Anisakis simplex: larval excretory secretory protein production and cytostatic action in mammalian cell cultures

Abstract

Excretory secretory proteins produced in vitro by Anisakis simplex larvae incubated in Medium 199 or phosphate buffered saline with dextrose are similar with respect to protein content and biological activity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the molecular weight of the component(s) responsible for inhibition of mitogen induced lymphocyte blastogenesis is between 66,000 and 95,000. In vitro production of excretory secretory protein, approximately 1 microgram/24 hr by a single larva, was sufficient to inhibit lymphocyte blastogenesis. Serum from a human anisakiasis patient reacted with these proteins in immunoblots, indicating that, during invasion of the gastric mucosa, enough of them are produced in vivo to induce an immune response. The excretory secretory proteins significantly inhibited proliferation of transformed mammalian cell lines of lymphoid (P3/X63-Ag8) and epithelioid (HeLa) origin. As in mitogen stimulated lymphocytes, the inhibitory effect was cytostatic rather than cytotoxic. These findings suggest that, in addition to being potent immunogens, larval excretory secretory proteins are produced in sufficient quantity to modulate the host response in anisakiasis.