Immunoaffinity purification of Schistosoma mansoni soluble egg antigens

Schistosoma mansoni egg antigens were purified from a heterogeneous mixture of soluble egg antigens (crude SEA) with an immunoaffinity column that consisted of the specific anti-SEA antibodies contained in 16-week S. mansoni-infected mouse serum bound to Sepharose 4B. On sodium dodecyl sulfate (SDS) gel electrophoresis, the purified antigen fraction yielded at least eight bands staining with Coomassie blue and at least five bands staining with Coomaisse blue and at least five bands reacting with periodic acid-Schiff (PAS). All of the proteins in the antigenic fraction appear to contain carbohydrate residues. Upon immunoelectrophoresis the antigen yielded four precipitin arcs. The antigenic fraction isolated by means of the immunoaffinity column was then compared to various fractions obtained from concanavalin A (Con A) chromatography of SEA. The results of Ouchterlony immunodiffusion and immunoelectrophoresis indicate that the antigenic fraction isolated by immunoaffinity purification of SEA contains the major antigens found in the fractions obtained from Con A chromatography of SEA. The results of SDS gel electrophoresis indicate that the major PAS-reacting bands of the antigenic fraction isolated by immunoaffinity purification are found in the 3rd peak (bound fraction) resulting from Con A chromatography of SEA, whereas the major Coomaisse blue-staining band in the isolated antigenic fraction is found in the 2nd peak (unbound fraction) from Con A chromatography of SEA.     

Schistosoma japonicum soluble egg antigens induce apoptosis and inhibit activation of hepatic stellate cells: a possible molecular mechanism

Hepatic stellate cells play a key role in the development of hepatic fibrosis. Activated hepatic stellate cells can be reversed to a quiescent-like state or apoptosis can be induced to reverse fibrosis. Some studies have recently shown that Schistosoma mansoni eggs could suppress the activation of hepatic stellate cells and that soluble egg antigens from schistosome eggs could promote immunocyte apoptosis. Hence, in this study, we attempt to assess the direct effects of Schistosoma japonicum soluble egg antigens on hepatic stellate cell apoptosis, and to explore the mechanism by which the apoptosis of activated hepatic stellate cells can be induced by soluble egg antigens, as well as the mechanism by which hepatic stellate cell activation is inhibited by soluble egg antigens. Here, it was shown that S. japonicum-infected mouse livers had increased apoptosis phenomena and a variability of peroxisome proliferator-activated receptor γ expression. Soluble egg antigens induce morphological changes in the hepatic stellate cell LX-2 cell line, inhibit cell proliferation and induce cell-cycle arrest at the G₁ phase. Soluble egg antigens also induce apoptosis in hepatic stellate cells through the TNF-related apoptosis-inducing ligand/death receptor 5 and caspase-dependent pathways. Additionally, soluble egg antigens could inhibit the activation of hepatic stellate cells through peroxisome proliferator-activated receptor γ and the transforming growth factor β signalling pathways. Therefore, our study provides new insights into the anti-fibrotic effects of S. japonicum soluble egg antigens on hepatic stellate cell apoptosis and the underlying mechanism by which the liver fibrosis could be attenuated by soluble egg antigens.     

Nutritional requirements of Schistosoma japonicum eggs

Newly laid eggs of Schistosoma japonicum were cultured in a serum-free, chemically defined medium, RPMI 1640, which contained 20 amino acids, glutathione, 11 vitamins, and glucose in a balanced salt solution. The requirements for these components in the nutrition of the eggs was investigated by the deletion of single component from the medium. The following 14 amino acids were shown to be essential for the full development of the egg in the medium: L-arginine, L-cystine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. Choline chloride was the essential vitamin. The omission of nicotinamide from the medium affected maturation adversely. Glucose was also required by the eggs. Minimal concentration of glucose for maturation of the eggs was 0.02 mM, but concentrations ranging from 0.16 to 20.00 mM gave better results while the concentration of the other elements of the medium were kept constant.     

The C-type lectin L-SIGN differentially recognizes glycan antigens on egg glycosphingolipids and soluble egg glycoproteins from Schistosoma mansoni

Recognition of pathogen-derived carbohydrate constituents by antigen presenting cells is an important step in the induction of protective immunity. Here we investigated the interaction of L-SIGN (liver/lymph node specific ICAM-3-grabbing nonintegrin), a C-type lectin that functions as antigen receptor on human liver sinusoidal endothelial cells, with egg-derived glycan antigens of the parasitic trematode Schistosoma mansoni. Our data demonstrate that L-SIGN binds both schistosomal soluble egg antigens (SEA) and egg glycosphingolipids, and can mediate internalization of SEA by L-SIGN expressing cells. Binding and internalization of SEA was strongly reduced after treatment of SEA with endoglycosidase H, whereas defucosylation affected neither binding nor internalization. These data indicate that L-SIGN predominantly interacts with oligomannosidic N-glycans of SEA. In contrast, binding to egg glycosphingolipids was completely abolished after defucosylation. Our data show that L-SIGN binds to a glycosphingolipid fraction containing fucosylated species with compositions of Hex(1)HexNAc(5-7)dHex(3-6)Cer, as evidenced by mass spectrometry. The L-SIGN "gain of function" mutant Ser363Val, which binds fucosylated Lewis antigens, did not bind to this fucosylated egg glycosphingolipid fraction, suggesting that L-SIGN displays different modes in binding fucoses of egg glycosphingolipids and Lewis antigens, respectively. Molecular modeling studies indicate that the preferred binding mode of L-SIGN to the respective fucosylated egg glycosphingolipid oligosaccharides involves a Fucalpha1-3GalNAcbeta1-4(Fucalpha1-3)GlcNAc tetrasaccharide at the nonreducing end. In conclusion, our data indicate that L-SIGN recognizes both oligomannosidic N-glycans and multiply fucosylated carbohydrate motifs within Schistosoma egg antigens, which demonstrates that L-SIGN has a broad but specific glycan recognition profile.     

Chicken egg yolk antibodies (IgY) for detecting circulating antigens of Schistosoma japonicum

BackgroundIgY isolated from egg yolk has been widely used in immunodiagnostic tests, including tests to detect circulating antigen (soluble egg antigen or SEA) of Schistosoma japonicum.ResultsA sandwich ELISA was established using a combination of anti-S. japonicum SEA-IgY polyclonal antibodies and IgM monoclonal antibodies. To explore sensitivity and specificity of the sandwich ELISA, serum samples from 43 patients infected with S. japonicum were tested. All acute cases and 91.3% of the chronic cases showed a positive reaction. Only 5% of the control sera from healthy persons gave a positive response. Cross-reactions with antibodies to nine other parasites were rare.ConclusionThe developed immunoassay is reasonably sensitive and specific. It could be used for field research and treatment efficacy assessments.     

Structural characterization of glycosphingolipids from the eggs of Schistosoma mansoni and Schistosoma japonicum

The granulomatous pathology in human intestinal schistosomiasisis induced primarily by the egg antigens of schistosome, a parasitictrematode. Glycolipids and glycoproteins were extracted fromthe eggs of the two major species which infect human, Schistosomamansoni and Schistosoma japonicum, for structural characterizationbased on highly sensitive mass spectrometric analysis coupledwith chemical derivatization. Here, we demonstrate that a seriesof uniquely multifucosylated glycosphingolipids constitute themajor egg glycolipids of S.mansoni but not of S.japonicum. TheS.mansoni glycosphingolipids were found to be extended by varyingnumbers of an unusual repeating unit,     

Cytokine regulation in experimentally-induced Schistosoma japonicum egg granuloma formation

The formation of granulomas in host tissues in response to trapped Schistosoma japonicum eggs is central to the etiology of schistosomiasis. However, analysis of the host hypersensitivity reactions that result in granuloma formation, in schistosome infection, is not without difficulty. This is due, in part, to the fact that the parasites continuously deposit their eggs as clusters. In order to synchronize host reactions, we established an experimental model of hepatic granuloma formation whereby in vitro laid schistosome eggs are implanted directly into normal and cytokine-deficient mice livers. This model, validated by comparison with an infection model, was used to analyze cytokine regulation of granuloma formation around S. japonicum eggs. Combined models of implantation and cercarial infection were also studied. With special reference to IL-4, IL-13, IFN-γ and IL-18, our in vitro schistosome egg implantation model has shed new light on the roles of cytokines in both the acute and chronic stages of schistosome egg-induced granuloma formation.     

Enzyme activities in Schistosoma mansoni soluble egg antigen

Schistosoma mansoni egg antigens are mostly responsible for the granulomatous pathology in human intestinal schistosomiasis. Several previous studies have indicated that the induction of an immune response against some parasite enzymes may protect against pathology. The present work was designed to identify enzyme activities present in a standard soluble egg antigen (SEA) preparation. Simple colorimetric analyses were performed incubating SEA with 2-naphthyl, 2-naphthylamide (2NA), or p-nitrophenyl substrates at different pHs in the absence of added effectors. Results showed prominent acid phosphatase (pH 5.4), alkaline phosphatase (pH 8.5), and N-acetyl-beta-glucosaminidase (pH 5.4) activities. Relevant peptidase activities were also detected at pH 6.5-7.5 against 2NA derivatives of (1) aliphatic (alpha-Ala > beta-Ala > Leu > Met > S-benzyl-Cys), polar (Ser > Gln), basic (Arg > Lys > ornithine), and acidic (Glu) amino acids; (2) dipeptides: X-Ala (X = Gly > Leu > Lys > Asp), X-Arg (X = Ala > Arg > Phe > Gly > Pro > Asp), Ser-Met, and Phe-Pro; and (3) tripeptides (Ala-Phe-Pro > Phe-Pro-Ala). The data demonstrated that S. mansoni SEA contains a rich set of hydrolases with different specificities that might play a role in the egg physiology and possibly also in the host-parasite relationships.     

Forensic study of Schistosoma japonicum eggs found in an autopsy case

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Immunoglobulins inside Schistosoma japonicum eggs from the livers of mice

Using fluorescent antibody techniques, immunoglobulins (Ig's), mainly IgG class, were detected inside Schistosoma japonicum eggs lodged in mouse liver. Ig's were observed as a focal pattern between the miracidium and the eggshell during early infection (5-7 wk), particularly in lightly infected mice (20 cercariae). With advancement of time of infection (8-18 wk), a diffuse pattern of staining over the miracidial body developed and became predominant. The diffuse pattern of staining could be observed in the eggs taken from heavily infected mice (50 cercariae), during early stage. Eggs showing the focal pattern in a restricted area appeared to be morphologically intact, whereas eggs showing the diffuse pattern exhibited some types of eggshell destruction. We conclude that the focal pattern reflects disintegration of eggs in the initial stage and the diffuse pattern in the advanced stage. This spatial relationship between Ig's and eggs is discussed in relation to destruction of eggs.     

Schistosoma mekongi and Schistosoma japonicum: Differences in the distribution of eggs in the viscera of mice

The difference in the distribution of Schistosoma eggs in the viscera has not been clearly elucidated in the two closely related species Schistosoma japonicum and Schistosoma mekongi. In this study, we quantitatively compared the distribution of eggs in mice infected with the two species. In S. mekongi-infected mice, 56.6% to 69.4% of total eggs were found in the distal small intestine 9 to 15 weeks after infection, while in S. japonicum-infected mice, 48.8% to 71.8% of eggs were found in the proximal small intestine during the same period. There were significantly more eggs in the liver in mice infected with S. japonicum than in those infected with S. mekongi. The number of adult worms recovered did not differ between the two species during the study period. The total number of eggs laid in the tissues also did not differ between the two species at 12 to 15 weeks postinfection, but in the earlier period the total number of eggs was significantly fewer in S. mekongi-infected than in S. japonicum-infected mice, suggesting the delayed maturation of the former compared with the latter. These results clearly show that S. japonicum and S. mekongi exhibit different oviposition behavior in their hosts.     

The ability of single sex infections of Schistosoma japonicum to induce resistance to reinfection in mice

In four experiments, mice harbouring an average 50, 76 or over 200 Schistosoma japonicum female worms were not resistant when challenged six to eight weeks after infection. The female worms from these single sex infections were stunted and immature (average length 4.6 mm) and induced no overt pathology in the host. Male worm burdens of 60, 135 or 140 also induced little or no resistance to challenge in the host. The males from these single sex infections were fully grown for their age (average length 9 mm) and burdens of 135 or 140 induced distended hepatic portal veins and marked deposition of pigment in the livers of infected mice.