematodes (22), and trematodes (23). 0.200 A-A aMSH

0.000

*-* Control active cells

-log10o [peptide conc. (M)] FIG. 1. Immunosuppressive effects of a- and 3-MSH on spon-

taneously asctive human PMN (A), hamster PMN (B), and B. glabrata

immunocytes Hu). immunocytes were incuoatea witn tne peptiues

The parasite ACTH can be converted to a-MSH by NEP from human, hamster, and invertebrate immunocytes (5, 6). This is highly significant, since a-MSH inhibits adherence and locomotory activity of PMN, monocytes, and invertebrate immunocytes (3, 4). Since the parasite ACTH can be converted to a-MSH, we surmise that it is also involved in the limitation of PMN and peripheral blood mononuclear cell adherence to adult worms, especially in their immediate

for 1 hr. Controls consisted of immunocytes treated with physiological saline. Variation for all mean values was not greater than 6%. Statistical analysis by Student's t test notes the point of significant difference between round and ameboid cells (SD = +0.12) as the result of drug.

0.900

ts

0

tion at the immediate cellular level, compatible with an inhibitory immune action in the vicinity of the sporocysts. Interestingly, the experiment reported in Fig. 2 indirectly demonstrates the participation of these POMC-derived factors in the inhibition of immunocytes. Immunocytes were freshly withdrawn from 14-day infected snails and immediately incubated with antibodies directed against ACTH or a-MSH. All cells were inactivated (rounded) at the beginning of incubation (form factor 0.800). Both antibodies reversed hemocyte inhibition to form factor 0.500 typical of activated cells. A plausible explanation is that immunoresponsive hemocytes are inhibited by the addition of both peptides, ACTH being converted into a-MSH by NEP which is present on the surface of B. glabrata immunocytes. In this experiment, only 37% ± 5% of the cells responded to antibodies, suggesting that other inhibitory factors might be operating.

O

0.800

0 U-

0~

AK A

0.700

* A

v

0.600 Control A * AB ACTH A-A AB-aMSH A-A AB-ACTH & -oMSH 0-C

*-

0.500

0.400 0

10

20

30

40

50

60

70

Time, min FIG. 2. Reversal of inhibition of immunocytes obtained from S. infected B. glabrata by antibodies (AB) directed against ACTH or a-MSH. The antibodies were diluted to a 1:100 with snail saline and added to the in vitro incubation medium, consisting of snail hemolymph. Immunocytes in this medium without the antibodies continued to be rounded for at least 1 hr. Variation for all mean values was not greater than 6%. mansoni

Immunology: Duvaux-Miret et al. vicinity. These peptides may be further implicated in the absence of cytolytic action by cytotoxic T lymphocytes, although adult worms acquire determinants of the host major histocompatibility complex on their tegument (24). We have demonstrated previously that the parasite P3-endorphin is very similar to human P-endorphin-(1-31) (1, 25). p8-Endorphin stimulates chemotaxis of monocytes and PMN (4, 9), but it inhibits the production of a T-cell chemotactic factor (26), augments suppressor T-cell activity (27), and enhances natural cytotoxicity (28). Concerning humoral responses, it inhibits antibody production (11) and B-lymphocyte conversion into immunoglobulin-secreting cells (29). Clearly, parasite 18-endorphin might be partly implicated in the up-regulation of the immune response toward schistosomula, and also in some immunosuppressive effects favoring parasite adaptation. 83-Endorphin has been shown to resist degradation better than ACTH or a-MSH and to be more potent, since it has the potential to initiate activity at 10-10 M (30). These characteristics lead us to surmise that ,8-endorphin may have an action that occurs at some distance from the site of parasitic infestation. Concerning the intermediate host, the freshwater snail B. glabrata, the present work demonstrates that a population of its hemocytes responds to ACTH and MSH as do other invertebrate immunocytes (4, 6). Moreover, a-MSH, and to a lesser extent ACTH and 13-endorphin, could be detected in the hemolymph of snails infected by S. mansoni. Whether these peptides are of parasitic or of molluscan origin remains to be determined. However, the consistent failure to detect them in hemolymph of control animals suggests that, at least in part, the parasite does contribute either directly or indirectly to the detectable levels found in infected animals. Penetration of noncompatible snails by S. mansoni miracidia results in rapid encapsulation and killing of the larvae. On the contrary, in a compatible snail, encapsulation is inhibited within the first hours following penetration of the miracidium and depends mostly on the inhibition of the capacity of the snail immunocytes to emit pseudopodia and adhere to the sporocyst surface (19), which could potentially be due to parasite a-MSH. Interestingly, Lie et al. (31) have demonstrated that circulating hemocytes from recently infected snails retain their capacity for phagocytosis or response to certain stimuli in the infected state. We feel that the report of Lie et al. (31) describes the physiological state of these cells in the entire snail rather than in the immediate vicinity of the parasite. Furthermore, this is in accordance with the low concentrations of circulating peptides that we observed. Another possible mechanism in this interaction may consist of peptide liberation by the snail immunocytes in the vicinity of the sporocyst. Supporting this hypothesis is the presence of ACTH and /8-endorphin in immunocytes of the closely related mollusc Planorbarius corneus (32). Several reports have described an increase of the number of circulating hemocytes in mollusc infestations by trematodes (33, 34). Inhibition of immunocyte locomotion by a-MSH has been shown to result in an increase of circulating cells as a result of inhibition of margination (35). Our results constitute an example of molecular mimicry by which parasites use phylogenetically conserved molecules to interfere with the host response. The results of the present work demonstrate the ability of schistosomes to release ACTH and 3-endorphin which can (i) act directly on immune cells of both the definitive and the intermediate hosts and (ii) be converted into immunosuppressive substances by NEP, an enzyme present on the surface of the same cells. This phenomenon constitutes parasitic interference with the normal autoimmunoregulatory activity of the host. We express our gratitude to Ms. Lisa A. Mallozzi and Mr. Patrick

Proc. Natl. Acad. Sci. USA 89 (1992)

781

Cadet of the Old Westbury Neuroscience Institute for excellent technical assistance. We thank Blandine Baratte and Han Vorng for technical help and Dr. Jean-Yves Cesbron for scientific advice. We also express our gratitude to Dr. Berta Scharrer for thoughtful comments and discussions. We acknowledge the following grant support: Alcohol, Drug Abuse, and Mental Health Administration MARC 17138, National Science Foundation INT 8803664, National Institute on Drug Abuse 47392, and the Research Foundation/State University of New York (to G.B.S.). E.M.S. was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 41034-01, Office of Naval Research Grant N0014-89-J1%2, and the John Sealy Memorial Foundation. 1. Duvaux-Miret, O., Dissous, C., Gautron, J. P., Pattoux, E., Kordon, C. & Capron, A. (1990) New Biol. 2, 93-99. 2. Robertson, B., Dostal, K. & Daynes, R. A. (1988) J. Immunol. 140, 4300-4307. 3. Van Epps, D. E. & Mason, M. M. (1990) in Comparative Neuropeptide Pharmacology, eds. Florey, E. & Stefano, G. B. (Manchester Univ., Manchester, U.K.). 4. Stefano, G. B., Smith, D. E., Smith, E. M. & Hughes, T. K. (1992) in Molluscan Neurobiology, eds. Boer, H. & ter Maat, A. (Elsevier/ North Holland, Amsterdam), in press. 5. Shipp, M. A., Stefano, G. B., D'Adamio, L., Switzer, S. N., Howard, F. D., Sinisterra, J., Scharrer, B. & Reinherz, E. (1990) Nature (London) 347, 394-396. 6. Smith, E. M., Hughes, T. K., Jr., Hashemi, F. & Stefano, G. B. (1992) Proc. Natl. Acad. Sci. USA 89, 782-786. 7. Boyum, A. (1968) Scand. J. Clin. Lab. Invest. Suppl. 21, 31-50. 8. Schon, C., Torre-Bueno, J. & Stefano, G. B. (1991) Adv. Neuroimmunol. 3, in press. 9. Stefano, G. B., Cadet, P. & Scharrer, B. (1989) Proc. Natl. Acad. Sci. USA 86, 6307-6311. 10. Stefano, G. B., Shipp, M. A. & Scharrer, B. (1991) J. Neuroimmunol. 31, 97-103. 11. Woloski, B. M. R. N. J., Smith, E. M., Meyer, W. J., III, Fuller, G. M. & Blalock, E. J. (1985) Science 230, 1035-1037. 12. Capron, A., Deblock, S., Biguet, J., Clay, A., Adenis, L. & Vernes, A. (1965) Bull. Organ. Mond. Sante 32, 755-778. 13. Johnson, H. M., Torres, B. A., Smith, E. M., Dion, L. D. & Blalock, J. E. (1984) J. Immunol. 132, 246-251. 14. Koff, W. C. & Dunegan, M. A. (1985) J. Immunol. 135, 350-354. 15. Pearce, E. J., Caspar, P., Grzych, J. M., Lewis, F. A. & Sher, A. (1991) J. &p. Med. 173, 159-166. 16. Grzych, J. M., Pearce, E. J., Cheever, A., Caulada, Z. A., Caspar, P., Heiny, S., Lewis, F. & Sher, A. (1991) J. Immunol. 146, 1322-1327. 17. Dessaint, J. P. & Capron, A. (1992) in Immunology and Molecular Biology of Parasitic Infections, eds. Warren, K. S. & Agabian, N. (Blackwell, Cambridge, MA), 3rd Ed., in press. 18. Capron, A. & Dessaint, J. P. (1989) Immunol. Rev. 112, 27-48. 19. Bayne, C. J. & Yoshino, T. P. (1989) Am. Zool. 29, 399-407. 20. Ackerman, S. B. & Seed, J. R. (1976) Experientia 32, 645-650. 21. Phares, C. K. & Watts, D. J. (1988) J. Parasitol. 74, 896-898. 22. Faubert, G. M. & Tanner, C. E. (1974) Immunology 27, 501-505. 23. Mazingue, C., Walker, C., Domzig, W., Capron, A., DeWeck, A. & Stadler, B. M. (1987) Int. Arch. Allergy Appl. Immunol. 83, 12-18. 24. Sher, A., Hall, B. F. & Vadas, M. A. (1978) J. Exp. Med. 148, 46-52. 25. Duvaux-Miret, 0. & Capron, A. (1992) Ann. N.Y. Acad. Sci., in press. 26. Brown, S. L. & Van Epps, D. E. (1985) J. Immunol. 134, 33843390. 27. McCain, H. W., Lamster, I. B. & Bilotta, J. (1986) J. Immunopharmacol. 8, 443-446. 28. Froehlich, C. J. & Bankhurst, A. D. (1984) Life Sci. 35, 261-265. 29. Morgan, E. L., McClurg, M. C. & Janda, J. A. (1990) J. Neuroimmunol. 28, 209-217. 30. Stefano, G. B. (1989) Prog. Neurobiol. 33, 149-159. 31. Lie, K. J., Heyneman, D. & Jeong, K. H. (1976) J. Parasitol. 62, 608-615. 32. Ottaviani, E., Petraglia, F., Montagnani, G., Gossarizza, A., Monti, D. & Franceschi, C. (1990) Regul. Pept. 27, 1-9. 33. Abdul-Salam, J. M. & Michelson, E. H. (1980) J. Invert. Pathol. 35, 241-248. 34. Van der Knaap, W. P. W., Meuleman, E. A. & Sminia, T. (1987) Parasitol. Res. 73, 57-65. 35. Stefano, G. B., Zhao, X., Bailey, D., Metlay, M. & Leung, M. (1989) J. Neuroimmunol. 21, 67-74.