Wilton E. Vannier

Surface antigens of Schistosoma mansoni

Abstract The outer tegument membrane of 18 h artificially prepared schistosomula of Schistosoma mansoni was labelled using the non-permeant diazonium salt of [ 125 I]iodosulphanilic acid. Eight iodinated surface proteins were identified by SDS-polyacrylamide gel electrophoresis. Three of these proteins, one of which is glycosylated, can be precipitated by immune serum.

Schistosoma mansoni: Analysis of surface membrane carbohydrates using lectins

Surface tegumental membrane sugars of cercariae, schistosomules, and lung- and liver-stage Schistosoma mansoni were examined using lectins specific for mannose, glucose and glucosamine (concanavalin A), N-acetylglucosamine (wheat germ agglutinin), N-acetylgalactosamine (soybean agglutinin), and fucose (Ulex europaeus agglutinin). Only concanavalin A (Con A) and wheat germ agglutinin (WGA) bound to schistosomules and lung and liver stages. Weak binding of Con A only was detected on cercariae. Binding of Con A to 18-hr schistosomules was highly specific, although nearly 40% of the binding of WGA to 18-hr schistosomules was nonspecific. The number of binding sites per schistosomule was estimated to be 6 × 106 for Con A and 150 × 106 for WGA. Competitive inhibition experiments indicated that Con A and WGA were bound to different receptors on schistosomules. Enzyme treatment with trypsin and Pronase failed to remove receptors for these two lectins from the schistosomule membrane, and exposure to neuraminidase did not alter lectin binding. These enzymes also failed to reveal cryptic receptors for soybean agglutinin and Ulex europaeus agglutinin. It is suggested that the sugar receptors for Con A and WGA are either inaccessible to enzymes or are incorporated into polysaccharides or glycolipids rather than glycoproteins.

ISOLATION AND CHARACTERIZATION OF SURFACE ANTIGENS FROM SCHISTOSOMA MANSONI. I. EVALUATION OF TECHNIQUES FOR RADIOISOTOPE LABELING OF SURFACE PROTEINS FROM ADULT WORMS

Radiolabeled surface proteins of adult Schistosoma mansoni were prepared by in vitro labeling of whole worms, and by labeling freeze-thaw surface membrane extracts. Incorporation of 125I into surface proteins was attempted using the lactoperoxidase, chloramine-T, iodosulfanilic acid, and Bolton-Hunter methods. Radiolabeling of whole worms with lactoperoxidase, chloramine-T and iodosulfanilic acid yielded a single protein peak (mol wt greater than 100,000) on SDS-PAGE, and showed considerable incorporation of label in the lipid fraction. Bolton-Hunter labeling of whole worms yielded four major peaks with molecular weights of 100,000, 60,000, 30,000 and 21,000, and minor peaks with molecular weights of 26,000, 36,000, 43,000, 68,000 and 78,000; three of the four major peaks corresponded to prominent bands in Coomassie blue-stained gels. Although carbohydrate-labeling techniques were not successful, a single carbohydrate band, molecular weight greater than 100,000, was detected was PAS staining. Radiolabeling of freeze-thaw extracts yielded results similar to those obtained with whole worms. Electron microscopy revealed the tegument to be left intact and undamaged after labeling with the Bolton-Hunter reagent.

A starch block electrophoresis study of aqueous house dust extracts.

Abstract 1.1. The approximate chemical compositions of four dust extracts, including the Hollister-Stier and Endo commercial preparations and two extracts prepared in our laboratory, were determined. These materials were found to consist of a heterogeneous mixture of acidic polysaccharides with varying amounts of polypeptide. 2.2. The materials were investigated by starch block electrophoresis in acetate buffer (pH 4.7, μ = 0.1). In general, a fast (negatively charged) and a slow peak were found. The allergen activity was predominately associated with the materials of slow or intermediate mobility. In some cases the fractions seemed to be more active than the unfractionated extract. 3.3. No simple relation was evident between the monosaccharide composition or the nitrogen content of the fractions and the allergen activity. 4.4. The color moved slightly faster than the fast peak and was definitely separated from the more active fractions. 5.5. Experiments were presented that indicated that the polypeptide moiety of the dust extract was linked to the polysaccharides by covalent bonds.

Isolation and characterization of surface antigens from Schistosoma mansoni. II. Antigenicity of radiolabeled proteins from adult worms.

Adult Schistosoma mansoni were radiolabeled in vitro with 125I Bolton-Hunter reagent. Surface membrane antigens were solubilized with non-ionic detergent, then reacted with infection or normal serum. The antigen-antibody complexes were then precipitated with staphylococcal protein A immunoadsorbent, eluted with urea and SDS, and fractionated by SDS-PAGE. The results indicated the presence of 6 to 8 tegument antigens, depending on the type of antisera used. Human antisera to S. japonicum and S. haematobium reacted with some but not all of the antigens identified with human S. mansoni infection serum; this implies the presence of species-specific tegument antigens. The molecular weights of the radiolabeled antigens ranged from 10,000 to 100,000. A large (greater than 100,000) molecular weight glycoprotein and an uncharacterized lipid fraction appeared to be precipitated nonspecifically. Immunoprecipitation methods with anti-mouse IgG and anti-mouse whole serum failed to detect the presence of hostlike antigens in the labeled extracts. Several of the labeled proteins from S. mansoni were found to react with serum from patients infected with either S. haematobium or with S. japonicum.

The isolation and characterization of a purified house dust allergen fraction

Abstract 1.1. An active house dust allergen fraction, low in nitrogen and colorless, has been isolated. 2.2. The material has been found to show activity with most, but not all, persons sensitive to less purified commercial preparations. This suggests the presence of more than one allergen specificity. 3.3. The dust fraction was found to be heterogeneous by electrophoretic and sedimentation analysis. Two main peaks were demonstrated by electrophoresis. The average sedimentation coefficient (Sw, 20) was found to be 2.6S. 4.4. The chemical composition was found to be roughly 5 per cent polypeptide and 95 per cent polysaccharide, with about equal amounts of uronic acid (probably glucuronic acid), D-glucose, D-galactose, and D-mannose as well as lesser amounts of L-rhamnose and L-arabinose. 5.5. The relationship of the composition and physical properties of the dust allergen fraction to current ideas concerning the origin of the house dust allergen was discussed.

Schistosoma mansoni: immunization of cynomolgus monkeys by injection of irradiated schistosomula.

Abstract Although the immunization of primates with irradiated schistosome cercariae has been demonstrated, no success has been reported by injection with the irradiated schistosomule stage. The present investigation was designed to test whether cynomolgus monkeys could be protectively immunized with 60 Co-irradiated Schistosoma mansoni schistosomula. Monkeys injected once with 10 4 irradiated schistosomula (50 krad at 4 krad/min) had 52% fewer challenge worms than the control group at necropsy. Four immunizations did not induce a higher level of resistance. At 50 days post-challenge, the immunized monkeys excreted 80% fewer eggs than did the control animals. An attempt to enhance irradiated schistosomule-induced protection with tetramisole · HCl was unsuccessful.

The preparation and properties of a hapten-cellulose antibody adsorbent

Abstract The preparation of a benzenearsonic acid cellulose derivative for use in the specific purification and fractionation of antibodies directed against the benzenearsonic acid determinant is described. The method involves the coupling of tyramine to insoluble carboxymethyl cellulose by the use of N , N ′ dicyclohexylcarbodiimide and the subsequent coupling of diazotized p -arsanilic acid to the tyramine. The adsorbents had a capacity of 5–10 mg of antibody per gram of adsorbent, depending upon the extent of hapten coupling. By the use of this adsorbent, essentially all the anti-benzenearsonic acid antibody could be removed from antisera. Elution with a phosphate-sodium chloride buffer, pH 3·0, and subsequent elution with 0·1 n hydrochloric acid removed approximately 75–80 per cent of the antibody from the adsorbent.

Sedimentation studies of skin-sensitizing antibody.

Abstract Serums from allergic patients have been fractionated by preparative and analytic ultracentrifugation into fractions devoid of the S19 components and fractions containing S19 components. Skin-sensitizing activity has been found to be associated with both kinds of fractions. The skin-sensitizing activity could not be correlated with the S19 globulin fraction alone.

Partial characterization of radiolabelled antigens from adult Schistosoma haematobium.

Previously, Bolton-Hunter labeled adult S. mansoni proteins were fractionated by SDSPAGE to determine molecular weights and immunoprecipitated using human infection sera to detect cross-reactivity (Hayunga et al., 1979, J. Parasitol. 65: 488-496; 497-506). These observations are now extended to S. haematobium. In order to provide a direct comparison, the conditions of radiolabeling, detergent extraction and molecular weight determination were kept identical to those of the previous study. Cercariae from an Egyptian strain of S. haematobium in naturally infected Bulinus truncatus were used to infect golden hamsters in the laboratory. (We are grateful to Drs. P. LoVerde, K. Murrell, and V. Schinski for collecting infected snails in the field, and to Miss K. Groover, Mr. D. Olson, and K. Holman for maintaining the life cycle in the laboratory.) Approximately 20 to 30 adult worms, obtained by portal perfusion of 12-wk infected hamsters, were radiolabeled with 0.5 mCi 125I-Bolton-Hunter reagent as described previously (Hayunga et al., loc. cit.); the worms were motile and appeared undamaged after this procedure. Tegumental proteins were solubilized in 0.5% NP-40 for 15 min at 37 C, and the lysate run directly on SDS gels or immunoprecipitated as follows: Aliquots (approx. 100,000 c.p.m.) of labeled lysate were incubated overnight at 4 C with 25 ,ul serum, followed by incubation for 2 hr with 500 ,ul of a 10% w/v suspension of staphylococcal protein A (Pansorbin?, Calbiochem); after washing 3 times with BBS, samples were eluted with 4% SDS-6 M urea and fractionated by SDS-PAGE on 10% tube gels. Serum from patients infected by either S. haematobium or S. mansoni was provided by Dr. E. Higashi, NAMRU-3, Cairo; rabbit serum was prepared by subcutaneous inoculation of a freeze-thaw preparation of adult S. mansoni. Fractionation of radioiodinated S. haematobium proteins on SDS gels revealed heavily labeled peaks of >100,000, 60,000, 30,000, and 13,000 mol. wt., and minor peaks of 85,000, 43,000, 36,000, 33,000, 25,000, 23,000, and 18,000 mol. wt. (Fig. 1A). Label also occurred in two peaks, presumed to contain lipid material, that migrated with and slightly ahead of the dye front. When samples were electrophoresed under reducing conditions using dithiothreitol, the large (>100,000) molecular weight peak dissociated but no additional peaks were found (Fig. 1B). In order to detect cross-reactivity, the labeled lysate from