Circulating immune complexes in schistosomiasis.

Circulating immune complexes (CIC) were investigated by the [125I]Clq binding test, the complement fixation test (CFT) and optical density measurement after redissolving 3% polyethylene glycol precipitates of serum from patients infected by Schistosoma mansoni. A highly significant correlation was obtained among these three techniques. More than 60% of the patients demonstrated significantly higher values than control individuals. The level of CIC was found to be higher in the mild than in the hepatosplenic form of the disease. Parasite antigen, IgG, IgM and IgE were characterized in these CIC. In experimental schistosomiasis in mice, maximum levels of CIC, evaluated by the CFT, were observed between the 40th and the 70th day of infection.     

Circulating immune complexes in acute schistosomiasis.

The sera of patients with acute and chronic schistosomasis were tested for the presence of circulating immune complexes with the 125I-Clq binding assay. Fourteen out of fifteen (93%) patients with acute schistosomiasis had elevated 125I-Clq binding activity, while only two out of eleven (18%) patients with chronic disease had C1q binding complexes. This difference was significant (P less than 0.001) and paralleled the degree of clinical didsease activity between the two groups of patients. IgG and IgM were readily detected in all of these circulating complexes but the specific parasite antigens initiating their formation could not be defined. The level of circulating immune complexes was inversely correlated with the absolute eosinophil counts for individuals in the acutely infected group, an observation compatible wiht the hypothesis that a functional role for the eosinophil is the destruction and elimination of immune complexes.     

Schistosoma mansoni: fractionation of polysaccharide egg antigens by lectin affinity chromatography.

Crude polysaccharide antigen was extracted from Schistosoma mansoni egg homogenate by 44% aqueous phenol. The aqueous soluble polysaccharide extract was subjected to affinity chromatography with concanavalin A-Sepharose 4B. Two fractions (bound and unbound) were obtained; both of them gave precipitin lines with serum obtained from mice infected with S. mansoni. These precipitin lines gave partial identity. Further fractionation with wheat germ agglutinin-sepharose of the unbound fraction resulted in three antigenic fractions. These different antigens were eluted with different N-acetylglucosamine molarities (0, 0.05) and 0.5) and gave lines of identity when reacted against infected mouse serum. When the four antigenic materials were subjected to polyacrylamide gel electrophoresis and stained for proteins and polysaccharides no migration bands were observed. The chemical analysis of the two initial fractions showed a small percentage of amino acids in both fractions. Sugar analysis with gas-liquid chromatography showed different sugar composition of the two initial fractions.     

Serodiagnosis of imported schistosomiasis by a combination of a commercial indirect hemagglutination test with Schistosoma mansoni adult worm antigens and an enzyme-linked immunosorbent assay with S. mansoni egg antigens

A commercial indirect hemagglutination (IHA) test using erythrocytes coated with Schistosoma mansoni adult worm antigens (WA) and an enzyme-linked immunosorbent assay (ELISA) with S. mansoni egg antigens (SEA) were assessed for their use in serodiagnosis of imported schistosomiasis (hereafter these tests are designated WA/IHA and SEA/ELISA, respectively). The sensitivity of the tests was evaluated with sera from 75 patients with proven S. mansoni infection, 25 with proven S. haematobium infection, and 10 with clinical Katayama fever. The specificity was assessed with sera from 283 patients with various parasitic, bacterial, viral, and fungal infections and sera containing autoimmune antibodies. Sensitivities of the WA/IHA with a cutoff titer of 1:160 (WA/IHA(160)) in detecting S. mansoni, S. haematobium, S. mansoni and S. haematobium combined, and clinical Katayama fever were 88.0, 80.0, 86.0, and 70.0%, respectively, with a specificity of 98.9%. The WA/IHA with a cutoff of 1:80 (WA/IHA(80)) showed sensitivities of 94.7, 92.0, 94.0, and 90.0%, respectively, with a specificity of 94.7%. The comparable values of SEA/ELISA were 93.3, 92.0, 93.0, and 50.0%, respectively, with a specificity of 98.2%. Combined use of ELISA and WA/IHA(80) gave sensitivities of 100% for S. mansoni, S. haematobium, and S. mansoni and S. haematobium combined and 90% for Katayama fever. The specificity of this combination in detecting schistosomiasis was 92.9%. Combination of SEA/ELISA with WA/IHA(160) gave sensitivities of 98.7, 96.0, 98.0, and 80% with a specificity of 97.2%. Our findings suggest that WA/IHA and SEA/ELISA are each sensitive and specific serological tests that are easy to use for the diagnosis of imported schistosomiasis. The combined use of these two tests enabled the serological diagnosis of schistosomiasis to be achieved with very high degrees of both sensitivity and specificity.     

Cytotoxicity of human and baboon mononuclear phagocytes against schistosomula in vitro: induction by immune complexes containing IgE and Schistosoma mansoni antigens.

Normal human blood monocytes, pre-incubated at 37 degrees C with sera from patients infected with Schistosoma mansoni, strongly adhered to S. mansoni schistosomula in vitro, whereas no significant adherence was induced by sera from uninfected individuals. Comparable adherence occurred with normal baboon blood monocytes or peritoneal macrophages when these cells were incubated with sera from S. mansoni-infected baboons. Adherence of macrophages to schistosomula was associated with damage to the larvae, as estimated by a 51Cr release technique. Neither adherence nor cytotoxicity was induced by pre-incubation of the schistosomula, instead of the monocytes, with immune serum. The relevant factor in immune serum was heat-labile, but was not a complement component. Absorption and ultracentrifugation experiments showed that immune complexes, containing S. mansoni-specific IgE antibody and soluble parasite antigens, produced monocyte or macrophage adherence and cytotoxicity. Similar observations have been reported previously in the rat model. Since the production of large amounts of IgE is a predominant feature of schistosome infections in man and experimental animals, it is possible that this new mode of mononuclear phagocyte activation could act as an immune effector mechanism against S. mansoni.     

Circulating antigens and immune complexes in Schistosoma mansoni-infected rats. Characterization by radioimmunoprecipitation-PEG assay (RIPEGA)

Circulating schistosome antigens (CSA) and circulating immune complexes (CLC) were investigated in rats infected with Schistosoma mansoni. The radioimmunoprecipitation-polyethylene glycol (PEG) assay (RIPEGA), with 125I-labelled anti-S. mansoni anti-serum, detected CSA during two distinct periods of the infection; the first between the 11th and the 14th week of infection and the second between the 11th and 14th week after infection. The CH50 deviation test revealed the presence of CIC in sera from infected rats, approximately at the two periods when CSA were detected. At 6 weeks of infection, the levels of CIC in infected rats were not different from those in control rats. However, a more sensitive method characterized IgG2a in C1q-binding C1C from infected rats. At weeks 5 and 6, IgE immune complexes were also detected in the serum from infected rats. In fact, the use of RIPEGA on the material eluted from infected rat serum after passage through an anti-IgE immunosorbent showed the presence of schistosome antigen at week 4, and at higher levels at week 6. Levels of 50% haemolytic complement in infected rat serum were lowered between the 2nd and the 4th week, the 5th and the 8th week and after the 12th week of infection. The possible role played by CIC in the protective mechanisms to a S. mansoni challenge infection in rats is discussed.     

Circulating antigens in schistosomiasis: detection of 31/32-kDa proteins in sera from patients infected with Schistosoma japonicum,S. mansoni,S. haematobium,or S. intercalatum

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect 31/32-kDa schistosome proteins as circulating antigens in sera from schistosomiasis patients. A monoclonal antibody was used as a capture antibody and rabbit antiserum raised against purified 31/32-kDa proteins was the detecting antibody. Positive results were obtained with patients infected with Schistosoma japonicum (88%; n=69), S. mansoni (80%; n=56), S. haematobium (100%; n=40), or S. intercalatum (94%; n=65). Sera from uninfected Chinese and African individuals and from Chinese patients with trichinosis, cysticercosis, or paragonimiasis did not react in the assay. This ELISA appears to be valuable in diagnosing infections by all major human schistosome species.     

Human immune response in schistosomiasis: the role of P24 in the modulation of cellular reactivity to Schistosoma mansoni antigens

Schistosome antigenic components are being tested as vaccine candidates with various degrees of success, but there are only few reports using multivalent antigens to stimulate an appropriate immune response that leads to resistance or granuloma modulation. We investigated the in vitro response of peripheral blood mononuclear cells (PBMC) from chronic intestinal schistosomiasis individuals to PIII, a multivalent antigen prepared from Schistosoma mansoni adult worm antigen, and response to P24, a single antigen obtained from PIII. Treatment of PBMC with either PIII or P24 caused significant decrease in cellular proliferation and granuloma formation induced by S. mansoni antigens, and a significant elevation in IL-10 and TNF-alpha but not in IFN-gamma production. Moreover, P24 promoted an elevation in TNF-alpha level on the in vitro granuloma reaction, when cocultured with polyacrylamide beads (PB) coupled to S. mansoni antigens. These findings suggest that, besides inducing protective immunity, PIII and P24 antigens seem to be important in the regulation of in vitro granuloma formation through stimulation of IL-10 and TNF-alpha production in human schistosomiasis. The more pronounced effect of P24 on reducing the in vitro granulomatous reaction could be associated with a balance between IL-10 and TNF-alpha production.     

Immunoblot analysis of Schistosoma mansoni antigens with sera of schistosomiasis patients: diagnostic potential of an adult schistosome polypeptide.

We compared the reaction in immunoblots of sera obtained from patients with parasitologically proven S. mansoni infections, with a suspected history of schistosomiasis infection, or with unrelated parasitic diseases. Several polypeptides from adult S. mansoni reacted with the schistosomiasis patients' sera in a heterogeneous manner. However, a component of approximately 31 kilo daltons (kD) reacted with all schistosomiasis sera and with several sera of suspected schistosomiasis cases. No reaction was ever observed with sera of patients harbouring other parasites. Thus, the polypeptide has potential diagnostic value. The use of sera of patients with recent infections demonstrated that: the earliest time of antibody formation against the 31 kD component was approximately 40 days post infection, the reaction with this polypeptide in immunoblots was exceptionally strong and antibodies directed against other schistosome proteins were barely detectable at this time. Identical results were obtained with sera of experimentally infected mice. The 31 kD component was present in parasites of either sex. It was apparently not a glycoprotein. Evidence suggests that the 31 kD polypeptide may originate from the schistosome gut.     

Circulating immune complexes in experimental syphilis: identification of treponemal antigens and specific antibodies to treponemal antigens in isolated complexes.

As a prelude to characterization of the host and treponemal antigens present in purified immune complexes from the sera of rabbits with disseminated syphilis, autoradiographic and immunoenzymatic analyses of solubilized extracts of Treponema pallidum, Treponema phagedenis biotype Reiter, and Treponema refringens were performed on electroblots of polypeptides first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electroblots of purified immune complexes were developed with the same panel of antisera so that protein profiles could be compared. Eight treponemal antigens were consistently present in isolated complexes; four of these cross-reacted with antisera prepared against avirulent treponemes. The average molecular weights of these antigens were 87,000, 76,000, 66,000, and 45,000. Antibodies dissociated from isolated immune complexes, when used for the development of T. pallidum electroblots, reacted with four antigens of comparable molecular weight. Antibodies to those polypeptides were also present in the sera of animals immunized with immune complexes. The demonstration of treponemal antigens in purified immune complexes convincingly argues that their occurrence in experimental syphilis is not merely due to tissue destruction and responses to endogenous host antigens.     

Differential responsiveness of humans with early-stage schistosomiasis haematobium to Schistosoma haematobium soluble adult-worm and egg antigens

Schoolchildren (7–8 years old) infected with Schistosoma haematobium were tested for lymphocyte proliferative responses, in vitro granuloma formation (IVGF), and cytokine release in T-cell Western assays and for serum antibody reactivity by enzyme-linked immunosorbent assay (ELISA) and immunoblotting against S. haematobium soluble adult-worm (SAWA) and egg (SEA) antigens. The lymphoproliferative response rate of individual subjects against 10 SAWA and 15 SEA electroseparated bands ranged from 0 to 33?% and from 11 to 66?%, respectively. The SAWA bands essentially failed to elicit significant IVGF, in contrast to the SEA bands, all of which were capable of inducing IVGF from peripheral blood mononuclear cells (PBMC) of 30–80?% of individual donors. The exclusive ability of SEA bands to induce IVGF could not be attributed to selective release of interleukin 2 (IL-2), IL-4, or interferon-gamma, as SEA and SAWA bands were capable of eliciting release of a similar array of cytokines in supernatants of 4-day PBMC cultures. The antibody response to SEA was stronger than that to SAWA, yet the proportion of SAWA bands binding humoral antibodies of individual donors was significantly larger than that observed for SEA. The study thus suggests that humans with early-stage S. haematobium infection respond poorly to SAWA but mount strong cellular immune responses to SEA that result in granuloma and antibody formation.     

Circulating immune complexes, complement and complement component levels in childhood Hodgkin's disease.

Serum levels of circulating immune complexes (CIC) assayed by the Raji cell radioimmunoassay, total haemolytic complement (TCH50), Clq and C3 were correlated with clinical stage, histological type, age, sex and treatment of eighty-six children with Hodgkin's disease over a period of 4 years. Most significant findings were the changes of levels of CIC, TCH50, Clq and C3 during disease activity and following treatment. Significant perturbations were also seen in association with relapse. Levels of C and CIC were significantly elevated (P less than 0.001) at the time of diagnosis prior to splenectomy and/or any treatment. In the group before treatment, 81 percent of CIC levels were above 16 micrograms/ml with a maximum value of 1120 micrograms/ml. During treatment 33 percent were still above normal with a maximum of 320 micrograms/ml. Within 1 year after cessation of treatment, 37 percent also remained above normal levels with a maximum of 240 micrograms/ml. At relapse prior to treatment, 63 percent were again elevated with a maximum of 1280 micrograms/ml. The most significant difference on TCH50 levels relates to treatment periods. Sera of patients with active disease who are previously untreated show elevation of TCH50 levels (P less than 0.001) (average 127 CH50 mu/ml. During and after treatment eht TCH50 levels drop to 96 and 102 CH50 mu/ml, as compared to normal control of 100 CH50 mu/ml. In sera of patients at the first, second or third relapse, the combined TCH50 levels are significantly different from controls and across treatment periods (P less than 0.005).     

Utilization of fractionated soluble egg antigens reveals selectively modulated granulomatous and lymphokine responses during murine schistosomiasis mansoni.

Worm eggs deposited in the livers and intestines of Schistosoma mansoni-infected mice secrete soluble egg antigens (SEA) and induce T cell-mediated circumoval granulomas. In the present study, we fractionated crude SEA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tested the fractions for granuloma elicitation and lymphokine production at different stages of the infection. SEA fraction-coupled beads were used to elicit artificial pulmonary granulomas. Acutely infected mice responded with granulomas to seven fractions (less than 21-, 25- to 30-, 32- to 38-, 60- to 66-, 70- to 90-, 93- to 125-, and greater than 200-kDa fractions) of SEA, whereas chronically infected mice responded to four fractions (60- to 66-, 70- to 90-, 93- to 125-, and greater than 200-kDa fractions). In response to both crude and fractionated SEA, granuloma T cells produced high levels of gamma interferon at the preacute (6-week) stage of infection, but production subsequently diminished. Interleukin-2 (IL-2) and IL-4 production peaked at the acute (8-week) stage of infection and concurrently decreased at the chronic (20-week) stage. At the acute stage of the infection, the granulomagenic SEA fractions also elicited IL-2 and IL-4 production; at the chronic stage, IL-2 production and, to a lesser degree, IL-4 production corresponded to SEA fractions that elicited granulomas. Isolated SEA proteins from the 32- to 38-kDa fraction demonstrated differential lymphokine responses: predominant gamma interferon and IL-2 production was elicited by the 32-kDa fraction, whereas the 35- and 38-kDa proteins elicited predominant gamma interferon and IL-4 production. However, all three proteins elicited granuloma formation. The present study reveals changes in granulomatous responses to SEA fractions during the acute and chronic stages of the infection as well as distinct phases of gamma interferon, IL-2, and IL-4 lymphokine production throughout the infection. Based on these results, it is concluded that granuloma formation and IL-2 and IL-4 production are interrelated.     

Soluble intercellular adhesion molecules in human schistosomiasis: correlations with disease severity and decreased responsiveness to egg antigens.

Granuloma formation, the principal pathologic consequence of infection with Schistosoma mansoni, is a complex process involving intricate cell-cell interactions in which intercellular adhesion molecules are likely to participate. To examine this possibility, sera of schistosomiasis patients in various clinical groups were assayed for the presence of soluble intercellular adhesion molecule 1 (sICAM-1) and soluble E-selectin (sE-selectin). Comparisons were made between groups with different infection intensities (as predicted by fecal egg count) as well as between groups with severe (hepatosplenic) or milder (intestinal) pathology. All groups had elevated levels of sICAM-1 compared with controls. Also, patients in the high egg-excreting and hepatosplenic groups had significantly higher levels of serum sICAM-1 than patients in the low-egg-excreting and intestinal groups, respectively. The levels of sE-selectin were significantly elevated in the sera of all patients except those in the hepatosplenic group compared with controls. Patients in the intestinal group had significantly higher levels of sE-selectin in their sera than did hepatosplenic group patients, but serum sE-selectin levels of high- and low-egg-excreting patients were comparable. A striking finding of this study was the inverse correlation observed between sICAM-1 levels and peripheral blood mononuclear cell responses to schistosome soluble egg antigens (SEA) but not with responses to other schistosome antigens, purified protein derivative, or mitogen. Because ICAM-1 can perform a costimulatory function in antigen-presenting cell-T cell interactions, it is possible that shedding of ICAM-1 in the granuloma microenvironment interrupts proper costimulation, leading to unresponsive SEA-specific T cells. In this way, sICAM-1 could be one factor contributing to the observed modulation of cellular responses to SEA in chronic human schistosomiasis.     

Anti-miracidial effect of recombinant glutathione S-transferase 26 and soluble egg antigen on immune responses in murine schistosomiasis mansoni.

The anti-miracidial potential of recombinant Schistosoma mansoni glutathione S-transferase 26 (rSmGST26) or native crude soluble egg antigens (SEA) was assessed. The associated dynamics of granuloma formation and immune responses were evaluated. Naive C57BL/6 mice were injected intravenously with multiple doses of either SEA (SEA-group) or rSmGST26 (GST-group) 7 days before cercarial infection. The immunized groups and the respective controls were sacrificed 6, 8 and 16 weeks postinfection (p.i.). Acceleration of ova destruction and reduction of granuloma diameter were greater in the GST-group than the SEA-group, mainly at 8 weeks p.i. However, the amelioration of hepatic pathology and function was more evident in the SEA-group. Concurrently, serum-specific IgG1 levels were elevated throughout the course of infection in the immunized groups compared to the infected controls. Initial rise of all splenic cytokines and serum anti-SEA IgE levels at 6 weeks p.i. was observed, followed by a dramatic drop in the levels of the proinflammatory cytokines IL-2, IFNgamma, IL-4 and TNF-alpha and IgE at 8 weeks of infection. IL-10 level was lower at 8 weeks p.i. than at 6 weeks, but was higher in immunized groups than in infected controls. Several responses may be implicated as an outcome of the present immunization protocol, such as increased levels of blocking antibody (IgG1) and IL-10 with decreased levels of proinflammatory cytokines and IgE.     

Protection against experimental Schistosoma mansoni schistosomiasis achieved by immunization with schistosomula released products antigens (SRP-A): role of IgE antibodies.

Schistosomula-released products (SRP-A) have been shown to induce preferentially a significant IgE response against Schistosoma mansoni schistosomula when injected into rats, in the absence of adjuvant. The present work provides additional evidence of the in vivo relevance of the anti-SRP-A target antigens. Two strains of rat (Brown Norway and Fischer) were immunized with SRP-A and infected percutaneously. A significant level of protection (up to 83% reduction in worm burden) was observed. Passive transfer experiments carried out with anti-SRP-A or IgE-depleted anti-SRP-A sera suggested the preponderant role of antibodies and particularly of IgE in the protective immunity developed by Fischer rats. Platelets and macrophages recovered from such immunized rats had surface IgE as demonstrated by immunofluorescence analysis with FITC anti-IgE, and have been shown to be directly cytotoxic for schistosomula. The chemiluminescence observed when the macrophages were incubated with anti-IgE suggested the presence of IgE on the surface of these cells.     

Schistosoma mansoni: impairment of the cell-mediated immune response in mice.

Skin-graft rejection in mice experimentally infected with Schistosoma mansoni is delayed when grafting is performed 60 days after the infection. In mice infected 30 days prior to the grafting, the grafts were rejected at the same time both in infected and in control animals. This observation indicates that impairment of cell-mediated immune response occurs in mice with mature S. mansoni infections.     

Circulating immune complexes in patients with usual interstitial pulmonary fibrosis: partial characterization and relationship with Thermoactinomyces vulgaris.

Sixty-six sera were analysed by solid-phase conglutinin binding assay, to detect the levels of circulating immune complexes (CIC), and by enzyme-linked immunosorbent assay (ELISA) to show a correlation with antibodies to Thermoactinomyces vulgaris. Sixty per cent of patients with usual interstitial pulmonary fibrosis (UIP), were positive for CIC; and T. vulgaris antibodies were detected in 60% of the same patients. In comparison, there was a low frequency of positive results in bronchitis patients (5% for CIC and 35% for T. vulgaris), and in normal blood donors (0% for CIC and 30% for T. vulgaris). Furthermore 31% of patients with lung cancer were found positive for CIC, but not for T. vulgaris. Immune complexes purified on Protein A-Sepharose and by sucrose density gradient from patients with UIP, showed a sedimentation coefficient higher than 19 S. The purified material was found to contain IgG and IgM as antibodies. Binding of immune complexes, purified by sedimentation on sucrose gradient, to conglutinin was inhibited by the presence of T. vulgaris antigen; thus suggesting that this antigen might be present in the complexes.     

Circulating immune complexes in six patients with trichinosis.

The sera of six patients infected with Trichinella spiralis were studied for the presence of circulating immune complexes (CIC). CIC were present in all six patients studied 1 month after infection. In two patients in whom serial serum samples were available, as clinical improvement occurred there was a decrease in the level of CIC and an increase in the fluorescent antibody titres.     

IgA and IgG immune complexes increase human macrophage C3 biosynthesis.

We have studied the effect of IgA- and IgG-containing immune complexes on the production of complement proteins C3, factor B and C2 by human monocyte-derived macrophages, using biosynthetic labelling, immunoprecipitation, sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and autoradiography. There was a consistent increase in C3 production and secretion with both IgA and IgG immune complexes. This increase appeared after a 24-hr incubation period of the macrophages in the presence of immune complexes. No change in the biosynthesis of factor B and C2 proteins was observed in these experiments. Concomitant with the enhanced C3 biosynthesis, the immune complexes caused an increase in macrophage tumour necrosis factor (TNF) production; 310 + 24 U/ml/5 x 10(5) cells and 430 + 51 U/ml/5 x 10(5) cells for IgA and IgG immune complexes, respectively, versus 12 + 8 U/ml/5 x 10(5) cells in the control cells. The presence of prednisolone (2 x 10(-5) M) or dexamethasone (1 x 10(-7) M) inhibited the immune complex-induced TNF production, but had no effect on C3-increased synthesis, suggesting that the effect of immune complexes was not mediated by endogenous TNF production. These findings may be relevant to the local inflammatory response in IgA immune complex-mediated diseases, including IgA nephropathy.