Taenia solium oncosphere adhesion to intestinal epithelial and Chinese hamster ovary cells in vitro

Abstract

The specific mechanisms underlying Taenia solium oncosphere adherence and penetration in the host have not been studied previously. We developed an in vitro adhesion model assay to evaluate the mechanisms of T. solium oncosphere adherence to the host cells. The following substrates were used: porcine intestinal mucosal scrapings (PIMS), porcine small intestinal mucosal explants (PSIME), Chinese hamster ovary cells (CHO cells), epithelial cells from ileocecal colorectal adenocarcinoma (HCT-8 cells), and epithelial cells from colorectal adenocarcinoma (Caco-2 cells). CHO cells were used to compare oncosphere adherence to fixed and viable cells, to determine the optimum time of oncosphere incubation, to determine the role of sera and monolayer cell maturation, and to determine the effect of temperature on oncosphere adherence. Light microscopy, scanning microscopy, and transmission microscopy were used to observe morphological characteristics of adhered oncospheres. This study showed in vitro adherence of activated T. solium oncospheres to PIMS, PSIME, monolayer CHO cells, Caco-2 cells, and HCT-8 cells. The reproducibility of T. solium oncosphere adherence was most easily measured with CHO cells. Adherence was enhanced by serum-binding medium with >5% fetal bovine serum, which resulted in a significantly greater number of oncospheres adhering than the number adhering when serum at a concentration less than 2.5% was used (P < 0.05). Oncosphere adherence decreased with incubation of cells at 4 degrees C compared with the adherence at 37 degrees C. Our studies also demonstrated that T. solium oncospheres attach to cells with elongated microvillus processes and that the oncospheres expel external secretory vesicles that have the same oncosphere processes.

FIG. 1.

(Panel i) T. solium oncospheres adhering to PIMS. Oncosphere adhesion was visualized by light microscopy using PAS. Magnification, ×40. Arrows A, oncospheres adhering to PIMS; arrows B, oncosphere hooks. (Panel ii) T. solium oncosphere binding to viable monolayer CHO cells. The arrows indicate secretory vesicles. Magnification, ×40. (Panel iii) T. solium oncosphere binding to fixed monolayer CHO cells visualized using PAS. The arrows indicate one pair of penetration glands. Magnification, ×100. (Panel iv) Light microscopy of 3-μm porcine small intestinal section embedded in paraffin and stained with H&E (20-min incubation period). An oncosphere (arrow) is interacting with epithelial cells. Magnification, ×400. (Panels v and vi) Photographs of frozen 8-μm sections, obtained using light and UV microscopy, showing oncospheres (arrows) identified by the presence of hooks and immunofluorescence (20-min incubation period; magnification, ×100). A light microscopy photograph with Evans blue (panel v) and a UV microscopy photograph (panel vi) show the same field of view. Material stained with a contrast agent appears to be blue under visible light and red under UV light, while fluorescence is green under UV light.

FIG. 2.

Ninety-five percent confidence intervals for oncospheres adherent on unfixed monolayer CHO cells after 30 min and 1.5, 3, and 25 h of incubation at 37°C.

FIG. 3.

(a) Ninety-five percent confidence intervals for the number of oncospheres adhering on unfixed CHO-K1 cells after 1.5 h of incubation at 37°C with binding medium with serum and binding medium without serum (RPMI), obtained using 24-, 48-, and 72-h mature monolayer CHO cells. (b) Curve and 95% confidence intervals for the number of oncospheres adhering on unfixed CHO-K1 cells after 1.5 h of incubation at 37°C with binding medium with different fetal bovine serum concentrations (0, 1, 2.5, 5, 7.5, 10, 15, and 20%), determined with 48-h mature monolayer CHO-K1 cells.

FIG. 4.

Curve and 95% confidence intervals for the number of oncospheres adhering on unfixed CHO-K1 cells after 1.5 h of incubation at different temperatures.

FIG. 5.

Scanning electron micrographs of T. solium oncospheres adhering to fixed monolayer CHO-K1 cells. (a) T. solium oncospheres attached to the surface of CHO cells by elongated microvilli. (b) Secretory vesicles (arrows) that are present outside the oncosphere membrane. Microvilli are on the surface membrane of the oncosphere and secretory vesicles. (c) Elongate microvilli that are attached to the surface of CHO cells. Hooks are present outside the oncosphere.

FIG. 6.

(a) Transmission electron microscopy of activated T. solium oncosphere. The arrows indicate microvilli (MV) that are under the oncosphere membrane (OM). (b) Transmission electron microscopy of activated T. solium oncosphere (Onc) adhering to pig small intestinal cell (IC). The large arrow indicates the microvilli (MV).

FIG. 7.

UV microscopy (magnification, ×100) of T. solium oncospheres adhering to viable monolayer CHO cells. (a and b) Activated T. solium oncospheres without oncosphere membrane attached to the surface of CHO cells by elongated microvilli. The arrows indicate the elongated microvilli and secretory vesicles that are present outside the oncosphere membrane. (c) Activated T. solium oncosphere with oncosphere membrane adhering to a viable monolayer CHO cell.