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Peritoneal exudate cells from guinea pigs with delayed hypersensitivity skin reactions were incubated with the specific antigen and ruptured by freezing and thawing. The supernatant of these ruptured cells was incubated with several primary and established tissue culture strains. Extracts of peritoneal cells from hypersensitive guinea pigs that were incubated with the specific antigen induced the tissue culture cells to overproduce acid in the medium. Similarly, extracts from nonsensitized guinea pigs did not induce a corresponding decrease in pH. The formation of "acid-stimulating activity" required specificity of the reactants because sensitive cells incubated with saline or heterologous antigen did not stimulate the tissue culture cells. "Acid-stimulating activity" was found to be independent of the tissue culture cell strain, the media used to grow the tissue culture cells, and the sensitized peritoneal cell-antigen system. No "acid-stimulating activity" was produced if frozen and thawed cells were incubated with the specific antigen. "Acid-stimulating activity" was produced by peritoneal cells capable of passively transferring delayed hypersensitivity, but not from sera of the same guinea pigs. Sera from sensitized guinea pigs did not confer "acid-stimulating activity" to nonsensitized peritoneal cells. KB tissue cells did not overproduce acid when specific precipitates were added to the media. The degree of hypersensitivity of the guinea pigs donating peritoneal cells corresponded with the ability of these cells to induce acid production in a KB cell culture. Guinea pigs, with lesser sensitivity to skin testing donated cells with lesser "acid-stimulating activity." It is believed that this phenomenon may be applicable to the measurement in vitro of delayed hypersensitivity uncomplicated by host factors.

Peritoneal cells from sensitized guinea pigs upon incubation with the specific antigen yield an agent that stimulates KB epithelial cells in tissue culture to overproduce acid—a phenomenon referred to as "acid-stimulation activity" (ASA). The formation of ASA varied directly with the concentration of peritoneal cells, the concentration of antigen, and the temperature of incubation. Its rate of production was dependent upon the concentration of antigen, but with every concentration of antigen the rate was greatest during the first hour of incubation. With less than maximal concentrations of antigen, ASA formation continued beyond 1 hour, whereas with maximal concentrations of antigen, it significantly decreased after 1 hour. Cell death, lack of extracellular nutrients, and subthreshold levels of antigen were eliminated as reasons for failure of further production after 1 hour. In order to explain the results it has been postulated that an intracellular antibody-like substance has been exhausted.