ARTICLE |
Correspondence to: Cecilia Thors, Swedish Institute for Infectious Disease Control (SMI), Solna S-171 82, Sweden. E-mail: cecilia.thors@smi.ki.se
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Summary |
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KLH (Keyhole limpet hemocyanin) is highly immunogenic, and crossreactive epitopes occur widely in nature. In schistosomiasis, infected hosts generate antibodies reactive with KLH. This is of diagnostic importance but we lack detailed information on the immunogen-carrying molecules and their distribution in the worm. We used anti-KLH antibodies to localize cross-reacting epitopes in the various developmental stages of the parasite in experimental Schistosoma mansoni infection. The staining results show KLH crossreactivity in the life stages of the parasite. By immunoblotting we show that KLH-crossreactive antigenic epitopes in the parasite eggs are carbohydrates, also recognized by antibodies against soluble schistosome egg antigens. The localizations in the larval stages and in adult worms suggest that crossreacting antigenic epitopes are secretory products.
(J Histochem Cytochem 51:13671373, 2003)
Key Words: Schistosoma mansoni, schistosomiasis, keyhole limpet hemocyanin, (KLH), crossreactivity, immunohistochemistry, carbohydrate epitopes, differentiation
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Introduction |
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KEYHOLE LIMPET HEMOCYANIN (KLH) is highly immunogenic, a property of interest from diverse points of view, ranging from use of KLH as an immunological adjuvant and in active immunotherapy of carcinomas (
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Materials and Methods |
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S. mansoni Infection in Mice
The schistosome life cycle was maintained as described previously (
Antigen Preparations
Frozen sections of adult schistosomes and egg granuloma- and worm-containing tissues mounted on microscope slides were fixed in acetone at room temperature (RT) for 10 min. Paraffin sections of formalin-fixed material were also used as antigen.
Preparations of cercariae, to be used as target antigen, were made in two ways. Living cercariae were added in aliquots of 10 µl on microscope slides, coated with human skin lipids (
For some experiments, suspensions of isolated eggs were allowed to "hatch" in water. Released miracidia and "hatch fluid" were used as antigen for IF staining. Schistosomula were prepared as described (
Antisera and Lectins
Commercially available rabbit anti-KLH antibodies (H-0892; Sigma) were used at a 1:40 dilution for IF staining. A mouse monoclonal antibody (MAb) against paramyosin (
Immunofluorescence Microscopy
Staining was performed as described previously (
IF microscopy was performed using a Leica DMRB fluorescence microscope equipped with filters for FITC fluorescence essentially as described previously (
SDS-PAGE and Western Blotting
SEA (48 µg per gel) and KLH (20 µg per gel) were separated in 10.6% SDS-PAGE with a 3% stacking gel under reducing or non-reducing conditions and transferred onto nitrocellulose membranes (
For deglycosylation of the antigens on nitrocellulose membranes, incubation with periodate was performed as described (
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Results |
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By immunofluorescence, rabbit anti-KLH antibodies reacted with the different developmental stages of the parasite (Table 1). The reaction was similar in paraffin and frozen sections. Schistosome eggs in both the liver and the gut wall were positive and the staining appeared rather homogeneous. Interestingly, the perioval tissue showed various degrees of diffuse staining in the close vicinity of the autofluorescent eggshells (Fig 1A and Fig 1B). Reactive material was frequently seen as granular deposits in the periphery of granulomas (Fig 1B). Eggs, hatched in vitro, showed anti-KLH reactivity mainly at the internal surface of the eggshell, and distinct staining of the miracidial surface could be seen (Fig 1C). The cercarial surface showed strong reactivity (Fig 2A) and the cercarial secretions also reacted with anti-KLH antibodies (Fig 2B and Fig 2C). Cercariae transformed into schistosomules retained strong surface reactivity with anti-KLH antibodies (Fig 3A). Control staining with anti-PM antibodies indicated that the schistosomular surface was intact (Fig 3B). In adult worms in both frozen sections (Fig 1E) and paraffin sections (Fig 4A4D), secretory ducts were seen in male and female worms. In addition, both the ovary and the testis were stained (Fig 4B and Fig 4C). In female worms, connective tissue stroma stained strongly, e.g., as septa surrounding groups of vitelline cells (Fig 4A and Fig 4B). In sections treated with periodate, staining was negative or significantly weaker (Fig 4E). Control sections stained with anti-SEA antibodies showed a similar staining pattern as anti-KLH antibodies (Fig 4F4J). Thin fluorescent threadlike structures seemed to be extensions of subtegumental cells localized close to strongly stained ducts and ended as knobs located at the center of tubercles in males (Fig 4D and Fig 4H).
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In Western blotting, rabbit anti-KLH and anti-SEA antibodies appear to recognize in part the same KLH components (Fig 5). However, all reactivity of anti-SEA antibodies with KLH was abolished after periodate treatment, whereas only a slight reduction in the staining intensity of KLH bands in the 5060-kD region was seen after staining with anti-KLH antibodies. When tested on periodate-treated SEA, all anti-KLH reactivity disappeared. A major 100-kD band in non-reduced SEA samples disappeared on reduction, and in the
50-kD region a band recognized by anti-SEA antibodies was periodate-sensitive.
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Discussion |
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KLH elicits a strong immune response in vertebrates. Interestingly, shared antigenic epitopes occur in schistosomes. These common epitopes of schistosomes are major immunogens in schistosomiasis, which is reflected by the use of KLH as a diagnostic antigen. In the present study we used anti-KLH antibodies to localize the KLH-crossreacting parasite components that may give rise to an immune response in schistosomiasis.
The major crossreacting immunogenic epitopes of KLH have been demonstrated in preparations of schistosome eggs ("soluble egg antigens," SEA) (1
3)GalNAc motif (
Considering the observed intra- and perioval distribution, KLH-crossreactive antigenic material may participate in granuloma formation by serving both as immunogen and as target for an immune response.
Our results show that KLH cross-reactive immunogens are localized at the surface of cercaria, cercarial secretions, and schistosomula, the invading larval stage.
Cercarial excretion products, originating from the penetration glands, "kissing marks" (
Based on the observed localization of KLH-crossreactive material, the antigen appears to be both surface-associated and excreted in the larvae, the cercariae/schistosomula, and miracidia. In contrast to the surface staining seen in larval forms, no surface staining was seen in the adult worms. This is consistent with the fundamental ultrastructural difference between schistosomula and adult schistosome surface membranes (
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Acknowledgments |
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Supported in part by the Swedish International Development Cooperation Agency.
We thank Aija Lassfolk for expert technical support.
Received for publication December 27, 2002; accepted May 7, 2003.
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