EXPERIMENTAL
47, 392-402
PARASITOLOGY
Schistosoma
( 1979)
mansoni: Circulating Antigens Complexes in Infected Mice
and Immune
F. SANTORO, B. VANDEMEULEBROUCKE, AND A. CAPRON Centre
d’lmmunologie
et de Biologie Parasitaire (INSERM U 167), Institut B.P. 3415,59019 Lille CQdex, France
(Accepted
for
Publication
1 February
Pasteur,
1979)
SANTORO, F., VANDEMEULEBROUCKE, B., AND CAPRON, A. 1979. Schistosoma mansoni: Circulating antigens and immune complexes in infected mice. Experimental Parasitology 47, 392-402. Circulating schistosome antigens (CSA) and circulating immune complexes (CIC) were investigated during the course of Schistosoma mansoni infection in mice. The radioimmunoprecipitation-polyethylene glycol (PEG) assay (RIPEGA) with [?]anti-S. “4” antibodies detected, respectively, total CSA mansoni antibodies or [‘“I] anti-antigen and antigen “4” in serum and in 3% polyethylene glycol-precipitated CIC from infected mice. Complement fixation test and [?I &-binding test revealed, respectively, an anticomplementary activity and the presence of &-binding CIC. All these substances appeared in infected mice at approximately the same period, i.e., between the 40th- and the 55thday postinfection. No correlation was observed between the detection of anticomplementary active substances and C&binding CIC. In contrast, a close relationship was noticed between CSA and complement-activating material during the course of the infection. This suggests that substances with anticomplementary activity in serum from infected mice could be one or various CSA. A close correlation was also observed between &-binding CIC and free or “complexed” antigen “4.” This observation supports well the possibility that antigen “4” is one of the major complexed circulating antigen present in schistosomiasis. The immunoglobulins G,, Gs,, M, and A were also characterized in 3% PEG-precipitated CIC from infected mice during the period in which we detected &-binding CIC. The roles played by specific S. mansoni CIC in either schistosomal nephropathy or protective mechanisms to a challenge infection in mice are discussed. INDEX DESCRIPTORS: Trematode; Schistosoma mansoni; Mice; Circulating schistosome antigens ( CSA ) ; Circulating antigen “4”; Radioimmunoprecipitation-PEG assay (RIPEGA) = test for circulating antigens that uses purified specific antibodies and the polyethylene glycol (PEG) to separate free from complexed radiolabeled antibodies; Circulating immune complexes ( CIC ) ; Complement fixation test; l”I-labeled &-binding test = assay for CIC that utilizes a subcomponent of C, labeled with the radioisotope 12jI.
INTF~ODUC~I~N
antigen-antibody In schistosomiasis, complexes have been suggested to be associated with the renal injury observed particularly in the hepatosplenic form of the disease (DeBrito et al. 1970, Andrade et al. 1971, Queiroz et al. 1973, Rocha et al.
1976). In addition, demonstration of glomerular capillary deposits of immunoglobulins, C3 component of complement, and schistosomal antigens (DaSilva et al. 1970, Falcao and Gould 1975, Hoshino-Shimizu et al. 1976) associated with the elution of renal antischistosome antibodies in patients
392 0014-4894/79/030392-11$02.00/0 Copyright A11 rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
Schistosoma mansoni: IMMUNE COMPLEXES infected with Schistosoma mansoni (Moriearty and Brito 1977) suggested an immune complex mechanism. Similar findings have been reported in murine schistosomiasis (Andrade and Susin 1974, Mahmoud and Woodruff 1975, Natali and Cioli 1976, Van Marck et al. 1977). There are now numerous reports of the presence of specific circulating schistosome antigens (CSA) in infected animals (Berggren and Weller 1967, Nash et al. 1974, Bawden and Weller 1974, Houba et aZ. 1976, Deelder et al. 1976, Deelder and Eveleigh 1978, Santoro et al. 1978a) and patients (Carlier et al. 1975, Madwar and Voller 1975, Santoro et ab. 1977b, 1978b). All these findings support the detection of circulating immune complexes ( CIC ) in human (Bout et al. 1977, Smith et al. 1977) and experimental schistosomiasis (Santoro et al. 1976, 1978a; Bout et al. 1977, Jones et al. 1977). In previous studies, Bout et al. (1977) showed the presence of a specific schistosome antigen called antigen “4,” in CIC from S. mansoni-infected patients. More recently, Santoro et al. (1977a) demonstrated antigen “4” in milk from infected mothers. Finally, the use of a new method, the radioimmunoprecipitation-PEG assay (RIPEGA) (Santoro et al. 1978a, 1978b) allowed the demonstration of CSA and circulating antigen ‘4” in serum from patients, rats and mice infected with S. mansoni. In the present study, we have used the RIPEGA to detect “complexed” CSA and antigen“‘4” during the course of S. mansoni infection in mice in comparison to the levels of CIC evaluated by the [?I Cl,binding test. Additionally, the immunoglobulins making up these complexes have been characterized. MATERIALS
AND METHODS
Parasite and host. A Puerto Rican strain of Schistosoma mansoni was maintained as previously described (Capron et al. 1974). Two groups of 30 C 57 black female mice (IFFA CREDO, L’Arbresle, France) weighing 18 to 20 g at the begin-
393
ning of the experiments, were exposed to 60 furcocercariae according to the ring method of percutaneous infection (Smithers and Terry 1965). Animals were bled through the retro-orbital sinus every 5 days for 100 days. Blood was allowed to clot at room temperature for 2 hr. Serum was separated by centrifugation at 4 C, pooled in its respective group and stored at -30 C. Thirty uninfected mice were maintained and bled under the same conditions as infected ones. During the bloodletting period ( 100 days), 8 out of 30 control and 28 out of 60 S. mansoniinfected mice were dead. Reagents. Anti-S. mansoni hyperimmune rabbit antiserum was prepared against a whole extract of adult schistosomes ( WSA) according to the technique of Capron et al. ( 1968). A monospecific rabbit serum against the antigen “4,” specific for the genus Schistosoma (Capron et al. 1968) was prepared according to Bout et al. ( 1978) and Santoro et aZ. (1977a). Briefly, the precipitation line (band 4) of this antigen, which supports a malate dehydrogenase activity, was identified by bidimensional immunoelectrophoresis, using WSA against the anti-S. mansoni rabbit serum. After 48-hr washing with phosphate-buffered saline (PBS), the agarose containing band 4 was excised, dispersed in saline, and emulsified in Freund’s complete adjuvant. This mixture was then injected intradermally into rabbits. The Ig fraction from these antisera was obtained by affinity chromatography with the soluble extract of S. mansoni antigen and radioiodinated following the method described by Morrison et al. (1971). C1, complement component was isolated from fresh normal human serum according to Volanakis and Stroud ( 1972) as modified by Zubler et al. ( 1976) and labeled with Y. PEG precipitation. An aliquot of mouse serum was precipitated with 3% polyethylen glycol (PEG, M, 6000) (Creighton et aZ. 1973) in order to separate free from
394
SANTORO, VANDEMEULEBROUCKE, AND CAF'RON
complexed antigens and immunoglobulins (Bout et al. 1977). After centrifugation, the pellet was dissolved in distilled water to restore the initial serum volume. Anticomplementary activity. The anticomplementary activity of mouse serum during the course of the infection was determinated according to Santoro et al. (1976) using the microcomplement fixation test described by Wasserman and Levine (1961). Cl,-Binding test. The radiolabeled Cl,binding test was performed according to Zubler et al. ( 1976). Briefly, ‘251-labeled Ci, was mixed with mouse serum, previously treated with 0.2 M EDTA. Free Ci, was separated from Cl, bound to complexes by precipitation with 3% PEG. Results were expressed as percentage lz51labeled C1, precipitated as compared with the protein-bound radioactivity precipitable with 20% trichloracetic acid. RIPEGA. The radioimmunoprecipitation-PEG assay (RIPEGA), previously described for the detection of specific CIC and circulating antigen in carriers of hepatitis B antigen (Santoro et al. 1977c) and recently applied to quantify circulating antigens in human and experimental schistosomiasis (Santoro et al. 1978b), was used to investigate complexed CSA and antigen “4” in serum from Schistosoma mansoniinfected mice. Briefly, to 0.2 ml of 3% PEG-precipitated sera diluted l/5 in borate buffer (0.1 M, pH 8.4), were added 0.2 anti-Schistosoma ml of radioiodinated mansoni antibodies (?-labeled anti-Sm anti-antigen “4” Ab) or radioiodinated antibodies ( lz51-labeled anti-F4 Ab) ( lO,000 cpm ). Free radioiodinated antibodies were separated from those bound to specific antigens fby precipitation with 7% PEG. Results were expressed as percentage 1251-labeled anti-Sm Ab or I*~Ilabeled anti-F4 Ab precipitated as compared with the protein-bound radioactivity precipitable with 20% trichloroacetic
acid.
Radial immunodifusion. The immunoglobulins Gl, G2a, G2b, M, and A were investigated in 3% PEG-precipitated serum by radial immunodiffusion as described by Mancini et al. (1965) on plates supplied by MELOY Laboratories (Springfield, U.S.A.). Results were expressed as percentage of 3% PEG-precipitated Ig as compared with the level of whole serum immunoglobulins. RESULTS
Circulating
Antigens
Results for the detection of specific schistosome antigens by the RIPEGA on serum from Schistosoma mansoni-infected mice were indicated in an earlier work (Santoro et al. 1978b). Total circulating schistosome antigens (CSA) appeared in infected mice at Day 45 after the infection and increased regularly until Day 100. However, a significant decrease was observed for these antigens at Day 75. As for CSA, the specific circulating antigen “4” was demonstrated in infected mice after the 45th day of infection and increased progressively until Day 95. Nevertheless, two important decreases were noticed at Days 80 and 100 postinfection. Circulating
Immune Complexes
Circulating immune complexes (CIC) were studied by the complement fixation test (anticomplementary activity) and the 1251-labeled C&-binding test on pooled serum from 30 normal and two groups of 30 Schistosoma mansoni-infected mice during the course of the infection. For anticomplementary activity results are reported as the reciprocal of highest positive dilution and are shown in Fig. 1. The maximal values (128-512) were observed between the 40th and the 70th day of infection. The titers for the control mice, which present naturally an anticomplementary activity, were always lower than 64 during the same period. It is worth noting that the anti-
Schbtosma
man.soni:
IMMUNE
1 10
20
30
40 Days
I 60
50 after
395
COMPLEXES
I
I 70
I
I 60
I
I 90
I
I 100
infection
FIG. 1. Anticomplementary activity in the serum of Schistosoma mansoni-infected mice ( l ). The maximal values, expressed as titer were observed between the 40th and the 70th day of infection. The results for the control mice (*) were always lower than 64 during this period.
complementary activity in infected mice appeared simultaneously with the first peak of CSA. Results obtained with the 1251-labeIed Cl,-binding test are reported in Fig. 2. C,binding CIC appeared significantly in S. mansoni-infected mice at 50 days after infection and increased progressively until
the end of the experiments. Moreover, a lower peak of CIC were noticed in the 15th day postinfection. Two insignificant decreases were also observed at Days 70 and 85. No relationship was remarked between the anticomplementary activity and the Cl,-binding material detected in infected mice. Nevertheless, a close correlation was
r :;:$;::: ::::::::: j::::::,: IY.II3 Days
after
infection
d uring the course of Schistosoma mansoni FIG. 2. Circulating immune complexes (CIC) CIq-binding test. The results for the control infection in mice detected by the =I-labeled mice (shaded area-mean f 2 SD) were lower than 15% during the same period.
396
SANTORO,
VANDEMEULEBROUCKE,
AND
CAPRON
22 2 P :; h $
20 18 16
u.? .; 14 6 F 12 L f
lo
B
8 10
20
30
40
50
Days after
60
70
90
80
100
infection
mice detected by the FIG. 3. “Complexed” antigen “4” in Schistosoma mansoni-infected RIPEGA with l”I-labeled antigen “4” antibodies on 3% PEG-precipitated semm. The results for the control mice were lower than 10% during the same period (shown by shaded areamean -C 2 SD).
seen between the presence of Cl,-binding CIC and circulating antigen “4” during the course of the infection. 3740PEG-Precipitated
Antigens
The involvement of specific S. mansoni antigens in the CIC present in infected
mice was investigated by the RIPEGA on 3% PEG precipitates from the pooled serum used earlier in the studies of CIC. Characterization of the antigen “4” is indicated in Fig. 3. There also, a close relationship was observed between C&binding CIC and “complexed antigen 4” during the course of the infection. Indeed, 3%
I
10
20
30
40
50
60
70
I
80
I
,I
90
I
100
Days after infection FIG. 4. “Complexed” schistosome antigens in infected mice detected by the RIPEGA with ‘“I-labeled anti-Schistosoma mansoni antibodies on 3% PEG-precipitated serum. The results for the control mice (shaded area-mean * 2 SD) were lower than 14% during the same period.
Schistosoma
10
20
T?lU?l.SOni:
30
40 Days
FIG. 5. Detection of IgGl and IgG2a serum from Schistosoma mansoni-infected 2 SD) for the control mice.
IMMUNE
50
60
alter
infection
397
COMPLEXES
70
60
90
100
by radial immunodiffusion on 3% PEG-precipitated mice. Shaded area represents the results (mean
PEG-precipitated antigen “4” was demonstrated in infected mice approximately at the period in which CIC were detected.
-C
3% PEG-precipitated CSA were also studied (Fig. 4). They were found in two distinct periods after infection: the first
90. d2 60:I” it L 70 . z P : a 600. 50 10
20
30
40
50 Days
olt*r
60
70
60
90
100
i”l.CliOll
FIG. 6. Detection of 3% PEG-precipitated IgM during the course of Schistosoma mansoni infection in mice. Shaded area represents the results (mean f 2 SD) for the control mice.
398
SANTORO, VANDEMEULEBROUCKE, AND CAPRON
tion (between the 15th and the 30th day); and the second and third, between the 60th and the 70th days and after the 80th day, respectively, were higher than the first. Between the 30th and the 55th day and at 75 days after infection significant decreases were observed. For the immunoglobulins G2b and A (Fig. 7) only much lower peaks were noticed approximately at the same periods as with the other immunoglobulins.
between the 5th and 15th day, which could correspond to the lower peak of CIC observed at the beginning of the infection, and the second, more unstable, between the 65th and 100th day after infection. 376 PEG-Precipitated
lmmunoglobulins
The characterization of the immunoglobulins involved in the CIC from S. munsoni-infected mice was performed by radial immunodiffusion on the 3% PEG precipitates used previously for the detection of “complexed” antigens. Results are expressed in percentage of precipitated Ig as compared with the respective immunoglobulin serum levels. Figures 5 and 6 show the detection of “complexed” IgGl, IgG2a, and IgM in infected mice. These immunoglobulins appeared in three distinct periods during the course of the infection: The first appeared at the beginning of the infec-
10
20
30
40
DISCUSSION
Both circulating antigens and CIC were detected during the course of Schistosoma mansoni infection in mice. RIPEGA with specific ?-labeled anti-Sm Ab or Ylabeled anti-F4 Ab demonstrated total CSA and antigen “4 in serum (Santoro et al. 1978b) and 370 PEG-precipitated CIC from Schistosoma mansoni-infected mice. The complement fixation test and the Ylabeled C&-binding test revealed an anti-
50
60
70
80
90
100
FIG. 7. Detection of 3% PEG-precipitated IgG2b and IgA during the course of Schistosoma mansont infection in mice. Shaded area represents the results (mean * 2 SD) for the control mice.
Schistosoma mansoni: complementary activity and the presence of (&-binding material in serum from infected animals, respectively. Finally, radial immunodiffusion allowed the characterization of IgGl, IgG2a, IgM, and IgA in the 3% PEG-precipitated immune complexes. No correlation was observed between the detection of substances with anticomplementary activity and the (&binding material in Schistosoma mansoni-infected mice. In fact, the maximal titers of complement deviation in infected mice (between the 40th and the 70th day postinfection) appeared before the maximal levels of &-binding material (after the 75th day). In contrast, in human schistosomiasis a close relationship was observed between these two procedures for the detection of CIC (Bout et al. 1977). Several hypothesis can be advanced to explain the discrepancy of the results obtained with these techniques in murine schistosomiasis. Among them, the activation of complement by certain CSA appears the most plausible. In fact, in the study of anticomplementary activity, all substances, which interfere with the complement reaction sequence at any level, such as natural polysaccharides, can lead to falsely positive results. Furthermore, the presence of polysaccharidecirculating schistosome antigens has been easily demonstrated by double diffusion method in experimental schistosomiasis (Nash et al. 1977, Carlier et al. 1978). In contrast, these antigens were never found by immunoprecipitation techniques in serum from patients infected with S. manson& Then, the higher amount of CSA in murine schistosomiasis could contribute in the observed anticomplementary activity. Moreover, the relationship noticed between CSA and anticomplementary active substances during the course of the infection is an argument for a complement activation by one or various CSA. In addition, in a recent unpublished study of our laboratory, we have demonstrated that several
IMMUNE
COMPLEXES
399
antigenic extracts from the schistosome are able, in absence of antibodies, to activate the alternative complement pathway. In contrast, the 1’51-labeled &,-binding test, which only uses the first component of the classical complement pathway, appears more specific and sensitive than the complement deviation test to the detection of CIC. Then, the difference observed between these two methods does not allow us to consider as CIC all the substances showing an anticomplementary activity in serum from S. munsoni-infected mice. The involvement of non-C&-binding CIC or other material of nonparasitic origin in this anticomplementary activity cannot be ruled out at this stage of our experiments and further studies are in progress to clear this point. Immunoglobulins and schistosome antigens were characterized in the 3% PEGprecipitated immune complexes from S. munsoni-infected mice. They must have been present in a complexed form since free specific S. mansoni antigen (Bout et al. 1977) and immunoglobulins (Creighton et al. 1973) are soluble in PEG concentrations up to at least 6%. Moreover, the detection of these immunologically active substances in 3% PEG-precipitated CIC at the periods in which infected mice serum presented a C&-binding activity, argues for the specificity of the characterized CIC. Indeed, in the first lower peak of Clq-binding CIC (at 19days postinfection), S. mansoni-infected mice also showed 3% PEG-precipitated IgG2a, IgM, IgA, and CSA. In the other periods of CIC (after the 45th day of infection), all the studied immunoglobulins and antigens were characterized in 3% PEG precipitates. A close relationship was observed between Clq-binding CIC and free or complexed antigen “4” during the course of the infection, This association should be related to the result of Bout et al. (1977) concerning the detection of antigen “4” in CIC from patients infected with S. man-
400
SANTORO,
VANDEMEULEBROUCKE,
soni. All these data suggest that antigen “4” is one of the major complexed circulating antigen present in schistosomiasis. It is now well established that mice experimentally infected with S. mansoni develop a renal injury similar to that observed in human schistosomiasis (De Rousse and Romero 1974, Andrade and Susin 1974). Demonstration of granular deposits on the glomerular basement membrane containing immunoglobulins, comcomponents (Mahmoud and plement Woodruff 1975, Natali and Cioli 1976), and schistosome antigens (Natali and Cioli 1976, Van Marck et al. 1977) suggested strongly the involvement of immune complexes in mm-me schistosomal nephropathy. The occurrence of CIC and circulating antigens at 6- to 7-week postinfection may account for this nephropathy, which appears approximately 7-8 weeks after infection ( Natali and Cioli 1976). On the other hand, CSA in a free or complexed form might be involved in the effector mechanisms of the immune response to schistosomes. In fact, it is worth noting that the last peak of CIC and complexed antigen “4” in infected mice was seen during the very period in which Sher et al. (1974) observed a significant immunity to a Schistosoma mansoni challenge infection in mice. This relationship suggests a possible role for CIC in immunity, probably in association with effector cells. Moreover, in rat schistosomiasis, CIC formed with IgE antibody have been shown to interact with normal macrophages in an in vitro cytotoxicity to schistosomula cc aPron et al. 1977). Further studies on the role played by CIC in immunity against a challenge infection in mice are underway. ACKNOWLEDGMENTS This work was supported by grants from the CNRS (ERA 422) and from the INSERM (U 167). The authors wish to thank T. Lepresle, M. C. Liebart, and A. Caron for their technical assistance.
AND CAPRON REFERENCES
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MOXIEARTY, P. L., AND BRITO, E. 1977. Elution of renal anti-schistosome antibodies in human schistosomiasis mansoni. A~~can ~~~1 of Tropicab Medicine and Hygiene 26, 717-722. MORRISON, M., BAYSE, G. S., AND WEBSTER, A. 0. 1971. Use of lactoperoxidase catalysed iodination in i~unochemic~ studies. Imm~no~h~isty 8,289-297. NASH, T. E., DIN, N. U., AND JEANLOZ, R. W. 1977. Further purification and characterization of a circulating antigen in schistosomiasis. ~0~~~ of immunology 119, 1627-1633. NASH, T. E., PRESCOTT, B., AND NEVA, F. A. 1974. The characteristics of a circulating antigen in schistosomiasis. Jou~nul of Immunology 112, 1509-1507. NATALI, P. G., AND CIOLI, D. 1976. Immune complex nephritis in Schbtosoma mansoniinfected mice. European Journal of Immunology 6, 359-364. QUEIROZ, F. P., BRITO, E., MARTINELLI, R., AND ROCHA, H. 1973. Nephrotic syndrome in patients with Schistosoma mansoni infections. American Journal of Tropical Medicine and Hygiene 22, 622-628. ROCHA, H., CRUZ, T. Bmro, E., AND SUSIN, M. 1976. Renal involvement in patients with hepatosplenic schistosomiasis mansoni. American Journal of Tropical Medicine and Hygiene 25, 108-115. DEROUSSE, A. R., AND ROMERO, M. J. 1974. Wltrastructural study of kidney schistosomiasis mansoni in mice. (Preliminary report). Rev&a do ~n~~tuto de Med~c~na TT~ical de S&J Pa&o
16, 362-367. SANTORO, F., BOROJEVIC, R., BOUT, D., TACHON, P., BINA, J. C., AND CAPRON, A. 1977a. Motherchild rela~onship in human schistosomiasis mansoni. I. Parasitic antigen and antibodies in milk. American Journal of Tropical Medicine andHygiene 26,1164-1168. SANTORO, F., BOUT, D., WATTRE, P., AND CAPRON, A. 1976. Immune complexes in schistoso~asis. I. The use of complement fixation tests for their detection. Revista do Institute de Me&&a Tropical de SZo Paul0 18, 152-156. SANTORO, F., CAPRON, M., JOSEPH, M., ROUSSEAUXPREVOST, R., AND CAPRON, A. 1978a. Circulating antigens and immune complexes in Schistosoma mansoni-infected rats. Characterization by radioimmunopre~ipi~tion-PEG assay (RIPEGA ) . Clinical and ~~e~rn~taZ immunology 32, 435-442. SANTORO, F., CARLIER, Y., BOROJEVIC, R., BOUT, D., TACHON, P., AND CAPRON, A. 1977b. Parasite “M” antigen in milk from mothers infected with Schistosoma mansoni. (Preliminary report).
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SANTORO,
VANDEMEULEBROUCKE,
Annals of Tropical Medicine and Parasitology 71, 121-123. VANDEMEULEBROUKE, B., AND SANTORO, F., CAPRON, A. 1978b. The use of the radioimmunoprecipitation-PEG assay (RIPEGA) to quantify circulating antigens in human and experimental schistosomiasis. Journal of Immunological Methods 24, 229-237. SANTORO, F., WATTRE, P., DESSAINT, J. P., AND CAPRON, A. 1977c. Hepatitis B circulating immune complexes. Characterization by radioimmunoprecipitation-PEG assay ( RIPEGA) . Journul of Immunological Methods 15, 201-209. SHER, A., MACKENZIE, P., AND SMITHERS, S. R. 1974. Decreased recovery of invading parasites from the lungs as a parameter of acquired immunity to schistosomiasis in the mouse. Journal of Infectious Diseases 130, 626-633. DASILVA, L. C., DEBRITO, T., CAMARGO, M. E., DEBONI, P. R., LOPES, J. D., AND GUNJI, J. 1970. Kidney biopsy in the hepatosplenic form of infection with S. mansoni in man. Bulletin of the World Health Organization 42, 907-910. SMITH, M. D., VERROUST, P. J., MOREL-MAROGER, L., GENITEAU, M., AND COULAND, J. P. 1977. A study of the presence of circulating immune complexes in schistosomiasis. Transactions of the
AND
CAPRON
Royal Society of Tropical Medicine and Hygiene 71, 343-348. SMITHERS, S. R., AND TERRY, R. J. 1965. The infection of laboratory hosts with cercariae of S. mansoni and the recovery of the adult worms. Parasitology 55, 695-700. VAN MARCK, E. A. E., DEELDER, A. M., AND GIGASE, P. L. J. 1977. Effect of partial vein ligation on immune glomerular deposits in Schistosoma mansoni-infected mice. British Journal of Experimental Pathology 58, 412-417. VOLANAKIS, J. E., AND STROUD, R. M. 1972. Rabbit Clq: Purification, functional and structural studies. Journal of Immunological Methods 2, 25-34. WASSERMAN, E., AND LEVINE, L. 1961. Quantitative micro-complement fixation and its use in the study of antigenic structure by specific antigen-antibody inhibition. Journal of Immunology 87, 290-295. ZUBLER, R. H., LANGE, G., LAMBERT, P. H., AND MIESCHER, P. A. 1976. Detection of immune complexes in unheated sera by a modified =I-Clq binding test. Effect of heating on the binding of Clq by immune complexes and application of the test to systemic lupus erythematosus. Journal of Immunology 116, 232235.
47, 392-402
PARASITOLOGY
Schistosoma
( 1979)
mansoni: Circulating Antigens Complexes in Infected Mice
and Immune
F. SANTORO, B. VANDEMEULEBROUCKE, AND A. CAPRON Centre
d’lmmunologie
et de Biologie Parasitaire (INSERM U 167), Institut B.P. 3415,59019 Lille CQdex, France
(Accepted
for
Publication
1 February
Pasteur,
1979)
SANTORO, F., VANDEMEULEBROUCKE, B., AND CAPRON, A. 1979. Schistosoma mansoni: Circulating antigens and immune complexes in infected mice. Experimental Parasitology 47, 392-402. Circulating schistosome antigens (CSA) and circulating immune complexes (CIC) were investigated during the course of Schistosoma mansoni infection in mice. The radioimmunoprecipitation-polyethylene glycol (PEG) assay (RIPEGA) with [?]anti-S. “4” antibodies detected, respectively, total CSA mansoni antibodies or [‘“I] anti-antigen and antigen “4” in serum and in 3% polyethylene glycol-precipitated CIC from infected mice. Complement fixation test and [?I &-binding test revealed, respectively, an anticomplementary activity and the presence of &-binding CIC. All these substances appeared in infected mice at approximately the same period, i.e., between the 40th- and the 55thday postinfection. No correlation was observed between the detection of anticomplementary active substances and C&binding CIC. In contrast, a close relationship was noticed between CSA and complement-activating material during the course of the infection. This suggests that substances with anticomplementary activity in serum from infected mice could be one or various CSA. A close correlation was also observed between &-binding CIC and free or “complexed” antigen “4.” This observation supports well the possibility that antigen “4” is one of the major complexed circulating antigen present in schistosomiasis. The immunoglobulins G,, Gs,, M, and A were also characterized in 3% PEG-precipitated CIC from infected mice during the period in which we detected &-binding CIC. The roles played by specific S. mansoni CIC in either schistosomal nephropathy or protective mechanisms to a challenge infection in mice are discussed. INDEX DESCRIPTORS: Trematode; Schistosoma mansoni; Mice; Circulating schistosome antigens ( CSA ) ; Circulating antigen “4”; Radioimmunoprecipitation-PEG assay (RIPEGA) = test for circulating antigens that uses purified specific antibodies and the polyethylene glycol (PEG) to separate free from complexed radiolabeled antibodies; Circulating immune complexes ( CIC ) ; Complement fixation test; l”I-labeled &-binding test = assay for CIC that utilizes a subcomponent of C, labeled with the radioisotope 12jI.
INTF~ODUC~I~N
antigen-antibody In schistosomiasis, complexes have been suggested to be associated with the renal injury observed particularly in the hepatosplenic form of the disease (DeBrito et al. 1970, Andrade et al. 1971, Queiroz et al. 1973, Rocha et al.
1976). In addition, demonstration of glomerular capillary deposits of immunoglobulins, C3 component of complement, and schistosomal antigens (DaSilva et al. 1970, Falcao and Gould 1975, Hoshino-Shimizu et al. 1976) associated with the elution of renal antischistosome antibodies in patients
392 0014-4894/79/030392-11$02.00/0 Copyright A11 rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
Schistosoma mansoni: IMMUNE COMPLEXES infected with Schistosoma mansoni (Moriearty and Brito 1977) suggested an immune complex mechanism. Similar findings have been reported in murine schistosomiasis (Andrade and Susin 1974, Mahmoud and Woodruff 1975, Natali and Cioli 1976, Van Marck et al. 1977). There are now numerous reports of the presence of specific circulating schistosome antigens (CSA) in infected animals (Berggren and Weller 1967, Nash et al. 1974, Bawden and Weller 1974, Houba et aZ. 1976, Deelder et al. 1976, Deelder and Eveleigh 1978, Santoro et al. 1978a) and patients (Carlier et al. 1975, Madwar and Voller 1975, Santoro et ab. 1977b, 1978b). All these findings support the detection of circulating immune complexes ( CIC ) in human (Bout et al. 1977, Smith et al. 1977) and experimental schistosomiasis (Santoro et al. 1976, 1978a; Bout et al. 1977, Jones et al. 1977). In previous studies, Bout et al. (1977) showed the presence of a specific schistosome antigen called antigen “4,” in CIC from S. mansoni-infected patients. More recently, Santoro et al. (1977a) demonstrated antigen “4” in milk from infected mothers. Finally, the use of a new method, the radioimmunoprecipitation-PEG assay (RIPEGA) (Santoro et al. 1978a, 1978b) allowed the demonstration of CSA and circulating antigen ‘4” in serum from patients, rats and mice infected with S. mansoni. In the present study, we have used the RIPEGA to detect “complexed” CSA and antigen“‘4” during the course of S. mansoni infection in mice in comparison to the levels of CIC evaluated by the [?I Cl,binding test. Additionally, the immunoglobulins making up these complexes have been characterized. MATERIALS
AND METHODS
Parasite and host. A Puerto Rican strain of Schistosoma mansoni was maintained as previously described (Capron et al. 1974). Two groups of 30 C 57 black female mice (IFFA CREDO, L’Arbresle, France) weighing 18 to 20 g at the begin-
393
ning of the experiments, were exposed to 60 furcocercariae according to the ring method of percutaneous infection (Smithers and Terry 1965). Animals were bled through the retro-orbital sinus every 5 days for 100 days. Blood was allowed to clot at room temperature for 2 hr. Serum was separated by centrifugation at 4 C, pooled in its respective group and stored at -30 C. Thirty uninfected mice were maintained and bled under the same conditions as infected ones. During the bloodletting period ( 100 days), 8 out of 30 control and 28 out of 60 S. mansoniinfected mice were dead. Reagents. Anti-S. mansoni hyperimmune rabbit antiserum was prepared against a whole extract of adult schistosomes ( WSA) according to the technique of Capron et al. ( 1968). A monospecific rabbit serum against the antigen “4,” specific for the genus Schistosoma (Capron et al. 1968) was prepared according to Bout et al. ( 1978) and Santoro et aZ. (1977a). Briefly, the precipitation line (band 4) of this antigen, which supports a malate dehydrogenase activity, was identified by bidimensional immunoelectrophoresis, using WSA against the anti-S. mansoni rabbit serum. After 48-hr washing with phosphate-buffered saline (PBS), the agarose containing band 4 was excised, dispersed in saline, and emulsified in Freund’s complete adjuvant. This mixture was then injected intradermally into rabbits. The Ig fraction from these antisera was obtained by affinity chromatography with the soluble extract of S. mansoni antigen and radioiodinated following the method described by Morrison et al. (1971). C1, complement component was isolated from fresh normal human serum according to Volanakis and Stroud ( 1972) as modified by Zubler et al. ( 1976) and labeled with Y. PEG precipitation. An aliquot of mouse serum was precipitated with 3% polyethylen glycol (PEG, M, 6000) (Creighton et aZ. 1973) in order to separate free from
394
SANTORO, VANDEMEULEBROUCKE, AND CAF'RON
complexed antigens and immunoglobulins (Bout et al. 1977). After centrifugation, the pellet was dissolved in distilled water to restore the initial serum volume. Anticomplementary activity. The anticomplementary activity of mouse serum during the course of the infection was determinated according to Santoro et al. (1976) using the microcomplement fixation test described by Wasserman and Levine (1961). Cl,-Binding test. The radiolabeled Cl,binding test was performed according to Zubler et al. ( 1976). Briefly, ‘251-labeled Ci, was mixed with mouse serum, previously treated with 0.2 M EDTA. Free Ci, was separated from Cl, bound to complexes by precipitation with 3% PEG. Results were expressed as percentage lz51labeled C1, precipitated as compared with the protein-bound radioactivity precipitable with 20% trichloracetic acid. RIPEGA. The radioimmunoprecipitation-PEG assay (RIPEGA), previously described for the detection of specific CIC and circulating antigen in carriers of hepatitis B antigen (Santoro et al. 1977c) and recently applied to quantify circulating antigens in human and experimental schistosomiasis (Santoro et al. 1978b), was used to investigate complexed CSA and antigen “4” in serum from Schistosoma mansoniinfected mice. Briefly, to 0.2 ml of 3% PEG-precipitated sera diluted l/5 in borate buffer (0.1 M, pH 8.4), were added 0.2 anti-Schistosoma ml of radioiodinated mansoni antibodies (?-labeled anti-Sm anti-antigen “4” Ab) or radioiodinated antibodies ( lz51-labeled anti-F4 Ab) ( lO,000 cpm ). Free radioiodinated antibodies were separated from those bound to specific antigens fby precipitation with 7% PEG. Results were expressed as percentage 1251-labeled anti-Sm Ab or I*~Ilabeled anti-F4 Ab precipitated as compared with the protein-bound radioactivity precipitable with 20% trichloroacetic
acid.
Radial immunodifusion. The immunoglobulins Gl, G2a, G2b, M, and A were investigated in 3% PEG-precipitated serum by radial immunodiffusion as described by Mancini et al. (1965) on plates supplied by MELOY Laboratories (Springfield, U.S.A.). Results were expressed as percentage of 3% PEG-precipitated Ig as compared with the level of whole serum immunoglobulins. RESULTS
Circulating
Antigens
Results for the detection of specific schistosome antigens by the RIPEGA on serum from Schistosoma mansoni-infected mice were indicated in an earlier work (Santoro et al. 1978b). Total circulating schistosome antigens (CSA) appeared in infected mice at Day 45 after the infection and increased regularly until Day 100. However, a significant decrease was observed for these antigens at Day 75. As for CSA, the specific circulating antigen “4” was demonstrated in infected mice after the 45th day of infection and increased progressively until Day 95. Nevertheless, two important decreases were noticed at Days 80 and 100 postinfection. Circulating
Immune Complexes
Circulating immune complexes (CIC) were studied by the complement fixation test (anticomplementary activity) and the 1251-labeled C&-binding test on pooled serum from 30 normal and two groups of 30 Schistosoma mansoni-infected mice during the course of the infection. For anticomplementary activity results are reported as the reciprocal of highest positive dilution and are shown in Fig. 1. The maximal values (128-512) were observed between the 40th and the 70th day of infection. The titers for the control mice, which present naturally an anticomplementary activity, were always lower than 64 during the same period. It is worth noting that the anti-
Schbtosma
man.soni:
IMMUNE
1 10
20
30
40 Days
I 60
50 after
395
COMPLEXES
I
I 70
I
I 60
I
I 90
I
I 100
infection
FIG. 1. Anticomplementary activity in the serum of Schistosoma mansoni-infected mice ( l ). The maximal values, expressed as titer were observed between the 40th and the 70th day of infection. The results for the control mice (*) were always lower than 64 during this period.
complementary activity in infected mice appeared simultaneously with the first peak of CSA. Results obtained with the 1251-labeIed Cl,-binding test are reported in Fig. 2. C,binding CIC appeared significantly in S. mansoni-infected mice at 50 days after infection and increased progressively until
the end of the experiments. Moreover, a lower peak of CIC were noticed in the 15th day postinfection. Two insignificant decreases were also observed at Days 70 and 85. No relationship was remarked between the anticomplementary activity and the Cl,-binding material detected in infected mice. Nevertheless, a close correlation was
r :;:$;::: ::::::::: j::::::,: IY.II3 Days
after
infection
d uring the course of Schistosoma mansoni FIG. 2. Circulating immune complexes (CIC) CIq-binding test. The results for the control infection in mice detected by the =I-labeled mice (shaded area-mean f 2 SD) were lower than 15% during the same period.
396
SANTORO,
VANDEMEULEBROUCKE,
AND
CAPRON
22 2 P :; h $
20 18 16
u.? .; 14 6 F 12 L f
lo
B
8 10
20
30
40
50
Days after
60
70
90
80
100
infection
mice detected by the FIG. 3. “Complexed” antigen “4” in Schistosoma mansoni-infected RIPEGA with l”I-labeled antigen “4” antibodies on 3% PEG-precipitated semm. The results for the control mice were lower than 10% during the same period (shown by shaded areamean -C 2 SD).
seen between the presence of Cl,-binding CIC and circulating antigen “4” during the course of the infection. 3740PEG-Precipitated
Antigens
The involvement of specific S. mansoni antigens in the CIC present in infected
mice was investigated by the RIPEGA on 3% PEG precipitates from the pooled serum used earlier in the studies of CIC. Characterization of the antigen “4” is indicated in Fig. 3. There also, a close relationship was observed between C&binding CIC and “complexed antigen 4” during the course of the infection. Indeed, 3%
I
10
20
30
40
50
60
70
I
80
I
,I
90
I
100
Days after infection FIG. 4. “Complexed” schistosome antigens in infected mice detected by the RIPEGA with ‘“I-labeled anti-Schistosoma mansoni antibodies on 3% PEG-precipitated serum. The results for the control mice (shaded area-mean * 2 SD) were lower than 14% during the same period.
Schistosoma
10
20
T?lU?l.SOni:
30
40 Days
FIG. 5. Detection of IgGl and IgG2a serum from Schistosoma mansoni-infected 2 SD) for the control mice.
IMMUNE
50
60
alter
infection
397
COMPLEXES
70
60
90
100
by radial immunodiffusion on 3% PEG-precipitated mice. Shaded area represents the results (mean
PEG-precipitated antigen “4” was demonstrated in infected mice approximately at the period in which CIC were detected.
-C
3% PEG-precipitated CSA were also studied (Fig. 4). They were found in two distinct periods after infection: the first
90. d2 60:I” it L 70 . z P : a 600. 50 10
20
30
40
50 Days
olt*r
60
70
60
90
100
i”l.CliOll
FIG. 6. Detection of 3% PEG-precipitated IgM during the course of Schistosoma mansoni infection in mice. Shaded area represents the results (mean f 2 SD) for the control mice.
398
SANTORO, VANDEMEULEBROUCKE, AND CAPRON
tion (between the 15th and the 30th day); and the second and third, between the 60th and the 70th days and after the 80th day, respectively, were higher than the first. Between the 30th and the 55th day and at 75 days after infection significant decreases were observed. For the immunoglobulins G2b and A (Fig. 7) only much lower peaks were noticed approximately at the same periods as with the other immunoglobulins.
between the 5th and 15th day, which could correspond to the lower peak of CIC observed at the beginning of the infection, and the second, more unstable, between the 65th and 100th day after infection. 376 PEG-Precipitated
lmmunoglobulins
The characterization of the immunoglobulins involved in the CIC from S. munsoni-infected mice was performed by radial immunodiffusion on the 3% PEG precipitates used previously for the detection of “complexed” antigens. Results are expressed in percentage of precipitated Ig as compared with the respective immunoglobulin serum levels. Figures 5 and 6 show the detection of “complexed” IgGl, IgG2a, and IgM in infected mice. These immunoglobulins appeared in three distinct periods during the course of the infection: The first appeared at the beginning of the infec-
10
20
30
40
DISCUSSION
Both circulating antigens and CIC were detected during the course of Schistosoma mansoni infection in mice. RIPEGA with specific ?-labeled anti-Sm Ab or Ylabeled anti-F4 Ab demonstrated total CSA and antigen “4 in serum (Santoro et al. 1978b) and 370 PEG-precipitated CIC from Schistosoma mansoni-infected mice. The complement fixation test and the Ylabeled C&-binding test revealed an anti-
50
60
70
80
90
100
FIG. 7. Detection of 3% PEG-precipitated IgG2b and IgA during the course of Schistosoma mansont infection in mice. Shaded area represents the results (mean * 2 SD) for the control mice.
Schistosoma mansoni: complementary activity and the presence of (&-binding material in serum from infected animals, respectively. Finally, radial immunodiffusion allowed the characterization of IgGl, IgG2a, IgM, and IgA in the 3% PEG-precipitated immune complexes. No correlation was observed between the detection of substances with anticomplementary activity and the (&binding material in Schistosoma mansoni-infected mice. In fact, the maximal titers of complement deviation in infected mice (between the 40th and the 70th day postinfection) appeared before the maximal levels of &-binding material (after the 75th day). In contrast, in human schistosomiasis a close relationship was observed between these two procedures for the detection of CIC (Bout et al. 1977). Several hypothesis can be advanced to explain the discrepancy of the results obtained with these techniques in murine schistosomiasis. Among them, the activation of complement by certain CSA appears the most plausible. In fact, in the study of anticomplementary activity, all substances, which interfere with the complement reaction sequence at any level, such as natural polysaccharides, can lead to falsely positive results. Furthermore, the presence of polysaccharidecirculating schistosome antigens has been easily demonstrated by double diffusion method in experimental schistosomiasis (Nash et al. 1977, Carlier et al. 1978). In contrast, these antigens were never found by immunoprecipitation techniques in serum from patients infected with S. manson& Then, the higher amount of CSA in murine schistosomiasis could contribute in the observed anticomplementary activity. Moreover, the relationship noticed between CSA and anticomplementary active substances during the course of the infection is an argument for a complement activation by one or various CSA. In addition, in a recent unpublished study of our laboratory, we have demonstrated that several
IMMUNE
COMPLEXES
399
antigenic extracts from the schistosome are able, in absence of antibodies, to activate the alternative complement pathway. In contrast, the 1’51-labeled &,-binding test, which only uses the first component of the classical complement pathway, appears more specific and sensitive than the complement deviation test to the detection of CIC. Then, the difference observed between these two methods does not allow us to consider as CIC all the substances showing an anticomplementary activity in serum from S. munsoni-infected mice. The involvement of non-C&-binding CIC or other material of nonparasitic origin in this anticomplementary activity cannot be ruled out at this stage of our experiments and further studies are in progress to clear this point. Immunoglobulins and schistosome antigens were characterized in the 3% PEGprecipitated immune complexes from S. munsoni-infected mice. They must have been present in a complexed form since free specific S. mansoni antigen (Bout et al. 1977) and immunoglobulins (Creighton et al. 1973) are soluble in PEG concentrations up to at least 6%. Moreover, the detection of these immunologically active substances in 3% PEG-precipitated CIC at the periods in which infected mice serum presented a C&-binding activity, argues for the specificity of the characterized CIC. Indeed, in the first lower peak of Clq-binding CIC (at 19days postinfection), S. mansoni-infected mice also showed 3% PEG-precipitated IgG2a, IgM, IgA, and CSA. In the other periods of CIC (after the 45th day of infection), all the studied immunoglobulins and antigens were characterized in 3% PEG precipitates. A close relationship was observed between Clq-binding CIC and free or complexed antigen “4” during the course of the infection, This association should be related to the result of Bout et al. (1977) concerning the detection of antigen “4” in CIC from patients infected with S. man-
400
SANTORO,
VANDEMEULEBROUCKE,
soni. All these data suggest that antigen “4” is one of the major complexed circulating antigen present in schistosomiasis. It is now well established that mice experimentally infected with S. mansoni develop a renal injury similar to that observed in human schistosomiasis (De Rousse and Romero 1974, Andrade and Susin 1974). Demonstration of granular deposits on the glomerular basement membrane containing immunoglobulins, comcomponents (Mahmoud and plement Woodruff 1975, Natali and Cioli 1976), and schistosome antigens (Natali and Cioli 1976, Van Marck et al. 1977) suggested strongly the involvement of immune complexes in mm-me schistosomal nephropathy. The occurrence of CIC and circulating antigens at 6- to 7-week postinfection may account for this nephropathy, which appears approximately 7-8 weeks after infection ( Natali and Cioli 1976). On the other hand, CSA in a free or complexed form might be involved in the effector mechanisms of the immune response to schistosomes. In fact, it is worth noting that the last peak of CIC and complexed antigen “4” in infected mice was seen during the very period in which Sher et al. (1974) observed a significant immunity to a Schistosoma mansoni challenge infection in mice. This relationship suggests a possible role for CIC in immunity, probably in association with effector cells. Moreover, in rat schistosomiasis, CIC formed with IgE antibody have been shown to interact with normal macrophages in an in vitro cytotoxicity to schistosomula cc aPron et al. 1977). Further studies on the role played by CIC in immunity against a challenge infection in mice are underway. ACKNOWLEDGMENTS This work was supported by grants from the CNRS (ERA 422) and from the INSERM (U 167). The authors wish to thank T. Lepresle, M. C. Liebart, and A. Caron for their technical assistance.
AND CAPRON REFERENCES
ANDRADE, Z. A., ANDRADE, S. G., AND SADIGURSKY, M. 1971. Renal changes in patients with hepatosplenic schistosomiasis. American Journal of Tropical Medicine and Hygiene 20, 77-83. ANDRADE, Z. A., AND SUSIN, M. 1974. Renal changes in mice infected with Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 23, 400-403. BAWDEN, M. P., AND WELLER, T. H. 1974. Schistosoma mansoni circulating antigen: Detection by complement fixation in sera from infected hamster and mice. American Journal of Tropical Medicine and Hygiene 23, 1077-1084. BERGGREN, W. L., AND WELLER, T. H. 1967. Immunoelectrophoretic demonstration of specific circulating antigens in animals infected with Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 16, 806-612. BOUT, D., DUPAS, H., CAPRON, M., EL GAZAWI, A., CARLIER, Y., DELACOURTE, A., AND CAPRON, A. 1978. Purification, immunochemical and biological characterization of malate dehydrogenase of Schistosoma mans&. Immunochemistry 15, 633-638. BOUT, D., SANTORO, F., CARLIER, Y., BINA, J. C., AND CAPRON, A. 1977. Circulating immune complexes in schistosomiasis. Immunology 33, 1722. DE BRITO, T., GUNJI, J., CAMARGO, M. E., PENNA, D. O., AND DA SILVA, L. C. 1970. Advanced kidney disease in patients with hepatosplenic manson’s schistosomiasis. Revista do Instituto de Medicina Tropical de Sa’o Paulo 12, 225-235. CAPRON, A., BIGUET, J., VERNES, A., AND AFCHAIN, D. 1968. Structure antigenique des helminthes. Aspects immunologiques des relations hateparasite. Pathologic Biologic 16, 121-138. CAPRON, A., CAPRON, M., DUPAS, H., BOUT, D., AND PETITPREZ, A. 1974. Etude in vitro des phenomenes immunologiques dans la schistosomiase humaine et experimentale. I. Etude comparative in vitro de l’activite l&hale d’immum&rums sur les formes immatures et sur les adultes de S. mansoni. International Journal for Parasitology 4, 613-623. CAPRON, A., DESSAINT, J. P., JOSEPH, M., TORPIER, G., CAPRON, M., ROUSSEAUX, R., SANTORO, F., AND BAZIN, H. 1977. IgE and cells in schistosomiasis. American Journal of Tropical Medicine and Hygiene 26 ( Suppl.), 3946. CARLIER, Y., BOUT, D., BINA, J. C., CAMUS, D., FIGUEIREDO, J. F. M., AND CAPRON, A. 1975. Immunological studies in human schistosomiasis. I. Parasitic antigen in urine. American Journal of Tropical Medicine and Hygiene 24, 949-954.
IMBUE CARLEER, Y., BOUT, D., AND CAPRON, A. 1978. Further studies on the circulating M antigen in human and experimental Schi~oso~ ma~oni infections. Annales d’Immunologie ( Institut Pasteur) 129C, 811-818. CREIGHTON, W. D., LAMBERT, P. H., AND MIESCHER, P. A. 1973. Detection of antibodies and soIubIe antigen-antibody complexes by precipitation with polyethylene glycol. Journal of Immunology 111, 1219-1227. DEELDER, A. M., AND EVELEIGH, P. C. 1978. An indirect haemagglu~ation reaction for the demonstration of Schiseosoma mans& circulating anodic antigen. Transactions of the Royal Society of Tropical Medicine and Hygiene 72, 178-180. DEELDER, A. M., KLAPPE, H. T. M., VAN DEN AARDWEG, G. J. M. J., AND VAN MEERBEKE, E. H. E. M. 1976. Schistosoma mansoni: Demonstration of two circulating antigens in infected hamsters. ~xper~~nta~ PaT~tolog~ 40, 189197. FALC~O, H. A., AND GOULD, D. B. 1975. Immune complex nephropathy in schistosomiasis. Annuls of l~e~l Medicine 83, 148-154. HOSHINO-SHIMIZU, S., DE BRITO, T., KANAM~RA, H. Y., CANTO, A. L., SILVA, A. O., CAMPOS, A. R., PENNA, D. O., AND DA SILVA, 1;. C. 1976. Human schistosomiasis: Schistosoma munsoni antigen detection in renal glomernli. Trunsactions of the Royal Society of Tropical Medicine and Hygiene 70, 492-496. HOWA, V., KOECH, D. K., STIJRROCK, R. F., BUTTERWORTH, A. E., KUSEL, J. R., AND MAH~~o~D, A, A. F. 1976. Soluble antigens and antibodies in sera from baboons infected with Schistoof Immunology 117, soma mansoni. Journal 705-707. JONES, C. E., RACHFORD, F. W. UZZCEL, M. A., AND LEWERT, R. M. 1977. Schistosoma japonicum: Semiquantitative assessment of circulating immune complexes, serum Clq and C3, and their rela~onship to renal pathology and hepatic fibrosis in rabbits. Experimental Parasitology 42, 221-234. MADW~, M. A., AND VOLLER, A. 1975. Circulating soluble antigens and antibody in schistosomiasis. Rr%ish LMedicul Journal i, 435-436. MAHMOUD, A. A. F., AND WOODRUFF, A. W. 1975. Renal lesions caused by immune complex deposition in schistosomiasis. Transactions of the Royal Society of T$op~~al Med~~ne and Hygiene 69, 187-188. MANCINI, G., CARBONARA, A. O., AND HEREMANS, J. F. 1965. Immunochemical quantitations of antigens by single radial immunodiffusion. Immunochemistry 2, 235-254.
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