The Fundamentals of Bacteriology

CHAPTER XXIX.
RECEPTORS OF THE THIRD ORDER.

CYTOLYSINS.

Before Koch definitely proved bacteria capable of causing disease several physiologists had noted that the red corpuscles of certain animals were destroyed by the blood of other animals (Creite, 1869, Landois, 1875), and Traube and Gescheidel had shown that freshly drawn blood destroys bacteria (1874). It was not until about ten years afterward that this action of the blood began to be investigated in connection with the subject of immunity. Von Fodor (1885) showed that saprophytic bacteria injected into the blood are rapidly destroyed. Flügge and his pupils, especially Nuttall in combating Metchnikoff’s theory of phagocytosis, announced in 1883, studied the action of the blood on bacteria and showed its destructive effect (1885–57). Nuttall also showed that the blood lost this power if heated to 56°. Buchner (1889) gave the name “alexin” (from the Greek “to ward off”) to the destroying substance and showed that the substance was present in the blood serum as well as in the whole blood, and that when the serum lost its power to dissolve, this could be restored by adding fresh blood. Pfeiffer (1894) showed that the destructive power of the blood of animals immunized against bacteria (cholera and typhoid) was markedly specific for the bacteria used. He introduced a mixture of the blood and the bacteria into the abdominal cavity of the immunized animal or of a normal one of the same species and noted the rapid solution of the bacteria by withdrawing portions of the peritoneal fluid and examining them (“Pfeiffer’s phenomenon”). Belfanti and Carbone and especially Bordet (1898) showed the specific dissolving action of the serum of one animal on the blood corpuscles of another animal with which it had been injected. Since this time the phenomenon has been observed with a great variety of cells other than red blood corpuscles and bacteria—leukocytes, spermatozoa, cells from liver, kidney, brain, epithelia, etc., protozoa, and many vegetable cells.

It is therefore a well-established fact that the proper injection of an animal with almost any cell foreign to it will lead to the blood of the animal injected acquiring the power to injure or destroy cells of the same kind as those introduced. The destroying power of the blood has been variously called its “cytotoxic” or “cytolytic” power, though the terms are not strictly synonymous since “cytotoxic” means “cell poisoning” or “injuring,” while “cytolytic” means “cell dissolving.” The latter term is the one generally used and there is said to be present in the blood a specific “cytolysin.” The term is a general one and a given cytolysin is named from the cell which is dissolved, as a bacteriolysin, a hemolysin (red-corpuscle-lysin), epitheliolysin, nephrolysin (for kidney cells), etc. If the cell is killed but not dissolved the suffix “cidin” or “toxin” is frequently used as “bacteriocidin,” “spermotoxin,” “neurotoxin,” etc.

The use of the term “cytolysin” is also not strictly correct, though convenient, for the process is more complex than if one substance only were employed. As was stated above, the immune serum loses its power to dissolve the cell if it is heated to 55° to 56° for half an hour, it is inactivated. But if there be added to the heated or inactivated serum a small amount of normal serum (which contains only a very little cytolytic substance, so that it has no dissolving power when so diluted) the mixture again becomes cytolytic. It is evident then that in cytolysis there are two distinct substances involved, one which is present in all serum, normal or immune, and the other present only in the immune cytolytic serum. This may be more apparent if the facts are arranged in the following form:

1. I. Immune serum dissolves cells in high dilution.
2. II. Heated immune serum does not dissolve cells.
3. III. Normal serum in high dilution does not dissolve cells.
4. II. + III., i.e., Heated immune serum plus diluted normal serum dissolves cells.

Therefore, there is something in heated immune serum necessary for cell dissolving and something different in diluted normal serum which is necessary. This latter something is present in unheated immune serum also, and is destroyed by heat. Experiment has shown that it is the substance present in all serum both normal and immune that is the true dissolving body, while the immune substance serves to unite this body to the cell to be destroyed, i.e., to the antigen. Since the immune body has therefore two uniting groups, one for the dissolving substance and one for the cell to be dissolved, Ehrlich calls it the “amboceptor.” He also uses the word “complement” to denote the dissolving substance, giving the idea that it completes the action of dissolving after it has been united to the cell by the amboceptor, thus replacing Buchner’s older term “alexin” for the same dissolving body.

AMBOCEPTORS.

The theory of formation of amboceptors is similar to that for the formation of the other types of antibodies. The cell introduced contains some substance, which acts as a chemical stimulus to some of the body cells provided with proper receptors so that more of these special receptors are produced, and eventually in excess so that they become free in the blood and constitute the free amboceptors. It will be noticed that these free receptors differ from either of the two kinds already described in that they have two uniting groups, one for the antigen (cell introduced) named cytophil-haptophore, the other for the complement, complementophil haptophore. Hence amboceptors are spoken of as receptors of the third order. They have no other function than that of this double combining power. The action which results is due to the third body—the complement. It will be readily seen that complement must possess at least two groups, a combining or haptophore group which unites with the amboceptor, and an active group which is usually called the zymophore or toxophore group. Complements thus resemble either toxins, where the specific cell (antigen) is injured or killed, or enzymes, in case the cell is likewise dissolved. This action again shows the close relation between toxins and enzymes. Complement may lose its active group in the same way that toxin does and becomes then complementoid. Complement is readily destroyed in blood or serum by heating it to 55° to 56° for half an hour, and is also destroyed spontaneously when serum stands for a day or two, less rapidly at low temperature than at room temperature.

Amboceptors appear to be specific in the same sense that agglutinins are. That is, if a given cell is used to immunize an animal, the animal’s blood will contain amboceptors for the cell used and also for others closely related to it. Immunization with spermatozoa or with epithelial or liver cells gives rise to amboceptors for these cells and also for red blood corpuscles and other body cells. A typhoid bactericidal serum has also some dissolving effect on colon bacilli, etc. Hence a given serum may contain a chief amboceptor and a variety of “coamboceptors,” or one amboceptor made up of a number of “partial amboceptors” each specific for its own antigen (“amboceptorogen”). Amboceptors may combine with other substances than antigen and complement, as is shown by their union with lecithin and other “lipoids,” though these substances seem capable of acting as complement in causing solution of blood corpuscles.

COMPLEMENTS.

As to whether complements are numerous, as Ehrlich claims, or there is only one complement, according to Buchner and others, need not be discussed here. In the practical applications given later as means of diagnosis it is apparent that all the complement or complements are capable of uniting with at least two kinds of amboceptors.

If complement be injected into an animal it may act as an antigen and give rise to the formation of anticomplement which may combine with it and prevent its action and is consequently analogous to antitoxin. If amboceptors as antigen are injected into an animal there will be formed by the animal’s cells antiamboceptors. As one would expect, there are two kinds of antiamboceptors, one for each of its combining groups, since it has been stated that it is always the combining group of any given antigen that acts as the cell stimulus. Hence we may have an “anticytophil amboceptor” or an “anticomplementophil amboceptor.” These antiamboceptors and the anticomplements are analogous to antitoxin, antiagglutinin, etc., and hence are receptors of the first order.

ANTISNAKE VENOMS.

A practical use of antiamboceptors is in antisnake venoms. Snake poisons appear to contain only amboceptors for different cells of the body. In the most deadly the amboceptor is specific for nerve cells (cobra), in others for red corpuscles, or for endothelial cells of the bloodvessels (rattlesnake). The complement is furnished by the blood of the individual bitten, that is, in a sense the individual poisons himself, since he furnishes the destroying element. The antisera contain antiamboceptors which unite with the amboceptor introduced and prevent it joining to cells and thus binding the complement to the cells and destroying them. With this exception these antibodies are chiefly of theoretical interest.

FAILURE OF CYTOLYTIC SERUMS.

The discovery of the possibility of producing a strongly bactericidal serum in the manner above described aroused the hope that such sera would prove of great value in passive immunization and serum treatment of bacterial diseases. Unfortunately such expectations have not been realized and no serum of this character of much practical importance has been developed as yet (with the possible exception of Flexner’s antimeningococcus serum in human practice. What hog cholera serum is remains to be discovered).

The reasons for the failure of such sera are not entirely clear. The following are some that have been offered: (1) Amboceptors do not appear to be present in very large amount so that relatively large injections of blood are necessary, which is not without risk in itself. (2) Since the complement is furnished by the blood of the animal to be treated, there may not be enough of this to unite with a sufficient quantity of amboceptor to destroy all the bacteria present, hence the disease is continued by those that escape. (3) Or the complement may not be of the right kind to unite with the amboceptor introduced, since this is derived from the blood of a heterologous (“other kind”) species. In hog-cholera serum, if it is bactericidal, this difficulty is removed by using blood of a homologous (“same kind”) animal. Hence Ehrlich suggested the use of apes for preparing bactericidal sera for human beings. The good results which have been reported in the treatment of human beings with the serum of persons convalescing from the same disease indicate that this lack of proper complement for the given amboceptor is probably a chief factor in the failure of sera from lower animals. (4) The bacteria may be localized in tissues (lymph glands), within cavities (cranial, peritoneal), in hollow organs (alimentary tract), etc., so that it is not possible to get at them with sufficient serum to destroy all. This difficulty is obviated by injecting directly into the spinal canal when Flexner’s antimeningococcus serum is used. (5) Even if the bacteria are dissolved it does not necessarily follow that their endotoxins are destroyed. These may be merely liberated and add to the danger of the patient, though this does not appear to be a valid objection. (6) Complement which is present in such a large excess of amboceptor may just as well unite with amboceptor which is not united to the bacteria to be destroyed as with that which is, and hence be actually prevented from dissolving the bacteria. Therefore it is difficult to standardize the serum to get a proper amount of amboceptor for the complement present.

COMPLEMENT-FIXATION TEST.

Although little practical use has been made of bactericidal sera, the discovery of receptors of this class and the peculiar relations between the antigen, amboceptor and complement have resulted in developing one of the most delicate and accurate methods for the diagnosis of disease and for the recognition of small amounts of specific protein that is in use today.

This method is usually spoken of as the “complement-fixation” or the “complement-deviation test” (“Wassermann test” in syphilis) and is applicable in a great variety of microbial diseases, but it is of practical importance in those diseases only where other methods are uncertain—syphilis in man, concealed glanders in horses, contagious abortion in cattle, etc. A better name would be the “Unknown Amboceptor Test” since it is the amboceptor that is searched for in the test by making use of its power to “fix” complement.

The principle is the same in all cases. The method depends, as indicated above, on the ability of complement to combine with at least two amboceptor-antigen systems, and on the further fact that if the combination with one amboceptor-antigen system is once formed, it does not dissociate so as to liberate the complement for union with the second amboceptor-antigen system. If an animal is infected with a microörganism and a part of its defensive action consists in destroying the organisms in its blood or lymph, then it follows from the above discussion of cytolysins that there will be developed in the blood of the animal amboceptor specific for the organism in question. If the presence of this specific amboceptor can be detected, the conclusion is warranted that the organism for which it is specific is the cause of the disease. Consequently what is sought in the “complement-fixation test” is a specific amboceptor. In carrying out the test, blood serum from the suspected animal is collected, heated at 56° for half an hour to destroy any complement it contains and mixed in definite proportions with the specific antigen and with complement. The antigen is an extract of a diseased organ (syphilitic fetal liver, in syphilis), a suspension of the known bacteria, or an extract of these bacteria. Complement is usually derived from a guinea-pig, since the serum of this animal is higher in complement than that of most animals. The blood of the gray rat contains practically as much. If the specific amboceptor is present, that is, if the animal is infected with the suspected disease, the complement will unite with the antigen-amboceptor system and be “fixed,” that is, be no longer capable of uniting with any other amboceptor-antigen system. No chemical or physical means of telling whether this union has occurred or not, except as given below, has been discovered as yet, though doubtless will be by physico-chemical tests, nor can the combination be seen. Hence an “indicator,” as is so frequently used in chemistry, is put into the mixture of antigen-amboceptor-complement after it has been allowed to stand in the incubator for one-half to one hour to permit the union to become complete. The “indicator” used is a mixture of sheep’s corpuscles and the heated (“inactivated”) blood serum of a rabbit which has been injected with sheep’s blood corpuscles and therefore contains a hemolytic amboceptor specific for the corpuscles which is capable also of uniting with complement. The indicator is put into the first mixture and the whole is again incubated and examined. If the mixture is clear and colorless with a deposit of red corpuscles at the bottom, that would mean that the complement had been bound to the first complex, since it was not free to unite with the second sheep’s corpuscles (antigen)—rabbit serum (hemolytic amboceptor) complex—and destroy the corpuscles. Hence if the complement is bound in the first instance, the specific amboceptor for the first antigen must have been present in the blood, that is, the animal was infected with the organism in question. Such a reaction is called a “positive” test.

On the other hand, if the final solution is clear but of a red color, that would mean that complement must have united with the corpuscles—hemolytic amboceptor system—and destroyed the corpuscles in order to cause the clear red solution of hemoglobin. If complement united with this system it could not have united with the first system, hence there was no specific amboceptor there to bind it; no specific amboceptor in the animal’s blood, means no infection. Hence a red solution is a “negative test.”

The scheme for the test may be outlined as follows:

Incubate one-half hour in a water bath or one hour in an incubator.

Then add the indicator which is

Incubate as above.

In practice all the different ingredients must be accurately tested, standardized and used in exact quantities, and tests must also be run as controls with a known normal blood of an animal of the same species as the one examined and with a known positive blood.

It should be stated that in one variety of the Complement Fixation Test, namely, the “Wassermann Test for Syphilis” in human beings, an antigen is used which is not derived from the specific organism (Treponema pallidum) which causes the disease nor even from syphilitic tissue. It has been determined that alcohol will extract from certain tissues, human or animal, substances which act specifically in combining with the syphilitic amboceptor present in the blood. Alcoholic extracts of beef heart are most commonly used. Details of this test may be learned in the advanced course in Immunity and Serum Therapy.

The complement-fixation test might be applied to the determination of unknown bacteria, using the unknown culture as antigen and trying it with the sera of different animals immunized against a variety of organisms, some one of which might prove to furnish specific amboceptor for the unknown organism and hence indicate what it is. The test used in this way has not been shown to be a practical necessity and hence is rarely employed. It has been used, however, to detect traces of unknown proteins, particularly blood-serum proteins, in medico-legal cases in exactly the above outlined manner and is very delicate and accurate.