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Protection against invasive amebiasis by a single monoclonal antibody directed against a lipophosphoglycan antigen localized on the surface of Entamoeba histolytica.

Marinets A, Zhang T, Guillén N, Gounon P, Bohle B, Vollmann U, Scheiner O, Wiedermann G, Stanley SL, Duchêne M - J. Exp. Med. (1997)

Bottom Line: Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A.Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess.Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.

View Article: PubMed Central - PubMed

Affiliation: Institute for Specific Prophylaxis and Tropical Medicine, A-1095 Vienna, Austria.

ABSTRACT
A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.

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Localization of the antigen binding  to antibody EH5 in E. histolytica trophozoites by  indirect immunofluorescence using confocal microscopy. (A) Amebas treated with methanol; (B)  amebas treated with Triton X-100 for stronger  permeabilization. Planes a, b, and c are horizontal  planes, and plane d is a vertical plane through the  middle of the ameba.
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Figure 5: Localization of the antigen binding to antibody EH5 in E. histolytica trophozoites by indirect immunofluorescence using confocal microscopy. (A) Amebas treated with methanol; (B) amebas treated with Triton X-100 for stronger permeabilization. Planes a, b, and c are horizontal planes, and plane d is a vertical plane through the middle of the ameba.

Mentions: To determine the cellular localization of the antigen recognized by the EH5 antibody in the amebas by independent means, immunolocalization experiments on the light and electron microscopic levels were performed. First, trophozoites were fixed and incubated with antibody EH5. Two different methods of fixation and permeabilization were used (see Materials and Methods). In method A, amebas were incubated in the presence of methanol. After labeling, trophozoites were analyzed by laser confocal microscopy (Fig. 5 A) at four different optical planes and revealed a labeling of the entire surface of the amebas. When fixed trophozoites were permeabilized by using Triton X-100 to solubilize the membrane lipids (method B), labeling with antibody EH5 and confocal microscopy (Fig. 5 B) revealed labeling of the periplasmic membrane as well as internal vesicle membranes. Secondary antibodies used alone in control experiments did not label the amebas (results not shown).


Protection against invasive amebiasis by a single monoclonal antibody directed against a lipophosphoglycan antigen localized on the surface of Entamoeba histolytica.

Marinets A, Zhang T, Guillén N, Gounon P, Bohle B, Vollmann U, Scheiner O, Wiedermann G, Stanley SL, Duchêne M - J. Exp. Med. (1997)

Localization of the antigen binding  to antibody EH5 in E. histolytica trophozoites by  indirect immunofluorescence using confocal microscopy. (A) Amebas treated with methanol; (B)  amebas treated with Triton X-100 for stronger  permeabilization. Planes a, b, and c are horizontal  planes, and plane d is a vertical plane through the  middle of the ameba.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199119&req=5

Figure 5: Localization of the antigen binding to antibody EH5 in E. histolytica trophozoites by indirect immunofluorescence using confocal microscopy. (A) Amebas treated with methanol; (B) amebas treated with Triton X-100 for stronger permeabilization. Planes a, b, and c are horizontal planes, and plane d is a vertical plane through the middle of the ameba.
Mentions: To determine the cellular localization of the antigen recognized by the EH5 antibody in the amebas by independent means, immunolocalization experiments on the light and electron microscopic levels were performed. First, trophozoites were fixed and incubated with antibody EH5. Two different methods of fixation and permeabilization were used (see Materials and Methods). In method A, amebas were incubated in the presence of methanol. After labeling, trophozoites were analyzed by laser confocal microscopy (Fig. 5 A) at four different optical planes and revealed a labeling of the entire surface of the amebas. When fixed trophozoites were permeabilized by using Triton X-100 to solubilize the membrane lipids (method B), labeling with antibody EH5 and confocal microscopy (Fig. 5 B) revealed labeling of the periplasmic membrane as well as internal vesicle membranes. Secondary antibodies used alone in control experiments did not label the amebas (results not shown).

Bottom Line: Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A.Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess.Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.

View Article: PubMed Central - PubMed

Affiliation: Institute for Specific Prophylaxis and Tropical Medicine, A-1095 Vienna, Austria.

ABSTRACT
A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.

Show MeSH
Related in: MedlinePlus