Predominance of IgA deposits in glomeruli of Schistosoma mansoni infected mice.

We found that IgA is predominant among the immune deposits in the renal glomeruli of mice infected with Schistosoma mansoni, and thus conducted an analysis of the deposition of different immunoglobulin isotypes in the glomeruli throughout the course of infection in mice. Both immunofluorescent and immunoperoxidase methodologies were employed and compared. The abundance of S. mansoni antigens and the isotypes of antibodies to these antigens were examined in the sera and kidney eluates of mice during the course of infection and the results were related to the findings of immunohistopathology. Our observations suggest that at least some immune complexes form in situ in the glomeruli of infected mice and support a possible role of liver damage in the pathogenesis of renal disease in schistosomiasis. Intestinal mucosal immune responses to schistosome antigens may be important in the evolution of renal disease. In addition, the relevance of the murine model to human schistosomal nephropathy is questioned.     

Immunofluorescent localization ofSchistosoma mansoni circulating cathodic antigen in tissues of infected mice using monoclonal antibody

In the present study the kinetics of the uptake and deposition of the major circulating cathodic antigen (CCA) ofSchistosoma mansoni in liver, spleen, and kidney ofS. mansoni infected Swiss mice was investigated in relation to the duration of infection and infection dose (50, 100, 200 cercariae). The presence of antigen was studied with a direct immunofluorescence reaction on frozen sections of the mouse organs, using a fluorescein isothiocyanate (FITC)-labelled mouse IgM monoclonal antibody recognizing a repeating epitope of CCA.CCA was demonstrable from 2 weeks post infection (p.i.) onwards in Kupffer cells in the liver, from 3–4 weeks p.i. onwards in macrophages in the marginal zones in the spleen and from 8 weeks p.i. onwards in kidney glomeruli. The immunofluorescence reactions on CCA in kidney glomeruli, however, remained relatively weak.     

Identification by monoclonal antibody of a major (28 kDa) surface membrane antigen of Schistosoma mansoni

Monoclonal antibody M.1 was generated from mice immunized with membrane enriched extracts of mechanically transformed schistosomula. M.1 bound to the surface membranes of cercariae and young (0-24 h post-transformation) schistosomula but did not bind to older schistosomula or cultured worms. M.1 immunoprecipitated an antigen of approximate molecular weight 27-28 kDa from schistosomula. Experiments using metabolic labeling showed that the antigen was actively synthesized by developing schistosomula. Further M.1 immunoprecipitated a similar 27-28 kDa antigen from membrane-enriched extracts of miracidia, lung and adult worms as well as from schistosomula.     

An erythroid species-specific antigen of swine detected by a monoclonal antibody.

A monoclonal antibody (1AC11) has been produced which recognized the glycophorin of swine red blood cells. 1AC11 was specific for swine membrane erythrocytes. No other swine cells (leukocytes, macrophages, kidney and testis cells) nor red blood cells from all the tested mammalian species (goat, human, sheep, cattle, horse, rabbit, cat and guinea pig) were recognized. There was no blood group activity detected. Immunocytochemical analysis of blood vessel in the swine pituitary tissue showed that besides membrane erythrocytes, cytoplasmic molecules were recognized in some cells. Immunoblot analysis of both membrane and aqueous phase of chloroform/methanol fractions from swine erythrocytes showed that the monoclonal antibody 1AC11 reacts with the major sialoglycoprotein of apparent molecular weight 45,000 daltons.     

Schistosoma mansoni: impaired clearance of model immune complexes consisting of circulating anodic antigen and monoclonal IgG1 in infected mice

The clearance of schistosome-specific model immune complexes (IC) consisting of circulating anodic antigen (CAA), a gut-associated excretory-secretory antigen, and radiolabeled monoclonal antibody (IgG₁) was investigated in mice with a light and heavy Schistosoma mansoni infection and in noninfected control animals. The size analysis of the in vitro prepared and injected IC, as determined by density gradient centrifugation, revealed a wide peak at 11S. In infected animals the injected IC were cleared at a significantly lower rate than in control mice. This was attributed to a decreased uptake of IC by the liver in infected mice. In heavily infected mice, 30 min after injection of 11S IC, 8S, 11S, and >11S IC were present in the serum, whereas only small 8S IC were detected in the serum of lightly infected animals and noninfected controls. Immune complexes were also present in the serum of heavily infected mice 30 min after injection of antibody and were detectable as 11S and >11S IC. The importance of this study is twofold. First, these results show that schistosome-specific monoclonal antibodies can be used in the production of model immune complexes applicable in clearance studies. Second, our findings might be of importance when the possible pathogenicity of circulating IC in schistosomiasis is considered.Abbreviations IC Immune complexes - CAA Circulating anodic antigen - SDG Sucrose density gradient - S Svedberg unit     

Serum levels of circulating anodic antigen and circulating cathodic antigen detected in mice infected withSchistosoma japonicum orS. mansoni

Serum concentrations of circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) were studied in mice infected with eitherSchistosoma japonicum orS. mansoni cercariae. Sera from uninfected mice were negative for both antigens. CAA was detectable in theS. japonicum-infected mice as early as at 2 weeks post-infection (p.i.), and levels were higher in these animals than in theS. mansoni-infected group during the full study period. At the moment of perfusion, 10 weeks p.i., a median of 9 and 29 worms, respectively, were recovered from theS. japonicum-andS. mansoni-infected mice, and the median CAA levels were 326 and 27 ng/ml, respectively. In contrast, CCA levels were much lower in theS. japonicum-infected group (27 ng/ml) as compared with theS. mansoni-infected mice (282 ng/ml). These results suggest an important difference betweenS. japonicum andS. mansoni infections in CAA and CCA production and/or clearance and indicate a significant role for CAA in the diagnosis of human schistosomiasis japonicum.     

Supertypic HLA-Bw4 antigen detected by a new monoclonal antibody

A monoclonal antibody, HLAO1, was prepared by immunizing BALB/c mice with human lymphocytes of known HLA genotype. The monoclonal antibody is cytotoxic, of the IgG 2b isotype and binds to a protein of Mr 43,000 noncovalently associated with the beta 2-microglobulin. Genetic analysis proved complete concordance in the expression of the antigenic determinant defined by this monoclonal antibody and the presence of a supertypic (public) antigen, the HLA-Bw4. As expected, the monoclonal antibody also reacted with the antigens of HLA-A locus, A23, A24, and A32. The computer analysis of HLAO1 antibody binding to the Bw4/Bw6 heterozygous lymphocytes gave the approximate number of antigenic determinants, n = 2.2 X 10(4)/cell, and Ka = (5.9 +/- 0.8) X 10(9) M-1. Besides its potential immunochemical applications, the HLAO1 monoclonal antibody can become a useful tool in routine cytotoxicity typing of HLA antigens on peripheral lymphocytes.     

Surface antigens on Schistosoma mansoni. II. Adsorption of a Forssman-like host antigen by schistosomula

A study was made of the nature of mouse (host) antigens adsorbed by schistosomula of Schistosoma mansoni. Using the mixed antiglobulin test, extracts of a number of individual mouse tissues were tested for their ability to coat schistosomula. All were effective to some extent, with the greatest activity being found in extracts of the lung and spleen. Antibodies against the schistosomulum-coating antigen as well as surface host antigens of adult Schistosoma mansoni were removed by absorbing with erythrocytes from a number of Forssman-positive but not Forssman-negative animal species. These antibodies were also absorbed by Forssman-positive guineapig kidney extract and methanol soluble (Forssman-positive) but not insoluble fractions of sheep erythrocyte stromata and mouse lungs. Schistosomula could be coated in vitro with methanol soluble fractions of mouse lung and erythrocytes and sheep erythrocytes. Though both mouse and sheep coating antigens reacted with anti-mouse and anti-sheep antibodies, reactions were stronger with the homologous antiserum. It was concluded that schistosomula of Schistosoma mansoni adsorb from mice an antigen similar but not identical to the Forssman antigen of sheep erythrocytes, and that this antigen is also found on the surface of adult worms.     

A monoclonal antibody blocking the Schistosoma mansoni 28-kDa glutathione S-transferase activity reduces female worm fecundity and egg viability

The protective effects of two different monoclonal antibodies (mAb) raised against the Schistosoma mansoni 28-kDa glutathione S-transferase (Sm 28 GST) were investigated. Two mAb of the same isotype (IgM) have been selected according to the blocking effect on Sm 28 GST enzymatic activity (S13) or the lack of blockade (H12). When passively transferred into Fischer rats, both S13 and H12 significantly reduced the worm burden. In BALB/c mice clear effects on female worm fecundity and egg viability were observed when the S13 mAb was transferred; these effects included significantly reduced loads of intestinal eggs, reduced egg hatching rates and an increased proportion of non-living eggs. No effect on egg production and egg hatching was observed in H12-treated mice. In addition, worm pairs recovered from S13-but not H12-treated mice laid significantly fewer eggs in vitro, and normal worm pairs incubated in vitro with the S13 mAb produced significantly fewer eggs than those incubated with H12 mAb. The impairment of egg hatching ability was also reproduced in vitro by the S13 mAb. These data suggest the existence of two different effector mechanisms induced by immunization with Sm 28 GST. The effect on the schistosome worm burden appears to be independent of GST activity whereas the effect on S. mansoni female fecundity and egg viability seems to be significantly linked to the inactivation of the enzymatic site.     

Preactivation of macrophages in mice acutely infected with Schistosoma mansoni

This paper shows that peritoneal murine macrophages become preactivated in vivo during the course of a Schistosoma mansoni infection. Thus, less macrophage-activating factor (MAF) was required to induce in vitro tumoricidal and schistosomulicidal activity in macrophages from S. mansoni-infected mice than in macrophages from uninfected control animals. Moreover, the respiratory burst activity, as measured by chemiluminescence, was enhanced in macrophages from S. mansoni-infected mice as compared to controls, whether or not lymphokine (LK) was present in the macrophage cultures. This response appeared at 3 weeks and persisted at least until 12 weeks after infection. Interferon-gamma (IFN-gamma) is most likely involved in the mechanisms leading to such an increased cytolytic and oxidative activity, since in vitro experiments showed: 1) that less IFN-gamma was required to induce tumoricidal activity in macrophages from infected as compared to macrophages from uninfected animals, 2) that the activity of (2'-5')-adenylate synthetase (2'-5' A-synthetase), an enzyme strongly induced by IFN, was elevated in cells from livers of S. mansoni-infected mice.     

Schistosoma mansoni: autoantibodies and polyclonal B cell activation in infected mice.

The appearance of autoantibodies was investigated during the course of Schistosoma mansoni infection in C57Bl/6 mice. Anti-liver autoantibodies or lymphocyte-reactive alloantibodies were detected respectively without cell-mediated immunity against liver antigen or lymphocytotoxic activity. Anti-liver, anti-DNA, anti-Ig and anti-lymphocyte antibodies were shown 6-7 weeks after the beginning of the infection concomitantly with the increase of immunoglobulin levels and circulating immune complexes. At this period, the antibody response to polyvinylpyrrolidone (PVP) was increased and the injection of spleen cells from day-45-infected mice to uninfected recipients increased the anti-PVP antibody response. Conversely, the injection of spleen cells from uninfected to infected mice did not modify the anti-PVP Ab response. After 6 weeks of infection, the basal thymidine incorporation of spleen cells was increased contrasting with the marked inhibition of spleen cell response to PHA. The present data are consistent with the induction of a polyclonal non-specific B cell activation by S. mansoni.     

Production of a mouse monoclonal antibody against the alkaline phosphatase of adult Schistosoma mansoni

This study describes the production and characterization of a monoclonal antibody (mAb) against the alkaline phosphatase of Schistosoma mansoni from splenocytes of chronically infected mice. Convenient selection of the mAb was achieved using the catalytic activity of the antigen in a developed enzyme-antigen immunoassay. The mAb was of the IgG1 subclass and it specifically recognized the alkaline phosphatase in adult worm sections by indirect immunofluorescence. Preincubation of the antibody with partially purified adult alkaline phosphatase did not result in inhibition of the enzyme activity and it did not mediate complement-dependent cytotoxicity against mechanically transformed schistosomula in vitro. The mAb was able to immunoprecipitate under reducing conditions a polypeptide of 65 kDa, similar in size to the monomeric subunit of the schistosome enzyme. The specificity of the mAb was assessed by competitive inhibition with antibodies of infected human sera in an immunoadsorption assay. Periodate treatment of the antigen resulted in altered electrophoretic mobility of alkaline phosphatase, which confirmed the presence of carbohydrate in the molecule, but this did not prevent binding by the mAb. Although the use of the mAb in capture assays for detection of circulating alkaline phosphatase in infected host sera was unsuccessful, the production of this antibody confirmed that the enzyme is exposed by adult worms to the host and that it is immunogenic; additionally, a monoclonal probe is available for further characterization of the structure and function of this important parasite surface molecule.     

Altered immunoglobulin isotype profile and anti-immature worm surface immunoglobulins in mice harboring a praziquantel-resistant Schistosoma mansoni isolate

After placement in mice of PZQ-sensitive and -insensitive S. mansoni isolates obtained from villagers responding and not responding to PZQ, parasitological criteria reflecting their biological development and also the host anti-immature worm immunoglobulin isotypes were examined 8 and 10 weeks post infection. Hepatic granuloma diameter, hepatic histopathological changes and immunolocalization of IgG and IgM on the surface of PZQ-sensitive and -resistant worms were also examined 10 weeks post infection. Data showed that parasitological criteria were not significantly different between mice infected with the PZQ-sensitive and -insensitive S. mansoni isolates. As regards serum immunoglobulins, in mice infected with the PZQ-insensitive S. mansoni isolate, IgG and IgG1 were significantly (p<0.05) lower 8 and 10 weeks post infection, respectively (1.41+/-0.07 and 1.08+/-0.10 and 1.35+/-0.06 and 1.09+/-0.07) than in mice infected with the PZQ-sensitive S. mansoni isolate (1.73+/-0.15 and 1.38+/-0.10 and 1.73+/-0.17 and 1.54+0.21) after the same observation periods. IgM level was nearly the same while IgE was lower than that recorded in mice infected with the PZQ-sensitive S. mansoni isolate. IgG immunofluorescence was also lower (60%+/-6.78) on the surface of resistant worms than that of sensitive worms (66.6%+/-5.27); meanwhile, hepatic granuloma diameter was significantly larger (296.5+/-3.0 vs 283.6+/-4.0) in mice infected with the PZQ-insensitive S. mansoni isolate with higher percentage of intact eggs. Differences in the immunogenic make up of PZQ-sensitive and -insensitive S. mansoni isolates qualitatively and/or quantitatively favoring a certain Th cell subpopulation response could be the underlying reason for such differences recorded in the host immunoglobulin isotype response and also the egg-induced hepatic histopathological changes.     

Rapid competitive enzyme-linked immunosorbent assay using a monoclonal antibody reacting with a 15-kilodalton tegumental antigen of Schistosoma mansoni for serodiagnosis of schistosomiasis.

A competitive enzyme-linked immunosorbent assay (CELISA) for antibody detection was developed by using a monoclonal antibody which reacts with a 15-kDa tegumental antigen of the adult worm of Schistosoma mansoni. This monoclonal antibody was not able to react with antigens of Schistosoma japonicum or Schistosoma haematobium in enzyme-linked immunoelectrotransfer blot (EITB) and indirect immunofluorescence tests. The assay was performed in a period of 1 h using an adult worm crude extract antigen. To evaluate the CELISA, a total of 73 serum samples was analyzed: 35 were from S. mansoni-infected patients, 23 were from individuals with parasitic infections other than schistosomiasis, and 14 were from healthy individuals. All serum samples from healthy individuals and from patients infected with other parasites were negative, as were two (6%) samples from patients infected with S. mansoni. EITB analysis showed that 32 of 33 CELISA-positive samples were positive in the EITB but with different patterns of reactivity. A 15-kDa protein reacted with 60% of serum samples, and a 60-kDa protein showed the highest level of reactivity (85%). The two samples from patients infected with S. mansoni that were negative in the CELISA reacted with 70-, 60-, 50-, 47-, and 38-kDa proteins. One sample, positive in CELISA, did not react with proteins of the antigenic extract.     

Extracellular matrix in hepatic granulomas of mice infected with Schistosoma mansoni. Qualitative and quantitative analysis

To investigate the role of extracellular matrix molecules in the granulomatous inflammation, cercariae of Schistosoma mansoni were injected subcutaneously into BALB/c mice. Well-organized granulomas consisting mainly of stimulated macrophages and epithelioid cells developed in the liver at 11 weeks after infection, thereafter showing a tendency to heal. Fibronectin and heparan sulfate proteoglycan deposition appeared around parasite eggs, then increased distinctly at 11 weeks after infection, and subsequently diminished. Quantities of glycosaminoglycans and hydroxyproline in the egg lesion increased significantly at 11 weeks after infection. Thereafter, the amounts of glycosaminoglycans decreased, whereas hydroxyproline content did not. The data suggest that fibronectin and other macromolecules interact to form granuloma extracellular matrix, and that these extracellular events participate in the development of granulomatous inflammation and subsequent fibrosis induced by schistosome eggs.     

Patterns of cytokine production and proliferation by T lymphocytes differ in mice vaccinated or infected with Schistosoma mansoni.

C57BL/6 mice vaccinated with irradiated cercariae of Schistosoma mansoni are highly resistant to challenge infection. To examine the role of T-helper (Th) activity in these vaccinated (V20) mice, cells from skin- and lung-draining lymph nodes (LN) and the spleen were cultured in vitro with soluble schistosomular antigen. Peak proliferation and release of T-cell growth factor (TCGF) by axillary LN cells on Day 5, and by mediastinal LN cells on Day 18, reflected the kinetics of parasite migration. High levels of interferon-gamma (IFN-gamma) were detected and production was prolonged, particularly in the mediastinal LN. The majority of the above activity was ablated with anti-CD4 antibody. IFN-gamma production by spleen cells increased, whilst proliferation and TCGF release remained low. Although levels of proliferation were similar, more IFN-gamma was released by LN cells from V20 mice than by those from mice infected with normal parasites (NI). This difference in IFN-gamma production was magnified by the greater number of cells in LN of V20 than NI mice. On Day 22 post-exposure, 24-fold more IFN-gamma was produced per pair of axillary LN in the former group. LN cells from V20 mice produced interleukin (IL)-2 and IL-4, whereas those from NI mice released IL-2 but negligible IL-4. Greater quantities of IL-3 were secreted by cells from V20 than from NI mice. These results support the conclusion that IFN-gamma-producing memory Th cells, generated in the LN of V20 mice, play an important role in protective immunity against S. mansoni.     

A histopathological and histochemical study of the ovaries of mice experimentally infected with Schistosoma mansoni.

Thirty-two female mice infected with Schistosoma mansoni and 16 non-infected control mice were studied. They were killed by cervical dislocation, dissected and their ovaries examined histopathologically and histochemically. Ovaries of infected mice showed definite structural damage. No ova, worms or specific granulomata were detected. The study points to a possible immunological mechanism producing such changes simulating those occurring in schistosomal nephropathy. Detection of immune complexes in such organs is recommended.     

A rat cytotrophoblast antigen defined by a monoclonal antibody.

A monoclonal antibody that recognizes specifically a cytotrophoblast antigen was obtained. The monoclonal antibody 22H6 was tested on rat choriocarcinoma (in vivo, in vitro), normal placenta, ectoplacental cone, blastocysts, and several normal organs. The antigen was detected on frozen sections and on tissue culture by indirect immunofluorescence. The monoclonal antibody 22H6 reacts with the cytotrophoblasts of rat choriocarcinoma. The giant cells do not display a positive reaction. It is not expressed on other tumors than choriocarcinoma. In adult rats the only cells revealing a positive reaction are the hepatocytes and the epithelial cells lining the small intestine. In the pregnant rat, the antigen is expressed on the cytotrophoblasts of the junctional zone in the placenta, but not on the giant cells. The mab reacts only with the small trophoblast cells of the ectoplacental cone, but not with trophectoderm of blastocyst. The mab has an IgG2b isotype and is not cytotoxic for choriocarcinoma cells in a complement-dependent cytotoxicity test. The described monoclonal antibody is to our knowledge the only known marker of rat benign and malignant cytotrophoblast.     

Use of a two-sited monoclonal antibody assay to detect a heat-stable malarial antigen in the sera of mice infected with Plasmodium yoelii.

Antigens, circulating in the blood during malarial infections, have been implicated in immune protection, immunosuppression, and immune-complex formation. We used a monoclonal antibody (MAb 7H8) to identify an antigen (Ag-7H8) in the sera of mice infected with Plasmodium yoelii. The major form of the antigen has a molecular weight of approximately 120,000 in P. yoelii, with minor components of 220,000; 65,000 to 75,000; and 45,000. Ag-7H8 remains antigenic after boiling for 5 min. A two-sited assay was developed with MAb 7H8 that demonstrated that the Ag-7H8 has at least two similar epitopes per molecule. The two-sited assay was used to follow Ag-7H8 in the blood of mice during lethal (strain 17XL) and nonlethal (strain 17XNL) P. yoelii infections. Ag-7H8 appeared on days 6 and 7 after infection with 10(6) and 10(4) 17XL P. yoelii parasites, respectively, and remained until the animals died. It was in plasma samples between days 6 and 14 after 17XNL P. yoelii injections in several inbred strains of mice, regardless of the course of parasitemia. Thus, the kinetics of antigenemia correspond with early stages of infection and not with the number of circulating parasites. Indirect immunofluorescence assays demonstrated that MAb 7H8 detects a cross-reactive antigen in other malarial parasites, including Plasmodium berghei and Plasmodium falciparum. Thus, this two-sited assay may have general application for the serodiagnosis of malaria and may be beneficial in determining the relationship of circulating antigens to malarial immunity.