When filtered alkaline extracts of pulverized bacteria of several varieties are precipitated with acid in the cold, boiled with acid, and all materials thrown down by these procedures removed, there remains a small amount of an alcohol-precipitable material which no longer gives any of the ordinary chemical reactions for proteins, such as the biuret, Hopkins-Cole, Millon, and sulfosalicylic acid reactions. The only protein reaction usually given by this material is a very weak xanthoproteic reaction. Nevertheless, the material, which is, as far as we can determine at present, free from coagulable protein, is specifically precipitable by homologous antiserum and gives specific complement fixation reactions.
Such material can also be obtained from organisms like the influenza bacillus, pneumococcus, and meningococcus by extraction without preliminary grinding of the bacteria, and is present in filtrates of young and old broth cultures of the organisms.
We believe that these acid- and heat-resistant antigenic materials are analogous to tuberculin and to the pneumococcus substances with which Dochez and Avery (6) made their observations some years ago.
The stability of these substances is considerable and was investigated particularly because we thought this represented an indirect method of eliminating the possibility of their protein nature. In all cases boiling in a reflux condenser at an acid reaction ranging from pH 5 to 6 for 1 hour failed to destroy the antigenic specificity of the residue antigens. After such treatment satisfactory and specific precipitation reactions could be obtained. Similar boiling in alkaline reactions, however, destroyed the precipitability of staphylococcus and influenza residues. Subjected to autoclave digestion at an acid reaction of pH 5.4 for 1 hour at from three to four atmospheres, none of the antigenic residues investigated, except that obtained from the influenza bacillus, were destroyed. The pneumococcus and tubercle bacillus residue antigens were resistant to boiling for 1 hour, both in acid and alkaline reactions (pH 5.4 and 9.4). In fact, none of the procedures resorted to made any difference with these two last mentioned substances. It would seem that these facts would add considerable weight to the assumption that the materials dealt with were not ordinary whole proteins.
On preservation in the ice box at an alkaline reaction of pH 9.4, the influenza residue deteriorated within 48 hours, but the other antigens withstood similar treatment for 6 days.
In spite of the fact that these residue antigens were precipitable by homologous sera produced by immunization with the whole bacteria or their unfractionated extracts, we have so far failed to produce antibodies in animals by injecting these residues. While this may be due to inability to inject sufficient amounts of the material it still suggests strongly the possibility that we may be dealing with substances that are antigenic only in the sense that they are able to react with antibodies, but are themselves incapable of inciting antibody production. We suggest, in this connection, the possibility of the relationship between the power of antibody production and molecular size. This phase of the work is being continued on a more extensive scale.
Our work on the reactions of the residue materials in infected animals indicates, as far as we have gone, that complete analogy exists in this respect between the conditions prevailing in guinea pigs infected with these organisms and those previously elucidated for tuberculous animals. This is in keeping with previous knowledge concerning the analogies between the mallein and tuberculin reactions and the studies on skin hypersusceptibility in Bacillus abortus- and typhoid-infected guinea pigs reported by Meyer and his coworkers.
It would seem from all these facts that, in guinea pigs infected with bacteria capable of forming foci in the body, infection is followed within a variable, but relatively short time (5 days to 2 weeks) by a type of hypersusceptibility which is distinct from protein anaphylaxis and which may be determined by intradermal skin reaction. It appears likely that the growing bacteria elaborate in the animal body a metabolic product, possibly not a whole protein, which, though practically non-toxic to normal animals, may become highly and specifically injurious to the infected ones.
Such a conception, if further confirmed, would lead to greater clearness in our comprehension of the toxic effects occurring in infections with organisms not true exotoxin producers and, judging by the cellular injuries observed in severe skin reactions, may easily explain focal necrosis and the deeper cellular degenerations observed in the course of many bacterial diseases.
The general bearing of this work upon conceptions of hypersusceptibility is obvious and has been briefly discussed in another paper. Its chief significance is in holding out the hope that we may be able to elucidate the mechanism of a type of specific hypersusceptibility in which the antigen concerned is not a coagulable protein and in which the laws of sensitization in regard to time and quantity differ from those recognized in true protein anaphylaxis.
It seems likely that a recognition of the fact that physical and chemical differences in the substances leading to various forms of specific hypersusceptibilities in the animal body must necessarily influence the mechanism of sensitization, may furnish a clue to further investigations. As such materials become simpler in structure, they fail to induce typical antibody production and by gradually increased diffusibility transfer the reactions from the cell surface to the interior of the cell. The extremes of the scale of differences would be represented by protein anaphylaxis, on the one hand, and drug idiosyncrasies, on the other. Although this suggestion is largely speculative, it has seemed worth mentioning as a line of reasoning suggested by our work.
Incidentally, these studies may indicate the usefulness of the residue antigens for specific precipitation and complement fixation reactions for routine purposes in laboratory investigations.
A THERMOPRECIPITATION REACTION IN TRYPANOSOMA EQUIPERDUM INFECTION IN LABORATORY ANIMALS