. ANISAKIS simplex antigens enhancer Th2 responses in animals and man (1±3). We conducted experiments to evaluate in vitro antigenspeci®c lymphocyte proliferation responses, as well as IL-4 production, with spleen cells from immunized mice. BABL/c mice were injected intramuscularly with 1 mg of crude A. simplex antigen in Freund's complete adjuvant. The animals were killed 10 days after the immunization, and spleen cells were placed in at-bottomed, 96-well microtiter plates (COSTAR). Samples containing 2.5310 cells per well were plated with 1, 2, 4, or 8 mg of antigen in 100 ml RPMI 1640 medium with 10% FCS. Control cultures were cells stimulated with concanavalin A (Con A; SIGMA) at 2.5 mg/ml or medium alone. The plates were incubated at 378C in a humidi®ed atmosphere of CO2 in air for 96 h, and assayed for proliferation by colorimetric MTT (data not shown), or for IL-4 determination in the culture supernatants. IL-4 levels were detected by a commercial ELISA system according to the manufacturer's speci®cations (mouse IL-4, Cytoscreen immunoassay kit, BioSource International, Inc., Camarillo, CA, USA). Because of the use of a biotinylated second antibody in the ELISA kit employed, and the high distribution of biotinyl enzymes in Anisakis (4), we tested the IL-4 levels of supernatants obtained at time 0, after the addition of the highest concentrations of antigenic extract (4 or 8 mg/well). We obtained reactivity levels (162.4 and 274.3 pg of IL-4/ml, with 4 and 8 mg of A. simplex antigen/well, respectively) much higher than those obtained after 3 days of culture (0.4, 65.1, 3.6, 7.5, 12.6, and 29.5 pg of IL-4/ml, with medium; Con A; or 1, 2, 4, and 8 mg of A. simplex antigen/well, respectively). For con®rmation of this ®nding, larval A. simplex extract was diluted from 5 mg/ml to 3.9 mg/ml and tested in duplicate by means of the mouse IL-4, Cytoscreen immunoassay kit with 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9, and 1.9 mg of antigenic protein per well. The concentrations of mouse IL-4 in pg/ml reached values from 297.8 to 48.5 pg/ml, as shown in Fig. 1, that were proportional to the dose of antigen used. IL-4 levels were also observed in the A. simplex antigen when tested by a system for human IL-4 determination (IL-4 endogen interleukin-4 ELISA, Endogen, Inc., Woburn, MA, USA) (data not shown). Work is underway to determine whether these parasite-derived molecules that share cross-reactive epitopes with the host cytokine IL-4 are merely artifacts or do have biologic signi®cance.
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