Eur. J. Imrnunol. 1990. 20: 221-227

Moshe Marikovsky, Miriam Parizade, Ruth Arnon and Zvi FishelsonA Department of Chemical Immunology, The Weizrnann Institute of Science, Rehovot

Complement regulation by S. mansoni

Complement regulation on the surface of cultured schistosomula and adult worms of Schistosoma mansoni* Cercaria and freshly prepared schistosomula of Schistosoma mansoni are highly sensitive to complement. However, early in their maturation, the schistosomula become resistant to complement killing. This conversion is preceded by a rapid and massive release of several acetabular proteases and of the glycocalyx coat. Thus, shedding of the glycocalyx which is a major immunogen and a strong activator of the alternative pathway of complement permits the parasite to escape immune damage. Mechanically transformed schistosomula, which were cultured in a defined synthetic medium and developed complement resistance, could be converted by proteolysis to complement sensitivity.Trypsin and pronase markedly increased the susceptibility of cultured schistosomula to complement. The trypsin-induced complement sensitivity persisted for at least 19 h without recovery of resistance. Similar treatment with trypsin produced complete killing of adult worms by complement in absence of antibodies. Efficient killing was obtained with normal human serum (NHS),with normal guinea pig serum (GpS), and with CCdepleted HS and C4-deficient GpS indicating that the killing was mediated by the cytolytic alternative pathway of complement. Larger quantities of C3b with intact a‘ chain could be demonstrated on trypsin-treated than on non-treated schistosomula. Antibodies which were raised in rabbits by immunization with the trypsin-released material bound to cultured (non-treated) schistosomula and t o adult worms, and induced their killing in GpS and C4-deficient GpS. These results suggest that following release of the glycocalyx, the transforming schistosomula of S. mansoni spontaneously express a complement regulatory protein(s). A similar regulator is postulated to be present on the surface of adult worms. Such regulatory molecules may serve as good targets for immunotherapy, since antibodies directed to them will inhibit their regulatory activity and thus potentiate in vivo the lytic action of complement.

1 Introduction Cercariae and freshly transformed schistosomula of Schistosoma mansoni are highly sensitive to the lytic action of C (reviewed in [l]). C activation in fresh normal sera is induced primarily by the glycocalyx which surrounds the cercariae and which is a strong activator of the alternative pathway [l-31. Sera obtained from immunized or chronically infected animals contain complement-fixing antibodies which may activate the classical pathway of C [4-61. Both activation pathways effect deposition of C3b molecules on the surface of the parasite [2, 7, 81. Upon transformation from cercariae and incubation in synthetic medium at 37”C, schistosomula become resistant to in vitro killing by both the classical [9-111 and the alternative [2]

[I 77821

* A

221

This work was supported in part by grants from the John D. and Catherine T. MacArthur and the Rockefeller Foundations. Incumbent of the Barecha Foundation Career Development Chair.

Correspondence: Zvi Fishelson, Department of Chemical Immunology, The Weizmann Institute of Science, P.O.Box 26, Rehovot 76100, Israel Abbreviations: aTRM: Antibodies t o trypsin-released material C4d: Complement C4 deficient DSM: Defined synthetic medium Gp: Guinea pig GpS: Normal G p serum NHS: Normal human serum SBTI: Soybean trypsin inhibitor 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

cytolytic pathways of C. Refractoriness to C-mediated killing is also expressed by in vivo derived skin, lung stage and adult worms [ll-141. Analysis of the transition of freshly prepared schistosomula from C sensitivity to resistance have demonstrated a rapid release of the C-activating substances (primarily the glycocalyx) [2] and of antibodybinding sites [15] from the schistosomula. Schistosomular serine proteases which are released from the acetabular glands may play a dual role in this conversion to resistance, both by accelerating the release of the glycocalyx and by cleaving C proteins [16, 171. Lack of C-activating substances and antigenic sites might have been sufficient to render the transformed schistosomula resistant to C-mediated killing. Nevertheless, the failure of antibodies which were bound to resistant stages of schistosomula through “transplanted” antigens to induce killing by C [18, 191 suggested that the schistosomula have acquired additional innate protecting mechanism(s). The present study shows that treatment of schistosomula and adult worms with trypsin converts them from being resistant to highly sensitive to killing by C in fresh normal human and guinea pig serum (NHS and GpS, respectively). Refractoriness to C is not recovered even after 19-h incubation of the treated schistosomula at 37°C in defined synthetic medium. Moreover, similar to trypsin treatment, antibodies raised in rabbits against the molecules cleaved by trypsin overcome the resistance of schistosomula and adult worms and allow their killing by the cytolytic alternative pathway of G p C. 0014-2980/90/0101-0221$02.50/0

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M. Marikovsky, M. Parizade, R. Arnon and Z. Fishelson

2 Materials and methods 2.1 Media and reagents DSM (defined synthetic medium) is a mixture of RPMI 1640 and nutrient mixture F-12 (1:l) obtained from Gibco (Grand Island, NY). To the DSM, 100 U/ml penicillin, 100 pg/ml streptomycin (Biological Industries, Kibbutz Bet Haemek, Israel), 2 mM L-glutamine (BioLab, Jerusalem, Israel) and 20 mM Hepes (Research Organics, Inc., Cleveland, OH), pH 7.2 were added. Acetylated trypsin, peroxidase-labeled affinity-purified rabbit anti-goat IgG antibodies, hydroxylamine, pronase E, proteinase K, elastase, papain type 111, a-chymotrypsin type I-S, TPCK-treated trypsin, soybean trypsin inhibitor (SBTI) and Tween 20 were purchased from Sigma (St. Louis, MO). Iodogen reagent (Pierce Chemical Co., Rockford, IL), Percoll (Pharmacia, Uppsala, Sweden), thrombin (Miles-Yeda, Rehovot, Israel) and lZI-Bolton-Hunter reagent (NEN DuPont, Boston, MA) have also been used.

C4d-GpS and incubated for 18 h at 37°C in a humidified incubator with 6% C 0 2 . Prior to that, they were treated or not with a protease for 30 min at 24°C and were washed three times with DSM. Antibody-mediated C-killing assay was performed in two steps. First, 3000 schistosomula or 50 adult worms were treated with 100 p1 of either immune or normal rabbit serum for 30 min at room temperature, washed three times with DSM and resuspended in DSM. Then, 200 schistosomula or 10 adult worms (20 pl) were mixed with 100 ~l of GpS or C4d-GpS diluted 1:2 in DSM and incubated at 37 "C as described above. Dead parasites were detected under a stereoscopic microscope by their lack of motility and granular and opaque appearance. Experiments were run in duplicates. Percent spontaneous mortality in control cultures (S) with heat-inactivated (30 min at 56°C) sera (usually < 15%) was subtracted from experimental (E) and was\ calculated according to the formula:

YO Mortality

=

E-S X l o o 100-s

2.2 Parasite A Puerto Rican strain of S. munsoni was maintained in Biomphaluriu glabrutu snails and outbred ICR mice. Cercariae were collected after a 90-min shedding period and mechanically transformed as previously described [20] except that the transformed schistosomula were washed immediately after transformation with ice-cold DSM. Bodies were separated from tails by centrifuging the transformed cercariae through a 65% Percoll solution. Cultured schistosomula were incubated at 37°C in DSM at a concentration of lOoO-loooO/ml in 24-well multidish tissue culture plates (Nunc, Roskilde, Denmark), 1 muwell in a humidified, 6% COz incubator. Adult worms were isolated by perfusion of the hepatic portal system [21] from mice infected 9 weeks earlier with 300 cercariae. The perfusion medium was DSM supplemented with 5% heat-inactivated FCS (Gibco) and 5000 U/mlheparin.

2.3 Sera and antisera NHS obtained from healthy volunteers was prepared and kept frozen in aliquots at -70°C. CCdepleted NHS was prepared by passing NHS over Sepharose-4B column (Pharmacia) to which goat anti-human C4 antibodies were bound. Sera were also obtained from normal guinea pigs (GpS) or from genetically C4-deficient guinea pigs (C4dGpS). Antibodies to trypsin-released schistosomular material (S'RM) were obtained by immunizing rabbits six times, in 2-week intervals, each time with the material released from 3x 105 6-h-schistosomula by trypsin (100 pg/ml) treatment for 30 min at 24 "C in DSM. In the last two immunizations, the injected material also contained SBTI which was added after trypsinization to stop the reaction. The first immunization was performed with CFA and the other five with F A . Monospecific antibodies to human C3 were prepared in goats.

2.4 Cytotoxicity assay Two hundred schistosomula or ten adult worms (in 20 pl DSM) were mixed with 100 pl of HS, NGpS, C4d-HS or

2.5 Analysis of C3 deposition by Western blotting C3b deposited on schistosomula incubated with NHS at 37 "C was released by treating them for 30 min at 37 "C with 1 M hydroxylamine in 0.05 M Tris, pH 7.0, containing 100 mM NaCl [22]. Proteins were exposed to SDS-PAGE on 7.5% gels under reducing conditions and transferred to a nitrocellulose paper [23]. The paper was treated first with goat anti-human C3 antibodies (whole antiserum diluted 1:400 in PBS) and then with rabbit anti-goat IgG antibodies conjugated to peroxidase (Sigma). The bands were developed with the peroxidase substrate diaminobenzidine.

2.6 Analysis of the trypsin-released material Schistosomula were iodinated using either Iodogen (Pierce Chemical Co.) and NaIz5I(Amersham, Bucks, GB) [24] or the 1251-Bolton-Hunter reagent (NEN, DuPont) [25]. Before radiolabeling, the schistosomula (25oooO) were washed with PBS, pH 7.0. After labeling, schistosomula were washed five times with ice-cold DSM and incubated for 30 min at room temperature with or without trypsin (100 pg/ml). Soybean trypsin inhibitor (SBTI, 500 pg/ml) was added to stop the reaction. The SN was analyzed by SDS-PAGE on 5%-15% gradient gels under nonreducing conditions. Gels were dried and X-ray films (Curix Agfa Gevaert, Leverkusen, FRG) were exposed to the dried gels.

3 Results 3.1 Killing of trypsinized schistosomula by C Fresh schistosomula of S. munsoni prepared by mechanical transformation are highly susceptible to killing by either NHS or NGpS but, as demonstrated in Fig. 1, upon incubation in DSM at 37"C, they acquire resistance to killing by NHS and by GpS. Refractoriness to NHS or GpS is obtained within 1-2 h or 3-4 h of incubation, respectively. As previously demonstrated, killing of freshly prepared

Eur. J. Immunol. 1990. 20: 221-227

Complement regulation by S. mansoni

schistosomula in normal sera is mediated by the cytolytic alternative pathway of C [2, 7, 121 and the conversion t o resistance follows the release of the glycocalyx which covers the body of the cercaria [2]. The acquired resistance to loot-

0

3

2

I

4

H o u r s at 37OC

Figure 1. Cultured schistosomula become resistant to C. Freshly transformed schistosomula were incubated in DSM at 37 "C. NHS or GpS was added at different times and incubation was continued at 37°C for 20 h. For further details see Sect. 2.4.

223

killing could be abrogated by treating the schistosomula with trypsin or pronase. Kinetics of the effect of trypsin at 100 pg/ml and at room temperature are shown in Fig. 2. Within 40 min, percent morality increased from below 20% to almost 100%. The effect of trypsin was concentration dependent; mild enhancement of sensitivity to NHS was observed after 30-min treatment with trypsin at 10 pg/ml and pronounced effect (75% mortality) at 50 pg/ml (not shown). The effect of serum dilution on C killing was studied next. Human C diluted to 16% still produced 98% mortality of 5-h schistosomula which had been pretreated for 30 min at 24°C with 100 pg/ml trypsin (Fig. 3). Non-trypsinized schistosomula were much more resistant to killing (20% mortality) even in 80% serum.Yet, control and trypsinized schistosomula differed only slightly in their capacity to activate and consume complement in the fluid phase. The degree of cleavage of C3 and Factor B in NHS incubated for 60 min at 37°C with 25000 cultured (20 h) schistosomula was determined by crossed immunoelectrophoresis as described before [26]. Forty-four percent of C3 and 35% of Factor B was cleaved in NHS treated with control schistosomula. Fifty-one percent of C3 and 46% of Factor B was cleaved in presence of trypsinized schistosomula (results not shown).

The ability of several proteases to render schistosomula sensitive to C killing after 20-min treatment at 37°C was also examined. Trypsin and pronase were highly effective (88% and 70% mortality), and proteinase K and elastase were less efficient (35% and 30% mortality, respectively), while thrombin, a-chymotrypsin and papain had no effect (Fig. 4). To determine whether trypsin binds due to its charge to the schistosomula and thus exerts its activity, the effect of acetylated trypsin was tested. Acetylated trypsin rendered the schistosomula sensitive to C killing (75% mortality), almost as efficiently as trypsin (88%, Fig. 4).

/ 10 20 30 40 50 60 Minutes ot 24°C

Fiaure2. Time course of the effect of trypsin on sensitivity of sckstosomula to C killing. Schistosomula-(C 3-h cultured) were incubated in DSM at 24°C with trypsin (100 pg/ml). A t different times they were washed, mixed with NHS and incubated for 20 h. Percent mortality was determined as explained in Sect. 2.4.

P rotease ( pg /m 1) Trypsin (loo) Acetylated trypsin (100)

I

,

Pronase (100) Trypsinized

Proteinase K (100) Elostase (200) a-Chymotrypsin (200) Thrombin (10) Papain

I /

2 o k L

(100)

Non-Trvosinized

Control

10

20

NHS

(Oh)

Figure 3. Killing of trypsinized and control schistosomula at various concentrations of NHS. Schistosomula (5-h cultured) were either exposed or not to trypsin (100 pg/ml) in DSM at 24°C for 30 min, washed three times and incubated for 20 h at 37 "C with NHS diluted with DSM.

( 1 1 I L I 20 40 60 80 100 Mortality

Figure 4. Effect of several proteases on sensitivity of schistosomula to C killing. Schistosomula (7-h cultured) were incubated in DSM for 20 min at 37°C with a protease, at the concentrations indicated in parentheses, washed three times with DSM and exposed to NHS.

224

Eur. J. Immunol. 1990. 20: 221-227

M. Marikovsky, M. Parizade, R. Arnon and Z. Fishelson kDa -

Moreover, addition of SBTI following trypsin treatment to block activity of trypsin which had remained bound to the schistosomula had no effect on parasite sensitivity to killing (not shown). Addition of SBTI together with trypsin prevented the trypsin effect.

c a'

110-

-P

67-

Ti-ypsin-induced sensitivity of schistosomula to C killing persisted for at least 19 h of incubation at 37°C in DSM (Fig. 5). As depicted, 5-h schistosomula pretreated with trypsin and exposed to NHS suffered 80% mortality as opposed to only 20% mortality of non-trypsinized worms, indicating 60% "net" mortality. Even after 19-h incubation at 37 "C,to allow for possible reappearance of the trypsinreleased molecules, percent net mortality was still 55.

43 -

Minutes: 2

5 10 20 Control

I

I

2

5 K) 20 Trypsin'ued

I

I

15

20

I

3.2 Effect of trypsinization on C3 deposition

Resistant schistosomula (5 h) were exposed to trypsin (100 pg/ml) for 30 min at 24 "C, washed, incubated for up to 20 min at 37°C with NHS and washed again. C3b molecules bound to the schistosomula by ester bonds [22] were released with hydroxylamine and analyzed by SDS-PAGE. As demonstrated in Fig. 6, the a' (110 kDa) chain of C3b which had deposited on the surface of control and trypsinized schistosomula was rapidly degraded to 68- and 43-kDa fragments indicating that most of the C3b was inactivated to iC3b. Nevertheless, more C3b with intact a' chain was found on the surface of trypsin-treated than of control schistosomula and it remained intact longer (Fig. 6, bottom). Thus, the area of the C3b a' band from trypsinized and control schistosomula incubated for 5 rnin with NHS was 2.2 and 0.4mm2, respectively.With 10-min incubation there was hardly any C3b left on the control schistosomula but still a substantial amount (1.8 mm2 band) on the trypsinized schistosomula. The areas calculated by the densitometer for the a' bands of the C3b bound to trypsinized schistosomula at 2 and 20 rnin of incubation were about 0.2 mm2. Analysis of the C3 p chain revealed that, following a similar initial rate of C3b deposition on

p chain T. - ,

.q/E

0

5

10

M i n u t e s at 37°C Figure 6. Analysis of C3b deposited on trypsinized (T) and control (C) schistosomula. Schistosomula (60000; 5 h) were incubated for 30 min at 24 "C with or without trypsin (100 kg/ml) in DSM,washed three times with DSM and incubated for various times with 500 p1 NHS.Then, they were washed five times with DSM and incubated for 30 rnin at 37°C with 1 M hydroxylamine, pH 7.0. The SN was subjected to SDS-PAGE on 7.5% gel under reducing conditions and then blotted to nitrocellulose paper. The C3 bands were visualized by monospecific antibodies as explained in Sect. 2.5. Upper: Autoradiogram; lower: relative amount of C3b a' and chains in the various lanes as determined by scanning densitometry.

Time 19h

Time10

trypsinized and control schistosomula, the C3b converted to iC3b was released from the control but not the trypsinized parasite. Therefore, after 20-min incubation there was two times more iC3b on trypsinized than on control schistosomula. Bands of higher molecular mass were also observed (Fig. 6).They could represent residual unreduced C3b or C3b deposited by a hydroxylamine-resistant amide bond [27] on certain surface membrane molecules which were shed during hydroxylamine treatment. 3.3 Analysis of the TRM Control Trypsin

Net

Control Trypsin

Net

Figure 5. Persistance of the trypsin-induced sensitivity of schistosomula to C killing. Schistosomula (C 5-h cultured) exposed to trypsin treatment (100 pglml in DSM for 30 rnin at 24°C) were washed and incubated with NHS immediately (time:O) or after 19-h incubation at 37 "Cin DSM (time: 19 h). Non-trypsinized schistosomula served as control. Net: subtraction of the percent mortality of non-trypsinized from trypsinized schistosomula.

To determine which molecules on the surface of cultured schistosomula are trypsin sensitive, 6-h schistosomula were surface iodinated by the Bolton-Hunter reagent and were then treated with trypsin at 24 "C for various times. SN from trypsin-treated and from control schistosomula incubated in DSM were analyzed by SDS-PAGE (Fig. 7). Several protein bands appeared in the trypsin-released crude mate-

Eur. J. Immunol. 1990. 20: 221-227

Complement regulation by S. mansoni

225

tive to killing by C4d-GpS or NGpS. Similarly, antibodies raised in rabbits against the material released by trypsin (ZTRM) from the surface of resistant schistosomula markedly increased the killing of resistant schistosomula and adult worms by either Gps (65% and loo%, respectively) or by C4d-GpS (56% and 62%, respectively). Since C4d-GpS lacks the activities of the classical pathway of C, it is probably the cytolytic alternative pathway of C which produces the killing of trypsinized worms or of worms treated with STRM followed by GpS or C4d-GpS.The same antibodies stained strongly both schistosomula and adult worms as examined by immunofluorescence but failed to induce killing of schistosomula by NHS.

4 Discussion

Figure 7. Analysis of the trypsin - released surface molecules by SDS-PAGE. 6-h schistosomula (250 OOO) were surface radiolabeled by the Bolton-Hunter reagent and exposed to DSM (-) or to trypsin (+) (100 pg/ml in DSM at 24°C). SN were collected at different times, and analyzed by SDS-PAGE on 5%-15% gradient gels under nonreducing conditions followed by autoradiography. Since the lower part of the gel contained much more radioactivity than its upper part, the presented autoradiogramm is a composite of different exposure times. Attachment line is between the 30- and the 43-kDa molecular mass markers.

rial, of which the 180,- 55,- 27- and 17-kDa major bands appeared already 10 min after treatment and were missing in control SN. Trypsin treatment of 5-h schistosomula 1251-labeledby the Iodogen technique resulted in the appearance of 180,- 8 5 , 27- and 17-kDa bands in the SN (not shown).

The results presented in this study suggest that in vitro cultured mechanically transformed schistosomula of S. rnansoni escape C-mediated killing by a spontaneous expression of surface membrane regulatory molecule(s). Treatment with trypsin or pronase converts the C-resistant schistosomula into sensitive and they can now be efficiently killed by NHS or GpS. C4-depleted HS and C4-deficient GpS are also lytic, indicating that in normal sera the trypsinized schistosomula activate and are killed by the cytolytic alternative pathway of C. The susceptibility of trypsin-treated cultured schistosomula to C killing is even higher than that of freshly prepared zero-time schistosomula. NHS diluted to 30% hardly killed fresh schistosomula [2] whereas NHS diluted t o 16% produced 95% mortality of trypsin-treated cultured schistosomula (Fig. 3). Trypsin had no effect on the susceptibility of fresh schistosomula (which are still covered by glycocalyx) to C killing (unpublished results). Adult worms obtained from infected mice by liver perfusion and treated by trypsin also became sensitive to the lytic action of C. Higher concentrations of trypsin were required to sensitize adult worms than schistosomula. This could be due to protection of the trypsin substrate molecules against cleavage by neighboring masking molecules or by protease inhibitors which may exist in adult worms, and not on schistosomula.

3.4 C-mediated schistosome killing induced by TRM Nine-hour schistosomula and adult worms were completely resistant to killing by NGpS or by C4d-GpS in absence of antibodies (Table 1). Trypsinization rendered them sensi-

The following findings support our suggestion that cultured schistosomula are equipped with C regulatory molecule(s): (a) despite their insensitivity to C, these schistosomula activate C and induce cleavage of C3 and Factor B in the

Table 1. Killing of schistosomula and adult worms by anti-trypsin released material and C

Pretreatment

Treatmenta) Antibody C

Percent mortality of: Schistowmula Adultworms

(9-h) ~

Trypsinizationb) None

-

GPS C4d-GpS

42 35

GPS

4

W-GpS

7

GPS W-GpS IRS(am) GpS IRS (SlXM) C4d-GpS

2 7 65 56

NRS NRS

65

ND 0 0 0 0 100 62

a) NRS: normal rabbit serum; IRS: immune rabbit serum;. b) Trypsinization: schistosomula, 100 pg/ml; adult worms, 500 pg/ml; 30 min at 24°C in DSM.

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M. Marikovsky, M. Parizade, R. Amon and Z. Fishelson

fluid phase (see Sect. 3.1). Following trypsinization, the schistosomula become sensitive to C, and their capacity to induce C3Ractor B cleavage is slightly increased. (b) Rabbit XRM can overcome the resistance of cultured schistosomula and adult worms and induce their killing by the cytolytic alternative pathway of C.This is in contrast to other rabbit antibodies of a different specificity which bind to the parasite but fail to sensitize it to killing by C. Various leukocytes, such as neutrophils and eosinophils, are capable of binding to and killing of schistosomula of S. mansoni (reviewed in [ l , 28, 291. Cell-parasite attachment may be mediated by Fc-R-antibody andor C3-R-C3 fragment interactions. This is followed by cell activation, degranulation and release of schistosomacidal substances such as the eosinophil major basic protein and cationic protein. Interestingly, leukocytic granules contain several proteases including elastase [30]. In this study we show that elastase increases the sensitivity of cultured schistosomula to killing by C. Therefore, it is tempting to propose that leukocytic granule content exerts a dual effect on target schistosomula, i.e. a direct toxic effect and an indirect potentiation of the humoral immune response. Trypsin-treated schistosomula are sensitive to C damage induced by both NHS and GpS. In contrast, a’RM induced parasite killing by Gp C (even in CCdeficient serum) but not by NHS. One of the possible explanations for this discrepancy in the results is that the schistosomula are equipped with distinct human-specific and Gp-specific C regulators; both are sensitive to trypsin but only the Gp-specific regulator remains immunogenic following trypsinization. Another, yet unresolved issue, is the reason for the lack of recovery of C resistance even 19 h following trypsinization. It is possible that the postulated regulatory molecule(s) have a very slow rate of turnover or that the culture conditions could not support protein synthesis and re-exposure. Alternatively, synthesis of the “regulator(s)” could be restricted to the early stage of schistosomular transformation. Pronase treatment is known to inactivate two human C regulatory molecules, CR1 (complement receptor type 1) and DAF (decay-accelerating factor) [31] which occur on erythrocytes, leukocytes and other tissues [32,33]. Treatment of tumor cells by pronase, trypsin and other proteases increased their sensitivity to killing by antibody and C [34]. Similarly,treatment of Trypanosoma Zewisi with trypsin [35] and of Trypanosoma cruzi with pronase [36] rendered their C-resistant trypomastigotes highly sensitive to killing via the cytolytic alternative pathway of C. Joiner et al. [37] have recently described a C regulatory molecule with DAF-like activity which is produced by trypomastigotes of T cruzi. Since the latter molecule is spontaneously shed from the parasite, it still remains to be determined whether the trypomastigotes also express this same molecule on their surface or whether they are equipped with another C regulatory molecule which is pronase sensitive. Preliminary experiments in our laboratory failed to detect CR1-like or DAF-like activities on schistosomula cultured in defined synthetic medium. However, the finding of larger amounts of C3b with intact a‘ chain on the surface of trypsinized than control schistosomula suggest the occurrence of a regulatory mechanism which is targeted at the step of C3 deposition. This is not necessarily the only or even principle

mechanism which enable cultured schistosomula to resist a C attack. Regardless of their mechanism of control, the postulated C regulatory molecules are excellent targets for immunotherapy. In vitro addition of antibodies specific for TRM produced substantial killing of otherwise resistant worms by C (Table 1). It can be envisioned that unlike antibodies which are directed to “naive” antigenic sites, antibodies to the regulators will block their activity and may thus potentiate in vivo the action of the host immune response. Recent results [38, 391 have suggested that schistosomula may acquire DAF molecules upon contact with host tissue. Thus, transforming schistosomula which develop in vivo are probably equipped with two sets of C evasion molecules, i.e. innate and acquired molecules. Future immunotherapeutic approaches will have to consider and “handle” both types.

Received July 10, 1989; in revised form September 1, 1989.

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529. Brandslund, I., Siersted, H. C., Svehag, S.-E. and Teisner, B., J. Immunol. Methods 1981. 44: 63. Law, S.-K. A., Minich, T. M. and Levine, R. P., Biochemistry 1981. 20: 1451. Capron, A., Dessaint, J.-P., Haque, A. and Capron, M., Prog. Allergy 1982. 31: 234. Butterworth, A. E., Taylor, D. W. ,Veith, M. C. ,Vadas, M. A., Dessein, A., Sturrock, R. F. and Wells, E., Immunol. Rev. 1982. 61: 5. Dewald, B., Rindler-Ludwig, R., Bretz, U. and Maggiolini,M., J. Exp. Med. 1975. 141: 709. Pangburn, M, K., Schreiber, R.D. and Muller-Eberhard, H. J., Proc. Natl. Acad. Sci. USA 1983. 80: 5430. Ross, G. D. and Medof, M. E., Adv. Immunol. 1985. 37: 217.

Complement regulation by S. mansoni

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Complement regulation on the surface of cultured schistosomula and adult worms of Schistosoma mansoni.

Cercaria and freshly prepared schistosomula of Schistosoma mansoni are highly sensitive to complement. However, early in their maturation, the schisto...
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