The protozoan parasite Entamoeba histolytica causes amebic dysentery and amebic liver abscess, diseases associated with significant morbidity and mortality worldwide. E. histolytica infection appears to involve the initial attachment of amebic trophozoites to intestinal epithelial cells, followed by lysis of these cells and subsequent invasion into the submucosa. A recent in vitro study (L. Eckmann, S. L. Reed, J. R. Smith, and M. F. Kagnoff, J. Clin. Invest. 96:1269-1279, 1995) demonstrated that incubation of E. histolytica trophozoites with epithelial cell lines results in epithelial cell production of inflammatory cytokines, including interleukin-1 (IL-1) and IL-8, suggesting that intestinal epithelial cell production of cytokines might play a role in the inflammatory response and tissue damage seen in intestinal amebiasis. To determine whether intestinal epithelial cell production of IL-1 and IL-8 occurs in response to E. histolytica infection in vivo and as an approach to studying the specific interactions between amebic trophozoites and human intestine, we used a SCID mouse-human intestinal xenograft (SCID-HU-INT) model of disease, where human intestinal xenografts were infected with virulent E. histolytica trophozoites. Infection of xenografts with E. histolytica trophozoites resulted in extensive tissue damage, which was associated with the development of an early inflammatory response composed primarily of neutrophils. Using oligonucleotide primers that specifically amplify human IL-1␤ and IL-8, we could demonstrate by reverse transcription PCR that mRNA for both IL-1␤ and IL-8 is produced by human intestinal xenografts in response to amebic infection. The increase in human intestinal IL-1␤ and IL-8 in response to invasive amebiasis was confirmed by enzyme-linked immunosorbent assays specific for human IL-1␤ and IL-8. Using immunohistochemistry, we confirmed that human intestinal epithelial cells were the source of IL-8 in infected xenografts and established that IL-8 production can occur at sites distal to areas of intestinal mucosal damage. These results demonstrate that human intestinal epithelial cells can produce inflammatory cytokines in response to infection in vivo and establish the SCID-HU-INT model as a system for studying the interactions between E. histolytica and human intestine.

Entamoeba histolytica is an enteric protozoan parasite that causes disease in humans by disruption and invasion of the colonic mucosa. A number of studies have shown that various soluble saccharide inhibitors will interfere with amoebic adherence to mammalian target cells in vitro (1), suggesting that amoebic lectins mediate amoebic adherence to mammalian cells. Based on this approach, two mechanisms of adherence of intact amoeba to mammalian cells have been described, one sensitive to inhibition with the monosaccharides N-acetylgalactosamine or galactose and a second inhibited by N-acetylglucosamine oligosaccharides .Studies on the binding specificity of various lectins have shown that sugars that are good lectin inhibitors in solution may not reflect the carbohydrate structure of receptors found on cell surfaces (2) . Furthermore, among lectins that interact with monosaccharides, several exhibit a pronounced preference for more complex oligosaccharides, suggesting that these lectins have extended binding sites (2). To obtain more information on the nature of the glycoconjugate receptors for E. histolytica, we measured the adhesion of E. histolytica to wild-type Chinese hamster ovary (CHO) cells and three lectin-resistant somatic cell mutants, WGAR 1021, WGAR 13, and RICK 15B (see Fig. 1). WGAR 1021 is deficient in membrane-bound sialic acid and has increased galactose residues at the nonreducing termini (3). WGAR 13 is deficient in both membrane bound sialic acid and galactose and has increased N-acetylglucosamine residues at the nonreducing termini (3). As a consequence of a deficiency in N-acetylglucosaminyltransferase I, RICK 15B cells lack Asn-linked complex (N-acetyllactosamine) units and accumulate Asn-linked oligomannosyl units in their glycoproteins (4, 5). We report here our analysis of E. histolytica trophozoite adherence to this panel of cells.

To model the initial pathogenic eflects of Entamoeba histolytica trophozoites on intestinal epithelial cells, the interactions of E. histolytica HM1-IMSS trophozoites with polarized human intestinal Caco-2 cell monolayers grown on permeabilized filters were examined. Trophozoites, when incubated with the apical surface of the monolayers at 37°C, induced a rapid decrease in transepithelial resistance over 15 to 60 min. The transmonolayer resistance response was not associated with changes in short-circuit current but was associated with an increase in mannitol flux, suggesting that the drop in resistance reflected a nonselective

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To study the relationship between carbohydrate-specific amebic cytoadherence and ameba-mediated cytotoxicity, we measured Entamoeba histolytica trophozoite-mediated cytolysis directed against a panel of four Chinese hamster ovary (CHO) cell lines that have defined alterations in their glycosylation patterns. We recently measured amebic trophozoite adherence to this panel of CHO cells and showed that trophozoites bind variant cells (RICR 15B), which are deficient in Asn-linked N-acetyllactosamine units, at 12% of the level observed for wild-type cells (E. Li, A. Becker, and S. L. Stanley, J. Exp. Med 167:1725Med 167: -1730Med 167: , 1988. Using a 51Cr release assay to measure trophozoite-mediated cytolysis, we demonstrate in this study that RICR 15B cells are less susceptible to trophozoite-mediated cytolysis than are wild-type cells. In addition, we found that N-acetyllactosamine, which inhibits trophozoite adherence to CHO cells, also inhibited trophozoite-mediated cytolysis of wild-type cells. These studies indicate that surface carbohydrates, on target cells can influence susceptibility to ameba-mediated cytotoxicity. This panel of CHO cells provides a useful model system for investigating the role of glycoconjugates in mediating amebic interactions with mammalian cells.