' '/ Professor of Bacteriology and Hygiene in Fordham University. N. Y. , Assistant Bacteriologist, Research Laboratory, Department of Health. \ City of New York -^ Jt ^ FIRST EDITION FIRST THOUSAND NEW YORK JOHN WILEY & SONS LONDON: CHAPMAN & HALL, Limited 1906 y I 8(" 4,V Copyright^ 1906 BV CHARLES BOLDUAN ROBERT DROMMOND, PRIHTER, NEW YORK To Dr. a L T H O F F Privy Councilor, Director in the Prussian Ministry of Education, Berlin, etc. The Able Friend and Promoter of Medical Science This Volume is Dedicated in Grateful Appreciation TRANSLATOR'S PREFACE. No apology is needed for presenting this translation of Ehrlich's classic studies in immunity, for a thorough knowledge of the master's work is indispensable to all workers in this field. Attention is called to the fact that the important work done since the publication of the German edition has been included by the addition of three chapters, two by Ehrlich and Sachs and one, written expressly for this translation, by Prof. Ehrlich. The subject is thus brought up to about March, 1906. Charles Bolduan. PREFACE TO THE GERMAN EDITION. The present volume embraces the greater portion of the studies: in immunity published during the past few years by myself and my collaborators. While the publication of these studies in a single volume meets the request of numerous workers in immimity, it is hoped that the collection will at the same time fulfill another purpose, namely, to show clearly that my theory of immunity rests on so broad an experimental basis that it is practically identical trith a summary of generalizations derived from an enormous mass of experi- mental data.i When Behring's great discovery of antitoxin opened new paths for the study of immunity it was at once clear that further progress could be attempted in two ways. The first of these, having practical therapeutic resiilts in mind, consists in bending all efforts to the pro- duction of various individual curative sera. The other method con- sists in seeking a deeper insight into the nature of immunity phe- nomena, and discovering the general principles imderlsdng the same,, for these in turn will md practical progres. By pursuing the latter method it has been found that the unmunity reaction is merely a repetition of certain processes of normal meta- bolism, and that what is apparently a wonderful adaptation to the purpose is nothing more than the ever-recurring manifestation of primeval wisdom inherent in the protoplasm. I have endeavored to establish this experimentally and to show that the bond between » With a view of giving the reader a better idea of the technique ordinarily employed, and thereby to facilitate his introduction to this subject, I have had my colleagues, Dr. Morgenroth and Prof. Neisser, present the result of their ex- tensive technical experiences with hemolytic and bacteriolytic test-tube experi- ments, in two special chapters. (Chapters XXIX and XXX.) VI PREFACE TO THE GERMAN EDITION. what are at first sight very dissimilar biological processes is really a conception of the simplest kind. The toxic metabolic products of bacteria, the artificially produced bacteriolysins, hsemolysins, and cytotoxins, and the majority of the ferments, probably always produce their eiTeots by the co-action of two active groups in the molecule. One of these effects the union with the substance to be acted upon, while the other really produces the characteristic effect. It is not surprising, in view of the enormous multiplicity of the vital phenomena, that this simple principle exhibits the greatest variations in individual cases. Certainly this corresponds entirely to what we constantly observe in the domain of biology. The cell, for example, occurs as a type in every living form, from the lowest plant to the highest animal. In principle it is ever the same; in the details of its structure, however, it is of endless variety. But even from such complex phenomena as are exhibited, for ■example, by the artificially produced hsemolysins, it is possible to develop the fundamental principles of my theory, and thereby give a harmonious uniform explanation of the manifold phenomena with their peculiar specific relations. My theory has developed essentially on the basis of chemical ■conceptions. I have been more and more forcibly impressed with the idea that in a study of the fundamental biological phenomena, the significance of morphological structure is far less than the sig- nificance of the chemistry involved. It is obvious that in order to effect a given chemical process certain mechanical conditions must be fulfilled. In other words the production of any chemical action necessitates the presence and the suitable arrangement of apparatus. The essential feature, however, is neither apparatus nor form, but the constituents involved; for without changing the apparatus hundreds of different combinations can be effected according to the components employed. Similarly in biology I believe that the morpho- logical arrangement of the organs and cells is not the essential feature, but that this is rather to be sought for in chemical differences of the constituents. I am convinced that the influence exerted by my theory will extend far beyond the limits of pure immunity studies, and that it is of considerable significance for an appreciation of vital phenomena. Furthermore, I believe that the theory is of great value in studying certain phenomena which dominate all life, namely, intracellular PREFACE TO THE GERMAN EDITION. Vli raetabolism, especially its two main phases, anabolism and catabolism. It has been shown that the substances obtained by immunization are nothing but the tools of normal cell-life, tools which we can thus isolate from their place of production and subject to an individual examination. This at once opens new paths for approaching the study of vital phenomena, which embraces not only the physiology and pathology of metabolism, but also certain other physiological problems such as those of secretion, heredity, etc. At the recent Congress for Hygiene and Demography (Brussels), in which the chief problems of immunity were discussed, it was seen that my theory is not yet accepted by all the workers in this subject, there being still a few opponents. This was to be expected. Cer- tainly nothing is more desirable in all scientific problems than the expression of different opinions, for as a result of experimental studies they lead to a deeper insight into the subject in question. Hence it is largely the opposition of Bordet and other distinguished workers in the Pasteur Institute that has spurred us on in our experi- mental labors, and caused us to establish the amboceptor theory more firmly than ever. On the other hand it is very annoying when such authors as Gruber, who have absolutely no personal experience in the main questions, wage a bitter war merely because they have made a few literary studies ; it is the more exasperating since they seek to make up the deficiencies in their arguments by the intensity and personality of their attacks. Such authors are in no position to correctly orientate themselves in the mass of true and false observations that each day's literature brings forth. It was a great pleasure, therefore, to see one of the founders of the doctrine of immunity, R. Pfeiffer, and that distinguished repre- sentative of Paltauf's Institute in Vienna, R. Kraus, express them- selves in favor of my theory. They confessed they had both really opposed the theory from the start, and that the main purpose in devis- ing their various experiments had been to show that it was untenable. Just these, however, had convinced them that the side-chain theory not only afforded the best explanation for their results, but had even enabled them to predict these results. The chief problems now under discussion are ; (1) the constitution of active cytotoxic sub- vui PREFACE TO THE GERMAN EDITION. stances, whether or not they are made up of two parts possessing different functions; (2) the union of specific amboceptors with the complements; (3) the plurality of complements. 1 am convinced that the near future will furnish so many additional arguments for the correctness of my views that all of these questions, as well as numerous others, will be decided in my favor. And the decision, I believe, will not be merely in favor of my views in general, but will extend even to the details. In a way, therefore, my position is like that of a chess-player who, even though his game is won, is forced by the obstinacy of his opponent to carry it on move by move until the final "mate." For the means to carry on these experiments, I am indebted first of all to the intelligent support which my scientific aims have received at the hands of my superiors, the Prussian Ministry of Education. I am especially grateful to the ministerial director. Dr. Althoff, who aided me in every way possible, and exerted himself to lighten my scientific labors. I may say that I was first spurred on to the im- munity studies contained in "Die Werthbemessung des Diphtherie- heilserums," and which have led to the formulation of the side-chain theory, by the remarks addressed to me by Dr. Althoff when the Institute was founded. It was he who begged that my first problem, be an exhaustive study whereby the difficulties which had arisen in titrating and standardizing diphtheria antitoxin might be overcome. To this kind and able friend I have therefore dedicated this volume aa a token of my gratitude and esteem. Paul Ehrlich. Frankfurt a. M., February 1904. CONTENTS. CHAPTBB PAGE) I. Contributions to the Theory op Lysin Action Ehrlich and Morgenroth. 1 II. Concerning Hemolysins. (Second Communication.) Ehrhch and Morgenroth. 1 1 III. Studies on H^emolysins. (Third Communication.) Ehrlich and M orgenroth. 23 IV. Contributions to the Stud of Immunity von Dungern. 36 New Experiments on the Side- chain Theory. Phagocytosis and Globulicidal Immuity. V. Contributions to the Study op Immunity von Dungern. 47 Receptors and the Formation of Antibodies. Milk Immune Serum. VI. Studies on Hemolysins. (Fourth Communication.) Ehrlich and Morgenroth. 56 VII. Studies on Hemolysins. (Fifth Communication.) Ehrlich and Morgenroth. 71 VIII. Studies on Hemolysins. (Sixth Communication.) Ehrlich and Morgenroth. 88 IX. Concerning the Mode of Action OF Bactericidal Sera M. Neisser. 120 X. The Deflection of Complements IN Bactericidal Test-tube Ex- periments Ldpstein. 132 XI. Active Immunity and Overneu- tralized Diphtheria Toxins Rehns. 143 XII, Is it Possible by Injecting Ag- glutinated Typhoid Bacilli to Cause the Production of an Agglutinin? M. Neisser. 146 XIII. Immunizing Experiments with Erythrocytes Laden wih Im- mune Body Sachs. 15f ix CONTENTS. CHAPTER PAGB XIV. The Escape of H«!moglobin from Blood-cells Hardened with Corrosive Sublimate Sachs. 163 XV. A CONTRIBCTION TO THE StUDY OF THE Poison of the Common Garden Spider Sachs. 167 XVI. A Study of Toad Poison Proscher. 175 XVII. Concerning Alexin Action Sachs. 181 XVIII. Concerning the Plurality op Complements of the Serum Ehrlich and Sachs. 195 XIX. Concerning the Mechanism op THE Action op Amboceptors Ehrlich and Sachs. 209 XX. Differentiating Complements by Means of a Partial Anticom- plement Marshall and Morgenroth. 222 XXI. Concerning the Complemento- PHiLE Groups op the Ambo- ceptors Ehrlich and Marshall. 226 XXII. Concerning the Complementi- bility of the Amboceptors Morgenroth and, Sachs. 233 XXIII. The Production op HiEMOLYTic Amboceptors by Means of Serum Injections Morgenroth. 241 XXIV. The Quantitative Relations be- tween Amboceptor, Comple- ment, and Anticomplement Morgenroth and Sachs. 250 XXV. The Hemolytic Properties of Organ Extracts Korschun and Morgenroth. 267 XXVI. Review op Besbedka's Study, ' " Les Antihemolysines Natu- RBLLES " Marshall and Morgenroth. 283 XXVII. The Mode op Action op Cobra Venom Kyes. 291 XXVIII. Further Studies on the Dysen- tery Bacillus Shiga. 312 XXIX. Methods op Studying Hemoly- sins Morgenroth. 326 XXX. The Technique of Bactericidal Test-tube Experiments M. Neisser. 348 XXXI. The Property of the Brain to Neutralize Tetanus Toxin Marx. 356 XXXII. The Protective Substances op the Blood Ehrlich. 364 XXXIII. The Receptor Apparatus of the Red Blood-cells Ehrlich. 390 CONTENTS XI CHAPTER PAGE XXXI V. The Relations Existing between Chemical Constitution, Distri- bution, AND Pharmacological Action Ehrlich. 404 XXXV. A Study op the Substances which Activate Cobra Venom Kyes and Sachs 443 XXXVI. The Isolation op Snake-venom Lecithids Kyes. 466 XXXVII. The Constituents op Diphtheria Toxin Ehrlich 481 XXXVIII. Toxin and Antitoxin: "a Reply to the Latest Attack of Gruber Ehrlich. 514 XXXIX. The Relations Existing between Toxin and Antitoxin and the Methods op their Study Ehrlich and Sachs. 547 XL. The Mechanism of the Action op Antiamboceptors Ehrlich and Sachs. 561 XLI. A General Review of the Recent Work in Immunity Ehrlich. 677 COLLECTED STUDIES IN IMMUNITY. I. CONTRIBUTIONS TO THE THEORY OF LYSIN ACTION.i By Prof. Dr. P. Ehrlich and Dr. J. Moegenkoth. One of the most important advances in the study of immunity is the discovery of Pfeiffer's phenomenon, and it is to Pfeiffer's splendid observations that we owe the first and most important insight into the mode of action of the bacteriolytic inimune sera. As is well known, the phenomenon of bacteriolysis, first demon- strated by Pfeiffer in a guinea-pig immunized against cholera, con- sists in the immediate dissolution of cholera bacilli introduced into the abdominal cavity of the animal. The same takes place when the bacilK together with a small amount of immune serum are intro- duced into the abdominal cavity of a normal guinea-pig. Subse- quently Metchnikoff (Annal. Inst. Pasteur, June 1895) showed that the phenomenon of bacteriolysis takes place also outside the animal body, in vitro, provided a small quantity of peritoneal exudate of a normal guinea-pig is added. Bordet (Arnial. Inst. Pasteur, June 1895) was thereupon able to show that the immune serum is able to effect bacteriolysis in vitro without any addition, provided that it is absolutely fresh. On standing it becomes inactive; but it may be reactivated by even very small amounts of normal serum. Pfeiffer's ideas as to the nature of bacteriolysis were formulated by him in a very clever theory which he published in 1896 (Deutsche med. Wo- ' Reprinted from Berl. klin. Wochenschr. , 1899, No. 1. 2 COLLECTED STUDIES IN IMMUNITY. chenschr., 1896, Nos. 7 and 8) and which is here reproduced only in its main features. The immunizing substances contained in cholera serum possess but feeble power to retard development. They are nothing but an antecedent form of substances developed in the peritoneum of the guinea-pig, specifically solvent for cholera vibrios. They are stored in the animal body in an inactive but stable form, somewhat as glycogen is stored in cell depots as an antecedent form of grape- sugar. When needed, these inactive substances of the serum can be converted into the specific active form through the active interference of the body-cells. This conversion can also be effected by the addi- tion of a suitable serum. In this added serum a certain "something," present in very small amounts, effects the change, but is very soon used up in the process. In the animal body, on the other hand, this constituent is produced by the body-cells as long as the stimulus, caused by the presence of the cholera bacilli, lasts. The action of this substance is ferment-like. Bacteriolysis is also regarded as a ferment action, caused by ferments of a very peculiar kind. These ferments are fitted in an absolutely specific manner each to a single bacterial protoplasm, acting on this exactly as pepsin or trypsin acts on coagulated albumin. According to Pfeiffer, a somewhat distant analogy is seen in E. Fischer's yeast ferments, each of which can only split up a sugar of a definite composition. If this theory be correct, these specific ferments must exist in an active and an inactive modi- fication. Recently Bordet (Annal. Inst. Pasteur, Vol. 12, No. 10) pub- lished a series of experiments in which he showed that the laws which govern the specific bacteriolytic action of immune sera govern also certain specific solvent phenomena seen in red blood-cells. Bordet treated guinea-pigs with repeated injections of defibri- nated rabbit blood. The serum of animals so treated possesses the property of dissolving rabbit blood in vitro rapidly and with great intensity, whereas serum of normal guinea-pigs is unable to do this. Solution is preceded by a marked agglutination of the erythrocytes. On heating the specific serum for half an hour to 55° C. the hsemolytic power is destroyed, while the agglutinating pov/er remains. The serum thus inactivated can again be rendered active by the addition of a certain amount of normal guinea-pig serum, and even of normal rabbit serum. The active guinea-pig serum has no effect on the red blood-cells of the guinea-pig itself or on those of pigeons, but CONTRIBUTIONS TO THE THEORY OP LYSIN ACmON. 3 acts, though to a less degree, on the blood-cells of rats and mice. The active guinea-pig serum injected into the ear-vein of a rabbit is highly toxic to that animal. The analogy existing between these phenomena and those of bacteriolysis is, as emphasized by Bordet, a very close one. This wUl be clear to the reader. Very hkely, therefore, the mechanism of haemolysis and that of bacteriolysis are very similar. The study of haemolysis thus gains considerable theoretical significance. Being so fortunate as to have at our disposal a considerable amount of appropriate serum, we have used this in order to gain a deeper in_ sight into the nature of haemolysis. This serum was derived from a goat which during eight months had been subcutaneously injected in somewhat irregular fashion with sheep serum rich in blood-corpuscles. The experiments were therefore made with sheep blood in the form of a 5% mixture of the defibrinated blood in 0.85% salt solution. By means of this great dilution certain sources of error arising from the constituents of the serum are avoided. These had manifested themselves in Bordet's experiments. The serum of our goat rapidly dissolves sheep blood-cells in vitro. The degree of action of this serum can be accurately determined as follows : To each 5 cc. of the above-mentioned blood mixtiu-e decreas- ing amounts of the goat serum are added. It is then found that at 37° C. the specimens containing from 1.5 cc. to 0.8 cc. serum will become completely laky. After allowing all the specimens to act for two hours in a thermostat they are placed in a refrigerator and allowed to settle. It will then be found that there is a regular decrease in the amount of solution effected until finally the limit is reached in the specimen containing 0.1 cc. of serum. The serum of normal goats (we tried the sera of a number of different animals) is unable even in large amounts to dissolve sheep blood-cells. It is to be remarked that in the use of this immune serum in the amounts mentioned no clumping was ever observed to precede hsemolysis, although this phenomenon was carefully looked for.^ ' The serum of normal goats in doses of 1.5 cc. and over possesses the prop- erty to agglutinate sheep blood-cells, but this property seems to be subject to great individual and chronologic fluctuations. This agglutination of foreign bloods by certain normal sera, and which probably corresponds to the normal agglutinating action of sera on bacteria, was observed many years ago by Creite (Z. f. rat. Med., Vol. 36) and later was again emphasized by Landois (Die Transfusion des Blutes, 1875). 4 COLLECTED STUDIES IN IMMUNITY. If the immune serum is heated to 56° C, it completely loses its solvent action. The addition of serum of normal animals to this inactivated serum causes it to be reactivated. For this purpose one can use not only normal goat serum but also normal sheep serum, though the latter acts somewhat more feebly. This power of the normal serum to reactivate an inactive immune serum is very readily lost. Even when the serum is kept on ice and protected against light it very soon shows a diminution of its reactivating power. In uantitative experiments, therefore, the inactive (stable) immune serum should always be reactivated by a perfectly fresh normal serum. In hemolysis, as in Pfeiffer's bacteriolysis, we are therefore forced to assume the existence of two substances. One of these, specific and quite resistant (stable), we shall call the immune body, following Pfeiffer's nomenclature. The other, normally present and highly labile (unstable), we shall for the present term addiment. Although our results in the main agree with those of Bordet, we must at once call attention to one difference in our observations. As already mentioned, the action of our goat serum on the sheep blood-cells is not preceded by any agglutination. From this we see that the agglutination cannot be considered a preparatory step neces- sary for the hsemolytic action, as Bordet seems to assume. The specific agglutinin has no relation whatever to the hamolytic immune body. Similarly, according to the views of eminent bacte- riologists, the specific bacteriolytic substances have no relation to the agglutinins. The lysins may exist independently of the agglu- tinins and these again independently of the bacterioloytic substances. The reader is reminded of the interesting observations of Pfeiffer and KoUe. These investigators described an immune serum which was strongly bacteriolytic but which did not at all agglutinate (Cen- tralblatt f. Bakt., 1896, Vol. XX, Nos. 4 and 5). On the other hand, E. Frankel and Otto state that if a young dog be fed on typhoid cultures, the dog's serum will acquire agglutinating but not bacte- riolytic properties. Similarly, if a frog is treated with typhoid bacilli, the frog serum will agglutinate such bacilli. They remain in the lymph sac of the animal, however, not only alive but virulent. (Widal and Sicard, Comptes rend. Soc. de Biol., XI. 27-97). Pfeiffer's original theory sought only to explain in general the mode of action of the specific bacteriolysins. It did not concern itself with the questions how or where they originated. It was in CONTRIBtniONS TO THE THEORY OF LYSIX ACTION. 5 order to throw some light on these problems that EhrUch devised his side-chain theory. At first Ehrhch's theory was appUed to the origin of the anti- toxins and to the chemical relation existing between the toxins and certain atomic groups of the protoplasmic molecule. Pfeiffer him- self appHed the theory to the substances specificallj- bacteriolj-tic for cholera bacOh, and was able to demonstrate experimentally that the source of these bodies was in the spleen, the bone-marrow, and the IjTnph bodies (Keiffer and Marx, Zeitschr. f. Hjg., Vol. 37, 1898). Wassermann, who in his weU-known tetanus experiments had fur- nished the first demonstration of the soundness of the side-chain theory, succeeded in showing the source of the specific tj-phoid bacteriolysin. The study of these bacteriolytic processes brought up a number of important questions directly concerning the side-chain theory, and we felt compelled to examine these experimentally. According to Ehrlich's theory, if any substance, be it toxin, toxoid, ferment, or constituent of a bacterial cell or of a blood- corpuscle, possess the property of combining with side-chains of the protoplasm, the possibDity is given for the formation of a corre- sponding antibody. The antibody, according to the theory, must possess such a group as will fit the haptophore (the specific com- bining) group of the invading substance. The soluble body, therefore, produced in response to the invading substance (toxin, toxoid, etc), must combine chemically with the latter. If the invading substance is in soluble form, as, for example, the toxins, the neutralization proceeds in the solution. If, however, it is not directly soluble, being originally an insoluble part of, say, a bacterial or blood cell, then the dissolved antibody in the blood will be abstracted from its solvent fluid and anchored by the cell particle. In the weU-known experiment of Wassermann on tetanus poison, the same thing is seen. In this the invading substance (tetanus toxin) is abstracted from its solution and anchored by the crushed brain cells. In order to maintain the analogy we shoiild expect that in our experiment the immune body dissolved in the goat serum would he anchored by the erythrocytes of sheep blood. The manner of procediu^ in this experiment is very simple and consists in the addition to sheep blood, or a dilution of the same, of inunune serum which has been heated to 56° C. in order to destroy its solvent properties. The mixture is then centrifuged to separate the cells and the fluid. In case the immune body has been anchored 6 COLLECTED STUDIES IN IMMUNITY. by the blood-cells, the clear fluid should be free from the same. To prove this we have merely to add to some of this clear fluid sheep blood-cells, and a sufficient amount of addiment in the form of normal serum. If the fluid is free from immune body, the blood-cells will remain undissolved. The centrifuged sediment must likewise be tested for the presence of immune body. The sediment, freed as much as possible from fluid, is mixed with salt solution and a sufS_- cient amount of addiment. If a corresponding amount of imutnune body has been anchored by the blood-ceUs, they will now dissolve. One of our numerous experiments follows: 4 cc. of a 5% mixture of sheep blood-cells are mixed with 1.0 •or 1.3 cc. inactivated serum from our immunized goat. This is allowed to stand for fifteen minutes at 40° C. and then carefully centrifuged. The supernatant clear fluid is poured off, mixed with 0.2 cc. normal sheeps blood and then with 0.8 cc. serum from a normal goat. This mixture after being kept in a thermostat at 37° C. for two hours and then allowed to settle in the cold, shows no trace of solution. The, centrifuged sediment, freed as much as possible from fluid by means of filter paper, is mixed with 4 cc. physiological salt solu- tion and with 0.8 cc. normal goat serum. This mixture after being kept for two hours in a thermostat at 37° C. is found completely dissolved or very nearly so. In this experiment in which a sufficient amount of immune body was used, we see that complete union took place between the immune body and the blood-cells, resulting in the entire abstraction of the former from the fluid. We have found that the same takes place at lower temperatures, even at 0° C. That this is a chemical union and not a mere absorption is seen by experiments with other species of blood. Thus the red blood-cells of rabbits and of goats have no affinity whatever for this immune body. As a result of these experiments, therefore, and in conformity with the side-chain theory, we must assume that the immune body possesses a specific haptophore group which anchors it to the blood-cells of the sheep. The next important question was that concerning the relation of the addiment to the red blood-cell. This was studied in a manner exactly similar to that of the previous experiment. Blood was mixed with addiment, the mixture centrifuged, and the two por- tions tested separately, by the addition of immune body, for the presence of addiment. We varied our experiments greatly so far CONTRIBUTIONS TO THE THEORY OF LYSIN ACTION. 7 as time and temperature conditions were concerned, but the result was always the same; the red blood-cells did not combine with a trace of addiment. This is in direct contrast to their behavior toward the immune body. Having now determined the behavior of the blood-cells to immune body and addiment separately, it remained to see what the affinities of the blood-cells were when both of these bodies were present at the same time. The solution of this problem offers many technical difficulties. Practically it will be best to make the mixtures so that there will be just the proper amount of the two ingredients to effect complete solution of the blood-cells. We found that if we mixed 1.0 to 1.3 cc. of our inactivated goat serum with 0.5 cc. normal goat serum, this would just suffice to dissolve 5 cc. of a 5% mixture (in saline) of sheep blood-cells. If this mixture is placed in the ther- mostat, complete solution will ensue; but because an excess of the solvent substances has been avoided, the process does not take place rapidly. Usually it is completed at the end of 1^ to 2 hours. If the mixture is kept at 0°-3° C, no solution occurs, and if it is then centrifuged and examined according to the methods just studied, the red blood-ceUs will be found to have loaded themselves with immune body, leaving the addiment in the fluid. The experiment shows that under the conditions mentioned, addiment and immune body exist in the fluid entirely independent of one another. It still remained to determine the combining afiinities at higher temperatures. A preliminary trial showed that if we used the pro- portions above mentioned and kept such mixtures in an Ostwald water-bath at 40° C. for six, ten, thirteen, and eighteen ' minutes respectively and then centrifuged, only in the first two tubes did the fluid remain colorless, while in the other tubes it was distinctly red. In the experiments at this temperature we therefore adopted a time limit of ten minutes. A tube of the above-mentioned mixture was allowed to remain in the water-bath at 40° C. for ten minutes and then centrifuged. The results were as follows: The sediment mixed with salt solution shows haemolysis of a moderate degree. (This occurs even if the sediment is mixed with ice-cold salt solution, centrifuged, and then again mixed with salt solution. By this manipulation the last trace of fluid originally adhering to the cells is removed.) Solution becomes complete when new addiment in the form of normal serum is added to the nuxture. The centrifuged fluid does not, by itself, dissolve blood 8 COLLECTED STUDIES IN IMMUNITY. added to it, or it does so in only a very limited degree. When, how- ever, new immune body is added, the blood-cells are completely dis- solved. From these experiments we conclude that the sediment this time contained both components, though not in equivalent proportion, for there was an excess of immune body which became manifest only on the addition of new addiment. Corresponding to this the centrifuged fluid contained only faint traces of immune body and an excess of addiment. The explanation of these phenomena presents no difficulties. It must be assumed that under certain circumstances the immune body and addiment enter into loose, readily dissociated chemical combi- nation. This combination is hastened by heat and retarded by cold in entire conformity to the views previously expressed by Ehrlich (Werthbemessung des Diphtherie-heilserums, Jena, 1897). On the other hand, the affinity existing between blood-cells and immune body must be very strong, for these combine completely even in the cold. We must therefore assume that the immune body possesses two different haptophore groups, one with a strong affinity for the corre- sponding haptophore group of the red blood-cell, and the other of feeble chemical affinity, which is able to combine more or less completely with the addiment present in the serum. At 30° C, therefore, the red blood- cell attracts to itself not only the free molecules of immune body, but also those which have already combined with the addiment in the fluid. In the latter case the immune body represents in a measure a link which ties addiment to the red blood-cells and subjects these to the action of the addiment. In agreement with Pfeiffer, we regard the phenomena appearing under the influence of the addiment as analogous to digestion, and we shall probably not err if we regard the addiment as having the character of a digestive ferment. Morgen- roth, by the experiments in which by immimization he successfully produced an antibody against rennin ferment, has made it very probable that the ferments, like the toxins, possess two groups, one a haptophore group and the other the actual carrier of the fer- ment action. With this preUminary analysis all the various phenomena are now readily explained. We assume that the immune body combines with the small amount of digesting fernient normally present in the blood, and then, by means of its other haptophore group, fitting, for example, to red blood-cells or bacteria, carries this digestive CONTRIBUTIONS TO THE THEORY OF LYSESf ACTION. 9' action over to these cells. From this we see also why the digestive action becomes manifest only on the addition of immune body. This brings the ferment, present in the serum fluid in such small quantity, to the blood-cells in comparatively large amounts, thus concentrating and increasing its action. It is possible and even probable that only a few substances with digestive properties exist. in the blood, perhaps only one; but that a countless variety of specific immune bodies can exist there, as Gruber, among others, assumes. In that case we must assume that in these immune bodies there is always one group which fits only to the cells or substances used to excite its production, but that all these immune bodies possess an atomic group in common which effects the combination with the digestive substance. On this assumption it is very easy to explain by means of the side-chain theory the otherwise difficult problem of the mode of origin of the lysins. According to Ehrhch's definition,, the side-chains possess definite atomic groups which are able to com- bine with certain other atomic groups and so increase the proto- plasmic molecule. As far back as 1885 (Sauerstoff Bediirfniss des. Organismus) Ehrhch had pointed out that the atomic groups thus anchored to the living substance were much more readily oxidized and that they therefore represent the nourishment (Kar e^oxrjv) of the cell. The study of immunity has considerably extended this view and taught us that the antibody represents such thrust-off side- chains; further, that the immunizing process consists in forcing the particular organism to produce these side-chains in surplus amount in conformity with Weigert's theory of cell injury. It is of course, very probable that these side-chains, according to their special func- tion, will be differently constituted. If a side-chain is designed to assimilate relatively simple substances, we may believe that the possession of a single combining group will suffice. Very Ukely the side-chains which anchor toxins are of this simple type. But it is entirely different when a giant molecule (albumin molecule) is to be assimilated. In this case the anchoring of the molecule is only a pre- liminary requisite. Such a giant molecule is useless to the cell and can only then be utilized when it is broken up by fermentative pro- cesses into smaller parts. It will be particularly advantageous to the cell if its "grasping arm" is at the same time a carrier of a fer- mentative group which can at once be brought to bear on the anchored molecule. We see such well-adapted contrivances (in which the grasping apparatus also possesses digesting properties) in a whole 10 COLLECTED STUDIES IN IMMUNITY. series of higher plants. For example, the tentacles of Drosera, which may be regarded as grasping arms in the widest sense, secrete a strong digesting fluid. If, then, we see that lysin action does not occur with toxins, but only when the contents of cells are absorbed, be these bacteria or blood-cells, we must conclude that in the latter case large-moleculed albuminous substances are concerned. These are much more complex in structure than the toxins, which represent mere cell secretions. For the assimilation of the highly complex bodies we therefore assume the existence of side-chains of a peculiar kind. These, besides their combining group, possess another group which by fixation with special ferments causes the digestion of the complex substances. If, by means of the immunizing process, one succeeds in having a surplus of these side-chains produced, they will be produced with both these functional groups and thrust off into the blood as immune body. This explains the wonderful contrivance whereby the injection of a bacterium is followed by the production of a substance which destroys this bacteriimi by dissolving it. This phenomenon is nothing but the reproduction of a process of normal cell life. n. CONCERNING H^MOLYSINS.i Second Communication. By Professor Dr. P. Ehelich and Dr. J. Moegbnroth. In a previous paper ^ we demonstrated the relations existing between the red blood-cells to be dissolved and the two components of a specific hsemolysin produced by immunization. It wiU be remem- bered that we termed the two components of the specific serum immune body and addiment. We were able to show that the immune body combines with the ery-throcytes of the species whose blood was injected, since it has a specific affinity for these cells. We showed further that the addiment, the unstable (labile) ferment-like body which effects the solution of the blood-cells, is tied to these cells indirectly by means of the inunune body. Proof was thus afforded that, in conformity with the require- ments of the side-chain theory, the immune body possesses one haptophore group by means of which it combines with the erythrocytes of the corresponding blood, and a second haptophore group with less ■affinity by which it combines with the addiment and transfers the action of the latter to the blood-cells. At that' time we availed ourselves of the serum of a goat which had been treated for some time with subcutaneous injections of a sheep serum rich in blood corpuscles. Corresponding to this treat- ment, the serum of the goat possessed a moderate degree of solvent action on sheep blood-cells. In order to continue these studies it seemed essential to make use of a serum derived from an animal treated for some time with full blood, a serum that would accordingly possess a higher degree of activity. For this purpose we began the immunization (Nov. 12 ' Reprinted from Berl. klin. Wocheneohr. 1899, No. 22. ' See pages 1-10 of this volume. 11 12 COLLECTED STUDIES IN IMMUNITY. and Feb. 24) of two male goats by injecting them subcutaneously with increasing amounts of defibrinated sheep blood. In a short time a strongly active serum was produced in both animals, and we were able to observe how, following the general laws of immu- nization, its activity increased. The course of the immunization did not manifest any peculiarities. It should, however, be remarked that on the days following the injection of a considerable amount of blood (350 cc.) not the least decrease in the activity of the serum could be observed, in contrast to the experiences with tetanus or diphtheria immunization. So far as the general method employed in the following experi- ments is concerned, it was the same as that mentioned in the first paper. The blood was always used in the form of a 5% suspension in physiological salt solution. At the time of these experiments the serum of buck I was able to dissolve the sheep blood com- pletely in the proportion of 0.2-0.3 cc. serum to 5 cc. sheep blood mixture; 0.03-0.07 cc. serum were able to produce a just noticeable amount of solution. Of the serum of buck II, 0.15-0.2 cc. suf- ficed for complete solution. It should be mentioned that the serum of buck II even before immimization possessed a slight solvent effect on sheep blood. This, however, was so slight that 4.0 cc. of the serum were not nearly able to dissolve 5 cc. of the 5% blood mixture, and 1.2 cc. serum produced only a just noticeable amount of solution. Heating the serum to 57° C. for half an hour destroyed this action, as it did also that for rabbit and guinea-pig blood. ^ With the sera of these two bucks we were now able to proceed with our experiments. The combination of the immune body with the erythrocytes of the sheep at 0° C. can be readily demonstrated, for at this temperatiu-e and by the employment of proper amounts of serum no solution takes place. The serum was allowed to act on the sheep blood for twenty-four hours, care being taken to keep the mixture at 0° C. The blood-cells were then separated by means ' On examining the sera of a large number of normal goats one will find some sera which possess this feeble . solvent power for sheep blood. Thus the normal goat sera which we employed for control tests in our first experiments, and which were used in great number, failed absolutely to show any solvent action, but at most manifested only a variable degree of agglutinating action. This will be seen from our -reports at that time. In our first communication we had already called attention to the great variability of the agglutinating property. CONCERNING HiEMOLYSINS. 13 of the centrifuge, and they showed by their behavior that they had combined with the immune body. They did not dissolve on the addition of physiological salt solution, but dissolved when addiment in the form of normal goat serum was added. In contrast to this, both components combined with the sheep blood-cells when the mixture was kept at room temperature (about 20° C.) even for only eight minutes. The blood-cells, separated by centrifuge and washed with physiological salt solution to free them from traces of serum, were mixed with more salt solution and placed in an incubator, where they dissolved in considerable quantity. These new and stronger immune sera therefore exhibited proper- ties in relation to the sheep blood-cells entirely analogous to those ■of the serum previously described by us. On the other hand in cer- tain respects their behavior was entirely different. The serum described by Bordet, as well as that of our goats, ^ lost its hsemolytic power when heated for half an hour to 56° C. This has been shown by Buchner to be true of all normal hsemolytic sera. The sera of our two bucks even when heated for three-quarters of an hour to 56° C. showed only a scarcely apjyreciable diminution of their ■solvent action on sheep blood, while their normal solvent action on guinea- pig blood and rabbit blood was entirely destroyed. Even when the serum was heated to 56° C. for three hours or when, after mixing with equal parts of water, it was heated for one and one-half hours to 65° C, it showed merely a reduction in its solvent action for sheep blood, but not a destruction of this action. Our preliminary experiments on the combining relations had shown us that the action of these haemolysins was due to the pres- ence in the serum of a specific immune body and an addiment. It was therefore clear that we were here dealing with an addiment of a very peculiar kind, which was distinguished from the addiments of aU haemolysins heretofore known by its extraordinary resistance to thermic influences. This property must pertain to the addi- ment itself and cannot be ascribed to the presence of another sub- stance in the serum increasing its resistance, for such a substance would have served to protect the hsemolytic bodies normally present. In order, however, to analyze these phenomena completely, it was absolutely essential to obtain the two components of the complex ' This refers to the female goats. The male goat is always designated ■"buck" by Ehrlich and Morgenroth. [Translator.] 14 COLLECTED STUDIES IN IMMUNITY. serum, the immune body as well as the addiment, in a free state. In the ordinary specific hsemolytic serum the former is usually readily obtained because the addiment is destroyed by slight heating. In. the case of our serum, however, heating proved ineffective, so it became necessary to adopt other means. Experience having taught us that the addiment is, as a rule, more readily destroyed than the immune body, we could expect to accomplish our purpose by using stronger destructive agents of a chemical nature. After a number of trials we have finally made use of the following procedure: One part of our serum is mixed with one-tenth part normal hydro- chloric acid, the mixture digested at 37° C. for 30 to 45 minutes, and then neutralized. It will be found that the serum has then lost its solvent power for sheep blood-cells; but that it still possesses immune body in scarcely decreased amount can be shown by re- activating the serum. The isolation of the immune body made it possible finally to demon- strate the combination of the immune body at higher temperatures, 20°- 35° C. This combination is seen to be quantitative, i.e., the sheep blood- cells are' able to combine with all the immune body -present in that quan- tity of serum which in its active state would just suffice for their com- plete solution. For example, to 5 cc. of the 5% blood mixture, 0.15 cc. of the serum inactivated with hydrochloric acid is added, it having been previously ascertained that this amount of active serum just suffices for complete solution. The mixture is allowed to stand for half an hour at room temperature and is then centrifuged. To the sediment 2.0 cc. normal goat serum are added, and to the clear fluid some additional sheep blood mixture and 2.0 cc. normal goat serum. The sediment thus treated will be seen to dissolve com- pletely, whereas the blood-cells added to the clear fluid remain intact despite the presence of the addiment. This shows that all the im- mune body combined with the sedimented sheep blood-cells. The addiment necessary for this reactivation is present in normal goat serum, as can be seen from the experiment. This is true for all goat sera thus far examined by us, although the amount varies. It will be recalled that we had found the original addiment which fltted the immune body was able to withstand heat. The question there- fore at once arises whether normal serum also contains such heat- resisting addiments. As a matter of fact this was found to be the case in a number of goats examined by us. When the serum of these goats was heated for i to f hr. to 56° C. and its normal hemolytic CONCERNING H.EMOLYSINS. 15 properties for other blood-cells were entirely destroyed, it was still able to typically reactivate the particular immune body here con- cerned.i In another series of goats, however, the result was different, for heating the serum to 56° C. destroyed its reactivating properties completely. These sera then contained exclusively a thermolabile addiment which, hke the thermostabile addiment, fitted the immune body. We must therefore conclude that the immune body developed by this immunization is capable of being activated by addiments of two kinds, which differ from each other by their resistance to thermic influences and which are both present in normal serum. It is probable that both kinds of addiment can be present in goat serum at the same time, but that in most cases only one, the thermolabile, is present. The varjdng behavior toward thermic in- fluences, manifested by the sera of our immunized animals, would thus be easily explained. We assume that the same immune body was present in both cases, but that the serum of the goat first immunized con- tained only the thermolabile addiment, while the sera of the animals examined later contained also the thermostahile addiment. In this connection, the fact that, previous to the commencement of immu- nization, we were able to demonstrate a considerable content of thermostabile addiment in the serum of the third animal Cbuck II) is of considerable interest. Having thus arrived at some understanding of the action of the hsemolytic sera produced by immunization it seemed essential that we extend our investigations to the hcemolytic properties of normal sera. These properties had long been known and had been studied particularly by Buchner and his pupils.^ The fact that the hsemolytic action of normal serum is destroyed by moderate heat led us to believe that the normal hsemolysins are ' As it is thus possible to destroy all the normal lysins (which interfere with the experiment) it ought to be possible to determine whether a similar heat- resisting addiment also occurs in the serum of other species. We succeeded in demonstrating its presence in varying amounts in the serum of a sheep and of a calf, but failed to find it in serum of a dog or rabbit. ' It is very probable that certain forms of hEemoglobiuuria originate through analogous hsemolysins. Many years ago Ehrlich showed that the hfemoglobi- nuria ex frigore was caused, not by any particular sensitiveness of the erythro- cytes to cold, but by certain poisons produced, especially by the vessels, as a result of the cold. Possibly also such autolysins play an important role in the convalescence of severe ansBmias. 16 COLLECTED STUDIES IN IMMUNITY. not of simple constitution; but the experimental solution of this problem was attended with great difficulties. The primary tests necessary to demonstrate the complex con- stitution of a lysin are very readily made on a number of series. They consist in this, that a serum which dissolves certain red blood- cells at ordinary temperatures is mixed with these cells at 0° and allowed to act at this temperature for some time. For example, goat serum is mixed with guinea-pig blood-cells, for which it is nor- mally hsemolytic. The mixture is kept at 0° and then centrifuged. The clear fluid is mixed with an additional amount of blood-cells and tested in the usual manner for its hemolytic power. In this way it was easily shown that through this procedure the serum had lost part of its power, but that this was completely restored by the addition of some of the same serum previously inactivated by heat. According to our previous experience these experiments show that this serum contains two substances: one, which we shall call interbody, possessing two haptophore groups and analogous to the immune body; the other, an addiment, which we shall hereafter term 'complmicnt. Further, they show that of these two bodies the blood- 'cells combine preponderantly with the interbody. The decrease in the power of the serum is thus explained by a lack of interbody, and this is supplied by the addition of inactive serum. In experiments of this kind we have succeeded with the following combinations : goat serum, sheep serum, calf serum, and dog serum, with guinea-pig blood. Although the demonstration of the lack of interbody is extremely simple, the counter-demonstration, that this interbody has combined with the sedimented blood-cells, is extraordinarily difficult; for in this demonstration a completely isolated comple- ment is essential. The production of a complement to fit the specific interbody obtained by heating the serum of our immunized goat is extremely easy, for it is found in all normal goat serum and can also be obtained from immune serum by means of elective absorp- tion. It will be well to analyze the conditions governing this elective absorption by means of which interbody and complement can be separated. Complete separation will be possible when, under the circumstances prevailing at the time, the affinity of the interbody's, haptophore group for the blood-cells is greater than the affinity of its haptophore group for the complement. A measure of the CONCERNING HiEMOLYSINS. 17 relative affinity is found in the degree of temperature at which combination occurs. In the case of the lysin obtained by immuniza- tion, which has already been described, the combination of the blood- cells with the corresponding haptophore group of the immune body took place at 0° C; the combination of the second haptophore group with the complement took place only at a higher temperature. At 0° C. the fluid would therefore contain immime body and comple- ment in a free state, i.e. imcombined. In this case, of course, it is possible completely to abstract the immune body from this mixture by means of the red blood-ceUs. This is the most favorable case. Its direct opposite will be one in which the affinity of the two hapto- phore groups is exactly equal. In that case the blood-cells will invariably combine with interbody + addiment in such a manner that equal amounts of the two components are withdrawn from the fluid. Naturally between these two extremes aU kinds of inter- mediate phases may exist showing variations in the degree of affinity of these two groups. It seems to us that the most frequent case is that in which the affinity of the hsemotropic group of the interbody is not much greater than that of the group fitting the addiment. In this case we are unable to produce free addiment by treating the mixture with erythrocytes; a certain amount of interbody always remains in the serum so that the latter does not completely lose its solvent property. Such sera, which stiU possess solvent property, cannot, of course, be used for experiments in activation. In our investigations on normal sera we met with this last case surprisingly often, and it was this circumstance that made the study of the complements so difficult. We therefore sought to find another method of procedure, one by which these difficulties could be avoided. For analytical purposes it is essential, as already stated, to have both components of the serum, viz., interbody and complement, in an isolated form. The interbody can at any time be obtained from the normal active serum by heating, but the production of the complement from the normal serum is not entirely successful because of the above-mentioned difficulties. We therefore proceeded on the assumption that every blood serum may contain a whole series of different ferment-like bodies, among which some would be capable of assuming the role of com- plement. It was of course clear that such a combination of circum- stances would only be a fortimate chance occurrence, and that only 18 COLLECTED STUDIES IN IMMUNITY. by examining a large number of separate cases wotJd such a favor- able combination be found. As a matter of fact after a rather long search, we succeeded in finding such cases. As is well known, dog serum dissolves guinea-pig blood with great energy. If it be heated to 57° C. it loses this power, in accord- ance with the usual rule. However if to the 5% guinea-pig blood mixture some of this inactive dog-serum is added, and also a sufficient quantity of normal guinea-pig serum (about 2 cc. to 5 cc. of the 5% blood mixture), complete solution takes place. This fact can be ex- plained only by assuming that the guinea-pig serum contains a complement which happens to fit the haptophore group of the inter- body derived from the dog, and that it thus reactivates this. In this case the proof is all the more convincing because solution is effected by the addition of serum of the same species from which the blood-cells are derived. This serum shoiild be the best possible preservative for the cells, for it represents their physiological medium.* By means of these experiments we regard it as positively proven that the hemolytic action exhibited by a serum, normally or in response to immunizing procedures, is due, in the cases examined by us, to the combined action of two substances. Now that we.had at our command the interbody of the hsemolysin solvent for guinea-pig blood, derived from dog serum, as well as a complement which reactivated this, we were ready to proceed to the last of our demonstrations. To each of two test-tubes containing 5 cc. 5% guinea-pig blood 0.2 cc. inactive dog serum were added, after it had previously been ascertained by experiment that 0.2 cc. dog serum previous to heat- ing were just sufficient completely to dissolve this amount of guinea- pig blood. The mixtures were allowed to remain at 20° for half an ' We succeeded also in finding other combinations in which an analogous relation in greater or less degree could be demonstrated. Of these we may mention: 1) guinea-pig blood, inactive calf serum, guinea-pig serum; 2) sheep blood, inactive rabbit serum, sheep serum; 3) goat blood, inactive rabbit serum, goat serum; 4) guinea-pig blood, inactive sheep serum, guinea- , pig serum. The fact that, such an interbody, i.e., one derived from one animal species, finds fitting complements not only in its own serum but also in that of different species, is of considerable importance in the question whether curative sera can be made harmless to man by means of pasteurization. Possibly this would serve to explain why heating of the diphtheria curative serum, introduced by Spronck, has not realized the expectations a priori held out for the procedure. CONCERNING HvEMOLYSINS. 19 hour and then centrifuged. The sediments thus obtained were washed with salt solution and again centrifuged. If now to one of these sediments physiological salt solution was added, and to the other 1.5 cc. guinea-pig serum, complete solution resulted in the latter, whUe the former remained undissolved. This proves that the interbody was completely anchored by the blood-corpuscles. The fluid obtained by centrifuging did not 'dissolve guinea-pig blood, even when considerable guinea-pig serum was added. It did not, therefore, contain any free interbody derived from the dog serum first added. By these experiments we became convinced that haemolysis in general is due, not to a simple body, but to the combined action of two distinct substances. At the present time we have no general method to demonstrate this for each individual case, and the solution of the problem therefore is now possible only under either of the above-mentioned favorable conditibns: (1) when the two hap- tophore groups of the interbody differ greatly in their affinity; and (2) when, by means of a combination whose discovery depends on chance, an activating complement is found. Where these conditions are not fulfilled, the solution of the problem, for the present at least, is impossible. This, for example, is the case with ichthyotoxin, the haemolytic constituent of eel serum. It is extremely easy to inactivate this eel serum, slight warming for fifteen minutes to 54° C. sufficing, but thus far we have been entirely unsuccessful in reactivating it, because we have been unable to find the requisite complement. Considering their multiplicity, it is but natural that we are only just getting a deeper insight into the nature of the substances in normal blood serum. It is obvious also that a great many questions whose solution is of importance present themselves, especially in connection with the substances discussed by us. The first question to be considered is that of the multiplicity of the hsemolysins contained in a given normal serum. According to our observations it is very probable that the ability of serum of one species to dissolve the blood-cells of various other species is de- pendent on the action, not of a single lysin, but of several lysins. If, for example, dog serum dissolves the blood-cells of guinea-pigs and of rabbits, it must be assumed that a multiplicity of interbodies and of corresponding complements effects this action. Some of the ways in which the solution of this problem can be approached are as follows: 20 COLLECTED STUDIES IN IMMUNITY. (1) The isolated destruction of single lysins by means of thermic and chemic influences. (2) The binding of the different lysins by means of corresponding species of blood, thus making their elective removal possible. With jed blood-cells this procedure, to which we shall return in a sub- sequent article, offers many technical difficulties. On the other hand, with a different kind of specific constituent of the serum, namely, the agglutinins, this method is easily appUed, as can be seen by the experiments of Bordet ^ made in connection with our first experiments and carried out by the methods employed by us. (3) A separation of the lysins also seems possible through im- munization, by means of which one is able to obtain antibodies against the normal lysins. Thus Kossel, Camus, and Gley, by treat- ing animals with the strongly globuhcidal eel serum, have obtained a serum which neutralizes the action of this eel serum, in other words, one containing an antilysin. Evidently this reactively formed anti- body thrusts itself into the hsemotropic group of the interbody and thus deflects this from the erythrocyte. Our attempts, based on these premises, to produce an isolated antibody for some of the lysins have thus far been unsuccessful. Thus a serum derived from rabbits after these had been treated with goat serum, protected the rabbit erythrocytes against solution by goat serum. At the same time, however, it protected the blood of guinea-pigs and rats against the same influence, and even prevented the hsemolytic action of dog serum on rabbit blood. From this fact we must conclude that immunization with one serum produces a whole series of different antilysins. Clearly this is to be explained by assuming that a serum contains a great number of different complexes possessing haptophore groups, of which many, whether they are toxic or not, are able to excite the production of corresponding antibodies. This surprising multiplicity of substances, present in the blood, which possess haptophore groups (hemolysins, agglutinins, ferments, antiferments) is very readily harmonized with Ehrhch's views. According to his conception all these substances represent side- chains of the protoplasm, which have been thrust off and have reached the circulation. The physiological object of the side-chains is, as Ehrlich stated in 1885,^ to bind assimilable substances to the protoplasm so that these may serve as nutriment for the latter. • Inst. Pasteur, March 1899. ' Ehrlich, Sauerstoffbediirfniss des Organismus. Berlin, 1885. CONCERNING H.EMOLYSINS. 2t A large part of these side-chains may, under suitable circumstances,, be thrust off and thus appear in the blood. Considering the large number of organs in the body and the mani- fold chemistry of their protoplasm, it should not surprise us that the blood, which represents all the tissues, can be filled with innumer- able side-chains; and it is not at all astonishing, considering the constantly changing chemistry of the organism (influenced by a large number of factors such as race, sex, nutrition, labor, secretion, con- ditions of the surrounding medium, etc.) that the serum should be subject to constant qualitative fluctuations. Such variations are seen in the examples already mentioned, showing the behavior of sera of normal animals. Goat serum at one time possesses a slight solvent action on sheep blood, at other times this is entirely absent. Dog serum in one case dissolves the red cells of cats very strongly, in another case it does not do so at all. The action of rabbit serum on guinea-pig blood shows a special variability. A very interesting example is afforded by lamprey serum, which, as is well known, possesses an extraordinarily toxic action for labora- tory animals in general and also for red blood-cells in vitro. Dr. Schonlein of Naples, whose recent death we lament, was kind enough to experiment with this for us. His investigations showed that the eerum of a not inconsiderable number of lampreys possesses no toxic action at all, so that it could be injected into rabbits intra- venously in amounts of 2 cc. without any damage whatever. It is clear that this extensive variability enormously increases the difficulties in investigating these sera. Thus on repeating the well- known experiment of Buchner, whereby a mixture, in certain pro- portions, of dog and rabbit sera loses its hemolytic property for guinea-pigs in the course of twenty-four hours, we were able to com- pletely confirm Buchner's results in three cases, while in five other cases the hsemolytic effect was only more or less lost. "We believe that all these investigations support the view we have already expressed regarding the nature of the complex poisons of the blood-sera. v. Dungern (Muench. med. Wochenschr., 1899, No. 14), basing his action on some new experiments of his, has accepted our views. We a > g a^o » -a S^„,rS V MVJ o- ^^ ^ o 2.3 e-aS CT-o if I ki ir S.p »=K 5-a-2 2. ^3gg n o ■a a sg O B o p- 8 c4-„. s-p p p i-» p £. o. cL S-m o p' hi u o r *"**§ r! B f;.^ C3 ^ CD 5 gp > S ^'3 a r 1 o 3 B- Pm £"3 3 2- B 03 "Coo B- _ f= St" m' o' a^ S'o TO "• 3 to It §1 |l 2.0 ClB t» ,^ p p" »B- m* 3 3s. crq ^d §2. O-B (5 -. P-o- 2.0- B '=-' n S IT* I P'S CD 2. p_c". 3 '». p-B 2 3 CD c B S=3 5;=3' 86 COLLECTED STUDIES IN IMMUNITY. complements circulating in the seriim do not cause the formation of autoanticomplements. Confirmation of this view is furnished by the fact that even in animal species possessing identical complements it is impossible to produce anticomplements by means of serum injections. Thus, neither sheep when injected with goat serum, nor, conversely, goats when injected with sheep senma produce any anti- complement, for these two species manifest an extensive similarity in their complements as well as in other serum constituents. When, then, in spite of this rule, we find that in our case auto- anticomplements have developed, only one explanation remains: that one or the other complement present in the goat serimi, although related, is not identical with the complement of the rabbit. If we assume that a certain goat complement possesses the same haptophore group as does a certain rabbit complement, but that it differs in the rest of its constitution, then the assumption that identical complements do not form anticomplements will not apply. In this case, by means of the haptophore group of the particular receptor of the rabbit cell, a foreign complex would be anchored which exerts a sufficient stimulus on the cell to cause an increased production and thrusting off of the corresponding side-chains which can fimctionate as anticomplements. We shall have to assume that the particular goat complement, because of its identical haptophore group, can be anchored at the same places as the idio complements with the same haptophore group. Foremost among these places we may consider the complex receptors which possess two haptophore groups (amboceptors). In this case, contrary to what we usually observe, the thrusting-off of an amboceptor would he effected through the anchoring of its complementophile group, and we should then have additional proof for our view that the com- plex receptors possess two binding groups. In any case it would seem to be of the greatest importahce to gain an insight into the conditions governing the disappearance of the idiocomplements. That they can be caused to disappear through injection of anticomplements produced by immunization follows as a matter of course from our definition of anticomplements. This, however, occurs only under artificial experimental conditions and so possesses but Httle significance pathologically. Of considerable importance for these occurrences under natural circmnstances are the vital conditions governing the disappearance of complement through internal metabolic processes. The origin of the autoanti- complements as it has jiist been presented by us surely belongs here, STUDIES ON HiEMOLYSINS. 87 and it has perhaps some practical significance, viz., that in the fre- quent injection of various .curative sera into man and animals, the possibility of autoanticomplement formation should be borne in mind. Another case belonging here has previously been described by us — the disappearance of part of the complements in a rabbit poisoned with phosphorus. In connection with this the following observation of Metalnikoff (1. c.) is of interest. He was immunizing a rabbit with spermatozoa and noticed that in consequences of a purulent process which developed during the course of the immuni- zation, the complement which activated the spermotoxin disappeared from the serum and did not reappear for a considerable time. These isolated observations seem to indicate that the com- plements can disappear during pathological conditions in conse- quence, perhaps, of a more rapid destruction or of a slower formation. The same holds true for the immune bodies (amboceptors) which in bacteriolysis as well as in haemolysis have at least as great a sig- nificance as the complements. Which of these two factors prevails in any single case cannot be decided by any general rule, but each case must be examined separately. Only through such investigation wiU we gain an insight into the nature of "natural predisposition" and its changes, "increased resistance," "loss of resistance," etc. Yin. STUDIES ON H^MOLYSINS.i Sixth Communication. By Prof. Dr. P. Ehelich and Dr. J. Mobgenroth. The steady progress of the investigations in immunity is rendered extremely difficult by the fact that in the immunization with living cells and in the study of the immune sera thus obtained a large niunber of different substances which exist simultaneously is concerned. In our second communication we pointed out that the hsemolysins present in normal serimi, which act on different species of blood, are not a single substance in the sense of Buchner's alexin; and in our fourth communication we showed that this could be demonstrated experimentally by means of elective absorption. It is possible that just as many interbodies come into action as the varieties of blood affected. We have also been tmable to accept Bordet's unitarian view of the complements. On the contrary, as a result of our own experiments we have become convinced that a large number of com- plements exist together in blood serum. In like manner Bordet's absorption experiments indicate a plurality of the bacterial agglutinins and those of Malkoff a plurahty of the normal haemagglutinins. The results of these experiments have been gathered together by M. Neisser ^ in a study in which, on the basis of the same principles, he demonstrates the variety of the antitoxic antibodies occurring in nor- mal serum. In conformity to this, the reactive antibodies produced by injections of serum of foreign species are most varied in their nature, and we are only just beginning to gain an insight into their constitution. Aside from the numerous coagulins and antiferments thus produced, it is of the utmost importance, so far as the discussion of immunity problems is concerned, to recognize the fact that the complements 1 Reprinted from the Berliner klin. Wochenschr., 1901, Nos. 21 and 22. 2 Deutsche med. Wochenschr., 1900, No. 49. 88 STUDIES ON HiEMOLYSINS. 89> formed through immunization, corresponding to the multipHcity of the complements present in the serum, are exceedingly manifold. Especially significant, however, is the fact that the cells possess. a great number of different kinds of groups, which groups can lead to the production of numerous different amboceptors (immune bodies).' Hence in immunzing an animal with cell material, the organism is injected, not with a single uniform substance, but with a multitude of the most varied receptors, each of which is more or less able tO' produce an antibody. In our fourth communication we defined our point of view on this basis as follows: "In view of our experiments on isolysins described in our third communication the occurrence of different immune bodies in a. haemolytic serum obtained by immunizing with red blood-cells is, not at all surprising. We have obtained a whole series of different isolysins by injecting goats with goat-blood. At present they number twelve. In the red blood-cells not merely a single group, but a large number of different groups, must be considered, which, provided there are fitting receptors, can produce a corresponding series of immune bodies. AH of these immune bodies, again, will be anchored by the blood-cells employed in immunization. We may assume that when an animal, species A, is immimized with blood-cells of species B, a hsemolytic serum will be produced which contains a great host of immune bodies. The immune bodies in their entirety are anchored by the blood-cells of species A." Durham 2 has adopted the same view for the bacterioagglutinins. He assumes a number of "components " (corresponding to our recep- tors) in the body substance of the bacteria, which can cause the production of a corresponding number of agglutinins. In this way each agglutinin which acts on a certain species of bacteria represents, the sum of different kinds of single agglutinins, a view entirely analogous to our assumption of a plurality of inmiune bodies. This view permits Durham to offer a sufficient and natural explanation of the varying degree of action of typhoid agglutinins on typhiod bacilli of different origin, and of the extension of the agglutinating action of specific sera to related species of bacteria. It would be ' Compare the thorough exposition by Ehriich in Vol. VIII of Nothnagel's Specielle Pathologie luid Therapie, Holder, Vienna, 1901. ' Durham, Journ. of Experimental Medicine, New York, Vol. V, No. 4, 1901. 90 COLLECTED STUDIES IN IMMUNITY. very interesting to see these as yet purely theoretical suggestions of Durham proved by means of experiments. The pluralistic standpoint adopted by us creates numerous difficulties for thorough analytical work in this field, but it leads to a deeper insight into the complicated problems and may perhaps also prove of value in the practical applications in immunity. I. Observations on the Pluralistic Conception of the Cellular Immunity Reaction. To begin, we shall briefly sketch one of the points of view yielded by the plurimistic conception, which seems to be of some practical value. Let us assume that a cell, e.g., a bacterial cell, possesses twenty different groups; then twenty different antibodies correspond- ing to these will be possible. Each haptophore group of the bacterial cell wUl then represent an isolated point of attack for one particular immune body. It is certainly most logical to conclude that the possibility of successfully combating a certain bacterial infection increases directly with the number of kinds of immune bodies which act on the bacterial cell.^ The ideal effect would obviously be attained if it were possible to produce a serum so constituted as to contain immune bodies for all the groups present in the bacterial cell. The phenomenon of antibody formation as it proceeds according to the side-chain theory is a very complex one, and is composed of a number of phases (binding, super-regeneration, thrusting-off) which are partly independent of each other. Hence a variety of circumstances may arise which exert an inhibitory action at certain points. To begin, the cell may be so severely damaged by the anchoring of certain poisonous substances that the formation of antibody does not occur at all, or occurs in only a very slight degree; for this antibody production, which is a kind of regeneration process, pre- supposes a certain degree of cell efficiency .^ This damaging effect will result especially with highly toxic sub- ^ It is, in fact, conceivable that the occupation of a single group only produces a certain amount of injury to the cell without being able to cause its death. The danger to the life of the bacterial cells would increase in proportion to the number of partial injmies, which again would correspond to the increase in the number of types of receptors. It is possible that the potent bactericidal sera so far ■obtained owe their success to a certain plurality of the immune bodies. 'Weigert has already called attention to this. See Lubarsch-Ostertag, Ergebnisse der Pathologie, 1897, page 138. STUDIES ON HiEMOLYSINS. 91 stances, provided the receptors fitting these are present exclusivelj' in vitally important organs, e.g., the central nervous system. This perhaps explains the circumstance that it is exceedingly difficult to produce an antitoxin in mice and guinea-pigs with unchanged tetanus poison, while this is easily effected when toxoids are used. On the other hand, an immunization of rabbits by means of imchanged tetanus poison is very easy to attain, because in these animals, as is shown by the investigations of Donitz and of Roux, the greater part of the receptors lies outside of the poison-endangered central nervous system. However, even without any development of illness it is not at all necessary that antibodies should be produced in every case in which an anchoring occurs. Metchnikoff, for example, has called attention to the fact that with frogs in whom every trace of Ulness is avoided by keeping them cool (as we know from Courmont's beau- tiful observations) it is impossible to produce any tetanus antitoxin. Investigations by Morgenroth have confirmed this result and shown further that even by treatment with toxoids under various conditions it is impossible to produce a trace of immunity. Probably in this particular case these results indicate that the regenerative powers of the frog's tissues are not equal to these extraordinary demands. Such an explanation for failure of the antibody to develop is, how- ever, much less probable in the case of warm-blooded animals; and as the number of experiments increases, these cases are becoming more frequent. Probably all who have busied themselves with the subject win have found, particularly with the artificially produced cell poisons, that in some cases it is extremely difficult if not impos- sible to effect the production of anti-immune bodies. Thus, Metchnikoff injected a series of guinea-pigs with specific spermo- toxin, a substance which certainly finds receptors in the guinea-pig's organism. Despite this, he found but two cases in which even a suggestion of antispermotoxin could be demonstrated. In the case of a dog injected with a specific dog blood immune body derived from a sheep, we have failed despite long-continued treatment to produce any anti-immune body. With this series of phenomena must also be classed the fact that it is extremely difficult if not impos- sible in a number of animal species to produce antienzymes by the continued injection of certain enzymes. The explanation of these facts presents two possibiUties: First, the receptors concerned in the particular case may be of peculiar 92 COLLECTED STTTDIES IN IMMUNITY. constitution in one respect, i.e. in being firmly boimd to the proto- plasm, so that a thrusting-off, which is essential for the formation of antibodies, does not occur even with an increased regeneration (sessile receptors). This leads to the conception that the regenera- tion of the receptors may take two courses: (a) a thrusting-off of the receptors ensues, and with this a formation of antibody ,- (6) in the case of sessile receptors, a hypertrophic process sets in comparable perhaps to a simple muscle hypertrophy, according to Weigert's conception. Second, it is conceivable, as Morgenroth^ has shown in the immunization against rennin, that normal pre- formed regulating contrivances come into action, for, in the case of enzymes (in contrast to toxins) we are dealing with substances nor- mally produced by the organism itself. Hence it is possible that the formation of an antienzyme is followed by the production of the enzyme itself, in consequence of an internal regulating contrivance. In any event these observations will show how the factors just discussed can make it possible, when cells possessing numerous different receptors are injected, that only a small number of the anti- bodies theoretically possible is actually produced. It is therefore very likely that the immunization of one animal species with a certain kind of cell or bacterium results in the production of only part of the possible antibodies. When, however, the same kind of cell or bac- terium is injected into a second animal species, it is highly probable that in this species the haptophore groups of the cells will find a receptor apparatus which in part at least is different from that of the first species The prerequisite for such a difference is the obvious assumption that the receptor apparatus of one species is not identical with the receptor apparatus of a second not very closely related species. For example, it is possible that a certain haptophore group a of the typhoid bacillus finds fitting receptors in the organism of the rabbit, but not in that of the dog, whereas another group, b, finds just the reverse conditions. If these presumptions are correct an important principle for the practical production of curative sera wiU follow, namely, that in any single case one wovM immunize a number of different animal species, select the sera containing different immune bodies, and by mixing these, produce a curative serum containing differ- ent types of receptors in as complete a form as possible. Owing to the importance of this subject we have first under- ' Centralblatt fiir Bacteriologie, Vol. 26, 1899. STUDIES ON HEMOLYSINS. 93 taken the experimental study of the preliminary question whether mmune sera derived by treating two different animal species with the same cells are identical so far as their antibodies are concerned, or whether they are partly or wholly different. Of these antibodies the most important are the bacteriolytic and hsemolytic immime bodies. According to our conception, as is well known, these possess two haptophore groups, one, the complementophile group ^ and the other (which anchors itself to the receptors of the cells causing the immtmity) which we can briefly designate the cytophile group. According to what has been said above it is this second group which possesses special significance in the question under discussion, and we may therefore formulate our problem as follows: To deterrmne whether, in the immunization of different animal species with cells of one kind, amboceptors {immune bodies) possessing different cytophile groups arise. The experimental study of this question can be pursued in the main in two different ways: 1, by means of the absorption test which, although it is very difficult, is applicable to bacteriolysins as well as to hsemolysins ; 2, by neutralization with antiamboceptors (anti- immune bodies). The latter way, the more elegant of the two, is, however, presumably applicable only to those immune bodies which are directed against cells of the organism. A hmmolytic or cytotoxic immune body, as is to be expected, always finds points of attack in the organism- of the corresponding animal species, for this is the first prerequisite for the possibility of an anti-immune body. As a matter of fact also, such anti-immune bodies have already been observed. On the other hand, the immime bodies of bacteri- cidal sera, since their natural counter groups are found in the bacterial cells, will in aU probability not find these groups in the cells of the higher animals. Hence it seems improbable, unless by chance they occur in an isolated case, that anti-immune bodies directed against the bactericidal immune bodies will be produced. II. Concerning the Variety of the Cytophile Groups of Homologous Immune Bodies. We selected immunization with ox blood-cells as being especially adapted for these experiments. Such immunization had already been carried out by von Dungern on rabbits. The production of immune bodies in high concentration succeeds particularly well in 94 COLLECTED STUDIES IN IMMUNITY. this case, so that later investigators (Buchner, Rehns, Bulloch) have also employed this iiseful combination. In many cases, most easily by means of intraperitoneal injections of the ox blood, a potent hsemolysin is produced of which 0.005-O.0005 cc. suffices to dissolve 1 cc. of the 5% ox-blood mixture. Since the production of the immime body is unaccompanied by any increase in complement (as von Dungem showed in just this case) it is always necessary, in order to bring the total amount of immune body into action, to add extra complement. This is found in large amounts in the serum, of rabbits and especially in that of guinea-pigs. Now we have observed that the serum of these rabbits which had been immunized with ox blood is able to dissolve not only the blood- cells of oxen, but also those of goats. The following table shows a comparison of the solvent action of several of these sera on the blood- cells of oxen and of goats. Guinea-pig serum (0.1 or 0.15 cc.) was used as complement since rabbit serum itself, in the doses required, often exerted a hsemolytic action on the goat blood-cells. TABLE I. Action of the Immune Body op the Rabbit Immunized with Ox Blood, on Ox Blood, and on Goat Blood. Number of the Rabbit Treated with Ox Blood. Complete Solvent Dose for 1 cc. of Ox Blood. Complete Solvent Dose for 1 cc of Goat Blood. Ratio of the Solvent Doses (Approximate). Complete Solvent Dose for Ox Blood = 1. No. 1 of 1-24-01 2"XII-14-00 3" II- 8-01 4 " II- 8-01 5" 1-21-01 6" XII-17-00 7"XII-14-00 8" II- 3-01 9"XII-15-00 10 " II- 9-01 0.0042 0.0035 0.002 0.003 0.0017 0.0014 0.00088 0.0051 0.00073 0.0035 0.0061 0.0061 0.0035 0.01 0.0061 0.0051 0.0042 0.05 0,0073 0.06 1.5 1.7 1.8 3.3 3.6 3.6 5 9.8 10 17 This table shows that the hsemolytic action of the immune body is always less on goat blood than on ox blood, and that the ratio of the solvent doses for the two species of blood is not constant but varies within fairly wide limits, as can be seen from the last column. STUDIES ON HEMOLYSINS. 95 This variable ratio indicates that the solvent action on the two species of blood-cells is not the simple function of one and the same immune body, but that two fractions of immune bodies are pres- ent in the serum, of which one acts exclusively on ox blood-cells, while the other fraction acts both on ox blood and on goat blood- cells. These relations can be studied directly by means of elective ab- sorption. If the immune body is treated with a sufficient amount of ox blood cells and the fluid is then separated by centrifuge, it wUl be found that the serum has lost its solvent action for both species Fig. 1. Blood-cell of an ox and of a goat, showing specific and common receptors of blood; for by means of the ox blood-cells, which as the original excitants of the immunity are carriers of all the receptors in question, both fractions of immune body have been bound. When the same experiment is performed with goat blood-cells, it can be shown that the fluid has lost its solvent power for goat blood, while that for ox blood remains. In favorable cases the solvent power for ox blood may remain almost unchanged. The conditions present can be readily understood by reference to Fig. 1. Let this represent schematically three portions of the combin- ing groups of the blood-cells, of which the first, a, is present only in ^6 COLLECTED STUDIES IN IMMUNITY. the ox blood-cells, the second, y, only in goat blood-cells, and the third, /?, in both. If a rabbit is injected with ox blood, the ambo- ceptors (immune bodies) corresponding to groups a and /? will be formed. Ox blood-cells, by means of their a and /? groups, will then be able to anchor all the immune bodies, whereas goat blood- cells will anchor only the immune body of portion ^, leaving the immune body of portion a in the solution. If, as this explanation assumes, the goat blood-cells possess a certain portion ofr eceptors which are common to goat and ox blood- cells, it is essential that by treating rabbits with goat blood an immune body should be obtained which likewise would act on both species of blood. This, in fact, is the case. And here, as in the first case, the solvent power for the two species of blood-cells usually differs, though of course the relations are reversed from those in that case, as can be seen by reference to Table II. TABLE II. Action of the Immune Body op the Rabbit which had been Treated with Goat Blood, on Goat Blood, and Ox Blood. (Reactivation with guinea-pig serum.) Number of the Rabbit Treated with Goat Blood. Complete Solvent Dose for 1 cc. of Goat Blood. cc. Complete Solvent Dose for 1 cc. of Ox Blood. cc. Ratio of the Solvent Doses (Approximate). Complete Solvent Dose for Goat Blood =1. No 1 of 11-28-01 0.01 0.0061 0.0012 0.0071 0.024 0.025 0.025 0.25 (almost complete) 1-2 4 " 2" 1-14-01 1:4 " 3 " II- 7-01 ' 1-20 " 4 " XII-18-00 1" <33 ' On employing the same serum on a different ox blood, 0.05 cc. produced no solution at aU, and 0.1 cc. merely a trace. This is evidently due to a casual, individual lack of receptors in the ox blood-cells in question, such as showed itself so frequently in goat blood when we studied isolysins. Because of these ratios we shall have to assume that the goat blood-ceUs in this case possess a second system of binding groups which is peculiar to them and represented in the above diagram by ;-. They possess these, of course, in addition to the receptors, /? which they have in common with the ox blood-ceUs. In accordance with this, in the elective absorption test in this case, the goat blood- cells will bind the entire lot of immune bodies; whereas when ox STUDIES ON HEMOLYSINS. 97 blood-cells are used, the group ;- will be left behind, for this possesses affinity only for the goat blood-cells. The following protocol shows the results of two series of experi- ments, which exhibits the effect of such reciprocal binding: To each 5 cc. of a 5% mixture of ox blood-cells or goat blood- cells (freed from serum by centrifuge) varying amounts of the immune body of a rabbit which had been immunized with ox blood are added. The amount of immune body is seen in the first column of the table; in the second and third columns the complete solvent doses (for ox blood -and for goat blood) contained in each specimen are given, as they were determined by tests made at the same time. The mixtures are made up to 6 cc. with physiological salt solution, kept at 37° C. for IJ hours and then centrifuged. Two equal por- tions of each of the decanted" fliiids are then taken and again mixed with corresponding amounts of blood-cells. Finally guinea-pig serum is added to activate -the mixtures. The hsemolytic action which the decanted portions exerted on ox blood-cells and on goat blood-cells is seen in the table. (See Table III.) The union of the immune body with the ox blood-cells has resulted in a considerable abstraction of both portions of immune body. On the other hand, the union with goaf blood-cells, by which the action of the fluid is considerably decreased for goat blood-cells, causes very little decrease in the solvent power for ox blood. In contrast to this experiment we here reproduce an analogous experiment which shows a directly opposite behavior of the two fractions of immune body of a rabbit immunized with goat blood. (See Table IV.) Here the goat blood-cells bind both portions of the immune body, while after treatment with ox blood-cells the fraction acting on goat blood is left almost intact. Hence by means of this crossed immunization and reciprocal elec- tive absorption we succeed in demonstrating that in the case of the rabbits treated respectively with goat blood and ox blood two large fractions of immune bodies can be separated. Of these, one fraction is common to both, sera; the other is peculiar to each of them. The main groups of receptors shown in the above illustra- tion and designated a and j3 for ox blood, and /? and -jr for goat blood, are thus to be differentiated. We have deemed it important to supplement this analysis by experiments on a second species of animal, and have therefore treated 98 COLLECTED STUDIES IN IMMUNITY. a goat with ox blood. Naturally the serum of the goat so treated dissolves ox blood-ceUs. Besides this, however, it manifests the ability to dissolve the blood-cells of a few other goats, and therefore contains true isolysins such as we have previously produced by treating goats with goat blood. Thus 0.025 cc. of the serum of TABLE III. Binding of the Immune Body of a Rabbit Treated with Ox Blood, with Ox Blood-cells, and Goat Blood-cells. Solvent Power of the Decanted Fluids. Amount of the Number of Solv- ent Doses Con- J. Ill LU. Hue Body Added. (Derived from tained Therein. A, after Binding with Ox Blood. B, after Binding with Goat Blood. a, Rabbit by Treating with Ox Blood.) (a) For Blood. (6) For Goat Blood. (o)On (,b)On (a) On (6) On Ox Blood. Goat Blood. Ox Blood. Goat Blood. No. cc. 1 0.001 i A 2 0.002 i tV trace 3 0.003 i Is very little : 4 0.004 f i very little to little 5 0.005 1 4 mod. to little 6 0.006 1 A moderate 7 0,007 U S I ( 8 0.008 IJ f aim' St comp. 9 0.01 If i i complete 10 0.012 2 8 t tt faint trace 11 0.016 2f f 1 1 faint trace 12 0.02 3i 1 faint trace very little tt very little,top 13 0.024 4 n very little it It 1 1 little, top 14 0.032 5J If lit. to mod. little to very little tt little 15 0.048 8 2f It little tt tt 16 0.06 10 3 aim' St comp. moderate tt It 17 0.08 13* 4 complete fair 1 1 tt 18 0.1 16§ 5 complete ti strong to al- most comp. tt little to mod. 39 0.14 23i 7 1 1 complete It mod. to little one of our goats, on the addition of complement, dissolved the usual amount of ox blood-cells. This serum, however, dissolved but two out of five different specimens of goat blood, and the isolysin con- stituent was present in only very small amounts, so that it required 0.75 cc. serum (thirty times the above amount) to effect complete hsemolysis of sensitive goat blood-cells. Hence in this case also the development of all such amboceptors as could find a point of STUDIES ON HEMOLYSINS. 99 attachment (receptor) in the blood-cells of the individual goat itself has been avoided, and the phenomenon which we have previously designated as a "horror autotoxicus " ^ is again presented. TABLE IV. Binding op the Immttne Body op a Rabbit Immunized with Goat Blood, ON Ox Blood and Goat Blood-cells. Number of Solvent Power of the Decanted Fluids. Amount of Solvent Doses the Immune Contained A. After Binding with B. After Binding with Body Added. Therein. Ox Blood. Goat-Blood. (Derived from a Rabbit by Treating with Goat Blood.) (a) For (6) For (a) For W For (o) For (6) For Ox Blood. Goat Blood. Ox Blood. Go.at Blood Ox Blood. Goat Blood. No. CO. 1 0.038 T*S 1 fair to mod- erate 2 0.05 tI i almost complete 3 0.062 i m complete 4 0.074 i 2 1 1 5 0.1 if 2f minimal trace 6 0.13 1 3i trace 7 0.15 4 1 1 8 0.2 5 very little 9 0.25 2 6i little 10 0.3 8 It 11 0.38 3 10 fair to strong From this experiment we can at once conclude that this receptor system ^ actually consists of different components, of which only those separate amboceptors (immune bodies) are foimd in the serum of goats treated with ox blood whose receptors are absent in the blood-cells of the goat itself. The most important result of these investigations — investigations: complete in themselves — is this: By treating animals with ox blood, two fractions of immune bodies are formed, of which one acts only on ox blood and the other also on goat blood; whereas by treatment with goat blood the contrary though entirely analogous result ensiies. These two frac- ' We were also able to observe that the immune body of the rabbits which had been immunized with ox blood and goat blood acted also on sheep blood. Closer investigation would probably show that this behavior is analogous to its action on goat blood. This corresponds entirely to our earlier observations on the extensive similarity of the receptor apparatus of goat and sheep blood as it was manifested particularly by the experiments on isolysins. 100 COLLECTED STUDIES IN IMMUNITY. tions do not correspond to two different single immune bodies, but each fraction includes several, perhaps an entire host of immune bodies. The experiments also lead to conclusions of considerable impor- tance in another direction, namely, as affecting our conception of cellular specificity and of the specificity of reaction products. Here- tofore it has been held that the injection of blood of species a results in a specific immune serum, i.e. one acting only on a; and even Metchnikoffi has recently expressed this view. We had already become acquainted with certain exceptions to this principle. The isolysin, for example, produced by injecting goats with goat blood, also dissolves sheep blood; and, vice versa, the immune body of goats which have been injected with sheep blood acts also as an isolysin. At that time we emphasized that these results are onlj' to be explained by assuming that certain types of receptors are com- mon to both species of blood. The same holds true in the case under discussion, von Dungem ^ has come to the same conclusion as a result of his experiments. He found that the immune body produced by injection of ciliated epithelium acts also on the blood- cells of the same species, and that conversely the haemolytic immime body produced by injection of blood-cells is partially boimd by ciliated epithelium. All these circumstances indicate that we must not regard the spe- cificity of the immune bodies from the conception of specificity employed in systematic zoology and botany. The immune sera, as we have often mentioned, are not of simple unitarian nature, but consist of a series of single immune bodies whose cytophile haptophore groups cor- respond to the receptors of the exciting cells. Hence such an irnmune serum will be able to affect all such elements which possess any one of the receptors whose type is common to those elements and the original cell "a." The influence exerted by the immune serum will be power- ful in proportion to the extent of this correspondence of receptors. Now we have reason to believe (cf. Ehrlich's deductions, 1. c, and Weigert's in Lubarsch-Ostertag's Ergebnisse der Pathologic, 1887, p. 141) that certain receptors are very widely distributed among various animal species. Thus the blood-cells of a large number of species possess receptors fitting ricin, abrin, crotin, and tetanolysin, and gan- glion cells of the most divergent animals possess receptors for tetano- ' Revue g(5n6rale des sciences, 1901, No. 1. ' See page 47. STUDIES ON HEMOLYSINS. 101 spasmin or for sausage poison. Within the animal organism, in like manner, certain receptors are evidently widely distributed in the most varied organs, as is shown, for example, by the experiments with tetanus poison. Looked at from this standpoint, the apparent deviations in specificity are comprehensible. We are convinced that in this field the near future will furnish us with extensive ma- terial of immense value in the analysis and study of the distribution of receptors. We are led to conclude, therefore, that in the produc- tion of immune bodies by immunizing with cells we can speak of specificity only in the sense that there is always a specific relation between the separate types of immune bodies and the receptors. The foregoing experiments constitute conclusive proof of the plurality of the immune bodies produced by injections of ox blood and goat blood. We next endeavored to extend these results by effecting a differentiation of various groups of immune bodies by means of the anti-immune bodies.^ The highest concentration of immune bodies at our disposal was the serum of a rabbit which had been immunized with ox blood. For various reasons we chose goats for these immunizing experiments, for we knew that their blood- cells already contained receptors capable of binding a portion of the mixed immune bodies. In treating these goats we used the inactive serum of a rabbit immunized with ox blood. This serum, which was of the highest possible strength, was injected subcutaneously. During the course of two months we had thus injected 120 cc. of an immune body serum, of which 0.005 cc. sufficed, when reactivated with guinea-pig serum, to completely dissolve 1 cc. of a 5% mixture of ox blood-cells. At the end of that time we were able to demon- strate the existence of an anti-immune body of considerable pro- tective power. That this was really an anti-immune body which inhibited the anchoring of the immune body to the red blood-cells, is seen by the following combining experiment. 0.5 cc. of the anti-immune body (inactive serum of a goat treated as just described) are mixed with varying amounts of the immune body (inactive serum) of a rabbit treated with ox blood. Thereupon 1 cc. of a 5% mixture of blood-cells is added to each specimen. These are then kept at 40*^ C. for one hour and centrifuged. The various sediments are then mixed with salt solution and 0.15 cc. normal guinea-pig serum . A parallel experiment (control test) is made in ' See Ehrlicli's recent study, page 573. 102 COLLECTED STUDIES IN IMMUNITY. which the anti-immune body is replaced by the same amount (0.5 cc.) of inactive normal goat serum. The degree of solution is shown in Table V. TABLE V. Amount of Izuiuune Body Added. Number of Complete Solvent Doses Therein. Degree of Solution After Addition of Complement. Degree of Solution of the Sediment in the Control Test. No. 0.00125 0.0025 0.00375 0.005 0.0075 0.01 0.023 1 2 3 4 6 8 20 no solution trace solution little solution almost complete solution complete tolution complete solution From these figures we see that a single solvent dose becomes available for combination with the red blood-cells only after eight times the solvent dose has been added, and that a triple dose is com- pletely neutralized, i.e., prevented from combining with the blood- cell. The control test shows that 0.5 cc. of a normal inactive goat serum does not prevent the combination of a single solvent dose of mmune body (0.00125 cc). The sediment in this case is compet«ly dissolved on the addition of complement.^ By this experiment the inhibiting substance is definitely characterized as an anti-immune body. The following example will show the exact quantitative relation of this anti-immune body. Each 0.4 cc. inactivated serum (anti-immune body) of the goat treated with immune body . are mixed with the given amount of inactive serum (immune body) of a rabbit treated with ox blood. The specimens are made up to the same volume by the addition of salt solution, kept at room temperature for half an hour, and then mixed with 1 cc. of a 5% suspension of ox blood, and with 0.15 cc. normal guinea-pig serum (complement). A control test is made in which normal inactive goat serum is used instead of the anti-immune body. (See Table VI.) 'We should like to remark that in the course of numerous experiments Tve have now and then found normal goat sera containing slight amounts of an anti-immune body acting on the immune body of rabbits treated with ox blood. This is to be brought into connection with the law (see also Neisser- 1. c.) that the artificially produced antibodies frequently represent only an increase of normal functions. STUDIES ON HEMOLYSINS. 103 TABLE VI. Experiment with 0.4 cc. Anti-immune Body. Control Test with 0.4 cc. Normal Goat Serum. Amount of Amoimt of Immune Body. Solvent Action. Immune Body. Solvent Action. cc. cc. 0.0175 complete solution 0.001 complete solution 0.0145 strong ' ' 0.00085 almost complete solution 0.012 fairly strong solution 0.0007 strong solution 0.01 moderate solution 0.0006 It It 0.0085 little solution 0.0005 moderate solution 0.007 ti It 0.006 trace solution 0.005 small trace solution 0.0044 ft 1 1 It 0.00375 tt it t i 0.003 minimal trace solution 0.0025 ( t I ( (t 0.002 ft It If 0.0018 This shows that 0.2 cc. of the anti-immune body are able to com- pletely inhibit the action of 1.8 times the solvent dose of immune body as determined by the control test, and that it almost neutral- izes the action of five times such a dose. If, however, we measure the protective power by comparing the complete solvent doses in the two cases, this appears much stronger. The ratio of the com- plete solvent doses in the presence of immune body and in the control test is then 1:17.5. We shall discuss the reason for this later. Since the inactive rabbit serum employed in immunization con- tained complementoids, the presence of anticomplements along with the anti-immune body is easily understood. The anticomplements at first were directed against rabbit serum. After the immunization had continued for some time longer anticomplements appeared directed against certain complements of guinea-pig serum. In these experiments, therefore, in order to overcome the anticomplement action (in reality insignificant) directed against the reactivating guinea-pig serum, it was merely necessary to employ a considerable excess of the latter. In contrast to these results are those obtained in an analogous series of experiments, in which, however, the complement was in the Jbrm of goat serum instead of guinea-pig serum. (See Table Via.) 104 COLLECTED STUDIES IN IMMUNITY. TABLE Via. Experiment with 0.4 cc. Anti-immune Body. Control Test with 0.4 oc. Norma] Goat Serum. Amomit of Immime Body. cc. Solvent Action. Amount of Immune Body. cc. Solvent Action, 0.051 0.042 0.029 0.02 0.017 0.014 complete solution almost complete solution moderate solution trace solution faint trace solution 0.051 0.042 0.029 0.02 0.017 0.014 complete solution almost complete solution moderate solution very little solution trace solution In this combination the anti-immune body exerts no action. Hence we must here he dealing with a particular type of immune body which effects a combination with a complement present in goat serum. This immune body enters into no relation with the complex of immune bodies here present; it must therefore possess a haptophore group which finds no fitting counter group therein. As a matter of fact the completion by means of goat serum occu- pies a special position, for the quantitative relations of the immune body are entirely different from those observed when guinea-pig serum is used. In order to effect complete solution when goat serum is used as complement, it is necessary, as a rule, to use from ten to thirty times the amount of immune body that would be required if guinea-pig serum were used as complement. This is well shown by Table VII. TABLE VIL No. Complete Solvent Dose of Immune Body when Complemented with Guinea-pig Serum 0.15. cc. Compete Solvent Dose of Immune Body when Complemented with Goat Serum 0.5. cc. Ratio of the Two Doses. 1 2 3 4 0.005' 0.0075 0.0075 0.0025 0.05 0.075 0.1 0.075 1:10 1:10 1:13 1:30 That this behavior is not due to a smaller content of complement in the goat serum can readily be determined by suitable experiments especially by increasing the dose of the latter. STUDIES ON HEMOLYSINS. 105 This can only be explained by assuming that only part of the total number of immune bodies find fitting complements in goat serum, and that this partial number varies, but is always less than the number of immune bodies activated by guinea-pig serum. The diagram presented below will best make this relation clear. We have made a further series of experiments in order to com- plete these studies, and have discovered that our anti-immune body also protected goat blood-cells against the immune body derived from a rabbit treated with ox blood. This of course is quite natural, for we have already shown that this action on a strange species of blood depends on a concordance of certain haptophore groups. Similarly, the anti-immune body protects ox blood-cells against the immune body of a rabbit immunized with goat blood. These experiments lead us to the following conclusions: The anti-immune body derived by injecting goats with immune bodies of rabbits is not a simple uniform [einheitlich] substance, but is made up of a whole series of partial immune bodies. In the ox blood used to immunize the rabbits we have already distinguished two main portions of receptors to which again two main portions of the resulting immune- body correspond. Each of the latter portions in all probability con- tains a host of partial immune bodies, and we must assume that, corresponding to this, the anti-immune bodies also possess a complex constitution. In the following diagram it is not sought to express that the immune bodies which can be activated by guinea-pig serum are all identical. On the contrary each group may represent a different kind of immune body. We have seen that there is a great difference between the dose, of immune body which is completely neutralized by a certain amount of anti-immune body, and that which in the presence of anti-immune body causes complete solution. This can be understood when we recall the above-mentioned distribution of partial immune bodies, and examine the diagram,. Fig. 2. In order to choose a simple illustration let us assume that, corre- sponding to the diagram, the immune serum of the rabbit immu- nized with ox blood contains only two different types of immune- bodies and these, furthermore, in unequal amounts. Let the main portion be represented by immune body type a, which is activated by a particular complement present in the animal's own (rabbit's) serum. ■ Further, let the second portion, present in much less amount,. 106 COLLECTED STUDIES IN IMMUNITY. be represented by immune body type b, which is activated by a different complement also present in rabbit serum but found in goat serum as well. Let the proportion of a: 6 be as 10: 1 ; i.e., a quan- tity of immune serum containing one complete solvent dose of b will contain ten solvent doses of a. In this case then it will require ten times as much of the immune serum to effect complete solution by means of immune body b (which is the case when goat serum, which contains complement only for b, serves for reactivation) used when immune body a is employed. The composition of this immune serum can be represented by the formula 10a +16. Fig. 2. Diagram to show the two types of immune bodies present in the immune serum of a rabbit treated with ox blood. Each immune body symbol corre- sponds to one solvent dose for the amount of ox blood employed in the ex- periment. Immune body type a is present in ten times the amount of type b. The complementophile groups of a and 6 differ; hence also the complements differ. The anti-immune body serum possesses anti-immune bodies only against a. As is seen by the experiments, an anti-immune body exists only •against immune body type a. If therefore to an amount of immune body which contains one solvent dose of immune body b and ten solvent doses of immune body a (i.e., 10a -|- 16) a large quantity of anti- immune body serum is added, and then sufficient suitable complement it will be found that solution always occurs, for the reason that a ■single solvent dose of 6 is present which is unaffected. by the anti- inmivme body although this was able to neutralize ten solvent doses of a. One-tenth of the above amount of immune body, on the other STUDIES ON BLEMOLYSINS. 107 hand, will be completely inhibited in its action. For this contains one complete solvent dose of immune body a which is neutralized by the anti-immune body, and only one-tenth of a solvent dose of b which, although not affected by the anti-immune body, is so slight as not to cause any appreciable solution. Only when larger amounts of immune body are employed in which b becomes active does solution occiu:, and this becomes complete only when that quantity is reached which contains 10a +1&. Naturally, if the ratio is 1:20, a quantity will be required which can be represented by the formiila 20a -|- 16. These explanations will perhaps suffice to make the above-men- tioned peculiarities in the action of the anti-immune body com- prehensible. They will perhaps also make clear that between the dose of immune body whose action is completely inhibited by the anti- immune-body serum and the dose which causes complete solution a large number of intermediary stages exist in which the degree of solution gradually increases. In reality the circumstances are much more complicated than this ; for with the increase in the dose of immune body a large number of new immune bodies, similarly superposed, come into action — immune bodies which find few or no corresponding anti-immune bodies in the antiserum. This brings us to another important question: Is it possible by means of the anti-immune body to demonstrate the difference of the immune bodies produced by injections of ox blood into different -species ? Our first experiments were undertaken with the serum of goats which had been immunized with ox blood. As will be seen by the following figures, our anti-immune body (derived by injections of an immune body obtained from rabbits) in this case exerts no action. The varying amounts of immune body mentioned are mixed with 0.4 cc. anti-immune body and then with 1 cc. 5% ox blood suspension and 0.5 cc. normal active goat serum to activate the mixture. In the control test 0.4 cc. inactive normal goat serum are used instead of the anti-immune body. (See Table VIII.) That this serum differed markedly with respect to its content of individual immune bodies was already shown by the fact that, in contrast to the serum of immunized rabbits, it did not possess a hsemolysin acting on all goat blood-cells in general, since such a haemoly- sin would have exerted a most injurious action in the form of an auto- lysin. As a matter of fact, the law already mentioned under the name " horror autotoxicus " applied here also, and hence merely an isolysin 108 COLLECTED STUDIES IN IMMUNITY. was developed which acted only on goat blood-cells of a few individuals and which therefore possessed only a few individtial special groups in its immune bodies. Against this isolysin, which represented a relatively small portion of the types of immune bodies found in the goat, our anti-immune body also proved entirely ineffective, as is seen in Table IX. TABLE VIII. Experiment with Anti-immune Body. Control Test. Amount of Immune of Immune Body, cc. Solvent Action. Body, cc. Solvent Action. o.orsi complete solution 0.051 complete solution 0.042 almost complete solution 0.042 almost completely dissolved 0.035 strong solution 0.035 almost dissolved 0.029 moderate solution 0.029 moderate solution 0.02 trace solution 0.02 very little solution 0.017 doubtful 0.017 trace solution 0.014 0.014 In this experiment the method was exactly similar to that of the previous ones. The blood was from goat No. Ill, 1 cc. of a 5% suspension being used. TABLE IX. Experiment with 0.4 cc. Anti-immune Body. Control Test with 0.4 cc. Normal Inactive Goat Serum. Amount of Amount of Immune Body, cc. Solvent Action. Immune Body, cc. Solvent Action. 1.5 complete 1.5 complete 1.0 strong 1.0 strong 0.88 strong 0.88 strong 0.61 moderate 0.61 moderate 0.51 little 0.51 little 0.42 trace 0.42 trace 0.35 0.35 We see from this that by treating a goat with ox blood-cells, immune bodies have been formed the main portion of which differs from those obtained by immunizing rabbits with ox blood or goat blood. A second species of animal in which we have been able to demon- strate a difference in the immune bodies is the goose. The immune bodies obtained by injecting a goose with ox blood-cells are also not in STUDIES ON ILEMOLYSINS. 109 the least affected by our anti-immune body. It may be that an entirely different receptor apparatus is present in the goose and that this effects a combination with different haptophore groups which leads to the formation of immune bodies of entirely different character. Our iurther experiments concerned themselves with the action exerted by our anti-immime body on immune bodies derived from rats, guinea-pigs, and dogs by treatment with ox blood. We foimd that the anti-immune body exerted a distinct protective action against all three sera, but that this was less strong than that against the immime body of the rabbit. The protection was least against the serum of the rat, for it did not even suffice to absolutely protect against one-half or one-third of the fatal dose. Complete solution ensued in the presence of 0.3 cc. anti-immune body even when only double the usual solvent dose of immune body was employed. This indicates that this serum contains a relatively large amount of the non-neutraliz- able types of immune bodies, in any case an amoimt far greater than is contained in the rabbit serum. In the guinea-pig the case is very similar, the proportion of double the solvent doses being as 1:3. The nearest approach to the ratio as found in the rabbit is seen in the serum of a dog treated with ox blood. In this it required six times the usual solvent dose to effect complete solution in the presence of the anti-immime body.^ AU this leads to the conclusion that in the immime serum of these three species the cytophile group of certain portions is identical with the cytophile group of certain immune bodies in the rabbit. Certain particular groups of the ox blood-cells therefore must fit equally into the receptors of these different animals. In view of this fact, the entire absence in the goat of that portion of immime body which can be neutrahzed by the anti-immune body is of special interest. As already stated, we are here dealing with an exception which is connected with the impossibility of autolysin formation. We must therefore conclude that in conformity with our assump- tion, the immune bodies formed in any single case by treating various ' It is perhaps of interest to know that the immune bodies derived from these three species (guinea-pig, rat, and dog) differed in their behavior toward goat blood-cells. It was found that while the immune bodies of guinea-pigs and rats acted on goat blood, those of the dog did not. This indicates that the dog, in contrast to rabbits, guinea-pigs, and rats, possesess no receptors for the groups (/3 of the diagram, Fig. 1) common to the blood-cells of oxen and goats. 110 COLLECTED STUDIES IN IMMUNITY. animals with ox blood-cells are not, as a matter of fact, of simple [einheitlich] nature. Those obtained from goats and geese are very markedly, if not entirely, different from those of rabbits, while those from guinea-pigs, rats, and dogs are partly so. We have already pointed out the significance of this circumstance in § II, page 92. In all probability similar conditions obtain for bacteria, and it would therefore be advisable not to attempt the pro- duction of bactericidal sera from a single animal species, as is now customary, but to make a preparation containing a mixture of immune sera derived from animals whose receptor apparatus are as divergent as possible. in. Concerning the Variety of the ComBlementophile Groups of Homologous Immune Bodies.' From the foregoing sections it will be seen that in combating infectious diseases we believe it advisable to employ simultaneously a great many bactericidal immime bodies which, in conformity with the multiplicity of groups in the bacterial ceU, will differ in their cytophile group. It will now be necessary to investigate the question of a difference in the complementophile groups of these immune bodies. However, the treatment of this question can at present only be fragmentary, since our methods in this field are still very incomplete and definite results can only be obtained in specially favorable cases. It will be advisable to commence this study with the immune serum of a rabbit treated with ox blood. In this it has already been pointed out that two portions of immune bodies are present, each of which again is to be regarded as composed of a number of partial-immune bodies. This view of the composition of the im- mune bodies is supported by the reactivating experiments in which a number of different kinds of sera furnished the complements. This brings us to our present topic. We have already mentioned that the most favorable results are achieved when our immime body is activated by rabbit or guinea- pig serum; the activation by means of goat serum, together with its peculiarities, has also been discussed at length. The following list of complements shows their action in the pres- ence of varying amounts of an immune body from a rabbit immunized with ox blood. The amount of complement employed was always ample. ' See also Ehrlich's later views, page 560. STUDIES ON H.^MOLYSINS. TABLE X. Ill Activating Serum. Amount of Immune Body with which Com- plete Solution Occurs, cc. Guinea-pig serum 0.0025 0.005 0.005 0.015 0.015 0.05 no complement action t i 1 C i I Rabbit serum Rat serum Goose serum Goat serum ' This horse serum, which had been freshly obtained, failed also to reacti- vate the immune bodies of a goat and a goose which had been immunized with ox blood. Yet it was not at all free from complement, for even in amounts of 0.15 cc. it dissolved guinea-pig blood completely. It did not act on rabbit btood. This shows that when different sera are used as complements there is a great variation in the amount of immune body necessary for solution. Especially the extreme cases make it seem probable that we are dealing with different types of partial-immune bodies, to which different complements in the serum of the individual species correspond. That the complements of different species are not iden- tical is admitted even by Bordet, although he recognizes only a single complement for each species. That these complements are anchored to the corresponding immune body by means of a haptophore group may practically be regarded as proven, (1) by our experiments with blood-cells which had been laden with immune body, and (2) by the demonstration of anti-complements which diverted the complements from the immune body. According to our view the point at which the haptophore group takes hold is situated in the complementophile portion of the immune body. Hence we formerly designated the latter as "interbody"; recently we term it " amboceptor." A number of special investi- gators have accepted this view, as can be seen from the designations used by them; thus P. Miiller, "copula"; London, "desmon"; Metchnikoff: "c2/tase" = complement; " pMocj/tose " = immune body. Consequently we arrive at the view that in the mixture of immune bodies in the case under discussion a number of different complemen- 112 COLLECTED STUDIES IN IMMUNITY. tophile groups come into play. With the means at present at our disposal it is impossible, except in a few favorable cases, to deter- mine whether this plurality of complementophile groups corresponds exactly to a like plurality of cytophile groups. A case in point is that of the partial immune body which is reactivated by goat serum, for which we could show that it was not diverted by our anti-immune body.^ The difficulty of a full analysis of these cases is due especially to the many possibilities that must be considered. It is possible that immune bodies with different cytophile groups possess the same complementophile group, or that those with the same cytophile group possess different complementophile groups; and finally it is possible that, besides a particular cytophile group, an immune body may possess two, three, or more complementophile groups {triceptor, quadriceptor) . In any case it may be considered a fact that in the immune-body mixture different kinds of complementophile groups come into play. Were we to assume that the serum of an animal species contains only a single complement, we should have to regard such a plurality of complementophile groups as evidently a useless arrangement. It seems incredible that a given organism should form haptophore groups in its cells (for the immune bodies are merely thrust-off cell derivatives) if these groups were never during life to come into action, but were only to be of service in case the organism were injected with foreign cells. It is much simpler and more natural to view these circumstances from our standpoint, namely, that the comple- ments of an animal are, from the first, of manifold variety. This assumption best harmonizes the results of the various experi- ments which we have made from the beginning of our studies in haemolysis. By filtering goat and horse sera through Pukall filters we were able to demonstrate two complements. One of these, fitting • In our fourth communication we have discussed analogous cases in ■detail, subjecting them to thorough experimental investigations. At that time, however, our studies were directed only to the complementophile groups. In that case the serum of guinea-pigs immunized with rabbit blood contained two immune bodies, of which one found its complement in guinea-pig serum but not in rabbit serum. These immune bodies were present in the propor- tion of 1:10. In another case mentioned at that time we observed consider- able chronological variations in the proportion of two immune bodies with ■different complementophile groups. STUDIES ON HiBMOLYSINS. 113 to an immune body acting on rabbit blood, passed through with the greatest difficulty; the other, fitting an immune body acting on guinea-pig blood, passed through in part completely isolated. We were further able to show that heating the serum of a buck treated with sheep blood caused all the complements excepting one to disappear. The one which withstood the heat fitted the immune body developed by the immunization. We were able to demon- strate the same thermostabile complement in greater or smaller amounts in the serum of normal goats and calves. To again call attention to these experiments is not superfluous, for Gengou (Annal. I'lnst. Pasteur, 1901) in spite of these proofs of the plurality of comple- ments, still maintains that the serum of each species contains only a single simple complement, " the alexin.'' It would be natural to conclude that there is a plurality of com- plements from the manifold variations observed in the comple- tion of various inactive sera by normal sera. The commonest, example of this, probably known to every one having experience in this field, consists in the fact that a certain immune serum can be activated by two different sera serving as complement, whereas other immune sera can be activated by only one of these sera. Never- theless from our standpoint we cannot regard this method of proof as at all conclusive because it rests on the assumption that for a certain species of blood a serum contains only a single interbody (or immune body). In our fourth communication we have already shown that this assumption does not hold, even for the interbodies of normal sera. The assumption of a plurality of complements in normal sera is supported by the fact that by injections of a normal serum (which, accord- ing to our view, possesses various active substances which may be present as complements, or, at times, in the form of complementoids) antisera are formed which act against the complements of various other sera. In a number of different animals by injecting various sera we have succeeded in obtaining anticomplements acting not only against the serum originally employed, but also against certain comple- ments of rabbits and guinea-pigs. According to Bordet's experi- ments it is possible, by injecting a rabbit with guinea-pig serum, to obtain an isolated anticomplement against a complement (able to act in this particular case) present in guinea-pig serum. From this it follows that in these sera, since they excite the production of different anticomplements, at least two different complements 114 COLLECTED STUDIES IN IMMUNITY. are concerned. In this connection it is particularly interesting to note that by long-continued treatment of a goat with rabbit serum we obtained an aniicomplement serum which acted also against guinea-pig serum. Table XI will make this clear. All of the experiments are made with an immune body derived from a rabbit by immunizing with ox blood. TABLE XI. Anticomple- ment Derived from Rabbit Goat Goat Goat Rabbit Rabbit Treated with Guinea-pig serum. Dog serum Horse serum Rabbit serum. . . . Goat serum Sheep serum Protection against Rabbit Complement. + + + + + + + + + + + + + + + Protection against Guinea-pig Complement. -!-■+-(■ + -I--I- + -H-t- + + + + -I- + + + + + -!-= strong protection; ++ = fairly strong protection; -I- = very slight protection. With the assumption of a plurality of complements we are led to the view that the various complementophile groups of the immune body here concerned (contained in rabbit serum) are complemented by a like number of partial complements. As a result of this fact the possi- bility exists that certain of these complements are not constant, occurring in the blood only from time to time. We may perhaps give another example of these partial com- plements, which concerns one of a number of rabbits treated with repeated injections of goat serum. As already described in a previous communication, this results in the disappearance of certain com- plements and their replacement by corresponding autoanticomple- ments. In the example mentioned, this disappearance manifested itself by the fact that large amounts of the rabbit serum were unable to activate the single or the double fatal dose of the immune body from a rabbit immunized with ox blood. How- ever, when thirty times the amount of immune body was employed complete solution ensued. Evidently the principal portion of the complements usually present had disappeared from this serum, but a partial complement had remained which acted on a partial-immune body present in relatively small amounts. The circumstances in this case therefore are entirely analogous to those above described in STUDIES ON HEMOLYSINS. 115 which we proved that a particular immune body present in small amounts and not diverted by our anti-immune body, finds a comple- ment in its own serum which, in contrast to the other complements, is present also in goat serum. Three things have thus been established: 1. Each normal serum contains a number of different com- plements; 2. In different animals a part of the complements present are either completely similar or at least similar in their hap- tophore groups; 3. The immune bodies obtained in an animal species represent a number of different complementophile groups. As a result of this demonstration the question whether or not the resultant immune-body mixtures obtained in different animals are identical in their complementophile portion loses in interest at least so far as the problems under discussion are concerned. Hence we should merely like to add to the results obtained by activating the immune body of a rabbit immunized with ox blood, the results of a parallel series of experiments made at that time with the same amounts of reactivating sera hut with the immune body from a goose immunized with ox blood. (See Table XII.) TABLE XII. Reactivating Normal Sera. Amount of the Rabbit Immune Body Sufficient to Effect Complete Solution. Amount of the Goose Immune Body Sufficient to Effect Complete Solution. Guinea-pig serum. Rabbit serum . . . . Rat serum Goose serum Chicken senmi. . . Goat serum Pigeon serum. . . . Horse serum 0.0025 0.005 0.005 0.015 0.015 0.05 no "completion" no "completion" 0.025 0.05 0.1 0.035 0.035 no "completion" 0.035 no "completion" This table again shows that the unitarian view, according to which each serum contains only a single complement, lacks all prob- ability, for it is to be expected that in that case the zoological rela- tionship of certain animal groups would manifest itself in their com- plements to a greater degree than it actually does. When, for example, we here see that the rabbit immune body is not reactivated 116 COLLECTED STUDIES IN IMMUNITY. by horse serum but is reactivated by goose serum, we should neces- sarily have to conclude that "the " complement of the goose is much more closely related to " the " complement of the rabbit than is that of the horse. From the unitarian standpoint also a more marked difference should be manifested by the complements of the goose, the chicken, and the pigeon, for the first two reactivate the immune body, while the last does not. A priori, therefore, the unitarian view is very improbable ; but aside from this the reactivat- ing experiment with the goose immune body (which shows this to be reactivated by all three avian sera) speaks against this view. All of these facts are readily explained if we accept the pluralistic view that each serum contains a large number of complements, and that certain types have a wide distribution in many classes of animals. In these they may be completely similar, or, what is of primary impor- tance, their haptophore groups may be identical. It may very ■well be that the avian sera are alike in the greater part of their partial complements, and that therefore all three sera may in certain cases — €.g., with the immune body of a goat immunized with ox blood — reactivate in like manner. But it is not necessary that these three species correspond in all their complements, and so it may happen that a certain partial complement which is absent in pigeon serum is present in the other sera. This occurs in the above case with the immune body of the rabbit immunized with ox blood (and with that of the goat similarly treated). I should like to emphasize one more point. The immune body of the rabbit immunized with ox blood is not reactivated by pigeon serum, whereas the immune body of the goat immunized with ox blood is thus reactivated. This fact in itself should occasion no sur- prise whatever. The tissue receptors which are present in the avian organism, and which constitute the matrix of the amboceptors in question, possess complementophile groups that fit complements widely distributed throughout the avian body. It is not at all remark- able, therefore, that the immune body obtained from the goose finds complements in various avian sera. In like manner it can readily be understood why pigeon serum is unable to reactivate the immune body of the rabbit immunized with ox blood. The general conclusion, however, that the avian complements in their entirety are different from those of mammals, cannot be drawn from this, as is shown by the reactivation of the rabbit immune body by goose and chicken sera. STUDIES ON HEMOLYSINS. 117 This brief analysis will show us that the complementophile groups of the immune bodies do not in general possess the great importance which we must ascribe to the cytophile groups. In order to obtain the greatest therapeutic effect from the immune bodies, their com- plementophile groups and the provision of suitable complements cannot, of course, be neglected. In this connection Donitz (Klin. Jahrbuch, 1897) first pointed out the importance, in the therapy of infectious diseases, of finding sufficient sources of complements. The conditions determining this have been more closely defined by Ehrlich in his Croonian Lecture ^ of March 22, 1900, as can be seen from the following extract: "Dr. Neisser at the Steglitz Institute sought to find an explana- tion of Sobernheim's experiments. He was able to determine that anthrax serum failed in mice even if large quantities of fresh sheep- serum (i.e., containing an excess of ' complement ') were introduced at the same time. The failure in this case appears to be due, oa the one hand, to the destruction, in the body of the mouse, of the 'complement' present in the sheep serum, and, on the other hand,, to the fact that the 'immune body' yielded by the sheep does not. find in the mouse an appropriate new 'complement.' "From this it appears that in the therapeutic application of antibacterial sera to man, therapeutic success is only to be attained if we use either a bacteriolysin with a ' complement ' which is stable in man (homostabile complement), or at least a bacteriolysin the immune body of which finds in human serum an appropriate ' com- plement.' The latter condition will be the more readily fulfilled the nearer the species employed in the immunization process is to man. Perhaps the failure which has as yet attended the employ- ment of typhoid and cholera serum will be converted into success if the serum be derived from apes and not taken from species so distantly removed from man as the horse, goat, or dog. However this may be, the question of the provision of the appropriate ' com- plement' will come more and more into the foreground, for it really represents the center round which the practical advancement of the bacterial immunity must turn." In view of the fact that every normal serum contains a great many complements, of which a larger or smaller part fits the most varied immune bodies, the need of artificially supplying complements ' Proceedings of the Royal Society, Vol. 66. 118 COLLECTED STUDIES IN IMMUNITY. would seem, to indicate that our therapeutic efforts he directed 'primarily to exciting the greatest production of the organism's own complements.^ The production of these complements can surely be increased by means of artificial procedures ; and this is borne out by a few experi- ences in this direction. Thus Nolf, by injecting certain foreign sera, and P. Mijller, by injecting pepton, have succeeded, in animal experi- ments, in increasing the production of complement. This increase may perhaps be referable to a hyperleucocytosis in accordance with the views held by Metchnikoff and Buchner. We are certain that at least the complements orgijially peculiar to the organism wiU be able to act when fitting complementophile groups present themselves ; this need not necessarily be the case, however, when foreign complements are introduced. In this question it is of no consequence whether the absence of complement action is due to destruction, to com- plementoid formation, or to a combination in the organism such as has been demonstrated by the ready binding of anticomple- ments.2 The question raised by Donitz, relative to the provision of really plentiful sources of complement, has not thus far been solved. It stiU remains to be seen whether the interesting in- vestigations of Wassermann^ on the completion of typhoid im- mune bodies with ox serum will lead to results which can be practically utilized. The amount of complement contained in the serum of the larger laboratory animals is not, as a rule, great enough to make the employment of these sera applicable for human therapeutic purposes. Wassermann, for example, found that with a method of procedure which excluded the above-mentioned diminu- tion of complements (since he injected bacteria, immune body, and complement mixed together into the peritoneal cavity) it required 4 cc. ox serum to achieve curative results. This amoimt of serum in itself causes severe injury to the animals experimented upon. Such being the case, it seems that in the matter of supplying com- plements, the method suggested by us, namely, the employment of 1 In his recent study (Zeitschrift fur Hygiene, No. 37) Wassermann also lays great stress on the increase of the individual's own complements. We were especially gratified to see that in regard to the multiplicity of the comple- ments Wassermann accepts our view completely. ' This is also supported by certain experiments of von Dungern (Miinch. medizin. Wochenschrift) concerning the binding of complement by certain cells in miro. ' Deutsche medizinische Wochenschrift, 1900, No. 18. STUDIES ON HjEMOLYSINS. 119 mixed sera which contain a great many different immune bodies would prove the most effective; for with the multiplicity of the immune bodies an increase of the different complementophile groups also takes place, and thus the probability increases that the normal complements present, especially those in the human organism, can come into action most effec- tively. IX. CONCERNING THE MODE OF ACTION OF BACTERI- CIDAL SERA.1 By Max Neisseb, Member of the Institute, and Dr. Fbedeeick Wechsbekq. Our experiences with diphtheria curative serum have taught us that in antitoxin the employment of a high dose of antitoxin is of primary importance. It is immaterial whether an excess of antitoxin is administered, since it may be regarded as certain that an excess does no harm and can on the contrary only be of benefit. Concerning the action of bactericidal sera, however, the litera- ture contains a number of examples which indicate that here an excess of immune serum is occasionally injurious. Thus several high authorities have published protocols of therapeutic experiments on animals which seem to contain paradoxical results ; for with the same infection and varying amounts of immune serum not only those animals died which had received the smallest amounts of serum but also those which had received the largest amounts. Only those animals were protected which received doses of immune serum lying between these extremes. Such a protocol, for example, was published by Lofiler and Abel ^ on their experiments with bacillus coli and a corresponding immune serum. Out of nineteen guinea-pigs which had been inoculated with the same amount of culture (^/lo loop) and had received varying amounts of the immune serum, only six animals were protected, those which had received doses of 0.25 to 0.02 cc. Eight animals with larger doses as well as five with smaller doses of serum died. A similar protocol is that of R. Pfeiffer,^ which states that of four guinea-pigs treated with virulent cholera and a corresponding im- mune serum only the two animals receiving the medium doses survived. ' Reprinted from the Miinch. med. Wochenschr., 1901, No. 18. ' F. Loffler and R. Abel, Centralbl. f. Bact., 1896, Vol. 19, page 51. ' R. Pfeifier, Zeitschr. f. Hygiene, 1895, Vol. 20, page 215. 120 MODE OF ACTION OF BACTERICIDAL SERA. 121 The same phenomenon was noticed by Leclainche and Morel '■ in their work on the bacillus of malignant oedema, and these authors had similar experiences with erysipelas of swine and with sympto- matic anthrax. As a result of this they concluded that there was a "dosis optima neutrcdisans " of the immune serum. Since we encountered the same phenomenon in bactericidal test-tube experiments it seemed advisable to undertake a study of these occurrences, especially because the question seemed to offer points of vantage important both theoretically and practically. None of the authors above mentioned has furnished an adequate explanation of the phenomenon. In our experiments the bactericidal action was determined in two ways, namely, with the aid of the bioscopic method previously described by us,^ and by means of plate countings. The methods gave identical results even in parallel series. In order, therefore, to facilitate looking over the results we shall here give only the results obtained by the counting method. The method of procedure was generally as follows: ^/sooo cc- of a one-day bouillon culture of the bacterium in question was put into each of a series of test-tubes. To this were added varying amounts of immune serum inactivated at 56° C. and equal amounts of the complementing active serum; or in another series, equal amounts of immune serum and varying amounts of the complementing serum. It was so arranged that all the tubes contained equal emounts of fluid, usually 2.5 cc. Dilutions were made with 0.85% salt solution. Furthermore three drops of bouillon were added to each tube, for we had convinced ourselves that this assured a good growth in the control tubes. Numerous control tests were necessary nevertheless, even if only to test the sterility of the sera employed. The specimens were kept at 37° C. for three hours and then plated on agar, using five drops from pipettes of uniform size for each plate. The results were noted by comparison and estimation, somewhat after the following scheme: 0, isolated, hundreds, thousands, infinite number. Omitting the very extensive preliminary tests the following example is given to show the phenomenon studied by us. The immune serum employed was obtained from a rabbit by treatment ' Leclainche and Morel, La S^roth^rapie de la septictoie gangreneuse, AnnaL de I'Inst. Pasteur, 1901, No. 1. ' Munch, med. Wochenschr., 1900, No. 37. 122 COLLECTED STUDIES IN IMMUNITY. with vibrio Metchnikoff. This serum was inactivated by heating to 57° C. for half an hour. Normal active rabbit serum served as complement. TABLE I. Inactive Im- Amount mune Rabbit Normal Active Number of of Serum against Rabbit Serum Colonies on the Culture. Vibrio MetchnikofE. cc. as Complement, oc. Plate. ^o f 1.0 0.3 00 "? „«■ 0.5 0.3 00 §1^ 0.25 0.3 Many thousands °I'S 0.1 0.3 Several hundred =3 g-S 0.05 0.3 About 100 "■2 . 0.025 0.3 About 50 °gg 0.01 0.3 S'-.2 0.005 0.3 g^ 0.0025 0.3 About 100 „g^-> 0.001 0.3 00 "to 0.0005 0.3 00 'Control I — — 00 II 0.01 — 00 " III 1.0 — " IV — 0.3 00 V 1.0 Three drops of bouillon to each tube. All the tubes filled to the same volume with 0.85% salt solution, then placed into the thermostat at 37° C. for three hours. Finally, five drops of each plated on agar. This experiment shows that the inactive immune serum alone is innocuous to vibrio Metchnikoff (Control II); also that 0.3 cc. of the active normal rabbit serum alone is innocuous. However when, for example, 0.01 cc. immune serum is mixed with 0.3 cc. normal active rabbit serum, the many thousand germs inoculated are killed. In the same way 0.005 cc. immune serum plus 0.3 cc. normal active rabbit serum also causes the death of all the organisms. With smaller amounts of immune serum (but with the same amount of the complementing serum as before) the destruction of the germs is incomplete, while with still smaller amounts there is no destruction whatever. But the destructive effect also becomes less when more than 0.01 cc. immune serum is used, so that with 0.5 cc. immune serum no destructive at all can be observed. Hence if we had tested only the mixture of 0.5 cc. of this immune serum plus 0.3 cc. normal active rabbit serum we should certainly not have supposed that we were dealing with a powerful immune serum. That this action MODE OF ACTION OF BACTERICIDAL SERA 123 is due only to the serum's content of immune body is shown by the following experiment in which inactive immune serum is compared with inactive normal serum of the same species, both sera being complemented with active normal serum. TABLE IL Amount of Culture. Amount of the Com- plementing Normal, Active Rabbit- serum. CO. Number of Colonies on a Plate on the Addition of Serum from a Rabbit Immunized against Vibrio Metchnii^off, the Serum having been Inactivated. — 1.0 CO. ice. A oc. Ace. ■g^iyj! cc. of a one-day bouil- lon culture of vibrio Metchnikoff , 1.0 ] i I 00 00 00 00 00 a few many thousands 00 00 00 Amount of Culture. Amount of Normal Active Rabbit- senmi. cc. Number of Colonies on a Plate on the Addition of Inactive Normal Rabbit-serum. 1 cc. i cc. Ace. Ace. ^^ CC. of a one-day bouil- lon culture of vibrio Metchnikoff r 1.0 ■ i CD 00 00 00 00 00 00 00 00 00 00 Control I. 3Tnnr cc. bouillon culture + 2 cc. 0.85% salt sol. -f3 drops of bouillon, planted as above, result oo . " II. Sterility of the immune serum, 0. " III. " " " inactive normal rabbit-serum, 0. " IV. " " " active normal rabbit-serum, 0. All the tubes made up to equal volumes with 0.85% salt solution, then placed into a thermostat at 37° C. for three hours. Finally, five drops of each specimen plated on agar. This experiment, too, shows that ^/le cc. immune serum plus 1 cc. or 1/3 cc. normal active rabbit serum kills the germs completely; while larger doses of the immune serum are less effective. The addi- tion oi. normal inactive rabbit serum has no effect. The same phenomenon can be demonstrated in another man- ner. For the complementing serum any active serum is used which by itself already possesses a slight destructive action. If to such a serum varying amounts of an inactive immune serum are added, it will at times be found that small quantities of the latter increase 124 COLLECTED STUDIES IN IMMUNITY. the action of the normal active serum, while somewhat larger quan- tities weaken the action. Still larger quantities may inhibit the action completely. In the following experiment an immune serum was employed which had been obtained by immunizing a goat with vibrio Nord- hafen. This serum was inactivated by heating it to 57° C. Normal active goat serum served as complement. (See Table III.) The first column shows that normal active goat serum by itself kills bacteria, even in doses of about 0.1 cc. The fourth and fifth columns show that this bacteriolytic effect of the normal active goat- serum is in no way affected by the addition of 1.0 cc. or 0.1 cc. in- active normal goat serum. From the third column we see, however, that if we add to the normal active goat-serum 0.1 cc. inactive tm- mune serum, the bacteriolytic effect of the former is lowered, and that it is almost neutralized when 1.0 cc. of the inactive immune serum is added. (Column 2.) TABLE III. Amount of Com- plement- ing Nor- 1 2 3 4 5 Number of Colonies Number of Colonies on a Plate on Addition of on a Plate on Amount of Inactive Goat Immune Serum against Addition of Inac- Culture. Goat Serum. cc. Vibrio Nordhafen. tive Normal Goat Serum. — 1.0 cc. 0.1 cc. 1.0 cc. 0.1 CO. tJtt CC- °f a r 1.0 about 50 one-day 0.5 many hundreds bouillon 0.25 00 culture 0.1 00 several hundred of vibrio 0.05 about 50 00 00 about 10 a f ew Nord- 0.025 00 00 00 00 00 hafen. ~~~ ~ 00 00 00 00 Control I. T^ cc. bouillon culture -I- 2 cc. 0.85% salt solution -1- 3 drops bouillon =00. " II. Sterility of the inactive immune serum, 0. ' ■ III. " " " " normal goat serum, 0. " rv. " " " active normal goat serum, 0. Three drops of bouillon to each tube. All the tubes made up to equal volumes with 0.85 % salt solution. Kept in the thermostat at 37° C. for three hours. Finally, two drops of each specimen plated on agar. MODE OF ACTION OF BACTERICIDAL SERA. 125 The same phenomenon is shown by the following protocol; TABLE IV. Amount of Amount of the Comple- menting Active Normal Guinea-pig Serum, cc. Number of Colonies on a Plate on the Addition of I-nadive Goat Immune Serum directed against Vibrio Nordhafen. Culture. — 1.0 CO. 0.1 cc. 0.01 CO. T^ CC. of a one-day bouillon culture of vibrio Nord- hafen. 1.0 0.5 0.25 • 0.1 0.05 0.025 a few several thou- sand 00 00 many thou- sands almost 00 00 00 00 00 00 a few about 100 several hun- dred 00 00 00 00 a few about 100 many hua- dred oo 00 Amount of Culture. Amount of the Comple- menting Active Nor- mal Guinea- Number of Colonies on a plate on the Addition of Inactive Normal Goat Serum. pig Serum, cc. 1.0 CO. 0.1 CC. 0.01 cc. T^ CC. of a one- day bouillon culture of vibrio Nordhafen r 1.0 0.5 0,25 0.1 0.05 0.025 about 100 a few hundred 00 00 00 oo a few a few hundred 00 00 00 a few several thous. 00 00 00 Control I. 1^5 cc. bouillon culture -1-2 cc. 0.85% salt solution -f 3 drops bouillon. Result, oo. ' ' II. Sterility of the goat immune serum, 0. ' ' III. " " " normal goat serum, 0. " rV. " " " " guinea-pig serum, 0. Three drops of bouillon to each tube. All the tubes made up to an equal volume with 0.85% salt solution. Kept in the thermostat at 37° C. for three hours. Finally, two drops of each plated on agar. We succeeded in obtaining similar results in such experiments with the following combinations : i ' We should like to call attention to a case which we encountered a number of times. We found that an immune serum obtained from a goat could be 126 COLLECTED STUDIES IN IMMUNITY. Typhoid + inactive immune serum (dog) + normal active guinea-pig serum. Vibrio Nordhafen + inactive immune serum (rabbit) + normal active horse serum; " " " " " " 4-normaI active goat << << " " " " serum; " " " " " " +normal active sheep serum; " " " " " " + normal active guinea- pig serum. In order to meet the objection that the agglutinins may possibly have interfered in the experiments we have devised the following method of demonstrating the phenomenon in question: Typhoid bacilli were subjected for one hour at 37° C to the action of inactive immune serum derived from a dog. As we know from the haemolytic experiments of Ehrlich and Morgenroth, this results in anchoring the interbody present in the immune serum to the bacteria. The mixture was then centrifuged and the fluid poured off. After care- fully shaking the sediment with a little fluid it was divided into two equal parts, to one of which inactive immune serum (dog) was added, while the other received some normal inactive dog-serum. Finally there was added to both portions the same amount of a complement- ing serum (normal active guinea-pig serum) which by itself was able to kUl the bacteria. At the end of three hours plate cultures were made in the usual manner. The results showed that no destruction had occurred in the tube containing the excess of immune serum, whereas the culture in the other tube had been killed. reactivated for Vibrio Nordhafen by a complement derived from rabbits. In this combination we again observed the phenomenon of deflection of com- plement by an excess of immune body. But even normal inactivated goat serum (which contains interbody) when used in exactly the same amounts manifested deflection of complement. Since no quantitative difference could be discovered between the immune serum and the normal serimi, we assume that in this case the deflection of complement has been effected by a substance in normal goat serum, as, for instance, another interbody of special affinity or perhaps a normal anticomplement. Not every complement can be used to reactivate a serum, for the complement may be deflected from the place of its intended action by any interbody, provided merely that this possesses sufficient affinity for the complement. It will be necessary to seek experi- mentally for combinations in which such disturbing deflections are absent and in which the difference in the affinity of the interbody which may be normally .present and of that produced artificially in large quantities becomes very mani- fest. MODE OF ACTION OF BACTERICIDAL SERA. 127 AU these experiments show that the effect produced by a given amount of complementing serum, just sufficient to reactivate a definite quantity of inactive immune serum, was diminished when large amounts of immune serum were employed. In like manner it was possible to inr- Mbit the activity of a normal serum, which was bactericidal by itself, by the addition of large amounts of the immune serum. It seems to us that an explanation of these important phenomena is possible only on the basis of the newer views of Ehrlich and Mor- genroth. From the work of these authors on hsemolysins and from our own bacteriolytic experiments we know that the immune serum contains a thermostable interbody (amboceptor) which while itself inactive renders the complement effective by linking itself, on the one hand, to the bacterium or erythrocyte to be dissolved, and on the other to the complement. The complements, as is well known, are thermolabile and are contained in normal sera. But the interbody may also be normally present in a serum. This follows from the side-chain theory, and has already been emphasized.^ An instance of this is shown in Table IV. The normal active guinea-pig serum contained complement and interbody. But besides this it contained additional complement, which became manifest when more inter- body, in the form of inactive immune serum, was added. In example II it was impossible to demonstrate an interbody in the normal serum, for this by itself did not kill the bacteria even though inactive normal serum was added. It did, however, contain complement, and this became manifest when inactive immune serum was added. These phenomena are exactly like those observed with hemolysins which have recently been so carefully studied. This one phenomenon, however, of the ineffectiveness of large doses of immune serum has not thus far been encountered in hsemolysins. This is apparently due to differences in the affinities of the interbodies, as we shall pres- ently show. In Fig. 1, on the next page, A II represents schematically a bac- terium a with a number of receptors; for there are many reasons why we should assume that each bacterium possesses a number of receptors of the same kind. According to the side-chain theor\r, if we inject this bacterium into an animal an overproduction of the corresponding group will occur, resulting in a serum which is rich in body b. This body 6, however, is not able by itself to injure the 'Deutsche med. Wochenschr., 1900, No. 49. 128 COLLECTED STUDIES IN IMMUNITY. ^gx5 &:^^C5 £:, 131 c3 133 164 a> 1:20 105 1:160 1:80 1:80 It may further be mentioned that examinations were also made on the 29th and 39th day ofter the injection, in which however a decrease of the agglutinating value was usually found. Investigations also showed that injections of physiological salt solution in bouillon caused no variation in the normal agglutinating values. A further question was whether and to what degree the serum of normal untreated rabbits possesses agglutinating properties on typhoid bacilli. Out of 17 rabbits which were examined for this pur- pose, 10 showed no agglutination in dilutions of 1:20, one serum agglutinated in the dilution 1:20, but no higher, 5 others in 1:40, but no higher, and only one agglutinated even in a dilution of 1 : 160. (See Table IV.) It is therefore a rare exception for normal rabbit serum to still manifest agglutinating powers on typhoid bacUli in a higher dilution than 1:40. It should be remarked that in the above table "0" has always then been put down when the agglutinating value of the serum in a dilution of 1 : 20 = 0; for the examinations began with this dilution. AGGLUTINATED TYPHOID BACILLI. 151 TABLE IV. Agglutinating Values of Normal Rabbit Serum. Dilution of the Serum. Number of the Animal. , . 1:20. 1:40. 1:80. 1:160. 1:320. 133 132 136 164 181 163 166 165 159 162 161 + 114 + + 160 + + 182 + + 117 + + 118 + + 134 + + + + We now come to the experiments proper. In the first of these (Table V) a series of rabbits was injected with agglutinated typhoid bacilli, while a control series was injected with the same or smaller amounts of non-agglutinated bacilli. This comparison shows a far higher agglutinating value of the serum of the control animals than that of the other animals. TABLE V. Agglu- tinating Num- Value ber of the Serum Injection of Aggluti - nating Average. Animal. previ- ous to the In- Value. jection. in ? "1 "0 xV ma.ss culture -t- \ agar culture 1:160 112 ? ta-o.™ ditto 1:160 103 ? .So .=3 ditto 1:160 1:147 104 ? ^■ag ditto 1:80 105 ? Is- ditto 1:160 106 ? J < ditto 1:160 108 ? i-i^^a ditto 1:1280 1 109 1 •||||-s ditto 1:2560 !• 1:1493 110 ? J^^SSi -^ mass culture -H \ agar culture 1:640 J 152 COLLECTED STUDIES IN IMMUNITY. The next question was whether rabbits really react at all to injections of agglutinated typhoid bacilli; in other words, whether the normal agglutinating value possibly present is at all increased by injections of agglutinated typhoid bacilli. The result was sur- prising, as is seen in Table VI. For while in four animals no increase occurred, in two others there was a very slight increase, and in four more the increase, though distinct, was insignificant in comparison with that in six animals injected with non-agglutinated typhoid. TABLE VI. Agglu- Maximum tinating Agglu- Number Value of tinating of the the Serum Injection of Value Average. Animal. previous to the Injection. after the Injection. 132 "3 2 agar cultures (intraperito- neally) 181 ■s. 2 agar cultures 116 ^.^ 1 mass culture 182 1:40 Td!^ ditto 1:40 164 ■-2§ J mass culture +i agar culture 1:20 1:106 163 !-§ ditto 1:40 166 '■§ ditto 1:320 11,5 be i mass culture 1:160 161 1:20 i mass culture + i agar culture 1:320 114 1:40 ■< 1^ mass culture 1:160 165 ^ A mass culture +4 agar culture 1:640 159 ■§,T3 i " " +i " 1:1280 162 119 ho a> 1:2560 1:160 1 : 1093 136 I? 1 agar culture 1:640 160 1:40 fiff mass culture +| agar culture 1:1230 Note. — 1 mass culture equals about 12 agar cultures. With this the main portion of the question had been answered; for these experiments already showed that the injection of agglutinated typhoid bacilli exerts an action which quantitatively is different from that following the injection of non-agglutinated bacilli. Never- theless even the agglutinated bacilli, although their injection is often wholly without effect, in many cases still exert a stimulus on the formation of agglutinins even though in a slight degree. This is due to individual peculiarities of the animals employed, and these we have not thus far been able to recognize in advance. The natural assumption that animals which already normally possess agglutinins react more readily to the injection of agglutinated typhoid bacilli AGGLUTINATED TYPHOID BACILLI. 153 than do those which do not normally possess agglutinins has not been confirmed, for out of seven animals (Table VI) in whose serum no typhoid agglutinin could be demonstrated previous to treatment, three did not react to the injection of agglutinated typhoid bacilli, two reacted feebly and two very distinctly. On the other hand, out of three animals in which, previous to treatment, a typhoid agglutinin could be demonstrated, two reacted distinctly to the injection of agglutinated bacilli and one not at all. Another assumption was, that in the animals which had reacted but feebly or not at all, an increase of the sensitiveness against agglu- tinated bacilli could be brought about artificially by repeated injec- tions of agglutinated bacilli. This also has not been confirmed. Thus three animals (Table VII) reacted to the second injection of agglu- tinated bacilli just as little as they did to the first, one animal reacted feebly, as it had done previously, and only two animals (Nos. 131 and 133), which had failed to react to the first injection, reacted distinctly to the second. The protocols of these last two animals, however, point out a peculiarity. On the first occasion these animals were injected intraperitoneally and it is noted that at this time the intestine was pricked. The first injection may therefore have mostly gone into the bowel and so produced no effect. The second injection would then have really been the only effective one. These two cases can- not therefore be used to prove that by means of a previous injection of agglutinated bacilli an artificial increase of the sensitiveness against a subsequent injection of agglutinated bacilli can be effected. The previous injection of agglutinated bacilli, however, in no way influences the sensitiveness against non-agglutinated bacilli, as is shown by the four control animals (Table VII). Finally experiments were made regarding still another assump- tion. It was conceivable that the previous injection of a certain amount of non-agglutinated bacUli would have sufficed to bring about a sensitiveness against a subsequent inoculation with agglutinated bacilli. This assumption also has not been borne out. Out of five animals (Table VIII) which, after a previous injection of non-aggluti- nated typhoid, received an injection of agglutinated typhoid, two showed a slight increase and three no increase in agglutinating value. It follows from all these experiments that there is a distinct dif- ference between the injection of agglutinated and of non-agglutinated typhoid bacilli. The injection of non-agglutinated typhoid bacilli is always followed by an increase of the agglutinating power. This 154 COLLECTED STUDIES IN IMMUNITY. ffl |> o s 1—1 s 1—1 00 1—1 ^ ' ~ ■ ' '^~ * 1 o o Q o o Q O o o w ■^ CO CO g s CD CD s 1—1 l-H ^ l-H ,, 1—1 1—1 f-1 l-H ■uoi^oaCai ^sjij; eq^ CO lO CD O CO ^ 1—1 1—1 1— ( (N ja^jB s^B(j p jsqinn^ '"' *"* 1—1 1—1 ^~' cq C^ C,i ^■> ■S-a I gg« O o o s" 1 «3 ■*^ -w -^s ■38 ■a£ ''B ^ ^ .'SE-' rt'^ ^ (U K O •»nannB3jj, aiojaq to ^ 2 J3MOJ 3miBin!jtn33v T— 1 T-H o o o ^ ■* N tP T-l C^ •IBraiuv aiR JO jaqratiH CO CO s 00 00 i-H rH W 156 COLLECTED STUDIES IN IMMUNITY. increase is usually very great and only rarely slight. The injection of agglutinated typhoid bacilU, provided that attention is paid to a sufBcient saturation with agglutinin, is frequently followed by. no reaction, often by a shght reaction, and rarely by a marked increase of the agglutinating value. This reacting power depends on the individuality of the animal and stands in no relation to the original agglutinating value, nor can it be influenced artifically. Furthermore, as we learned from a special experiment, it is immaterial whether the immune serum used to agglutinate the typhoid bacilli is derived from the same or from another animal species. The explanation of these facts is not difficult provided one pro- ceeds on Ehrlich's theory. According to this the agglutinin consists of thrust-off cell-receptors. As a result of their seizure by the bacterial receptors they have been produced in excess and give off to the circulation. They, therefore, possess a definite relation to the corresponding bacterial receptors. Hence when we fully saturate typhoid bacilli with agglutinin, we cause the bacterial receptors to be occupied, and are then as little able to cause a reaction with these bacteria as we are to cut with a sword in its scabbard. If then, in spite of this, certain animals react to such "occupied" typhoid bacilli, we shall have to assume that these animals possess the power to dissolve the combination of agglutinin and bacterial receptor and thus set the latter free. This action, however, never proceeds to the full extent. Incomparably more important, and, as it appears to us, explicable only with the aid of Ehrlich's chemical views, is the main phenomenon, that in many animals no reaction whatever follows the inoculation of agglutinated typhoid bacilli; that therefore in many cases it is possible to dispose of the bacterial group giving rise to the agglutinin, by causing this group to be occupied by the corresponding agglutinin. Subsequent Note. R. Pfeiffer and Friedberger,' through recent experiments on cholera vibrios and cholera amboceptors, have obtained results which are in gratifjang accord with those obtained by v. Dungern, M. Neisser and Lubowski, and Sachs. ^ In earlier experiments R. Pfeiffer ' had found that the bacterial substance dissolved in the peritoneum through the influence of the cholera immune serum ' R. Pfeiffer u. Friedberger, Berl. klin. Wochenschr., 1902, No. 2j. ' See the following article. 'R. Pfeiffer, Deutsche med. Wochenschr., 1901, Nos. fO-51. AGGLUTINATED TYPHOID BACILLI. 157 ■usually still excited an extraordinarily strong immunity reaction, a phenomenon seemingly in contradiction to Ehrlich's theory. Further experiments, however, showed that when very high doses of an active cholera goat serum were em- ployed, the immunizing action was almost entirely lost. Of especial importance for future methodical investigations of this kind is the fact determined by these authors, that a real saturation of the receptors of the cholera vibrios requires s, surprisingly high multiple of the amount of immune serum sufficient to dis- solve the same amount of cholera vibrios. 7500 times this amount does not yet satisfy all the affinities and it requires enormous doses, up to 3-4 million times, to completely saturate all the receptors. XIII. IMMUNIZING EXPERIMENTS WITH ERYTHRO- CYTES LADEN WITH IMMIJNE BODY.i By Dr. Hans Sachs, Assistant at the Institute. The interesting experiments of v. Dungern^ have furnished further proof that the same group (receptor) of the blood-cells which in hemolysis combines with the specific immune body causes the production of this immune body within the organism, v. Dungern injected rabbits with ox blood to which a plentiful amount of an immune body (obtained from rabbits by inununizing with ox blood) had been added, and found, as was to be expected, on the basis of the side-chain theory, that animals so treated failed to produce any immune body whatever. The results of the investigations of M. Neisser and Lubowski^ show that the complete inactivity of such saturated receptors — agglutinated typhoid bacilli — in the animal body is not at all a general rule, but that, on the contrary, a moderate development of the immunity reaction occurs even with such mixtures and that this depends on certain individual differences. Hence at the suggestion of Prof. Ehrlich I have extended the experiments of v. Dungern and undertaken blood-immunization experiments on a large series of animals. The results obtained lead to certain modifications of von Dungern's conclusions. The method of these experiments must be guided by two prin- ciples. To begin, it is important that the receptors of the injected blood are really saturated, for even a very slight free residue might effect an immunity reaction in the animal body. And yet it is es- sential to remove any possible excess of immune body, because this ' Reprint from the Centralblatt fiir Bacteriologie, Parasitenkunde und In- fection Krankheiten, Vol. XXX, 1901, No. 13. ' V. Dungern, Muench. med. Wochenschr., 1900, No. 20. See also page 66. ^ See the preceding article, page 146. 158 IMMUNIZING EXPERIMENTS WITH ERYTHROCYTES. 159 could passively reappear in the serum of the injected animal and so simulate an active new formation of immune body. In accordance with this the experiments were made as follows : Ox blood was treated with an excess of inactive serum from a rabbit which had been im- munized with ox blood, the mixture digested at 37-40° C. for half an hour and then centrifuged. The decanted fluid was then tested for its content of immune body. Only when this test proved posi- tive, and it could therefore be assumed that all receptors had been saturated, was the blood so treated employed for injections. But it was previously repeatedly washed with physiological salt solution in order to remove all free immune body. Finally the centrifuged sediment was made up to its original volume. The course of such an experiment is illustrated in the following: 100 cc. ox blood are mixed with 25 cc. inactive immune serum of a rabbit which has been immunized with ox blood. Of this im- mune serum, 0.0025 cc. suffice, when complement is added, to just completely dissolve 1 cc. 5% ox blood; the amount employed, there- fore, represents five times the amount necessary to dissolve the 100 cc. ox blood. After the mixture has remained in the thermostat for half an hour it is filled up to 300 cc. with 0.85% salt solution and centrifuged. The first decantation is tested by adding it in decreas- ing quantities to 1 cc. 5% ox blood plus 0.4 cc. normal rabbit serum (as complement). The following results are obtained : 1st.. Decantation: 1.5 cc. complete haemolysis. 1.0 cc. almost complete haemolysis. 0.5 cc. " " 0.25 strong haemolysis. 0.1 no From this it must be concluded that the blood-cell receptors are incapable of further absorption; in other words, that they have been saturated. The second decantation tested in this same manner yields the following result : 2d. Decantation : 3.0 cc. strong. 2.0 " moderate. 1.0 " little. 0.5 " trace. It therefore contains only very httle immune body. 160 COLLECTED STUDIES IN IMMUNITY The blood is once more washed and centrifuged and then filled up to 100 cc. The blood-cells thus saturated with immune body are in- jected in rabbits intraperitoneally, each animal receiving 25 cc. of the mixture. At the same time control animals are injected with the same amounts of normal ox blood. Usually on the tenth day after the injection, as this had shown itself the most favorable time, serum was withdrawn, inactivated and tested for its content of immune body by adding it in decreasing quantities to 1 cc. 5% ox blood plus sufficient complement. Either rabbit serum, 0.4-0.5 cc, or guinea-pig serum, 0.1-0.15 cc, served as complement, for these are equally well adapted for this purpose. The results of the experiments are as follows: Out of eight rabbits injected intraperitoneally with ox blood saturated with immune body, only three corresponded to the requirements which follow from von Dungem's results. Their serum, tested exactly like the immune body, failed even in amounts of 1.0 cc. to produce a trace of haemolysis, whereas when the serum of the corresponding control animals was tested, 0.025 and 0.05 cc. respectively sufficed to effect complete hsemolysis. These results are approached very closely by the serum of a fourth animal. The haemolytic action of this serum compared to that of the serum of the corresponding control animal was 1 : < 135, i.e., was exceedingly slight. The remaining four rabbits had pro- duced an immune body in greater or less amounts, though this amount was always far less than that produced by the corresponding control animals. When the absence of a zone of marked complete solution rendered it irnpossible to make an exact determination, the compari- son of the immune body values of the sera in parallel tests was accom- plished by comparison of tubes whose colors corresponded. The amount of immune body possessed by these animals compared to that of the corresponding control animals was as follows : (1) 1:5; (2) 1:7; (3) 1:10; (4) 1:10. I have supplemented these experiments with a smaller series of experiments made with intravenous injections. In these, of course, very much smaller amounts of blood were used for injection because when the blood-cells loaded with immune body are injected directly into the circulation, they suffer haemolysis through the action of the com- IMMUNIZING EXPERIMENTS WITH ERYTHROCYTES. 161 plement present in the serum, caiising serious symptoms or, with larger amounts of blood, fatal results. This accords with the phenomena observed by Rehns ^ when he injected rabbits which had been immu- nized with ox blood, intravenously with normal ox blood. Only two of the animals 1 employed, namely those injected with 7-8 cc. blood, re- mained aUve sufficiently long. In one of these only traces of immune body were found in the serum, whereas the serum of the other animal effected complete solution in doses of 0.05 cc. In the serum of a control animal the limit of complete solution was 0.01 cc. These few experiments confirm the results obtained with intraperitoneal injections, that blood-cells saturated with immune body have not by any means always lost the power to excite a certain degree oj immunity reaction in the organism. Our results, therefore, show that in half of the animals, in con- formity with the results obtained by von Dungern, the power of the blood to cause an immunity reaction is lost, owing to the blocking of that particular group in the blood-cell which unites with the immune body. In the remaining cases, however, the specific immune body was produced, though always in decidedly less amount, since only a fifth to a tenth part of the amount appeared that was produced by the control animals. This apparently unfavorable portion of the ex- periment shows at least that saturation with immune body exerts a marked restricting influence. These results agree with those obtained by Neisser and Lubowski with injections of agglutinated typhoid bacilli. Furthermore, like Neisser and Lubowski, in an animal which had not reacted to the injection of saturated blood, we found after injec- tion of the same amount of normal blood that an immune body of considerable power had developed in the serum. The complete solvent dose for 1 cc. 5% ox blood amounted to 0.005 cc. serum These last experiments, which have been done on a much larger scale by Neisser and Lubowski on typhoid bacilli, indicate that the failure of antibodies to form is not due to possible individual differences in the reacting capacity of the organism. Considering the uniform appear- ance of immune body in rabbits treated with ox blood such an assump- tion would have lacked all probability. That portion of the experiments in which the injection of saturated blood-cells was borne by the animals without producing any reaction, can be regarded, as has been done by von Dungern, as a complete demon- ' Rehns, Comp. rend de la Soc. biol., 1891, No. 12. 162 COLLECTED STUDIES IN IMMUNITY. siration that the groups exciting the production of immunity are actually the same as those which in haemolysis anchor the immune body. We have seen, however, that there is not always an absence of reaction and that even the injection of the same saturated blood, which in one animal fails to cause a production of immune body, is followed in another animal by a certain low-grade production of immune body.. The cause of these phenomena can only be that certain animals possess the individual capacity to anchor the saturated receptors in spite of this saturation. We do not know the mechanism of this action. Two factors in particular come into consideration; a part of the immune body may perhaps be destroyed in the animal body through special agencies (oxidation?) and the receptors thereby set free. It is also possible, however, without assuming a destruction of immune body to explain the phenomenon in the sense of Ehrhch's views, by assuming a higher affinity of the tissue receptors present in the animal body, which receptors then would be able to break up the union of blood-cell receptors and immune body, and draw the blood- cell receptors unto themselves.^ Whichever of these explanations is the correct one, our experiments certainly show one thing, that the dissolution of the blood-cell receptor combination is never a complete one. Merely a portion of the groups is concerned, for only by this partial dissolution is the fact (determined by Neisser and Lubowski, as well as by us), to be explained that a very sUght degree of immunity reaction is produced by the injection of saturated receptors. Hence even in the cases running an apparently unfavorable course, only a part always of the receptors exert their action. This portion of the experiments may therefore also be used as a sup- port for the side-chain theory. ' A similar assumption must be made in order to explain certain forms of over-sensitiveness studied particularly by v. Behring, in which, despite a large excess of antitoxin, very small doses of toxin cause death. The most ready explanation is that here, in contrast to the behavior in normal animals, the toxinophile receptors possess a pathologically increased avidity by which they are enabled to break up the neutral toxin-antitoxin mixture (which cannot be: broken up by normal cells) and take up the toxin thus set free. XIV. CONCERNING THE ESCAPE OF HAEMOGLOBIN FROM BLOOD CELLS HARDENED WITH CORROSIVE SUBLIMATE.i By Hans Sachs, Assistant at the Institute. The following study was undertaken on reading the results of investigations carried on by Matthes ^ on the role of the immune body (amboceptor) in haemolysis. The peculiarity of his very inter- esting results demands a thorough study of the factors concerned. The facts there brought out have been confirmed by us, but the results of our study have led us to regard these facts in an entirely different light. As a result of numerous earlier experiences with pepsin, pancreatin and papain we can confirm the observation that normal as well as sensitized red blood-cells (i.e. cells loaded with immune body) cannot be attacked by digestive ferments.^ With digestive experiments with pepsin and pancreatin, to be sure, the difficulty exists that the amounts of HCl and alkali respectively which represents the optimum of action, are in themselves not indifferent for the blood-cells. With these ferments one is therefore forced to work under relatively unfavorable conditions. Matthes killed the blood-cells by means of Hayem's solution (which, as is well-known, contains J% mercuric chloride) and found that hlood-cells so treated were readily dissolved by means of active pancreas fluid. These fixed blood-cells, which are no longer sus- ceptible to the destructive action even of distilled water, are dissolved by the specific hsemolytic serum and even by their own normal serum. ' Reprint from the Mueuchener med Wochenschr , 1902, No. 5. ' M. Matthes, Experimenteller Beitrag zur Frage der Hamolyse, Muenoh. med. Wochenschr., 1902, No 1. ' According to recent investigations of Dr, Morgenroth, the interesting intes- tinal ferment, erepsin, described by Cohnheim and by him kindly placed at our disposal, is also not able to attack sensitized blood-cells. 163 164 COLLECTED STUDIES IN IMMUNITY. Although we can entirely confirm these statements, we cannot accept Matthes' view, according to which the solution of the fixed blood- cells by pancreatin is conceived as a digestion, the Hayem solution acting somewhat like an immune body. The striking fact that the fixed blood-cells dissolve even in their own serum appeared to us rather to be the result of the union of the mercuric chloride (which adhered to the blood-cells and prevented this solution) with the albumin of the serum. The experiments made in this direc- tion at the suggestion of Prof. Ehrlich have completely confirmed this view. Following the procedure of Matthes, I employed rabbit blood which, freed from serum, was mixed with Hayem's solution in the proportion of 1:4. After standing a short time, the blood was cen- trifuged and then washed three or four times with 0.85% salt solu- tion. Finally a 5% suspenson of the fixed blood-cells in .85% salt solution was prepared. The corresponding control was made with normal 5% rabbit blood. In the experiments 1 cc. of the 5% blood mixtures was used; the fluid, after the addition of the reagent being made up to 2 cc. with physiological salt solution. It was found that not only fresh rabbit serum, but even rabbit serum which had been inactivated by half an hour's heating to 56° C, as well as rabbit serum which had been diluted with ten volumes of •physiological salt solution and then boiled one hour, was still able to cause solution of the fixed rabbit blood-cells; 0.075 cc. serum causing complete and almost instantaneous solution. In this case the toxic action of the serum can hardly be thought of. The experiment indicated rather that other kinds of influences are the cause of this curious phenomenon. If the conception is correct that we are dealing with a combination of the mercury with the serum, it should be possible also, with other means which abstract the mercury, to cause a solution of blood-ceUs fixed with Hayem's solution. As a matter of fact this can very easily be done. I chose potassium iodide and sodium hyposulphite for this test and found that extremely small amounts of these substances cause immediate solution of the fixed blood. 0.00075 cc. of a 20% KI solution in physiological salt solution or 0.00025 cc. of a similar hyposulphite solution sufficed to completely dissolve 1 cc. of our 5% fixed blood suspension.! This positively shows that the function of the serum 1 With normal rabbit blood, 1000-2000 times the amount of KI or of hypo- sulphite solution still acts indifferently. ESCAPE OF HAEMOGLOBIN PROM BLOOD CELLS. 165 albumin in the experiments made by Matthes is that which we assumed. It must therefore be concluded that the blood-cells treated with Hayem's solution do not dissolve in water because the mercuric chloride with which they have combined prevents the escape of the haemoglobin. The cause of this may be that the soluble substances, e.g. the haemoglobin, form an insoluble combination with the mer- curic chloride; it is sufficient, however, to assume that the limiting membrane of the discoplasma becomes denser through the deposited mercurj' , salt and so prevents the diffusion of the blood coloring- matter. Be this as it may, certainly all agencies which break up the mercury combination will cause an immediate solution of the hae- moglobin. The reason for this is that the discoplasma, which in the living state hinders the diffusion of haemoglobin, has been killed by the sublimate treatment, From this it is easUy seen that the solution of the fixed blood by means of pancreatin as it is described by Matthes, is not to be regarded as a species of digestion. Every such ferment solution contains enough albumin to explain the action according to our view. I was able to confirm this by the experiment in which the haemolytic action (observed by us also) of neutral pepsin and pan- creatin solutions was exerted in like manner when the solution had previously been heated to 95° C. for 1 hour. In conclusion it may be remarked that, after fixation with \%. mercuric chloride solution in physiological salt solution instead of with Hayem's fluid, the blood-cells behaved in exactly similar fashion, as was a ■priori to be expected. The control tests made at the same^ time with normal blood gave negative results in all the experiments. On the other hand with solanin, a substance which dissolves normal blood even in enormous dilutions, haemolysis of fixed blood-cells could not be effected even though large doses were employed. In this substance the necessary albumin is wanting and the dead blood- cells are no longer vulnerable to the action of the blood poisou. To sum up, we may say that in the blood-cells hardened with Hayem's solution it is merely the chemically bound mercuric chloride which hinders the escape of the haemoglobin. All agents which are capable of attracting this salt to themselves, i.e. to " de-harden " the blood-cells, cause the immediate escape of hcemoglobin. Hence, although the observations of Matthes are extremely inter- esting in themselves, they possess no value for the doctrine of hae- molysis. On the other hand it would seem as though they might 166 COLLECTED STUDIES IN LMMUNITY. be applied to a method of detecting smallest amounts of mercury. Subsequent Addition. — In a recent communication (Muench. med. Wo- chenschr. 1902, No. 17) Matthes has completely confirmed the results of our experiments so far as mammalian blood-cells are concerned. The fact that other species of blood, such as frog blood studied by Matthes, after hardening with mercuric chloride, do not give up their haemoglobin even in fluids rich in albumin does not affect our view, but only points to a high degree of hardening of the frog-blood stromata which does not permit the escape of the haemoglobin even in the presence of substances abstracting mercury. We did not deny that the stromata could be digested by means of proteoljrtic ferment. Our objection was directed only to regarding the escape of haemoglobin, an indi- cation of a digestion, or of digesting complements. XV. A CONTRIBUTION TO THE STUDY OF THE POISON OF THE COMMON GARDEN SPIDER.^ By Dr. H^ANS Sachs, Assistant at the Institute. The studies in hsemolysis, constantly keeping pace with the develop- ment of the doctrine of immunity, have shown that besides the usual blood poisons sharply defined chemically, there is another group of hsemolysins of animal or vegetable origin which exert their damag- ing influence like the toxins, by combining with certain definite groups of the protoplasm. Included in this are snake venom, numer- ous bacterial secretions such as tetanolysin and staphylolysin, tox- albumins of higher plants, such as crotin. Besides this there is the endless series of haemolysins, both normal and those produced at will by immunization, which are found in the blood serum. Of the highest importance for the conception of the similarity of these blood poisons was the fact that only such blood-cells are sen- sitive to these hcemolysins which are capable of anchoring them. This fundamental law, which was first recognized and clearly formulated by Ehrlich and Morgenroth^ has constantly been confirmed, espe- cially in the study of the serum haemolysins artificially produced. As a resuU of this the mode of action of these poisons as well as of the toxins has been conceived from the standpoint of the side-chain theory. " * * * the prerequisite and the cause of the poisonous action in all these cases is the presence in the blood-cells of appropriate receptors (side chains) which fit into the haptophbre groups of the toxin; con- versely, therefore, there is an intimate connection between natural immunity and the absence of receptors." (Ehrlich.) It is evident that the study of the combining relations of the toxin-like blood poisons is of great significance for the study of the ' Reprint from Beitrage zur chemischen Physiologie u. Pathologie, Vol. II, No. 1-3. ' See page 1 et seq. 167 168 COLLECTED STUDIES IN IMMUNITY. causes of this poisonous action. Such a study, moreover, is calcu- lated to extend our knowledge of the receptors and their physio- logical distribution in the animal kingdom. While examining an extract derived from the common garden spider (Epeira diadema) I found in it a hsemolysin which showed itself particularly well adapted to researches in this direction. The description of a complete experiment will give an idea of the method of obtaining and testing this poison. A garden spider weighing 1.4 grams is rubbed up with 5 cc. toluol water containing 10% NaCl and the fluid kept in the refrigerator for twenty-four hours. Then water is added to make the total volume 25 cc. and the mixture filtered (or centrifuged). The hsemolytic experiments are made in the usual manner with this cloudy, brownish-yellow filtrate. Decreasing amounts of the poison solution are placed in a series of test-tubes, each of which is then fiUed up to 1.0 cc. with physiological (0.85%) salt solution. Each tube now receives one drop of undiluted blood or 1 cc. of a 5% suspension of blood in physiological salt solution. The specimens are kept in the incubator at 37° C. for two hours, and then in the refrigerator until the following day when the amount of solution is determined. The blood employed was always centrifuged and washed in order to remove the adherent serum and so exclude any possible disturbance from that source. The Arachnolysin, as we may designate the active principle of the poison solution, causes solution of the sensitive blood-cells even at room temperature; when present in certain proportions, solu- tion occurs almost instantaneously. In this respect, arachnolysin is somewhat analogous to snake venom, while it differs therein from the haemolysins of blood serum, in which, as is well known, actual haemolysis is preceded by a longer or shorter period of incubation. The more exact determinations on different species of blood were made in the usual manner and yielded the results shown in the fol- lowing table. The amounts of arachnolysin given in the table refer to the original solution, containing 28% of spider substance. As can be seen from the table we are here dealing vrith a hcBmolysin of extraordinary power, the action of which on the indimdual species of blood, however, is very variable. Thus a number of species of blood are destroyed even in dilution of 1 : 1000 or 1 : 10000 (this refers to the original poison solution) ; others remain unaffected even by large amounts of poison. Next to rat blood, the most sensitive was rabbit blood, for 0.0001 cc. of the original solution, i.e., 0.000028 g. spider substance, sufficed to completely dissolve 0.05 cc. blood (=200,000,000 blood-cells). A garden spider weighing 1.4 g. there- THE POISON OF THE COMMON GARDEN SPIDER. 169 Arachnolysin. Hsemolytic Action on the Blood of Rabbit. Hat. Mouse. Man. 1/1000 1/10000 CO. 1.0 0.75 0.5 0.35 0.25 0.15 1.0 0.75 0.5 complete almost complete strong complete almost complete strong complete almost complete do. 1 1 strong complete almost complete do. moderate little trace Arachnolysin. Hsemolytic Action on the Blood of Ox. Goose. Guinea- Pig. Horse. Sheep. Dog. cc. 1/1000 1.0 75 complete almost complete strong little trace strong moderate 0.5 0.35 0.25 0.15 1/10000 1.0 0.75 0.5 No haemolysis even with larger amounts fore contains sufficient poison to completely destroy 2.5 liters rabbit blood. Remembering that only an extremely small part of the spider's weight is made up by the active poisonous constituent, and even assuming that the content of arachnolysin amounts to 1%, we see that this enormous activity indicates that the arachnolysin belongs to the class of blood poisons which exert a powerful action after the man- ner of the toxins. The .same is indicated by the marked instabilty of the active principle. Heat readily destroys the arachnolysin, although a higher degree is necessary than for other haemolysins. Heating to 56° C. for 40 minutes does not affect the poison solution, and at 60° C. only a very slight reduction of action is noticed. Complete destruc- tion does not occur until the poison is heated to 70°-72° C. for 40 minutes. Arachnolysin is easily preserved by the addition of glyc- erine, showing no reduction in activity even after months. Experiments, designed to show whether normal sera possess an inhibiting action on haemolysis due to spider poison, have had nega- 170 COLLECTED STUDIES IN IMMUNITY. tive results; the sera of man, rabbit, horse, pig, dog, rat, guinea- pig, goat, sheep, ox, goose, and pigeon, inactivated by heating to 56° C. in order to eliminate any possible solvent action, were unable ■even in amounts of 1.0 cc. to protect rabbit blood against just a com- plete solvent dose of arachnolysin. On the other hand, the study of the poison's behavior toward .sensitive and insensitive cells has yielded results of special interest in connection with the receptor theory. Certain species of blood, such as dog or guinea-pig blood, have shown themselves immune to the spider poison. This presents the most favorable conditions for studying the relations between the binding of poisons and their action. This point, as we have seen, is of the greatest importance for the view that serum hsemolysins are toxin-lite bodies. If arachnolysin is a blood-poison whose action is due to the anchor- ing of a certain haptophone group to a receptor of the sensitive blood- cell, and if, corresponding to this, the immunity of certain species of blood is due to a lack of appropriate receptors, it follows that the sensitive blood-cells must be able to bind the active principle of such a poison solution, while the insensitive cells leave it entirely unaffected. So far as the insensitive bloods are concerned, the method of making the experiment is very simple. Dog blood is mixed with a certain quantity of arachnolysin, kept in the incubator for an hour and frequently shaken. Thereupon the blood, which, of course, is unchanged, is separated by means of a centrifuge. The decanted fluid, compared with the original material, shows not the least diminu- tion of its solvent power on rabbit blood-cells. This shows that the .insensitive dog blood is not able to hind the arachnolysin. In the case of the sensitive blood-cells, the demonstration of the combining power is much more difficult, for these, when tested in ^ similar manner are dissolved, so that it is impossible to separate blood-cells and fluid. We can then only operate with the laky blood solution, the inactivity of which permits of no direct conclusion that a binding of the poison by means of receptors had occurred. Furthermore, if the poison solution has lost its power as a result of the action already exerted, there is no means by which this can be determined. It was necessary, therefore, to employ blood-cell material which had been made stable so far as the vital influences of the haemolysis were concerned, without, however, losing its chemi- ■cal character. For this purpose we iised blood-cell stromata, by THE POISON OF THE COMMON GARDEN SPIDER. 171 which we mean blood-cells deprived of their hsemoglobin by swell- ing and then again condensing the blood-cell residues. Ehrlich^ had already (in 1885) pointed out the importance of this true pro- toplasm of the blood-cells, and had termed it " discoplasma " because of its peculiar character. According to Ehrlich, the main function of this discoplasma is to prevent the escape of the hsemoglobin, and he therefore ascribed the diffusion of the blood coloring-matter to ■death of the discoplasma. In agreement with this is the fact first ■described by Bordet^ and afterward confirmed by Nolf,^ that it is the stroma ta which bind the specific serum haemolysins. We ■could therefore assume that in our case, in all probability, the arach- nolysin would be bound, if bound at all, by the stromata. In this Institute a method for the production of the stromata, which differs somewhat from the one commonly employed, has proven particularly valuable, especially in studying the receptors. With the usual solution of the blood in distilled water, the separation by ■centrifuge of the stromata condensed with salt is extremely dif- ficult; and even with suitable species of blood only a small yield is ■obtained. By previously heating the blood we have found that the subsequent centrifugation is made considerably easier (perhaps because of a kind of coagulation of the blood-cells) and that a plentiful sediment of stromata is thereby assured. The blood employed is heated on a water-bath at 50°-60° C. for half an hour (depending on the species of blood, ox blood 60° C, rabbit and guinea- pig blood about 54° C.) until, dark brown in color, it just begins to become laky. Thereupon the blood, made up to 6 to 10 volumes by the addition of water and shaken, is mixed with so much salt that this amounts to 1% of the total amount. The mixture is then strongly centrifuged. The stromata remain at the bottom of the vessel in the form of yellowish-white masses, and can be washed by repeatedly adding NaCl solution and centrifuging. The stromata so obtained have preserved their receptor property; they hind specific serum hcemolysins, and also, when introduced into the organism, excite the production of specific hcemolytic immune todies.^ ' Ehrlich, Zur Physiologie und Pathologic der Blutscheiben, Charity Annalen, X, 1885. ^ Bordet, Les Serums h^molytiques, etc., Annales de I'lnstit. Pasteur, 1900. ' Nolf, Le Mecanisme de la globulyse, Annal. de I'lnst. Pasteur, 1900. ' It may be recalled that immunization with heated bacteria has been suc- cessfully practiced even from the beginning of the study of immunity. 172 COLLECTED STUDIES IN IMMUNITY. The fact that they have suffered a certain quantitative loss in these properties, owing to the extensive manipulation to which they have been subjected, in no way affects their utility for combining ex- periments. In the qualitative demonstration of specific affinity the employment of an excess of receptors answers all requirements. In order, furthermore, to meet the objection of a mechanical absorp- tion of the poison by the stromata, exactly similar combining experi- ments were made simultaneously with a blood of the sensitive class, and with one of the insensitive class. As a representative of the former, rabbit blood, which is highly sensitive, was used. For the control, guinea-pig blood, which is not dissolved by arachnolysin, was used. The degree of activity of the poison solution before and after binding was measured by means of rabbit blood. The stromata sediments derived from each of 40 cc. rabbit blood and guinea- pig blood, are mixed each with 10 cc. of an arachnolysin solution of which 0.026 cc. suffice to just completely dissolve 0.05 cc. rabbit blood. The stromata so treated are digested for half an hour in the water-bath at 40° C, being re- peatedly shaken. They are then centrifuged. The decanted fluid from the stromata of guinea-pig blood, like the original material, still completely dis- solves 0.05 cc. rabbit-blood in amounts of 0.025 cc; the decanted fluid from the rabbit blood stromata, on the other hand, has entirely lost its poisonous action. Even in amounts of 1.0 cc. it is unable to exert the least action on rabbit blood. Hence the stromata obtained from the sensitive blood have actually bound the arachnolysin, and this combination must be regarded as a chemical one because the control test with guinea-pig blood shows that the insensitive cell-material exerts no attraction whatever on the arachnolysin. Such behavior, however, is most easily explained by assuming, in accordance with the side-chain theory, the presence of appropriate receptors in the sensitive cells as a prerequisite for the action of the arachnolysin. The natural immunity of certain species of blood will then correspond to an absence of appropriate receptors. We see from this that the distribution of receptors capable of binding arachnolysin, at least so far as the blood is concerned, is not universal throughout the animal kingdom, but confined to certain species. While the experiences already mentioned lead us to regard arachnolysin as a poison belonging to the class of toxins, the evidence will be made absolutely conclusive by demonstrating the ability of the poison to produce antitoxin, the most important criterion for the THE POISON OF THE COMMON GARDEN SPIDER. 173 toxin nature of any substance. Owing to the scarcity of material the immunizing experiments were somewhat delayed; they will, however, be dealt with in detail at the proper time. Nevertheless I can announce that shortly before the conclusion of this work we succeeded, by means of a short immunization of guinea-pigs ^ with garden-spider poison, to produce a high-grade antitoxic serum, of which 0.0025 cc. sufficed to fully protect 0.05 cc. rabbit blood against a complete solvent dose. This proves the toxin nature of arachnolysin. In conclusion I should like to refer to the relations which arachnoly- sin bears to what we know about spider poisons in general. In doing so I shall follow Kobert,2 who made the fundamental studies in the toxicology of animal and vegetable poisons, and to whom we owe most of our knowledge concerning spider poisons. In addition to the true secretion of the poison gland, Kobert distinguishes "a toxal- bumin which permeates the entire body of the spider, even the legs and eggs, but which bears no necessary relation to the poison gland." In some species of spiders this substance mixes with the gland poison. According to Kobert, the more toxalbumin gets into the woimd, the stronger are the constitutional symptoms ; the more true gland poison, the stronger the local changes. The latter is especially the case in the lathrodectes species (malmignatte, karakurte) whose sting produces most fearful general symptoms, even being able to kill human beings. In these the gland secretion becomes dangerous only when mixed with toxalbumin derived from the body. In contrast to this, the sting of the garden spider produces only local symptoms of irritation, although the spider's body contains a toxalbumin whose action is analogous to the preceding; but this substance does not become mixed with the gland secretion. This being the case, it is very hkely that the hsemolysin described by us is identical with the toxalbumin already known to Kobert; for we also obtained it from the body substance of the garden spider, and foimd its properties to be those of the toxin. Addition on REVisiON.^Since sending in the manuscript of this study I have learned of a monograph by Kobert (Beitrage zur Kenntniss der Giftspinnen, Stuttgart, 1901) which has just appeared. In this Kobert also reports on the hsemolytic action of the poison of Karakurtes and of garden spiders. He states ' Hence although guinea-pig blood is insensitive to arachnolysin, appropriate receptors capable of binding the poison must be present in the guinea-pig organism outside of the blood. ' Kobert, Lehrbuch der Intoxicationen, Stuttgart, 1893, p. 329. 174 COLLECTED STUDIES IN IMMUNITY. that although he found the hsemolytic action to be present in the latter, "it was much less than that of Karakurtes poison." It is possible, however, that Kobert made these experiments on one of the species of blood found by us to be insensitive to arachnolysin (horse blood, dog blood?). At any rate our garden-spider extract far exceeds in hsemolytic action the Karakurtes poison tested by Kobert in this respect. I should also like to point out that for the hsemolytic experiments with Karakurtes poison, Kobert used dog blood, which according to our table belongs to the class of blood species immune to garden- spider poison. Perhaps in conformity with the extensive analogy between, these two spider poisons, the Karakurtes poison possesses a far greater hsemo- lytic action on other species of blood. Kobert's observation that a tolerance can be established against Karakurtes poison as well as against garden-spider poison, agrees very well with the idea of a strong antitoxic serum, a fact actually observed by us. Since then we have obtained such a serum also in rabbits. XVI. A STUDY OF TOAD POISON.i By Dr. Fr. Proscheh. The numerous investigations concerning toad poison which have been made especially by French and Italian workers, have not yet come to a definite conclusion as to whether this substance is alkaloid- like or toxin-like. The skin secretions of the different varieties of toads contain a number of bodies which have not thus far been studied. In the garlic toad, for example, there is a substance of garlicky odor, which has not been more closely identified. Besides this, according to Calmels, toad secretion contains methylcarby laminic acid and methylcarbylamin, which are said to act intensely on the nervous system. Kobert applied the name "phrynin" to a substance which irritates the mucous membranes very intensely. Phisalix and Bertrand claim to have isolated an alkaloid from the blood serum of the common toad, but it remains doubtful whether the substance was not a toxin, for they were unable to produce it in chemically pure form. At the conclusion of their investigations they themselves say that the poisonous action is not due entirely to the "alka- loid." In like manner Jornara and Casali claim to have isolated "bufidin" from dried toad poison. They say that this forms crystal- line salts and must therefore be an alkaloid. The alcoholic extract: of toad skin is said to have an action similar to digitalis. Pugliese found that toad poison changes hsemoglobin into methsemoglobin, and that it also dissolves the blood-cells outside the body. Pugliese has not attempted any more detailed investigation. From the abstracts of his study at my disposal I was unable to determine the species of toad used in his experiments. The object of the following investigation is to fiu'nish a small contribution to our knowledge of toad poison. At present there can be no thought of any exact analysis of the poison. ' Reprint from Beitrage zur chemischen Physiologie u. Pathologie, Vol. 1^ Nos. 10-12. 175. 176 COLLECTED STUDIES IN IMMUNITY. METHOD OF OBTAINING THE POISON. The toad poison used in my experiments was derived from bombinator igneus, the fire-toad, and from bufo dnereus, the common garden toad. In order to obtain the poison, the skin of the abdomen and back of a freshly captured toad was used, for the poison is present in largest amounts in the skin. The muscles and blood serimi of the fire-toad also contain the poison, but in smaller quantities. After the toads were thoroughly rinsed with physiological salt solution they were decapitated and skinned. The skin was again rinsed with salt solution and then rubbed to a paste, as homogeneous as possible, with powdered glass. After adding 2 to 3 cc. physiological salt solution the mixture was filtered or centrifuged. The resulting fluid had a feebly acid reaction, a greyish white color and a peculiar, garlicky odor. Toluol was added as a preservative, and the fluid stored in the refrigerator. In the same manner I prepared an extract from the skin of the garden toad. The extract of the skin of the fire-toad showed strong hsemolytic properties; that of the garden toad the same, though only in traces. (See Table III.) The following experiments refer only to the fire- toad poison which, for short, we shall call "phrynolysin." The poison of the garden toad was used merely for comparison. PROPERTIES OF PHRYNOLYSIN. Phrynolysin is an exceedingly labile body. Heating to 56° C, exposure to light, the addition of alcohol, ether, chloroform, min- eral acids, strong potash lye, pepsin and trypsin, all destroy it in a short time. Drying the phrynolysin over anhydrous phosphoric acid at room temperature weakens it materially. It does not dialyze. Since, as already mentioned, the extract from the toad skin pos- sesses a faint acid reaction, requiring 1 to 1.3 cc. decinormal lye for neutralization, it could be assumed that the acid reaction slowly destroys the toxin. The destruction of the toxin, however, proceeds in the same time in neutral as in feebly acid solution, so that the acid reaction cannot possess any great influence. The hsemolytic action is the same in acid as in neutral solution. The best preservative for this substance is toluol, first employed by Ehrlich for preserving the toxins. Cold storage is also good. After a time the fluid becomes cloudy, owing to the separation of •albumin, but it maintains its hsemolytic power unimpaired for a A STUDY OF TOAD POISON. 177 considerable time. After from one to two months the phrynolysin gradually becomes inert. Owing to the extreme lability of the toxin there can, for the present, be no thought of obtaining the substance pure, for even drying at room temperature weakens the poison con- siderably. Owing to lack of material, a pharmacological examination of the poison could not be undertaken. BEHAVIOR OF THE PHRYNOLYSIN TOWARD DIFFERENT SPECIES OF BLOOD. The method of testing was such that a series of test-tubes was prepared, each containing 1 cc. of the dilution 1:10, 1:20, etc., i.e. decreasing amounts of the poison. The dilutions were made with 0.85% salt solution. To each tube 1 cc. of the 5% blood suspension in 0.85% salt solution was added. Thereupon the tubes were kept at 37° C. for two hours and in the refrigerator overnight. A " com- plete solution " is one that on shaking shows no body elements of any kind: "almost complete " if there is still a slight sediment; and "incomplete " when numerous blood-cells are undissolved. This is followed in order by "red," "top," "trace," "0." Commencing with Table III all the experiments are made on sheep blood. As can be seen from Table I, sheep blood is most strongly dis- solved, frog and toad blood not at all. The limits of solution for sheep blood are a dilution of 1 : 10240 in the case of phrynolysins I and II, and 1:5120 in phrynolysin III. In Table IV, decreasing amounts of the poison are added to 1 cc. 5% sheep blood. Of phry- nolysin I, 0.0025 cc. sufficed to effect complete solution; of II and III, 0.00025 cc. sufficed, and of IV, 0.005 cc. By determining the amount of dry residue in poison solution II it is seen that 0.0000022 g. of organic substance suffice to completely dissolve 1 cc. 5% sheep blood. Of poison solution III, 0.0000015 g. have the same effect. If we assume that one-tenth of this organic substance (probably it is still less) represents true phrynolysin, the rest being merely indifferent albimiinous bodies, we find that ^/iq mg. are sufficient to completely dissolve one liter of sheep blood. The yield of phrynolysin is subject to individual fluctuations. Animals freshly caught yield a stronger haemolysin than those which have been kept in captivity for some time. 178 COLLECTED STUDIES IN IMMUNITY. TABLE I. Dilution. Sheep Blood. Goat Blood. Rabbit Blood. Dog Blood. Ox Blood. 1:20 complete complete complete complete top 1:40 1:80 ti red tt 1:160 tt '?P trace 1:320 red 1:640 tt tt 1:1280 1 1 tt 1:2560 incomplete trace tt 1:5120 almost complete top 1:10240 red trace 1:20480 trace 1:40960 Dilution. Chicken Blood. Guinea-pig Blood. Rat Blood. Pigeon Blood. 1:20 1:40 1:80 1:160 1:320 incomplete tt red tt top trace red top trace trace Dilution. Pigeon Blood. Goose Blood. Frog Blood. Toad Blood. 1:20 1:40 1:80 trace red top TABLE n. Phrtnolysin op the Common Garden Toad. Dilution. Sheep Blood. Goat Blood. Dog Blood. Rabbit Blood. Guinea-pig Blood: Ox Blood. 1:20 1:40 1:80 red trace tt red top TABLE III. Behavior op Dipperent Pheynolysins toward Sheep Blood. Dilution. Phrynolysin I. Phrynolysin II. Phrynolysin III. 1:640 1:1280 1:2560 1:5120 1 : 10240 1:20480 complete tt tt almost complete top complete tt tt almost complete red complete tt almost complete top red A STUDY OF TOAD POISON. 179 TABLE IV. Behavior op Different Phrynolysins toward Sheep Blood. Phrynolysin I. Phrynolysin II. Phrynolysin III. Phrynolysin IV. 0.005 0.0025 0.001 0.00075 0.0005 0.00025 0.0001 complete incomplete red top complete incomplete complete red complete incomplete top ATTEMPTS AT REACTIVATING A PHRYNOLYSIN WHICH HAD BECOME INACTIVE AT 56° C. The investigations of Ehrlich and Morgenroth have shown that the haemolysins of the higher vertebrates are of complex constitu- tion. They consist of two portions, the complement and the immune body. By heating to 56° C. the complement is destroyed, while the immune body remains intact. The immune body by itself can- not exert any hsemolytic action; a fitting complement must first be added. It would be quite comprehensible for the phrynolysin likewise to consist of two parts. Heating to 56° C. would destroy the com- plement, while the thermostable interbody would be preserved. I therefore attempted to reactivate the toxin which had become inactive at 56° C. and tried the addition of a number of different normal serum for this purpose, such as goat serum, sheep serum, pigeon serum, horse serum, guinea-pig serum, and rabbit serum, all without success, no solution taking place. Unfortunately, owing to lack of material, I was unable to obtain 1 or 2 cc. of serum from the fire-toad in order to employ this for reactivation. Experiments with the normal sera of the higher vertebrates are not conclusive, because the complement sought for may possibly be contained only in the serum of the fire-toad. For the present therefore the ques- tion as to the complex character of the phrynolysin must still be kept open. DO NORMAL SERA CONTAIN ANTIBODIES AGAINST PHRYNOLYSIN? A number of normal sera, which had first been inactived at 56° C. in order to avoid solution of the sheep blood added, were tested for this purpose, e.g., pigeon serum, sheep serum, guinea-pig serum, horse serum, rabbit serum, and goat serum. None of these sera. 180 COLLECTED STUDIES IN IMMUNITY. even in amounts up to 1 or 2 cc. was able to prevent solution, although only the single solvent dose of phrynolysin was added to the mix- ture of blood and serum. IMMUNIZATION WITH PHRYNOLYSIN. In order to furnish conclusive proof that phrynolysin is a true toxin, a number of rabbits were immunized with the same. The poison was injected subcutaneously, commencing with i cc. and increasing to 5 cc. in the course of eight days. The dose of 5 cc. was then injected every 5 to 6 days for two or three times so that in the course of three weeks about 30-35 cc. had been given. It is not advisable to give more than 5 cc. at once because other- wise the animals die in one or two days. The anatomical findings in an animal which has died of toad poison are negative, excepting a marked hyperaemia of the abdominal viscera; no macroscopical changes of the organs are demonstrable. As already mentioned, normal rabbit serum does not contain a trace of anti-body against phrjmolysin. The production of anti- toxin commences about fourteen days after the injection of the toxin and reaches its maximum in three weeks. Of the strongest serum which I obtained, 0.025 cc. protected against double the solvent dose of phrynolysin for 1 cc. 5% sheep blood. LITERATURE. VuLPiAN, Comp. rend, de la soc. de biol., 1854, p. 133; 1856, p. 124. DoM. JoENABA, Sup les effets physiol. du venin de crapaud., Journ. de Thdrap., 4, p. 833 et 929. G. Calmels, Comp. rend., 98, 536 (1883), and Archiv. de physiol., 1883. Capparelli, Archiv. ital. de biol., 4, 72 (1883). KoBERT, Sitzungs-Bericht der Dorpater Naturforschenden Gesellschaft, 9, 63 (1891). Phisalix and G. Behtrand, Toxische Wirkung von Blut und Gift der gemeinen Krote, Comp. rend., 116, 1080-1082, Arch, de physiol., 25, 511, and 517, Comp. rend. soc. biolog., 45, 477, 479. D. JoRNARA and Casali, Das Gift der Krote und das Bufidin, Revista clinica Bologne, 1873. A. PuGLiESE, Die Methaemoglobinbildende Wirkung des Krotengiftes, Archiv. di farmac. e terap., 1898. Ehrlich and Morgenroth, Uber Hsemolysine, Berl. klin. Wochenschr. 1899, Nos. 1 and 22; 1900, Nos. 21 and 31; 1901, Nos. 10, 21, and 22. XVII. CONCERNING ALEXIN ACTION.^ By Dr. Hans Sachs, Assistant at the Institute. After the fundamental studies of Bordet, and of Ehrlich & Morgenroth had shown that the hsemolysins produced in serum by immimization with blood-cells owed their effect to the combined action of two substances (amboceptor and complement) it seemed very natural to suppose that the hsemolysins of normal sera, which had been known for some time, were also of a complex nature. Buchner, who was the first to recognize the significance of the bac- tericidal and globulicidal properties of blood serum, had conceived these actions from a unitarian standpoint and referred them to the " alexin " of each particular serum. Recent investigations, how- ever, have shown that Buchner 's alexin is not a single simple sub- stance, but the sum of an infinite number of combinations, whose more thorough analysis has been rendered possible only by the methods of the newer haemolysin investigations. The credit of applying the experiences gained with the hsemoly- sins artificially produced to the study of hsemolysins of normal serum, belongs to Ehrlich and Morgenroth. They made use of a method which they had already employed in the analysis of hsemolytic immune sera, the separation by means of cold. This depends on the fact that at 0° under favorable circumstances only the interbody, and not the complement, is bound by the blood-cells. Accordingly by appropriate treatment it could be shown that the serum had lost part of its power, but that it could be regenerated by the addi- tion of the same kind of serum previously inactivated by heat. This confirmed their view of the complex nature of normal hsemolysins. They were further able to activate inactive hsemolytic normal sera by the addition of other kinds of sera which served as complements, and which by themselves did not dissolve the particular blood-cells ' Reprint from the Berl. klin. Wochen. 1902, Nos. 9 and 10. 181 182 COLLECTED STUDIES IN IMMUNITY. used. This showed conclusively that the glohulicidal yroperty of normal serum is due to the co-action of two bodies, one which withstands heating (thermostable) and the other which does not {thermohhile .) These views have been accepted by the majority of investiga- tors, and numerous observers, P. Miiller,! London,^ E. Neisser, and DoringS have constantly added new facts, the analysis of which in every instance demonstrates the complex nature of nor- mal hsemolysins. Nevertheless it does not seem to me superfluous to thoroughly discuss this question once more, since such eminent authorities as Buchner * and Gruber,^ because of the negative result of part of their experiments, hold that Ehrlich and Morgenroth's conception of the nature of normal hsemolysins is erroneous. Ehrlich and Morgenroth had from the beginning stated that the solution of this problem in any particular case was only possible by their method under certain favorable circumstances. Now, although Buchner and Gruber have employed this method, so that a negative result proves nothing whatever, in consideration of the importance of the matter I have followed the suggestion of Prof. Ehrlich,^ and undertaken a critical study of the negative findings of these authors. The results of this have already been briefly alluded to elsewhere. Buchner sought to discover the presenee of thermostabile bodies (his " Hilfskorper ") on the occurrence of haemolysis, by reactivat- ing normal sera, which had been inactivated by heating to 60° C, with fresh serum of a different species. But out of the large number of possible combinations he chose only one and used as a source of complement only that serum which was derived from the same species that fiu-nished the blood-cells. In an address on the protective bodies of the blood, delivered at the Hamburg Congress of Natu- ralists, Ehrlich pointed out that this procedure was inapplicable. It can surely not be expected that every serum contains a fitting ' P. Muller, tjber Antihamolysine, Centralblatt fiir Bacteriologie, Vol. 29, 1901. ^ E. S. London, Contribution k I'^tude des htoolysines, Arch, des Sciences biolog. (Inst, imperial de m&l. exper. k St. Petersbourg), T. VIII, 1901. ' E. Neisser u. Doring, Zur Kemitniss der hamolytischen Eigenschaften des menschlichen Serums, Berl. klin. Wochen. 1901, No. 22. * Buchner, Sind die Alexine einfache oder complexe Korper? Berl. klin. Wochenschr. 1901, No, 33. ' M. Gruber, Zur Theorie der Antikorper, II, Uber Bacteriolyse u. Hsemolyse, Munch, med. Wochenschr. 1901, 48 and 49, ° Ehrlich, Vortrag im Verein fur innere Medicin, Dec, 16, 1901. CONCERNING ALEXIN ACTION. 183 complement for any given amboceptor. In testing a series of com- binations, therefore, the finding of a suitable complement will to a certain extent be merely a coincidence. In all the cases studied at this Institute, however, even though often only after consider- able labors, this has always led to a certain realization of the com- plex nature of the hsemolysin. Buchner was successful in two of his cases in activating the combi- nation chosen by him: blood-cells A + inactive serum (amboceptor) B + active serum {complement) A. (Guinea-pig blood and ox serum; goat blood and rabbit serum.) In three other cases, however, he was unable with a corresponding mode of procedure to restore the solvent power which had been lost by inactivation. Guinea-pig blood and sheep serum (Case I); sheep blood and rabbit serum (Case II); guinea-pig blood and dog serum (Case III). These results, to be sure, are contrary to those of Ehrlich and Morgenroth, who observed more or less marked haemolysis in these same combina- tions. These opposing results are, however, explained first by the fact that the amount of complement contained in the serum of the same species is subject to individual and chronological variations within wide limits. Beside this, recent experiences, which we shall subsequently discuss in detail, have shown us that the temperature at which the serum is inactivated is not indifferent for the function of the amboceptor. Hence it appears significant that in these experi- ments Buchner inactivated the sera by heating to 60° C, whereas ordinarily this is done at 56°-57° C. As a matter of fact, Buchner's experiment No. 6 shows that, dog serum, in this experiment inac- tivated by heating only to 57° C, is activated in its haemolytic action for guinea-pig blood by rabbit serum. In view of this, the nega- tive findings of Buchner in Case III lose their significance. In the three cases looked upon by Buchner as negative, I tried, by separation by means of cold, to convince myself of the presence of two substances effecting the haemolysis. My method of pro- cedure was as follows: Two parallel series of tubes of blood containing decreasing amounts of active serum were prepared, kept at 0° C. for 2-3 hours and then centrifuged. The decanted fluid of one series was then allowed to act on the sediments of native blood, that of the other series on the sediments of blood which had been treated with a like quantity of inactivated serum. The amount of blood, as in all our experiments, was 1 cc. of a 5% suspension in .85% salt solution. 184 COLLECTED STUDIES IN IMMUNITY. In two combinations (Cases I and II) the separation of the two components was effected without any trouble. The following pro- tocol will also show the technique of the experiment. Negative case I of Buchner. 0.5 cc. sheep senmi is still just able to completely dissolve guinea-pig blood. To each 1 cc. of a 5% guinea-pig blood suspension varying amounts of active sheep serum are added and the volume of fluid made up to 2 cc. with physio- logical salt solution. Two parallel series like this are kept at 0° C. for two hours and then centrifuged. The clear decanted fluids from the one series are allowed to act each on the sediment of 1 cc. native 5% guinea-pig blood; the fluids from the other series, each on the sediment of 1 cc. 5% guinea-pig blood, which had previously been treated with the same varying amounts of inactive sheep serum. The haemolytic action of the decanted fluids is shown by Table I. TABLE I. Absohption of Sheep Serum by Guinea-pig Blood at 0° C. Amount of the Sheep Serum Added. 00. Solvent Power of the Deoanted Fluids for A, Native Guinea-pig Blood. B, Guinea-pig Blood Previously Treated with Inactive Sheep Serum. 1 0.7 2 0.6 3 0.5 4 0.4 5 0.35 6 moderate it little trace It complete tt almost complete strong Buchner's second negative case deals with the combination sheep blood and rabbit serum. In the following experiment, entirely ana- logous to the preceding, the complete solvent dose of rabbit serTun for sheep blood was 0.2 cc. See Table II. These experiments, which are confirmed by numerous parallel experiments, show that in these two cases, as a matter of fact, hcemolysis depends on the presence of two substances. One of these, thermostable, is bound by the blood-cells at 0°C., the other, thermolabile, is left behind at this temperature. The latter, however, is only then able to effect haemolysis when it acts on blood-ceUs which have previously anchored the thermostable substance, the amboceptor. A comparison of Tables I and II also shows how much the combining relations between amboceptor and blood-cell on the one CONCERNING ALEXIN ACTION. 18& hand, and amboceptor and complement, on the other, may vary from case to case. Whereas in Case II the decanted fluids were TABLE II. Absorption or the Rabbit Sertjm by Sheep Blood at 0° C. Amoiint of Rabbit Serum Added. cc. Solvent Power of the Decanted Fluids for A, Native Sheep Blood. B. Sheep Blood Previously Treated with Inactive Rabbit Serum. 1 0.6 2 0.45 3 0.35 4 0.25 5 0.2 6 trace complete almost complete moderate absolutely inactive against native blood (i.e., aU the amboceptor had been bound at 0° C. by the blood-cells) in Case I the decanted fluids were then still active when the amounts of serum added were less than the solvent dose. This indicates that in this case the affinity of the amboceptor's cytophile group for the receptor of the cell is relatively slight at 0° C. In like manner the columns B of the tables show a certain difference of affinity between amboceptor and com- plement. In Case I the decanted fluid still contains the entire com- plement; in Case II, on the other hand, a portion of the complement must have combined with the amboceptor, for the decanted fluid shows a distinct loss of complement. The separate examination of the sediments of the specimens to which active serum was added agrees with this; in Case I these sediments mixed with physiological salt solution and placed into the incubator showed no trace of solution, while in Case II the sediments of the first three tubes showed mod- erate, little, and trace of solution respectively. Both normal hcsmolysins (Buchner's negative Cases I and 11) therefore correspond in their main behavior. They consist of two components {readily separable by the "cold method") which in their mutual relations manifest a certain variation in the hehmnor of their receptors. The conditions in these two combinations were favorable for analysis of the mode of action by means of our method. In the study of Buchner's third negative case, however (guinea-pig blood and dog serum), difficulties presented themselves which at first ap- 186 COLLECTED STUDIES IN IMMUNITY. peared to be insurmountable. Despite numerous variations iii the conditions of the experiment we did not succeed with appropriate procedures in effecting a separation by means of the "cold method." The fluids decanted from the mixture of guinea-pig blood-cells and active dog serum manifested the same behavior, so far as hsemolytic action was concerned, on normal guinea-pig blood and such as had previously been treated with inactive dog blood; and yet they showed slight differences so that we did not feel justified in drawing any conclusion. However, we soon became convinced that a separation of two substances causing haemolysis had nevertheless been effected by the absorption in the cold. We allowed the fluid decanted from the ^inea-pig blood-cells previously treated with active dog serum, which fluid only slightly dissolved native guinea-pig blood, to act on guinea- pig blood sediments, which also had previously been mixed with active dog serum. We were then able to determine that these sedi- ments were strongly, in appropriate quantities completely, dissolved by the decanted fluid, although when mixed merely with salt solution and placed into an incubator they did not dissolve at all, or dissolved tjnly in traces. An experiment of this kind is shown in Table III. TABLE III. Absorption of Dog Serum by Guinea-pig Blood at 0° C. HEemolysis of the Sediments Sus- pended in Salt Solution at 37°. Solvent Power of the Decanted Fluids for Amount of Dog Serum Added. oc. A, Native Guinea- pig Blood. B, Guinea-pig Blood Previously Treated with Inactive Dog Serum. C, Guinea-pig Blood Previously Treated with Active Dog Serum at 0°. 1 0.25 2 0.2 3 0.15 4 0.1 5 0.075 trace faint trace almost complete strong moderate little trace complete almost complete moderate little trace complete IC CI strong Hence by means of the absorption with guinea-pig blood in the cold, the active dog serum was separated into two components each of which by itself was incapable of effecting solution. One of these became attached to the red blood-cells, the other remained in the fluid. The former there- fore corresponded in its behavior to the amboceptor, and it was only a coincidence that dog serum inactivated by heating to 60° C. was unable also to assume that role. We hoped to discover more about the nature ■of this curious behavior by employing a different method of inactivat- CONCERNING ALEXIN ACTION. 187 ing the dog serum, and therefore in this case turned first to the com- pletion method. By means of completion of the' variously inactivated dog serum with other sera which do not dissolve guinea-pig blood, we hoped to obtain an insight into the circumstances here presented. In this way we were able to convince om'selves that dog serum which had been inactivated by half an hour's heating to 60° C. according to Buchner's procedure, is no longer activated in its hsemolytic action on guinea-pig blood, by the addition of guinea-pig serum. When the dog serum, however, was heated only to 55° C. or even only to 50° C. it was always possible to activate such an inactivated serum by means of guinea-pig serum. This was the more readily effected, the lower the inactivating temperature employed. It need hardly be mentioned that in particular cases we always determined whether the serum really was inactive; and this showed that dog serum loses its hsemolytic property for guinea-pig blood completely, even when merely warmed for half an hour to 49° C. We must therefore regard it as a fortunate coincidence that the complement of dog serum is so markedly thermolabile, for only under this condition could it be possible to preserve the amboceptor intact, i.e., capable of react- ing, for that body is but little more stable. Whether the amboceptor heated to 60° has been damaged in its cytophile or complementophile affinity is still undetermined. One could perhaps also think of a blocking of the complementophile group of the amboceptor due to a binding of the comp ement taking place at the higher temperature. Be this as it may, these experiments certainly show that the power of a dog serum (inactivated at a suitable temperature, e.g., 50° C.) to be activated by guinea-pig serum is lessened by heating the dog serum to 55° C. and destroyed at 60° C. Table IV shows such an experiment: TABLE IV. Completion (with Guinea-pig Serum) op Dog Serum Inactivated at Different Temperatures. Amount of the Activated Guinea- pig Serum. Degree of Solution of the Guinea-pig Blood Mixed with 0.15 cc. Dog Serum and Inactivated by Half an Hour's Heating to cc. A, 60°. B, 55°. C, 50°. 1 0.5 2 0.25 3 0.1 4 moderate little trace complete strong little 188 COLLECTED STUDIES IN IMMUNITY. We now repeated the experiment of separation in the cold by allowing the fluid which was decanted from the guinea-pig blood-cells after these had been treated with active dog serum at 0°C., to act on guinea-pig blood sediments previously mixed with dog serum. Our results were in accord with the above and led to a clear under- standing of our previous negative findings. See Table V. TABLE V. Absorption of Dog Serum by Gtjinea-pig Blood at 0° C. (0.075 oc, dog serum just completely dissolves 1 cc. 5% guinea-pig blood.) Solvent Power of the Decanted Fluids on Amount of Dog Senun Added. A, Native Guinea-pig Blood. B, Guinea-pig Blood Previously Treated with Dog Serum Inactivated at cc. I, 60°. II, 55». III, 50°. 1 0.15 2 0.1 3 0.075 complete moderate little complete almost complete moderate complete strong complete In this case, therefore, we have demonstrated a thermolability of the amboceptor 1 which shows itself especially in the activa- ting experiment with guinea-pig complement, but also in that with its own dog (complement). Only through this thorough analysis was it possible to furnish for Buchner's third negative case also positive proof of the complex constitution of normal hsemolysins. After having determined that certain amboceptors will only ' It is therefore not at all permissible to define the two components of the hBemolysin, as Gruber would do (Discussion of Gruber's Address, Wiener Klin. Wochensch. 1901, No. 50), only according to the temperature, and to say that at a certain degree of heat the amboceptor remains intact while the complement does not. As long ago as their second communication Ehrlich and Morgenroth described a thermostable complement of goat serum which remained intact at 56° C; and according to our experiences here described a general definition of amboceptors as bodies which withstand heating to 55° C. is absolutely impos- sible. The influence of temperature on amboceptor and complement varies from case to case. Hence that these two factors act together in haemolysis we know only from this, that two substances, in themselves not capable of causing solution, when combined, effect hcemolysis; and that one of these substances (the complement) can never alone be bound by the blood-cells but always only through the intervention of ike other {the amboceptor). CONCERNING ALEXIN ACTION. 189 bear slight warming, in order to remain capable of reacting, we had to abandon our custom of inactivating sera by simply heating them to 60° C. Thereafter we had always first to determine the minimal inactivating temperature for each individual case. The limits of temperature can usually be determined accurately; for dog serum it is 49° C. We have also tried by means of other complements to activate dog serum inactivated at 50°, and have found a suitable complement not only in guinea-pig serum but also in human serum. In this case also, the thermolability of the amboceptor showed itself, for heating to 60° C. destroyed the reactivatibility. In two cases, however, the power to reactivate was preserved to a greater or less extent even after heating to 60° C. In like manner dog serum could be activated by the complements described when it had been deprived of its solvent power by other means. Thus the comple- ments of dog serum were absorbed by means of yeast, and by means of an anticomplement serum (from a goat) whose normal amboceptor for guinea-pig blood had been removed by washing with guinea-pig blood. The dog sera so inactivated manifested their amboceptor properties when they were appropriately activated. In the first two negative cases of Buchner, separation in the cold had shown the presence of amboceptors in the sheep and rabbit serum. I now sought by means of activating experiments to find fitting complements for these amboceptors in other sera. Naturally, after the above experiences, it was necessary here also to first determine the minimal inactivating temperature. For sheep serum this is 50° C, for rabbit serum 51° C. If sheep serum is inactivated by half an hour's heating to 50° C, it is easy to restore the hemolytic action on guinea-pig blood (Buchner's Case I) by the addition of fresh human serum. In this way the complex nature of the normal hemolysin of sheep serum can be demonstrated. One can also acti- vate with guinea-pig serum, although then a feebler solvent action is obtained. In both cases the thermolability of the amboceptor is readily demonstrated; for by heating the sheep serum to 60° C. this can no longer be activated, or only in very much less degree.^ ' In addition to this I have also demonstrated a thermolability of the am- boceptors of goat serum which are activated by horse serum and act on rabbit and guinea-pig blood. Repeated investigations by Dr. Morgenroth have shown that a markedly thermolabile amboceptor is contained also in horse serum. This amboceptor, which fits guinea-pig blood, and can no longer be 190 COLLECTED STUDIES IN IMMUNITY, The combination, sheep blood and rabbit serum (Buchner's second case) presents entirely analogous conditions. Both guinea- pig serum and human serum, the latter only in a moderate degree contain a complement which activates the amboceptor of rabbit serum. The rabbit amboceptor, however, is evidently of more stable constitution; for even after heating to 60°, its solvent power can be completely restored. I can therefore confirm the facts found by Buchner in this case, namely that sheep serum is incapable of restoring the solvent power for sheep blood. This, however, accord- ing to the above statement, is naturally no argument against the complex nature of the hsemo ysin because not every serum need con- tain a fitting complement for every particular amboceptor. Provided that sufficiently numerous combinations are examined, the " completion method " as a rule leads to the positive demon- stration of the amboceptors. The " separation in the cold " on the contrary, owing to the peculiarity of the combining relations of the separate components, is entirely inapplicable in a number of cases. Gruber, the second author to come out against the conception of the complex nature of normal serum hsemolysins, sought to demon- strate amboceptors in a number of normal sera, by means of " sepa- ration in the cold." In view of the preceding it is not surprising that he failed in a number of cases to effect a separation of the hemolysin . Ehrlich and Morgenroth in their second communication on hae- molysins have already analyzed the conditions for separating the interbody by means of absorption, emphasizing " that the solution of the problem therefore is now possible only under either of the two above mentioned favorable conditions; (1) When the two haptophore groups of the interbody differ greatly in their affinity; and (2) when, by means of a combination whose discovery depends on chance, an acti- vating complement is found." The limitations of the two methods applicable to an analysis of the complex nature of hsemolysins, are therefore sharply defined. In any individual case when one method fails, it will always be be necessary to make use of the other in order to gain an insight into the constitution of the hemolysins at all commensurate with the means at our disposal. The schematic application of only one activated after heating to 55° C. can be shown to exist in active horse serum (which does not dissolve guinea-pig blood) by combining and completing it with guinea-pig serum. CONCERNING ALEXIN ACTION. 191 method can lead to the greatest errors. In this respect a comparison of the results obtained by Buchner and Gruber, is very instructive, for among their cases are two combinations which are designated by the one as positive, and by the other negative. The amboceptor of rabbit serum for sheep blood, which Buchner, because of the failure to reactivate this with sheep serum, regarded as absent, Gruber, by means of the cold separation method, could demonstrate as present; and for ox serum, whose amboceptor Buchner had already demonstrated by the activation with guinea-pig serum, Gruber, through the failure of his cold absorption method, arrived at the view of a pure alexin action. In Gruber's negative cases, which embrace the following com- binations: I, rabbit blood — dog serum; II, rabbit blood — ox serum; III, gui:ea-pig blood — ox serum, IV, rabbit blood — guinea-pig serum; I have systematically sought for sources of fitting complements and have found these in abundance. Naturally in view of the experi- ences above mentioned the inactivation of the sera was effected at the lowest temperatures possible; thus dog serum and guinea-pig serum at 50°, ox serum at 52° C. In the following sera (in part in agreement with other previous experiences) I have found comple- ments suitable for activation: I. For the amboceptor of dog serum, acting on rabbit blood; in guinea- pig serum, ox serum, goat serum, and sheep serum. II. For the amboceptor of ox serum, acting on rabbit blood; in guinea-pig serum, rabbit serum, and rat serum. III. For the amboceptor of ox serum, acting on guinea-pig blood; — ■ in guinea-pig serum, human serum, rat serum, horse serum, and to a slight extent also in sheep serum Naturally in all the experiments, control tests were made with the active serum, which served as complement. In the cases desig- nated as positive completion, this serum by itself had to exert no hsemolytic action or at least to act in a very much smaller degree. Gruber's fourth negative case, rabbit blood and guinea-pig serum, offered considerable difficulties because the combination is very little or not at all effective, and it is probably because of this that Gruber speaks of " concentrated guinea-pig serum." Among a large number of guinea-pig sera examined for this purpose, we found only two sufficiently haemolytically active. But here also, through the successful activation by means of human and ox sera (sera, to be sure, which by themselves dissolve rabbit blood, but which still 192 COLLECTED STUDIES IN IMMUNITY. effect complete haemolysis as complements in amounts in which alone they are entirely inert) we could furnish positive proof of the presence ■oi amboceptors. Buchner and Gruber have therefore described a total of seven cases said to show pure alexin action; and these cases were held by them to be sufficient to decide in the negative the entire question •of the complex nature of the normal serum haemolysins. Against ■this we have in all these cases brought positive proof that the " alexin," conceived by Buchner to be a simple unit, always produces its effects through the co-action of two components, the existence of which is demonstrable in different ways. We must therefore uphold Ehrlich and Morgenroth's view, that normal and artificially produced hcemo- lysins exert their action according to exactly the same mechanism. We do not yet possess a method generally applicable to demon- strate the complex nature of the hsemolysin, and even a thorough analysis, therefore, need not necessarily achieve the desired result in •every case. The method adopted by Muller^ for demonstrating the amboceptors in chicken serum, which is haemolytic for rabbit blood, is of interest in this connection. When the usual methods iailed he found that bouillon injections caused an increase in the amount of complement in the chicken serum without affecting the amount of amboceptor. This led him to recognize the complex nature of the hemolysin, a fact confirmed by the successful activa- tion of heated chicken serum by means of pigeon serum. When therefore in isolated cases the separation does not succeed accord- ing to the methods heretofore employed, such results, the product of incomplete methods, most certainly do not argue for a simple alexin .action. We hope that the employment of the lowest possible tem- peratures in inactivation will result in increasing " completion " possibilities and make the demonstration of the complex consti- tution of the hsemolysins easier in difficult cases. At present this demonstration has failed only in the case of eel serum (which, to be sure, is very peculiar in its hsemolytic behavior), for thus far no fitting complements have been found for this serum. In all other cases of haemolysis through normal sera, which have been investi- gated for this purpose, according to our experiences positive proof of the presence of the amboceptors has been furnished. The normal bactericidal sera also owe their bactericidal power 'PMiiUer, 1. c. CONCERNING ALEXIN ACTION. 193 to the co-action of two substances. Pfeifferi furnished the first observations which led to this view when, in 1895, he succeeded in restoring the bactericidal power of inactivated goat serum in the peritoneal cavity of a guinea-pig. Moxter^ subsequently demon- strated the presence of normal bacteriolytic amboceptors by means of reactivating experiments in vitro. And according to the numerous investigations of M. Neisser and Wechsberg in this Institute, all of the bacteriolysins of normal sera which they investigated, are of complex constitution. This is natural because in the cell-destroying properties of normal serum, as in the development and increase of these properties through immunization the mechanism is exactly the same in principle, although, owing to the multiplicity of the reaction products, the action of the latter appears more complex. In my investigations of the cytotoxic properties of normal serum I have included the widely distributed spermotoxic function. Accord- ing to the unanimous opinion of all authorities the specific spermo- toxin produced by immunization consists of two substances. Thus far, however, this has not been demonstrated for normal spermo- toxin, and Metalnikoff^ has regarded the impossibility of reacti- vating the heated normal spermotoxic serum as an important diag- nostic means to differentiate the latter from the specific immune serum. In opposition to this, by means of suitable mixtures, I was able, here also, to convince myself of the complex nature of the normal spermotoxin. After the spermotoxic property of rabbit serum for guinea-pig spermotozoa had been destroyed by heating to 56° C, I was able to restore this by the addition of guinea-pig or horse serum provided I mixed the inactive rabbit serum and guinea- pig serum in the proportion of 3:1 or 3:2. In that case the guinea- pig spermatozoa were killed after 12-15 minutes, whereas, in the control test with inactive rabbit serum or active guinea-pig serum alone, the spermatozoa showed lively movements even after IJ-IJ hours. The proportion of amboceptor and complement employed by me is in direct contrast to that recommended for immune sera by Metchnikoff and his co-workers. The reason for this will be under- ' R. Pfeiffer, Weitere Mittheilungen iiber die Spezifischen Antikorper der Cholera, Zeitschr. f. Hygiene, XX, 1895. ' Moxter, Uber die Wirkungsweise der bacterienauflosenden Substanzen der thierischen Safte, Centralbl. f. Bacteriol., XXVI, 1899. ' Metalnikoff, Etudes sur la Spermotoxine, Annates de I'lnstitut Pasteur, 1900. 194 COLLECTED STUDIES IN IMMUNITY. stood when the high degree of amboceptor concentration in immune sera is considered. In my case larger amounts of guinea-pig serum must be avoided, because in large doses the guinea-pig serum by itself finally exerts a toxic action on guinea-pig spermatozoa. This agrees with a statement of London (1. c.) that most all normal sera contain autospermo toxins. Subsequent Note. — In the meantime the French translation of a study by London, which had already been published in Russian, has appeared (Contribu- tion k I'^tude des spermolysines. Archives des Sciences Biologiques, T. IX, 1902), which shows that this investigator had also already recognized the complex con- stitution of the normal spermotoxin. Our views concerning the complex nature of haemoylsins have recently been confirmed by Flexner and Noguchi through the successful separation of am- boceptor and complement in the cold (Snake venom in relation to haemolysis, bacteriolysis, and toxicity, Journal of Experimental Medicine, vol. VI, 1902). XVIII. CONCERNING THE PLURALITY OF COMPLE- MENTS OF THE SERUM.i By Professor Dr. P. Ehrlich and Dr. H. Sachs. The continued study of the hsemolysins of blood serum has not only considerably extended our knowledge of the origin and mechan- ism of the immunity reaction directed against cells, but has revealed to us an unsuspected complexity of cellular metabolism to which the numerous protective bodies circulating in the blood owe their existence. It is probably everywhere conceded at the present day that the specific cytotoxins produced through immunization consist of two substances, amboceptor and complement; and we must regard it as proven that the cytotoxic substances in normal serum are also of complex constitution.^ A sim-ple alexin action, in Buchner's sense, does not exist. But even within the limits of this complicated field, Ehrlich and Morgenroth through their experimental work, have come to a further pluralistic conception, so that the closer analysis of the factors making up the cytotoxic function of a serum is enor- mously complicated. Thus it has been found in immunization with cells, that not merely a single kind of amboceptor is developed in the blood serum, but that a large number of different types of ambo- ceptors appear, which vary both in their cytophile and complemento- phile groups. Furthermore, a number of facts and theoretical con- siderations (discussed in detail in the Sixth Haemolysin commu- nication) could be satisfactorily explained only by the assumption of a plurality of complements, and were absolutely irreconcilable with the unitarian assumption of only one complement in each serum. After all this one might well regard the pluralistic conception as well founded, and abandon all further theoretic argument along this line. But Bordet,^ the strongest supporter of the unitarian ' Reprint from the Berl. klin. Wochenschr. 1902, Nos. 14 and 15. ^ See the previous study. ' Bordet, Sur le mode d'action des scrums cytolitiques, etc. Annales de rinstit. Pasteur, May, 1901. 195 196 COLLECTED STUDIES IN IMMUNITY. character, in a recent work especially 'designed to refute the plural- istic view of the complements, has published a series of experiments, which in his opinion necessarily point to a simple alexin. Bordet's argument is based on the discovery of the interesting fact that blood corpuscles or bacteria treated with an inactive immune serum specific for themselves were able to deprive a normal active serum of all its complement activity. Bordet sensitized blood corpuscles with appropriate amboceptors, and then exposed them to the action of a freshly drawn normal serum. If now he waited for the occurrence of haemolysis and then added sensitized cells, bacteria, or blood corpuscles of different species, they remained totally unchanged, although the serimi that had been used as complement was capable in its original condition of destroy- ing these also. When fresh serum was first brought into contact with sensitized bacteria, similar results were obtained. The blood corpuscles subsequently added did not then undergo haemolysis. // such an action on one of the sensitive substrata has once taken place, the active sera, as a rule, are deprived of all their complement functions, from which Bordet concludes that the destruction of the most varied elements by one and the same serum must be due to a single complement. It must be acknowledged that these experiments, which we have been able to verify in numerous cases, at first sight seem to sup- port Bordet's view. If one assumes that a certain serum A, which is capable of complementing two different bodies B and C, one bac- tericidal and the other haemolytic, contains only a single comple- ment, Bordet's results would then most readily be explained by assuming that the two immune bodies are identical in their com- plementophile groups. In that case, of course, owing to the previous exercise of the one function, the available complement will have been used up, so that nothing is left for the exercise of its second function. But a closer examination shows us that this view is an artificial one, and does not correspond to the facts observed. For if it be assumed that this particular serum A contains two different complements, both of which can be absorbed by the amboceptors B and C, Bordet's experiment will find an entirely different explana- tion. Now previous investigations ^ have shown that the artifi- cially developed immune sera are not of simple constitution, but contain a number of different amboceptors possessing different com- ' Ehrlich and Morgenroth, p. 56. PLURALITY OF COMPLEMENTS OF THE SERUM. 197 plementophile groups. To one, therefore, conversant with this con- ception, Bordet's conclusion cannot appear otherwise than forced. The unity of the complement would only then be demonstrated by Bordet's experiment if in the immune serum employed for absorp- tion but a single complementophile group came into action, and not a plurality of groups. Despite these objections raised against Bordet's evidence, and in spite of Ehrlich and Morgenroth's previous positive demonstration of the plurality of the complements, it seemed advisable, owing to the importance of the question, to enter once more upon a thorough investigation of the subject. We at first confined ourselves to the complements which effect the hsemolytic actions, and have been able to bring forward a large number of new and more conclusive proofs for the diversity of these complements in the same serum. These investigations have in part already been mentioned by Ehrlich at the Congress of Naturalists in Hamburg. The method of the experiments was guided by the following considerations. If only a single complement is present in a cer- tain serum, it follows that all the complement actions of this serum would be weakened equally by any given influence, chemical, physi- cal, or thermic. If, on the contrary, our view of the plurality of complements is correct, it should be possible through appropriate experimental conditions to influence the serum in such a way that only a part of the complements will be destroyed, while others remain intact. Not only the absolute inhibition of the action of a few com- plements, but also marked quantitative differences in the impair- ment of the individual completions can only be satisfactorily explained by the assumption of different substances as carriers of these prop- erties. A single complement would have all its functions impaired equally. We have especially subjected the complementing property of goat serum to a thorough analysis, using for this purpose five different combinations which can be activated by goat serum. For simplicity's sake, we shall designate them by the following numbers : Case I. Guinea-pig blood — inactive normal goat serum. Case II. Rabbit blood — inactive normal goat serum. Case III. Rabbit blood — inactive serum of goats immunized with rabbit blood. Case IV. Ox blood — inactive serum of goats immunized with ox blood. 198 COLLECTED STUDIES IN IMMUNITY. Case V. Dog blood — inactive serum of goats immunized with dog blood. The various means by which we have succeeded in a separation of the single complements are as follows: 1. Digestion with papain. 2. Partial destruction with an alkali. 3. Partial destruction by heating to 50° C. 4. Combination with blood-cells. > We discovered that invariably under the influence of papain digestion four complementing actions disappeared, or were more or less strongly diminished. Only a single complement remained intact, namely, that fitting the amboceptor developed in goat serum through immunization with rabbit blood. In these experiments 20 cc. goat serum mixed with 3 cc. of a 10% papain solution were placed ifi an incubator in order to digest the complements. We found that the proper time to interrupt the digestive process was usually thirty to forty-five minutes later, when an examination i demonstrated complete preservation of the complements for Case III with complete disappearance or consider- able diminution of the others. Of the large number of our experi- ments made in this connection three examples may be cited. See Table I. TABLE I. Digestion of Goat Serum by Means op Papain. Solvent Power of the Goat Serum. Example I. Examole II. Example III. (a) Digested. (b) Normal. (a) Digested (6) Normal. (a) Digested. (6) Normal. Case I I 0.5 0.25 0.5 0.15 0.5 0.25 moderate complete trace complete moderate complete Case II 1 1.0 trace 0.5 complete 1.0 0.25 complete 1.0 ft. trace 0.5 complete Case ni 1 0.2 0.15 0.15 0.15 0.15 0.15 complete complete complete complete complete complete Case IV 1 0,3 0.06 0.3 0.07 0.5 0.08 little complete little complete strong complete Case V I 0.5 trace 0.06 complete — — 0.3 alm't c'm'te 0.05 complete ' In all our experiments the amount of blood used as a reagent was 1 cc. almost nothing 1 difference in the diminution suffered by complement V and that suffered by complement III. This is so marked that merely a com- bination of the above three experiments already furnishes positive proof that the complement actions in III, IV, and V proceed inde- pendently of one another, and are effected by three different comple- ments. But against this method of proof the objection might be made that in the end we may still be dealing with simple [einheitlich] com- plements and that the results of the experiments mentioned do not necessarily indicate a plurality of complements. It could be assumed that the view we have expressed concerning the plurality of the complements was true only in a certain restricted sense. Thus it would be possible that the complements possessed but one hapto- phore group, but a plurality of zymotoxic groups of which one effected the damaging action in any individual case. It could then easily be imagined that the various zymotoxic groups differ from one another in their behavior toward chemic or thermic influences, so that per- haps one was injured by papain, and another by an alkali. In order to decide this possibility either one way or another it seemed advis- able to undertake absorption experiments. In case of a simple complement with different zymotoxic groups, the complement would be absorbed as a unit, whereas in the other case, differences such as we have already observed on heating, etc., would be expected to occur. Because of the great significance of obsorption, we regard these experiments as particularly valuable. Our first experiments were made to see if the complements, like so many bodies known to chem- istry would adhere to granular substances of various kinds by virtue of surface attraction. Bone charcoal, skin powder, lycopodium, PLURALITY OF COMPLEMENTS OF THE SERUM. 201 and diatom earth, which we employed for this purpose, all proved more or less unsuitable for the absorption of complement. A stronger absorbent power on the other hand was exhibited by organized mate- rials, thus confirming the statements of von Dungern.' Suspensions of staphylococci, when used in sufficient quantity, were able to abstract the complements quite energetically .^ In like manner yeast powder is an excellent means to deprive a serum of its complement prop- erties. A separation of the complements, however, was not achieved by these experiments. We are inclined to believe that in these cases the fixation of the complements is due to physical absorption and not to definite chemi- cal union. This view is the outcome of the positive results obtained in the absorptions when we employed blood-cells which had been mixed with suitable amboceptors, and which, according to our views, were able to bind complements chemically. If blood-cells which have been saturated (sensitized) with a normal immune body or with one artificially produced are shaken with a certain amount^ of complementing serum, it is very easy to determine that in con- formity with the results of Bordet's experiments, the complement properties possessed by the normal serum have in most cases com- pletely disappeared with the onset of hemolysis. It was just this phenomenon that led Bordet to his unitarian conception. Yet even in this absorption it is possible by means of suitable methods to convince one's self of the diversity of the complements, for by making the time as short as possible only those complements are absorbed which possess the strongest affinity for corresponding complementophile groups. Naturally experiments of this kind are difficult and require considerable variation. But it is usually possible to finally devise a suitable method of procedure. An interesting case studied by us in this respect is the combination rabbit blood and goat serum (Case II). With sufficiently rapid digestion (2 to 3 minutes at the most, possibly with the aid of gentle heat) the decanted portion showed a considerable loss of complements for Case IV or V, or for both, without suffering any injury in the rest of its complement ' See p. 36. 'The same results were obtained by Wilde (BerL klin. Wochenschr. 1901, No. 34) in absorption tests with anthrax, cholera, and typhoid bacteria; but to conclude from this that the alexin is a simple unit, as Wilde does, is not per- missible in view of our above statements. " This amount must be determined separately for each case. 202 COLLECTED STUDIES IN IMMUNITY. functions. "We were able to observe this behavior repeatedly and reproduce the following as an illustration. 10 cc. goat serum are shaken with 8 cc. rabbit blood for a very short time and then rapidly centrifuged. The following table shows the solvent power of the decanted fluid and of normal goat serum. The figures, I-V, correspond to the blood-cell amboceptor com- bination employed in the previous tables. TABLE IV. Bhief Absorption op Goat Sebtjm with Rabbit Blood. Solvent Power of the Goat Serum. (.a) After the Absorption. (6) Normally. . Case I " II " III " IV V . 25 complete 0.5 0.04 " 0.35 complete 0.2 . 25 complete 0.5 0.04 0.08 complete 0.03 Complements I, II, and III have been completely preserved, IV and V have been reduced to one-fourth and one-seventh respec- tively, thus furnishing another proof for their diversity. It is of special interest that in this brief action the particular activating principle (complement II) which we shall term the " dominant com- plement " has not at all combined with the cell, whereas other com- plements, which are of no consequence so far as the solvent process is concerned, have already been subjected to a distinct absorption. With the absorptions are also to be classed the experiments con- cerning Case I, which we have made with guinea-pig blood stro- mata obtained after the method of H. Sachs i by heating the blood to 55° C. In these stromata the receptors which bind the ambo- ceptors present in normal goat serum have been preserved capable of reacting. These experiments demonstrated the absorption of the comple- ments for the two normal hsemolysins (Cases I and II) while the rest of the complements were in the main preserved.^ An experi- ment of this kind is shown in Table V. ' See page 167. ' In this also it is necessary first to determine the favorable conditions governing the experiment. Thus, in order to completely bind the guinea-pie PLURALITY OF COMPLEMENTS OF THE SERUM. 203 20 cc. goat blood are treated with the stromata from 53 cc. guinea- pig blood. After absorption has occurred the mixture is centrifuged and the complement action of the fluid compared with that of nor- mal goat serum. (See Table V.) TABLE V. Absoeption of the Goat Serum by Guinea-pig Blood Strom.4TA. Solvent Power (a) Of the Decanted Fluid. (6) Of tUe Normal Goat Serum. Case I " II " III " IV " V 1.0 faint trace 1.0 " 0.1 complete 0.15 complete 0.15 complete 0.15 complete 0.25 0.1 complete 0.04 complete 0.15 comprete Hence after the absorption, the complements of the normal hsemo- lysins had almost completely disappeared, while complements III and V were entirely preserved. Complement IV occupies a place between these, for in this case also a partial absorption could not be avoided. Its behavior very prettily confirms the demonstra- tional ready made by us of this complement's peculiar isolated position. Entirely analogous results are obtained when, instead of using guinea-pig blood stromata, a series of experiments is made with red blood-cells, using the red fluid obtained when the red blood-cells have dissolved directly as complement for another combination. In such experiments we could show that the blood solution thus obtained had lost complements I and II and contained only the complements for cases III, IV and V. This method of procedure blood haemolysin (amboceptor+ complement) of normal goat-blood serum, it is necessary to absorb with a large excess of guinea-pig blood stromata. It then readily happens that some complements other than those belonging to the two normal hsemolysins suffer injury to a greater or less extent. This was observed especially in several cases in which, in order to render easier the •complete binding of the complements for the normal haemolysins, the guinea- pig blood stromata had been sensitized with a large amount of inactivated normal goat serum. In that case, evidently, several partial amboceptors present in the goat serum in relatively small amounts and possessing affinities also for the other complements come into play. 204 COLLECTED STUDIES IN IMMUNITY. therefore confirms the separation effected by means of the stromata, whereby the complements of the normal haemolysins I and II are separated from the rest. Bordet himself, by the way, has described such a case concerning the combination rabbit blood — guinea-pig serum. This experiment, of course, was not to be reconciled with his unitarian view, and he therefore sought to explain this inconvenient result in accordance with his view by assuming a special law of distribution for the normal hsemolysins, together possibly with an inhibiting action exerted by the products of the destruction of the red blood-cells first used, on further solution of the same.^ Against this we should like to emphasize that in our case the result has been confirmed by the experiment with blood stromata. By means of this, since the stromata plus the anchored complement is removed by centrifuging, Bordet's assumptions can be entirely excluded. Our absorption experiments therefore show that of the two possi- bilities, namely, of a complement vnth several different zymotoxic groups, or of a plurality of different complements, the latter assumption must be accepted. Regarding the number of complements to be assumed for normal goat serum, as based on our experiments, this can best be seen from the following table: TABLE VI. Complementing Power of Goat Serum after (a) m (<;) (d) Absorption Absorption (/) Absorption Digestion The Action Heating with with with with of Soda. to 500. Rabbit Guinea-pig Papain. Blood. Blood. Blood Stromata. Case I + " II + " III + + 4 + + + " IV + i + i " V nV 1 + + ' This objection, moreover, is entirely incomprehensible to us. According to our view, normal and artificially produced haemolysins manifest their action by means of the same mechanism; for when the normal amboceptors are re- placed by the host of amboceptors present in an immune serum, new comple- mentophile groups come into action, and with these, of course, new partial complements. PLURALITY OF COMPLEMENTS OF THE SERUM. 205 This shov/s us that the two complements I and II (normal haemoly- sins) cannot by these experiments be differentiated from each other, that the other three complements, however, can absolutely be distinguished by their behavior, not only from one another but also from the first group. Hence in the five different combinations the existence of at least four different complements is positively demonstrated. And that the two normal hemolytic functions of goat serum are also effected by two different complements follows from a previous experiment of Erhlich and Morgenroth.i These authors showed by filtering a normal goat serum through Pukall filters, that the filtrate contained exactly the same amount of complement for guinea-pig blood, whereas the com- plement for rabbit blood was almost entirely absent. E. Neisser and Doring ^ have confirmed this result in the case of human serum. The necessary consequence, therefore, of our experiences with goat serum is the demonstration of the fact that in the five completions examined, five different complements of the goat serum come into play.^ We have also examined the complementing properties of the sera of other animal species, and have arrived at results which abso- lutely contradict the unitarian view of the complements. These experiments concern first the serum of rabbits. We shall proceed from the fact determined by Schutze and Scheller* under Wasser- mann's direction, that, following intravenous injections of goat blood, the rabbit serum completely loses its property to dissolve goat blood. The question now was whether the rabbit serum had been deprived merely of this one complementing function, or whether it had also suffered loss in the rest of its complement properties. We therefore tested the power of rabbit serum, before and after the injection of goat blood, to activate the immune body obtained by immunizing rabbits with ox blood. As the essential result of our numerous investigations we established the fact that the com- ' See page 56. 'E. Neisser and Doring, Berl. klin. Wochenschr. 1901, No. 22. ' Through the courtesy of Dr. Wendelstadt in Bonn, we learn that that investigator, by means of an interesting method, has also succeeded in demon- strating a number of complements in goat serum. He immunized a goat with several species of blood and was then able by means of chemical and thermic influences to separate the complements fitting the immune bodies produced.' See Centralblatt f. Bacteriologie, in which this study is about to appear. * Schutze and Scheller, Experimentelle Beitrage zur Kenntniss der im normalen serum vorkommenden globuliciden Substanzen, Zeitschrift f. Hygiene, Vol. 36, 1901. 2n6 COLLECTED STUDIES IN IMMUNITY. plement for goat blood disappeared after the injection while that for the immune body sensitizing ox blood remained intact. The following test may serve as an example: A rabbit of 1900 g. is injected intravenously with 22 cc. goat blood. The change in the solvent power of the goat serum which results from the injection may be seen from the following table: TABLE VII. Solvent Power of the Rabbit Serum. Blood Species, (o) Before the Injection. (6) After the Injection. Goat blood — inactive normal rabbit serum Ox blood — inactive serum of a rabbit im- munized with ox blood . 35 complete 0.05 " . 1.0 no solution . 25 complete Similar results are obtained in the absorption of rabbit serum by means of goat blood in vitro, so that this experiment already justi- fies us in assuming two different complements in rabbit serum. In one of these experiments with goat-blood injections the hae- molysis of pig blood by means of rabbit serum was also tested, and it was found that the complement of the normal hsemolysin for pig blood, like that for sensitized ox blood, had remained unchanged. Neither was it possible by means of intravenous injection of pig blood to separate these two complements of rabbit serum, for in this case, contrary to their previous behavior, both were absorbed, while the complement for goat blood remained in the serum. For the present we must therefore content ourselves with the knowledge that we have brought forward positive proof of the existence of two different complements in rabbit serum; a proof which is strongly cor- roborated by the divergent behavior of the two complements in the absorption with goat blood and pig blood respectively. The difference between the two complements also manifests itself in their different vulnerability to papain. While the com- plementing power of rabbit serum toward the artificially produced immune body for ox blood suffers considerable diminution under the influence of papain digestion, the complement of normal haemolysin for goat blood is hardly affected, so that this experiment also sub- stantiates our demonstration of at least two complements in rabbit serum. Some rather cursory tests were finally made with dog and guinea- PLURALITY OF COMPLEMENTS OF THE SERUM. 207 pig serum with the view of separating the complements by care- fully heating the sera. In the dog sermn a half hour's heating to 49.5° and in the guinea-pig serum to 49° was sufficient to enable us, by means of the differences of the weakening of the various com- plementing functions, to recognize here also the plurality of the com- plements. The results of these experiments are shown in Tables VIII and IX. TABLE VIII. Half an Hour's Heating of Dog Serum to 49°.S C. Blood-cell— Amboceptor Combination. Solvent Power of the Dog Serum. Solvent Power Still Preserved. (a) Heated. (5) Normal. 0.5 0.25 complete 0.5 0.1 0.5 0.08 " . 5 moderate 0.15 " less than J 0.35 complete 0.06 " * . 5 strong 0.045 " less than -jx I. Rabbit blood — inactive dog serum Guinea-pig blood — inactive dog serum Sheep blood — inactive dog serum Human blood — inactive se- rum of goats immunized with human blood Ox blood — inactive serum of goats immunized with ox blood VI. Ox blood — inactive serum of rabbits immunized with ox blood II. III. IV. V TABLE IX. Half an Houe's Heating of the Guinea-pig Serum to 49° C. Blood-cell — Amboceptor Combination. I. Rabbit blood — inactive guinea-pig serum II. Ox blood — inactive guinea- pig serum III. Ox blood — inactive serum of goats immunized with ox blood IV. Ox blood — inactive serum of rabbits immunized with ox blood V. Sheep blood — inactive se- rum of goats immunized with sheep blood VI. Dog blood — inactive serum of goats immunized with dog blood Solvent Power of the Guinea-pig Serum. (o) Heated to 49° 1.0 . 5 trace 0.008 complete 0.025 " 0.025 0.5 (&) Normal. . 5 complete 0.5 0.008 0.025 0.006 0.25 Solvent Power Still Preserved. almost 1 1 208 COLLECTED STUDIES IN IMMUNITY. If we review all our observations, they show that in the ques- tion of the complements the unitarian conception leads to a con- fused mass of inexplicable contradictions, and that it must there- fore be abandoned. All experiences, on the other hand, harmonize best with the assumption of a number of different complements in the same serum. Sober consideration, in fact, makes this appear as the necessary consequence of such a multiplicity as has been demon- strated anew by these experiments. It is a satisfaction to know that in the Institut Pasteur a high authority (Metchnikoff ) i has also given up the Buchner-Bordet conception of the simplicity [einheitlichkeit] of the alexines, and has come to the conclusion that there are at least two complements in the same serum. Metch- nikof5f found that the exudates rich in macrophages acted hsemo- lytically, but were unable to effect bacteriolysis. On the other hand the exudates rich in microphages exerted a marked bactericidal action, but were incapable of dissolving even sensitized red blood-cells. Metchnikoff concludes that these two kinds of cells produce two different complements, one, which he terms microcytase, effects the bacteriolytic actions, the other, macrocytase, is the carrier of the functions which destroy animal cells. He emphasizes that the demonstration of the duality of complements does not affect the correctness of Bordet's experiments, and he says in explanation of Bordet's results: " II n'y a qu'a admettre que les elements figures, une fois qu'ils sont impr^gn^s de fixateurs specifiques, deviennent capables d'absorber non seulement la cytase qui les digere, mais aussi une autre qui, sans les dissoudre, se fixe simplement sur eux." So far as this is concerned we should like again to emphasize that we also have not questioned the correctness of Bordet's experi- ments, but have merely objected to the unitarian conception deduced therefrom. The old controversy concerning the two views would thus be ended, and definitely decided in favor of our view. ' Metchnikoff, L'Immunit6 dans les maladies infectieuses, page 206, Paris, 1901. XIX. CONCERNING THE MECHANISM OF THE ACTION OF AMBOCEPTORS.' By Prof. Dr. P. Ehruch and Dr. H. Sachs. I. Blocking of the Amboceptor by Complementoids. The complements -which activate the amboceptors of blood serum are, as is well known from the experiments of Ehrlich and Morgen- roth, like the toxins characterized by two groups in the molecule, viz., the haptophore group, which combines with the complemento- phUe group of the amboceptor, and the zymotoxic group, which represents the actual carrier of the complement's specific function. In complete harmony with this, Ehrlich and Morgenroth^ could show through the production of anticomplements by heating inac- tivated sera, that the complements, like the toxins, under certain circumstances are changed into inactive modifications. These mod- ifications are stiU able to excite the production of antibodies, and must therefore possess their haptophore group intact; in analogy with the toxoids, therefore, they are called complementoids. Although the presence of the complementoids could easily be shown by means of animal experiments, it was impossible to demonstrate their react- ing power by means of haemolytic test-tube experiments. The reason for this was that a decrease of the complement action, such as was to be expected in the inactivated sera (which really con- stitute a mixture of amboceptor and complementoid) , did not occur, even when the complementoid was present in large amounts. Ehr- lich and Morgenroth have therefore assumed that in the change from complement to complementoid, the affinity of the complement's hap- tophore group suffers a diminution. A similar assumption has been made by Myers ^ for the toxoids of cobra poison. ' Reprint from the Berl. klin. Woohenschr. 1902, No. 21. 2 See page 79. ' Myers, Cobra Poisons, etc., The Lancet, 1898. 209 ^10 COLLECTED STUDIES IN IMMUNITY. It is, of course, not at all necessary that such a diminution of affinity occur with all complements; and, considering the great dis- tribution and multiplicity of the substances included in the con- cept " complement," this is a friori but little probable. We have therefore hoped that in the course of our investigations we would discover a suitable combination in which, on the formation of com- plementoid, the diminution of affinity does not occur, or occurs only to a slight degree. As a matter of fact, such a case has recently presented itself to us. As is well known, normal dog serum dissolves guinea-pig blood energetically. If this dog serum is inactivated, it is easy to restore the hsemolytic property with active guinea-pig serum; the inacti- vation, however, must be effected at suitable temperatures, 50-51° C, for at higher temperatures, as Sachs ^ has demonstrated, the ambo- ceptor of dog serum shows itself thermolabile. That is why Buchner in his experiments could not activate the amboceptor of dogs, for at the inactivating temperature employed by him, 60° C, the completion with guinea-pig serum is no longer possible. Continuing the analysis of this interesting case we made a curious observation: If guinea-pig blood-cells were treated with appropriate amounts of inactive dog serum for one hour in an incu- bator and the mixture then centrifuged, it was found that, con- trary to all expectations, the sediments could no longer be activated with guinea-pig serum, whereas when the three constituents were mixed simultaneously, prompt haemolysis occurred. (See Table I.) Our first thought was that the amboceptor, despite the relatively long contact with the blood-cells (one hour), had perhaps not been bound by these. Such behavior, to be sure, although conceivable and, as we shall see later, sometimes actually occurring, would be exceptional. In this case, however, we could readily convince our- selves that this suspicion was groundless. For when by means of guinea-pig serum we attempted to activate the guinea-pig blood- cells digested with dog serum as above described, vnihout first removing the fluid medium, no hsemolysis took place. And we could see by the behavior of the fluid obtained by centrifuging the blood mixture as described that the amboceptor was not present in the fluid. When this was allowed to act on native guinea-pig blood to which active guinea-pig serum (complement) had been added, no solution could • See pages 181 et seq. THE MECHANISM OF THE ACTION OF AMBOCEPTORS. 211 TABLE I. Inactive Dog Serum. cc. Solvent Action on the Guinea-pig Blood.' (A) Blood + Inactive Dog Serum kept at 37° for One Hour, then Centrifuged. To the Sediments 0.5 cc. Guinea-pig Serum. (B) Blood -1- Inactive Dog Serum +0.5 cc. Guinea-pig Serum Mixed Simultaneously. 1. 1.0 2. 0.5 3. 0.35 4. 0.25 5. 0.15 6. complete ( ( almost complete * The amount of blood used in our experiments is always 1 cc. of a 5% suspension in .85% salt solution. be effected. Hence the amboceptor must have been bound by the blood- cells. How then, through this previous binding, had the amboceptor lost its power of being activated? After excluding all other possible explanations we were forced to conclude that the phenomenon observed is due to a blocking of the complementophile groups of the dog serum's amboceptor by the complementoids still present in the inactive serum. The correctness of this view has to our minds been confirmed: 1. By the isolated binding of the amboceptor at 0° C. 2. By the subsequent blocking of the amboceptor bound at 0° C, by means of free complementoids. 3. By the behavior of dog serum inactivated by shaking with yeast. 4. By the combining experiment with inactive dog serum (inac- tivated by heat) when the salt content of the fluids was increased. We shall take these up in order. 1. If we repeated the combining experiment above mentioned, modifying it, however, so that the amboceptor was anchored by the blood-cells, not at 37° C, but at 0° C, we could show that the guinea- pig blood-cells, treated in this way at 0° C, were all activated by guinea- pig serum. (See Table II.) Now we know that at 0°, as a rule, only the amboceptor is bound by the blood-cells, and that the complement for the most part is uninfluenced. It is, therefore, perhaps quite natural in those cases in which the complementoids, like the complements, are bound by 212 COLLECTED STUDIES IN IMMUNITY. TABLE II. Guinea-pig Blood. Inactive Dog . Serum. cc. Amount of Solution of ttie Sediments on the Addition of 0.4 co. Guinea- pig Serum, after Previously liaving been Treated. (A) At 0°. (B) At 37°. 1. 1.0 2. 0.5 3. 0.35 4. 0.25 5. 0.15 6. complete ti It almost complete the amboceptors, that this binding will not take place if the experi- ment is performed at 0° C. These considerations confirm our view that the impossibility of activating the blood-cells sensitized at 37° C. is due to a blocking of the complementophile amboceptor groups of the dog serum by the complementoids of the same serum. 2. It still remained to show that the substance which prevented the binding subsequent to the binding effected at 0° C, was really present in the fluid medium. This could easily be shown in the following manner. Two parallel series of tubes with guinea-pig blood were treated at 0° C. for one and one-half hours with inactive dog serum (i.e., containing amboceptor + complementoid) . The tubes of series A were then centrifuged and the sediments, freed from fluid, suspended in physiological salt solution; the tubes of series B were left unchanged. All the tubes were now placed into the incubator for one hour, then centrifuged, and the sediments mixed with active guinea-pig serum and physiological salt solution. In the tubes of series A solution ensued, the blood-cells of series B remained undis- solved, as can be seen from Table III. The substance which caused the blocking of the ambocepters was therefore contained in the fluid portion of the blood sensitized at 0°; for in series A, in which the fluid medium was decanted, the blood- cells although subsequently kept at 37° C, could still be activated. In series B, on the contrary, the complementoids still remaining free at 0° C, were bound when subsequently kept in the thermostat, and so prevented the "completion" with active serum. From all this it follows that we can be dealing only with complementoid action in the test-tube, and the correctness of this view is confirmed in another way. THE MECHANISM OF THE ACTION OF AMBOCEPTORS. 213 T.\BLE III. Inactive Dog Serum. cc. Amount of Solution of Guinea-pig Blood on the Addition of 0.4 cc. Guinea-pig Serum. Series A. Series B. 1. 1.0 2. 0.5 3. 0.35 4. 0.25 5. 0.15 6. complete strong moderate I c 3. We know from the studies of v. Dungerni and Erhlich and Sachs 2 that yeast constitutes an excellent means of removing the complements of a serum. If we prepared an inactive dog serum by treatment with yeast instead of with heat, or if we allowed the com- plementoids of a serum inactivated by heat to be absorbed by yeast, it was found that a dog serum so treated was no longer capable of causing this "blocking" phenomenon. Haemolysis occurred in like manner whether we added the activating guinea-pig serum at once, or first kept the blood-cell — dog-serum mixture in the thermostat for an hour. (See Table IV.) TABLE IV. Dog Serum, 1.0 0.5 0.35 0.25 0.15 Amount of Solution of Guinea-pig Blood on the Addition of 0.4 cc. Guinea-pig Serum after Remaining at 37*" C. for One Hour, Dog Serum Inactivated. (A) By Shaking with Yeast.* complete almost complete strong „ (B) , -By Heating.- (a) Shaken with Yeast.* complete almost complete strong (6) Employed Directly. * 6 cc. serum are shaken with 0.2 grams yeast. The complementoids had simply been removed by the yeast and the isolated amboceptors reacted in normal fashion. ' See page 36 et seq ' See page 195 et seq. 214 COLLECTED STUDIES IN IMMUNITY. 4. A further proof of the correctness of our view was furnished by the results of the combining experiment when the molecular con- centration of the fluid medium was increased. As is well known the hsemolytic action of the sera is retarded and even entirely prevented by an increase in the amount of salts present. The investigations of Markl ^ have shown that under these circumstances the amboceptor is bound by the red blood-cells, whereas the complement is unable to take hold.2 Through extensive investigations, not yet published, we have been able to verify this. Under these circumstances, provided the view developed by us is correct, it should naturally be possible to prevent the blocking with complementoids by means of suitable concentrations of salts. Two parallel series of tubes with guinea-pig blood to which inactive dog serum had been added were therefore kept at 37° C. for one hour, ammonium sulphate having first been added to one of the series in the strength of 1.3%. This addition, as special tests showed us, suffices to entirely prevent the hsemolytic action even of large amounts (1 cc.) of active dog serum. The result of the experiment corresponded exactly to our expectations. The sediments of those guinea-pig blood-cells which had been treated with ammonium sulphate could be complemented with guinea-pig serum, whereas in the other series no solution whatever occurred. (See Table V.) The analysis of this case furnishes the first proof by means of test- tube experiments that complementoids, the inactive modifications of the complements, actually exist in the inactive serum. To be suje, even heretofore their existence could not appear doubtful, for, in our opinion, through the possibility of producing antibodies, proof had been furnished of the preservation of the complement's haptophore group in the inactivated serum.^ ' Markl, Uber Hemmung der Hamolyse durch Salze. Zeitsehr. f. Hygiene, Vol. 39, 1902. 'These conditions by the way, in our judgment, have no connection with the osmotic conditions of the cell membrane, as Markl believes. It seems to us that the action of the salts is most readily explained by assuming that the increased concentration hinders the chemical union of amboceptor and com- plement. That the salts can exert such an antireactive action is seen by the fact pointed out by Knorr (Munch and Wochensch. 1898, Nos. 11 and 12) that tetanus antitoxin and toxin are absolutely prevented from combining by the addition of 10% NaCl. " In view of this new confirmation I should not want to deprive the reader of an exposition of the complementoid theory from the standpoint of an opponent THE MECHANISM OF THE ACTION OF AMBOCEPTORS. 215 TABLE V. Inactive Dog Serum. CO. Amount ot Solution of tlie Gumea-pig Blood Sediments (Centrifuged, after the Mixtures had been kept for One Hour at 37°) on the Addition of 0.5 cc. Guinea-pig Serum, the Mixtures having been Previously Treated with (A) 0.15co.20%(NH,)2S04 (B) 0.15 cc. 0.85% NaCl. 1. 1.0 2. 0.5 3. 0.35 4. 0.25 complete moderate little trace 1 [ ° In contrast to the usual behavior we must assume that in the case described the affinity of the complement has not suffered any con- siderable decrease through the formation of complementoid. This is supported also by an experiment which we made in order to deter- mine the lowest temperature at which the anchoring of the com- (Proctocoll der k.k. Gesellschaft der Aerzte in Wien, Wiener klin. Wochenschrift, 1901, No. 51): "If an animal is injected with inactive serum of the same foreign species instead of active serum, it is found that its serum likewise becomes charged with anticomplement ; proof that the alexin also — like everything else in this world — contains a haptophore group and an active group, the latter this time termed zymotoxic. As a result of the inactivation the zymotoxic group is destroyed; the haptophore group remains intact. Hence a continuance of the assimilation of complementoid and the production of the anticomplement. So far, so good. Now, however, we come to a questionable point. If the complement deprived of its zymotoxic group still possesses its haptophore group, it must still be able to satisfy and bind its amboceptor. How then does it happen that an inactivated antiserum again becomes lytic on the addi- tion of suitable complement (active normal serum), a phenomenon which, according to Ehrlioh (despite Dr. Wechsberg), is due to the formation of lysin from amboceptor and complement. If the haptophore group of the amboceptor has already been bound by the remains of the old complement, the 'comple- mentoid,' it surely is unable to bind new complement. Hence by heating (inactivating) the serum the haptophore group of the complement cannot have remained unchanged; it must have completely lost its affinity for the amboceptor. Now, gentlemen, I should like to know what is left of the com- plement after this heating? The zymotoxic group is destroyed, the haptophore group so changed that it is not recognizable. Nothing remains of the comple- ment except Ehrlich's fervent wish that a little of it might be left, because other- wise it would not harmonize with the theory! It is this wish that floats around in the inactive serum under the name of complementoid." Thus far Gruber! I shall refrain from any personal remarks for which the 216 COLLECTED STUDIES IN IMMUNITY. plementoid still takes place. In this way we sought to find an approx- imate criterion for relative affinity of the complementoid. From the power to be reactivated possessed by the guinea-pig blood-cells previously treated at different temperatures with inactive dog serum it was seen that even at 3° C. a moderate binding of complementoids takes place, and that complete blocking phenomena can already be obtained at 8° C, as is seen in the following experiment: TABLE VI. Inactive Amount of Solution of Guinea-pig Blood on the Addition of 0.5 cc. Guinea-pig,Serum after Preliminary Treatment at CO. (A) 0°C. (B) 3" C. (C) 6° c. (D) 8°C. 1. 1.0 2. 0.75 3. 0.5 4. 0.35 complete almost complete strong moderate moderate It it little ■ faint trace Nevertheless we believe that even in this case a certain, though slight, decrease in the affinity has occurred in the complementoid formation. At least the fact speaks in favor of this, that with the simxiltaneous addition of inactive dog serum (i.e., amboceptor -|- com- plementoid) and active guinea-pig serum solution of the guinea-pig blood occurs. Under these circumstances, in which the amboceptor has both complement and complementoid to choose from, the former is preferred. When, then, we find that it is possible by previous treat- ment with complementoid to block the complementophile group of the amboceptor for the complement subsequently added, we shall explain this most readily by assuming that after the complementoid has been anchored, the union becomes firmer. Analogous phenomena are common in immunity. Thus Donitz ^ has shown that the union unusual tone of this attack surely offers sufficient provocation, merely expressing my astonishment at the fact that Gruber's exposition disregards the most important and explanatory point, namely, as Morgenroth and I have emphasized, that in the change into complementoids, the complements must usually suffer a decrease in their affinity, for only in this way can the absence of all disturbing inter- ference on the part of these complementoids in test-tube experiments be explained. If, however, Gruber assumes a complete destruction of the complements by inactivation how does he explain the fact easily verified by every one, namely, the production of anticomplements by injection into the organism of serum which has been heated? Surely a mere wish floating around in the serum cannot suffice to produce anticomplements. — ^Ehrlich. ' Donitz, tjber die Grenzen der Wirksamkeit des Diphtherieheilserums. Arch internat. de Pharmacodynam., Vol. V, 1899. THE MECHANISM OF THE ACTION OF AMBOCEPTORS. 217" of diphtheria poison in the animal body, at first a loose one, soon becomes more and more firm so that it cannot be broken up even by very large amounts of antitoxin. Madsen's ^ experiments, to liberate, by means of antitoxin, tetanolysin which had been anchored by the blood-cells, also confirm this. Blocking by means of, complementoids is also of value for the- technique of demonstrating the presence of amboceptor. Suppose, for example, that in doubtful cases one seeks to show the existence of the amboceptors in the usual manner, by sensitizing the red blood- cells and subsequently complementing with a different kind of serum. In this case, owing to the blocking action of the complementoids, an absence of the amboceptors could be simulated. In this connec- tion it is of considerable interest to know that so capable an investi- gator as Buchner^ employed the above method for analyzing the haemolysin in just the case here described. His attempts to demon- strate the arnboceptor by this method (inapplicable in this particular instance), as well as by means of the amboceptor's thermolability ^■ (already discussed), were unsuccessful. II. Amboceptor or Sensitizer? In another case we have met with a different complication- equally fatal to the successful demonstration of the amboceptor by routine procedures. This is of especial interest for the theory of hsemolysin action, and concerns the hsemolytic property of ox serum for guinea-pig blood. If the ox serum is inactivated, this property can readily be restored by the addition of active horse serum. If, however, one tries by means of active horse serum to complement blood-cell sediments (obtained by centrifuging guinea-pig blood after ' Madsen, Uber Heilversuche im Reagensglas. Zeitschr. f. Hygiene, Vol. 32,, 1899. ' H. Buchner, Sind die Alexine einfache oder complexe Korper? Berl. klin. Wochenschr. 1891, No. 33. ' According to the newer researches already mentioned it would be con- ceivable that the thermolabUity of the amboceptors is simulated by this, — that the complementoids, in themselves possessing a relatively high affinity, become firmly anchored to the amboceptors. However, as special experiments have shown us, such is not the case, for dog serum which has been inactivated by shaking with yeast, and which therefore contains no complementoid, likewise loses its ability to be activated when it is heated to 60° C. It does not lose. this power when heated only to 50°-51° C. 218 COLLECTED STUDIES IN IMMUNITY. these have been treated at 37° C. for one hour with inactive ox serum), it will be found, just as in the previous case, that hsemolysis does not occur. The reason for the non-activatibility in this case differs essen- tially from that in the case previously described. The chief differ- ence manifests itself in the behavior of the decanted fluid medium. If the centrifuging is omitted, and active horse serum is added to the sensitized blood-cells without previously removing the fluid medium, it will be found that solution occurs. If the centrifuging is not omitted, it will be seen that the decanted fluids behave in an analogous manner, for when mixed with active horse serum they will dissolve native guinea-pig blood. A complete experiment is reproduced in Table VII. TABLE VII. Amount of Solution of Guinea-pig Blood on the Addition of 0.5 cc. Horse Serum to: Inactive Ox Serum. (A) The Sediments on Centrifuging after the Mixture had been kept at 37° C. for One Hour. (B) The Decanted Fluids from (A) Added to Native Guinea-pig Blood. (C) The Uncentrifuged Mixture of Blood and Ox Serum. cc. (a) After Remaining at 37° C. for One Hour. (6) Immediately. 1. 0.5 2. 0.35 3. 0.25 4. 0.15 5. 0.1 6. faint trace complete It tt strong moderate complete ii almost complete strong complete << tl almost complete strong In contrast, therefore, to the behavior in the first case described by us, the amboceptor has remained in the decanted fluid, and has therefore not been bound by the blood-cells, or only to a very slight degree. Our attempts by means of horse serum to activate the guinea-pig blood-cells which had previously been treated at 0° C. with inactive ox serum and then centrifuged, failed as a matter of course; and the result was the same when the ox serum had been freed of complementoid by shaking with yeast. This peculiar behavior, namely, that the amboceptor by itself does not unite with the cell at all, and acts only after it has com- Tjined with the complement, is of special significance for the method THE MECHANISM OF THE ACTION OF AMBOCEPTORS. 219 of analyzing hsemolysins. For, entirely aside from the fact that under these circumstances the attempt to activate the centrifuged, and presumably "sensitized," blood-cells necessarily fails, it will be seen that the occurrence of this complication considerably limits the application of the second method employed to discover the com- plex nature of hsemolysins, namely, separation in the cold, a method already markedly restricted. This method, it will be recalled, depends upon the fact that at 0° C. usually only the amboceptors are bound to the blood-cells, not the complements to the amboceptors. In the case just described, however, the union of amboceptor and cell depend on the combining of amboceptor and complement. How, then, can a separation of the two components be effected if, on the one hand, the conditio sine qua non for the union of amboceptor and cell, a condition which obtains here, cannot be fulfilled at low temperature, and if, on the other, it in itself precludes any sepa- ration whatever? No wonder, therefore, that Gruber i failed with the cold separation method in just this case (guinea-pig blood + active ox serum). The two atypical cases here described are, however, peculiarly adapted to throw light on the mechanism of hsemolysin action. In the first case the fact that blood-cells " sensitized " in the usual manner withstand the action of the complement is hard to explain in accordance with Bordet's view. But the behavior shown in the second case becomes entirely inexplicable if, like Bordet, we believe the action of hsemolysins to consist in this, that the amboceptors (substance sensibilatrice) sensitize the blood-cells and so render them vulnerable to the action of the complements (Bordet's alexins) exerted directly upon them. For here we have demonstrated that a sensitization does not take place; the amboceptor by itself is not at all bound, and becomes effective only on the addition of comple- ment. If, however, we were to assume that in our case the com- plement nevertheless attacks the cell directly so that then the ambo- ceptor can be found, we should arrive at a theory as unlike Bor- det's as that held by Ehrlich and Morgenroth. But such a theory, strange to say, would apply only to this and perhaps a few other cases, that is, only to a few exceptions. Although superfluous, a suitable experiment was also made in this case and, as might have • Gruber, Zur Theorie der Antikorper. Munch, med. Woohenschr. 1901, No. 49. See also H. Sachs, 1. c. 220 COLLECTED STUDIES IN IMMUNITY. been expected, it was found that the complement as such was not bound by the cell. The facts, however, are very readily explained if, following Ehrlich and Morgenroth, we regard the amboceptor as a coupler possessing two haptophore groups. Owing to a mutual combination this trans- mits the action of the complement to the cell. In the case just described, it follows at once that the cytophile group of the ambo- ceptor possesses a very slight affinity to the cell receptor. We have therefore only to assume that, in contrast to the usual behavior, the amboceptor in this case, while itself unable to combine with the cell, by combining with the complement takes on increased affinity and so becomes capable of action. The significance of the variations in affinity will be discussed connectedly at a subsequent time. We shall content ourselves here by pointing out that an understanding of the phenomena of immunity is impossible without the assumption that certain hapto- phore groups become increased or decreased in their chemical energy, owing to changes in the total molecule. Chemically, such an assump- tion is a matter of course. We believe that the observations described above constitute additional proof that amboceptor and comple- ment combine with each other. In the main this question has already been decided by the beau- tiful investigations of M. Neisser and Wechsberg i on the deflection of complement by an excess of amboceptor. The objections raised against these experiments by Gruber^ and by Metchnikoff^ have been completely met by the recent investigations of Lipstein.* The case last described by us is to a certain extent an experi- mentum crucis for the correctness of the views formulated by Ehrlich and Morgenroth for the mechanism of hsemolysin action. We there- fore believe that Bordet's sensitization theory has become unten- able, and that now this question, just as that concerning the plurality of complements, is definitely closed. Subsequent Addition. — According to recent investigations of Dr. Sachs, guinea-pig blood-cells, which, because of treatment with inactive dog serum, can no longer be dissolved by guinea-pig serum, owing to blocking by com- ' See page 120 et seq. ' Gruber, Protocol! der k.k. Gesellschaft der Aerzte in Wien, Wiener klin. Wochenschr. 1901, No. 50. ' Metchnikoff, l'Immunit6 dans les malad. infect., page 313, Paris, 1901. * See page 132 et seq. THE MECHANISM OF THE ACTION OF AMBOCEPTORS. 221 plementoid, are still dissolved by the complements of dog serum. The source of the dog serum complement was the fluid decanted from guinea-pig blood- cells which, by treatment with active dog serum at 0° C, had abstracted as much of the amboceptor from the latter as possible. These experiments there- fore show: 1. That the complement of dog serum suffers a diminution of affinity in changing to complementoid. 2. That the complement present in guinea-pig serum possesses a weaker affinity than the complement of dog serum with analogous action. XX. DIFFERENTIATING COMPLEjMENTS BY MEANS OF A PARTIAL ANTICOMPLEMENT.i By H. T. Marshall, Fellow of the Rockefeller Institute, and Dr. J. Mohgen- ROTH, Member of the Frankfurt Institute. The question whether the serum of one and the same species contains a plurality of complements or only a single one seems to us to have been positively decided in favor of the pluralistic concep- tion. This decision has been brought about mainly by the observa- tions of Ehrlich and Morgenroth,^ of Wassermann,^ Wechsberg* Wendelstadt,^ and by the recently published studies of Ehrlich and Sachs.^ Nevertheless, we shall briefly describe an experiment which, in a single instance at least, constitutes a proof for the plurality of the complements. Our object in doing this is not that the number of arguments may be further increased, for they are already amply sufficient, but that we may call attention to a method of demonstra- tion which has not heretofore been employed. Because of purely technical difficulties the most rational and simplest method of differentiation, namely, by means of anticomple- ments, has not thus far been employed in this question. As is well known, it is very easy by immunizing with serum containing com- plement or complementoid to obtain potent anticomplements. Such anticomplement sera, however, usually contain anticomplements cor- responding to the sum of all the complements originally injected,' and are, therefore, not adapted to the separation of complements. 1 Reprinted from the Centralblatt f. Bact. Original Vol. XXXI, No. 12, 1902. 2 See pages 11, 56, 86. 'Wassermann, Zeitschr. f. Hyg., Vol. XXXVII, 1901. * Wechsberg, Sitzung d. k. k. Ges. d. Aerzte in Wien, Wiener klin. Wochen- schr. 1901, No. 48, ' Wendelstadt, Centralblatt f. Bact. Part I, Vol. XXXI, No. 10. ' See page 195 et seq. ' See page 63 et seq. 222 DIFFERENTIATING COMPLEMENTS. 223 At least this had been the case thus far, for a partial anticomplement, one acting only against a single complement, had not been observed. Through the courtesy of Dr. Cnyrim we detained a normal anti- complement which possessed the desired properties, and we there- fore gladly availed ourselves of this favorable opportunity to demon- strate, by means of the elective binding of anticomplement, the difference between two complements in one and the same serum, a difference that .had not heretofore been demonstrated.^ This normal anticomplement was an ascitic fluid derived from a case of cirrhosis of the liver; it exerted a marked antihaemolytic action in one particular case. By means of an experiment we first determined that this action was due to the presence of an anticomple- ment and not of an anti-immune body. This showed us that the ascitic fluid exerted practically no influence on the anchoring of the immune body in question to the red blood-cells. The serum whose complements we examined was guinea-pig serum, which activated two amboceptors obtained by immunization. These amboceptors were contained in the inactive serum of a rabbit, A, which had been immunized with ox blood; and in the inactive serum of a goat, B, which had been immunized with sheep blood. Corresponding to this, ox blood was used for case A, and sheep blood for case B. The inactive ascitic fluid does not dissolve these species of blood even after the addition of guinea-pig serum To begin, we saturated ox blood-cells with the specific amboceptor by adding 0.01 cc. immune body A to each 1 cc. of a 5% suspension of the cells. This is about ten times the amount which on the addi- tion of sufficient complement (0.1 cc. guinea-pig serum) effected complete solution. The mixture was placed in the incubator and frequently shaken. At the end of one hour it was centrifuged, the fluid poured off, and the blood-cells, loaded with amboceptor, sus- pended in salt solution. In exactly the same manner sheep blood- cells were treated with the inactive serum B, 0.2 cc. for each 1 cc. of the 5% Suspension. On the addition of guinea-pig serum to these blood-cells, haemolysis ensued very quickly in the thermostat; in both cases it required 0.008 cc. guinea-pig serum to fully dissolve 1 cc. of the suspension, while 0.0065 cc. caused incomplete solution and 0.002 ' In the following, for the sake of simplicitj-, we shall speak only of two complements, whereas we wish here to remark that two groups of complements are probably to be understood, each group made up of a host of single comple- ments which it is impossible thus far to analyze. 224 COLLECTED STUDIES IN IMMUNITY. only a slight degree of solution. For the sake of clearness it was especially fortunate that the complementing amounts should happen to be identical in the two cases. A parallel series of experiments was then undertaken with these two cases, as follows: Varying amounts of the guinea-pig serum were mixed each with 0.4 cc. of ascitic fluid inactivated at 56° C, and the mixtures kept at room temperature for half an hour, after which the binding was entirely completed.^ Thereupon the blood-cells -loaded with amboceptor were added. The result of these experiments js shown in the following table: Case A (Ox Blood + Amboceptor). Guinea-pig Serum Alone. Guinea-pig Serum +0.4 cc. Ascitic Fluid. . 008 complete solution 0.0066 vestige 0.005 strong 0.0055 considerable 0.003 0.0025 moderate . 1 almost complete 0.08 " ^' . 065 considerable 0.05 fairly little 0.035 very little 0.03 trace 0.025 " 0.02 Case B (Sheep Blood -|-Amboceptob). Guinea-pig Serum Alone. Guinea-pig Serum + 0.4 cc. Ascitic Fluid. 0.008 complete solution 0.0065 almost complete 0.005 0.0035 strong 0.008 complete .0065 almost complete 0.005 " ^' 0.0035 strong We see, therefore, that in case A the complement protects com- pletely against 2^ times the complete solvent amount of complement, Tvhile the amount of serum required to effect complete solution increases more than twelve times. In case B, on the contrary, the complete solvent dose of guinea-pig serum remains unchanged, and the series proceeds just as though there had been no addition of anticomple- ment. These experiments, which were repeated many times, therefore ' The union of complements and anticomplements, analogous to the behavior of certain toxins and antitoxins, is dependent on the time. Hence here also this had to be considered and sufficient time allowed for the mixture to aot DIFFERENTIATING COilPLEMENTS. 225 show that the ascitic fluid contains an anticomplement ^ which fits into that complement which is activated by amboceptor A, whereas anticomplements for the complement of amboceptor B are absent. Hence we are justified in differentiating in guinea-pig serum at least two complements with different haptophore groups. It may be hoped that continued investigations of normal body fluids will bring to light numeroiis other favorable cases which wiU make possible differences along the lines indicated. For although in normal serum the complication of haptins present, such as ambo- ceptors, complements, complementoids, antiamboceptors, and anti- complements, is very great, the conditions here are certainly simpler than in the serum of immimized animals; for in the latter there are also present innumerable primary, and (owing to internal regulative processes) secondary reactive products. ' Erhlich and Moigenroth have discussed the nature of anticomplements at length in the BerL klin. Wochenschr. 1901, No. 10. They conclude that the origin of these bodies is this, that foreign complements combine with the complementophile group of certain cell receptors. According to this view the anticomplements are nothing else than thrust-off ambocepters whose com- plementophile groups possess a higher a£Snity than is usually the case. It is curious, therefore, that Gruber, nine months later (Sitzg. der ki. Ges. der Aerzte in Wien, Wiener klin. Wochenschr. 1901), presents this view, which had been recognized as a natural consequence of the receptor theory, as an entirely new objection against just this theory. XXI. CONCERNING THE COMPLEMENTOPHILE GROUPS OF THE AJMBOCEPTORS.i By Prof. Dr. P. Ehblich and H. T. Maeshall, M.D., Fellow of the Rockefeller Institute of Medical Research. The studies of the past year, especially the recent conclusive work of Ehrlich and Sachs ,2 show that we may regard it as definitely proven that, in contrast to the unitarian conception of Bordet, there is a plurality of complements in the serum. This knowledge largely supplements our views concerning the mechanism of lysin action, and is in complete harmony with the principles of the amboceptor theory. The latter, in contrast to the untenable sensitization theory of Bordet, has become still more firmly established through the recent experiments carried out in the Institute by M. Neisser and Wechsberg,^ Lipstein,^ and Ehrlich and Sachs. ^ If we consider that, as is shown especially by Bordet's experi- ments,^ an amboceptor, after having been anchored by cellular ele- ments, can almost completely rob a serum of its complement, and if, further, we regard what we now know about the plurality of comple- ments, we shall of necessity be led to a view concerning amboceptors according to which an amboceptor is capable of binding a number of different complements simultaneously. Attention was called to such a possibility by Ehrlich and Morgenroth '' when they stated: "Finally, it is possible that an immune body, besides one particular cytophile ' Reprint from the Berl. klin. Wochenschr. 1902, No. 25. 2 See page 195. ' See page 120. ' See page 132. ' See page 209. ' Bordet, Annal. de I'Institut Pasteur, May 1901. ' See pages 88 et seq. 226 COMPLEMENTOPHILE GROUPS OF THE AMBOCEPTORS. 227 group, contains two, three, or more complementophile groups." According to this latter view, therefore, it is to be assumed that an amboceptor possesses one haptophore group specifically related to a certain receptor of cell or of a foodstuff, and that it also possesses a number of complementophile groups. The term amboceptor would thus indicate that two different substances, foodstuff and comple- ment, are anchored by this body and brought into close relation with each other. This is illustrated in the following diagram. Fia. 1. (a) receptor of the cell; (t) haptophore group of the amboceptor; (c) domi- nant complement; (d) non-dominant complement. Complementophile groups of the amboceptor: (a) for the dominant com- plement; (|8) for the non-dominant. The next question to be considered is whether it is necessary, in order to get the specific lysin effect, for all these complements to come into action. Recent experiments indicate that this is not the case but that among the number of complements only a few are necessary in any single instance in order to obtain the effect. These comple- ments are termed "dominant complements," the rest are " nonndominant complements." 228 COLLECTED STUDIES IN IMMUNITY. A case described by Ehrlich and Sachs makes this clear, and we shall therefore briefly reproduce it here; ^ Two amboceptors are concerned, namely, the normal ambocep- tor of goat serum for rabbit blood, and an amboceptor obtained bj' immunizing goats, which is anchored by ox blood-cells. We shall for the sake of simplicity designate these amboceptors as A and B. Naturally both these amboceptors are activated by goat serum, in which we shall have to assume at least two complements x and h. For immune body A, x is the dominant complement; for £ it is 6. If in one of the two combinations, for example, in that of rabbit blood-cells loaded with immune body A, the serum is allowed to act long enough, both complements will be bound; that is, dominant and non-dominant. The result, however, is entirely different if the action be made as short as possible. In this case the fluid obtained on centrifuging the blood-cells still contains the dominant comple- ment X, while it has for the most part lost the non-dominant com- plement h. We observe the surprising result that the immune body A with which the blood-cells are loaded combines with the non- dominant complement before it combines with its own dominant complement. In this case, therefore, amboceptor A's complementophile groups which combine with the complement must possess a higher affinity for the non-dominant complement h than for the dominant comple- ment X. Here then the binding of the non-dominant complement is independent of the binding of the dominant complement. Such a behavior, of course, is not a general rule; it was not long before a case was found in which the contrary was true, i.e., in which the non-dominant complement does not combine until after the dominant complement has been bound. The demonstration of this relation succeeded only because in a certain human ascitic fluid an anticomplement was present which acted only against part of the complements of a serum. The peculiar behavior of this anticomplement has been described in a recent com- munication by Marshall and Morgenroth,^ and is also readily seen in the following experiment. The complements here concerned are ' For the sake of clearness the case has here been somewhat simplified. The details of this experiment are found in Ehrlich and Sachs, page 195. ' See page 222. COMPLEMENTOPHILE GROUPS OF THE AMBOCEPTORS. 229 present in normal guinea-pig serum. This serum reactivates two immune bodies, of which one, immune body A, was obtained by treat- ing rabbits with ox blood, and the other, immune body B, by treat- ing a goat with sheep blood. These immune bodies, naturally, acted respectively on ox blood-cells and sheep blood-cells. This anti- comjilement is strongly active in case A, while it is entirely without effect in case B. From this we may conclude that the complements concerned in these two cases, and which we may designate as x and 6, are unlike. A further question was whether immune body A binds other com- plements in guinea-pig serum besides its own dominant complement. In order to determine this the following experiment was made : First, ox blood-cells and sheep blood-cells were saturated with their re- spective amboceptors A and B, and then to each cubic centimeter of the 5% blood suspension varying amounts of guinea-pig serum were added as complement. In the first case 0.0075 cc. guinea-pig serum sufficed to cause complete solution; in the second case 0.005 cc. was required. Thereupon another test was made exactly like the preceding with ox blood and immune body A. After the mixture had remained in the thermostat at 37° C. for IJ hours and haemolysis was practically completed, the same quantity of ox blood-cells laden with immune body (0.05 cc. ox blood freed from serum and made up to the original volume) was added anew and the mixtures kept in the thermostat for two hours longer. The haemolysis which had then taken place, observed by allowing the mixture to sediment in a refrigerator, indi- cated the amount of complement x left after the first haemolysis and available for the case A. At the same time a similar experiment was made in which, after the first haemolysis, sheep blood-cells saturated with ambo- ceptor were used in the place of the ox blood-cells. In this case, after determining the amount of complement originally present, that of complement 6, left after the first haemolysis, could also be found. In this a considerable loss of complement is observed for both cases; for it now requires 0.075 cc. of the complementing guinea- pig serum to cause complete solution for case A and 0.025 cc. for case B, so that 1/10 and 1/5 respectively of the original complement are still preserved. This shows that the binding of complement a, dominant for case A, is accompanied by a binding of complement j9, 230 COLLECTED STUDIES IN IMMUNITY. dominant for case B but non-dominant for case A. It was next necessary to determine whether or not in case A the absorption of the non-dominant complement ^ is dependent on the binding of the dominant complement a. Owing to the peculiar nature of the anti- complement it is possible to prevent the binding of complement a for case A, whereas the binding of complement /? for case B is not affected. On the addition of 0.4 cc. of the anticomplement serum the amount of complement necessary for complete solution increases from 0.0075 cc. to 0.2 cc, i.e. 26 times, whereas no change occurs for case B, 0.005 of the guinea-pig serum still causing com- plete solution. If, therefore, the binding of the complement ^ by ox blood-cells laden with amboceptor A is dependent on the binding of the domi- nant complement a, it must be possible by the addition of the fluid containing the anticomplement to prevent this binding. The ex- periment is made as follows: First, 0.4 cc. anticomplement serum is mixed with varying amounts of guinea-pig serum. After this mixture has remained at room temperature for half an hour the ox blood-cells laden with amboceptor are added and the whole kept in the thermostat for IJ hours, when the undissolved blood-cells are centrifuged off. The decanted fluid is mixed sheep blood-cells loaded with their ambo- ceptor. The result shows that in this case a decrease of complement h for B has not occurred, for the tube containing 0.005 guinea-pig serum shows complete solution. The following table will make the results plain: Complete Solvent Amounts of Guinea-pig Serum. I. II. III. IV. Absolute Deter- After Bmding the After Binding the Amount of Com- mination of ttie Complement by- Complement by plement Used by Complement. Means of Ambo- Means of 0.4 cc. Amboceptor -H ceptor -t Blood- Anticomplement Blood-cells (Case cells (Case A). A ) after Binding of the dominant Complement by Means of 0.4 cc. Anticomplement Case A 0.0075 0.075 0.2 Case B 0.005 0.025 0.005 0.005 By means of this experiment, therefore, it has been proved that in this case binding of the non-dominant com,plement ensues only after the corresponding complementophile group of immune body A has an- COMPLEMENTOPHILE GROUPS OF THE AMBOCEPTORS. 231 chored the dominant complement a. We shall probably not be wrong if we assume that in this case, owing to the occupation of the com- plementophile group for a, there is an increase in affinity of the com- plementophile group for /?. The subject of hsemolysins contains many analogies for such a behavior. Thus it is quite common that not until the haptophore group of an amboceptor is bound to a cell does the complementophUe group of the same possess sufficient affinity to anchor the complement. Such an arrangement, whereby a single amboceptor is able to blind a number of different complements, is certainly not useless. Owing to their Z3'motoxic groups the complements can manifestly exercise quite different actions, so that the digestion of highly com- plex food molecules — in which, of course, we must see the physiological function of the amboceptor mechanism — is surely made easier. Such an arrangement seems still more adapted to the purpose when we consider that the cytophilic haptophore group of an amboceptor is fitted, not to the entire food molecule as such, but only to a partial group of the food molecule. The possibility is thus given for a par- ticular amboceptor to anchor foodstuffs, which are almost entirely different but happen to agree in the possession of this one partial group. Granted that this is the case, the presence of only a single complement, acting only in one or the other possibility, would be dysteleological, whereas a plurality of complements would insure the greatest possible effect on the most varied foodstuff molecules. Re- cent investigations have brought to light a great many examples which show that in extracellular and intracellular digestive processes ■various ferments act together or in sequence. Thus, as Hofmeister ^ states, we already know of ten different ferments in the liver-cell: "A maltase, a glycase, a proteolytic ferment, a nuclein-splitting ferment, an aldehydase, a lactase, a ferment which converts the firmly bound nitrogen of amido acids into ammonia, a fibrin ferment, and, with some probability, a lipase and a rennin-like ferment." Even in so simple an organism as the yeast-cell, according to Delbriick.^ at least five endoferments are demonstrable. If one cares, one can regard an amboceptor whose various comple- mentophUe groups are occupied by different complements as a kind ' Hofmeister, Die chemische Organisation der Zelle. Vortrag. Braunschweig, 1901. ^Delbriiek, Jahrbuch des Vereins der Spiritusfabrikanten, Vol. II, 1902. 232 COLLECTED STUDIES IN IMMUNITY. of polyenzyme. Analogous views have been expressed by Nencki^ for the ferments of the digestive tract. Even though his conception, that pepsin is a single ferment with different active groups (pepsin group, rennin group, plas tin-forming group), does not entirely apply, we must say that his conception of such polyenzymes is fully justi- fied. The properties of the amboceptor above demonstrated will, we believe, speak in favor of the essential soundness of the view of this eminent chemist. ' Nencki and Sieber, Zeitschr. f. physiol. Chem. 1901. XXII. CONCERNING THE COMPLEMENTIBILITY OF THE AMBOCEPTORS.' By Dr. J. Mobgenhoth, Member of the Institute, and Dr. H. Sachs, Assistant at the Institute. I. A Presumptive Law Concerning the Complementibility of Normal Amboceptors and those Obtained by Immunization. Gruber2 believes he has discovered an essential difference in the complementibility of the normal amboceptors of blood serum and those produced by immunization. He says: "The amboceptors* of the normal sera never seem to make the erythrocytes of another species sensitive to their own serum, . . . and I think I can say before- hand that the specific amboceptors regularly make the erythrocytes soluble in their own serum. This would constitute an essential difference between the two." If Gruber believes Ehrlich has ever maintained that the ambo- ceptors of normal and of immune sera are identical, this is a mis- understanding. On the contrary, the studies at this Institute ^ have emphasized that the immune sera, owing to the manifold variety of the reaction products developed in the immunization, contain a great host of different partial amboceptors whose cytophile and complementophile groups can vary greatly. Normal serum, on the contrary, possesses only few types of amboceptors identical with those of the immune serum. Hence if there is to be any question at all as to the identity of normal and artificially produced amboceptors, this can only be a partial identity. Special proof by Gruber of their non-identity in order to controvert the opposite view was therefore unnecessary. However, since what Gruber advances is incorrect and in ' Reprint from the Berl. klin. Wochenschr. 1902, No. 27. ' Gruber, Miinch. med. Wochenschr. 1901, No. 49. ' Gruber terms this " preparator." * See especially pages 88 et seq. 233 234 COLLECTED STUDIES IN IMMUNITY. contradiction to our experimental results, let us examine his evidence somewhat more closely. In support of his first assertion, that "the amboceptor of the normal sera seems never to make the erythrocytes of another species sensitive to their own serum," he advances the following eight com- binations (see Table I): TABLE I. Number. Species of Blood, and Complement. Amboceptor. 1 2 3 4 5 6 7 8 rabbit guinea-pig rabbit guinea-pig 1 1 I ( rabbit OX C( dog sheep rabbit chicken sheep It seems entirely to have escaped Gruber that only a few lines previously he denies the existence of amboceptor in the first three of these combinations. Hence he should not have included these as evidences of the amboceptor's non-activatibility, for his own experiments had shown him that in these hsemolysins no ambo- ceptors could have been present.^ From our own experiments we know that the next three combinations (4r-6) usually lead to solution of the blood; there remain therefore only two cases (7 and 8) which we may consider as evidence of Gruber 's contention. ^ Against these two cases can be placed a single case described by Gruber, one which he advances to support his second statement, "that the specific amboceptors make the erythrorytes soluble in their own serum." Gruber believes he can say in advance that this is regularly the case. ' Since then, however, amboceptors have also been demonstrated in these cases. (See H. Sachs, page 181.) ' One of these cases deals with the combination guinea-pig blood + chicken serum. From Ehrlich and Morgenroth's earlier communications (see pages 88 et seq.) Gruber could have seen that between animal species so far removed as chicken and guinea-pig the chances of complementibility are not as great as they are between mammalian species. If Gruber therefore employs as evi- dence such distantly related species he must necessarily also have used widely separated species when complementing the inmiune sera. We have no doubt at all that by immunizing distantly related species (birds) with guinea-pig blood, amboceptors can be obtained which are not complemented by guinea- pig serum, or at least not regularly so. COMPLEMENTIBILITY OF THE AMBOCEPTORS. 235 Gruber has prophesied correctly. To one who has familiarized himself with the plurality of the amboceptors it will, to be sure, appear a matter of course that the erythrocytes loaded with specific amboceptors usually find suitable complements which cause their solution, as in most other sera, so also in their own serum. As a matter of fact, according to our own experience, the amboceptors of the immune sera seem as a rule to make the blood-cells sensitive to their own serum. But the far-reaching difference between the im- mune sera and normal sera which Gruber sees in this fact does not exist. In the following table we have collected, either from personal knowledge or from the statements of other authors,^ those cases in which the combinations blood-cells o -I- inactive normal serum (am- boceptor) 6 + complement a lead to hemolysis, in contradiction to their behavior as stated by Gruber. (See Table II.) TABLE II. Number. Species of Blood and of Complement. Amboceptor. 1 guinea-pig dog 2 calf 3 goat rabbit 4 sheep 5 guinea-pig sheep 6 horse 7 { 1 ox 8 rabbit It 9 (I man 10 gumea-pig rabbit This table, which makes no pretense at completeness, shows that the solubility, in their own serum, of blood-cells loaded with normal amboceptor is quite common. This becomes still more evident when we consider that the combinations mentioned include only a limited number of the most common experimental animals, and that by using other species Still more combinations would be found. Gruber's statements therefore are all the more surprising since a large part of the cases here reproduced have already been described in the literature. Just this activatibility of normal amboceptors ' Erhlioh and Morgenroth, page 11 ; Neisser and Doring, Berl. klin. Wochen- schr. 1901, No. 22; H. Buohner, Berl. klin. Wochenschr. 1901, No. 33; H. Sachs, page 181. 236 COLLECTED STUDIES IN LMMUNITY. by means of serum corresponding to the blood-cells employed has very recently been employed by Buchner ^ exclusively as a reaction for the presence of normal amboceptors. Although the principle advanced by Gruber as an invariable means of differentiation has failed, we are far from identifying normal and specific amboceptors. As already stated, we believe that in the sense above described it has been proved that they vary. Here we should like to emphasize that, despite individual multiplicity, all amboceptors belong essentially to a common class of similarly react- ing substances. To us these observations appear of interest also in another direc- tion. Baumgarten^ ascribes the haemolysis in a' foreign serum entirely to the influence of the amboceptors, which he identifies with the agglutinins. He says that "while in themselves incapable of effecting haemolysis, they put the red blood-cells into such a condition that they allow their haemoglobin to escape even on relatively slight osmotic disturbances." Just these slight osmotic disturbances, according to Baumgarten, are caused by the foreign sera whose osmotic tension is changed by heating (inactivation). Hence Baum- garten regards the assumption of complements as entirely unnecessary. In opposition to this we would like to call to mind the numerous combinations described by us (even Bordet has described such for the haemolysins obtained by immunization), in which the blood- cells dissolve in their own serum^ i.e. in the ideal isotonic medium, if they have previously been treated with an inactive serum (ambo- ceptor) of a different species. Such cases clearly indicate that hffi- molysis by means of blood serum has nothing to do with isotonic conditions; that it is rather due to a poisonous action which depends on the coaction of two components — amboceptor and complement. II. Concerning the Variability of the Complements. The plurality of the complements contained in a serum has been proved by the most varied experiments. A separation of the indi- vidual complements of the serum has been undertaken in various sera by means of chemical or thermic influences,^ by binding with ' Buchner, Beri. klin. Wochenschr. 1901, Xo. 33. ' Baumgarten, ibid., No. 50. ' Ehrlich and Morgenroth, see pages 11 et seq.; Ehrlich and Sachs, pages 195 et seq.; Wendelstadt, Centralblatt f. Bact. 1902, Vol. 31, No. 11. COMPLEMENTIBILITY OF THE AMBOCEPTORS. 237 Wood-cells loaded with amboceptors/ by filtration through porous filters,^ and by the action of a partial anticomplement.^ But it does not in all cases require even these methods of separation; all that is necessary is a thorough and continued study of the constituents of the native serum of a given species. Variations can thus be observed therein which lead at once to the view of a plurality of complements. After several years' observation we found horse serum to be of especial interest in this respect, and we shall therefore briefly ■discuss the complements of this serum. Horse serum is particularly well adapted for complementing •experiments, because, as a rule, it exerts but slight hsemolytic effect lay itself. Sheep blood, ox blood, goose blood, and others, so far as we know, are not dissolved at all by horse serum, while so far as guinea-pig blood and rabbit blood are concerned there is an extraor- dinary amount of variation, some horse sera exerting considerable hsemolytic effect on one or both of these blood species, others having no effect whatsoever. In this respect not only did the sera of^different horses behave quite differently, but we also observed marked chrono- logical variations in the serum of one and the same normal horse. These show how much the ha^molytic properties of an individual's serum can vary. The behavior of the serum (always examined in the fresh condition) on the different days is seen in the following table: TABLE III. Date. Amount of Serum. Haemolysis of Rabbit Blood (5% 1.0). Guinea-pig Blood (5% 1.0). June 19. June 22. July 15. 2.0 1.5 0.5 2.0 1.5 1.0 0.5 2.0 0.6 0.3 very little trace trace minimal complete ( t strong complete little ' Ehrlich and Sachs, 1. c. ^ Ehrlich and Morgenroth, page 56; E. Neisser and Doriug, Berl. klin. Wochenschr. 1901, No. 22. ' Marshall and Morgenroth, pages 222 et seq. 238 COLLECTED STUDIES IN IMMUNITY. Hence within three days the serum of the horse has become strongly hsemolytic for guinea-pig blood without altering its hsemo- lytic property for rabbit blood, whereas within a further three weeks its- properties have almost become reversed, since now it does not dissolve guinea-pig blood at all, and dissolves rabbit blood (which at first was but slightly affected) very strongly. It is worthy of note that in almost every horse serum which we examined for the purpose we found a considerable amount of amboceptor for guinea- pig blood. This amboceptor was characterized by a particularly high degree of thermolability, being invariably destroyed by heat- ing to 55° C. A complement for the same is very often absent, and even when present it is only on the addition of considerable amounts of fresh guinea-pig serum that this amboceptor becomes manifest. The cause of this varying hsemolytic property of the horse serum, which is in contrast to the extraordinarily constant amount of normal haemolysin present in other sera, e.g. goat serum and dog serum, is perhaps due in part to the unusual lability of the complements here concerned. We often observed that a horse serum which dissolved guinea-pig or rabbit blood completely lost this property, or nearly so, by keeping the serum on ice for twenty-four hours, a behavior which we never met with in other sera. In a similar manner horse serum shows its variability when it is employed purely as a source of complement. We have frequently used horse serum as complement in the following combinations: Number. Blood. Amboceptor. 1 2 guinea-pig rabbit goat serum dog serum 3 ( t ox serum 4 5 gumea-pig goat serum dog serum 6 ox serum 7 S sheep dog serum serum of a goat immunized with sheep blood Of all these cases only the complement for 6 and for 8 was present in considerable amounts. So far as the other six complements were concerned we observed a fundamental difference between the ex- periments which we had made some years ago in Steglitz and those made during the past two years in Frankfurt. Whereas formerly COMPLEMENTIBILITY OF THE AMBOCEPTORS. 239 all of the completions of normal amboceptors succeeded, we found in Frankfurt that we obtained negative results in the great majority of the experiments. The complements necessary for the completion of almost all normal amboceptors were absent, while complements were present for a certain normal amboceptor (guinea-pig blood, ox serum), and for one obtained by immunizing a goat with sheep blood.i This behavior indicates clearly enough a plurality of the comple- ments in a serum, and we do not doubt that further investigations will show the same to be true for the partial complements of other sera. The occasional absence of one or the other complement will most easily be discovered just in the completion of normal amboceptors, for here but few amboceptors have to be considered. Of the numerous amboceptors produced by immunization in many cases, at least a few will find fitting dominant complements. According to our observa- tions, conclusions can be drawn only with the greatest care from isolated negative completion experiments. One cannot conclude that an amboceptor is absent from the impossibility to reactivate normal inactive sera by means of several other active sera. For the evaluation of bactericidal sera in animal experiments we believe it to be especially important to consider cases of this kind. The entire absence or a marked diminution of complements ^ which functionate as dominant complements for certain bactericidal amboceptors may lead to a disturbance in the regularity of a series of experiments, disturbances which show themselves in the fact that now and then an animal dies of the infection even though in the zone of sufficient immune serum to protect the animal. Such irregularities are quite common in the usual test series and manifest themselves frequently in the evaluation of bactericidal sera, where they then are very disturbing. ' In respect to its complements horse serum occupies a special place among most other sera used in the laboratory. Thus, for example, we were rarely successful in complementing the amboceptor of a rabbit immunized with ox blood; we never found a complement in horse sera for the amboceptors of geese or goats immunized with ox blood. That the locality plays a certain role in these phenomena follows from our observations that here, in contrast to the statements of so reliable an observer as P. Miiller in Graz, rabbit blood is not dissolved by duck serum to any appreciable extent. ^ Another abnormal phenomenon which is often observed in this connec- tion, the disturbing action of large amounts of the immune serum, is explained by the peculiar deflection of complements by an excess of amboceptor, as has been determined by M. Neisser and Wechsberg (see pages 120 et seq). 240 COLLECTED STUDIES IN IMMUNITY. It is hardly to be doubted that such variations of the complement are responsible for the occasional failures of bactericidal sera in practice, especially if we consider that in diseased conditions a marked diminution or a disappearance of the complements can take place (Ehrlich and Morgenroth, MetchnikofE, Wassermann, Schiitze and Scheller). XXIII THE PRODTTCTION OF HEMOLYTIC AMBOCEP- TORS BY MEANS OF SERUJl INJECTIONS.i A Contribution to Our Knowledge of Receptors. By J. MoRGENEOTH, Member of the Institute. As a result of the side-chain theory of immunity, and especially in consequence of the conception of "receptor" which this theory brings with it, our views concerning the cytotoxins have to a great extent been emancipated from the morphological point of view and placed on a chemical basis. This is seen most clearly by looking at the complex hsemolysins of serum, for of all the various cytotoxins these have been most clearly analyzed. As is well known, if an animal is injected with erythrocytes of a foreign species, there develop in the serum of this animal new sub- stances, the hcemolytic amboceptors (immune bodies). The ambo- ceptors are bound, above all, by the red blood-cells of that species whose blood was used for the injection, and it is through this binding that the amboceptors make possible the hsemolytic action of the complement contained in fresh serum. According to the side-chain theory the anchoring of the amboceptors is the result of chemical processes, which again are based on the existence of certain groups of the blood-cells' protoplasm, the receptors. If on the basis of this theory one has once clearly seen that the specific binding is strictly a chemical reaction between receptor and amboceptor (or rather between their haptophore groups) , it becomes quite evident that the morphological structure of the cell concerned in the reaction is some- thing quite secondary. This is, of course, apart from certain prac- tical points which are mainly the indicators of the deleterious action exerted by the coaction of amboceptor and complement. Among these would be, in this case, escape of hsEmoglobin; in the cases of other cytotoxins, disintegration and solution of the cell, cessation ' Reprint from the Munch, med. Wochenschr. 1902, No. 25. 241 242 COLLECTED STUDIES IN IMMUNITY. of the motion of flagella and cilia. The specific binding of the am- boceptors is therefore not dependent on a coarser or finer morpho- logical structure : it can occur wherever the specifically related receptors are present. For the doctrine of immunity these views constitute a new and reaUy concise definition of specificity. The latter thus loses the systematic character originally given it by botany and zoology and must from now on be regarded purely chemically, as absolutely dependent on the conceptions as to the nature of the cell's receptors. Every product of immunization is specific for those receptors by which it was called forth, irrespective of where the receptors may he} When injected into an animal the receptor produces antibodies, and these again, when they meet the receptor under suitable conditions, are boimd by the receptor. This binding, in our conception, always remains specific. It matters not whether the receptor is peculiar to the protoplasm of that species of cell which originally excited the immunity, or whether it belongs to a different kind of cell of the same species or to one of a strange species. Hence the principle of specificity of the amboceptors produced by immu- nization is not violated when, for example, v. Dungem obtains haemolytic amboceptors by injections of ciliated epithelial debris, such as is contained in goat milk. v. Dungem ' has very properly pointed out this fact in emphasizing the community of the receptors. The same holds true for the hsemolytic am- boceptors obtained by Moxter ' by injections of spermatozoa. Several different zoological species, such as goat, sheep, and ox, possess a number of common receptors in their blood-cells.* On the basis of the side-chain theory as it has just been laid down it is almost a matter of course that these receptors of the protoplasm which excite the production of the amboceptors are normally present dissolved in the body fluids, a physiological proto- type of what occurs to such a high degree in consequence of immu- nization.^ * See the explanations by Ehrlich concerning the receptor apparatus of the red blood-ceUs in Schlussbetrachtungen, Vol. VIII, of Nothnagels spezielle Pathol, und Therapie, Vienna, 1901. ' v. Dungem, Miinch. med. Wochenschr. 1899, No. 38. ' Moxter, Deutsche med. Wochenschr. 1900, No. 1. * Ehrlich and Morgenroth, page 88. * It has already been shown that as a result of injection of amboceptors into sensitive animals a considerable number of ceU receptors are thrust off, which PRODUCTION OF HEMOLYTIC AMBOCEPTORS. 243 The extraordinary multiplicity of such dissolved substances in blood serum has already been pointed out by Ehrlich.i "The chief tools of the internal metabolism are the receptors of the first, second, and third order. They are constantly being used up and produced anew, and can readily therefore, when overproduced, get into the circulation. Considering the large number of organs and the com- plexity of the protoplasm's chemistry it need not be surprising if the blood, the representative of all the tissues, is filled with an infinite number of the most diverse receptors. Of these we have thus far learned to distinguish the various kinds of lysins, agglutinins, coagu- lins, complements, ferments, antitoxins, anticomplements, and anti- ferments." These free receptors when injected into a suitable foreign animal species should therefore show their identity with those of the cells by the fact that, like the latter, they produce immune bodies identical with those produced in the usual way. A few isolated observations have been made in this direction, but the conclusions following therefrom according to the theory have not been drawn. Thus v. Dungem^ has observed the development of a hsemolysin directed against chicken erythrocytes as a result of injections of chicken serum into guinea-pig serum, and Tschistovitsch ^ has observed the formation of a haemolysin (besides agglutinins) on injecting rabbits with horse serum.* For some time past I have made experiments of this kind to demon- strate the existence in goat serum of free receptors identical with receptors of goat erythrocytes. These studies were prompted by the observation that a few normal goat sera exerted a slight inhibiting action on the amboceptors of rabbits immunized with ox blood, an action which Ehrlich and Morgenroth had shown to be due to an anti-immune body.^ I am led to publish these experiments now then functionate as anti-immune bodies. See Ehrlich and Morgenroth, pages 23 and 88. ' Ehrlich, Schlussbetrachtungen, 1. c. ■■ V. Dungern, Miinch. med. Wochenschr. 1899. ' Tschistovitsch, Anna!. Inst. Pasteur, 1899. * The increase in hasmolytic action of rabbit serum for chicken blood after the injection of chicken blood-plasma, described by Nolf (Annal. Inst. Pasteur, 1901), rests apparently only on an increase of complement, not on the develop- ment of new amboceptors. ' See pages 88 et seq. 244 COLLECTED STUDIES IN IMMUNITY. because of a rather important contradiction which exists between them and certain experiments recently published by Schattenfroh.^ This author found that one can produce homiolytic immune bodies for goat blood by injecting rabbits with goat urine. He was unable, however, to obtain these immune bodies by injection of the corre- sponding serum. It must at once be regarded as extraordinary that immune bodies which evidently are excreted through the kidney regu- larly and plentifully should be absent from the serum itself. It would, of course, have been possible to say that the concentration of the receptors in the serum was small compared to that in the urine, as is the case, for example, with urea, uric acid, and other substances. But the casual antiamboceptor action of the serum prevented this, and pointed to the presence in this of the dissolved receptors. As a matter of fact, therefore, the "interesting contradiction" described by Schattenfroh as existing between the action of the urine and the serum does not obtain; for it is possible by injecting rabbits with goat serum completely deprived of blood-cells to produce specific amboceptors. These amboceptors, to be sure, do not become mani- fest if the usual methods of investigation, such as have been em- ployed by Schattenfroh, are followed. They are, however, readUy and surely demonstrated if one attends to certain fine details. As a rule a hsemolytic serum obtained by specific immunization will, when fresh, dissolve the corresponding blood-cells; for, as v. Dungern has shown, in immunization with blood-cells the comple- ments usually do not in any sense suffer a change. Only one excep- tion is thus far known in this respect, namely, the injection of goat serum into the organism of a rabbit. Ehrlich and Moregnroth ^ have shown that the injection of goat serum into rabbits is followed by the loss of certain complements of the rabbit serum, a loss which is caused by the development of anticomplements directed against the complements of their own serum. These anticomplements are therefore to be regarded as auto-anticomplements. They not only suffice to neutralize the complements present in the serum, but are able to bind complement subsequently added. Thus the amboceptor of a rabbit mixed with goat serum is completely obscured; for if the immune serum is employed fresh, the fitting complements enabling it to act are lacking, while if the serum is inactivated and one seeks ' Munch, med. Wochenschr. 1901, No. 31. * See pages 71 et seq. PRODUCTION OF HEMOLYTIC AMBOCEPTORS. 245 to activate it by the addition of normal rabbit serum, the comple- ments of the latter will be made inert by the auto-anticomplement present. Since these auto-anticomplements, however, have no in- fluence on the binding of the amboceptor, the rational mode of pro- cedure is at once indicated. The blood-cells are mixed with the serum of the immunized rabbits and the mixture allowed to stand until the amboceptors present have been bound by the blood-cells. The latter are then separated by centrifuge, the supernatant fluid which contains the cause of the trouble, the auto-anticomplement, being removed. If the blood-cells are now mixed with fresh normal rabbit serum, the hasmolysis which ensues in the incubator will show the presence of the anchored amboceptor. Should this method, which guards against all errors, prove successful, one can also get round the difficulty in an easier manner by using guinea-pig serum as com- plement. Against this serum, according to our experience, the auto- anticomplement is ineffective. This method, however, does not suffice if we wish to obtain results which permit of only one inter- pretation. In order surely to avoid another source of error it is well to modify the test still further. It has been found that normal rabbit serum possesses a con- siderable though variable hsemolytic action for goat blood (see Table I) . The question whether we are dealing with an amboceptor artificially produced or with one which was originally present requires detailed preliminary examination and control tests, and even then is very uncertain because the amboceptor normally present finds a supply of complement in guinea-pig serum more plentiful even than that in rabbit serum itself, as can be seen on reference to the table. This difficulty is avoided without further trouble if the amboceptors produced by immunization and which it is desired to find are taken out of the fluid by means of ox blood-cells instead of goat blood-cells. Because of the partial community of receptor of these two blood- cells this is perfectly allowable. As a rule, too, normal rabbit serum dissolves ox blood only verj' little, even though considerable comple- ment is present. (See Table I.) The experiments from which the conclusions are drawn in this study were therefore always made with ox blood. One cc. of a 5% suspension of ox blood-cells is mixed with varying amounts of serum from a rabbit immunized with goat serum, the mixture kept at 38° C. on a water-bath for one hour, then centrifuged, and either fresh rabbit serum added after the supernatant fluid had been decanted, or acti- 246 COLLECTED STUDIES IN IMMUNITY. TABLE I. HEMOLYSIS OF Goat Blood (1 co. 5%) by Fresh Serum op Normal Rabbits. Rabbit Serum. I. II. III. IV. V. VI. VII. 0.25 0.1 0.05 strong moderate very little moderate little trace little moderate very little complete very little little .0 fair HiEMOLYSIS OF GoAT BlOOD BY THE SaME RaBBIT SerA ACTIVATED WITH 0.15 GuiNBA-PiGf Serum. 0.25 complete complete complete complete complete complete complete 0.1 " " strong -j almost complete 1 f strong " 0.075 •' *' — strong — strong 0.05 •' ] alm.ost complete f - — " — — 0.025 ~ ~ ~ HAEMOLYSIS OF Ox BlOOD BY THE SaME RaBBIT SeKA ACTIVATED WITH 0.15 Guinea-pig Serum. 0.5 0.25 0.1 trace faint trace faint trace faint trace trace faint trace very little trace fair moderate little The freah rabbit sera, even in amounts of 0.5, do not by themselves exert any hsemolytic ■effect on ox blood. vation was effected by the addition of normal guinea-pig serum. The hsemolytic action of the immune sera is seen in Table II. Rabbits were treated with goat serum which had been carefully freed from all blood-cells by continued centrifuging. Usually the serum was inactivated by heating it to 55° 0. for half an hour, then it was injected intraperitoneally. As a rule the animals received two to three injections of increasing doses of serum, in all about 35-90 cc. More frequent injections caused no greater formation of amboceptors, a behavior which corresponds to that seen with the injection of ox blood or goat blood. These experiments show that specific amboceptors were developed in aJl the rabbits treated with goat serum. Quantitatively this was subject to individual fluctuations just as is seen following the injec- tion of blood-cells; in some cases the development was quite con- siderable. Most of the sera were examined fresh for their action on ox blood, and invariably showed themselves without action even in doses of 0.5 cc.i The addition of large amounts of normal rabbit • The method here employed to disclose amboceptors whose presence is masked can often be used with success. Dr. Marshall and I shall shortly report an analogous case dealing with the amboceptors of n. pathological exudate. PRODUCTION OF H^EMOLYTIC AMBOCEPTORS. 247 TABLE II. 1.0 cc. 5% Ox Blood. A. Blood + amboceptor are kept at 37° C. for one hour. After centrifuging the fluid is decanted and the sediment mixed with 2 cc. physiological salt solution and 0.2 cc. rabbit serum as complement. Complete Hsemolysis. Serum rabbit I . 05 cc. II 0.05 " " " III 0.25 " B. Blood+amboceptob+0.1-0.2 Guinea-pig Sbkum as Complement. Serum rabbit IV 0.1 cc. V 0.05 VI 0.05 " " VII 0.028 " VIII 0.013 " " IX more than 0.25 X 0.05 " " XI less than 0.05 serum does not suffice to overcompensate the auto-anticomplement present. For example, the serum of rabbit III shows the following solvent action after the addition of 0.6 cc. rabbit serum: 0.5 cc 0.25 " trace 0.15 " " 0.1 " very little 0.075 cc very little 0.05 " " " 0.025 " trace The abnormal course of this slight hsemolysis shows very well the interference of anticomplement on the one hand and of the amboceptor on the other. The similarity of the amboceptor produced by injections of goat serum to that produced by injections of blood is more plainly seen by the fact that the anti-immune body obtained by immunization acts against the former amboceptor just as well as against the latter. Table III shows this behavior very well. The anti-immune body ■ used was contained in the inactivated serum of a goat which had been injected several times with the serum of rabbits immunized with ox blood. 0.3 cc. of this anti-immune body serum were mixed with varying amounts of the amboceptor sera to be tested and the mixtures kept at room temperature for one hour. Thereupon 1 cc. of a 5% suspension of ox blood-cells was added to 248 COLLECTED STUDIES IN IMMUNITY. each specimen, which was then kept on a water-bath at 38° C. for one hour, after which the mixtures were centrifuged. The blood- cell sediment was again suspended in salt solution and 0.15 cc. guinea- pig serum added as complement. The solution which then ensued was a measure for the bound amboceptor, or for the deflection by the antiamboceptor. Control tests were made with 0.3 cc. normal in- active goat serum in parallel experiments. TABLE III. A. Inhibition of the Amboceptor of the Rabbit Teeated with Goat Serum. Amount of Amboceptor. + 0.3 Antiamboceptor. + 0.3 Normal Inactive Goat Serum. 0.25 0.15 0.1 0.075 0.05 0.025 complete solution strong little very little complete solution It t( 11 It It It tt tt strong B. Inhibition op the Amboceptor of the Rabbit Treated with Goat Blood. 0.2 complete solution complete solution 0.15 strong 0.1 little 0.075 trace 1 1 it 0.06 tt tt 0.05 moderate 0.025 little 0.012 trace 0.009 The antiamboceptor is thus seen to offer exactly the same pro- tection against the amboceptors obtained as a result of goat-blood injections and those resulting from goat-serum injections, whereby their identity is demonstrated. The presence of free receptors in the urine and serum leads to the conclusion that an active receptor metabolism exists in the organism of the goat; in other words, that receptors are constantly reaching the serum from the cells and are then excreted by the kidney. Whether one is here dealing with decomposition products or with the products of some secretion or other cannot be determined. The PRODUCTION OF HEMOLYTIC AMBOCEPTORS. 249 fact that free receptors leave the serum to reappear in the urine seems to make it probable that they have no significance for the organism itself. On the contrary, one may suspect that these are products of regressive metabolism which are eliminated from the body as useless. The explanation that the free receptors originate from the breaking down of red blood-cells or other cells is entirely sufficient. It may be, however, that there is a physiological thrusting-off of the same which bears some relation to their nutritive function. In view of the elimination through the urine, it seems improbable that this constitutes a regular function as anti-immune body against the action of a possible autolysin. That certainly would be an unsuita- ble process. In fact the free receptors evidently do not generally possess the character of antiautolysins, as Besredka i believes, for by injecting a rabbit with ox serum it was impossible to obtain any hsBmolytic amboceptors. This corresponds to the negative results obtained by London ^ on injecting guinea-pigs with rabbit serum. One thing is clearly shown by the presence of dissolved substances capable of producing amboceptors, namely, that without the idea of "receptors" a universally applicable conception of the origin and mode of action of the cytotoxins is impossible, as is also a clear con- ception of the nature of "specificity." Svbseqiient Note. — In a recently published study (Miinch. med. Wochen- schr. 1902, No. 32) P. Th. Mliller reports on the production of hEemolytic amboceptors by treating pigeons with guinea-pig serum, and he accepts the views here developed. • Besredka, Annal. de I'Institut Pasteur, Oct. 1901. > London, Arch, des Sciences biologiques, St. Petersburg. XXIV. THE QUANTITATIVE RELATIONS BETWEEN AMBOCEPTOR, COMPLEMENT, AND ANTICOMPLE- MENT.i By Dr. J. Moegenkoth, Member of the Institute, and Dr. H. Sachs, Assistant at the Institute. I. Amounts of Amboceptor and Complement Required. Every laboratory in which systematic quantitative studies are ^made on haemolysis will have had encountered the relations exist- ing in different cases between the amounts of amboceptor and com- plement necessary for haemolysis. Attention was first called to these relations by v. Dungern,^ who described a hsemolytic experiment with ox blood + amboceptor from a rabbit immunized with ox blood -|- rabbit serum as complement. In this case he noticed that in order to accurately find the minimal amount of a completing serum neces- sary for haemolysis, it was necessary to employ a high multiple of that amount of amboceptor which is sufheient to effect complete solution when a large excess of complement is present. In determining the amount of complement required, v. Dungern therefore employed sixteen times the required amount of amboceptor. Gruber also says recently that "highly prepared (sensitized) human blood-cells," in consequence of their preparatory treatment, are dissolved by a mini- mum of active normal serum. In the following we wish to describe several interesting observations .made by us in the course of several years. We shall begin by describing a number of different cases in which the relations between the amount of amboceptor necessary for com- plete solution and that of the completing serum were studied. In the experiments 1 cc. of a 5% suspension of the blood-cells is always used. Especial emphasis is laid on the fact that in the comparative tests all the test-tubes contained the same volume of fluid. The first experiments were made with sheep blood + amboceptor of a goat immunized with sheep blood + guinea-pig serum as com- plement. (See Table I.) ' Reprint from the Berl. klin. Woehensohr. 1902, No. 35. ^ See page 38. 250 AMBOCEPTOR, COMPLEMENT, AND ANTICOMPLEMENT. 251 TABLE I. 1 cc. 5% Sheep Blood + Amboceptor of Goats Treated with Sheep Blood +GtriNEA-PiG Serum as Complement. Amount of Amboceptor. Proportion of the Amounts of Amboceptor. Amount of Complement Sufficient for Complete Solution. Proportion of the Amounts of Complement. I 0.05 IX 0.008 1 0.2 4X 0.0025 1 3.2 0.4 8X I] 0.0014 1 5.6 - 0.025 IX 0.04 1 0.038 1.5X 0.025 1 1.6 0.05 2X 0.025 1 1.6 0.075 3X 0.02 1 2 0.1 4X 0.016 1 2.5 0.2 8X 0.01 1 4 0.5 20 X II 0.004 I. 1 10 0.05 IX 0.1 1 0.1 2X 0.03 1 3.3 0.2 4X 0.01 1 10 0.4 8X 0.01 1 10 I^ '_ 0.05 IX 0.08 1 0.1 2X 0.015 1 5.3 0.2 4X 0.004 1 20 252 COLLECTED STUDIES IN IMMUNITY. The figures in Table I show that in the four similar cases here examined the relation between the amount of amboceptor and of the complement required is such that in the presence of larger amounts of amboceptor smaller doses of complement suffice for complete hcemolysis. The relation is not exactly the same in the separate cases, as can readily be seen from the figures of columns 2 and 4. In one case (I) increasing the amboceptor eight times reduced the amount of com- plement required only to p-^, whereas in another case (IV) increas- ing the amount of amboceptor only four times reduced the comple- ment required to ^. This shows us at once that there is no definite ratio between the two factors. The causes of this varying relation will be discussed later. The phenomenon in question is much less marked in the cases reproduced in Table II, in which the combination was ox blood + the amboceptor of specifically immunized rabbits + guinea-pig serum or rabbit serum as complement. TABLE II. A. 1 cc. 5% Ox Blood + Amboceptok op Rabbits Treated with Ox Bi-ood+ GuiNEA-piG Serum as Complement. Amount of Amboceptor. Proportion of the Amounts of Amboceptor. Amount of Complement Sufficient for Complete Solution. Proportion of the Amounts of Complement. 0.002 IX 0.035 1 0.005 2JX 0.015 1 2.3 0.01 5X 0.01 1 3.5 0.05 25 X 0.008 1 4.4 0.1 SOX 0.008 1 4.4 0.2 100 X 0.008 1 4.4 0.4 400 X 0.01 1 3.5 AMBOCEPTOR, COMPLEMENT, AND ANTICOMPLEMENT. 253 TABLE 11— Continued. B. The Same, but Rabbit Serum as Complement. Amount of Amboceptor. Proportion of the Amounts of Am^boceptor. Amount of Complement Sufficient for Complete Solution. Proportion of the Amounts of Complement. 0.005 0.01 0.05 0.1 0.2 0.4 0.005 0.01 0.05 0.1 0.2 0.4 0.005 0.0075 0.015 0.03 0.06 0.12 IX 0.5 1 2X 0.17 1 2.9 10 X 0.12 1 4.2 20 X 0.14 1 3.6 40 X 0.14 1 3.6 SOX I 0.15 I. 1 3.3 IX 0.6 1 2X 0.17 1 2.5 10 X 0.12 1 5 20 X 0.14 1 4.3 40 X 0.14 1 4.3 SOX 0.15 1 4 II I. IX 0.75 1 liX 0.6 1 1.25 3X , 0.14 1 5.3 6X 0.17 1 4.4 12 X 0.14 1 5.3 24 X 0.12 1 6.3 254 COLLECTED STUDIES IN IMMUNITY. Here we see that the employment even of very high multiples of the amboceptor effects a reduction in the amount of complement required of one-third to one-sixth at the most. But what is particularly char- acteristic for this case is the fact that the minimal amount of com- plement is almost reached with a small multiple of the "amboceptor unit," ^ and that it does not materially change with a further in- crease of the amboceptor. Thus, in Table II, A, we see that when five times the amboceptor unit is employed the amount of comple- ment required is 0.01; when 25, 50, or 100 times the unit is employed the complement is 0.008. Table II, B, shows that with the employ- ment of two to three times the amboceptor unit the maximum of complement action is already attained. An entirely analogous behavior is shown by the cases in Table III, in which the same blood and the same amboceptor are used as in Table I, but in which different kinds of complement are added, namely, sheep serum and horse serum. These cases constitute the transition to those reproduced in Table IV which deal with ox blood + the amboceptor of goats treated with ox blood + three different complements, namely, guinea-pig, rabbit, and sheep serum respectively. In these also a limit is reached beyond which the decrease of complement required is but slightly or not at all affected by an increase in the amount of amboceptor. We see therefore that vrith an increase of the amount of amboceptor the amount of complement required at one time drops to a greater or less degree, at another time it remains unchanged. Upon what does this phenomenon depend? In order to explain this we must consider three factors which may be combined with one another, and which must be considered in each individual case. These are: 1. The receptors present in the red blood-cell. 2. The conditions of afhnity. 3. The plurality of the amboceptors. So far as the first point is concerned we know that the amount of receptors of the red blood-ceUs may exhibit great differences in any individual case .2 • "We use the term ." amboceptor unit " to specify that amount of amboceptor which on the addition of the optimal amount of complement just suffices for com- plete hsemolysis. In the same sense R. Pfeiffer uses the tei-m "immunity unit" when speaking of bactericidal sera. Corresponding to the amboceptor unit the "receptor unit" is that amount of receptor which binds the amboceptor unit. ' See Ehrlich, Schlussbetraehtungen in Nothnagels spec. Pathologie und Therapie, Vol. VIII, Vienna, Holder, 1901 ; and Ehrlich and Morgenroth, page 71. AMBOCEPTOR, COMPLEMENT, AND ANTICOMPLEMENT. 25,'i^ TABLE III. A. 1 cc. 5% Sheep Blood + Amboceptor of Goats Treated with Sheep- Blood + Sheep Sbr0m as Complement. B. The Same, but with Horse Sebum as Complement. Amount of the Amboceptor. Proportion of the Amount of Amboceptor. Amount of ComplemeDt ■which Suffices for Complete Solution. Proportion of the Amounts of Complement. A. 0.1 1 X 0.15 1 0.25 2.5X 0.035 1 4.3 0.5 5 X 0.05 1 3 0.75 7.5X 0.05-0.035 1 ^ 1 3*° 4:3 B. 0.1 0.2 IX 2X . 5 almost [complete 0.1 1 1 5 0.4 4X 0.1 1 5 0.8 8X 0.1 1 5 One erythrocyte may possess just so many receptors for a cer- tain poison as are necessary to bind a single solvent dose, i.e. there is present just a receptor unit, whereas in other cases such a multiple of the receptor unit may be present that a hundred times the ambo- ceptor unit is bound. In bacteria the latter condition is present to a still very much greater degree: agglutinins (Eisenberg and Volk) and bacteriolytic amboceptors (R. Pfeiffer) are bound in enormous excess, frequently as high as many thousand times the effective amount. It is therefore entirely clear that these conditions must exercise a deciding influence on the fact whether an increased amount of immune serum decreases the amount of complement required or not. It may be regarded as self-evident that in all those cases in which only the single effective dose can be bound, i.e. in which only one amboceptor unit is anchored, an excess of amboceptor can never exert a favorable influence; on the contrary an increase in the 256 COLLECTED STUDIES IN IMMUNITY. amount of complement can readily result owing to the deflection phenomenon whose significance was first pointed out by M. Neisser and Wechsberg.i TABLE IV. A. 1 cc. 5% Ox Blood + Amboceptor op Goats Treated with Ox Blood + Guinea-pig Serum as Complement. B. The Same + Rabbit Serum as Complement. C. The Same + Sheep Serum as Complement. Amount of the Amboceptor. Proportion of the Amounts of Amboceptor. Amount of Complement which Suffices for Complete Solution. Proportion of the Amounts of Complement. A. 0.1 IX 0.01 1 0.2 2X 0.01 1 0.4 4X 0.01 1 0.8 8X E 0.01 1 0.1 IX 0.15 1 0.2 2X 0.15 1 0.4 4X 0.15 1 0.8 8X C 0.15 1 0.1 IX 0.1 1 0.2 2X 0.1 1 0.4 4X 0.1 1 0.8 8X 0.075 1 1.4 The problem is more difficult in those cases in which the red blood- cells contain a plurality of receptor units and therefore bind a mul- tiple of amboceptor units. In these cases the result of the experi- ments will depend mainly on the following factors. We know that as a rule the affinity of the amboceptor's comple- mentophile group is increased when the cytophile group is anchored by the receptors. If this relative increase of affinity is very large, the added complement will combine exclusively with the anchored amboceptor, and in certain doses will effect solution. In this case ' M. Neisser and Wechsberg, see page 120. AMBOCEPTOR, COMPLEMENT, AND ANTICOMPLEMENT. 257 the required equivalence will already be reached with the amount of complement just sufficient for solution, and an increase of the com- plement action by loading the blood-cells with additional ambo- ceptor will not occur. The conditions, however, are entirely different if the affinity of the complementophile group of the anchored amboceptor for the complement is only very slight; in other words, when we are dealing with an easily dissociated combination in a reversible process. In that case, in accordance with a well-known chemical law, the more of one of the elements is in excess, the more of the completed combination will remain intact. Hence if there are very few receptor units in the blood-cells, it will be necessary to add very much complement in order to diminish the amount of dissociation and to cause the formation of an effective unit of hiemolysin; if more receptor units are present, less complement will suffice. The tables here given present numerous considerations which show that little amboceptor + much complement and much amboceptor + little complement lead to the formation of the same amount of complement-amboceptor combination (haemolysin unit) anchored by the receptors. A most conspicuous role, however, is played by the fact that the immune serum is not a simple substance, but is made up of partial ambo- ceptors to which various dominant complements of the sera correspond. Of especial importance in this respect are partial amboceptors present in immune serum in small amounts (and which therefore can only come into action when high multiples of the immune serum are employed), but which, for their completion, find a partial complement which is particularly plentiful in the completing serum. Such a partial amboceptor present in these small amounts (such, for example, as has been demonstrated in the serum of rabbits treated with ox blood) constitutes one of the main explanations for the phenomena above described. From these considerations we see that the various phenomena which we observe in the interdependence of the amounts of ambo- ceptor and complement required may have entirely different causes, but that, by regarding all of the three above-mentioned factors, these phenomena can be explained very naturally. Under these circum- stances it is, of course, not permissible to generalize from one particular case. 258 COLLECTED STUDIES IN IMMUNITY. II. Amount of Amboceptor and Anticomplement Required. The following observations deal with the quantitative relations existing between the amount of amboceptor and that of the anticom- plement required to prevent haemolysis. In a number of cases we determined the amount of anticomplement which just suffices to prevent the solution of red blood-ceUs loaded with varying amounts of amboceptor, when that amount of complement was present which always just sufHced for complete solution. The majority of our experiments again refer to the solution of sheep blood by an immune serum (derived from a goat) whose ambo- ceptor is complemented by guinea-pig serum. This, it will be re- called, is the case in which with large amounts of amboceptor the complement required decreases considerably. For the anticomple- ment we made use of the serum of a goat which had previously been treated with repeated injections of rabbit serum. This serum, as can be seen from a previous conmiunication, does not only protect against the complement of rabbit serum, but also against those of guinea-pig serum. To begin, the amount of completing guinea-pig serum was deter- mined which, with varying amounts of amboceptor, sufficed for the complete solution of 1 cc. 5% sheep blood. After this the quan- tity of anticomplement required in each instance to effect neutrali- zation was determined, whereupon complement and anticomplement were mixed and kept at 37° C. in an incubator for half an hour. Blood and amboceptor were then added. Such an experiment is reproduced in Table V. As shown in the table by the degree of haemolysis, the peculiar behavior is observed that with small amounts of amboceptor 0.015 cc. anticomplement serum neutralize the complement of 0.05 in guinea- pig serum, whereas with large amounts of amboceptor 0.35 cc. anti- complement serum are required to neutralize 0.006 guinea-pig serum. If we calculate the amount of complementing serum neutrahzed in both cases by 1 cc. anticomplement serum, we find that in one case it is 3.3 cc, in the other 0.017 cc. Hence when large amounts of ambo- ceptor are employed the anticomplement acts 195 times weaker. The required amount of anticomplement is therefore absolutely dependent on the quantity of the amboceptor employed. This becomes most evident by the fact that even with equal amounts of AMBOCEPTOR, COMPLEMENT, AND ANTICOMPLEMENT. 259 complement required, but with varying additions of amboceptor (see columns a and 6 of Table V), different amounts of anticomplement (corresponding to the amount of amboceptor present) are required to neutralize the complement, more being required with larger amounts of amboceptor. In these cases, therefore, the amount of anticomplement required is far from heing a simple function of the amount of comple- ment, but is dependent on the amount of amboceptor present. TABLE V. A. Amount of the Amboceptor. Amoimt of the Complement Sufficient for Complete Solution. 0.3 0.05 0.01 0.005 0.005 0.005 0.01 0.035 B. Amount of a b c d Anticomple- Amboceptor, 0.3. Amboceptor, 0.05. Amboceptor, 0.01. Amboceptor, 0.005. ment. Complement, 0.006 Complement, 0.006 Complement, 0.01. Complement, 0.05.. 0.35 0.25 faint trace 0.15 trace 0.1 t c 0.075 moderate faint trace 0.05 complete trace faint trace 0.035 moderate little 0.025 tt complete 1 ( 0.015 n complete 0.01 ' * faint trace It complete In several other combinations, which we analyzed in a similar manner, we met with the same behavior to a greater or less extent. In Table VI such an experiment is reproduced; it deals with the solution of ox blood by an amboceptor derived from rabbits and complemented by guinea-pig serum. As in the previous case, inactive serum of a goat treated with rabbit serum served as anticomplement. In this case when small amounts of amboceptor are present 1.0 cc. of the anticomplement serum neturalizes 1.0 cc. guinea-pig serum; with larger amounts of amboceptor it neutralizes only 0.067 cc; i.e., about fifteen times less. 260 COLLECTED STUDIES IN IMMUNITY. TABLE VI. Ox Blood + Amboceptor of an Ox-blood Rabbit + GtriNBA- pig Sekum. Amount of Amount of Complement Sufficient to Amboceptor. Effect Complete Solution. 0.2 0.05 0.004 0.075 Anticomple- Amboceptor, 0.2. Amboceptor, 0.004. ment. Complement, 0.05. Complement, 0.1. 0.75 0.5 strong 0.3S almost complete 0.25 complete 0.15 0.1 0.075 trace 0.05 little 0.035 moderate 0.025 strong 0.015 almost complete 0.01 complete The study of the phenomena of immunization has taught us that nothing is so liable to error as premature generalization. Hence we were not at all surprised to find that there are cases in which, in contrast to that above described, the quantity of anticomplement required appeared exclusively to be a function of the amount of complement, and in no way dependent on the degree of occupation of the receptors by amboceptors. Curiously enough this case con- cerns the combination first described, namely, sheep blood, ambo- ceptor of goats treated with sheep blood, and guinea-pig serum as complement, with this difference, however, that in this case the anti- complement was not the same, since it was derived from a rabbit treated with guinea-pig serum. This anticomplement, therefore, so far as its relation to guinea-pig serum was concerned, can be termed "iso- genic" in contrast to the anticomplement previously used, which can be termed "alloiogenic," since it was derived by injecting rabbit serum. The experiment is shown in Table VII. Here we see that neutralization of the ten times larger amount of complement, such as is made necessary by the smaller amount of amboceptor, requires ten times as much anticomplement as it does with one-tenth the quantity of complement when larger amounts of amboceptor are used. AMBOCEPTOR, COMPLEMENT, AND ANTICOMPLEMENT. 261 TABLE VII. Amount of Amboceptor. Amount of Comple- ment Sufficient for Complete Solution. Amount of Comple- ment in the Anti- complement Test. Amount of Anticom- plement Required for Complete Neu- tralization. 0.1 0.2 0.02 0.0025 0.025 0.0035 0.04 0.005 The results of the experiments in the various cases are diametric- ally opposite, for in one case there is a relation between complement and amount of anticomplement required with different quantities of amboceptor, in other cases there is a wide divergence. How are these phenomena to be explained? To begin, let us assume for the sake of simplicity that comple- ment and anticomplement are of simple constitution. In that case, if, as all our experiments show, the affinity of complement is much greater for anticomplement than for amboceptor, the neutralization of complement and anticomplement should follow stoichiometric laws. As a matter of fact this is what we found in the last case (Table Vll). In the first two cases, however, the results diverge so widely from this, and are moreover so far beyond the limits which might be caused by errors, that from this fact alone it necessarily follows that con- ditions of affinity cannot by themselves suffice for an explanation. We are therefore compelled to call to our aid another factor, one which we have already emphasized, namely, the plurality of the comple- ments and anticomplements. Let us assume that in this case two dominant complements, A and B, came into play in the complementing serum. The serum serving as anticomplement must therefore contain the corresponding anticomplement a or /?. It is self-evident that the corresponding anticomplements are present in the isogenic serum; that they may also appear in the serum obtained by injection of a different serum, e.g. of rabbit serum, is shown by previous experience. It is not at all necessary to assume that rabbit serum contains exactly the same complements A and B present in guinea-pig serum; it suffices to assume a partial identity for the rabbit serum's complements {A-^ and Bi), namely, an identity in the haptophore group. Following the terminology of the theory of numbers in which "friendly numbers" (numeri amicabiles) are spoken of, one could designate complements of different species which correspond in their haptophore groups, as "friendly complements." 262 COLLECTED STUDIES IN IMMUNITY. Now if one injects any serum containing two different comple- ments, the production of partial anticomplements will to a great extent depend on the relative amount of the two complements. For example, if in one case there is considerable complement A and but little B, while in another case there is considerable B and little A, the anticomplement will be directed for the greater part against A in the one case, and against B in the other. It is therefore readUy understood that with isogenic sera the yield of anticomplements can correspond fairly well to the mixture of complements present in the injected material, for the average composition of this mixture is quite constant. A serum thus results which to a certain extent is fitted to the complements of the serum injected. Since, however, a serum contains, not two complements as we have assumed for the sake of simplicity, but a large number of com- plements, it can, of course, happen even with isogenic anticomple- ments that a disharmony will occur so far as certain fractions of complements are concerned. The following case shows that even with an isogenic anticomplement the relative proportion between complement and anticomplement with different amounts of ambo- ceptor is not maintained. (See Table VIII.) TABLE VIII. -Human Blood + Amboceptor op a Human-blood Rabbit + Rabbit Seeum+ Anticomplbment from the Goat Treated with Rabbit Serum. Amount of Amboceptor. Amoimt of Complement Necessary for Complete Solution. 0.2 0.2 0.05 0.05 0.05 0.075 Anticomplement. Amboceptor, 0.2. Complement, 0.05 Amboceptor, 0.1. Complement, 0.05. Amboceptor, 0.05. Complement, 0.1. 0.1 0.075 0.05 0.035 0.025 0.015 0.01 trace little moderate almost complete complete trace little complete trace moderate complete In this case 1.0 cc. anticomplement neutralizes 4.0 cc. complement when 0.5 cc. amboceptors are present, 1.42 cc. when 0.1 cc. amboceptor is present, and only 0.67 cc. complement with 0.2 cc. amboceptor. AMBOCEPTOR, COMPLEMENT, AND ANTICOMPLEMENT. 263 A priori, it is, of course, conceivable that in the rabbit the complements Ai and Bi exist exactly in the same proportion as do complements A and B in the guinea-pig, but we must admit that this would be a coincidence. In all probability the development of the aUoiogenic anticomplement will result in a serum in which the proportion of the two anticomplements is absolutely different, so that, for example, anticomplement B will be present in much smaller amount than in the isogenic anticomplement serum. The behavior of this will then be as follows: A certain quantity of the isogenic anticomplement serum produced by guinea-pig serum (presupposing that its constitution is uniform) will neutralize guinea-pig serum in such a way that complement A and complement B of this mixture are neutralized at the same time. If we proceed to do the same with the alloiogenic anticomplement serum, we find that in the mix- ture of anticomplement and guinea-pig serum, complement A is completely neutralized, but that a larger or smaller excess of com- plement B is stiU unsaturated. In those cases in which comple- ment A is the dominant complement both mixtures will prove neutral; when amboceptors are employed for which B is the dominant com- plement, only one of the mixtures will be neutral, the other will still be active. Now we shall assume that with the employment of large amounts of amboceptor, a partial amboc.eptor comes into action which is present in the immune serum in relatively small quantity. This partial amboceptor is complemented by complement B contained in guinea-pig serum, whereas the preponderating amboceptor is sensitized by comple- ment A. Complement B finds a plentiful amount of anticomple- ment in the isogenic immune serum, but not in the aUoiogenic serum. In the latter case, therefore, disproportionately much serum contain- ing B anticomplement will be required in order to inhibit the com- plement action when large quantities of amboceptor are present. If the difference becomes so great that the anticomplement against complement B is present only in very slight amounts, we shall have a condition like that described by Marshall and Morgenroth (see page 222). They found an ascitic fluid which was effective only against a particular complement of a serum, while it was entirely inert against another serum of this same species. We have endeavored to establish this point of view on a wider experimental basis. With this end in view we first used small amounts of amboceptor, adding various multiples of the complementing dose 264 COLLECTED STUDIES IN IMMUNITY. of serum and then determining the amount of anticomplement required in each case. In one of the experiments we made a parallel test with a large excess of amboceptors. The results showed that imder these circumstances, for each of the cases and with a certain amount of amboceptor, the anticomplement required is proportionate to the amount of complement. This is shown in Table IX. TABLE IX. 1 cc. 5% Sheep Blood + Amboceptor of Goats Immunized with Sheep Blood + GTTINEA-PIG Serum as Complement. The serum of a goat treated with rabbit serum, as anticomplement. Amount of Amboceptor. Amoimt of Complement. Amount of Anticomplement Necessary for Com- plete Neutralization. A. Little Amboceptor ( = 1 Amboceptur Unit). 0.005 0.1 0.22 0.005 0.2 0.4 B. Much Amboceptor ( = 25 Amboceptor Units). 0.125 0.125 0.125 0.006 0.012 0.024 0.24 0.42 0.8 1 cc. 5% Ox Blood -(-Amboceptor op a Goat Immunized with Ox Blood -f- Rabbit Serum as Complement. The serum of a goat treated with rabbit serum as anticomplement. Amoimt of Amboceptor. Amount of Complement which is just Fully Neutralized. Amount of Anticomplement. 0.15* 0.15 0.15 0.2 0.1 0.05 0.1 0.05 0.025 * = about 2 amboceptor units. Here, then, we are dealing with the same phenomenon which in the domain of antitoxin immimity we know as the multiplication of the Lq dose. From our standpoint this is easily explained, for if at any point in the saturation of the blood-cells' amboceptors a certain amount of the complement dominant in this case is neutral- ized by a certain quantity of anticomplement, the other conditions will in no way be altered by a doubling, quadruphng, etc., of the AMBOCEPTOR, COMPLEMENT, AND ANTICOMPLEMENT. 265 complement', and the amount of complement and that of anticom- plement required remain in the same ratio. A definite relation there- fore exists in every grade of amhoce'ptor saturation between the amount of complement and that of anticomplement required. This is in con- trast to the great differences which appear when the occupation with amboceptors varies. The relation just described indicates that we are here dealing with a chemical process following stoichiometric laws. We should like to mention further that this peculiar behavior observed by us is of some importance in refuting an objection made by Gruber (1. c.) against Wechsberg. As is well known, Gruber believed he had shown that in the bactericidal sera anticomplements were present produced by the immuniza- tion. This he held to be very important, since according to his view it showed that the defiection of complements by excess of amboceptors, which had been described by Neisser and Wechsberg, was incorrect. This is not the place to enter into the great improbability of Gruber's deductions, for this has already been well pointed out by Wechsberg, by Lipstein,' and by Levaditi.^ Wechs- berg ' repeated Gruber's experiments, but was unable to confirm his results. Sachs also was unable to do this. Gruber has now objected to Wechsberg's work on the score of a gross error, saying that Wechsberg worked with weakly sensitized blood-cells, whereas he had used strongly sensitized blood-cells. Wechsberg had therefore used considerably more complement than he, and had in consequence required much more anticomplement for neutralization, so that the presence of small quantities of anticomplement could easily have escaped Wechsberg. From what has been said above, however, just the contrary occurs; with allmo- genic sera larger amounts of anticomplement are used. That the anticomple- ment which would be produced artificially by injections of bacteria (even if that be regarded as conceivable) would eminently be aJloiogenic need not further be emphasized. It is shown by Table VIII that the conditions which Gruber assumed to exist do not obtain, even with an isogenic anticomplement, in Gruber's case (human blood + human-blood rabbit 4- rabbit serum). It is unnecessary to enter further into Gruber's objections, for Wechsberg * has succeeded through the demonstration of complementophile amboceptoids in finding the soiirce of the differences. These amboceptoids have meantime been found independently by E. Neisser and Friedemann ' and by P. Th. MiiUer.' It is immaterial in judging of this phenomenon whether in the anticomple- mentary sera used by Gruber the diverting amboceptoids developed as a result of long standing or under the influence of too high an inactivating temperature. The main thing is that even the phenomenon observed by Gruber and used ' Lipstein, see pages 132 et seq. ' Levaditi, Compt. rend. Soc. de Biol. 1902, No. 25. ' Wechsberg, Wiener klin. Wochenschr. 1902, Nos. 13 and 28. «Ibid. ' Neisser and Friedemann, Berl. klin. Wochenschr. 1902, No. 29. » P. Th. MiiUer, Miinch. med. Wochenschr. 1902, No. 32. 266 COLLECTED STUDIES IN IMMUNITY. by him as an objection constitutes a new and telling demonstration of the correctness of the amboceptor theory. Thus we see that the anticomplement experiments give us a further insight into the mechanism of hsemolysin action. This in its turn shows that the simple imitarian conception must be aban- doned to be replaced by the view maintained by us that the exciting substances as well as the reaction products arising in immunization are exceedingly manifold in character. XXV. THE BLEMOLYTIC PROPERTIES OF ORGAN EXTRACTS.^ By Dr. S. Korschun, of Charkow, and Dr. J. Mokgenboth, Member of the Institute. The first observations concerning the hsemolytic properties of organ extracts were published, so far as we are aware, by Metchni- koff.2 Proceeding from his observation that in the peritoneum of the guinea-pig goose blood-cells are taken up by certain phagocytes, the macrophages, and digested intracellularly, Metchnikoff sought to demonstrate digestive actions in vitro in extracts of such organs which are rich in macrophages. He regarded the hsemolytic function as an indicator of this digestive action. He found that extracts of certain organs of guinea-pig (but not guinea-pig serum) exerted a hsemolytic action on goose blood; the lymphoid portion of the omen- tum showed this action quite regularly, the mesenteric glands fre- quently, and in a limited number of cases the spleen. Of the other organs the pancreas showed a marked, and the salivary glands a weak hsemolytic action; the bone marrow, liver, kidney, brain and spinal cord, ovaries, testicles, and adrenals were inert. Metchnikoff found the hasmolytic substance to be a soluble ferment contained in the macrophages; he termed it "macrocytase" to dis- tinguish it from the bactericidal ferment derived from microphages, which he calls "microcytase." It shows itself to be a "cytase"^ " Reprint from the Beriin. klin. Wochenschr. 1902, No. 37. 'Metchnikoff, Annal. de I'lnstit. Pasteur, Oct. 1899; see further references in Metchnikoff, I'lmmunit^, Paris, 1901. 'Metchnikoff and his pupils use the term "cytase'' for our complements as well as for the complex oytotoxins (haemolysins, bacteriolysins, etc.) of normal sera. It is«to be regretted that although in numerous instances these have been shown to consist of amboceptor and complement this fact has not been sufficiently regarded by this school (see especially the recent studies by Sachs, pages 181 et seq., and Morgenroth and Sachs, page 233). 267 268 COLLECTED STUDIES IN IMMUNITY. by its behavior toward heat, completely losing its action on being heated to 56° C. for three-quarters of an hour. Observations in this same direction have been made by Shibayama ^ and Klein 2 and a comprehensive study by Tarassevitsch ^ has recently appeared from Metchnikoff's laboratory. Shibayama, working in Kitasoto's laboratory, studied the action of extracts of guinea-pig organs on dog blood and obtained haemolysis with those of spleen and lymph glands, but not with those of bone marrow and other organs. Without further analysis he classes as identical the hsemolytic substances of the organs and the specific hsemolysins which appear in the serum after immunization with dog blood-cells. This leads him to the following conclusion: "From the facts mentioned it can readily be seen that the hsemolytic side-chains of the guinea-pig are already physiologically present in the spleen and lymph-glands and that the injection of dog blood aids their hyper- production." Klein prepared the organ extracts by crushing them with quartz gravel, then mixing with an equal amount of physiological salt solu- tion and filtering in the cold. The only constant effect was the hsemolytic action of the extract of pancreas; in a few cases the ex- tract of kidney and of intestinal mucosa also dissolved the red blood- cells. Metchnikoff's experiments were continued in his laboratory by Tarassevitsch, who studied principally the organs of guinea-pigs, rabbits, and dogs. Corresponding to Metchnikoff's first experiments, he tested the hsemolytic action mostly on avian blood-cells, but also on those of mammals. In the guinea-pig, in the great majority of cases, he found the extracts of omentum, mesenteric lymph-glands, and spleen to be hsemolj'tic. Besides this pancreas extract and in many cases salivary gland extract were hsemolytic. In general the hsemolytic action of the organ extracts of rabbits is weaker than that from the organs of guinea-pigs. Omentum, spleen, and mesenteric glands frequently were hsemolytic; the salivary glands acted feebly; bone marrow, liver, and thymus were not hsemolytic. According to Tarassevitsch, therefore, only the macrophagic organs and the digestive glands possess a hcemolytic action. ' Shibayama, Centralblatt f. Bact., VoL 30, 1901, No. 21. ' Klein, K. k. Ges. der Aerzte in Wien, Sitzung von Dec. 20, 1901, reported in Wiener klin. Wochensehr. 1901, No. 52. ' Tarassevitsch, Sur les Cytases, Anna!, de I'lnst. Past. 1902. THE HiEMOLYTIC PROPERTIES OF ORGAN EXTRACTS. 269 If the organ- extracts are heated to 56° C. for half or one hour the hsemolytic property disappears in many cases; in other cases it is diminished; very rarely it remains unchanged. According to Tarassevitsch, this variation from the "cytases" (which in general .are destroyed by heating for half an hour to 56° C.) is only an apparent one. In the organ extracts the "macrocytase" is not completely liberated, but is held back to a great extent by the cell detritus pres- ent in the emulsion. It leaves the detritus only very slowly and incompletely, as is shown by the fact that the entire emulsion is always more active than the fluid portion obtained by centrifuging, and also that by filtering through paper the clear fluid is deprived of the greater part of the properties which the entire emulsion possesses. This filtered fluid, in which, according to Tarassevitsch, all the "cytases" present are in dissolved form, is said to behave toward thermal influences like haemolytic serum. Finally according to Tarassevitsch the thermostability of the entire extracts is not very great. If he heated his extracts a little higher, one to two hours, to 58.5°, 60°, 62°, the hemolytic property disappeared com- pletely. From this behavior toward thermic influences Tarassevitsch concludes that the relationship of the hsemolytic substances of the organ extracts to the "cytases" of serum is perfectly clear, and that it is incorrect to ascribe a hsemolytic property which can be de- stroyed at such low temperatures, to osmotic phenomena or to the presence of "de quelques substances chimiques." Hence, as Metchni- koff assumed, the organs in question contain a macrocytase, and this circumstance proves that the macrophagic organs must play a r61e in the formation of the natural and the artificial haemolysins. In the following pages we shall describe certain experiments in which we have reached essentially different results from those obtained by Metchnikoff and Tarassevitsch. The emulsion of the organs was prepared as follows: The organs removed from the exsanguinated animals are rubbed up very finely with sea-sand which has first been purified with hydrochloric acid. Then 5 to 10 times their weight of physiological salt solution is added and the mixture thoroughly shaken in a shaking-machine for two hours, whereupon the coarser particles are re- moved through several hours' centrifuging. A more or less uniformly clouded fluid remains. The organ extracts were employed as fresh as possible, though it was found that they could well be preserved by freezing them at -10° to -15° C ' On thawing them out we often observed the appearance of mmierous 270 COLLECTED STUDIES IN IMMUNITY. In studying the hsemolytic action blood-cells were used which had been freed from serum as much as possible. The series of tubes was kept in the thermostat at 37° C. for two to three hours and overnight in the refrigerator at 8° C. In the presence of large amounts of organ extracts hsemolysis proceeds rapidly; with small amounts it is veryslow> The tubes must be frequently shaken while being kept at 37°; the result can only be judged of on the following day. To begin we sought to gain a general idea of the hsemolytic action of several organ extracts on various species of blood. The extracts of intestine and of stomach of the mouse as well as that of the stomach of guinea-pigs and of the pancreas of oxen always showed a strong hsemolytic action on all species of blood which we examined, 1.0 cc. to 0.5 cc. of the extracts sufficing to completely dissolve 1 cc. 5% blood of rabbit, guinea-pig, mouse, rat, goat, sheep, ox, pig, horse, dog, or goose. The rest of the organ extracts examined, namely guinea-pig intestine, rat intestine, rat stomach, varied in their hsemo- lytic property with different bloods, qualitatively as well as quanti- tatively. Extract of guinea-pig spleen dissolved only dog blood and guinea-pig blood ; extract of mouse spleen possessed a feeble hsemolytic action on guinea-pig blood and pig blood. Extract of guinea-pig adrenals dissolved both the blood species examined in this case, viz. guinea-pig blood and goose blood. We found the extract of spleen, mesenteric lymph nodes, pancreas, stomach, intestine, and adrenals of one dog to be strongly hsemolytic for guinea-pig blood, whereas in another case the spleen showed itself absolutely inert, although the pancreas was strongly hsemolytic. This variation in the hsemo- lytic action on various blood-cells has already been noticed by other investigators, and we therefore desire merely to call attention to a point which thus far has not been regarded, namely, that the organ extracts are able to dissolve the blood-cells of the same species and even of the same individual from which they are derived. Thus according to our experience emulsions of guinea-pig stomach spleen, adrenal, kidney, and intestine, of mouse intestine and stomach, of rat intestine and stomach, of ox pancreas, dissolve the red blood- cells of their own species. The relation existing between this action on the blood of the same species and haemolysis of foreign species of blood is shown by the following two experiments. (See Table I.) clumps in the organ extracts which before had been free from visible particles These clumps could be separated by centrifuge, and exhibited a ha;molytie action when suspended in salt solution. THE HEMOLYTIC PROPERTIES OF ORGAN EXTRACTS. 271 TABLE I. Emulsion of Mouse Intestine (10%). 1 cc. 5% Ox Blood. Ice. 5%Gmnea- pig Blood. 1 CO. 5% Mo bacterium. This action is so perfectly adapted to the purpose and is apparently so novel that it seems to fall beyond the pale of the normal functions of the body. It was therefore of the highest importance to explain, from the standpoint of cellular physiology, the origin of these sub- .stances also. The. solution of this problem offered considerable diffi- THE PROTECTIVE SUBSTANCES OF THE BLOOD. 379 culties and did not succeed until the haemolysins were used in the experiments in place of the bacteriolysins. Hsemolysins are peculiar poisons which destroy red blood-cells. Such hsemolysins are found in part in certain normal species of serum, in part they can be produced artificially, as will be subsequently described. In their fundamental properties they correspond entirely to the bacteriolysins, but possess the great advantage over the latter in that they readily permit the employment of test-tube experiments whereby the individual variability of the animal body is excluded, and so allow accurate quantitative determinations. Belfanti and Carbone discovered the curious phenomenon that the serum of horses, after they had been treated with blood-cells of rabbits, contains substances which are highly toxic to rabbits, and •only to these animals. Bordet showed that the cause of this toxicity is a specific haemolysin directed againt the rabbit blood-cells. He showed further that such haemolysins, derived by injection of foreign blood-cells, lose their power to dissolve blood when heated for half an hour to 55° C. Bordet found also that the haemolytic property of such inactivated sera is again restored if certain normal sera are added. These important observations showed a complete analogy between these phenomena and those observed with bacteriolysins by Pfeiffer, Metchnikofi, and especially by Bordet. In the case of bacteri- olysins it was found that serum freshly drawn from a goat immunized against cholera is able to effect solution of cholera vibrios, i.e., to give the so-called Pfeiffer reaction. Apparently this property disappears spontaneously if the serum is allowed to stand ; it disappears rapidly when the serum is heated to 55° C. The cholera serum rendered inert by heating exerts its protective power in the animal body un- -changed; and in test-tube experiments it attains its original solvent power on the addition of small amounts of normal goat or guinea- pig serum, although the latter do not by themselves injure cholera vibrios. These experiments show that in bacteriolysis two substances act together; one, contained in immune blood, is relatively stable and represents the carrier of the specific protective action ; the other, pres- ent in every normal serum, is easily destroyed. For the present the former is called the "immune bodj," while the latter, since it complements the action of the immune body, is called the " com- plement." Since the hsemolysins are by far the most convenient for experi' 380 COLLECTED STUDIES IN IMMUNITY. mental study, Dr. Morgenroth and I have endeavored in these to dis- cover the mode of action of these two components on the susceptible object, the red blood-cells. For this purpose we first prepared solu- tions containing either only the immune body, or only the complement. These solutions were then brought into contact with the appropriate blood-cells, after which the fluid and blood-cells were separated by means of the centrifuge. The two portions were then tested to determine whether these substances had been taken up by the blood- cells. These experiments showed that the blood-cells are incapable of taking up complement alone, whereas they eagerly take up the immune body. If, however, the serum contains both components they are both bound by the blood-cells in question. A confirmation of this fact was furnished by Bordet, who showed that blood-cells or bacteria which by previous treatment have become loaded with immune body, abstract the complement from fluids con- taining the same with great avidity. These facts have been confirmed from all sides. They show that the blood-cells, or the bacteria, anchor the immune body but not the complement, but that the complement is also bound as soon as the immune body has been anchored. Morgenroth and I have made these relations more easily com- prehensible by means of the following assumptions concerning the constitution of the immune body and complement. We believe it necessary to assume that the immune body possesses two kinds of haptophore groups, one of high affinity which combines with a corresponding receptor group of the red blood-cell or bacterium; the other a group of less affinity which combines with the complement exerting the deleterious action on the cell. Hence the immune body is a kind of intermediate element which links complement and red blood-cells. In order to denote this function I have proposed the name "amboceptor," which is to express this two-sided grasping power. According to our conception the complement possesses a con- stitution analogous to that of the toxins. Thus it possesses a hapto- phore group which effects the specific combination with the ambo- ceptor. The presence of this is confirmed by the existence of analogues of antitoxins, namely, corresponding anticomplements. Besides this the complement possesses a second group, the cause of the injurious action, which is analogous to the toxophore group of the toxins. In view of the properties of this group, partly toxic, partly ferment- like, I have decided to name it the "zymotoxic" group. If one cares THE PROTECTIVE SUBSTANCES OF THE BLOOD. 381 to illustrate the action of the two components by means of a crude comparison, the action of gun and cartridge may be taken. The complement in itself is harmless, like a cartridge, whicn only acquires destructive power by being introduced into the gun. In like manner only by the exclusive mediation of the amboceptor is the injurious action of the complement called forth and transmitted to certain particular elements. In opposition to this conception Bordet maintains the view that complement and immune body do not combine as we believe, but that the entrance of the immune body into the cell substance exerts a specific injury to the latter, an injury which manifests itself by the fact that now the cells succumb to the action of the simple pro- tective substance present in blood serum, namely, Buchner's "alexin." In other words, by means of the immune substances the blood- cells are made susceptible, "sensitized," to the action of the alexin. In conformity with this Bordet terms our immune body or amboceptor the "substance sensibilatrice" and our complement the alexin. Although this view is also shared by Buchner, there are many reasons why I cannot accept it, especially in view of the observation made by M. Neisser and F. Wechsberg concerning the peculiar phe- nomenon of deflection of complement through an excess of immune body. To begin it is absolutely impossible to picture to one's seli the nature of this sensitization. If Bordet believes that the sensitizer acts after the manner of a safety-key which, when introduced into a par- ticular lock, makes the introduction of a second key possible, I must say that I cannot understand this comparison. It can positively be proven that the red blood-cell possesses no complementophile groups, since neither in the normal state nor after death does it lay hold of complement. The living blood-cell, as well as that killed by heating, however, through the occupation with the immune body, acquires the property to anchor complement. It surely is much more natural to believe that the immune body itself, the amboceptor, is the carrier of the group which binds the complement, than to assume that new complementophile groups arise owing to the action of the sensitizer. Finally, one can conceive of such a process in a living cell, one therefore capable of alteration, but in the case of dead cells which have been treated by heat or all sorts of chemicals, in the case of stabilized albumin as one might say, this assumption cannot be allowed. Bordet's assumption furthermore does not explaiii the fact that 382 COLLECTED STUDIES IN IMMUNITY. an immune body derived from a particular species is most surely activated by the serum derived from the same species. From the standpoint of Bordet's theory it would be most puzzling to under- stand why an anthrax immune body derived from a sheep should sensitize the bacilli against just the sheep alexin, one derived from a rabbit against just the rabbit alexin. From the standpoint of the amboceptor theory, however, such a phenomenon does not offer the least difficulty, since it is natural that the amboceptors circulating in every animal species are fitted to their own complements. I wish to mention still one more point which plays a great r61e in Bordet's views. Bordet assumes that the alexin is a simple [ein- heitlich] substance, whereas I maintain that there is a plurality of complements. Some very interesting experiments have recently been published by Bordet which appeared to support the unitarian view. He first determined that a certain serum, e.g. guinea-pig serum, was able to activate two different immune bodies, e.g., a cholera- immune body and a hsemolytic immune body. To this guinea-pig serum Bordet added sensitized blood-cells, i.e., blood-cells eager to take up, and susceptible to complement. If now he waited until haemolysis had begun, he found that the guinea-pig serum had lost its property to dissolve sensitized cholera vibrios. The same thing occurred if he reversed the experiment. Although it was easy to confirm the experiment of this distin- guished investigator, I found it impossible to accept Bordet's con- clusions. This experiment is only then positive proof for a simple alexin (in this case for the identity of bacteriolytic and hsemolytie alexin) if it can be shown that the two immune bodies in question are acted on by only a single complementophile group and not by a plurality of such groups. Previous investigations, however, have shown th"at the immune sera artificially produced are not simple in character but are made up of a number of different amboceptors possessing different complementophile groups. Nevertheless I consider Bordet's experiments so important that I have once more had this question thoroughly studied by Dr. Sachs and Dr. Morgenroth. These gentlemen were able to establish positive proof for the existence of different complements. Dr. Sachs, for instance, studied these conditions in goat serum, employ- ing for the purpose five different combinations of immune body, each of which could be complemented by goat serum. If goat serum THE PROTECTIVE SUBSTANCES OF THE BLOOD. 383 contained only a single complement, the course of the five series of tests should have been identical when the complement was affected. It was found on the contrary that under the influence of digestion, for example, one completion remained intact, while four others dis- appeared. By means of absorption further analogous differences were manifested which made the assumption certain that in this case four different complements come into action. Since these results positively prove the existence of a plurality of complements I think it will be unnecessary here to bring forward additional evidence in support of this. A r6sum6 of these observations confirms my view that the mech- anism of haemolysis and bacteriolysis is most easily explained by the amboceptor theory. So far as the brgin of the two components which take part in this reaction are concerned there is not the least doubt that they are of cellular origin. I assume that, in addition to the ordinary receptors which serve to take up relatively simple substances, the cells contain higher kinds of receptors designed to take up large-moleculed albuminous substances, as, for example, the contents of living cells. In this case, however, the fixation or anchoring of the molecule constitutes only a prerequisite for the cell's nutrition. Such a giant molecule in its natural state is useless for the nutrition of the cell and can be utilized only after it has been broken down into smaller constit- uents by fermentative processes. This will be accomplished most readily if the grasping group of the protoplasm is also the carrier of one or several fermentative groups which will immediately come into close relation with the molecule to be assimilated. It seems as though the economy of cell life finds it advantageous for the re- quired fermentative groups to come into action only temporarily, perhaps only in case of need. This purpose is effected most simply if the. grasping group possesses another haptophore group which can anchor the ferment-like substances present in the serum, the comple- ments. Hence such a receptor of the higher order possesses two hapto- phore groups of which one anchors the foodstuff, while the other is complementophile. It is obvious that when, as a result of immuniza- tion, such receptors reach the blood, they will exhibit the properties which we have found to belong to the receptor type. In regard to the second constituent, the complements, we shall not err if we regard these as simple cell secretions, designed to serve 384 COLLECTED STUDIES IN IMMUNITY. internal metabolism. In accordance with the conception of Metch- nikoff we must for the present beheve that the leucocytes are pri- marily concerned in their production. From these points of view the organism's immunity reaction loses the mysterious character which it would have if the protective sub- ■stances artificially produced represented a constituent originally for- eign to the organism and to its physiological economy. But we have seen that immunity represents nothing more than a phase of the general physiology of nutrition, a view in which I ■agree entirely with that distinguished investigator Metchnikoff. Phenomena entirely analogous to those of the formation of anti- bodies are constantly occurring in the economy of normal metabolism; in all kinds of cells in the organism the absorption of foodstuffs, or of products of intermediate metabolism, can lead to the formation or the thrusting-off of receptors. Considering the large number of organs and the manifold chemistry of their cells it need not be surprising that the blood, which is representative of all the tissues, contains •an innumerable number of such thrust-off receptors. To these I have given the collective name of "haptins." Only in recent years, thanks to these very theoretical considerations, have we reached a point where we can get some idea of this enormous multiplicity. In addition to the true ferments and those ferment-like sub- stances, the complements, already mentioned, the blood normally ■contains a number of substances which act specifically against cer- tain substances present in solution. Chief among these I may mention the normal antitoxins, and as «xamples of these the diphtheria antitoxin and antitetanolysin of normal horse serum, the antistaphylotoxin of normal human serum, and the anticrotin of pig serum. Next come the antiferments, such as antirennin, antithrombase, anticynarase, and others. We ■also normally find substances which prevent the action of specific hsemolysins and bacteriolysins, being directed in one case against the amboceptor, in another agamst the complement. For example in goat blood I discovered an antiamboceptor which was directed against a goat-blood haemolysin obtained in accordance with Bordet's procedure. In the blood of one animal species P. Miiller of Graz iound antibodies directed against certain complements of other species of animals, and which may, therefore, be termed normal anticomplements. Of still greater interest, however, are those haptins which are THE PROTECTIVE SUBSTANCES OF THE BLOOD. 385 directed against living cells of all kinds, thus, against vegetable cells, such as bacteria, and against animal cells, such as red blood- cells, leucocytes, spermatozoa, epithelia, and others. The haptins which are so antagonistic to cells are divisible into two large groups: (1) the agglutinins, which cause the bacteria or other cells to stick together, and which through the researches of Gruber, Durham, and Widal have attained such great diagnostic significance; (2) the bactericidal or cytotoxic substances, and these are intimately related to natural immunity. In case the substances not only kill but also exert a solvent action we call them lysins, and speak of haemolysins, bacteriolysins, etc. Thus a certain blood serum, e.g. dog serum, will simultaneously exert antitoxic, antifermentative, agglutinating, bacteriolytic, and cytotoxic effects against the appro- priate substances. If we consider one of these functions by itself, e.g., the agglutinating function of a certain serum, we shall be met with the question whether or not this property is due to one simple substance, the agglutinin. Numerous experiments have shown that this is not so, but that in this precipitating process just exactly as many different agglutmins take part as there are present different agglutinable substances. It is easy to demonstrate this plurality by means of the principle of specific union introduced by me. If, for example, a certain serum is able to agglutinate two varieties of blood- cells, say rabbit and pigeon blood-cells, and two kinds ot bacteria, as cholera and typhoid, it should be found, in case this plural effect were produced by a single simple agglutinin, that absorption by one of these elements, e.g. the cholera vibrios, would remove the other three actions also. As a matter of fact, however, the serum which has been shaken with cholera vibrios, while it will no longer agglutinate cholera vibrios, is still able to produce agglutina- tion in the other three elements, and vice versa. In this case, therefore, tour different agglutinations take part. Results entirely analogous to these are obtained if the other functionating groups contained in blood, e.g. the antitoxic, bacterio- lytic, etc., are examined in a corresponding manner. These facts confirm the pluralistic view first maintained by me, according to which every blood serum contains many hundreds, or even thou- sands, of effective haptins. All of these, with the exception, per- haps, of ferments and complements, owe their origin to an excessive assimilative metabolism. Their peculiar action on certain substances foreign to the body may be regarded as due to an incidental meeting. To a large extent, therefore, they are to be looked uron 386 COLLECTED STUDIES IN IMMUNITY. as luxuries which are not in themselves of any significance for the life of the organism. Of what use is it to a person or to an animal to have circulating in his blood a great variety of substances directed against heterogeneous materials which under normal cir- cumstances never come into account, and which at the most are brought into relation with these substances only by the experi- menter? Of what use is it to a goat to have in its blood certain substances which are directed against the red blood-cells or the spermatozoa of other animals, since these do not normally get into the circulation? Furthermore every experimenter finds that the blood serum is subject to constant change in most of its haptins, a fact which argues strongly against the assumption that all of these substances in a free state play an important or even necessary r6Ie in the organism. I cannot and do not deny that with such a superabundance of combinations in every serum substances will also be present which either 'by themselves or in conjunction with complements are able to destroy invading injurious bodies, especially bacteria. These substances then may be regarded as acting as defensive agents. In spite of tills, however, I believe it is wrong to group this most com- plex system of haptins under the collective name alexin, because this leads to an incorrect unitarian view which cannot help scientific progress. These remarks are in no way intended to detract from the very valuable work of Buchner; his study on alexins, viewed in the light of that time and according to the then state of science, must be regarded as a masterpiece which has been of enormous value in the development of this subject. Still another difference of opinion existing between Buchner and myself concerns the bactericidal and haemolytic power of nor- mal blood serum, and these properties Buchner again ascribes to the action of his alexin conceived as a simple substance. In oppo- sition to this I have demonstrated that the conditions in normal hsemolysins are exactly the same as in the artificial haemolysins, for here again two different components act together: one of them is thermostable while the other corresponds to the complements. This fact has been confirmed by a large number of observers, among whom I may mention v. Dungern, Moxter, London, P. Miilier, Meltzer. All these authors, like myself, have come to the conclusion that the thermostable substance necessary for the lytic process corresponds in every way to the artificially produced immune bodies or ambo- THE PROTECTIVE SUBSTANCES OF THE BLOOD. 387" ceptors. The haemolysins occurring naturally and those artificially produced manifest their action according to exactly the same mechan- ism. According to the observations of Pfeiffer and of Moxter, as well as to certain experiments of Wechsberg and M. Neisser, still to be published, the same holds tnie for the bactericidal substances. Against this view Buchner, while in general he confirms our find- ings of fact, maintains that the thermostable substances of normal sera are not analogous to the immune bodies, but are something apart by themselves. He therefore gives them a distinct name, " Hilfskorper " [= aiding body]. Such a separation of the con- nection between the physiological and the pathological is opposed to the teachings of Virchow. Aside from this, however, I regard the proof which Buchner advances for placing these " Hilfskorper " by themselves as insufficient. It is entirely negative and consists in this, that, according to Buchner, proof has not been offered that in normal haemolysis a " Hilfskorper " does not always come into action. Against this I should like to point out that, in the very large number of cases of normal haemolysis studied during the past years by myself and fellow workers, we have always succeeded in discovering the amboceptor effecting the action. At times, of course, this required a great deal of labor and trying all sorts of sources for complement. Experiments like those recently published by Buchner, in which only one combination chosen at random from the many possible ones is employed, do not argue against the pres- ence of amboceptors in case the experiment results negatively, for no one versed in this subject would assume that every amboceptor must find a fitting complement in every serum used. Hence Buchner does not furnish any proof that haemolysis can be produced by the alexin alone. In connection with this I should like to call attention to the fact that the alexin or complement action possessed by normal serum is due to a plurality of substances, not to a single one. Each comple- ment by itself is harmless, for only through the intervention of the amboceptor is its injurious action carried over to certain tissues. When this occurs, however, the action is the same on its own as on foreign tissues. It is surprising to watch how guinea-pig blood-celte which have been loaded or sensitized with certain amboceptors at once dissolve if their own serum is added, this serum now acting as a deadly poison. There is very little ground, therefore, to regard the complements as playing the role of defenders against foreign invaders. 388 COLLECTED STUDIES IN IMMUNITY. Tbat they appear to play this role is due to the action of what I have termed the "horror autotoxicus," which prevents the production within the organism of amboceptors directed against its own tissues. In this " horror autotoxicus " we are dealing with a well-adapted regulatory contrivance which it may be well to discuss briefly. The investigations of numerous authors have shown that by injecting animals with any kind of foreign cell material cytotoxic substances can be produced directed exactly against the material used for im- munization. Thus if a dog is immunized with an emulsion of goose brain, it will be found that the dog's serum will be highly toxic only for geese, killing these animals with cerebral symptoms. In the same way we can produce other poisons, hepatotoxins, nephrotoxins, etc., each of which acts only on a certain organ of a particular species. In human pathology, however, we must consider the absorption of the body's own constituents and not of those of other bodies. The former may occur under many conditions; for example, in haemorrhages into the body cavities, in the absorption of lymph-gland tumors, in the febrile waste of body parenchyma. It would be dysteleological to the highest degree if under these circumstances poisons against the body's own parenchyma, autotoxins, were to arise. I have attempted to solve this question by injecting goats with the blood of other goats. The sera of animals so treated did not dissolve their own blood-cells, but dissolved those of other goats. Hence it did not contain an autotoxin, but an "isotoxin," in conformity with the law to which I give the name "horror autotoxicus." I believe that the isotoxins may perhaps come to play an im- portant role in diagnosis and pathology. In the serum of dogs in which he had produced a chromium nephritis, Metchnikoff found that an isonephrotoxin had developed, for when this serum was injected into normal dogs it produced a nephritis. It is more than probable that in man also the greatest variety of isotoxins is formed. In the case of the blood this has already been positively demonstrated by a number of authors, such as Landsteiner, Ascoli, etc. With the exception of the red blood corpuscles we cannot, of course, undertake any studies in man concerning the isotoxins of the parenchyma. Many considerations, however, indicate that it will be possible to carry out these experiments on monkeys and so gain a new foundation for pathology and therapy in man. The number of combinations present in the blood serum and making up the ever-changing haptin apparatus is infinitely great. THE PROTECTIVE SUBSTANCES OF THE BLOOD. 389 Of these especially the substances of the amboceptor type are in most intimate relationship to the processes of natural immunity, for it is they which, in conjunction with the complement, effect the de- struction of the injurious bacteria. Hence if there is a loss of natural immunity, it will next be necessary to inquire whether there is a lack of complement or of amboceptor. I am convinced that these haptin studies open up a new and important field of biological investigation and will add to our knowl- edge concerning the process of assimilation. Clinically they should be of even greater importance. Since I am not in the position to make such chemical investigations on an abundance of material, I have thought it my duty to clearly define my point of view, thus furnishing to others the basis for a proper study of this subject. The significance of this method for pathology and therapy will not perhaps be fully realized until after the lapse of years. XXXIII. THE RECEPTOR APPARATUS OF THE RED BLOOD-CELLS.i By Professor Dr. P. Ehrlich. We know of a large number of agents which are able to injure the red blood-cells or kill them. In a study entitled " Zur Physiologie und Pathologie der rothen Blutscheiben " (Charite Annalen, Vol. 10) I have shown that solution of red blood-cells is brought about by all agencies (mechanical, chemical, or thermic) which kill proto- plasm. At that time I had already expressed the hypothesis that the erythrocytes possessed a peculiar protoplasm, the discoplasma, whose chief function consists in preventing the escape of the haemo- globin into the blood plasma. If the discoplasma is killed, the haemo- globin will immediately diffuse, i.e., the blood becomes laky. This process is in no way connected with conditions of osmotic tension, for in many blood poisons, such as digitoxin, veratrin, solanin, cor- rosive sublimate, etc., this destruction takes place in very high dilu- tions which hardly change the molecular concentration at all. The ordinary blood poisons, and they are very numerous (saponin bodies, helvellic acid, aldehydes, polyphenols, etc.), are chemically clearly defined substances; they exert their deleterious action in exact accordance with the principles which we have already studied in connection with the distribution of pharmacological substances, isuch as alkaloids, etc. Recently, however, we have come to know another group of blood poisons which exert their injurious action after the manner of toxins, i.e., through the agency of special hapto- phore groups which fit into suitable receptors. All of these sub- stances are highly complex derivatives of living animal or vegetable ' Reprint from: Schlussbetrachtungen; Erkrankiingen des Blutes; Noth- ^nagel's Specielle Pathologie und Therapie, Vol. VIII, Vienna, 1901. 390 THE RECEPTOR APPARATUS OF THE RED BLOOD-CELLS. 391 cells; for the present at least their chemical nature is unknown. Into this class, to mention only the simplest types, belong the following: 1. Poisonous phytalbumoses: ricin, abrin, crotin, phallin; 2. Bacterial secretions; tetanolysin (Ehrlich, Madsen), staphylo- toxin (van de Velde, M. Neisser, and F. Wechsberg), pyoeyaneous poison (Bulloch), streptococcus poison (v. Lingelsheim), cholera poison, and probably many others. 3. Poisonous animal secretions, especially the various snake venoms. The majority of these substances, especially all of the bacterial products, produce ordinary haemolysis. In contrast to this, as Kobert has shown, abrin and ricin cause a rapid clumping of the erythrocytes, a process which is analogous to the agglutinative phe- nomena studied by Gruber, Durham, and Widal. However, in the case of the poisonous phytalbumoses we cannot assume that there is an essential difference between haemolysis and agglutinatin, be- cause one of them, crotin, has been shown by Elfstrand to exert a pure agglutining action on certain species of blood (sheep, pig, ox) and a pure solvent action on others (rabbit) .^ Of especial importance, however, is the fact that all these poisons on being introduced into the animal body produce specific antitox- ins (antiricin, antiabrin (Ehrlich); anticrotin (Morgenroth) ; anti- tetanolysin (Madsen); antileucocidin (van de Velde). In view of what we have already discussed this fact alone is sufficient to ascribe to these substances the possession of a haptophore group through which they exert their toxicity. Furthermore, just like the true toxins, they possess a second group which is the cause of the toxic action. As Madsen has shown in the caseof tetanolysin, and M. Neisser and F. Wechsberg for staphylolysin, it is possible to change these poisons into modifications which have more or less completely lost their toxicity but which preserve unchanged the properties dependent on the possession of the haptophore group (affinity for the anti- body, production of immunity). These modifications, first recognized ' Even ricin, which is apparently purely agglutinating, exerts an action on the discoplasma which causes hemolysis. In the ordinary technique of the experiment this action is obscured by the fact that in the agglutinated masses the conditions are very unfavorable for diffusion. If these condition.^ are made more favorable by breaking up the clumps by shaking, one can easily observe the escape of the haemoglobin. 392 COLLECTED STUDIES IN IMMUNITY. by me in diphtheria poisons, depend on the separate dtstruction of the very unstable toxophore group. In passing now to the substances contained in blood plasma I shall discuss first the agglutinins. Even normal serum frequently contains substances which clump certain bacteria and erythrocytes. Although at first, in accordance with Buchner's views, one single substance was made responsible for the different actions, I believe that at present the pluralistic standpoint first maintained by me is generally accepted. The plurality of normal agglutinins was at once proven as soon as my principle of specific combination was applied to this question, as was done by Bordet and Malkow. The latter showed that if goat serum which agglutinates the erythrocytes of pigeon, man, and rabbit is shaken with the red cells of one of these species, e.g. pigeon, it will be found that the centrifuged fluid still contains the two other agglutinins unchanged, whereas the agglutinin for pigeon blood is absent. These substances can be obtained artificially by following the procedure of Belfanti and Carbone, who injected animals with con- siderable amounts of foreign red blood-cells (blood-cell immunization). They are readily separated from the hsemolysins developing simul- taneously by heating for half an hour to 56° C. As a result of this the action of the amboceptor lysins is destroyed while the agglutinins themselves are unaffected. To be sure if the temperature is increased to 70° C. it is possible to destroy also the agglutinating action. In that case, however, the addition of normal serum no longer exerts a reactivating action. From this it follows that the agglutinins ^ are not of such complex constitution as the amboceptor lysins; analogous to the toxins they contain a haptophore group and a zymophore which causes the coagulation process. In accordance with this I believe that the agglutinins are nothing more than receptors of the second order? ' The agglutinins here described, in contrast to ricin and abrin, give rise to no further injurious action on the discoplasma. 2 In the first part of "Schlussbetrachtungen" I have distinguished: 1. Receiptors of the first order, which concern themselves with the assimilation of simple substances (toxins, ferments, and other cell secretions). For this purpose a single haptophore group suffices. When thrust off into the blood in consequence of the introduction of toxins, these receptors constitute the antitoxins (antiferments). 2. Receptors of the second order, which in addition to the haptophore group possess a second group which effects the coagulation. After they have been THE RECEPTOR APPARATUS OF THE RED BLOOD-CELLS. 393 m ^ Fio. 1. — The Various Types op Receptors accobding to Ehklich. I, Receptors of the First Order. — This type is pictured in a. The portion e represents the haptophore group, whilst 6 represents a toxin molecule, which possesses a haptophore group c and a toxophore group d. This represents the union of toxin and antitoxin, or ferment and antifer- ment, the union between antibody and the toxin or ferment being direct. II. Receptors of the Second Order are pictured in c. Here e represents the haptophore group, and d the zymophore group of the receptor, / being the food molecule with which this receptor combines. Such receptors are possessed by agglutinins and precipitins. It is to be noted that the zymophore group is an integral part of the receptor. III. Receptors of the Third Order are pictured in III, e being the haptophore group and g the complementophile group of the receptor. The com- plement k possesses a haptophore group h and zymotoxic group z; whilst / represents the food molecule which has become linked to the receptor. Such receptors are found in haemolysins, bacteriolysins, and other cytolysins, the union with these cellular elements being effected by the amboceptor (a thrust-off receptor of this order). It is to be noted that the digesting body, the complement, is distinct from the receptor, a point in which these receptors therefore differ from those of the preceding order. 394 COLLECTED STUDIES IN IMMUNITY. Next we come to the \-ery important substances in serum which "Cause haemolysis. I have previously dwelt in detail on the fact that in this the action is always due to amboceptors which attract both :blood-cells and complement. Hence I may limit myself at this time to some supplementary remarks. It has long been known that the blood serum of one species injures and dissolves the erythrocytes of -other animal species. This is the case not only in distantly related types, such as fish and mammals, but, as was shown by therapeutic blood transfusions, occurs also in comparatively near relatives. Buchner was the first to appreciate the significance of this phenomenon, ;and assumed that the serum contained a substance innocuous for its own body but acting destructively on foreign elements (bacteria and blood-cells). This substance he therefore terms alexin. Not until, in later years, the mechanism of artificially produced lysins became clear was this unitarian view shown to be untenable. First it was found that the lysins contained in normal blood are not simple in nature, but are composed just like those artificially produced, of two components, the amboceptor and the fitting complement. Further- more, corresponding to the results in the case of agglutinins, and by means of the same methods, it was found that a given serum can con- tain a large number of different amboceptor lysins. If a certain .serum (e.g. dog serum) dissolves the erythrocytes of different species, the specific combining method has shown that this property is due to the presence of different amboceptors, each of which is related to only one of these species of blood-cells. In fact it even seems as if different complements may correspond to these amboceptors. In view of what has been said we are fortunately able to regard these different agents which injure the blood from a common point of view. Whether we are dealing with vegetable or animal prod- ucts, whether with lysins or agglutinins, whether with substances of toxin-like nature or of the complex amboceptor type, — in all of these cases the prerequisite and cause of this poisonous action is the thrust off into the blood they constitute agglutinins and precipitins. The toxins also are to be regarded as receptors of the second order thrust off by bacteria. 3. Receptors of the third order, which possess two haptophore groups, one of which effects the union with the foodstuff, whereas the other lays hold on certain substances circulating in the blood plasma, the complements, which cause ferment^like actions. After they are thrust off these receptors con- sit ute the "amboceptors." THE RECEPTOR APPARATUS OF THE RED BLOOD-CELLS 395 same, namely, the presence of suitable receptors on the blood-discs, i.e., receptors which fit the haptophore groups of the toxin or the corre- sponding groups of the amboceptor. This view, already generally- accepted for the toxin poisonings, is supported by considerations of two kinds. First is the positive proof in the case of the manifold blood poisons, that their injurious action is always preceded by the anchoring of the poison to the blood-cell. Only such species of blood-cells are susceptible to a certain haemolysin which are able to anchor the same. This has been confirmed again and again in the case of amboceptor lysins. Conversely, therefore, there is the closest connection between natural immunity and absence of receptors. That the fixation of the poisons is not due to mechanical effects, such as surface attraction, but to a true chemical process, is at once shown by the strict specificity which obtains. This is observed especially in the amboceptor lysins produced artificially. This specificity is in marked contrast to the many-sided and non-selective action of surface attraction (charcoal, etc.). The second point which supports the above view is the fact that the action of a certain poison, and only of this one, is inhibited by the correspond- ing antitoxin. According to my views, the action of antitoxins is explained by assuming that they occupy the haptophore groups of the toxin molecule and so prevent these from combining with the receptors of the tissues. It is quite incomprehensible to me how the specificity of the antitoxins can more easily be explained on the basis of the mechanical conception. This brings us to a very important point, namely, the surprising plurality of receptors. Even in the blood poisons each antiserum protects only against the substance through which it was produced by immunization. This law of specificity, which has so repeatedly been confirmed in the infectious diseases, is thus seen to apply here without any change. Antiricin serum protects the blood-cells only .against ricin, antitetanolysin only against tetanolysin, every anti- amboceptor only against a corresponding amboceptor. Hence in every species of blood-cell we shall have to assume the existence of as many different kinds of receptors as there are poisons. This is obviously a very large number. Thus if the blood- ■cells of rabbits are injured by riein, crotin, abrin, phallin, by the most diverse products of bacterial metabolism, and by a large num- ber of sera of other species, we shall have to assume a certain recep- tor (ricin receptor, etc.) for each case. Almost every day, however 396 COLLECTED STUDIES IN IMMUNITY. we are coming to know more such blood poisons; the number of different receptors which we can determine, therefore, continues to increase. In this connection I should like to present the results which Dr. Moigenroth and 1 have obtained in attempting to produce auto- lysins by immunizing goats with blood from the same species instead of blood from foreign species. In 9nly one single instance were we successful, i.e., in obtaining a solution of the animal's own blood- cells. In all other cases we obtained merely an isolysin, which dis- solved the blood-cells of other goats but not those of the goat immu- nized. If the blood of a large number of goats is tested with a par- ticular isolysin, it would be found that of some goats the blood is highly susceptible, of others it is feebly susceptible, and of still others the blood is not at all susceptible. In the case of the susceptible bloods it can be shown that the isolysin consists of the amboceptor which is anchored, plus a complement of normal goat serum. In course of time we have produced thirteen such isolytic sera, and found to our surprise that they all differed from one another, i.e., that they represented different isolysins. Thus the first serum dissolved the blood-cells of A and B; a second serum those of C and D; a third A and D, etc. By means of this one experiment we have, therefore, come to know thirteen different lysins, to which, of course, a similar number of receptors must correspond. It was fortunate for us that in the blood-cells of an animal all the receptors were not present, but only a part of the same, for it was only owing to this fact that a separation of the different kinds was possible. It is worthy of note that many receptors may be present in the blood-cells in relatively large amounts. If we designate as the single lethal dose (L.D.) that amount of a certain amboceptor which when supplied with sufficient complement just suffices to completely dis- solve a constant amount of blood, we can, by employing different amounts of amboceptor solutions inactivated by heat, readily deter- mine how many L.D. can be anchored by the amount of blood in question. As a result of this it has been found that in some cases only just the single L.D. is bound. More frequently the combining power of the erythrocytes is much higher, so that two to ten and even fifty times the L.D. is bound. In such cases, therefore, we are deahng with a marked excess of these particular receptors. An analogous case, by the way, has long been known as a result of Wasseimann's experiment concerning the power of brain substance THE RECEPTOR APPARATUS OF THE RED BLOOD-CELLS 397 to bind tetanus poison. In virtue of such an excess of tetanus receptors, the brain also absorbs a considerable multiple of the L.D. Hence in test-tube experiments it is still possible to neutralize con- siderable quantities of poison with the brain ct a guinea-pig which has died of tetanus. All of these tacts lead to the conception that the red blood-cells possess an enormous number of receptors which probably belong to hundreds of different types. Of these, again, a few may be present in relatively large quantities. This fact is surprising; for in a way it is opposed to the view held until now concerning the function of the red blood-cells. It is inconceivable that the simple inter- change of oxygen, a purely chemical function of the haemoglobin, would require so complex an arrangement as that just described. In my opinion, therefore, this enormous apparatus indicates that the red blood-cells actually exercise properties which we have thus far overlooked. If we consider that the receptors in general serve to take up foodstuffs, or in some cases the products of internal metabolism, we may easily assume that the receptor apparatus of the erythrocytes fulfills this same purpose. Since, however, we know that the vita propria of the blood-cells is very limited, we shall have to assume that the substances taken up are not for the blood-cells' own consumption, but are designed to be given off to other organs. The red blood-cells may therefore be regarded as storage reservoirs in the sense that they temporarily take up the most varied substances derived from the food or from the internal metabolism, provided these substances are supplied with haptophore groups. I may be permitted to call attention to the fact that the erythrocytes contain chiefly receptors ot the first order, i i.e., recep- tors which take up substances but do not further digest them. After these explanations I feel justified in believing that the study of receptors has opened up a new and important field ot bio- logical investigation. In order to make my meaning clearer I should like to quote the following paragraph from Verworn (Beitrage zur Physiologie des central Nerven-Systems, I. Thiel, page 68) in which our present knowledge is reviewed: "The living substance of every cell, so long as it actually is living and manifests vital phenomena, is constantly decomposing automatically and constantly forming new substances. Dissimilation and assimilation are the fundamental ' See note, page 392. 398 COLLECTED STUDIES IN IMMUNITY phenomena of metabolism, while they are also at the same time the two phases of the vital process. " As a result of a large number of facts we have, as is well known, arrived at the conclusion, confirmed chiefly by Pfliiger, that the mid- pomt of metabolism is represented by complicated combinations of egg albumin called by Pfliiger living albumin. Such combinations are exceedingly labile, decomposing to a certain extent sponta- neously, and to a greater degree in response to stimuli In these combinations we are dealing with chemical substances whose mole- cules, just because of this easy decomposition, disclose a chemical constitution quite different from the lifeless albuminous bodies which we know. 1 have therefore proposed to replace the name 'living albumin molecule' by the term 'biogen molecule.' The decomposi- tion and production of the biogens is therefore the corner-stone of the vital process in every living cell. The substances given off by the cell are derived from the decomposition of the biogens; the material for the formation of new biogen molecules is furnished by the food taken up and transformed by the cell. 1 have, however, called attention to the fact that this view needs to be extended in one direction (Allg. Physiologie, Jena, 1897). A number of facts indi- cate that the decomposition of the biogen molecule is not complete and that all of the atomic groups thus arising are not given off by the cell." In view of these explanations Verworn assumes that in the de- composition of the biogens a residue is always left which again takes up food substances and so regenerates the biogen molecule. It seems to have entirely escaped Verworn that I had expressed entirely analogous views in much greater detail twelve years pre- viously (" Uber den Sauerstoffbediirfniss des Organismus," Berlin, 1885). I assumed that the specific function of the cell is depen- dent on a central group in the living protoplasm, of peculiar structure; furthermore, that atoms and atomic groups are attached to this central group as side-chains. These side-chains are of subordi- nate importance for the specific cell function, but not so for the life itself. 1 also said that everything indicated that it was just through these indifferent side-chains that physiological combustion was effected for one portion of these side-chains effects combustion by giving off oxygen, the other portion being thus consumed. On page 11 of this monograph I expressed myself as follows: "The question as to the manner in which the side-chains constantly being consumed THE RECEPTOR APPARATUS OF THE RED BLOOD-CELLS. 399' are regenerated must, of course, excite the greatest interest. It can be conceived tliat certain portions of the functional central group [Leistungskern] can fix combustible molecular groups, and that- these groups are thus rendered more susceptible to complete com- bustion." It is at once clear that these fixing portions, which I now term receptors, correspond exactly m their nature to the biogen residues of Verworn. Probably no one who has seriously studied these questions will . question the importance of these deductions. In spite, however,. of the decades which have elapsed since Pfiiiger's publication we have not advanced one step in our experimental knowledge of this sub- ject, a fact which is due to the endless difficulties occasioned by the nature and instability of the living material. I hope that my theory is destmed finally to bridge this wide gap. The knowledge that the numerous antibodies are nothing more than thrust-off receptors of the cell should make it possible to get at the nature of assimilating processes. By means of immunization we can compel the thrusting- off of certain particular receptors which then collect in the serum. Free from the disturbing connection with the protoplasm, they no longer offer any difficulties for biochemical investigations. Viewed in this light, I believe that the facts which I have determined con- cerning the action of uniceptors and amboceptors constitute a new step toward a true conception of the vital processes. It can hardly be doubted that the red blood-cells, owing to their relatively simple structure and the ease with which they can be manipulated, are better adapted for these purposes than other cellular elements. I also believe that clinical investigations are destined to play a leading role in the solution of these problems, simply because the various types of disease offer a much greater variation in the vital conditions than we can attain by means of experiments. Even aside from the gain to pure biological science, clinical medicine should derive the greatest advantage from such studies, for, as already men- tioned, they deal with the true conception of the pathology of the red blood-cells. In order somewhat to facilitate such a study it may perhaps be well to give a brief sketch of the facts which in conjunction with my colleague, Dr. Morgenroth, I have discovered regarding the physiology of the receptors. Considering the large number of receptors which each species 400 COLLECTED STUDIES IN IMMUNITY. of blood-cell possesses, it is not surprising that certain types are common to the majority if not to all the vertebrate species. In this connection I shall only point out the fact that receptors for ricin, abrin, ichthyotoxin (which injure a large number of different erythro- cytes) are widely distributed in the animal kingdom. Side by side with such generally distributed groups, however, there are types which are limited to a comparatively small group of animal species. Thus by means of cross immunization we have demonstrated that the blood-cells of goat and sheep possess several special receptors in common. This was shown by the fact that the isolysins obtained by injecting goats with goat blood usually effected solution of sheep blood-cells, although to a less degree. In making the counter ex- periments, immunizing goats with sheep blood-cells, we obtained in addition to sheep lysin the isolysin acting on goats. Besides this there are groups of receptors which are specific for each animal species. This is best shown by the normal course of the Belfanti-Bordet experiments. In these as a rule only specific hsemolysins are formed, i.e., hsemolysins directed against the erythro- cytes exciting the immunization.^ Such variations in the zoological distribution of certain recep- tors (also of the complements, etc.) is readily explained by the very natural assumption that the metabolic processes, whose indicator the receptors really are, show corresponding variations. It is just as little to be doubted that certain assimilative processes are specific for only one species of animal as that others occur in exactly the same manner in man and in the frog. It is also of considerable importance that in any given animal species a considerable individual variation of the receptors may occur, a fact first observed in experiments with crotin on rabbits. The strongest confirmation of this point is the result of our experiments on goat isolysins. As already stated, out of- the goats we used there were always only a few which reacted to one of the thirteen different isolysins. Through the opportunity so offered we convinced ourselves of another important fact, namely, that the susceptibility of a given individual can change in a comparatively short time. We found that a goat which reacted to a certain isolysin became unsuscep- ' We have obtained entirely analogous results also with other constituents of blood serum, e.g., with complements. THE RECEPTOR APPARATUS OF THE RED BLOOD-CELLS. 401 tible after several weeks, and further that in this case there had been a disappearance of the special receptors previously demon- strated as present. We have also encountered the reverse of this, namely, the appearance of receptors previously absent. Evidently this coming and going of certain receptors reflects internal metabolic processes which may be dependent on a large number of external or internal factors. In this connection a fact observed by Kossel is especially interesting. This observer found that during the course of immunization with eel blood the blood- cells of rabbits acquire a high degree of resistance against the poison, a fact which we should perhaps ascribe to a lack of receptors. In this case we are dealing with something which is specific for the immunization with eel blood, for we could not obtain these results with two other blood poisons, crotin and tetanolysin. To a certain extent the experiments of Kossel, Gley, and Tschis- towitsch furnish a clue to the mechanism of these phenomena. They show that the first phase of immunization is that of antitoxin formation, and that the unsusceptibility of the red blood-cells is not developed until later. The way in which blood-cells which have previously been sus- ceptible to a certain poison become unsusceptible to this can very readily be explained. We have seen that those blood-cells, which are susceptible to the action of a poison (e.g., eel blood) possess appropriate receptors. Under physiological conditions the ofiice of these is to anchor a certain particular product of metabolism, x. If now through treatment with the poison the specific antitoxin is produced, it is clear that this antitoxin when present in the circu- lation is able to anchor not only the poison but also the normal meta- bolic product, X, thus preventing the latter from combming with the ■erythrocytes. Since this, however, renders the corresponding recep- tors permanently useless, the possibility of their disappearance is at once given — after the manner of atrophy through disuse. This will occur most readily in those cases in which the substance x can readily be spared by the cell, i.e., cases in which (as in sugar) the substance can be replaced by some other kind of material (e.g., fat). A disappearance of the receptors can, however, occur without the development of such a deflecting antibody, as is shown by the isolysin experiments. The most natural conclusion is that the lack of receptors in this case is produced by an inconstant, perhaps only 402 COLLECTED STUDIES IN IMMUNITY a temporary, metabolic product. Perhaps this can be brought into connection with the interesting observation of Gley that the blood- cells of new-born rabbits are highly resistant against eel poison, acquiring the normal high susceptibility only in the course of weeks. Be this as it may, everything indicates that there is an organic harmonious connection between the metabolism of any given period and the nature of the receptors present. This connection depends on the fact that substances with haptophore groups exert a stimulus on the protoplasm which excites the production of the receptors in question. In conclusion I wish to point out that many facts indicate that the species of receptors found in the erythrocytes may also be present in the cells of other organs. Thus, mentioning only one example, tetanolysin is anchored not only by the erythrocytes, but also by the brain and other organs. This phenomenon also shows itself in the immunizing test. Von Dungern, for example, found that serum of rabbits which had been treated with tracheal epithelium of oxen exerted a marked hsemolytic action on ox blood in addition to its injurious action on epithelium. Metchnikoff's objection that this was due to an error in technique (the injection of admixed blood- cells) was controverted by von Dungern, who showed that injections of cow milk, a material absolutely free from blood-cells, produced the same hseraolysins. It follows that certain receptors must be common to the red blood-cells and the epithelial tissue or the milk derived from this. The wide distribution of a particular combining group harmonizes very well with the assumption discussed above concerning the func- tions of the receptor apparatus of the red blood-cells. According to Miescher's comparison the red blood-cells serve as a sort of bank of deposit where the metabolic products in excess at any given time may be stored temporarily. In this case the sub- stances will be yielded up only to organs possessing suitable receptors. This process will be all the more complete if the affinity of the tissue receptors is greater than that of the blood receptors. There are many reasons for. believing that the affinity of the tissue receptors is not constant, and that it can be considerably increased through certain stimuli (assimilative stimuli). It is obvious that hunger, if we may apply the term to purely cellular processes, must constitute one of the most important assimilative stimuli. This functional in- THE RECEPTOR APPARATUS OF THE RED BLOOD-CELLS 403 crease of affinity would constitute a wonderful illustration ot how well the process of assimilation is adapted to its purpose. Note — Subsequent addition to page 400; Calmette also has recently reported (Compt. rend, de l'Acad6mie des sciences, T. 134, No. 24, 1902) that the blood-cells of animals highly immunized with cobra poison preserve their sensitiveness completely against the hsemolysin of the cobra poison. In a goat highly immunized with ricin, Jacoby (Hof- meister's Beitrage z. chem Physiologic und Pathologic, Bd. II, 1902) was unable to discover any increased resistance ot the red blood-cells agamst the action of the ncin. XXXIV. THE RELATIONS EXISTING BETWEEN CHEM- ICAL CONSTITUTION, DISTRIBUTION, AND PHARMACOLOGICAL ACTION.i (An Address delivered in the "Verein fiir innere Medicin," Dec. 12, 1898.) By Professor Dr. P Ehrlich. Until recent years the relations between chemistry and medicine were in general confined to purely scientific questions. In the last decade, however, a change has taken place, such as has rarely been seen in the history of medicine. One is justified in saying that at the present time the chemical view constitutes the axis about which the most important views in medicine turn, and that the two poles are the synthetic construction of new therapeutic agents on the one hand, and the discovery of specific therapeutic products of hving cells on the other. The contrast between these two methods is very pronounced. In the first case, one makes use of the retort and simple, definite reactions; in the other, of the mysterious powers of living nature so infinitely well suited to their purpose. A greater contrast cannot be imagmed than that existing between the modern medicaments, whose constitution is known down to the finest details, and diphtheria antitoxin, which we know only through its specific action and about whose chemical constitution we know absolutely nothing. Thus far the genius of the most eminent chemists has not availed to produce these bodies in a pure form and get. an insight into their chemical nature. All that this endless study has brought forth is the conviction that we are_ dealing with atomic groups of the utmost complexity, which for the present are entirely beyond our chemical researches and which, so far as we can see, will long remain so. ' Reprint from the v. Leyden Festschrift, Vol. I. 404 CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION. 405 As a result of this and other considerations the view has become prevalent that the chemo-therapeutic and the bio-therapeutic ten- dencies are absolutely different from each other. As late as two years ago a certain high authority said that the antitoxins act after the manner of specific forces (in a physical sense). If this theory of "forces" were to be upheld every possibility of bridging the con- tradictions would be completely lost, for then every tertium compara- tionis would be lacking. If instead of this we assume that both kinds of substances exert their power by purely chemical means, we shall find that certain questions arise which are of great significance for the further develop- ment of therapeutics. Convinced that this is correct 1 have busied myself during the past ten years with attempts to prove the chemical theory of toxins and antitoxins experimentally. I believe I am justified in claiming that I have caused the chemical conception to be accepted among ever-widening circles. This 1 have accomplished : 1. By the introduction of the test-tube experiments. 2. By systematic investigations concerning the mutual satisfying affinities. 3. By the demonstration of toxoids and their various modifications. I. If then the medicaments of known constitution and the biothera- peutic products, both act only in a chemical manner, i.e., if both effect the organism chemically, the first problem to be solved is to determine on what factor the very dissimilar action of these two classes of bodies depends. It will be well to begin with the simplest condition, and to study first the mode of action of bodies whose chemical constitution is well known. It is particularly desirable to gain an insight into the relations exist- ing between chemical constitution and pharmacological action. Dur- ing the last few decades these have come to play an important role in the modern synthetic tendencies. The history of this tendency is comparatively recent, dating from the year 1859 when Stahlschmidt demonstrated that strychnine loses its tetanizing action when a methyl group is introduced, being transformed into a curare-like poison. In view of the fact that this methylation forms an ammo- nium base, Fraser and Braun studied a number of other ammonium bases derived from various alkaloids and found that all of these bodies 406 COLLECTED STUDIES IN IMMUNITY. possessed a curare-like action. Since that time a large number of ammonium bases derived from the most varied alkaloids have been investigated, most all of which showed the same action. The final step was achieved only recently when Bohm showed that curarin is itself an ammonium base. He found that the curares contain a tertiary alkaloid, curin, which is of slight toxicity. If this curin was subjected to methylation an ammonium base was formed which corresponded completely in properties and actions with the natural curarin, but was about 260 times as toxic as the original substance. Since this time these questions have been studied on many different combinations by a large number of investigators, among whom may be mentioned Nencki, Jaffe, Filehne, Mering, Brunton, Brieger, Gibbs, and Aronson. I cannot, however, go into details and must confine myself to giving a short epitome of what has been done in the development of synthetic remedies. First in importance are the artificial antipyretics, of which the main types are the antipyrin series and the phenacetin series. The history of the origin of these two groups .is absolutely unlike. In one case the starting-point was the fact that quinine contains a hydrated chinolin derivative; by means of simpler combinations it was attempted to obtain the same end. Finally, after chinoHn, kairin and thallin had proved of such little value, antipyrin was obtained and found most useful. The second group, which includes phenacetin and its numerous relatives, owes its discovery not to theoretical speculations but to a coincidence, the result of an error. Of the other therapeutic agents the discovery of the hypnotic action of sulfonal by Baumann has proven of great practical and theoretical significance. The same holds true of the production of the new anaesthetics (orthoform and eucain), which was closely con- nected with the discovery of the constitution of cocaine. In recent years efforts are constantly being made to do away with the by- effects possessed by certain remedies, such as guaiacol and formal- dehyd. These efforts, first undertaken by Nencki, seek by means of suitable combinations and cleavages to give rise to a gradual liberation of the active component. While of great practical value they have but little interest in the question concerning the connection between constitution and action. When now we come to inquire what "conclusions we can draw from the study of the large number of therapeutic agents, which now embrace many hundreds of different remedies, conclusions which CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION. 407 will apply to the study of the relation between constitution and action, we find that the results are still very meagre. In the main they are as follows : 1. The discovery that the antipyretic action of the anilin and amidophenol derivatives (phenacetin) is proportional, within cer- tain limits, to the amount of p-amidophenol split off in the organism (Hinsberg). Hence all such combinations in which, through im- proper substitution of the amido group or of the main group (p- amidoacetophenon, NH2 • C6H4 - CO ■ CH3) , the liberation of p-amido- phenol is prevented cannot be used as antipyretics. 2. The discovery by Kendrick, Dewar, Filehne, that in the pyxi- din series the hydrated products act more strongly than the parent substance. Thus piperidin, C5H10NH, is a much stronger poison than pyridin, CsHsN. In this the transformation of the tertiary nitrogen atom in the imin group plays a certain role, as is shown especially by the observations of Filehne on the tetra-hydro-chinolin series. According to these the replacement of the imid's hydrogen atom by alcohol radicals reduces the irritant action. 3. The demonstration that the antipyretic power of antipyretics is destroyed by the introduction of salt-forming acid radicals, such as SO3H, CO2H (Ehrlich, Aronson, Nencki, Penzoldt). Hence so far as this action is concerned acetanilido-acetic acid, C6H5N(COCH3)CH2C02H, is inert. So also are acetanilin sulfonic acid, CeHs • NH • CO • CH2SO3H, the carbonic and sulfonic acids of phenacetin, and the ethoxy-phenylglycin which is similar to phenacetin. p XT /OC2HS UW4\NH-CH2-C02H. 4. The demonstration by Filehne, Einhorn, Ehrlich, and Poulson, of the ansesthesiophore character of the benzoyl radical. Homo- logues of cocaine, such as are obtained when other acid radicals, such as succinic acid, phenylacetic acid, cinnamic acid, are intro- duced into the ecgoninmethylester, lack these anaesthetic properties. This discovery resulted in the production of new potent anaesthetics containing the benzoyl group as the active agent, e.g. eucain (Marling) and orthoform and nirvanin (Einhorn). 5. The function of the ethyl group. This has been brought out very clearly by Baumann's discovery that the hypnotic action of certain disulfons is due exclusively to the presence of ethyl groups 408 COLLECTED STUDIES IN IMMUNITY. and that it increases with the number of these groups: thus sulfonal, (CH3)2-C-(S02C2H5)2, and trional, CH3C2H5-C-(S02C2H5)2- Of the other hypnotics which owe their action in part to the ethyl group I may mention amylenhydrate, C(CH3)2(C2H5)0H, and ethyl ur- ethan, NH2 • CO ■ OC2H5. The influence of the ethyl radical is further- more clearly shown in another series of combinations. In an artificial sweetening substance, dulcin, which is about two hundred times sweeter than sugar, this influence is very evident. This substance is phenyl urea ethoxylated in the para position, C2H50C6H4-NHCONH2. Since neither simple phenyl urea nor the methoxy combination, CH3 •0-C6H4-NH -CO •NH2, analogous to dulcin, possesses any sweet taste whatsoever, we are forced to conclude that this is due to a function of the ethyl radical. Of the remedies containing the ethyl radical there may still be mentioned phenacetin, C2H5 • • C6H4 • NH ■ CO • CH3, and two anaesthetics, holoeain, C2H5-0-C6H4-NH-C(CH3): N ■ C6H4 • OC2H5, and acoin, all three of which are derived from phenetidin. It is significant that of the entire series of alcohols only ethyl alcohol has become established as a beverage, and that since the earliest time attention was directed to producing it as pure as possible, i.e., to free it from higher and lower relatives. In all of these examples we are dealing with an influence on the nervous system, the central system (sulfonal ethylurethan, amylen hydrate, alcohol), as well as the peripheral endings (dulcin, anaesthetics) . Hence we shall probably not err if we assume that the ethyl group possesses a certain relation to the nervous system. In this con- nection an observation which I made in conjunction with Dr. Michaelis may perhaps be of some significance. We were studying a blue-green azo dye which is formed by the combination of diazotated diethyl- saffranin and dimethylanilin, and which therefore is expresed by the formula (C2Hs)2Nr Y I |-N=N N(CH3)2 It was found that this substance has the power, somewhat like methylene blue, to stain the nerve endings of living (?) tissue organs, CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION 409' whereas the corresponding dyes derived from saffranin, tolusaffranin, and dimethyl-saffranin do not possess this property. Some time after this we received a second dyestuff, of unknown constitution, which possessed the same neurotropic properties, and we therefore at once assumed that this body also contained a diethylanihn radical. On inquiry of the manufacturer we found our conjecture verified. This staining experiment may perhaps afford valuable confirmation of the view expressed above concerning the function of the ethyl radical. This synopsis will show that our actual knowledge concerning the relation between constitution and action is still in its very infancy. Hence the expectation to be able to construct new remedies of pre- determined action on the basis of theoretical conceptions will prob- ably have to be deferred for a long time. To the initiate the lack of sufficient positive knowledge is revealed by the inactivity which now characterizes a field once entered upon with so much promise. The innumerable remedies which have overwhelmed medicine in the past few years, of which only a few are of any value, and thus denote any real progress, have sufficed speedily to allay the original enthu- siam. A feeling of indifference has thus been engendered which is constantly being increased by the advertisements which are daily becoming more and more evident. Aside from these evils, however, this line of study is at present suffering especially from two other evils : 1. The habit, when a remedy has been partly accepted, of imme- diately following it with a dozen rivals of similar composition. 2. The exclusive preference given to remedies acting purely symptomatically, which are not true curative agents. A change for the better will only then occur if pure biological points of viiew are adopted, i.e., if the initiative is transferred from the chemical to the biological laboratory. As physicians we must stop remaining content with the auxiliary role of counsel in these important questions. In this subject, our very own since time immemorial, we must insist on taking first place. Just now it is essential that we gain more general, biological conceptions, and it is therefore every one's duty to contribute his mite to the develop- ment of this therapy. 410 COLLECTED STUDIES IN IMMUNITY. II. One of the main causes which has made an insight into the rela- tion between constitution and action so difficult to obtain is to be found in the fact that these relations were considered to be much simpler than they really are, and in the further fact that purely chemical conceptions were applied arbitrarily to biological processes. In pure chemistry there is an abundance of material for observing the relations between physical properties and chemical constitu- tion. In such a study it is first necessary to determine which proper- ties, to follow Ostwald's terminology, are "additive" and which "constitutive " by nature. The question arises what are the essential properties which are still found in the combinations. Evidently they are such as per- tain to the substance of the elements and are independent of the arrangement of these. These properties accompany the elements in their combinations, assuming therein values which represent the sum of the values of the elements. In other words these are "additive" properties. Real additive properties are not known apart from mass. The nearest approach to them are perhaps the specific heat of solid com- binations, and in a less degree the refraction of organic substances and their property to occupy space. In these, however, another factor becomes evident, namely, the arrangement of the elements in their combinations. This factor is of paramount importance in deter- mining such properties as color, boiling- and melting-point, form of crystals, etc. The properties which are under the mutual control of the nature of the elements and their arrangement are called "constitutive" properties. The extreme in this direction is made up of those properties which are no longer in any way dependent on the nature of the substances but only on their arrangement j these are called " colligative " properties. To which group, then, do the properties of affinity, i.e., the power of elements to effect chemical reactions, belong? Evidently to the constitutive, for daily experience teaches us that the nature as well as the arrangement of the elements is a factor. Acetic acid, lactic acid, and glucose contain the same elements in the same propor- tions by weight, yet they manifest entirely different reacting capaci- ties. Butyric acid and acetic ester are not only of the same con- CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION. 41J stitution but have the same molecular weight, yet their affinitiea are different. ^ There is probably no doubt that those properties of organic sub- stances which interest us as therapeutists are constitutive in nature, R. Meyer has published a most interesting article on certain re- lations between fluorescence and chemical constitution. In this he calls attention to the fact that the relations between the color of chemical combinations and their constitution have not up to the present time been studied with the exactness with which charac- teristics less apparent have been examined, such as rotation and the refractive inclex. The reason for this is that the refractive index of a body is a definite number, the specific rotation an angle whose size can be exactly de.termined, whereas color is more qualitative in character, and, strictly speaking, is not a physical .but a physio- logical characteristic. A body which possesses strong ultraviolet absorption bands is colorless to our eyes, yet it may appear colored to a visual organ differently constituted than ours. We see, therefore, that even in so conspicuous a property as color the physiological factor interferes with our gaining a clear insight into the relations existing between constitution and action. It will at once be con- ceded that this is true to a still greater degree in the complex processes which underlie pharmacological action. But it is just because of this intermediate position that the chem- istry of dyestuffs affoi'ds so good a point of vantage for our con- sideration. I may therefore perhaps be permitted to briefly outline what has thus far been learned concerning the relations between color and constitution, especially in view of the fact that 1 shall frequently have to touch on the biology of dyes in the succeeding chapters. In 1868 C. Graebe and C. Liebermann demonstrated that color was in some way associated with a certain denser' combination of the atoms. If this is overcome by the addition of hydrogen the color will disappear, the dye passing into the "leuco" combination (thus indigo into indigo white), out of which it can again be produced by oxidation. A great advance was then made by 0. N. Witt, who showed that the color properties of a dyestuff are due to the presence of a certain unsaturated group of atoms which he terms the color-producing cr ' Ostwald, Grundriss der allgemeinen Chemie. 412 COLLECTED STUDIES IN IMMUNITY " chromophore " group. Concerning the deatils of the various types of chromophores I refer the reader to the admirable work of Nietzki. 1 may, however, say here that, as a rule, the action of the chromophore groups as such does not become manifest if the group is part of a molecule very poor in carbon atoms. Hence colored combinations- are rare in the fatty series; they belong almost exclusively to the aromatic series (Nietzki). The presence of a chromophore group does not, however, by itself suffice to produce true dyes. Thus- azobenzol, which possesses the chromophore azo group, N=N, is no dye, because it possesses no affinity for tissues. For this reason Nietzki terms azobenzol a "chromogen," i.e., a combination which becomes a true dye when suitable groups are introduced. Radicals which have the power to develop the nature of a dye are called " auxochrome " radicals (Witt). Thus far we know but two, namely, the OH group which produces dyes of an acid character, and the amido group which produces basic dyes. In contrast to this it is rfound that other salt-forming groups are not auxochromic. This holds true not only lor acid complexes, such as the carboxyl group and the radical of sulpho acids, but also for certain basic radicals as NH4, CH2-NH2, CH;,-N-(CH3)2, and O-CH^ N (CH3)2. From every chromogen, therefore, two series of dyes may be de- rived, acid and basic, each acid derivative having an analogous basic one. Thus Acid Basic Oxyazobenzol Amidoazobenzol Dioxyazobenzol (resorcin yellow) Diamidoazobenzol(chrysoidiii\ Rosolic acid Rosauilin Thionol Thionolin Aposaffranon Aposaffranin If several similar auxochromes are introduced into a chromogen it will be found that up to a certain point the intensity of the shade and the affinity for the tissues increases with the number of groups in- troduced; thus, amidoazobenzol — yellow; diamidoazobenzol— orange; triamidoazo benzol — brown. Witt's observations extended only to the question whether and under what conditions a body is colored. Nietzki went a step fur- ther and showed that the simplest azo bodies, as also all the most simply constituted dyes, possess a yellow color. He showed that the tint deepens not only with the increase in auxochrome groups just mentioned, but also with the accumulation of carbon atoms in CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION. 413 the molecule. In many cases the color thus passes through red into violet, in other cases it passes into brown. Besides this the chemistry ■of the rosanilin dyes furnishes many examples of change in tint through the introduction of substituting groups; thus, rosanilin — red; tri- methylrosanilin — red violet; hexamethylrosanilin — blue violet; tri- phenylrosanilin — blue. I may add that in several cases these views have been applied also to bodies possessing physiological action. In cocaine, for ex- ample, the ester-like benzoyl radical, (CO-CeHs), undoubtedly repre- sents the ansesthesiophore group; the tertiary amin contained in the basic portion representing an analogue of the auxochrome group, and hence called auxotox. This is borne out by the fact determined by me that cocaine loses its ansesthetizing properties when through methylation the tertiary amin is converted into a quaternary ammo- nium base. Analogous to this is the fact that through complete methylation tertiary groups lose the property to act as auxochromes, for the ammonium radicals thus formed merely give rise to an in- creased solubility. Thus through the introduction of a methyl group, hexamethyl violet, which possesses three dimethylamido radicals, passes over into the soluble methyl green, which possesses two di- methylamido groups and one ammonium group. Hence methyl green is a tripheny'-methan dye which contains two dimethylamido groups as auxochromes. In this it is like malachite green, which it therefore matches entirely in tint. The third portion of the cocaine molecule, the carboxylmethyl ^roup, COOCH3, on the other hand, is probably of but little im- portance, as can be seen from the strong anaesthetic action of benzoyl- pseudotropein, which does not possess this group. III. Having thus briefly sketched some of the more important points concerning the relation between chemical constitution and action, I pass on the pharmacological side of the subject, in which, to be sure, the conditions are far more complex. It will be well to com- mence with a very simple example. We know a large number of poisons which through appropriate substitution are practically de- prived of their deleterious action. As was shown by Aronson and myself, this is true, especially of the radicals of sulphuric and carbonic acids. Independently of us, Nencki came to the same conclusion. 414 COLLECTED STUDIES IN IMMUNITY Thus by allowing sulphuric acid to act on anilin, which, as is well known, is highly toxic, the toxicity is completely destroyed, for the result- ing sulfanilic acid can be taken in large doses .without injury. In like manner the amidobenzoic acids are non-toxic; so also the meta- and para-oxybenzoic acids derived from phenol, while the ortho isomer (salicylic acid) still exhibits the familiar toxic effects, although they are far less intense than those of phenol. These surprising results cannot be ascribed to purely chemical effects, as, for example, by assuming that the acid derivatives are more difficult to oxidize than the original substance and that they therefore do not abstract oxygen from the tissues. Certain observations, however, which I had made many years previously in connection with vital staining furnish a very simple explanation. I found that the power to stam gray nerve tissue is possessed by only a small number of dyes, and especially by certain basic dyes (chrysoidin, Bismarck brown, neutral red, phosphin, flavanilin, methylene blue), whereas of the acid dyes, in which OH constitutes the auxochrome group, only one, alizarin, possesses this property. All dyes which contained a sulphuric acid radical were absolutely negative, and I examined a very large number. What is especially significant is that even neurotropic stains lost this property entirely if sulfonic acids were introduced, a fact demonstrated in the flavanilin sulfonic acids, the alizarin sulfonic acids, and the sulfonic acids derived from methylene blue. From this it follows that the introduction of the above-mentioned acid group changes the dis- tribution in the organism and causes especially a complete destruc- tion of neurotropic properties. The central action of a poison is to be explained logically by an accumulation of the toxic substance in the central nervous system. Since, therefore, the central part of the toxic action has been completely destroyed by the introduction of a sulfonic acid radical we find that the reduction in toxicity is readily explained. It is obvious that under these conditions other toxic properties, which do not depend on the central nervous system may be preserved intact. Thus according to my observations the blood destructive properties of phenylhydrazin and benzidin are still present in their monosulfonic acids. ^ ' The action of these combinations is not as strong as the original sub- stance, but this is probably due to the fact that the sulfonic acid radical (and even t'he neutral sulfonic radical) by itself reduces the toxic power of the amido group. This mitigating action explains why sulfanilic acid which is derived from anilin is no blood poison; this power of the sulfonic acid group, however. CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION 415 From these considerations it is at once clear that there is a link between chemical constitution and pharmacodynamic action, namely, the distribution in the organism. In this we are dealing with a prin- ciple which has long been known, and which, I might say, is almost self-evident, but which nevertheless is clearly expounded in but few text-books on therapeutics (see Stockvis, de Buck, and especially H. Schulz). Unfortunately we have been satisfied with a mere theoretical acknowledgment of this principle, and have practically made no efforts to gain a deeper insight into the laws governing this distribu- tion. This is esepcially true of the new synthetic tendency, which labors exclusively for symptomatic effects and leaves questions con- cerning localization absolutely untouched. To my mind just this neglect is to blame for the insufficient progress thus far made, and I believe that new points of vantage can easily be gained if the distributive views are given greater prominence. In this connection I may call attention to the fact that through the application of the principle of localization, which I have attempted, new and promising paths have been opened up in the domain of bacteriology, although this subject was already beginning to become barren under the sche- matic application of the doctrines of immunity. To be sure it must be admitted that there are enormous difficulties attending the determination of the distribution of chemical substances with the necessary degree of precision. We are here confronted with a problem whose solution is simple in only a few special cases. These we shall discuss in a moment. In the great majority of chemical compounds, however, only a combination of various methods gives us any definite knowledge. Animal experiments, as such, do not give us complete informa- tion concerning the distribution in the organism; they only mark the regions most susceptible to the poison, and then usually only for those systems, such as the nervous or muscular system, in which disturbances of function are recognizable. The animal experiment, however, furnishes but little information concerning the processes in the vital parenchyma, for to these graphic or other ordinary physio- logical methods are inapplicable. The assistance afforded by pure chemical analysis is very slight. is insuflBoieut to destroy the powerful NH-NHj group of phenylhydrazin, or the two amido groups of benzidin. 416 COLLECTED STUDIES IN IMMUNITY. It can be carried out exactly with only a very small number of readily determinable substances, hence primarily with inorganic combinations. Besides, the demonstration that a poison, for example arsenic, occurs in a certain organ, as the brain, is of little value, for this does not tell us what is really of the greatest importance, namely, the localisa- tion in the separate cell constituents of the various organs. The pathological and histological findings are of far greater importance. To be sure, if one turns the pages of the text-books, one will not have very great hopes in this direction, for the same banal changes, fatty degeneration of the liver, nephritis, destruction of the blood, are always given. Nissl's investigations, however, demonstrated that exact histological studies on the central nervous system allow the points of attack to be recognized. He showed that certain poisonings always affected certain groups of ganglion cells. How fruitful these points of view may be was shown by the pretty investigations of Goldscheider, through which he showed that the motor ganglion cells had already suffered demonstrable lesions from tetanus poison at a time when even the slightest clinical symptoms were absent. In many other cases also, most valuable information may be furnished by minute histological investigations; in this connection I may mention that with cocaine I have found in mice an absolutely specific foam-like degeneration of the liver cells in a form which I have seen with no other substance. In general, I may add that the chronic poisonings extending over several days, and not the acute poisonings, are best suited for the demonstration of specific injuries to certain organs, a point which has already been emphasized by Nissl. In my pharmacological investigations, which far antedate Nissl's publications, I have given this method special preference. I also described a method (Deutsche med. Wochensch. 1890, No. 32) by which these otherwise laborious experiments can be carried out with ease. This method depends on feeding mice with biscuit which con- tains a certain amount of the substance in question. It is then very easy to find a dose which will kill the animals in the desired period of time. Although the results of these anatomical-pathological investiga- tions are most valuable, it cannot be gainsaid that through them one only discovers the injury to the most susceptible organs, but that the general distribution of a certain substance within the entire organism remains unknown. CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION 417 In my opinion, however, this general distribution is a very im- portant problem, for just these facts furnish the most valuable in- formation concerning the chemical functions of the organs, and of the elements which compose them. At present this problem can only be solved by the employment of dyes whose distribution we can readily follow both macroscopically and microscopically. It is to be ■deplored that these investigations, which possess such a high didactic value should thus far have found so few adherents; they are only exceptionally studied and then for some particular purpose. If rabbits are injected with dyes it will be found that even macro- scopic study yields most interesting pictures. There are certain •dyes, although not very common, which stain only a particular tissue, €.g. fat tissue; these are called " monotropic." Usually a dye possesses an affinity for a number of systems of organs, although frequently it then happens that one particular organ is stained in an especially conspicuous manner. Very often one finds that the maximum staining is in the kidney (especially in the cortex) and in the liver. Other dyes, such as acridinorange and dimethylamido- methylene blue, exhibit their stain particularly in the thyroid gland ; still others, as dimethylphenylene green, stain especially the fat tissue; some, such as alizarin blue, the submaxillary gland, etc. Alizarin blue, besides staining brain and kidneys, stains the sub- maxillary gland with especial intensity. As examples of polytropic stains we may mention neutral red and a basic dye, brilliant cresyl blue, for these stain the majority of body parenchyma intensely and apparently uniformly. It is particularly significant that the majority of basic dyes which stain the brain are also stored up by fat tissue. As we shall soon see neurotropism and lipotropism are related to one another. The variation in the localization of dyes frequently corresponds to certain peculiarities in their excretion; the chief points of excre- tion are probably kidney cortex, liver, and intestine. In contrast to the great majority of dyes which, like methylene blue, fuchsin, alizarin, indigo carmine, and many others, gain access to the urinary secretions very easily, there are several which seem incapable of doing this and which therefore seem by preference to be excreted through the bile or through the intestinal juices. An example of this is benzopurpurin, a very large-moleculed cotton dye which is made from diazotated toluidin and naphylaminsulfonic acid.' 1 __ , ' It IS possible that this phenomenon can be fully explained by this that we 418 COLLECTED STUDIES IN IMMUNITY Besides this, however, one could assume that analogous dyes also effect a loose combination with the blood albumin, which makes, excretion through the kidney impossible. In that case the condi- tions would be analogous to those which we see with many metals,, e.g. iron or lead, and to those which obtain in the excretion of a poisonous albuminous substance, ricin, as they have been deter- mined by investigations in the Pasteur Institute. None of the sub- stances which occur in the circulation in the form of albumin com- binations pass into the urine, since the albumin molecule is unable to pass through the intact kidney filter. In contrast to this, how- ever, the intestinal glands or liver allow even these large-moleculed substances to pass through. The salivary glands do not play any important part in elimina- tion, as is shown by the fact that with the majority of dyes the saliva is not at all colored, and with certain others, e.g. alizarin blue, is but slightly tinged. This is apparently because of the fact that the salivary glands are not well adapted to the secretion of substances with large molecular weights. In the excretion of substances of small molecular weights, however, they may play a prominent r6le, as can be seen from the behavior of various salts, e.g., potassium iodide, rodan combinations, and the salts of mercury. In the aro- matic series it is particularly paraphenylendiamin, dimethylpara- phenylendiamin, trihydroparaoxychinolin , and related substances, which are excreted through the submaxillary gland of rabbits and there give rise to marked inflammatory changes (oedema, necrosis). The least important r61e is that taken by the sweat glands. So far as I am aware the only dyes excreted on the body surface are those of the phosphin series, as is shown by Mannabeig's researches concerning the therapeutics of malaria. Much greater significance, however, attaches to the possibility of exactly determining the distribution of the dyes by means of the microscope. I need only call to mind the vital staining of nerve endings by means of methylene blue, a procedure which has found are here dealing with large-moleculed substances which are soluble with diffi- culty and which therefore must be regarded more like colloids. In contrast to methylene blue, methyl violet, and many other dyes, benzopurpurin ia ab- solutely non-difiusible. According to the researches of Krafft (Bericht der deutsch. chem. Gesell. 1899) solutions of benzopurpurin (raising of the boiling- point) showed an apparent molecular weight of 3000 instead of 774 reckoned out from the formula. CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION. 419 extensive application in the histology of the nervous system. Then there are the wonderful vital stains which the majority of granules give with neutral red; and the beautiful stains of these same bodies which can be effected with brilliant cresyl blue (oxazin dye). I cannot here enter into still other interesting and important vital stains. Besides this each stain possesses its own peculiar characteristics. Thus methylene blue, besides staining the nerve endings and a number of the most diverse granules, stains intensely the cell protoplasm of the islands of Langerhans of the pancreas, and, further, also muscle cells of a certain particular function, striped as well as smooth. I am practically convinced that in the vascular system certain muscle fibres which can be stained with methylene blue cause a marked narrowing and perhaps even a complete closure of the lumen after the manner of a ligature. These muscle fibres never form a con- tinuous lining of the vessel wall but only occur singly and separated from one another by comparatively wide intervals. The uniform calibration of the tube would then fall to the lot of the evenly dis- tributed muscle lining which takes no stain. We should thus have what is surely of great significance, namely, the fact that vessel calibration and vessel closure are two functions which are absolutely distinct anatomically and biologically. In a description so general in character as this one I cannot enter into still other interesting groups of dyes, e.g., those that stain nuclei vitally, etc. Exactly the same differences which we have observed in the case of dyes manifest themselves if we introduce other kinds of sub- stances into the body, it matters not whether they are well defined, organic or inorganic combinations, or whether they constitute chem- ically unknown and highly complex bacterial products. In general we shall probably have to assume that substances which are chemically nell defined are to a great extent polytropic in character. In my studies with several substances readily demonstrable by means of color reactions and whose distribution can therefore readily be followed, I have convinced myself that the aromatic bases as a rule' have an affinity for many different kinds of parenchyma. If in spite of this the clinical injury manifests itself in only one tissue, this in no way contradicts the polytropic character of these substances. It merely proves, what is really a matter of course, that among a number of tissues there are some that are particularly susceptible to an equal injury. To what extent other circumstances, such as saturation of 420 COLLECTED STUDIES IN lMMUNlf\: the tissues with oxygen, reaction of the tissues (nephritis in chromium poisoning), conditions of alkalinity, peculiarities of elimination, etc., affect the result in any given case cannot now be discussed. We find exactly the same conditions to hold with bacterial poisons. Tetanus poison, for example, as is shown by the experiments of Donitz, Roux, and others, is monotropic in highly susceptible animals, whereas in other animals, rabbits, pigeons, etc., the tetanus-binding groups are present not only in the brain but also in a number of other organs of less biological importance. This explains why, for instance, in guinea-pigs the lethal dose is the same whether the poison is in- jected subcutaneously or intracerebrally, whereas in the pigeon, and to a certain extent also in the rabbit, much larger doses are required for subcutaneous poisoning. Under these circumstances part of the poison is laid hold of by the body parenchyma and thus deflected from the endangered organs. We may perhaps regard it as a matter of course, that these laws of mutual deflection play an important role in all polytropic sub- stances, and that we shall gain a real insight into the action of drugs only if we regard this factor suflaciently. If, for instance, as is so often the case, a poison is both neurotropic and lipotropic, if the same amount of poison per kilo body weight is injected into a lean animal as into a very fat one, it is clear that the share of poison which falls upon the brain in the former case is much greater than in the latter. IV. We now take up the question as to how this varied distribution -occurs. As a rule the poisons reach the tissues through the circu,- lation, and we shall therefore first study the influence of the vascular system on this distribution. A moment's consideration, however, shows that although the circulation may be the prerequisite, it can in no way be the cause of the varied distribution discussed above. According to the views held by the majority of investigators and also by me this localization in certain organs depends ift every in- stance on causes within the tissues and not on the vascular distri- bution. For example, if in a case of jaundice we find that the brain shows not a trace of bilirubin coloration, while many other tissues, such as kidney, liver, etc., are saturated with bile pigment, this, in my opinion, is due to the chemistry of the brain substance. The brain lacks all such substances which attract bilirubin, that is to CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION 421 say bilirubin is not neurotropic. In recent years a different view has been pronaulgated, especially by Biedl, who ascribes a decisive role in the distribution of poisons to the vessel wail. As a result of my own long experience with the greatest variety of substances I am unable to assume that the vascular endothelium as such exer- cises different functions in different organs, so that, for example, a liver capillary is permeable for certain substances which will not pass through other capillaries.^ On the other hand the vascular system plays a very important r61e in a different direction, as can be seen from the following strik- ing example. Mice are fed according to my "biscuit method " with derivatives of paraphenylendiamin (acetylparaphenylendiamin, thio- sulfonic acid and mercaptan of paraphenylendiamin). On autopsying the animals very peculiar changes are observed in the diaphragm. The parts surrounding the central tendon are stained intensely brown, while the peripheral portions are usually unstained. Frequently the margin of the stain is wavy and marked by a more intense colora- tion. At times I have observed similar changes in other muscular regions, namely, in those of the eye, larynx, and tongue. Micro- scopical examination shows that this is not a case of infarct, but that there is apparently a uniform brown staining of the muscle areas in question. The cross striation is preserved intact, and a moderate degree of fatty degeneration is not infrequently observed. Usually also there is a certain amount of hypersemia. We are not dealing with a derivative of haemoglobin; on the contrary it is much more probable that we are dealing with a highly complex oxidation product of the paraphenylendiamin.^ The question which now arises is why, in this feeding, only part of the muscles, a very small part, show this vital staining. It was soon seen that the groups of muscles affected were analo- gous in other respects. Thus with injections of methylene blue it ' It was especially gratifying to note that Bruno, as a result of the investi- gations which he made under the direction of R. Gottlieb, is also very skeptical regarding Biedl's views (Deutsche med. Wochensch. 1899, No 23). ' This assumption has subsequently been clearly confirmed by the work of Dr. Rehn? (Archiv internat. de Pharmacodynaraie, Vol. VIII, p. 203) It was found in animals poisoned acutely with paraphenylendianiin that the muscles which were saturated with the poison assumed the typical brown color when brought in contact with air I would also call attention to the fact that both paraphenylendiamin and paramidophenol are employed, by oxidation, for true brown and black dyes for hair and fur (Ursol dye). 422 COLLECTED STUDIES IN IMMUNITY. is just in these areas that the motor nerve endings take a more or less complete stain. In comparative pathology also we find this group in evidence, for trichinae invade by preference diaphragm, and the muscles of the eye and larynx. These facts are very readily explained. In accordance with a principle discovered by Robert Mayer, the blood-supply of the muscles is dependent on their biological importance. Muscles, such as the diaphragm, which labor continuously and whose failure to act would constitute a ma.rked disturbance of health are far better supplied with blood than others of less importance. Naturally in this group of "most favored" muscles, correspond ing to the greater supply of blood, there will also be a maximum supply of oxygen, foodstuffs, 9.nd all other materials present in the circulation. Hence such a muscle cell will be more highly charged with oxygen and can therefore exert a more energetic oxidizing a,ction, as is manifested in the brown staining with paraphenylen- diamin. The staining of the muscle end-plates is explained in exactly the same way, through the increased supply of methylene blue on the one hand, and the saturation with oxygen and the alkaline con- stitution of the nerve endings on the other. An important principle governing the distribution of substances in the organism can be deduced for these experiments, namely, that myotropic and neurotropic substances can produce an isolated injury to certain systems solely through the character of the blood-supply. It would, however, be wrong to assume that all muscle and nerve poisons must always injure only the most favored system of muscles as described above. That would be disregarding the fact that the poisonous action is dependent not only on the supply of poisons but also on the capacity of the tissues to take up the poison. A nerve ending of neutral or acid reaction will take up other substances (e.g. alizarin) than one of alkaline reaction (methylene blue) ; a muscle loaded with oxygen will oxidize certain substances and so overcome their poisonous action, whereas this same poison will re- main intact in muscle tissue deficient in oxygen. I believe that the various nerve endings — motor, sensory, and secretory — are made up of the same chemical material. If, however, we consider the manifold and specialized actions of the alkaloids, for example, the very different actions of digitalis, curare, pilocarpin, and atropin, and if we ascribe the toxic action to an accumulation, we shall be forced to conclude that the nerve endings, though com- CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION 423 posed of the same chemical substances, are subjected to different ■conditions in the various tissues, conditions which may possess a decisive influence. Foremost among these I regard variations in the reaction and in the degree of oxygen saturation to which I have already referred. As a result of my experiments in biological stain- ing I assume that certain nerve endings, central and peripheral, are ■characterized by a particular complex of such determining factors, and that this "chemical milieu " represents the resultant of the normal physiological functions. Whether these views possess any heuristic value for the further development of the science, I do not know. For the present I shall content myself by remarking that the isolated 'disease of nerve or muscle apparatus, so far as it affects certain par- ticular groups (lead paralysis, arsenic paralysis), is readily explained from this point of view. We shall have to assume the existence of just as many different types of nutrition as we can demonstrate different types of disease. This brings me to a further question which concerns this dis- tributive therapy, and that is whether it is possible simply by chem- ieal means to change the type of distribution of a given substance. This question can readily be answered ia the affirmative. If, for ■example, a frog is injected with methylene blue, the nerve endings, as is well known, will be stained in the living state. However, if an easily soluble a.cid dyestuff, e.g. orange-green, is added to the methylene blue solution so that a clear green solution results, it will be found that the injection of such a mixture no longer produces staining of the nerve endings. Hence we see that the conditions are entirely analogous to those which we find in the staining of dry prepa- rations. The basic dyes by themselves stain nuclei, whereas the combination of basic dyes with acid dyes, which I introduced into histological technique under the name of "triacid dyes," lack this property to a greater or less degree. In both cases we are dealing with a distribution of the methylene blue between the acid dye and the tissue constituents. The tissues as well as the acid dyestuff have an affinity for the methylene blue. If the affinity of the tissues is greater, they will be stained blue; if that of the acid dye is the greater, the staining will not occur.i ' Naturally this phenoraenon will occur conspicuously only in those cases in which the tissue substances possess an affinity for the base only and not for the acid dye. If the latter condition obtains the mixture of both components 424 COLLECTED STUDIES IN IMMUNITY. In the deflection of methylene blue by means of orange we thus have presented a phenomenon which in its essential features reminds, us of the mode of action of the antitoxins. The opposite behavior, however, also occurs, namely, that the localization of a certain substance in a particular tissue becomes possible only through the simultaneous introduction of a second combination, even though the latter effects no union whatever with the first combination. Naturally these complicated phenomena can be demonstrated with certainty only by the aid of vital stainings, fox in these can the microscopical distribution be positively determined. The following examples are the result of this method of investigation : Bismarck brown, the well-known basic azo dye, exhibits a certain amount of neurotropy manifested especially in the staining of the gray matter of the brain. This affinity, however, is insufficient to give rise to a staining of the peripheral nerve endings in a frog, particularly a staining of the taste bulbs. If, however, a frog is injected with a mixture of methylene blue and Bismarck brown it will be found that the terminal apparatus is stained a mixed shade. The blue very readily loses its color through reduction, and in a preparation mounted on a slide and sealed with a cover-glass the blue color can be seen to disappear rapidly, leaving only a pure brown stain. The other example is still more striking: If a rabbit is infused with a solution of methylene blue, one always finds well-marked stain- ing of the pancreas, due especially to a staining of the granules and protoplasm of the islands of Langerhans. In no case have I ob- served a staining of the nerve endings under these conditions. If, however, one adds certain dyestuffs of the triphenylmethane series to the fluid infused, dyes which in themselves do not stain the nerve endings, a truly beautiful staining of the nerve apparatus frequently occurs. In these and other similar cases I believe that we can only assume that the favoring substances cause a modification of the function of the apparatus in question, and that this carries with it a change in the '"chemical milieu" defined above, and so in the ab- sorbing power. It is possible that similar factors also play a certain r61e in many abnormal actions of drugs, especially in inherited or acquired hypersensitiveness. (i.e. the neutral stain) will come into play, a fact which is so well observed in the staining of the neutrophilic granules. CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION. 425. The question now arises as to how we conceive this selection of the tissues to occur. It is very probable a -priori that we are dealing with chemical affinities in the widest meaning of the term. We must, however, discuss in detail the nature of these affinities. In this, I must emphasize, we are dealing primarily with substances which, like the various natural and artificial drugs, are foreign to the body, not with foodstuffs capable of assimilation. The latter will be treated by themselves subsequently. The simplest case is that in which the organism is injected with indifferent substances, neither acid nor basic in character, to which, corresponding to their constitution, we can ascribe no great chemical affinities, but which nevertheless exert marked and often highly toxic effects. In this category belong especially the various hydro- carbons, e.g. toluol, benzol; a number of ketones, such as acetophe- non; many sulfones, which are characterized by their chemical in- difference; also various kinds of ethers, alcohols, and a large number- of other narcotics. The best opinion seems to be that in these cases no direct chemical affinities come into play on the part of thfr organism, and that the molecule is always present in the tissue constituents unchanged and chemically uncombined. That is to. say, the phenomenon is one of contact action. In spite of this it can readily be shown that all these compounds possess a typical localization in the tissues, the cause of which we shall soon discuss. First, however, I should like to say a few words concerning the historical side of this question. The idea that chemical substances, can act solely through contact was first affirmed many years ago, thus by Buchheim in 1859, Schmiedeberg in 1883, Harnack in 1883, and by Geppert. The latter's investigations may be found in the Zeitschrift fiir klin. Medicin, Vol. XV, and deal with the nature of prussic-acid poisoning. He showed that in this highly interesting case the hydrocyanic acid acts as such. He explained the result of the toxic action in the following manner: " We know that chemical processes are retarded simply through the presence of minimal amounts of prussic acid. Thus iodic acid does- not yield up its oxygen to formic acid under conditions otherwise favorable if even a minimal amount of prussic acid is present. It is quite natural, I suppose, that in the poisoned organism, highly 426 COLLECTED STUDIES IN IMMUNITY oxidized substances (the analogues of iodic acid) are no longer able to yield up their oxygen to oxidizable combinations when prussic acid is present. (One must think of these highly oxidized substances as transmitters or carriers of oxygen.) Prussic acid poisoning is therefore an internal suffocation of the organs." This discovery of contact action constituted the first step toward penetrating the mystery of the action of drugs. This, however, afforded no explanation as to why the substances mentioned ex- hibited an elective action. That was because the link was missing which, according to modern views, is absolutely indispensable, namely the connection between action and distribution in the tissues. I think I am justified in claiming to be the first to recognize the right path, for in 1887, in my article on " The Therapeutic Significance of the Substituting Sulphuric Acid Group" (Therap. Monatshefte, March, 1887), I demonstrated that neurotropic stains are deprived of this property on the addition of the sulfonic-acid group. Even at that time I compared the localization of the dyes and of the alkaloids in the brain with the principle of the shaking-out procedure devised by Stas-Otto, expressing myself as follows: "The principle of 'shaking-out' poisons devised by Stas-Otto depends on the fact that basic substances, e.g. alkaloids, etc., are generally firmly combined in acid solutions, and hence extracted •with difficulty, whereas the same substances can readily be shaken out of alkaline solutions. Acid substances, of course, exhibit exactly the opposite behavior: they are held back by alkaline media, but readily given up by acid media. If we apply these experiences to the question under discussion we can readily understand why basic dyes (which are not held back by the blood through any chemical affinities) are especially laid hold of by the brain, whereas the acid dyes and the sulfonic acids (which are bound by alkalies of the blood to form salts, and are thus anchored, as it were) show exactly the opposite behavior." Besides this I showed that fat tissue behaves like the brain, for a large part of the substances taken up by the brain are taken up also by the fat tissue. In 1891 this question received a fresh impetus, for Hofmeister, Pohl, and also Spiro, called attention to the significance of loose combinations which could readily be dis- sociated. Thus in 1891 Pohl showed that the ability of the red blood-cells to take up chloroform, a fact which Schmiedeberg had 'demonstrated in 1867, was due to the cholesterin and lecithin which CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION. 427 the cells contain Both substances can be shaken out with chloro- form. He also referred the union of chloroform in the brain to similar fat-like bodies in that organ, as 1 have done for the color- ing matter of the alkaloids. A basis was thus secured from which to study the action of the above-mentioned substances in the brain. These substances, it will be seen, are most all readily soluble in fats and fat-like bodies, corresponding to their physico-chemical nature.^ The conditions, however, were far more complex in the large number of bodies which, like many medicinal substances (e.g., the antipyretics), and the most varied basjc substances (among these the alkaloids), phenols, aldehydes, and many others, in contrast to the indifferent bodies, do not seem incapable of combining synthetically with the tissues. In numerous articles Low assumes that most of the bodies in question are able to unite synthetically with constituents of the cell or with the living protoplasm. It is obvious that we must assume the protoplasm to contain many different kinds of atomic groups possessing very strong affinities, and it was certainly very plausible when Low ascribed a leading r6Ie in the phenomena of poisoning, to groups so well able to act. His experiments and re- searches lead him to conclude that in the cell it is particularly alde- hyde groups or labile amido groups which play this anchoring or grasping r61e. According to Low all substances which can combine with these two radicals are poisons for the protoplasm; the greater the affinity the stronger the poisonous action. Against this view of a substituting action of the poisons a large number of easily verified facts can be brought forward. If benzalde- hyde and anilin (or phenylhydrazin, etc.) are mixed, the two sub- stances will condense to form a new substance, benzylidenanilin, water separating at the same time. This benzylidenanilin is a single ' It is impossible to do more than refer to the great advances made since my address, especially through the labors of Hans Meyer and Overton. In three studies on the theory of alcohol narcosis (Archiv f. experim. Pathologie 1899-1901), Meyer has shown in the most exact manner tor a large number of chemical substances that the mode of action of the indifferent narcotics is not dependent on their other chemical properties but is governed exclu- sively by the partition coefficient which determines their distribution among water and certain fat-like substances (brain and nerve fat), H Overton came to the same conclusion regarding the causal relation between solubility in fat and narcotic action. His investigations, which have been gathered together in a work entitled "Studien iiber die Narkose," Jena, 1901, dealt especially with vegetable cells and small animals present in the fluid. 428 COLLECTED STUDIES IN IMMUNITY body which does not give up either anilin or benzaldehyde to indif- ferent solvents. It requires chemical splitting in order to form the- two original substances. In this way the question can very readily be decided whether or not a certain substance is anchored to a cell synthetically, for the material in question need simply be treated with indifferent solvents possessing strong extractive properties (alcohol, ether, etc.). If animals are injected with the most varied poisons, alkaloids, phenols, anilin, dimethylparaphenylendiamin, antipyrin, thallin, etc., and if one waits until the distribution is completed (which usually occurs in a moment), it is easy to extract the unchanged poison by means of suitable methods of extraction, and, provided the substance is easily detected, like thallin or dimethylparaphenylendiamin, to discover it in the tissues by means of staining reactions. Naturally these experi- ments are carried out most strikingly with dyestuffs, for in these the extractive decolorization of the methylene-blue brain cortex or of the fuchsin kidney can very easily be followed. The experiments with dyestuffs furnish still another argument against a process of substitution. In the basic dyes when one or several amido groups are replaced by aldedyde radicals a change in color often takes place. Thus by means of aldehyde, fuchsin red is made to yield violet dyes. In accordance with Low's theory one would have been led to suppose that when suitable dyestuffs were employed a change of color due to substitution should occur in some case or other and in some organ or other. In spite of experiments, specially devised for the purpose I have never observed this to occur, either with dyestuffs which, like those mentioned above, unite with, aldehyde, or with certain basic dyes (e.g., the azonium base which Kehrmann produces from safranin) which take up amido radicals, of the most varied kinds and cause an intensification and change of the color characteristics. Many other reasons can be adduced which speak against the correctness of Low's theory. I may merely mention the transitory character of the action, a point which is so often noted, especially in the alkaloids; furthermore, in the case of many drugs, the rapid elimination, which argues against a firm synthetic combination; another fact, one which may perhaps be of practical importance, is this: that in the construction of new therapeutic substances efforts were directed particularly to the elimination (by appropriate sub- stitution) of groups which could effect syntheses. This is the case. CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION. 429 for example, with phenacetln, in which by the introduction of the methyl radical and of the acetyl group the powerful OH and NH2 groups of paramidophenol are occupied. A4I this has led me to conclude positively that Low's theory of the substituting action of therapeutic substances is untenable. Ey this I do not in the least wish to say that groups capable of xeacting, such as Low presupposes to exist in the living protoplasms, cannot occur there. It must be borne in mind, however, that condensation phenomena are not produced merely by the presence of two substances capable of condensing, but that the combining affinity must usually first be increased through appropriate means, such as increase of temperature, the addition of substances abstract- ing water, etc. Even in the practice of the synthetic chemist, who allows the substances to act on one another either directly or in con- centrated solutions, such direct condensations are not especially fre- quent. The numbe.- of these, however, is still more limited if tiie synthesis is to occur under conditions corresponding to those in the living organism, i.e. in dilute solutions, at low temperature and in the absence of suitable auxiliary substances. Dimethylamidoben- zaldehyde unites with indol, for example, even in dilute solutions, at room temperature, forming a red dye, but only when the solution contains small amounts of f ee mineral acid. If this is absent, or if the solution is even faintly alkaline, no combination of any kind occurs. VI. These considerations lead at once to the view that in certain cases apparently it still is possible to effect a substitution within the organism by the introduction of chemical substances. In order to accomplish such a synthesis the selection of suitable substances will be prerequisite, and these substances must be of such a chemical constitution that they can exert chemical influences of the most powerful kind. I have made extensive experiments with many hundreds of different combinations, and in all of these I have only discovered one substance to which I am inclined to ascribe such a substituting action on protoplasm. This substance, vinylamin, discovered by Gabriel and described by him in a masterly manner, is formed by abstracting bromine from bromethylamine by means of potassium. 430 COLLECTED STUDIES IN IMMUNITY. CH2 Bromethylamine = ll/H Since then, however, Marckwald has positively shown (1900- 1901) that this substance cannot, as was at first supposed, contain a double bond (ethylene combination), for it does not reduce per- manganate at ordinary temperature nor take up bromine. It caa therefore only possess the constitution of a dimethylenimin: CH2\ I >NH CH2/ In view of this a complete analogy exists between the ethylenimim and the ethylenoxid: CH2^ >0 CH /^ In conformity with Bayer's tension theory we must ascribe ao extraordinary tension to the three-sided ring contained in the di- methylenimin. This manifests itself also in the fact that this sub- stance shows a marked tendency, through the addition of acid radicals and the breaking of the ring, to pass over into a substituted ethyl- amin of the chain series. Thus, as Gabriel showed, HCl is added with the formation of chlorethylamin, and sulphurous acid with the forma- tion of taurin. These reactions proceed with great energy, as is shown by the fact that even in dilute watery solutions of the freshly prepared hydrochloride an alkaline reaction develops within a few minutes, due to the formation of free chlorethylamin which reacts alkaline. Ethylenoxid behaves in an analogous manner. This is shown in surprising fashion by the fact that this neutral body precipitates magnesia out of chlormagnesium, iron oxide out of iron chloride, entirely after the manner of free alkalies. In doing so it adds the acid radical and becomes transformed into chlorethylalcohol. These two substances, ethylenimin and ethylenoxid, are highly toxic combinations as has been shown by the researches of Levaditi and myself. The pathological changes excited by dimethylenimin ' I have taken the liberty of somewhat modifying the text of this chapter in accordance with the positive advance of our knowledge, which we owe to the labors of Marckwald. CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION 431 are especially interesting. Administered to a great variety of ani- mals (mouse, rabbit, dog, goat, guinea-pig, rat) in doses which cause death after IJ to 2 days or more, this substance causes total necrosis of the kidney papilla. In the rabbit Levaditi found, besides this, marked changes extending from the pelvis of the ureter to the urethra, and consisting of necrosis of the lining epithelium, hemorrhages, and cedema. (Archives internat. de pharmacodynamie. Vol. VIII, 1901.) Every one who has learned to know these changes — changes absolutely unique in pathology— will be forced to the assumption that this localization is dependent on a direct attack of the vinylamin on the affected epithelia, an ethyl amido group entering the proto- plasmic molecule. This assumption is supported by the fact hat only the active three-sided ring is able to produce this phenomenon, not the ethylene combination (CH2^CH2), furthermore, the fact that neunn (trimethylvinylammonium hydroxid) which can be obtained by an exhaustive methylation of the dimethylenimin, acts in an entirely different manner. That we are dealing with a typical ethylene com- bination is shown by the behavior toward bromine and permanganate of potash. It has, of course, long been known that neurin is a highly toxic substance. Aside from its clinical toxicological mode of action it is characterized by an exceedingly evanescent action in contrast to dimethylenimin. The toxic phenomena develop rapidly and dis- appeai equally so without leaving behind any permanent injuries, especially destruction of the papillse. In contrast to this, vinylamin is characterized by a slowly developing action, which in small doses may show several hours' incubation period and leaves the organism permanently damaged. I have compared this action with that of several other compounds which I have studied; thus camphylamin, which according to Duden has the composition /C-NHg CsHiZll allylamin with a double bond (ethylene radical) : CH II CH \NH2 432 COLLECTED STUDIES IN IMMUNITY. and propargylamin, which contains the acetylen group, C— H III C All of these substances were found to possess the evanescent ^general symptoms together with an absence of permanent organic injuries. Hence I believe that the chemical avidity of the double and triple combinations is insufficient to effect substitutive reac- tions with the protoplasm. I am strengthened in this view by the CH fact that prussic acid, which owing to its threefold combination |{| N' can be classed with the most active substances known to chemistry, is nevertheless not anchored in the animal body, as can be seen from Geppert's findings already referred to. If we consider that substances which possess double or triple bonds are usually much more poisonous than the corresponding saturated combinations, ^ and if we bear the above considerations in mind, we shall ascribe this increased toxicity not to a combining ■capacity but to the fact that the unsaturated groups possess auxotoxic properties, i.e., that they are able to increase the toxicity when they -enter into complexes which in themselves already possess certain toxic properties. I must emphasize the fact that all observations thus far made ■are only to be applied to organic substances foreign to the body We must, however, assume that all substances which enter into the construction of the protoplasm are chemically fixed by the proto- plasm. A distinction has always been made between substances capable of assimilation, which serve the nutrition and enter into a permanent combination with the protoplasm, and substances foreign to the body. No one believes that quinine and similar substances are assimilated, i.e., enter into the composition of the protoplasm. The foodstuffs, however, are bound in the cell, and this union must be regarded as a chemical one The sugar molecule cannot be ab ' Neunn is twenty times as toxic as cholin (trimetbylethyilammonium hydroxide); allylalcobol fifty times more toxic than propyl alcohol; ct also Low, NatiJrliches System der Giftwirltungen 1S93, page 95. CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION 433 stracted from the cells with water; it must first be split off by means of acids in order to set it free. Such a chemical union, however, just as every synthesis, presupposes the presence of two combining groups of maximal chemical affinity which are fitted to one another. Those groups in the cell which anchor foodstuffs I term "side-chains" or "receptors;" the combining group of the food molecule the "hap- tophore group." Hence I assume that the living protoplasm pos- sesses a large number of such "side-chians" and that these in virtue of their chemical Constitution are able to anchor the greatest variety of foodstuffs. In this way the cell's metabolism is made possible. This view of the constitution of the protoplasmic molecule has made it possible to get a much clearer insight into the action of the toxins and into the hitherto mysterious phenomenon, the formation of antibodies. I assume that the toxins, just like the food mole- cules, possess a particular haptophore group, which, by fitting into the receptor of the cell, gives rise to the poisonous action. Putting this receptor out of action causes a formation of new receptors to replace it, and these are finally thrust off into the blood. The re- ceptors thus present in the blood constitute the antitoxin. This theory, known as the "side-chain theory," has proven its worth in the hands of numerous investigators, for by its means the manifold reactions of immunity are all led back to the simplest processes of cellular life.^ Hence I assume the presence of a haptophore group only in such combinations which, like the foodstuffs, enter into the substance of the protoplasm, or which, like the large number of poisonous and non-poison- ous metabolic products of living cells, effect a union similar to that of the foodstuffs. The marked difference between the two classes of substances becomes plainly evident by the fact that only those substances possess- ing haptophore groups are able to excite the production of antibodies through immunization. And despite the most painstaking eflorts neither other investigators nor I have ever succeeded in producing any appreciable production of antibody with alkaloids, glucosides, or drugs of well-known chemical constitution. ' I content myself here with these brief remarks and refer the reader to my more recent detailed articles: 1. On Immunity, etc., Croonian Lecture, Proceedings of the Royal Soc, Vol 66, 1900. 2. Schlussbetrachtungen zur Anaemic, in Nothnagel's Handbuch, Vol. VIII, 1901. pages 555 et seq. 3 Die Schiitzstoffe des Blutes, page 364 of this volume. 434 COLLECTED STUDIES IN IMMUNITY. VII. In the case of the chemically defined poisons, drugs, and dyes discussed above, incorporation into the protoplasmic molecule does not, barring a few exceptions, take place by means of synthesis. Since, however, almost the greater part ot ail substances foreign to the body exhibit a typical selective action in the tissues, it becomes neces- sary to study the reasons for this action. Here again we shall do best to begin with a consideration of the phenomena which takes place in staining reactions. A cotton fibre placed in a dilution of picric acid of one to a million takes up the dye, becoming intensely stained. Methylene blue introduced intra vitam into the organism is taken up by the nerve endings. In poisoning by alkaloids certain nerve centres may react specifically and alone. All of these phenomena are obviously analogous in their nature. It seems necessary, therefore, to discuss briefly the views held concerning the nature of the staining process. The purely mechanical conception which refers it all to physical processes, such as surface attraction and absorption, can probably be discarded for the staining of substances in general. This leaves only two other explanations, either of which may be the cor- rect one for certain cases. The first of these, maintained particularly by Knecht, proceeds from the assumption that certain constituents of the fibre substance form with the dye insoluble salt-like combinations usually termed laky combinations. This conception is supported by the fact that by treatment with alkalies an acid can be obtained — lanuginic acid derived from wool, and nucleic acid from nuclear substances — which possesses the property of precipitating the salts of basic dyestuffs even out of very dilute solutions. Analogous conditions are found to a great extent in vital stainings. I need only remind the reader of the investigations of Pfeiffer. These show that in the vital staining of plant-cells one can frequently observe that the staining is due to conspicuous granules of the almost insoluble tannate of methylene blue. Naturally in the higher animals secretion substances present in the cells and constituting precipitants which form laky combina- tions can play a part in localization. The second theory, one which associates the staining process with the phenomenon of solid solutions, we owe to the researches of O. N. Witt. This investigator starts with the fact that silk dyed CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION 435 with rhodamin exhibits a beautiful fluorescence. Rhodamin itself, however, shows fluorescence only when in solution; when in the dry state, even in the finest possible form, it merely shows a pure red color. . Because of this fluorescence Witt assumes that the dye forms a homogeneous mixture with the fibres of the silk, i.e., it is in the form of a solution. Since the fibre, however, is a solid substance this solution must be what Van't Hoff terms a "solid solution." We know that the same dye often produces different tints in various kinds of fibres. This is analogous to the fact that the same substance often dissolves in different solvents in entirely different tints, as is the case, for example, with iodine. Witt therefore believes that the process of staining proceeds exactly the same as the distribution of a substance in two different solvents. Thus, if we dissolve anilin in water, we find that we can shake all the anilin out with ether, because the solvent power of the ether is greater than that of water. In the staining process such a. vast difference in solvent power shows itself by the fact that the materials introduced entirely exhaust the staining-bath. If, however, the difference in solvent powers is less than this, e.g. in the combination water, ether and resorcin, we shall find that the resorcin is distributed between both fluids in accordance with a law of distribution which can be figured out mathematically for every case. In dyeing this type corresponds to the dyes which are said to "take" poorly. In these the staining-bath does not become exhausted under ordinary conditions. Exhaustion can be effected only through the addition of certain substances which limit solution (salt dyes, etc.). In the introductory chapter I have already mentioned that all neurotropic and lipotropic substances lose the property to stain brain substance and fat by the introduction of the sulfonic acid radical. If these substances are examined in a test-tube it is found that this substitution has caused them to lose also the solubility in ether or in fats. Thus, although flavanilin is easily taken up by ether from an alkaline solution, not a trace of flavanilinsulfonic acid is taken up. Another interesting case may be mentioned, one which concerns staining with neutral red. This has the following formula: NHa N N(CH3)2 436 COLLECTED STUDIES IN IMMUNITY This substance has the property of staining the granules of cells most intensely, and the same holds true of a number of derivatives, e.g. violet dimethyl neutral red, in which the two hydrogens of the second amido group are replaced by two methyl groups; further, also, the golden-red diamidophenazin: NH2 N N(CH3)2 In contrast to this, however, the combination in which one of the central amin radicals contains an ethyl group which gives to the group the character of an ammonium base, is absolutely unable to effect the staining. All phenazin derivatives which stain granules can be completely shaken out of weak alkaline solutions by means of ether, whereas not even a trace of the ammonium base belonging to the safranin series is thus taken up by the ether A very intimate connection, however, exists between solubility in the test-tube and ability to be absorbed in the organism, a connection which 1 observed as long as fifteen years ago. Hence we must assume that certain fat-like substances of the nervous system as well as the fat of fat cells possess a high solvent power by means of which these substances are anchored or stored up in the tissue in question, just as the alkaloids are taken up by the ether in the Stas-Otto pro- cedure. ^ If we bear in mind not only the extraordinary multiplicity of Substances foreign to the body, but also the varying chemistry of the tissues which make up the organism, we shall not expect that a single principle can be rigidly applied to the phenomenon of ' This behavior has been studied especially by Overton, He terms the substances of the bram which serve as extracting agents "lipoids" Chief among these are cholesterin and lecithin Among the alkaloids Overton dis- tinguishes feebly basic and more strongly basic substances. The former can be shaken out — for example, the indifferent narcotics; whereas the more strongly basic unite with constituents of the cell to torm salt-like combinations which are very easily dissociated. According to Overton's conception therefore Knecht's explanation would apply at one time and Witt's at another CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION. 437 selective action. For a large number of substances which localize in fat or fat-like bodies during life, it will probably be difficult to prove whether a pure shaking-out process occurs or a formation of but slightly soluble salts. Furthermore, both processes may occur toegther, as Knecht as- sumes in dyeing, the lake-forming components being contained in the tissues in the intimate molecular mixture characteristic of solid solutions. In that case the resulting selective action will be due to a combination of salt formation and solid solution. In many instances, however, it will be extremely difficult to decide whether one is dealing with solid solution or salt or double-salt formation, especially since chemistry often finds it impossible to decide this question in the case of pure bodies. This is seen, for example, in the study of mixed crystals which are looked upon mostly as crystalline solutions. '^ In any case we see that even without the intervention of a chemic- synthetic union the conditions necessary for a selective storage of a substance in the organism are present and are sufficient both in extent and in variety .^ That these conditions in the case of the salt-like combinations are essentially chemical in nature is self-evident; in the case of the solid solution the enormous mass of evidence which I have merely touched makes this extremely probable. If we regard the principles governing distribution in the organism from these standpoints we shall no longer be surprised that in the localization ' If two combinations of somewhat similar chemical constitution (for ex- ample, benzole and pyridin; stilben, benzylidenanilin, and azobenzole; fluoren and diphenylenoxid) form mixed crystals with each other, one can readily comprehend this in view of their close chemical relationship, and can ascribe it to "isomorphogenous" groups. Frequently, however, substances crystallize together which exhibit the greatest divergence in the configuration of their molecules, as, for example, phenol and urea, chloroform and salicylid, triphenyl- methan and benzol. The crystalline fiery-colored combinations which picric acid is able to effect with a large number of hydrocarbons are especially im- portant. Certain investigations concerning the basic properties of oxygen (Baeyer) and of carbon (Kehrman and Baeyer) seem to show that such crys- tallizations, as, for instance, of ferrohydrocyanic acid with ether, etc., are anal- ogous of salt formation. ' I must here refer the reader to the extremely interesting investigations of Spiro (IJber physikalische und physiologische Selection, Habilitationsschrift, Strassburg 1897). In these, although starting from entirely different stand- points +he author reaches many of the views held by me. At the time of my address I was unaware of this study, as it is not to be had in the bookshops. 438 COLLECTED STUDIES IN IMMUNITY of substances foreign to the body synthetic processes play practically no r61e whatever. If we take methylene blue as an example, we see at once that we can easily find a large number of different fluids which are able to shake it out. On the other hand, we know of a large number of acids, like picric acid, phosphomolybdic acid, hyper- sulphuric acid, which are able to precipitate the methylene blue in insoluble form even out of very dilute solutions. This dyestuff, how- ever, is practically useless for synthetic processes; all the efforts of the chemists to introduce other groups into the completed molecules (with one exception, nitro-methylene blue) have absolutely failed. When we stop to consider that in such chemical procedures the strongest possible agents can be used, sulphuric acid, high tempera- tures, etc., we shall at once see that methylene blue cannot at all be synthetically bound in the organism. The extensive distribution of methylene blue, however, is very easily explained by the plentiful opportunities offered for localization. Synthetic processes, such as occur in the absorption of foodstuffs, in assimilation, and in the growth of living matter, are connected with the existence of certain chemical groups, the "receptors." These receptors are able to synthesize with fitting haptophore groups of the foodstuffs or of the toxins, the two groups fitting specifically to each other (like lock and key: E.Fischer). The eagerness with which the living protoplasm lays hold of the foodstuff which it re- quires is in marked contrast to the manner in which it resists taking up substances foreign to itself. This was observed even in the begin- ning of histology, for at that time it was regarded as an axiom that living cells could not possibly be stained. Gerlach, for example, had shown that an amoeba does not take up any coloring matter from a solution of carmine, whereas it stains immediately when it is dead. Since then, to be sure, largely through my efforts, we have come to know a number of important vital stains (neutral red, methylene blue, brilliant cresyl blue), but closer analysis of these phenomena have shown that that which can be demonstrated in the living cell by the various dyes is not the functionating protoplasm but its lifeless (paraplastic) surrounding medium and the granules, etc., present therein. In this point I agree entirely with Galeotti. CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION 439 VIII. What practical conclusions can be drawn from these considera- tions? We see that drugs, such as the majority of narcotics — in fact the large number of neurotropic and lipotropic substances — ^be- come localized through a shaking-out process. It follows from what has already been said that only such substances can be anchored at any particular part of the organism which fit into the molecule of the recipient combination as a piece of mosaic fits into a certain pat- tern. Such configurations, however, are not confined to a single substance, but usually include a large group of related substances. In this connection the investigations which Einhorn ^ and I made concerning the action of cocaine are most important. Cocaine is a derivative of ecgonin, whose molecule contains two groups differing in function: a hydroxyl group, which combines with acid radicals, and a carboxyl group, which forms esters with alcohol radicals. All derivatives of ecgonin in which both groups are thus occupied represent bodies of the cocaine series. Thus in the cocaine ordinarily used in medicine the acid radical is that of benzoic acid, the ester former is a methyl group. By means of the methods of modern chemistry it has been possible to introduce the greatest variety of radicals into ecgonin, leading to the formation of a large number of homologous substances. It was soon found that the substitution of other alcohol radicals, such as ethyl, propyl, etc., for the methyl radical did not cause the least change in the physiological effects of the cocaine,- as Falk proved. On the other hand, the acid radical is of prime importance for the anaesthetic action of the cocaine. Pouls- son, Liebreich, and myself studied the various cocaines with other acid radicals (cinnamyl cocaine, phenacetyl cocaine, valeryl cocaine, phthalyl cocaine) and found only one, the phenylacetic acid derivative which possessed even feeble anaesthetic properties. As a result of these toxicological experiences one could have assumed that this benzoyl cocaine was in every way unlike all other acid derivatives. But this is not the case, for I was able to show that so far as another toxic action is concerned all of the various cocaines show the same ' Einhorn is one of the best authorities on alkaloids known to me. The studies referred to, appear in the Deutsche med. Wochensch. 1890, No. 32, and in Berichte der deutschen chem. Gesellschaft 1894, Vol. 27, page 1870. 440 COLLECTED STUDIES IN IMMUNITY. behavior, namely, in mice they all produce a peculiar fo4.m-like degen- eration of the liver-cells which I have observed only in substances belonging to this series. From this it follows that all bodies of the cocaine series are alike so far as the liver is concerned. Considering that the substances which precipitate and dissolve these bodies are the same and that the liver findings are identical, we may perhaps assume that all cocaines are taken up by the liver in the same way and therefore probably also by the other parenchyma. And since the benzoyl derivative is the only one which possesses anaesthetic action we sliall have to assume that the rest of the molecule is only the carrier which brings the benzoic acid radical to the proper place. (The ansesthesiophore character of this group had already been made very probable by the earlier investgations of Filehne.) Let us go back to our illustration of the mosaic in order to get this idea clearly before us. In order for a piece to help complete a given figure it is first necessary that it possess a particular form, but in order that the pattern be really completed the piece must also possess certain material properties, such as hardness, color, lustre, etc. It will be one of the problems of the future to extend our knowledge concerning the active toxophore groups. The first fundamental experiments in this direction were made by Ladenburg, who showed that the two substances obtained on splitting atropin, namely, tropin and tropic acid, could readily be recombined and the atropin molecule thus be reconstructed. As a result of this demonstration that atropin represents an acid ester of tropin it was possible to produce a number of homologous combina- tions, Ladenburg's "tropeins," e.g., benzyltropein, salicyltropein, phenylglycoltropein (homatropin). A comparative study of the these substances showed that for mydriatic purposes aromatic oxyacids were the most favorable — and especially those in which the hydroxyl is in aliphatic combination, as in tropic acid and phenylglycolic acid. In cocaine, Einhorn and I attempted to determine the function of the benzoyl group by introducing various side-chains. It was found that the introduction of a nitro group in the meta position had a marked influence on the anaesthetizing property of cocaine without preventing the injurious action on parenchyma described above. The introduction of a hydroxyl group in the same place acted still more strongly in this direction, for the anaesthetizing property had dis- appeared, the toxic action on the liver decreased. Meta-amido cocaine was entirely inert. CHEMICAL CONSTITUTION AND PHARMACOLOGICAL ACTION. 441 What was extremely interesting was the fact that by the intro- duction of suitable radicals into this inert amido cocaine the alka- loidal action could be restored. Thus when acetyl and benzoyl groups are introduced into amido cocaine, cocaines are formed which, although they are not anaesthetic, again possess this property of acting on the liver. It is especially interesting, however, that the cocaine urethane obtained by the action of chlorcarbonic acid on amido cocaine again acts ansesthetically, in fact much more so than the original cocaine. That is to say, if we nitrify cocaine, reduce it to amido cocaine, and finally condense it to a urethane, we find that the ansesthesiophore group is first diminished in power, then its action is entirely lost, and finally heightened. We already know the function of the toxophoie group in a number of "alkaloids, in atropin for a single group, in strychnine for two. If only we had a deeper insight into this function we might hope by means of substitutive action on the toxophore groups (such as Einhorn and I have car- ried out on the benzoic acid radical of cocaine) to modify the action of the alkaloids to suit our purpose. In the synthetic field of pharmacology, however, a knowledge of the groupings on which the selective distribution in the organs depends would appear to be far more important. In the case of foodstuffs and toxins I assume that the union is effected by a single definite group, the " haptophore '' group. Substances foreign to the body, as already explained, lack such a single group and the laws of dis- tribution in the organism are dependent on the combined action of the separate components. In their distribution, therefore, the entire constitution of the substance is the deciding factor. This we have seen to be true with substances belonging . to one group. Within this group type, as we have described it in detail with the cocaine series, modifications of the separate components can then be made within wide limits. Starting from this point of view we obtain a new method of synthetic-chemical pharmacology. If one is desirous of studying organ therapy in this sense' it will be necessary first to hunt up bodies which possess a particular affinity for a certain organ. Having found such bodies one can then use them, so to speak, as a carrier by which to bring therapeutically active groups to the organ in question. It is self-evident that in the selection of these groups one is bound by definite limits ; so also is the fact that all substituting groups which themselves influence the distributive character (e.g. acid radicals) must be avoided. All these are problems which ex- 442 COLLECTED STUDIES IN IMMUNITY. tend far beyond the powers of single individuals and make it desirable that chemists and pharmacologists work together in some definite plan. That is one reason why 1 have gone into such detail concerning my views on the connection between constitution, dis- tribution in the organs, and pharmacological action. 1 shall indeed be happy if these views, the gradual development of ten years of study, will advance the study of pharmacology. Translator's Note. — See also the recently published study by Bechhold and Ehrlich on the relation of chemical constitution to disinfecting power. of a saturated solution of cholestenni in hot methyl alcohol, with 9 cc. 85% salt solution A third difference between cobra amboceptor and the finished lecithid is seen in the behavior toward high temperature. The aqueous solution both of the primary and the secondary cobra lecithid is far more stable than solutions of the amboceptor alone. The former can be heated to 100° C. for six hours without any particular loss in power, while the amboceptor of cobra venom loses its action if heated to 100° C. for only thirty minutes. Obviously this is to be explained THE ISOLATION OF SNAKE VENOM LECITHIDS. 477 by assuming that the combination has become firmer by the entrance into it of the lecithin molecule. There is a fourth pomt of difference, the behavior toward the snake-venom serum discovered by Calmette. The finished lecithid is affected far less by this serum than is the cobra amboceptor. We shall discuss this in a later article. In contrast to these differences the behavior of cobra lecithid and cobra amboceptor + lecithin toward cholesterin is similar. We have already mentioned that cholesterin possesses the power to inhibit the haemolysis by means of cobra venom. The same is true in haemolysis by means of the finished lecithid, although quantitatively to a less degree. (See Table 111 opposite.) IV. The Lecithids of Several Other Poisons. Naturally it was of considerable interest to see whether this peculiar formation of lecithid (thus far without parallel in chemistry) was confined to cobra venom, or extended also to other poisons. The following poisons, which we owe to the courtesy of Dr. Lamb, Prof. Calmette, Dr. Kinyoun, Dr. Dowson, and Prof. Kitasato, have there- fore been studied by us for this purpose: 1. Bothrops lanceolatus; 2. Daboia Russellii; 3. Naja haye; 4. Kerait; 5. Bungarus fasciatus; 6. Trimeresurus anamalensis (Hill viper) ; 7. Trimeresurus Riukiuanus (Japan); 8. Crotalus adamantus. In a subsequent article we shall discuss the behavior of these poisons toward different species of blood-cells. For the present, how- ever, we may say that all of these poisons, on the addition of suffi- cient lecithin, dissolve the blood-cells examined by us, namely, those of man, guinea-pig, rabbit, ox. With the exception of two poisons (Bothrops lanceolatus and Trimeresurus anamalensis) the absolute quantity of poison necessary to effect solution, an excess of lecithin being present, is about the same for all species of blood examined; 0.003 grm. are sufficient to dissolve 1 cc. of a 5% suspension. The 478 COLLECTED STUDIES IN IMMUNITY. Bothrops poison is ten times weaker, and that of Trimeresums anama- lensis twenty-five times. This observation made the formation of a lecithid seem probable. As a matter of fact it was easy, by means of the method above described, to prepare a solid lecithid which contained the entire haemolytic power of the poisons.^ Hence we believe that in general all hijemolytic snake venoms are of the am- boceptor type and possess a lecithinophile group, the occupation of which by lecithin gives rise to the haemolytic action. In fact it seems as though in the last analysis the factor which determines the type of the haemolytic action of snake venom was principally this, lecithinophile group. A fact which goes to support this view is the observation that several of the poisons examined by us probably differ in their hapto- phore group, which unites with the receptor of the blood-cells. Thus Lamb 2 has shown that the Daboia amboceptor, unlike the cobra amboceptor, is not inhibited in its action by Calmette's serum. The same is true for Bothrops, Crotalus, and Trimeresurus Riukiuanus, whereas the poisons of BungariLS and Naja haye are similar to the cobra venom so far as their behavior toward the serum is concerned. It is quite possible, therefore, that the differences in the various types of poison are only differences in the haptophore group, while the characteristic lecithinophile group is identical in all cases. It was important to see whether in animals other than snakes poisons are present which are capable of forming lecithids. We therefore next studied the poison of the scorpion, choosing this because Calmette ^ had already shown that the acute fatal action of scorpion poison could be inhibited by the snake-venom serum, a fact indicating a certain analogy between the toxic components of scorpion poison and snake venom.* We were able to determine that the scorpion poison by itself exerts only a slight haemolytic action on guinea-pig blood-cells, leaving other species of blood-cells unaffected. On the addition of lecithin, however, it exerts con- ' In conformity with its weaker action Bothrops poison yields only a tenth the lecithid obtained from the other poisons, and the poison of Trimeresurus anamalensw only one twenty-fifth. ^ Lamb, Scientific Memoirs, Medical and Sanitary Depts., Govt, of India, 1903, No. 3. ' Calmette, Ann. de I'Instit. Pasteur, 1895, No. 4. ' For this scorpion poison we are much indebted to Prof. Treub, Director of the Botanical Garden in Buitenzorg. THE ISOLATION OF SNAKE VENOM LECITHIDS. 479N siderable solvent action on all the different species of blood examined by us. Its action is about one twentieth as strong as that of cobra -venom. (See Table IV.) TABLE IV. Action op Scobpion Poison with and withodt the Addition of Lecithin;. Amounts of the 0.2% 1 CO. 5% Ox Blood. Solution of Scorpion Poison cc. + 0.2 0C. 0.1% Control without Lecithin. Lecithin. 1.0 complete 0.75 I 0.5 1 0.35 t 0.25 t 0.15 1 0.1 t 0.075 i 0.05 t 0.035 t 0.025 ' 0.015 almost complete 0.01 moderate 0.0075 little 0.005 trace 0.0035 tt 0.0025 faint trace 0.0015- Corresponding to this behavior we succeeded in actually pro- ducing a typical lecithid from scorpion poison by following the usual procedure.! All this leads us to the view that the essential character of the hsemolytic cobra venom is due not to the haptophore group, but finally to the lecithin anchored by the blood-cells by means of a lecithinophile amboceptor. Now we know that lecithin is present in every red blood-cell, and this seems apparently to contradict the fact determined by us experimentally that the lecithin is the cause of haemolysis. This contradiction, however, is merely apparent, for we need only assume that by the aid of the cobra venom ' It is probable that the poison of a fish, Trachinus draco (see Briot, Journ. de Physiol, et de Pathol. g4n. 1903, No. 2), is also capable of forming a lecithid; at least a statement of Briot speaks in favor of this, namely, that the haemolytic agent in the Trachinus poison can be activated by a serum which has been heated to more than 60° C. 480 COLLECTED STUDIES IN IMMUNITY. the lecithin is brought into proximity with cell constituents other than those normally in its proximity. In other words, we are dealing with the deleterious action of a vitally important substance which has been forced into the wrong place. This conclusion is made plain if we bear in mind the fact that in the blood-cells primarily suscep- tible to cobra amboceptor, the hsemolytic action depends not on the addition of new lecithin, but on a transposition of the lecithin pre- iormed in the cell, due to the anchoring of the cobra amboceptor. XXXVII. THE CONSTITUENTS OF DIPHTHERIA TOXIN/ By Paul Ehblich. The Festschrift, published at the opening of the Serum Institute in Copenhagen, contains a study by Arrhenius and Madsen ^ which deals mainly with the neutralization phenomena of toxin and anti- toxin. We must all rejoice that Madsen has succeeded in interest- ing so excellent a physical chemist in this question, especially as I had tried unsuccessfully for years to secure the interest of physical chemists in Germany. In the present state of scientific knowledge we shall for the present have to give up oUr attempts to isolate the toxins in pure form. For the same reason also in the analysis of the relations between toxin and antitoxin we cannot conform to the ordinary methods of the chemist working with the balance. On the other hand, the study of toxin and antitoxin is of too great practical importance for us to wait idly for years or decades until chemistry is so far advanced. We must, therefore, content ourselves with the slight means at our disposal, applying these, however, in all direc- tions in order to gain as great an insight into this complicated subject as the present state of our knowledge permits. I had ap- plied, myself to this problem for years and come to the conclusion that the only way to approach it was by an exact quantitative study of the neutralization phenomena. Particularly in partial neutraliza- tion I believed I had found a method by which we could gain an insight into the most intricate constitution of the toxins. To my regret high authorities pronounced this method as incorrect and of no avail. I am all the more pleased, therefore, to see that so high ' Reprint from the Berl. klin. Wochensohr. 1903, Nos. 35-37. ^ S. Arrhenius and Th. Madsen, Physical Chemistry applied to Toxins and Antitoxins, Festkrift ved indvielsen af Statens Serum Institute, Kopenhagen, 1902. (This is to be had in English text, Kopenhagen, 1902.) 481 482 COLLECTED STUDIES IN IMMUNITY an authority as Arrhenius recognizes my method as correct in prin- ciple and proceeds along the same lines. The study of Arrhenius and Madsen deals principally with tetano- lysin, the hsemolytic poison discovered by me in tetanus toxin. Tetano- lysin and tetanospasmin differ from each other in their haptophore groups, as a result of which each possesses a particular antibody in the tetanus serum of the market. Madsen studied this tetano- lysin in my Institute, and found that when it is gradually neutralized with increasing amounts of antitoxin, the same definite amounts of aptitoxin first added neutralize far more poison than subsequent additions. Because of this and also because of other reasons (atten- uation, phenomena during neutralization) Madsen concluded that several poisons of different affinities were present. On taking up these studies in tetanolysin Arrhenius and Madsen obtained practically the same results, and these authors succeeded in constructing a formula for the action of antitetanolysin on tetano- lysin which conforms to the law of Guldberg-Waage. Based on this they next attempted to determine similar relations in the case of very simple blood poisons. This had already been done by Danysz, but the method was open to criticism. Arrhenius and Madsen ("hose a weak base and an acid (ammonia and boric acid) as hsemolysin and antihaemolysin. It was found that in these the neutralization phenomenon is very similar to that of tetanolysin and antilysin, from which they concluded that in the neutralization of toxins and antitoxins we are dealing with reactions between simple substances of weak affinities. In this connection they express themselves as follows: "The last-mentioned curve gives a fairly accurate picture of the neutraliza- tion of ammonia with boric acid. In the investigation of ammonia as haemolysin a spectrum analogous to that of toxin or tetanolysin (Fig. 3) could have been constructed ; the following conclusion could also perhaps have been drawn: One part of boric acid (antitoxin) added to ammonia neutralizes 50% of this base; if two parts are added it neutrahzes 66.7%; if three parts, 75%; and four parts, 80%. From this it follows that since the respective amounts 50, 16.7, 8.3, and 5% are each time neutralized by the same amount of boric acid, the amount first neutralized is three times as toxic as the amount next neutralized, this again twice as toxic as the next after it, which in its turn is one and one-half times as toxic as the following, etc. In other words, ammonia is not a simple substance, THE CONSTITUENTS OF DIPHTHERIA TOXIN 483 but consists of several constituents of different toxicity (and these toxicities bear a simple reciprocal relation to each other). Of these constituents the toxin possessing the highest chemical affinity is neutralized first. A similar conclusion has actually been drawn in the case of toxin; the toxin first neutralized (the strongest) has been called prototoxin, the next deuterotoxin, the next tritotoxin, etc. The final very weak toxins are called toxones." The findings of Arrhenius and Madsen are thus seen to be directly opposed to my statement that diphtheria poison is composed of several constituents. In view of the exceeding importance of the subject I cannot avoid entering the discussion and state the reasons which cause me to maintain my views absolutely and without any modification.! The new views of the authors in question will doubtless lead many to wonder how 1 could err in so simple a matter and employ compli- cated theories when the simplest conceptions of chemistry would have sufficed. It must seem strange that I, who have followed this sub- ject for years and have busied myself especially with chemical studies, should have failed to discover this very ready explanation. As a matter of fact, however, 1 too began with the conception, now held by Arrhenius and Madsen, that in the union of toxin and antitoxin we were dealing with a phenomenon of incomplete neutralization. A more thorough analysis of diphtheria poison (my publications refer only to this poison) compelled me, however, to adopt more complex explanations. At the very outset of my investigations I discovered that tetano- lysin and its antitoxin possess weak affinities, and I devised the tech- ' Gruber, whose experiments especially devised to refute my theory I was able to show were incorrect, has employed the opportunity to side with Arrhenius and Madsen, and to announce that their observations "will give this entire spook of side-chain theory its quietus." No one who knows any- thing about this subject needs be told that the question as to whether diph- theria poison is made up of one or more substances has nothing to do with the side-chain theory. When 1 formulated this theory I too believed the diph- theria poison to be a simple substance, and when subsequently 1 felt compelled to differentiate several components in the poison 1 always emphasized that the separate components differed only in their toxophore group and were similar so far as the haptophore groups were concerned, the groups which give rise to antitoxin formation (see my reply to Gruber in Miinch. med. Wochenschr 1903, Nos. 33 and 34). 484 COLLECTED STUDIES IN IMMUNITY. nique of my experiments accordingly. At that time I stated in connec- tion with this tetanolysin that the union of toxin and antitoxin pro- ceeds more slowly in dilute solutions than in concentrated, and that the process is hastened by heat. How feeble the combining affinities of tetanus toxin and antitoxin are can be seen from the following experiment devised over eight years ago: If a given, not very con- centrated, mixture of serum and toxin is allowed to stand for two hours it will be found that the action of the serum is forty times as great as when the mixture is employed immediately. Whether the optimum of neutralization is thus reached is difficult to say. The determination of the exact limits fails because of the fact that the poison rapidly decomposes in watery solutions, especially if these be dilute. One constantly faces either of two difficulties : insufficient union on the one hand and decomposition of the poison on the other. i With diphtheria poison, on the other hand, the affinity of the toxin for the antitoxin is much greater. As is well known, these substances unite so rapidly that even the time for combination prescribed in the test — fifteen minutes — is still unnecessarily long. Hence, even if I admit that the union of tetanolysin and antilysin is comparable to the neutralization of a weak base by a weak acid, I shall in the following pages show that the affinity of diphtheria toxin and antitoxin is very great, comparable perhaps to that between ,a strong acid and base. In accordance with this also I am convinced that the neutralization of diphtheria toxin by antitoxin proceeds in the form of a straight line and not in that of a curve. This, then, constitutes my first objection to the general deductions drawn by Arrhenius and Madsen from their particular findings. Just as it is impossible to apply the results of the neutralization of boric acid and ammonia to every combination of acid and base, so it is impossible to apply the experiences with tetanolysin to the doctrine of toxins in general .2 '■ When, then, years ago, in spite of these unfavorable conditions, I proposed the study of tetanolysin to Thorvald Madsen, this was but a makeshift neces- sitated by the lack, at that time, of suitable haemolysins. At present a number of such substances are available, such as arachnolysin and snake venom. These are very stable and far better suited for exact determination since the factor of decomposition is absent. ^ 1 should like to mention that recently Dr. Kyes has discovered that in snake venom also the neutralization with antitoxin proceeds with high affinities .and in a straight'lme. THE CONSTITUENTS OF DIPHTHERIA TOXIN. 485 If, therefore, the affinity between diphtheria toxin and antitoxin is so great, we shall have to ascribe the irregular course of the neutraliza- tion process to other factors thau those assumed by Arrhenius and Madsen. Diphther^ toxins. In order to understand what follows it will be necessary to speak of some of the ■main principles of toxin-antitoxin analysis. As is well known diphtheria toxin is the bouillon fluid in which the diph- theria bacilli have grown, and to which they have given up their toxic secretory products. In order to determine the toxicity we make use of guinea-pigs. The lethal dose (L. D.) is that amount of poison which will surely kill a guinea-pig weighing 250 grammes on the fourth day. In order to determine the relations between toxin and antitoxin it is best to start from the serum because this can be preserved constant by means of the methods devised by me (vacuum, drying). This dry serum also serves as the standard for the ofKcia titration. The immune unit (I. E. = Immunitats Einheit) is, of course, an arbitrary quantity which originated by terming that amount of antitoxin a unit which just neutralized 100 L. D. of a poison that happened to be available at the time, so that the mixture when injected did not produce even the slightest trace of illness (either general or local reaction). If one mixes one immune unit of serum with graduated amounts of poison, two limits may be obtained. One of these is termed limit zero (Lq), and corresponds to the quantity of poison which is completely neutralized by 1 I. E. The other is limit death (Lf) and corresponds to that quantity of poison which on the addition of 1 I. E' is so far neutralized that only just one L. D. remains. Of these two limits the Lt is very easily and accurately determined so that it serves as a measure in testing the potency of the diphtheria serum. This limit signifies nothing more than that of a; L. D. present, 1 I. E. neutrahzed x—1 L. D., so that just 1 L. D. remains free and leads to the death of the guinea-pig in four days. A priori one might have expected that the number of lethal doses which are neutralized by 1 I. E. is always the same in poisons from different sources. The only difference which one wpuld have ex- pected would be that in different poison solutions, the volume in 486 COLLECTED STUDIES IN IMMUNITY. which a given number of L. D. were contained would vary from case to case, depending on the varying quantity of poison produced by the bacilli. Closer investigations, however, showed that in reality the con- ditions are entirely different, the number of L. D. contained in Lt varying enormously in different toxic bouillons. In poisons which have been analyzed the figures have fluctuated between 15 and 160. Since it had been shown, especially by myelf, that the neutrahzation of toxin-antitoxin rests on a chemical basis, this result could only be explained by assuming that the diphtheria bouillon, in addition to the toxins, contained other non-toxic substances which were able to combine with antitoxin just like the diphtheria toxin. I deemed it to be of the highest importance to clear. up this mystery experi- mentally, and therefore subjected a number of different poisons (some freshly derived, others precipitated with ammonium sulphate, and still others which had been kept for a long time) to comparative analyses. In the course of these it was found that the non-toxic . substances, which still possess combining properties, increase as the toxic bouillon ages, and I therefore studied these changes in the poisons genetically at various stages. I emphasize this part of my method because the casual remark by Arrhenius and Madsen i that my results were derived mainly from a study of decomposed poisons might readily be misconstrued and give one the impression that in my investigations I had not been espe- cially careful. I may at once add, however, that my most valuable results were obtained by studying the course of this decomposition, but this, of course, corresponds entirely with the methods of chem- istry. It is impossible to gain an insight into the constitution of highly complex combinations by means of an analysis which leads only to the compact formula. This can only be gained by the careful decomposition of the substance to be studied. Whatever knowledge we possess regarding the constitution of sugars, uric acid derivatives, alkaloids, etc., is due mainly to the decompositions intel- ligentljr carried out, and a careful study of their products. Of course, the decomposition must not give rise to secondary reactions which could obscure the results ; this might be the case if strong acids or a high temperature were employed. The decomposition must be quantitative and of moderate intensity. The following observa- tions will show that this is especially the case in the spontaneous THE. CONSTITUENTS OF DIPHTHERIA TOXIN. 487 attenuation of the toxins, which occurs at room temperature and without any further chemical manipulation.'^ It has been found that the bouillon on standing can preserve its neutral- izing property intact, and often actually does so, while the toxicity is considerably decreased. Observations of this kind have been made by myself and Madsen for diphtheria poison, by Jacoby for ricin, by Myers for snake venom, and recently by Arrhenius and Madsen for tetanus poison. This phenomenon, which in many cases is quanti- tative, is most readily explained by assuming that the poison molecule contains two functionating groups. One, the "haptophore group," combines with the antitoxin and in the animal body effects the com- bination with the tissues; this group is quite stable. The other, the "toxophore group," effects the true poisonous action; it is com- paratively readily destroyed. In my opinion the transformation of toxin into toxoids by the destruction of the toxophore group is the key to a correct understanding of my conception of antitoxic im- munity and the subject of toxins.^ If we see, for example, that in spite of decreased toxicity the ■constants of neutralization L-f and Lq remain entirely unchanged, it follows, in my opinion, that two important deductions can be made. The first is one which I have always drawn, namely, that in normal toxoid formation not brought about by chemical additions, the num- ber of haptophore groups suffers no loss. This behavior, however, also seems to indicate that in toxoid formation the affinity of the hapto- phore groups for the antitoxin is in no way changed. I may be per- mitted to elucidate this by means of a chemical example. Tetra- methylammoniumhydroxid is a very strong base (like KOH) which through suitable procedures (heating, etc.) is transformed into the ' Obviously these poisons can also be attenuated through chemic or thermic influences, but the decomposition in that case takes place rapidly and with destruction. In my investigations, therefore, I have never made use of these methods, but have kept to the moderate changes which occur spontaneously in the toxic bouillon on standing. ' At the outset of the modern study of immunity, von Behring, Aronson, and others had observed that an active immunity could be brought about particularly through attenuated, modified poisons. At that time, however, it was very difficult to appreciate these relations, and so in the year 1894 we find a high authority, as a result of his investigations, denying the existence of modified poisons, although he had previously assumed their existence. The results, which had been obtained with immunization, he ascribed, not to the presence of modified poisons, but exclusively to a dilution of the poison. 488 COLLECTED STUDIES IN IMMUNITY. far less basic trimethylamin, methyl alcohol being split off in the process. Let us take a certain definite quantity of tetramethylam- monium hydroxid, say 20 molecules, and determine the quantity of boric acid which will just suffice for complete neutralization, as shown by a suitable indicator. On changing the ammonium base into the tertiary amin (a change which we shall assume to be com- plete) we shall find that a larger quantity of boric acid is necessary for neutralizing the tertiary amin. In other words, there has been a change in the position of the neutral point, although the number of basic radicals remains the same. This necessarily follows from the decrease in affinity brought about by the transformation. The reverse will take place if a weak base is transformed into a stronger one. A change in the position of the neutral point will occur even if the transformation is only a partial one, i.e., does not affect the entire number of molecules. If, however, in spite of an extensive formation of toxoid, we find the test limits unchanged, we can only conclude that any considerable change in affinity has not occurred. We shall subsequently learn of another fact, which affords conclusive evidence of the correctness of these views. Our next problem will be to study the influence of the toxoids on the neutralizing process. To begin, it should be remarked that the bacterial poisons with which we are deahng are not, as a rule, pure poisons. By this, of course, I do not mean to deny that pure poisons can occur. If the toxophore group possesses considerable resistance fo that it is not affected by the processes used in its pro- duction (keeping in the incubator for weeks, etc.), it will be possible to obtain poisons which contain only toxins and no toxoids. Such a result, however, can probably only be counted on in a small number of isolated cases, and is not obtained as a rule. So far as diphtheria poison is concerned, of which I have made a special study, I have never yet, among a large number of specimens examined, found a single one free from toxoids. In estimating the degree of purity one proceeds by finding in various poisons how many fatal doses (L. D.) are neutralized by one immune unit (I. E.). The maximum value in the poisons at my disposal was 130, but Madsen has described a poison in which the Lf dose contained 160 L. D. But even this poison, as I shall show later,i merely approached the character of a pure poison. ' It is especially important that even diphtheria poisons which have been THE CONSTITUENTS OF DIPHTHERIA TOXIN. 489 Naturally the poisons whose toxophore groups are very labile will be the least pure. This is especially true in tetanus poison, which is far more readily destroyed than diphtheria poison. In the former, several hours' standing of an aqueous solution suffices to give rise to toxoid formation. It is all the more probable, therefore,, that the toxin produced in the usual manner by keeping the culture in the incubator for eight days contains a considerable admixture of toxoids. In the precipitation with ammonium sulphate these tox- oids, of course, are present in the resulting solid product. A dry poison of this kind, such as I placed at Madsen's disposal for his experiments, can, of course, keep for a long time unchanged provided it is carefully preserved; the primary content of toxoid, however, also remains unchanged. For this reason I believe that the assumption of Arrhenius and Madsen, that the tetanus poison used by them was a pure poison, since it did not change, is entirely unwarranted. It is even possible that this particular specimen contained far more toxoids than the old toxin solutions which I had employed. In pure chemistry in carrying out exact mathematical determina- tions it is a general principle that the substance be either absolutely pure or at least that its degree of purity be exactly determined by analysis. In determining the molecular weight of an element, a great deal of preliminary work (recrystallization, etc.) is required in order to obtain the original material as pure as possible. If this cannot be done, as, for example, in the case of hydrogen peroxide, or ozone, a quantitative study requires at least that the exact percentage of pure substance contained in the mixture be known. It is hardly necessary to say that these principles should, as far as possible, be applied to the study of toxins. In these substances also one should know the degree of purity before attempting any exact investigations. But just in this domain, where it is impossible to isolate the substances, this task is uncommonly difficult. It required a year's most tiresome and monotonous labor before I was able, by means of very exact deter- minations of all kinds of poisons, to approach this problem. At that produced in a very short time (three to four days in the incubator) are not free from toxoids. In one such poison (No. 9 of the titration series) I found 123 L. D> in L^. I was therefore greatly pleased recently to hear from Dr. Louis Martin, who has had such wide experiences in this direction at the Pasteur Institute, that in his fresh poisons he never saw the figure 200 L. D. in Lf reached. 490 COLLECTED STUDIES IN IMMUNITY time I gained the impression that a pure poison must oe so consti- tuted that one 1. E. fully neutralizes exactly 200 L. D.^ Later on I shall show that by means of the "spectrum" analysis I have suc- ceeded in verifying this figure.^ The discovery of this number, 200, led me to represent the con- stitution of diphtheria poison by means of a "spectrum" which is divided into 200 segments, each of which corresponds to a toxin, toxoid, or toxon equivalent. This scheme is not, as some have as- sumed, a mere makeshift, but is the expression of knowledge labori- ously attained. This graphic reproduction shows at a glance how much toxin or toxoid is neutralized by each combining unit of anti- toxin. Such a reproduction possesses so many advantages over the curve used by Arrhenius and Madsen that I shall not hesitate a moment in retaining the spectrum method for diphtheria poison. By its means one obtains a view of the entire process of neutralization.^ It may be well at this point, by means of a suitable chemical illustration, to elucidate the influence which such admixtures of toxoid exert in the titration of alkaloids. In doing this it will be best to proceed on the following assumptions. An alkaloid acts hsemo- lyticaliy when in the form of free base, but not when in the form of a salt.* The base would then correspond to the toxin. The ana- logue of the toxoid would then be an alkaloid which exerts no dele- terious action either as such or in the form of a salt. The antitoxin would be represented by any acid, e.g., hydrochloric acid. Under these conditions the mixture of the two alkaloids can be titrated bio- logically (by determining the haemolytic power at any point) by means of an acid exactly, as a toxin solution containing toxoid by means of its antitoxin. Let us assume that the toxic alkaloid A as well as the atoxic B possesses so strong an affinity for hydrochloric acid that neutraliza- tion is effected to within a very small fraction. A solution of a mole- cules A would then correspond to the pure toxin, while mixtures of ' It is self-evident that each toxin-combining unit can be replaced by an equivalent amount of less toxic or non-toxic substances possessing combining properties (toxones, toxoids). ' The poison studied by Madsen, therefore, which contained 160 L. D. in Lf, corresponded to a purity of four-fifths. ' See also page 552. ' This is probably the case with solanin, whose hsemolytio power is inhibited by the addition of acid salts (Pohl) or ot free acids (H^don, Bashford). THE CONSTITUENTS OF DIPHTHERIA TOXIN. 491 A and B: ^+4 or j+-^ represent analogues of solutions containing also toxoids. In all of these mixtures the end point of neutralization will be practically constant. If, however, the affinities of A and B for hydrochloric acid are not exactly equal the neutralization will proceed in a straight line only if we are dealing with the pure alkaloid. In all other cases it will follow the course of a curve whose character, of course, is dependent on the relative amounts of the two com- ponents. This problem of the simultaneous neutralization of two alkaloids has been studied in suitable cases by J. H. Jellet. Let us take the neutralization of quinine and codein with hydrochloric acid, in which the coefficient of equihbrium K = 2M. For the sake of simplicity I have assumed this to be 2.0. In order, furthermore, to have the conditions as simple as possible, let us take as an example a mixture of 100 molecules quinine and 100 molecules codein. These will then be neutrahzed by 200 molecules hydrochloric acid. By means of the formula devised by Jellet one next determines how much quinine is transformed into the salt by each successive addition of one-tenth the entire neutralizing dose (20 molecules HCl). It will be found that the first tenth neutralizes 13 and the last tenth 7 molecules of quinine, while the course of the neutralization of the quinine is itself entirely uniform. If another combination is taken, in which the second alkaloid possesses a weaker affinity, so that K=Vi, it can easily be calculated that under these circumstances the first tenth hydrochloric acid neutralizes 17.8, the last tenth only 3 molecules of quinine. On representing these reactions graphically we shall obtain curves entirely similar to those representing the neutralization of a weak base with a weak acid, and it would probably not be difficult to find a combination of alkali and acid whose curve corresponds to the alkaloid curve mentioned. Hence, if such a mixture of alkaloids together with the appro- priate neutralizing agent (acid) were given one for a biological titra- tion, and if, furthermore (to make the analogy with toxin-antitoxin determination complete), the employment of any additional chemical aids was barred, the neutralization curve obtained under such stringent conditions could easily give the impression that one were dealing only with the neutralization of two substances possessing weak affini- ties. Nevertheless, even under these limitations, it is possible to learn the true conditions if, as I have done, one does not confine one's 492 COLLECTED STUD1E8 IN IMMUNITY. self to a single mixture, but analyzes a great many different mixtures in which the relation of toxin-alkaloid and toxoid-alkaloid varies.' It is all the more surprising that in the analysis of the constitu- tion of poisons Arrhenius and Madsen have not studied the question from this point of view because they do not at all neglect the exist- ence of toxoids. Apparently this is because of a slight misunder- standing, for these authors proceed exclusively on the assumption that in toxoids one is dealing with protoxoids, i.e., with toxoids which possess a higher affinity for the antitoxin than does the toxin. In fact, one can easily observe that the formation of prototoxoids affects the end point of the titration but little. This I had predicted in my first study on the evaluation of diphtheria serum. Let us assume, for example, that a mixture of 1 equivalent hydrochloric acid (proto- toxoid) and 3 equivalents prussic acid (toxin) is neutralized by a strong base. In that case the hydrochloric acid will be neutralized first, after which the neutralization of the prussic acid will proceed very much the same as though only prussic acid were present. We must now see whether diphtheria poisons, such as I have investigated, contain other toxoids besides prototoxoids. The ma- terial at hand makes the decision of this point very simple. In four poisons containing a prototoxoid zone (of which two were published by myself and two by Madsenj I have calculated the relation of proto- toxoid and toxoid to toxin. In doing this I have regarded exclusively the Lt dose, and so eliminated the toxons which would otherwise still more increase the toxoid figure. ' In the very simple example of two alkaloids just mentioned two determina- tions of different mixtures would permit the calculation. In my opinion no definite conclusions as to the constants of the toxin can be drawn from the analysis of one particular toxin containing toxoid. Arrhenius and Madsen analyzed two different tetanus poisons, one of which had undergone toxoid modifications through years of preservation as a dry substance, while the other had suffered similar modifications through several days' standing of the solu- tion. The authors calculated from their experiments that in the one case the constant of dissociation had been increased 50%, in the other ten times. In view of what has just been stated this calculation, which leaves out of account the presence of toxoids, cannot be regarded as conclusive. The divergence o£ the constants could easily be due exclusively to the presence of toxoids, and these, in view of the different methods by which the poisons were attenuated could be different in the two cases. I may also add that in the toxoid for- mation of diphtheria toxins I am convinced that the toxin groups which remain do not suffer any change in their affinity. THE CONSTITUENTS OF DIPHTHERIA TOXIN 493 Poison, For 100 Parts of Toxin there are Prototoxoid, Parts Toxoid, Parts. A Madsen C Madsen IV Ehrlich V Ehrlich (4th phase) 160 79 82 77 400 59 200 131 This table shows that the four poisons contain considerable amounts of toxoids in addition to the prototoxoids. The affinity of these toxoids is more or less small, as can be seen from the curves plotted l)y Madsen and myself. From this it follows that in the interpreta- tion of the results obtained by neutralizing diphtheria poison due attention must be paid to the decisive influence exerted on the course of the partial neutralization by the toxoids notoriously present in such considerable amounts. It is incorrect, therefore, to refer the decreased binding of a.ntitoxin, such as is seen in the tri to toxoid zone, to the boric acid-ammonia scheme. It will be well, by means of a concrete example, to study some- what more in detail the course of this toxoid formation. For this purpose 1 shall select a poison which I have already described in my publication on the constitution of diphtheria poison i as Poison No. 5 At that time I briefly gave the spectrum and the constants based on the investigations which I and my friend Donitz had carried out. In this poison the conditions were most interesting and yet extremely simple: The Lq dose was 0.125 cc; the Lt dose 0.25 cc, that is, just twice as much. The L.D. was 0.0025 cc, so that the Lq dose contained exactly 50 L. D. and the Lt dose exactly 100 L. D. These facts caused us to make the thorough analysis. This poison, as is so often the case, suffered certain transformations, whereby it became weaker. These changes occurred in three phases characterized by the formation of different kinds of toxoids. The spectra of these phases are as fol- lows (Fig. 1). The phases in which the content of toxin shows itself are I, II, and IV; phase III, which deals with the toxons, will be considered in a separate chapter. As a result of all my experiences with similar poisons, as well as Deutsche med. Wochensch. 1898, No., 38. 494 COLLECTED STUDIES IN IMMUNITY. from a direct determination, it follows that the first phase must have represented a pure hemitoxin which reached exactly to 100 (see illus- tration). Accordingly each —I. E. ( = 1 combining unit) succes- sively added to the L dose takes away i L. D. from the fatal doses ,„ Phase I 1i 10 20 30 40 50 60 70 80 90 100 00 120 130 140 150 160 170 180 190 20O Phase II lO-r 10 .20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 Phase in 7i 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 Phase IV 10 20 30 40 50 60 70 90 100 no 120 130 140 150 160 170 180 190 200 Fig. 1 contained inLo, and this all occurs within the first hundred antitoxin doses added. Amounts of antitoxin beyond this have no further influence on the toxin (death, necrosis), but afi'ect only the toxon. A fact to which I attach particular significance is that the hemi- THE CONSTITUENTS OF DIPHTHERIA TOXIN 495- toxin reaches just up to the 100 limit and shows no trace of any- gradual decline. This follows from the determination of the Lt dose, as can be seen from the following analysis. Given a poison in which, in the Lq dose, the hemitoxin zone reaches exactly to 100, how large will the Lf dose be? Lf, i.e., the amount of poison which on the addition of 200 combining units still leaves 1 L. D. free, will be reached when 200 equivalents of hemitoxin are present. We shall therefore have to multiply the Lq dose of the 202 poison by — r in order to obtain the Lf dose. If we carry out this multiplication we obtain an Lt dose of 0.253, which agrees very well with the value actually found, 0.25 cc. Thus the important fact is demonstrated that in this case the neutralization of the diphtheria poison by antitoxin proceeded exactly the same as the neutralization of a strong acid by a strong base. Here then the course of the reaction is represented by a straight line and not by a curve. Further evidence for the view that in this poison the hemitoxin extended right up to the limit 100 is furnished by phase 11. Here we see a simultaneous increase of the L-f dose and a decrease of the toxicity manifesting themselves by the fact that the L. D. increases from 0.0025 to 0.003 cc, so that the number of L. D. contained in the Lo dose has decreased from 50 to 42. This increase of the L-f dose amounted to about 0.26 cc. and from it, by means of the simple calculation already mentioned, it can be shown that toxoid formation took place in the end zone of the toxin the "tritotoxoid zone," as I term it. Let us assume that the end zone (which before as well as after the second phase extended to 100) contains a toxoid mixture of — toxicitv 10 •' instead of the hemitoxin. In order to reach the Lt dose in this case we must multiply the Lq dose by — - and not by — -^, as was ^00 *- 200 the case with hemitoxin. On carrying out this calculation, Lq being -^„. ,0125X210 _„„-, ^ 0.125, we get -p— — = 0.2625 = Lt. In the determination made at that time I actually found the Lt dose to be 0.26, but noted "a little over." That the tritotoxoid zone possessed a toxicity of — was shown by the subsequent analysis by means of partial neutralization, for near the end, a zone of 18-20 496 COLLECTED STUDIES IN IMMUNITY. tritotoxid of exactly — toxicity was found. It should be emphasized . that the fatal doses which disappeared in the deterioration were found in the form of toxoids in the tritotoxoid zone. These investigations show that these changes are due exclusively to the fact that a part of the toxin has become transformed into toxoids ; in fact into toxoids which are neutralized after the main portion of the toxin, and which, therefore, must possess less affinity. If we were to represent this phase by means of a curve according to the method of Arrhenius and Madsen, we should observe a marked flattening of the curve in the tritotoxoid zone. This, however, is not the expres- sion of the weak affinity of the diphtheria toxin, or of the neutraliza- tion dependent thereon. It is to be ascribed with absolute certainty solely to the presence of toxoids and their appearance in place of toxin molecules which have disappeared. I shall discuss phase III later, merely remarking at this time that in this phase, 80 out of 100 parts toxon have disappeared. The Lq dose of 0.125 cc. now contains only 120 combining units instead of the 200 units (toxin and toxon) originally present. Corresponding to this, therefore, the Lq dose, which must contain 200 combining units, increases from 0.125 cc. to 0.21 cc. In this third phase the toxin zone has not suffered any essential change. The L-j- dose has accord- ingly remained constant at 0.26 cc. Because of the new Lq dose made necessary by the loss of toxon, the spectrum representing this phase shows a much wider toxin zone than the previous one. The toxin- toxon boundary has been moved from 100 to 166. In phase IV, Lf remained 0.26 cc, but the toxicity decreased, the L. D. increasing gradually from 0.003 cc. to 0.004 cc. During the course of these changes 22 L. D. had disappeared from the Lq dose of phase III. The fate of these 22 L. D. is made plain by the spectrum which I constructed at that time. In this I found an extended prototoxoid zone which included the first 40 combining units of the spectrum, sufficient, as can be seen, to explain the loss of toxin which had oc- curred. I desire to call particular attention to the fact that no loss of combining groups had occurred despite the shght increase of the L.|- dose.^ ' A superficial glance might lead one to suppose that the fact that the Lf dose of 0.25 cc. in the first phase had become increased to a little over 0.26 cc, THE CONSTITUENTS OF DIPHTHERIA TOXIN. 497 This behavior shows that on standing there is not, for example, a marked destruction of the poison, but merely a slight chemical change affecting only the toxophore and not the haptophore group. It would be improper, therefore, to speak of the poison "spoiling." The observations on the origin in the various forms of toxoid are particularly important. In the first phase of toxin formation, there was a development of toxoids of weaker affinity for the antitoxin, while during the second stage, toxoids of greater affinity developed. Occupying a position between these two opposing poison modifications is the hemitoxin fraction, and this has remained intact. We are thus really forced to arrange these three poison constituents, according to their affinity, as prototoxoid, deutero toxoid, and tritotoxoid. This brings me to the crux of my views concerning the constitution of diphtheria poison. In titrating and evaluating the diphtheria antitoxic serum I began with the simplest assumption, namely, that the poison was a simple uniform substance. In the formation of toxoids, therefore, I con- sidered three possibilities: 1. That the affinity of the haptophore becomes increased; 2. That it remains the same, and 3. That it decreases. Which of these possibilities will apply in any given case will, of course, depend upon the stereochemical circumstances, especially upon how far one functionating group is removed from the other. If, in what we must conceive to be a very large molecule, these groups are quite far apart, it may be assumed a priori that the destruction of the toxophore group will probably not exert a marked influence on the haptophore group. In other words, syntoxoids will be formed. If the two groups are nearer together a change in the affinities, either positively or negatively, can readily occur. As a matter of fact, the possibility of an increase or decrease of affinity as a result of this transformation into inert modifications has also been observed in con- nection with related subjects. Researches conducted by myself and Sachs have shown that in the formation of complementoid the hap- was the expression of a certain loss of combining groups. This, however is merely apparent; in the second phase a greater excess of the poison (containing, as it does, more toxoid) is required to produce death than is the case with the haemitoxin. Bearing this consideration in mind it is easy to convince one's self that not a single one of the combining groups present has been lost and that the change which the poison has undergone was a quantitative one. 498 COLLECTED STUDIES IN IMMUNITY. tophore group suffers a decrease in affinity. Complementoids, it will be remembered, result from the destruction of the zymotoxic group, the analogue of the toxophore group. Eisenberg and Volk by their discovery of proagglutinoids have shown that in the formation of agglutinoids an increase in affinity can take place. Hence in diphtheria poison the possibility had to be considered that similar conditions obtain in toxoid transformation. In this case, however, it was remarkable that this toxoid formation did not always follow the same scheme, the poison, of course, always being thought of as a simple uniform substance. I was finally able to solve this, problem in the following manner. My earlier investigations had given me the impression that 1 I. E. (immune unit) should neutralize 200 fatal doses of a pure toxin, one consisting only of toxin molecules and therefore free from toxoids. I am quite ready to admit that I did not at that time furnish any absolute proof for this view. My first effort was therefore directed to a study concerning the correctness of the figiue 200. I began by analyzing a large number of different toxins in the hope that sooner or later I would find an ideally pure toxin. I have already men- tioned that the highest purity thus far obtained, a toxin obtained by Madsen, corresponds to only four-fifths purity, L-f containing 160 L. D. Nevertheless by means of the method of neutralization I was able to find poisons which fulfilled my requirements, at least in part. This was the case, for example, in my Poison No. 2 (see spectrum, Fig. 2). In this the Lo dose contained 84 L. D. The first third of the 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 .160 .170 .180 190 200' Flo. 2. spectrum was taken up by a zone of hemitoxin not quite pure, i.e., each combining unit added ( ^^ I. E. j decreased the toxicity by about -^ L. D. In the next zone, on the other hand, stretching from 72 to 115,^ each combining unit took away exactly 1 L. D. The spectrum is, here reproduced. It shows the zones of hemitoxin, pure toxin, trito- toxoid, and toxon very clearly. THE CONSTITUENTS OF DIPHTHERIA TOXIN 499 Madsen, too, has described a poison "C," the constitution of which is very interesting because prototoxoid and pure toxin are distinctly marked off from one another. During the phase at which Madsen examined it the pure toxin zone occupied the zone 50 to 100 of the spectrum. Before the formation of tritotoxoid this zone may, however, have extended to 150. From these observations we see that for certain portions of the spectrum (which lie in the middle and not at the commencement i) it has been possible to prove that -^r-r I. E. combines with exactly 1 L. D. This argues strongly in favor of the correctness of my assumed figure 200. In these zones of pure toxin only toxin molecules, are neutralized and no toxoids. Although it is rare to find zones of pure toxin in poisons which have been kept some time, it is extremely common, or even constant, to. find in these older poisons zones in which -—r. I. E. neutralizes exactly J L. D. Manifestly under these conditions equal parts of toxin and toxoid must always be neutraUzed; for this reason I have termed such a poison a hemitoxin. The following scheme represents such a changed poison: Toxin : Pure Toxin Toxoid : Hemitoxin Fig. 3. It needs no further explanation to show that in this hemitoxin zone the affinity of toxin and toxoid to antitoxin has remained un- changed. The entire process of toxoid formation takes place in two phases, as can readily be seen from the initial zones of suitable spectra (see Fig. 3). The pure toxin first changes iiito hemitoxin; in the second phase, however, the hemitoxin changes into pure toxoid, especially in the first part of the spectrum. This is illustrated by the following scheme : ' In the curve of ammonia-boric acid and of tetanolysin the maximum combining power always occupies the very first portions of the curve. 500 COLLECTED STUDIES IN IMMUNITY. Toxitt < Toroia / T Pure Toxin Hemitoxin (Hemi- toxoid). Pure Toxin Fig. 4. I must again emphasize that this sketch of the decomposition of the poison is not at all hypothetical, but merely the expression of the facts observed. The regular course in two phases points di- rectly to the fact that the individual toxins are not simple uniform substances but are composed of two modifications present in equal amounts in the toxin solution and behaving differently on decompo- sition. One, the more unstable of the two, the a-modification, decom- poses rapidly and so gives rise to the stage of hemitoxin. The subse- quent destruction of the more stable ^-modification leads to pure toxoid. It is, of course, somewhat remarkable that exactly equal parts of two toxin modifications should develop in diphtheria bouillon. This is readily understood, however, if we remember that E. Fischer has made it extremely probable that the active groups of ferments (groups exhibiting a great similarity with the toxophore group) pos- sess an asymmetrical constitution. If then in accordance with this we assume an asymmetrical constitution of the toxophore group, there will be nothing remarkable in the fact that the diphtheria bacilli produce both asymmetrical components simultaneously. Nor is it surprising that both are produced in equal amounts if we consider, for example, that optically inactive tartaric acid consists of equal parts of dextro and Isevo tartaric acid. If optically active combina- tions (of which a large number can be made artificially) are produced in the retort, the rule holds that exactly the same number of mole- cules of the two components are produced by the reaction. Ever since Pasteur showed that in the fermentation of tartaric acid by moulds the dextro tartaric acid is decomposed first, it has been found possible to demonstrate a similar behavior in numerous other instances ; thus by the aid of moulds, yeasts, and bacteria it was found possible to isolate one of the optically active components from racemic THE CONSTITUENTS OF DIPHTHERIA TOXIN 501 combinations. Looked at in this way the formation of hemitoxin is explained in very simple fashion.^ It can readily be shown that in the first stage of toxoid formation which leads to hemitoxin no change in affinity takes place, and this holds true also for all the toxoid formation, for if an increase in affinity occurred there could be no hemitoxin zone; a prototoxoid zone would again be followed by a zone of pure toxin. Conversely if there were a decrease in affinity a zone of pure toxin would precede the toxoid portion. The following scheme will serve to make these conditions clear: These considerations at once show us that in the formation of toxoid no change in affinity can take place. As a matter of fact, however, the pro- totoxoid possesses a much stronger, and the trito- toxoid a much weaker, affinity than the toxin or hemitoxin occupying the central portion of the spectrum. This we saw in our analysis of the poison mentioned above. We must, therefore, conclude that this difference is not produced by the formation of toxoid, but exists in the toxic bouil- lon from the beginning, the initial portion of toxin, which subse- quently passes over into prototoxoid, already possessing a higher affinity for the antitoxin. The poison of diphtheria, for example, could be represented by the following rough diagram, in which the degree of affinity is expressed schematically by the length of the lines : Pure Toxin Increased AflSnity Decreased Affinity Affinity Unchanged Fig S. Erototoxin Deuterotoxin Pig. 6. Tritotoxin ' See E. Fischer, Zeitschr. f. physiol. Chemie, Vol. 26. 502 COLLECTED STUDIES IN IMMUNITY. Certain other considerations have convinced me of the pluraUty of the toxins. Chief of these is the behavior of the poisons on long standing. As is well known, poisons freshly produced rapidly deterio- rate in toxicity until a point is reached beyond which the constants of titration, especially L-j-, remain unchanged. Such "ripened" poisons are made use of in the official testing of diphtheria antitoxin, and we have therefore had abundant opportunity to convince ourselves that they remain constant. From the standpoint of physical chemistry this fact (that the toxicity after a time becomes constant) could perhaps be ascribed to an equilibrium between toxin and toxoid. Such an equilibrium, however, is found only in reversible reactions, i.e., in chemical proc- esses, which also proceed in the reverse direction. Toxoid formation, however, is not a reversible reaction; no one has yet discovered even a suggestion of a toxoid passing over into toxin. Another point which speaks against a condition of equilibrium is the fact that through artificial influences— heat, chemicals — any desired proportion of toxin and toxoid can be produced. Only one other explanation therefore remains, namely, that various toxins are present, of which some are more resistant, others less so. 1 have thus presented in detail the reasons which led me to assume the existence of preformed varieties of toxins. As a result of my ex- periments I must emphatically deny the assumption that the phe- nomena observed by me in diphtheria poison are only the expression of a weak affinity between diphtheria toxin and antitoxin. I have demonstrated that the observed deviations can only be due to the admixture of toxoids with different affinity, and have further made it probable that these different degrees of affinity exist preformed in the toxin and do not arise with the formation of toxoid. It must however, be distinctly understood that the points of view here laid down are not applicable to the relations between toxins and antitoxins in general. They apply only to diphtheria toxin and its antitoxin. The important researches of Arrhenius and Madsen on tetanolysin show that neutralization proceeds in an entirely different fashion ■when the two components possess a weak affinity for one another. The studies of these authors clearly indicate the errors in the interpre- tation of neutralization phenomena when dissociation is disregarded. My results were obtained by the long and tedious experimental method. I can assure the reader that the experiments upon which all this is based, experiments carried out by my fellow workers (espe- THE CONSTITUENTS OF DIPHTHERIA TOXIN 503 'daily Geh.-Rath Donitz and Dr. Morgenroth) and myself, have been most exact, and I venture to say that in medicine but few investiga- tions exist which have been carried out with such precision and on such abundant material. II. Toxons. Thus far we have dealt only with the true toxin portion of the 'diphtheria poison, and have entirely disregarded another constant secretory product of the diphtheria bacillus, namely, the toxons. On testing a diphtheria poison and determining the two limits, Lq and Lf, we should expect that the difference, L-f-L =D, would correspond exactly to one lethal dose, provided the poison were a simple uniform substance. Thus if L , for example, contains a lethal doses these, according to our definition of Lq, will exactly be neutralized by 1 I. E. Assuming that the two substances have a strong affinity for 'each other, the "addition of one L. D. would suffice to transform this neutral Lq mixture into Lf, i.e., L-j- should contain (a+1) L. D. and the difference, D, should equal 1. As a matter of fact, however, it was found that with the exception of one poison examined by me, the difference between Lt and Lo is much greater. In the poisons de- scribed in my first communications the difference D ranged from 5 to 50 L. D. At first, when I still held to the unitarian conception, I had interpreted these results' §,s Indicating the existence of a toxin derivative of very little toxicity and possessing less affinity than the toxin. For this reason I termed the derivative "epitoxoid." In my second communication, however, I abandoned this assumption, and stated that we were evidently dealing with a primary secretory prod- uct of the diphtheria bacilli — the "toxon." The reasons which led me to this view will be presented in a moment. The toxon possesses the same haptophore group as the toxin, but a weaker affinity for the .antitoxin. The main difference is in the toxophore group, for even when given in large doses the toxon does not produce death, but only paralyses which develop after a long incubation of fourteen days or more.i ,_j„,,. Arrhenius and Madsen have doubted particularly the existence of ' It may be remarked in passing that such additional or "by-poisons" with a long period of incubation are not limited to diphtheria bacilli. According to the observations of Sclavo on animals infected with anthrax it is highly probable that anthrax bacilli also produce poisons having a toxin-like action. 504 COLLECTED STUDIES IN IMMUNITY the toxons. According to them the long-drawn-out toxon zones are the expression of the incomplete combination of toxin and antitoxin, the neutralization of which they believe follows the ammonia-boric acid type. There are, however, a number of weighty reasons why this view cannot be accepted. It was but natural at first to ascribe the toxon stage to phenomena such as Arrhenius and Madsen now have in view. It had already been noticed by others that often a considerable interval exists be- tween Lt and Lq. Knorr, in referring to this, had spoken of "un- neutralized poison residue." The assumption, however, that we are here dealing with the result of an mcomplete neutralization is con- troverted by the analysis of a poison which I encountered during the course of my investigations. This was Poison No. 10 (of my series), whose Lq and Lf values were very close together. Lo contained 27. & and L-f 29.2 L. D. Hence D = 1.7 L. D., which is a close approach to the figure demanded by a simple diphtheria poison. The following considerations will show that this value, 1.7. should be cor- rected so as to be still lower. The original calculations were based on my earlier assumption that toxins and toxoids are uniformly mixed. This however, has been superseded by the spectrum method of representing the neutralization of poisons. Experience has taught us that such deteriorated poisons usually consist of a small zone of hemitoxin and a more or less pronounced zone of tritotoxin-toxoid, in which as a rule nine toxoid equivalents fall on one toxin equivalent. Several times 1 have observed tritotoxin-toxoid zones containing V,o toxin, and Madsen also has described such a poison. As can be seen from our calculations given above, the theoretical change from L , to Lt is influenced solely by the tritotoxoid zone. If we therefore assume that our poison pos- sessed a tritototoxin-toxoid portion whose strength was '/lu (and this is extremely probable) we shall find that by a little calculation that the poison probably contained no toxon whatever. Very likely the tritotoxoid zone reached to the end (200) of the spectrum. On the assumption of a '/.o tritotoxin-toxoid, if we multiply L„ by ^'Vjog we shall obtain Lt = 28.9 L. D. This agrees very well with the figures obtained experimentally, Lt = 29.2 L. D. We may therefore very well assume that we were dealing with a poison free from toxon or one which contained only very small traces of toxon. This fact is hard to reconcile with the theory of Arrhenius and Madsen, for if toxin and antitoxin neutralized each other like ammonia and boric acid, all poisons should show a long zone of in- complete neutralization. The independent existence of the toxons is further corroborated by the fact that the toxon zone varies enormously in different sped- THE CONSTITUENTS OF DIPHTHERIA TOXIN 505 mens of poison. In one it may amount to about one-fifth of the toxin portion, in another I have seen equal parts of toxon and toxin. Dreyer and Madsen in fact have recently described a poison which contained three times as much toxon as toxin. According to our present ex- periences, therefore, the amount of toxon calculated on the toxin can vary from per cent to 300 per cent. Hence I find it impossible to assume that we are dealing with neutralization phenomena such as are observed with ammonia and boric acid, for such neutralizations would show at least some agreement. This still left undecided whether the toxon is a primary bacillary secretion or a secondary modification of the toxin. A study of the development of one poison finally gave me the clue to this. This was poison V, whose constitution has been described in the Deutsche med. Wochenschrift 1898. It will be recalled that this poison pos- sessed the following limits in the second phase: Lo=0.125; Lt=0.26; L. D.=0.003. During the course of three weeks Geheimrath Donitz made con- tinuous determinations of Lq and U, using very uniform animal material. The protocol of this experiment is reproduced in full because the precision of the methods will thereby also be exhibited (see table on page 506). From the table we see that in the course of three weeks Lq has increased from 0.15 to 0.20. After this an insignificant increase brought this to 0.21; from then on lio remained constant. During this time the L^ dose (0.26) had suffered no change whatever, for on the 16th of July a mixture of 0.25 poison + 1 I. E. killed in six days and 0.275+1 I. E. in three days. L-f, which according to our defi- nition is the mixture that will just kill on the fifth day, must have been about midway between these two values, a little over 0.26. This agrees very well with the value obtained in the beginning. To repeat, during the course of this stage L-f has remained constant, but Lo has increased considerably (from 0.125 to 0.21). This fact is easily explained. The toxin portion has remained absolutely unchanged in its end zone, as can at once be seen from the constancy of the Lt dose. On the other hand in the toxon por- tion, which is expressed by the difference between L-f and Lq, 80 toxon equivalents out of 100 have apparently disappeared. This eliminates the possibility of a transformation of toxin into toxon, for if that assumption were correct one would expect that on allow- 506 COLLECTED STUDIES IN IMMUNITY. ing the bouillon to stand, the toxin zone would decrease and the toxon zone become considerably greater. In this case, however, we see that the toxin zone remains constant while the toxon zone is reduced to one-fifth.^ Determination of L„ Dose. Guinea-pigs are Injected with 1 I. E. + Varying Amounts of Poison. Amount of Poison. CO. June. July 21 25 29 1 4 6 10 0.125 0.1275 0.13 0.14 faint trace almost — — — — — slight but distinct 0.15 — — ■ — just neutral — — — 0.16 — — — slight but distinct — — — 0.17 — — — — little slight — 0.18 — — — 1 1 << . 0.19 — — — — more slight oedema — 0.2 — — — — more oedema almost neutral 0.215 — — — — more oedema some oedema 0.23 marked oedema "Faint trace," "slight," etc., denote the degree of infiltration. It is difficult to say a priori what has become of the toxon which has disappeared. On account of certain facts which I shall mention later, 1 have assumed that we are here dealing with the formation of an analogue of toxoid, namely, a substance which I term "toxo- noid." I conceive this to be a toxon in which the toxophore group has become modified. ' The entire course of the decomposition, in which from day to day we could observe the toxon becoming weaker and weaker speaks against the possibility (in itself very remote) that the varying composition of the bouillon is respon- fible for the variation in the number of toxons in the individual poisons. In the poison here described the decomposition has taken place in the same bouillon and in so short a time that very great alterations in the bouillon appear to be excluded. THE CONSTITUENTS OF DIPHTHERIA TOXIN 507 Another fundamental difference, one which in my opinion argues in favor of the individuality of toxin and toxon, consists in the differ- ent action of the two constituents. The action of diphtheria toxin, as is well known, is such that the animals die with symptoms of hydrothorax, ascites, congestion of thesuprarenals, necrosis of the skin. Somewhat smaller doses kill guinea-pigs in from six to seven days, the animals showing ulceration and extensive necrosis. Still smaller doses, J, i, I, ^ L. D., no longer produce death, but regularly cause necroses which are surrounded by an extensive area of total loss of hair. Small fractions of the fatal dose always produce emaciation of the animals. In contrast to this, the toxon, i.e. a serum-poison mixture in which only the toxin fraction is completely neutralized, never kills animals acutely, even in high doses. The inflammatory properties may be entirely absent in small doses, while in large doses they are present to only a slight degree. The cEdema disappears ■completely in the course of a few days, there are no necroses, and the loss of hair, if it occurs at all, is only partial. On the other hand the paralyses are very characteristic, and these appear at any time between the fourteenth and twentieth day, depending upon the dose, usually in the third week. Frequently the animals do not show even a trace of local reaction and maintain their weight ; then suddenly they are attacked with the paralyses and may die from these within a few ■days. I have never seen such a result in animals inoculated with a pure diphtheria poison. Now and then a guinea-pig was observed ■which showed these paralytic phenomena. It was usually one that had received a considerable fraction of the L. D. Invariably it showed extensive necroses, was generally very sick from the beginning, and had suffered considerable loss of weight. In view of the sUght amount of toxon which I found in these poisons, such animals were ■evidently supersensitive to the toxon. Dreyer and Madsen have succeeded in differentiating toxin and toxon qualitatively, as follows: They found that mixtures of a diph- theria poison and antitoxin in which the limit of complete toxin neutralization was nearly approached, exerted only toxon effects when given in small doses. If, however, the mixture was increased tenfold, death was brought about by the toxin. This is readily ■explained. The determination of toxon by means of 1 I. E. natu- rally cannot be absolutely exact, for a small residue of toxin, e.g. i/io L. D., can readily escape observation. If, however, a sufficiently large multiple of this mixture, e.g. ten times the original quantity, is 508 COLLECTED STUDIES IN IMMUNITY injected, this will now contain i°/io L. D. unneutralized. If now the amount of antitoxin was also somewhat increased, Dreyer and Mad- sen found that even with this multiple amount only toxon effects were observed, the toxin now being comipletely neutralized and only toxon remaining free. Dreyer and Madsen ^ thereupon subjected this same poison to a thorough study, using rabbits for the purpose. They foimd if 0.6 cc. poison was mixed with 1 I. E., that this mixture, which represents the Lo dose for guinea-pigs, is still highly toxic for rabbits. In order to render this dose of poison completely innocuous for rabbits it is 240 necessary to add more antitoxin, in this ca^ — - I. E. The state- ments concerning the behavior of mixtures between these two limits are also of considerable importance. A mixture of 0.6 cc. poison + 210 ^TT^ I. E. injected into a rabbit causes death on the twenty-second day with paralytic symptoms. The incubation period is sixteen 232 days. Even a mixture of -^r^ I. E. with the same amount of poison caused paralyses, which appeared on the sixteenth day and con- tinued for several weeks. This behavior is so important for our view concerning the existence of different poisons that I must enter a little more fully into the subject. According to our definition of the 232 Lo dose, mixtiu-es like the one containing jr— - I. E., and therefore possessing a considerable excess of antitoxin, are absolutely innocuous for guinea-pigs and can be injected in any quantity. In virtue of the excess of antitoxin such mixtures suffice to passively immunize the animal and to protect it, provided suitable doses have been in- jected, against diphtheria poison and diphtheria bacilli. If then such mixtures are still toxic for rabbits only one possibihty remains, namely, that the diphtheria poison in question contains a substance which is non-toxic for guinea-pigs but toxic for rabbits. This sub- stance I term toxonoid.^ • See also my article in Miinch. med. Wochensch. 1903, Nos. 33, 34. ^At the outset of my investigations I made entirely similar observations. My very extensive but unpublished studies made at that time convinced me that this property is not common to all diphtheria poisons, for I also found some in which the Lq dose was exactly the same in rabbits and in guinea-pigs. This fact furthermore refutes the assumption that the phenomenon described THE CONSTITUENTS OF DIPHTHERIA TOXIN. 509 So far as the behavior of partially neutralized mixtures is con- cerned, the observations of these authors show that mixtures which exert only toxon effects on guinea-pigs produce death in rabbits with symptoms of diphtheria poisoning. I believe that all these phenomena are best explained by the assumption that there are at least three different varieties of poisons, and that these possess differ- ent affinities and different actions. These poisons are : 1. Toxin, possessing the highest affinity, kills rabbits and guinea- pigs acutely, but is more toxic for the former. 2. Toxon, killing rabbits acutely and guinea-pigs with symptoms of paralysis. 3. Toxonoid, producing paralyses in rabbits, non-toxic for guinea- pigs. The fact that all three poisons act more strongly on rabbits than on guinea-pigs is explained by the absolute higher susceptibility of the former. Dreyer and Madsen have recently described a diphtheria poison in which toxoid effects could be demonstrated even on the injection of sublethal doses of the pure poison. This behavior is at once under- stood if we study the constants of this poison as they were determined by these authors, for whereas in the other poisons examined there were 33 toxon equivalents to 167 toxin equivalents (toxon : toxin = 1:5), in this poison the proportion was just the reverse, there being three times as much toxon as toxin. No wonder therefore that with the toxon fifteen times more concentrated even sublethal doses of the pure poison should suffice to make toxon effects evident. In view of the high theoretical significance which attaches to the poison described by Dreyer and Madsen, I cannot refrain from giving briefly my conception of its constitution. The authors have repre- sented the poison in the form of a curve, one which at first sight seemed rather strange to me. As soon, however, as I transformed their graphic representation into a spectrum by the aid of their figures the constitution of the poison was found to agree very well with •other well-known diphtheria poisons. The only difference is the very is due to an incomplete neutralization, such as Arrhenius and Madsen, for exam- ple, have demonstrated in the case of boric acid and ammonia, and in the union of tetanolysin with its antitoxin. If that were the case one would expect to see the phenomenon in all diphtheria poisons in equal degree, and this is not the case. 510 COLLECTED STUDIES IN IMMUNITY. large content of toxon. The spectrum, which corresponds to the curve obtained by the authors, is here reproduced (Fig. 3, Phase II). From this we see that a zone of hemitoxin in the beginning of the spectrum is followed by a zone of almost pure toxin, and this in turn by a zone of tritotoxin-toxoid. Then comes the very long toxia fraction. To one employing this mode of representation, such a spectrum not only pictures the present constitution of the poison but also frequently permits him to reconstruct itfe previous constitution. In this case, for example, it was possible to do so with the aid of several statements by the authors concerning earlier and later stages. According to these figures I would assume that in the first phase the poison contained a pure toxin in the initial zone. In the second phase, the period at which the poison was studied by Dreyer and Madsen, this had become transformed into hemitoxin. In the third phase it may become pure prototoxoid. A fourth phase would then show the transformation of the pure toxin in the above spectra into hemitoxin and the poison would then have reached the point which we have so frequently observed in other poisons. The spectra of these various phases is as follows (Fi^. 7) : I shall now present the figures which Madsen and Dreyer ob- tained when they started with double the Lq dose (0.1 cc. poison). In the first phase, their statement that the lethal dose was 0.0015 cc. shows that 0.1 cc. poison contains 66 L. D. Calculation from the spectrum gives 65 L. D. The second phase, of course, agrees entirely with the statements of the authors, since the spectrum was constructed according to these. In the third phase the formation of the prototoxoid zone from the previous zone of hemitoxin is readily seen from a second neu- tralization test, one made with normal horse antitoxin. In phase IV the lethal dose had risen to 0.0027, corresponding^ to 37 L. D. in 0.1 cc. Calculating this from my spectrum I obtain 35 L. D., which is but 2 L. D. smaller than would correspond to the final stage. Perhaps this stage had been nearly but not yet completely attained. It is probable that if the examination had been made a little later the figure would have been exactly 35. The figures obtained from my reconstructed spectra harmonize so well with those obtained experimentally by the authors that it seems almost impossible to doubt the correctness of my assumptions concerning the constitution of the p&ison and the process of its trans- THE CONSTITUENTS OF DIPHTHERIA TOXIN. 511 formation. This proves that in this poison the toxin zone behaved exactly the same in its transformation as it did in the other diph- theria poisons examined. I beHeve it will be seen from my explanations that my mode of procedure in the study of diphtheria poison has been exceedingly Phase I \ a "'- S 65 ?0 S 90 lOO llo 120 130 140 150 1&) itO iSO iSO 8IJQ- Phase n -1 . 3 . . , , . , . , . . . . 1 ) 60 TO80 90 100 110 130 130 140 150 160 170 180 190 200.. Phase in l l^y ■ m^^-J.^^^^^^^SS^^•w^ 1 1 1 1 n ■ ■ ■ i i ■ , , , 1 20 30«i 50 60 TO80 90 iOO liO iSO iSO liO 150 l60 170 iSO 190 200 Phase IV ) 60 '10 SO 90 100 liO Fig. 7. 140 150 160 ItO «1S0 idO 200' careful, and that the objections raised against my results do not apply. I must therefore continue to maintain my original standpoint, andl deem it well therefore to once more define my views concerning the: poison of diphtheria. 512 COLLECTED STUDIES IN IMMUNITY. 1. The diphtheria bacillus produces several kinds of poisons, especially toxins and toxons. 2. The affinity of diphtheria toxin to the antitoxin is very great. 3. The deviations from a straight line as they manifest themselves in the graphic representation of the neutralization of the poison cannot be explained by the assumption of a single poison possessing a weak affinity. They are rather the expression of the fact that the poison bouillon contains admixtures of various kinds of substances of a toxoid character. 4. The varied affinity of the toxoids cannot be explained by the assumption that a simple toxin when transformed into toxoid suffers a change in affinity either positively or negatively. Rather does this indicate that the toxic bouillon contains, preformed, various toxins of different affinities. 5. There is no change in the haptophore group in the formation of toxoid. 6. The absolute number of combining units contained in the immune unit or in the Lq dose of poison is 200.^ I have finished. If the results of the first encounter of two such different methods of study as the mathematico-physical and the bio- logical have not shown complete agreement we should not be at all surprised. The natural aim of physical chemistry must always be ' Bordet has recently attempted to explain the toxon phenomena by the assumption that the toxin molecule can combine with antitoxin in varying proportions. One would accordingly have to assume that the toxin molecule contains several haptophore groups. The complete occupation of these groups causes the toxicity to be entirely lost, whereas partial saturation causes a de- crease in toxicity. That is to say, amounts of antitoxin which do not com- pletely neutralize the toxin would weaken it in such fashion that it would exert a different action. It is strange that so eminent an investigator as Bordet should not have attempted to convince himself of the correctness of this hy- pothesis by means of the experiment. He would then have found that the facts are irreconcilable with such an assumption. We have shown at great length that the toxon actions are nothing less than constant phenomena and have called attention to the great extent of the quantitative variations (0-300). If one were to follow Bordet it would then be necessary to assume an enormous multiphcity of haptophore groups in the toxin molecules, and this would lead to a hypothesis far more comphcated than mine, although the latter harmo- nizes all the experimental results. In support of his conception Bordet refers to experiments with complement and anticomplement. I must say, however, that in these we are dealing with such complicated relations that it is not per- missible to apply the conclusions drawn from them to the far simpler relations existing between toxin and antitoxin. THE CONSTITUENTS OF DIPHTHERIA TOXIN 513 to introduce as few factors as possible for purposes of calculation, whereas biological analysis always seeks to pay due regard to the wonderful multiplicity of organic matter. However, I believe that these two methods can readily be combined and that this will be very desirable. The biologist will have to content himself in so far yielding to the economy of the mathematical view that he restricts his assumptions to the smallest possible number. The physical chemist, on the other hand, cannot escape the obligation of paying due heed to this minimal multiplicity, the result of experimental research. Naturally the problem is thus made extremely difficult, so that success will require that the greatest authorities in physical chemistry work hand in hand with the best biological talent. For this reason I regard it as a great gain to science that so eminent a leader as Svante Arrhenius is taking a lively interest in our work, and has joined hands with my friend and pupil, Thorvald Madsen. XXXVIII. TOXIN AND ANTITOXIN :i A REPLY TO THE LATEST ATTACK OF GRUBER.' By Paul Ehrlich. In a domain that is open to experimental investigation it is neither easy nor without danger for one to express criticism merely as a result of literary studies. This is especially true in that most difficult field in the entire study of immunity, namely, the subject of toxins. Only one who has devoted years of unprejudiced study at the laboratory table to this subject and gathered a host of observations and experiences will be in a position to orientate himself in the confused mass of true and false statements contained in the literature. The outsider will find it very difficult to correctly analyze all this material. Hence it is all the more remarkable that Gruber^ should choose the subject of toxins for the main portion of his attack upon me, for according to his own admissions that is the field which he knows merely from literary studies. Against such critics I am in the unpleas- ant position of a man who is compelled to discuss colors with the blind. Nevertheless I cannot well escape the thankless task of replying, at least to the main points in Gruber's polemic, for it is indisputable that this attack, addressed chiefly to those without special training in this field, is capable of causing wide-spread con- fusion, owing to its positive tone and its severity. Gruber's first important error lies in the assumption that a con- troversion of the plurality of poisons, to which I hold, signifies the downfall of the side-chain theory without further ado. The side- chain theory, however, proceeds from the assumption that the toxin- ' Reprinted from the Miinch. med. Wochensch. 1903, Nos. 33 and 34. ' M. Gruber and CI. v. Pirquet, Toxin und Antitoxin, Miinch. med. Wochensch. 1903, Nos. 28 and 29. 514 TOXIN AND ANTITOXIN. 515 like poisons are characterized by a haptophore and a toxophore group, of which only the former effects the anchoring of the tOxin. Practically therefore only this group is important for the produc- tion of antitoxins. This view is only the logical consequence of the fact that on long standing the poison bouillon undergoes modi- fications, resulting in the production of what 1 term toxoids. These substances are characterized by this, that the haptophore group has remained intact, while the toxophore group, depending on cir- cumstances, has suffered partial or complete modification. Not infrequently it can be shown that the formation of toxoid is quan- titative, the combining power of the toxic bouillon being unchanged despite a considerable loss of toxicity. Gruber, by means of certain calculations, appears to question this fact; he refers exclusively to my very earliest publications in which, naturally, the evidence was still incomplete. It would have been better if Gruber had studied instead my later publications, for then he could easily have convinced himself that my statement is entirely correct. I shall mention but one of my poisons ^ as an example. In this the L dose was originally 0.25 cc, the lethal dose 0.0025 cc. At the end of the investigation L-f had increased to 0.26 cc, the lethal dose, however, to 0.004 cc. The number of lethal doses, therefore, in approximately the same amount of Lf had been reduced from 100 to 65. Madsen ^ describes a poison in which the neutralizing power remained constant during the course of two years, while the toxicity was reduced one-half, from 0.02 to 0.04. Furthermore Arrhenius and Madsen in their most recent work * describe the toxoid modification of a tetanus toxin. These consist in the fact that the combining power remains intact while the toxicity is decreased to one-sixth. It is seen therefore that the doubt thrown upon my quantitative statements is due entirely to a disregard of readily accessible facts. This quantitative transformation consti- tutes a somewhat annoying fact for Gruber, and he therefore seeks to explain it as follows: " Imagine, if you will, that ^/lo of the toxin molecules present are changed into toxoids, the minimal lethal dose will then be increased ' Described in Deutsctie med. Wochensch. 1898, No. 38. ^Annales de I'Institut Pasteur., T. 13, 1899. ^ S. Arrhenius and Th. Madsen, Physical Chemistry applied to Toxins and Antitoxins, Festskrift ved. indvielsen af Statens Serum Institut, Kopenhagen, 1902; German i.T Zaitsch. f.ir physiol. Chem. 1903. 516 COLLECTED STUDIES IN IMMUNITY. tenfold whereas the Lq value will remain unchanged; this is Ehr- lich's hypothesis. If ^/lo the toxin molecules had lost their toxicity, without there being any formation of toxoids capable of combining with antitoxin, the Lq value would be increased ten times, li, how- ever, simultaneously with the loss of ^/lo the toxicity, the fluid were to lose ^/lo the reaction rapidity for antitoxin, so that the constant of the reaction would be decreased ^/lo, it would be found that the Lq value would manifest itself unchanged." Gruber would have done better to have made some of these com- paratively simple experiments himself than to advance such an untenable assumption. We are here dealing with experiments ■which constitute, in fact, the very beginning of the technique of testing poisons. Thus, when in 1897 ^ I formulated the law that the combination of poison and antibody takes place more rapidly in concentrated solutions than in weak solutions, it was as the result •of just such studies made on diphtheria and tetanus toxin. In these studies I convinced myself that the affinity between diphtheria anti- toxin and diphtheria toxin is far greater than that between tetanus antitoxin and tetanus toxin; The union of diphtheria toxin and its antitoxin is effected very quickly, so that at the end of five to ten minutes one may be sure that complete union has taken place. It is entirely immaterial whether one is dealing with fresh poisons or with poisons poor or rich in toxoids. I shall here reproduce an experiment which I have recently made because Danysz ^ insisted that the neutralizing power of the diphtheria poison changes when the poison is allowed to stand for some time. The experiment was performed with the standard serum and standard toxin used in the official standardization. Both substances had therefore been very accurately titrated. The mixture was allowed to stand fifteen minutes and twenty-four hours and the result showed that in this time not the least change had taken place in the constant. In the experiments of Danysz, therefore, some error has probably crept in. In any event there is no change in the reaction time on the decrease of toxicity of the diphtheria toxin. Guinea-pig I receives 1 I. E. serum -I- 0.78 cc. poison (L^j) fifteen minutes after mixing. It dies on the fourth day. Guinea-pig II receives the same mixture twenty-four hours after mixing. It dies on the fourth day. ' Die VVerthbemessung des Diphtherieheilserums, Jena, 1897 '' Annales de I'lnstitut Pasteur 1902. TOXIN AND ANTITOXIN 517^ Guinea-pig III receives 0.8 cc. poison, otherwise same as I. It dies in three and one -half days. Guinea-pig IV receives 0.8 cc. poison, otherwise same as II. It dies in three and one-half days. Another thing which is entirely irreconcilable with Gruber's assumption is the fact that there exist prototoxoids, i.e., toxoids which possess a higher affinity for the antitoxin than the toxifl. itself does. The existence of these was first pointed out by me and has since been confirmed by Madsen and also by Arrhenius. The exist- ence of the prototoxoids becomes clearly manifest by the fact that one can add a certain quantity of antitoxin to the toxin solution without affecting the toxicity in the slightest degree. Mention must also be made of the fact that similar phenomena, have been observed in a large number of other poisons. It will suffice here if I remind the reader that toxoid changes have been, observed in ricin (Jacoby), abrin (Romer), staphylotoxin (Wechs- berg, Neisser), cobra venom (Meyers, Flexner). Furthermore Mor- genroth and I showed that in complement also there is a destruction of the real active portion, the zymotoxic group, while the hapto- phore group remains intact. The existence of complementoids has been demonstrated decisively by Sachs and myself,^ although Gruber had termed them " merely fervent wishes floating about in the serum." Furthermore it will be remembered that similar phenomena are observed in the agglutinins and coagulins (precipitins), the hap- tophore group of the agglutinin or the precipitin remaining intact, while the agglutinophore group is destroyed. This phenomenon was first pointed out in the excellent study rnade by Eisenberg and Volk in Paltauf's laboratory. Since that time a large mass of liter- ature has grown up around this subject so that now there is not the least doubt concerning the existence of these substances, which normally occur in the form of proagglutinoids. A recent study by Korschun ^ makes it probable that something similar to this occurs in ferments, particularly in rennin. In all these various eases it seems to be the rule that the real functionating group is far more labile than the one which effects combination, namely, the haptophore group. Hence I believe that the formation of such ' See page 209. 2 Zeitsch. f. physiol. Chemie, Bd. 37, 1903. 518 COLLECTED STUDIES IN IMMUNITY, modifications must be classed with tlie positively demonstrated facts in medicine. It is entirely incomprehensible how Gruber could believe that the possible controversion of the plurality of poisons assumed by me denotes the downfall of the entire side-chain theory .1 How false such a conclusion is can be seen from the fact that when 1 devised the side-chain theory I believed the diphtheria poison to be a simple substance. My later studies, however, convinced me that the poison consists of several modifications: prototoxin, deuterotoxin, tritotoxin, and toxon. It can easily be seen from my publications, however, that I ascribe the same combining group to all of these; they differ merely in their toxophore groups. In the production of diphtheria antitoxin all of these modifications act in exactly the same way. It shows a deplorable lack of com- prehension, therefore, when Gruber says that the refutation of the plurality of toxins will " give this side-chain-theory spook its quietus." However, let us see what proofs Gruber advances against the plurality of the poisons. On a previous occasion when Gruber brought forward these same arguments I allowed them to pass with- out specially controverting them, for I felt that his faulty mode of reasoning would at once be apparent to the specialist. Now that Gruber, however, returns to this subject I think it may be well to discuss the facts somewhat in detail. In the majority of poisons it is probably a fact that the toxicity depends upon the animal species, a certain poison being more toxic for one species than for another. In chemically definite poisons, alkaloids, etc., this behavior is usually a constant one, so that in text-books on toxicology the fatal doses per kilo of body weight > Arrhenius and Madsen (1. c.) in their very interesting study have ques- tioned whether the phenomena of neutraUzation, which I described and referred to a plurahty of poisons, are due to a difference in the poisons or whether, as they think probable, they are merely the expression of a neutralization between two substances of weak affinities. For the present I shall merely point out that my own statements refer only to diphtheria toxin, which possesses a much higher affinity for the antitoxin than does tetanus toxin. The investigations of these esteemed authors have disclosed one source of error which could easily creep into neutralization experiments. Nevertheless I believe that their con- ception does not apply to the toxin of diphtheria which I have studied so closely. I shall go into these questions more fully elsewhere, and hope then to show .that the standpoint maintained by me is entirely correct. TOXIN AND ANTITOXIN. 519 are usually given for various animal species. In the beginning it was thought that the same conditions held true for the bacterial poisons and several such scales of toxicity were given out by high authorities. As soon, however, as different toxin solutions of the same species were examined, e.g. diphtheria toxins obtained from different cultures or in different laboratories, it was found that, unlike the alkaloids, the scale of toxicity was a variable one. In the case of one poison, for example, I found that a guinea-pig of 250 grammes was uniformly killed by a dose of 0.00375-0.004 cc, and a rabbit of 1800 grammes by a dose of 0.009 cc. This corre- sponds to a ratio of 1:2:2-2.4. In another poison the figures were 0.003 for guinea-pigs and 0.004 for rabbits, corresponding to a pro- portion of 1:1.3. This showed that in two different poisons the susceptibility of rabbits varied more than half. The conditions, however, are far more interesting and instruc- tive in the case of tetanus poison. For a long time a controversy existed between v. Behring and Tizzoni. The former stated that tetanus poisons act 150 times weaker on rabbits than on mice, whereas Tizzoni declared that a poison prepared by him was just as toxic for rabbits as for mice. From the papers of these authors it is cer- tain that the two poisons when tested on mice were identical. A definite amount of either poison — for example, a single fatal dose for mice — was neutralized by the same quantity of antitoxin. So far as mice were concerned, therefore, the two poisons were identical. As soon as the poisons were tested on rabbits, however, the above- mentioned enormous- difference in toxicity becomes apparent. This at once shows that these two poisons cannot possibly be identical. Wherein, then, does the difference consist? We have seen that the two poisons are neutralized by the same antitoxin, and that fur- thermore immunization with one of the poisons is followed by the production of an antitoxin, which acts also on the other poison. From this it follows that the haptophore group must be the same in both. Hence we must be dealing with a difference in the toxo- phore group, v. Berhing's poison possessing a toxophore group which is highly virulent for mice and only slightly so for rabbits, whereas Tizzoni's poison contains a group which acts equally on both ani- mals. This difference would be very like that which I have demon- strated in the case of diphtheria toxin and toxon. One might, how- ever, think of an entirely diiferent explanation, namely, that the strain of bacteria with which Tizzoni worked secreted an entirely 520 COLLECTED STUDIES IN IMMUNITY. different kind of poison than the Marburg culture. But this proved not to be the case, for v. Behring demonstrated that his tetanus poison when injected into rabbits in large quantities suffers a considerable diminution in toxicity. On testing the properties of the poison contained in the serum of the poisoned animals he found that this residual poison possessed the same constants as Tizzoni's poison. From this it follows that v. Behring's poison contained also a cer- tain proportion of the Tizzoni variety. The Marburg culture must therefore have produced two varieties of poison at the same time. Naturally by mixing the two poisons one can obtain new poisons which, while they manifest the same action on mice, will have any desired relative toxicity for rabbits; this, of course, within certain limits. If one were to take the time and trouble to examine a large number of native poisons from different laboratories, corresponding differences between them would probably be encountered. If we recollect that various specimens of the chemically simple poisons manifest the same relative toxicity on different animals, and then consider the behavior of tetanus toxins as just described, we shall conclude that bacterial poisons of different origin, which manifest a variation in their relative toxicity, are not of simple con- stitution, but are made up of several different constituents. It shows very little knowledge of the subject therefore when Gruber says: " v. Behring shows that two toxin solutions, which in a given unit of volume contain equal f Ms., i.e., whose unit of volume kills a like number of grammes of mouse in four days, may have an entirely different content of f rabbit, f pigeon, f goat^ and f horse. This at once disposes of Ehrlich's conclusions." It is just such phenomena which argue in favor of the plurality of poisons; they do not speak against it. , Gruber bases another of his objections on the interesting obser- vations made by Madsen and Dreyer on toxons (Zeitsch. f . Hygiene, Vol. 37, page 251). In his dictatorial manner he says that " these observations demonstrate conclusively that Ehrlich's method of analyzing toxins is absolutely useless. Only a person ignorant of chemistry could maintain that the different results in guinea-pigs and in rabbits are sufficiently explained by the different suscepti. bility of the animals to the toxins." To begin, Gruber's premise is absolutely misleading, when he says: " But if the poison is neutralized it will be without effect even TOXIN AND ANTITOXIN. 521 on the most susceptible animals. Let us imagine, for example, a mixture of sulphuric and acetic acids, neutralized by the gradual addition of baryta water. Once all the sulphuric acid is neutralized, even the most sensitive reagent to free strong mineral acids will be unable to detect any trace of it." Let us see just what Gruber means by this comparison. The sulphuric acid corresponds to the toxin ; the antitoxin is represented by the alkali. In accordance with the comparison the receptors of the cells are represented in the animal body by the alkali of the tissues. If now we inject an animal with sulphuric acid previously neutralized with ammonia, i.e., a solution of ammonium sulphate, it will depend mainly on the affinity of the tissue alkali, whether or not the neutral ammonium sulphate will be decomposed and sul- phuric acid allowed to enter the tissues, ammonia being set free. If we assume, for instance, that the tissue alkali is comparable to a strong base like sodium hydroxid or barium oxid, the ammonia introduced in combination with the sulphuric acid will be absolutely unable to prevent the poisoning; the weak base will be forced out of the salt and replaced by the stronger base. In general we must assume that the antitoxin possesses a higher affinity to the toxin than do the tissue receptors, for only on this assumption can we explain the protective action of the antitoxin. Numerous phenomena, however, indicate that the affinity of the tissue receptors can become increased. 1 had reached these conclusions long before the pub- lication of my theory, which as many know I formulated years before it was published. The cause of this long delay was the phenomenon of hypersusceptibility, i.e., the peculiar fact that immunized ani- mals, despite a colossal excess of antitoxin, succumb to the action of the poison. The first light on this subject was the study of Donitz, in which it was shown that the poison shortly after its union with the tissues is but loosely bound. In the course of a few hours the union becomes firmer and firmer so that after a certain time, which may vary from a ffew minutes to six hours, according to the dose, the poison can no longer be abstracted from the tissues by the anti- toxin. This fact seemed to indicate that under the influence of the poisoning the aflSnity of the tissue receptors gradually becomes Increased and that when a certain point is reached a cure by means of antitoxin is impossible. This, however, furnished me with an explanation of hypersusceptibility and removed the obstacle which had kept me from publishing my theory. 522 COLLECTED STUDIES IN IMMUNITY. I should also like to mention that Kretz,i many years later and lentirely independent of me, reached exactly the same conclusions as I had. His very interesting study was based on experiments with diphtheria-immune horses. Following his usual tactics, Gruber will, of course, draw the conclusion that the increase in the tissues affinity, since it agrees with my theory, cannot really occur, and he will therefore regard the entire subject as utterly fallacious and best not discussed. The unprejudiced observer, however, need hardly be told that it is impossible for chemical groups attached to living protoplasm to maintain their affinity unchanged as though they were made of stone; especially is this true if we consider the varying function of the protoplasm. Let us take anilin as an example, and determine the combining heat of the NH2 group for a certain acid. We shall then find that nearly all substitutions of the benzol nucleus, as, for instance, the introduction of an amido group, a nitro group, a sulfo group, etc., markedly change the affinity either positively or negatively. Thus even the introduction of what is conceivably the most indifferent group, the methyl radical causes a distinct and marked diminution of the combining heat. Under these circumstances any one who thinks chemically would consider it peculiar if a change in the affinity of the cell constituents were to be regarded as something absolutely inconceivable and beyond the pale of discussion. Since Gruber has given only that part of Madsen and Dreyer's experiments which fits into his polemic, it will be necessary for me to supplement this with some additional' data from their study. These authors employed a diphtheria poison of which the fatal dose for a guinea-pig of 250 grammes was 0.009, and for rabbits -of 1200-1600 grammes, 0.0076. Calculated per kilo this shows that the rabbits were about six times as f.usceptible as guinea-pigs. The Lq dose, i.e., that amount of poison, which is just completely neutralized by one immune unit, was 0.6 cc. for guinea-pigs. Right here I must emphasize that the Lq dose, as I conceive it, refers exclu- sively to guinea-pigs, since according to my experiences this is the only animal in which, thanks to the pecuhar susceptibility, the con- stants of the poison can accurately be determined. In the serum mixture Lq all the constituents of the poison, toxin, and toxon are -completely neutralized, so that not only the single amount but also ' Zeitsoh. f. Heilk., Vol. 23, 1902. TOXIN AND ANTITOXIN. 523 iiigh multiples of this can be injected into guinea-pigs without causing a, trace of local or general reaction. If the same amount of poison, 1 fi7 0.6 cc, was mixed with xp^ I. E. instead of with one I. E. it was found that the toxin fraction had practically been completely neu- tralized, leaving only the toxons, characterized by the develop- ment of paralyses. Just in this poison Madsen and Dreyer have shown that the difference between toxin and toxon is qualitative and not quantitative. They found that mixtures of poison and antitoxin, which were near the limit of toxin neutralization, showed •only toxon action when given in small doses, whereas when the mix- ture was increased tenfold, death occurred from toxin.i //, however, the quantity of antitoxin was also slightly increased, even the tenfold multiple showed only toxon action. From these data we see that the poison consisted of about 167 units toxin-toxoid and 33 units toxon. This same poison was subjected to a thorough investigation on rabbits by Dreyer and Madsen and gave the following results: If 0.6 cc. poison are mixed with one I. E., it will be found that this mixture, which represents the Lq dose for guinea-pigs, is still highly toxic for rabbits. In order to render this amount of poison com- pletely innocuous for rabbits it is necessary to add more antitoxin; 240 as a matter of fact it requires ktjk I- E. Their statements concern- ing the behavior of mixtures between these two limits are also very 210 interesting. A mixture of 0.6 cc. poison + ^^ I. E. given to a rabbit gives rise to paralytic phenomena appearing on the fifteenth day and ending fatally on the twenty-second day. Even a mixture of 232 the same dose of poison with — -r I. E. produced paralysis com- mencing on the sixteenth day and continuing for several weeks. In view of the importance of these facts for the conception of a plu- rality of poisons, I cannot pass on without discussing them more fully. According to our definition of the Lq dose, such over-neu- ' The explanation of this is that the toxon determination by means of 1 I. E. naturally cannot be an absolutely exact one, small residual amounts of toxin, e.g., 1/10 lethal dose, readily being overlooked. If, however, an appropriate multiple, say ten times this mixture, be injected, this will contain ten times 1/10 fatal dose. 524 COLLECTED STUDIES IN IMMUNITY. (232\ like the mixture ;r^\ possess a considerable excess of antitoxin, are absolutely innocuous for guinea-pigs and can be injected in any desired quantity. In fact, owing to the excess of antitoxin, such mixtures furnish the animal with passive immunity and protect it, provided suitable amounts have been injected, against diphtheria poison and diphtheria bacilli. If such mixtures, how- ever, are still toxic for rabbits, only one possibility remains, namely, that the diphtheria poison in question contains a substance which is non-toxic for guinea-pigs, but still toxic for rabbits. This is my toxonoid.'^ So far as the behavior of partially neutralized mixtures is con- cerned, the investigations of the two authors show that mixtures which exert only toxon effects on guinea-pigs cause death and symp- toms of diphtheria poisoning in rabbits. In my opinion the phe- nomenon described can best be explained by the assumption that at least three varieties of poison are to be distinguished, possessing different affinities and different actions. Such an assumption, I believe, will best harmonize the actual facts. These poisons are: 1. Toxin, possessing the greatest affinity, kills rabbits and guinea- pigs acutely, but is much more toxic for the former. 2. Toxon, killing rabbits acutely and guinea-pigs with paralytic symptoms. 3. Toxonoids, producing paralyses in rabbits but innocuous for guinea-pigs. That all these poisons act more powerfully on rabbits than on guinea-pigs is explained by the absolute higher susceptibility of these animals. So far as the behavior of the toxonoids is concerned, in which enormous differences in rabbits and guinea-pigs are mani- fested, such behavior finds numerous analogies in toxicology, espe- cially in the study of toxins. Thus heroin, an acetyl derivative ' Almost at the outset of my investigations and long prior to Madsen and Dreyer I obtained results entirely similar to these. My unpublished but very extensive studies showed that this property is not possessed by all diphtheria poisons, for I also encountered poisons in which the L,, dose was exactly the same in guinea-pigs and rabbits. This tact controverts the assumption that perhaps the described phenomenon is due to an incomplete neutralization, such as Arrhenius and Madsen have demonstrated in the union of boric acid and ammonia, and in that of tetanolysin and antilysin. If this were the case one would expect the phenomenon to be present in all diphtheria poisons to the same extent, and this is not the case. TOXIN AND ANTITOXIN 525 •of morphine, is far less toxic for rabbits than is morphine; for asses on the other hand it is far more toxic than the latter substance. In the case of toxins v. Behring long ago showed that for different species of animals certain toxins are very differently affected by trichloriodine. As I suggested in my address at the International Medical Congress in Paris we are evidently dealing here with incom- plete toxoids, i.e., with toxoids whose toxophore complex is not yet completely destroyed. Portions of this complex still left to the poison possess a high toxicity for one species of animal and little ■or no toxicity for another. The toxophore groups of the tetanus poisons mentioned above (Tizzoni and v. Behring) afford a sufficient analogy. A consideration of these facts will show that Gruber's statement, that the facts observed by Madsen and Dreyer reduce my theory to an absurdity, is absolutely incorrect. On the contrary, 1 may say that the facts brought out by these authors are most readily explained on the basis of my theory. I shall now take up Gruber's recent experiments. These were first published in the Wiener klin. Wochenschrift ^ in a form strongly suggestive of the comic supplement of a newspaper. The discussion takes the form of a letter purporting to be written by a certain " Phantasus," and is really very cleverly conceived. Only I would protest against publications of this sort appearing in the columns of a scientific journal. Two series of experiments come into question. The first series is so curious that I have not felt any desire to repeat the experi- ments. These deal (a) with the property of sulphuric acid to act as a poison on cane sugar, and (6) with the antitoxic action which water exerts on this property. Any one with even the faintest knowl- edge of chemical processes knows that the sulphuric acid as such is not deprived of this poisonous action by water; this is effected only by an alkali which, by forming a salt, neutralizes the acid. 1 am able to furnish an additional case which shows the " detoxitizing " effect of water. A highly concentrated sulphuric acid, containing considerable anhydride, acts destructively on iron. If H2O is added until the solution contains the monohydrate it will be found that the addition of the water has reduced this capacity to attack iron ' Wiener klin. Wochenschr., No. 27, 1903. '526 COLLECTED STUDIES IN IMMUNITY. to practically zero. In this case then, just as Gruber states, the water has acted as an antitoxin. On the addition of more water to the mixture, however, the iron is again attacked. In fact the more water now added the stronger becomes this action. We thus obtain the curious result that in small doses water acts as antitoxin, while in large doses it increases the action of the poison, surelj' an interesting problem for Dr. Phantasus! This is merely one of the special instances of the fact thus far unexplained, that the different hydrates of sulphuric acid, or their mixtures, manifest a most extraordinary variation of properties- I may refer the reader to the minute and fundamental study of Knietsch,! [^ which the variations of the properties of sulphuric- acid at different concentrations have been represented in the form of a curve for many of these properties, thus specific heat, electric resistance, boiling point, vapor tension, viscosity, capillarity, action on iron, etc. A glance at this chart gives one the impression of chaos, and at once shows that on these complicated problems only deep studies can lead to any results, and that the ten-minute experi- ments made by Phantasus-Gruber-Pirquet are absolutely worthless. This is especially true in Gruber's case, which deals with an obscure reaction in which oxidation, abstraction of water, cleavage and sul- phurization take part. Hence I deny that crude experiments of this kind can be used to gain an insight into such an entirely different subject, or that the conditions there observed can even be com- pared to the minutely differentiated processes of toxin-antitoxin combination. We shall next take up Gruber's experiments which deal with the haemolytic action of water, since to persons at a distance these might give the impression that they really have something in com- mon with studies in hsemolytic toxins. The experiments are sup- posed to show that water is composed of an infinite number of differ- ent poisons. Let us listen to Gruber for a moment: " Pure water exercises a very great osmotic pressure on red blood-cells, leading to their swelling and to the escape of haemoglobin. Hence water is a toxin for the erythrocytes, salt is an antitoxin. When successive amounts of salt are added to the water this toxicity is gradually lost, for the affinity of the water, and with it the osmotic pressure, is thus gradually decreased." ' Bericht d. deutsch. chem. Gesellschaft, 1901, page 4069. TOXIN AND ANTITOXIN 527 We see therefore that Gruber-Pirquet assume that pure water possesses a high osmotic pressure and that salt diminishes this. The very foundation of the doctrine of osmotic tension, however, is the fact that water as such possesses NO osmotic pressure, and that such pressure is produced by salts dissolved in the water. I can- not refrain from pointing out this woful ignorance of the most ele- mentary principles on the part of authors who do not hesitate to- accuse me of " complete lack of insight into chemistry," although for years I have endeavored, and not unsuccessfully, to apply the great discoveries in chemistry to medicine. The solution of erythrocytes by means of water is one of th& best studied subjects in medicine. It is generally recognized that the water as such is no poison whatever, but that its action is due to the fact that water abstracts the salts and other soluble substances from all living cells, including, of course, the red blood-cells. These substances are abstracted in such considerable amounts that this alone suffices to bring about the death of the cell. The swelling of the red blood-cells is due to the penetration of water and this again depends on the permeability of the limiting membrane on the one hand and the power of the water to abstract water on the other. With the same right that Gruber regards water as a poison one could call nitrogen a poison and oxygen as the counter poison for the nitrogen, for animals die in pure nitrogen, but live if oxygen is added. At any rate nitrogen poison can be recommended to Dr. Phantasus for extended study. Perhaps some day he will also work out its spectrum for us. Despite the fact that the premises from which their experiment proceeds are based on a complete misconception of the idea of poison, I have repeated the experiments of Gruber and Pirquet. The results show that their statements concerning the experiment are entirely incorrect. I first determined the concentration of salt and of sugar, in which the ox blood-cells remained completely intact; for NaCl this was found to be 0.63%, for cane sugar 6.4%. By diluting with water, various degrees of this isotonicity (1/10, 2/10, etc.) were produced. Each tube contained altogether 2 cc. of fluid and one drop of defibrinated ox blood. The result is shown in the form of a " spectrum," which may be compared to that obtained by Gruber in his experiments. This comparison shows us that Gruber's experiments are abso- 528 COLLECTED STUDIES IN IMMUNITY lutely incorrect, and that they contradict all that is thus far known concerning solution of the red blood-cells. Gruber states that in a 1/10 isotonic solution, one containing about 0.07% NaCl, about one-fifth of the blood-cells remain undissolved. All other authors, however, have found that even in a solution of 0.3% NaCl, the blood-cells of all warm-blooded animals are still completely dis- solved, so that the solution appears uniformly laky, and microscopical examination shows not even a trace of red-blood corpuscles. In Gruber's spectrum, however, we find that with this percentage more than half of the blood-cells remain undissolved. This indicates that in Gruber's experiments the grossest sort of errors abound. With Salt Decrease of Hamolyse ia Percent With Sugar Decrease of Hamolyse ia Percent 30 y„ Ji'o fo y,a *f. ^-Jfo %, %o X Isotonicity Xo % r,« 'A, Sfo Jf. Jlo "/lo % % Isotonicity Fig. 1. — "Poison spectrum" of water according to Gruber. What can we deduce from these spectra? The fact that a cer- tain amount of NaCl can be added to the " poisonous " water with- out inhibiting haemolysis, would lead authors holding Gruber's views to conclude that this " poisonous " water contains a prototoxoid ■whose neutralization has no effect whatever on the toxic action. A single glance at the detailed literature on this subject should, how- ever, have convinced these authors that their curve, as such, has nothing whatever to do with toxic actions, but is merely the expres- sion of the specific differences in the red blood-cells. It is well known TOXIN AND ANTITOXIN 529 that the blood represents a mixture of cells of various ages, and it is not at all surprising, therefore, that these should behave differently toward different injurious influences. We are here dealing with a property of the erythrocyte's protoplasm, which protoplasm will possess a different degree of vulnerabihty according to its age. Are Gruber- Pirquet entirely unaware that an important and much-employed procedure for determining the resistance of the blood rests on just With Salt Decrease 35 r 30 ■ 10 No Decrease With Sugar Decrease 35 r No Decrease >fo ^Ji, SIl^ % % 51. %. % «o % iKo y„ % '4, Ko 51o Jlo ' Isotonicity Isotonicity Fig, 2. — " Poison spectrum" of water according to Ehrlich. this principle? Every text-book on haematology teaches that we distinguish blood-cells of maximum, minimum, and intermediate resistance, and that the extent of resistance is merely the difference between the maximum and minimum. Instead of this, however, Gruber feels compelled to draw from his curves conclusions having such far-reaching consequences as, for example, that water is full of poisons, of haptophore and toxo- phore groups, etc. But if he believes that this proves the folly of my conception of toxin neutralization, so much the worse for him and his authority Phantasus. 530 COLLECTED STUDIES IN IMMUNITY. If one conducts experiments that have nothing to do with the problem under discussion, further, if the method of these experi- ments is grossly at fault, and it, finally, the results thus obtained are given an utterly false interpretation, it is not surprising that the most fantastic results are obtained. Finally Gruber describes one more experiment which he illus- trates by means of a curve. According to him this too demonstrates that my theory is untenable. The experiment shows that the haemol- ysis of ox blood, by means of a certain quantity of specific hsemolytie serum within half an hour, is dependent on the dilution. I need hardly remind my readers that 1 have always laid stress on the chemi- cal nature of the toxin and antitoxin combination. 1 can assure them that the factor of the degree of concentration has ever been sufficiently regarded. If Gruber will refer to my first study on this subject, " Die Werthbemessung des Diphtherieheilserums," he will find the statement: "that the union of poison and antibody pro- ceeds much more rapidly in concentrated than in dilute solutions," and further also " that heat hastens the union and cold retards the same." The behavior which Gruber describes is all the less surprising since he is dealing with a complex process depending on the action of the amboceptor-complement combination. How readily this combination is dissociated has repeatedly been pointed out by us. Perhaps Gruber thinks that this experiment is new to me; every one versed in the subject, however, knows that we are here deal- ing with the most commonplace phenomena, with which every beginner is well acquainted. I should like to point out, however, that this phenomenon, namely, that dilution with water inhibits the action of hsemolysins, is not at all constant. On the contrary it is limited to those cases in which the affinity between amboceptor and cell, or between amboceptor and complement is relatively slight. If one employs poisons in which the affinity between receptor and cell is great it will be found that within the limits mentioned the addition of water is practically without effect. Thus, in working with cobra venom, I found that a given quantity of this poison exerted exactly the same effect whether the volume of water used was 1 or 15. It would lead us too far to enter into all the distortions and mis- conceptions contained in Gruber's polemic. To do this would require almost a complete reprint of all my articles, as well as of many others- TOXIN AND ANTITOXIN. 531 emanating from the Institute— with all of which -Gruber seems quite unfamiliar. 1 shall content myself therefore with a brief discussion of Gruber's conclusions. Gruber states: 1. " There is no warrant for assuming that the bacterial toxic solutions contain a number of poisons possessing qualitatively simi- lar actions but differing in intensity and in their affinity to the anti- toxin." In the preceding pages I have conclusively shown that his view cannot be harmonized with the actual facts. But even a priori there is no reason to assume that bacterial cells always produce only a single poisonous metabolic product. Thus, to mention only a few examples, we know that cinchona bark contains about twenty different alkaloids, opium about the same number; Flexner and Noguchi's researches show that snake venom contains at least four different poisons (haemotoxin, leucotoxin, neurotoxin, endothelio- toxin), and the yeast cell, we know, contains a number of different ferments. Furthermore, 1 may again call attention to the fact that the secretion of tetanus bacilli contains four distinct poisons, namely, two varieties of tetanospasmin, my tetanolysin, and the poison which, according to Tizzoni, causes the cachexia. So far as diphtheria poison is concerned the reader is referred to my previous statements. My assumption of the existence of at least two poisons, toxins, and toxons, is borne out by the clincal observation that in certain epi- demics there is a large percentage of paralyses.^ 2. " There is no reason for assuming that the mode of action of the toxins is absolutely unlike that of other organic poisons." Nevertheless, the fact remains that the principal characteristic of the toxins, namely, the production of antibodies, does differentiate them from all other poisons, Gruber to the contrary notwithstand- ing. Two years ago Gruber could have found an ally in Pohl, who ' In animal experiments as a rule, the toxons do not manifest themselves until the toxins (which possess a greater affinity) have been neutrahzed by the antitoxin. Dreyer and Madsen, however, have described a diphtheria poison (Festskrift, Kopenhagen, 1902), in which the toxons could be demon- strated even by the mjection of sublethal doses, the injections being followed by paralytic phenomena. In view of the constants of this poison, as they were determined by Dreyer and Madsen, this behavior is not at all surprising, for while old diphtheria bouillons ordinarily contain about 33 toxon equivalents to 167 toxin equivalents, this poison contained about 500 toxon equivalents for that amount of toxin. •'532 COLLECTED STUDIES IN IMMUNITY had apparently succeeded in immunizing against solanin. Since then, however, the researches of Bashford ' and of Besredka'' have shown that it is impossible to produce antibodies against either solanin or saponin. Pohl himself no longer maintains the existence of a specific antisolanin. Of the various poisons, which seemed to promise the best for successful immunization, morphine should be mentioned first. Recently Hirschlaff ^ claimed actually to have produced an antimorphine serum. Morgenroth,* however, was able to show that the results obtained by Hirschlaff were merely apparent, not real, and that they depended on the fact that the doses of poi- son employed by Hirschlaff were not surely fatal, especially owing to the increased resistance of the animal following the serum injection. Hence the statement still holds true that all poisons chemically well defined do not possess the property of producing antitoxins. So far as other differences between ordinary poisons and toxins are concerned, I may refer particularly to my detailed articles in von Leydens Festschrift * and to the excellent monograph by Over- ton .^ From these it will be seen that the action of the chemically defined poisons, alkaloids, glucosides, etc., on parenchyma is the result of a solid solution or of a loose salt formation. In accordance with the loose character of the combination, the action of these poisons is a transitory one. The firm union and prolonged action peculiar to the toxins is entirely absent. Besides this the period of incubation is wanting in most ordinary poisons, although there are a few exceptions like arsenic, phosphorus, tartrate of tin and sodium, and vinylamin. In the toxins, on the other hand, a period of incubation is the rule. Entirely in accordance with the views of Emil Fischer concern- ing ferments, I have ascribed the specific combining processes of toxins to certain stereochemical groups of atoms (haptophore groups). These unite only with such other atomic groups which fit to them as does a key to a lock. The ordinary chemical groups of organic chemistry possess affinities for a large number of other groups. Thus ' Archives Internationales de Pharmacodynamics, Vols. 8 and 9. 'See Metchnikoff, L'Immunite, Paris, 1901. ' Berliner klin, Wochenschritt 1902. 'Ibid.. 1903, No. 21. « Von Leydens Festschrift. .August Hirscbwald, Berlin, 1902. ' Studien iiber die Narko.se, .lena IPOl. TOXIN AND ANTITOXIN 533 the aldehyde group can unite with amido groups, hydrazin groups, methylen groups, etc. In this group therefore the combining prop- erty is not specifically hmited, but extends to a large number of combinations. On the other hand the one characteristic of toxins and ferments is just this specific combining property. 3. " The transformation of toxins into non-poisonous combina- tions (toxoids), possessing the same affinity for the antitoxin is pos- sible, but has not been definitely proven." I have already clearly shown that the doctrine of toxoids, now generally accepted, is one of the best-established foundations in the entire subject of immunity. However, with critics like Gruber, who blindly condemn the views of others, one ought to be satisfied if they recognize at least a possibility. 4. " Toxin and antitoxin have feeble chemical affinities and therefore unite with one another to form dissociable combinations or perhaps molecular combinations in varying proportions. These con- ditions explain the long incubation of the poisonous action and other marked phenomena." To be sure the affinity between toxin and antitoxin may in some instances be a feeble one, but this is by no means always the case. The affinity between tetanus toxin and antitoxin is slight, and so is that between complement and amboceptor. On the other hand, however, there are poisons, such as diphtheria toxin and snake venom, in which the reaction proceeds under strong affinities, so that the process of neutralization takes the course of a straight line and not of a curve. Gruber's statements might also give one the impression that he is the first to introduce dissociation as an explanation of some of the phenomena in immunity. I have always emphasized the fact that amboceptor and complement are loosely bound, uniting at high temperatures, but dissociating at low temperatures .^ But this is all wrong according to Gruber,^ for a year and a half ago he > I shall cite a passage from Ehrlich and Morgen roth's First Communi- cation Concerning Hsemolysins (see page 7 of this volume), a passage which Wechsberg has already called to Gruber's attention (Wiener kiln. Wochenschr. 1901, No. 51). "This experiment clearly shows that under the conditions present complement and immune body exist in the serum independently of one another "; further also, " under certain circumstances the immune body enters into a loose chemical union with the complement, one which is easily dissociated." In view of this I cannot understand why Gruber still main- 534 COLLECTED STUDIES IN IMMUNITY laid down the dictum, " There is no dissociation by means of cold." It seems not to have mattered to him that his statement is opposed to even the most elementary principles of chemistry. As a matter of fact we have always paid due attention to disso- ciation and to the reversibility of the reactions. I should like to call Gruber's attention to the fact that the sentence: " In the union of the amboceptors we are dealing with a reversible process " occurs in one of Morgenroth's studies ^ from this Institute. Further than this such questions do not affect the Side-chain Theory, as such. The whole discussion is evidently designed to hide the fact that Gruber's position is really based on my theory. So far as the mode of action of the toxins is concerned, Gruber's standpoint and mine are essentially the same. Thus Gruber states that: ''All poisons must be 'anchored' by the cells and the anchoring group of atoms is probably always different from that group which gives the substance its toxicity." I spent many years in establishing this view and it is now everywhere accepted as axiomatic. I defy Gruber to show me the text-books of toxi- cology in which, previous to my work, this conception appears, a conception which dominates the laws of the distribution and action of poisons. If he should again refer to S. Frankel's book^ I can only remark that while the account of my views is very admirable, it is nothing more than a resume of the points which I had previously developed. Perhaps I can even aid Gruber's memory and let him speak for himself. A year before his declaration of war he spoke of " the brilliant hypothesis of that genius Paul Ehrlich, the greatest of living pathologists." In a little work * published at that time, and quite enthusiastic over my theory he states: " According to Ehrlich only such substances are poisons which unite chemically with some constituent of the organism." And yet this same Gruber to-day says: " These are merely new words for what has long been known." I should not like to deprive the reader of hearing still another tains that my view of the production of anticomplements, according to which amboceptor and complement are prmly united, is absolutely incomprehensible. ' Miinch. med. Wochenschr. 1901, No. 48. ' Ibid., 1903. ' Die Arzneimittelsyntbege, Berlin, 1901. ' Max Gruber. Neuere Forschungen ijber erworbene Immunitat, Vienna, J900. TOXIN AND ANTITOXIN. 535 authority often cited by Gruber, namely von Behring. Shortly after my theory was formulated this author expressed himself as follows : ' " It seemed about hopeless to attempt to penetrate these mysteries, ■when recently Prof. Ehrlich published a theory which is destined to illuminate even this subject." But even now Gruber does not doubt " that the toxins are very complex bodies and that the toxic action is connected with certain atomic groups; that possibly it is necessary for certain atomic groups to be present so that the poison molecule can be anchored and the toxicity manifest itself." One will at once ask why then Gruber attacks my theory if he is satisfied with its fundamental principle, namely, the assumption of an independent haptophore and toxophore group m the poison molecule? That I cannot answer. To be sure further along one encounters the warning, " But one must not too highly personify these different atomic groups, and think of this entire poisoning as a drama with four long intermissions between the acts." I cannot see what is to be gained by such idle talk. As a matter of fact the majority of infectious diseases as well as the poisonings do proceed in three phases, and these have always been separated, namely, incubation, the disease itself, recovery. Hence to explain these, as we do, through the independent action of toxophore and haptophore groups seems the most natural thing to do. It is strange that Gruber should now speak of the anchoring of the poison by the elements susceptible thereto as something per- fectly obvious, for in his first attack he laid especial emphasis on " his being the first to furnish the important demonstration that the specific immune substances are bound by the bacteria." How- ever, Gruber's claim cannot be allowed, for all that he demonstrated was that the agglutinins' are used up in the reaction. The signifi- cance of a chemical union, however, was first pointed out by us. This union, as Morgenroth's studies on the behavior of anchored amboceptors show, need in no way be connected with toxic action or with a using up of the substance. Gruber's statement that the long period of incubation is explained by the feeble affinities I must emphatically deny. The studies of Donitz 2 and of the Heyman school ^ show that the injected toxins ' Deutsche med. Wochenschr. 1898. 'Ibid., 1897. ' Decroly et Rouse, Arch, de Internat. de Pharmacodynamie, Vol. VI. 536 COLLECTED STUDIES IN IMMUNITY disappear from the circulation in a few minutes. It is therefore idle to talk of a slow union such as would correspond to weak affini- ties. But, says Gruber, " it is impossible to understand why the toxophore groups, after they have been brought into proximity to the protoplasm, do not at once commence their activity, but always «top to consider the matter for several hours." One cannot seriously discuss the subject with such a questioner. Gruber might just as well ask that all chemical reactions proceed rapidly, and deny the possi- bility of a slow reaction. The slow action of the toxophore group is not at all remarkable, especially in the domain of toxins. This is particularly true if we remember that with certain poisons (e.g. botulism toxin), one part of toxin to 500 million parts of body weight suffices to cause death, and that the rapidity of action is dependent to a high degree on the amount of the active substance. Is Gruber possibly of the opinion that in the paralysis of diph- theria, which as is well known usually develops after the lapse of weeks, the toxon courses about free for twenty days or more before entering the tissues and then suddenly exerts its action? To the unprejudiced critic the importance of the separation of toxin bind- ing and toxin action for the proper understanding of the period of incubation, is conclusively demonstrated by Morgenroth's i experi- ments with tetanus in frogs. Courmont and Doyon, as is well known, discovered that the frog is susceptible to tetanus poison only at higher temperatures, and not when the animal is kept cold. Mor- genroth was able to show that at low temperatures the tetanus poison is bound, but exerts no toxic action. Frogs are injected with tetanus toxin and then kept on ice for days. If then they are subjected to higher temperatures, it will be found that they behave exactly as if they had just been inoculated. And yet the toxin has been bound by the central nervous system even at the low temperature; for if after several days at low temperature the animal be injected with an amount of antitoxin, even much more than sufficient to neutralize the poison, tetanus will still develop if the frog is subjected to a higher temperature. But this is not all. If frogs, after being injected with tetanus, are subjected to a high temperature for one day, and then placed in the refrigerator, they will not become sick. But on bringing the animals back into higher temperatures after ' Arch. Internat. de Pharmacodynam., Vol. 7, 1900. TOXIN AND ANTITOXIN. 537 the lapse of weeks or months, it will be found that they sicken after a shortened period of incubation. Are any further proofs of the slow action of the toxophore group required? It is not easy to meet all of Gruber's statements because he fre- quently makes use of misleading tactics. He often reaches the same conclusions as 1 myself, and grants that certain of my views are permissible or probable. In some things, he says, I am correct jn the main, in others 1 may be right, but have not strictly proved my point. All these statements are but a clever contrivance to give the reader the impression that my theory is but a product of the imagination when as a matter of fact is it really a hypothesis developed experimentally. This brings me to Gruber's fifth con- clusion. 5. " The development of antitoxin has no connection whatever with toxic action or cell immunity." It will suffice for me to call attention to the fact that I have always insisted on distinguishing between the haptophore and toxophore groups in the toxin molecule and also between the anchoring and the action of poison. I might add that this absolute independence of toxic action and antibody production is a principle which 1 formu- lated, not Gruber. As far back as 1898, Weigert i rightly • pointed out that my demonstration ^ of antitoxin production through non- poisonous toxoids was sufficient to demonstrate th-e independence of antitoxin production and toxic action. Furthermore I have repeatedly pointed out that the development of antitoxin depends on the haptophore group. Over 1^ years ago Paltauf^ called Gruber's attention to the weak points in his objection and one might therefore have expected that Gruber would not again bring forward this old fairy-tale. In the future I shall not reply to perversions of this kind. So far as the reasons are concerned, which Gruber gives in sup- port of the above statement regarding the development of anti- toxin, I may at once say that I can assent to them word for word- Thus the statement that: (a) " Many substances which are entirely innocuous lead to the formation of antibodies '' is the first consequence of my views and experimental labors. The fact that ' Lubarsch-Ostertag, Ergebnisse der pathologischen Anatomie, IV Jahrgang ' Werthbemessung des Diphtherieheilserums, Klin. Jahrbuch. 'Wiener klin. Woclienschr. No. 49, 1901. 538 COLLECTED STUDIES IN IMMUNITY. (b) " Certain animals non-susceptible to certain toxins never- theless produce antibodies '' needs no further explanation according to my theory. Certain species of animals may possess suitable receptors for binding the toxin and producing antitoxin although their cells are insensitive to the action of the toxophore group. Accord- ing to Metchnikoff this seems often to be the case with tetanus toxin in crocodiles. As already pointed out years ago by Weigert > accord- ing to my theory, the production of antitoxin need not at all be preceded by any injury in a clinical sense. In fact, too strong an injury may cause the cell to lose its power of regeneration, owing to the toxic action on the vital group [Leistungskern], For example, if a specific nerve poison is anchored by a fitting receptor of an indiffer- ent cell (liver) we should expect the production of an antibody by the liver, even if the liver-cell does not become tetanized. In my address at Hamburg' before the Congress of Naturalists I pointed out that the local origin of antitoxin, which Romer deduces from his splendid experiments with abrin, will often make it possible to transfer part of the antitoxin production from the vital organs to the indifferent connective tissue, by means of subcutaneous injec- tion of poison. Gruber's next statement is: (c) " Despite a plentiful production of antibody, the suscep- tibility to the poison may remain, or even increase." 1 have already discussed the principle of hypersensitiveness and mentioned the fact that this objection restrained me for a long time from publishing my theory, iiut even these phenomena were satisfactorily explained in accordance with the side-chain theory, by the assumption of an increase of affinity and a rupture of the toxin-antitoxin comVjination. To be sure it is possible that our explanation touches but part of the subject, and that in reality the phenomena are far more complex. But this is no reason for seek- ing to overthrow the theory; to do so would be to completely mis- apprehend the purpose of a theory. Surely one cannot demand that a theory will at once explain all the complex phenomena of so difficult a subject as this. A theory ought primarily to possess heuristic value, pointing out new paths into a complex subject; it should smooth the way. The actual research must be left to the scientific investigator. Science can be advanced only by means I- c. ' Deutsche med, Wochenschr, 1901, TOXIN AND ANTITOXIN. 539 of experimental analysis, and not by high-flown words of a mis- leading dialectic. (d) "Cell immunity can be acquired without the formation of antibodies." This statement, too, does not surprise me. All that the side- chain theory aims to do is to explain how the production of anti- bodies may be conceived. But I have never yet claimed that this is the only means by which the organism can defend itself against deleterious influences. I would call attention particularly to the Sixth Communication on H£emolysins,i in which Morgenroth and I pointed out that not all substances capable of being anchored need necessarily excite the production of antibodies. We have always emphasized, however, that immunity may be developed despite this, chiefly through a disappearance of receptors.^ In our isolysin ■experiments we observed that the blood-cells became insusceptible and we demonstrated that this was due to a lack of receptors. The interesting fact observed by Kossel and by Camus and Gley that •during the course of immunization with eel blood, the blood-cells -of rabbits acquire a high resistance against that poison, is probably most easily explained by assuming that the cells acquired immunity in the way above mentioned. This, of. course, does not exhaust the possibilities of the origin ■of immunity not due to antitoxins. Thus under the influence of the anchored poison new receptors may be formed which are so firmly united to the protoplasm that they are not thrust off. Such receptors Morgenroth and I have therefore termed "sessile receptors." If the production of such an excess of receptors takes place in a rather indifferent tissue, as in connective tissue, it will readily be seen how the receptors can serve to deflect the poison, and produce a more ■or less marked immunity. In that case on comparing a normal animal with an immunized one, the conditions would be like those ■observed with tetanus poison in normal guinea-pigs and normal rabbits, respectively. The studies of Donitz and Roux have shown that the guinea-pig possesses receptors for tetanus toxin only in the brain, whereas, rabbits, in addition to the receptors in the cen- tral nervous system, possess about thirty times as many such recep- tors outside this system. ' See page 88. ' Schlussbetrachtungen in Nothnagel's Handbuch., Vol. ^'III. 540 COLLECTED STUDIES IN IMMUNITY. Another possibility of cell immurity is that the protoplasm of cells which are ordinarily susceptible is no longer affected by cer- tain poisons. This kind of immunity, which to be sure I consider very rare, would correspond to mithridatism or acquired tolerance in the old sense. A fourth possibility, finally, is the adaptation of the phagocytic apparatus in Metchnikoff's sense. It is obvious, of course, that all the sevarious subordinate kinds of immunity occur alone as well as in manifold combinations. Thus, as already mentioned, immunization with eel blood is followed by antitoxin immunity and tissue immunity. In the lower animals, however, which as Metchnikoff has shown are but little adapted to the production of antitoxin, other defensive contrivances leading to cell immunity will predominate From this point of view there- fore the condition described by Gruber, namely, that frogs can be immunized against abrin without their showing any antitoxin, offers no difficulty. So far as the frog is concerned the only question is which kind of cell immunity is present, i.e., whether there is a dis- appearance of receptors, or whether there are sessile receptors, etc.^ In view of the detailed statements given above I presume I need add nothing to the following passage in Gruber's conclusion: (e) "The production of antibodies takes place at entirely different localities than does toxic action." The discerning reader will at once see that this statement does not in the least contradict my views. In fact it is merely another way of expressing what is really the nucleus of my theory. The generalization, however, is false, that the production of antibody necessarily takes place in localities different from those in which toxic action occurs. If Gruber therefore believes that this riddles my theory it is evident that he understands the principles under- • Gruber cites, as a serious objection to my theory, that Madsen observed immunity in a. rabbit which had been immunized with diphtheria toxin, and yet was unable to find antitoxin in the blood. I will only say that Madsen did not find the blood entirely free from antitoxin since he tested the serum only to 1/10 I. E. Small quantities of antitoxin could be very well have been present and these, of course, would be of considerable importance for the ques- tion as to whether this was a case of entire absence of antitoxin. Besides this I may add that in diphtheria poison the case reported by Madsen must be extremely rare. During the course of many years the different Serum Institutes have immunized thousands of different animals against diphtheria. In all this time, however, I have never learned of a case analogous to Madsen's, either from the literature or from private sources. TOXIN AND ANTITOXIN 541 lying my views no better than he did two years ago. At that time Paltauf 1 tried in vain to make this elementary consequence of the side-chain theory comprehensible to him. Gruber's sixth conclusion is as follows: 6. " The specific antibodies are not normal body constituents. They are newly formed only after the introduction of foreign sub- stances. This new formation has the character of an internal secre- tion." So far as the first point is concerned one cannot help being amazed at the lack of literary knowledge which permits an author to make such statements. 1 need only refer to the studies of Pfeiffer, Bordet, Flexner, Kraus, Bail, Peterssen, etc., or to the comprehensive resume by M. Neisser ^ concerning the antibodies found in normal serum, The literature on normal antibodies of various kinds is very large, and yet has been entirely ignored by Gruber. Thus amboceptors against different bacteria (cholera, typhoid, anthrax), antiambo- ceptors, anticomplements, antitoxins, antiterments, etc., have been observed. 1 shall, however, mention merely a few points which may be of special interest. I. The very frequent occurrence of diphtheria antitoxin in horses (Meade, Roux, Bolton, Cobbett). in view of the high percentage bf this occurrence, the attempts to ascribe this antitoxin in normal horse serum to a diphtheria running a latent course must be regarded as failures. Since this phenomenon has been observed in about 30% of the horses, it is surely not reasonable to assume that an occurrence of diphtheria in horses should so frequently have entirely escaped the large number of excellent observers representing animal pathology. Such a frequency of the disease should, of course, also have manifested itself epidemiologically. The fact that in one single instance Cobbett observed a diphtheritic infection in a horse cer- tainly does not alter the circumstances. II. I must mention the interesting observations made by v. Dun- gern ^ that normal rabbit serum contains an antibody against that substance in star-fish eggs which is toxic for sea-urchin spermatozoa. 1 am sure that no one, just to please Gruber, will assume that there is any connection between rabbits and star-fish and their eggs III. Laveran has found that the blood of healthy human beings 'Wiener klin. Wochenschr. 1901, No 49. ' Deutsche med. Wochenschr. 1900. ' Zeitschr. f. aligemeine Physiologie, Vol. 1, 1901. 542 COLLECTED STUDIES IN IMMUNITY contains a substance which kills trypanosomes, whereas this is not present in the blood of other aninaals and cannot be produced in so- large an amount even by immunization. This might be the reason why (aside from sleeping sickness of Central Africa) man is so refrac- tory toward trypanosome infection. But if such a wealth of facts is disregarded in statements con cerning " our certain knowledge," it must be admitted that a scientific discussion is entirely out of the question, and had best be avoided in the future. Furthermore, so far as conceiving the production of antitoxin to be a secretion is concerned, I may say that this part of the paper is nothing but another way of stating what 1 have always held. Paltauf,! for instance, pointed this out to Gruber some years ago, " In passing I may say that an ' escape ' of particles of protoplasm into the blood really denotes a secretion." In an address delivered in 1899 (!) I expressed myself in a way that shows that I have always considered the production of antitoxin to be a secretory process.^ " Or, s'il y a lieu de croire que les Antitoxines doivent leur origine a une sorte de fonction secretoire des cellules et ne sont par conse- quent nuUement 6trangeres a I'organisme, le rapport specifaque qui les unit avec leurs toxines n'en devient que plus strange." This point has been demonstrated especially by the researches of Salomonsen and Madsen, and of Roux and Vaillard. But just this secretory character of antibody production is abso- lutely at variance with the older view that antitoxins are merely transformation products of the toxins. This was the view defended by Buchner and held to be possible by Gruber even in his last attack. It is just as impossible to believe that antitoxins arise from toxins as it is to believe that lipase is transformed fat, or amylase, trans- formed starch. Thus we see that the various points brought up by Gruber are nothing but reproductions of my views, the little that deviates is incorrect or is based on misconceptions of an inflated knowledge of the literature. Gruber's last two conclusions contain so little that is new that It hardly pays to discuss them. For completeness' sake, however, I shall append them. ' Weiner klin. Wochenschr. 1901. No 49. ' This appeared only as an abstract in La Semaine Medicale, 1899. TOXIN AND ANTITOXIN. 543 7. "The power to excite the formation of antibodies is due to certain peculiarities in the chemical structure of the substance which excites this antibody production. A prerequisite for this produc- tion as well as for toxic action is the chemical union of the foreign substance with certain particular constituents of the cells." This, I may say, is a short, though not particularly good, r6sum6 of the side-chain theory. 8. "The non-poisonous toxin-antitoxin combination also lacks the power to excite the production of antitoxin. The entire chemi- cal character of this combination is different from that of the uncom- bined substances." This, too, is one of the fundamental principles of my theory, and is most readily explained by the assumption that the antitoxin fits into the same group which effects the uaion of the toxin with the susceptible cells. Furthermore, I really see no reason why Gruber should make a special point of the fact that the chemical character of the toxin-antitoxin combination has changed. That is merely a trick of speech which will make but little impression on the scientific reader. That the antitoxins are nothing but thrust-off receptors capable of uniting with the poison — this assumption, together with its immediate consequence that the toxin-antitoxin combination must be non-poisonous, is the key to my entire theory. We are, in fact, dealing with an extremely important law which Weigert and I compared to the principle of the lightning-rod and which V. Behring briefly expressed as follows: "The same substance in the hving body which, when in the cell, is the prerequisite of a poison- ing, becomes the healing agent when it is present in the blood." This law applies not only to the toxins but possesses general applic- ability. I may here refer the reader to Ransom's experiments, which show that the cholesterin in the red blood-cells causes haemolysis by saponin, while at the same time the cholesterin of the serum causes an inhibition of this poisoning. Gruber, however, thinks that it has not been proved that the haptophore group, which anchors the toxin to the vital constituent of the protoplasm, is the same which anchors the toxin to the anti- toxin. A year and a half ago he expressed this quite clearly as follows : 1 "Ehrlich may have demonstrated that the toxin is bound to ' Wiener klin. Woohenschrift, 1901, No. 50. 544 COLLECTED STUDIES IN IMMUNITY the antitoxin by a combining group which differs from the toxo- phore group. But where and how has he shown that tiie toxin in addition to its toxophore group possesses only one haptophore group, namely, the one which combines with the antitoxin? How has he shown that the same haptophore group acts in all chemical reac- tions of the toxin? On the contrary it can positively be stated that the toxin must necessarily be a very complex molecule possessing many different haptophore groups. Here, gentlemen, lies the root of the evil. All this misconception of the side-chain theory would have been impossible but for the mistake in the choice of an article; i.e., if Ehrlich instead of speaking of the haptophore group had said o haptophore group." So this is my great fault, the choice of an article! I may leave it for the reader to decide how weighty this objection is. Never- theless let us see what Gruber really means. Let us assume, for example, that a poison, in addition to the toxophore group, possesses two different groups with haptophore functions. One of these, group a, corresponds to what my theory demands, since it is able to combine with a receptor of the cell. As a result of this combination, however, there is to be not an over- production of a receptor fitted to a, but the production of a differ- ent substance, fitting the second haptophore group, h, of the toxin. It will at once be seen that this entire premise of Gruber is very artificial and unnatural. One can easily understand that the blocking of a given group can cause a new development of the same group. This corresponds to Weigert's fundamental law of regenera- tion. But it is very difficult to comprehend how the blocking of one group, a, would always lead to the development of a different group, h. Furthermore, it is incomprehensible why at least part of the poison by means of its haptophore group h should not be anchored by a combining substance preformed in the cell, a substance which can therefore act as a receptor. If the toxin really possessed two haptophore groups, a and 6, it would be possible and probable that two different antitoxins would be developed by the cell. But that is a question easily decided experimentally, and one which has been studied in this Institute for years. During all this time we have never discovered even the slightest reason for believing that diph- theria serum, obtained from different animals and by means of differ- ent cultures, possesses any such complex constitution as Gruber's view would require. TOXIN AND ANTITOXIN 545 We see, therefore, that the first step taken with the aid of Gruber's hypothesis leads us astray. But when we attempt to see how the antitoxin could act according to Gruber's scheme, we find ourselves lost in a maze. The antibody secreted by the cell is to combine with a collateral group b, of the toxin, leaving group a, which pri- marily effected the anchoring of the poison, intact. How then is any antitoxic effect to take place? One might perhaps assume that by the occupation of group b, the toxin loses its toxicity through some influence exerted on the toxophore group. The poison would thus in a certain sense be changed into a toxoid by the occupation of group b. In that case, however, the toxin with group b neutralized, should still be able to excite the production of antitoxin, just as toxoids do. As a matter of fact, this is not at all the case, for we know that toxin neutralized with antitoxin has completely lost both its toxic property and its power to produce antitoxin. This fact, which is absolutely irreconcilable with a plurality of the haptophore groups, is easily explained by my theory by a blocking of the haptophore group of the toxin. We see, therefore, that Gruber's assumption leads to consequences which are absolutely untenable. It certainly is far from being an improvement on my theory. In general, also, the principles of scientific investigation demand that we restrict ourselves to the simplest explanations possible and only make use of more complex ones if it is absolutely necessary. But there is not the least reason for Gruber's assumption of several haptophore groups; on the con- trary there are a large number of objections to it. By this I do not mean to say that in addition to the haptophore and toxophore group the toxin molecule contains no other chemical groups, such as amido or aldehyde groups, which are able to com- bine with other bodies. I merely contend that these atomic groups do not influence the specific immunizing process. To take a chemical example, it is possible by diazotizing all kinds of amins to transform these into diazo combinations which, corre- sponding to the original substance employed, contain other radicals capable of reacting, thus COH, CN, OH, NO, etc. The specific prop- erty of these substances, that is, the property of forming azo dyes, is, however, connected exclusively with the N-N group. The reac- tions which the other groups can enter into have nothing to do with this specific reaction. I conceive the constitution of the toxins to be similar in character. 546 COLLECTED STUDIES IN IMMUNITY. A few words, now, concerning the side-chain theory and immunity Gruber himself has found that this theory is constantly gaining ground, while I am gratified to see it treated in detail in the best text-books as well as in excellent digests compiled by a large num- ber of my colleagues.! Jq addition to this hundreds of separate studies have been based on the side-chain theory so that I may well believe that it best serves to explain the facts already observed as well as to allow new facts to be predicted. Gruber's appeal,^ there- fore, that "EhrUch's theory is a great mistake, and is bound soon to disappear from the scientific arena," has had but little success; in fact it seems to have had the contrary effect. The large number of investigators, who are constantly eagerly working on the prob- lems of immunity know what is best for them, and will not be dictated to against their own experience and conviction by one who seeks to make up his own lack of experimental work in this complex domain, by superficial studies of the literature. Gruber, for instance, says that his original failure was due perhaps to the fact " that a few of his experiments proved not to be quite sufficient." This is a mild expression in view of the fact that every one of Gruber's experi- ments directed against my views has been shown to be fallacious.. The studies in which his errors were pointed out and demonstrated experimentally have all been published in detail.^ The result, as usual, was, that after the corrections had been made, Gruber's attacks proved to be additional supports for my theory. Gruber has not replied to these articles, despite the long time since their publication. Perhaps he thinks the less said the better. 1 have finished. I must almost wonder why this detailed reply to an attack whose virulence and unusual tone are almost a con- firmation of my views. But I have thought it my duty to guide the reader through the intricate maze of Gruber's statements because I feel that, owing to the large number of misconceptions and mislead- ing arguments which they contain, a field of investigation full of promise might become discredited. ' I may mention those of Aschoff, v. Dungern, Griinbaum, Levaditi, Sachs Tavel, Wassermann, Welch, Bruck. 2 Wiener klin. Wochensch. 1901, No. 44. s Sachs, Berl. klin. Wochensch. 1902, Nos. 9 and 10; Ehrlich und Sachs, same journal, 1902, No. 21; Morgenroth and Sachs, same journal, 1902, Nos. 27 and 35; Marx, Zeitsch. f. Hyg., Bd. 40, 1902; Wechsberg, Wiener klin. Wochensch. 1902, Nos. 13 and 28. XXXIX. THE RELATIONS EXISTING BETWEEN TOXIN AND ANTITOXIN AND THE METHODS OF THEIR STUDY.i BY Prof. Paul Ehrlich and Dr. Hans Sachs. The subject of toxins and antitoxins, although representing one of the best studied domains of biology, is still the subject of lively- controversy. The difficulties which beset exact studies are obvious^ We are dealing with substances which, for the present at least, are- of unknown chemical constitution and which we are compelled to- employ in the form presented by the life activities of vegetable or animal organisms, i.e., in an impure state and mixed with countless other products of the living body. All attempts to isolate these bodies and discover their chemical character encounter endless diffi- culties, so that, if we consider their great significance in practical medi- cine, it almost seems ironical for nature to offer these substances to man in such an unstable and variable form. In spite of this, however, scientific investigations have been able to obtain a deep insight mto the nature and mode of action of toxms and antitoxins; and since chemical means could not be employed, it remained for the experi- mental biologist to undertake these studies. In place ot chemical analysis, therefore, we have the biological reaction, which in the case of toxins is the characteristic toxic action, in the case of antitoxins the property of specifically influencing or inhibiting this action. An event of considerable importance was the introduction of the quantitative method of study by EhrUch, a method which opened the way for the present development of immunity studies. At the same time Ehrlich's introduction of test-tube experiments (hsemag- glutination, hsemolysis), by avoiding the individual fluctuations of ' tjber die Beziehungen zwischen Toxin und Antitoxin und die Wage ilirer Erforschung, Leipzig, 1905, Gustav Fock. 547 548 COLLECTED STUDIES IN IMMUNITY animal experiments, furnished a more exact basis, so that the mathe- matical harmony of toxin-antitoxin experiments in vivo and in vitro became very convincing. At the present time, therefore, we may regard it as almost axiomatic that toxin and antitoxin act on each other chemically and without the intervention of vital forces. These quantitative biological studies, however, have not merely thrown light on the relations existing between toxin and antitoxin but have also given us valuable information concerning the constitu- tion of the poisons themselves. Almost at the outset it was found that the two properties of toxins which could be analyzed, namely, poisonous action and the property to bind antitoxin, do not at all go hand in hand. In this connection the continuous study of toxin solutions which are allowed to stand for some time proved particu- larly instructive, for it was found that while the power to bind anti- toxin remained constant, the toxicity gradually dminished. This study gave us one of the fundamental conceptions underlying the modern view of toxins, namely, that toxicity and combining power are two distinct and independent properties of the toxin molecule. As is well known, this fact is expressed by the side-chain theory by assuming that the toxin molecule possesses two specific atomic groups, one of which is toxophore, the other haptophore. Destruction or loss of the toxophore group gives rise to the non-toxic toxoids which are still capable of binding antitoxin. As a result of the high degree of lability of the toxophore group, this transformation into toxoid is a spontaneous process. And since the production of effective bacterial toxin solutions takes a certain time, it is obvious that we can practi- cally never obtain a pure toxin consisting entirely of similar molecules. All our work must be done with toxic solutions which, even if we assume that the bacteria have produced only a single primary toxin, represent a mixture of toxin and toxoid. But do the bacilli secrete only a single, homogeneous poison? This question has come more and more to be the subject of an ani- mated discussion. Closely associated with it is the further question as to the nature of the reaction which occurs when toxin and anti- toxin unite. The study of these problems was made possible by an important extension of quantitative toxin analysis, namely, Ehrlich's method of partial neutralization, This consists essentially in mixing a constant amount of poison with varying amounts of anti- toxin and then determining the toxicity of the various mixtures, i.e., the decrease in toxicity brought about by each successive addi- TOXIN AND ANTITOXIN: METHODS OF THEIR STUDY 549 tion of antitoxin. By means of -a graphic representation ot tlie figures thus obtained, we can get a deeper insight into the details ot the combining phenomena. Even now, after physical chemistry has taken such great interest in the reactions between toxin and antitoxin, all the various statements concerning the subject are finally based on the method of partial neutralization. From the outset Ehrlich felt sure that toxin and antitoxin could not be simple substances of strong affinities which combined, for instance, like caustic soda and hydrochloric acid. This was evi- denced particularly by the phenomenon which has often been termed the "inequality" of serum experiments. Thus if varying amounts of toxin are added to a constant amount of antitoxin (an immune unit), two distinct limits will be obtained: Lq ( = Limit zero) is the quantity of toxin in which the mixture is just completely non-toxic, i.e., physiologically neutral. Lt ( = Limit death) is the quantity of toxin in which the mixture is still just able to exert all its character- istic toxin action, i.e., in the case of diphtheria poison to just kill the guinea-pig acutely. Now if toxin and antitoxin behaved like caustic soda and hydrochloric acid, the difference between L-j- and Lq, which we shall term D, should correspond to one lethal dose (L D ) As a matter of fact, however, D is usually considerably larger, so that our first inequality becomes Lt-Lo>L. D. Hence only two possibilities exist. Either toxin and antitoxin react with one another like a weak base and a weak acid (e.g., am- monia and boric acid), in which case the high value of D is the expres- sion of an incomplete neutralization, or else the poison solution,, besides the real toxin, contains a second substance of less affinity. This substance, while unable to produce the characteristic toxin effects, gives rise to certain mild toxic phenomena. In the case of diphtheria poison (owing to the practical importance ot diphtheria antitoxin, the discussion has usually centered around this poison) human pathology had long taught that acute diphtheria infection is often followed by a second set of intoxication phenomena, namely, the peculiar paralyses which develop after the acute disease has dis- appeared. A priori, therefore, the assumption was highly probable that the high value of D was due to different components of the poison . And when the results of clinical experience and animal experiments harmonized so perfectly, the probability became almost a certainty It has been found that the toxicity of mixtures whose toxin content lies between Lq and Lt is not quantitatively diminished, but is actually 550 COLLECTED STUDIES IN IMMUNITY. different qualitatively. Guinea-pigs injected with such mixtures sicken, after a long period of incubation, with typical paralyses and show no local reaction. The hypothetical toxic constituent which gives rise to these paralyse sis termed "toxon." Why then is it impossible to demonstrate the action of the toxon in native diphtheria poison? This is readily explained by the relative concentration of toxin and toxon in the toxic bouillon. Quantitative analysis has shown that the toxin is usually much more (about 5 times) concentrated than the toxon. Hence the fractional parts of the lethal dose which allow the animal to hve long enough to manifest toxon effects usually contain too little toxon to produce the typical paralyses. If, however, a large amount of poison is so far neutralized with serum that all the toxin, with the higher affinity, is just bound and the toxon is still free, a mixture will be obtained which practically represents a pure toxon solution, for the neutral toxin-antitoxin molecules play no role in an animal experiment. It is at once appar- ent that, in view of the individual multiplicity of vital phenomena, the poisons of all strains of diphtheria bacilli will not contain both components in the same relative concentration. As a matter of fact, we find that the number of lethal doses contained in the difference Lf — Lo varies enormously, and so far as the toxon content is con- cerned the variations were from to 300% figured on the basis of the toxin content. If will be well to enter somewhat more into a study of these two extremes, for these striking exceptions to the typical <;onditions argue strongly in favor of the views here presented. One of the poisons in question was studied by Ehrlich, and was remarkable in that the difference L-f — Lq represented only 1.7 lethal doses. We may therefore assume that the poison was free from toxon or nearly so, for the value of D was actually quite near one lethal dose, the figure demanded of a toxon-free poison, provided toxin and anti- toxin combine like a strong base with a strong acid. The opposite extreme was manifested by a poison described by Dreyer and Madsen. The constants of this showed that it contained three times as much toxon as toxin. This poison, moreover, gave rise to toxon effects when sublethal dose of the native poison, without serum addition, were injected into animals. In view of what we have said above, this is readily understood, the relative concentration of toxon in this case was so great that even sublethal doses sufficed to make the toxon effects manifest. In most native poisons this demonstration fails because of the slight relative content of toxon. TOXIN AND ANTITOXIN: METHODS OF THEIR STUDY, 551 The existenee of the toxons which has been deduced mathematic- ally from the biological experiments is, however, no longer based merely on these calculations. At the present time their existence is a proven fact, for quite recently van Calcar succeeded in separately isolating toxin and toxon from the native poison solution by means of a ingenious dialyzing procedure. Owing to its smaller molecular volume, toxin diffuses through a suitable membrane under less ten- sion than toxon. In this way one obtains toxon-free toxin on one side and toxin-free toxon on the other. This direct confirmation of the conclusions drawn from the bio- logical analysis of the toxins shows how a mathematical study, pro- vided biological facts are carefully regarded, can get at the nature of the phenomena in question, despite the failure of chemical methods. To be sure the mathematical treatment of biological problems must be undertaken very carefully. The phenomena of animate nature are so manifold, and subject to so much change, that they cannot all be forced into the limits of a formula. It is particularly dangerous to build up formulas and laws on the basis of too simple assumptions. For them one can easily be deceived by the apparent exactness of figures, and arrive at conclusions which do not sufficiently regard the complexity of the actual phenomena. Unfortunately these warnings are much needed at the present time, for certain high authorities are striving energetically to explain the most complex phenomena, like those which occur in the union of toxin and antitoxin, as though they were simple and readily cal- culated reactions between simple substances. In opposition to the plurality of the pojson constituents demon- strated by Ehrlich, Arrhenius and Madsen, as is well known, uphold a unitarian standpoint. Their deductions are based entirely on the method of partial neutralization introduced into toxin study by Ehrlich and referred to above. Up to this point they differ only in the method of representing their results graphically. For this purpose they use a system of coordinates, laying off the amounts of antitoxin contained in each mixture on the abscissas. But whereas in Ehrlich's scheme the ordinates represent the amounts of toxin which each addition of antitoxin causes to disappear, Arrhenius and Madsen use the ordinates to represent the toxicity which each mixture still retains. In their work these authors observed that now and then in a num- ber of poisons, especially in tetanolysin, the line connecting the points plotted possessed a certain similarity to curves obtained when weak 552 COLLECTED STUDIES IN IMMUNITY. bases are neutralized by weak acids (ammonia and boric acid). This similarity constitutes the basis for their mathematical work, which leads them to conclude that toxin and antitoxin are simple substances whose reaction is reversible. This reaction finds its expression in the curve just mentioned. Let us examine their conclusions and see whether they are justified. The two graphic methods referred to are equally correct. Never- theless it cannot be denied that the one employed by Ehrlich, the so-called "poison spectrum," has certain advantages, for it brings out more clearly any deviations from the regular curve. Speaking mathematically we say that the "poison spectrum" is the graphic representation of the differential quotients of Arrhenius and Madsen's curve. In this sense, the ordinates of the spectrum represent the direction of the neutralization curve, i.e., the trigonometric tangent of the angle which the tangent forms at every point with the axis of the abscissas. Hence, if the course of the neutralization curve is that of a straight line, the direction therefore being the same at all points, we must represent the poison spectrum as a rectangle. If, as is often the case, the addition of a small amount of antitoxia causes no decrease in toxicity (prototoxoids), so that the neutraliza- tion curve in this part of its course lies parallel to the axis of the abscissas, we must represent the poison spectrum as having a gap at this point, for the angle between tangent and axis of abscissas' is 0°. This brief statement should make it clear that in the poisoa spectrum, by representing the direction of the separate parts of the curve as ordinates, deviations from the regular curve-like course will be more clearly shown. It may be well to study these conditions by means of a diphtheria poison investigated by Madsen.^ See Figs. 1 and 2. These figures show that the deviations from the hyperbolic curve demanded by Arrhenius and Madsen's views are much more clearly shown in the representation employed by Ehrlich. Entirely aside from the question whether the sharply defined zones of the poison spectrum actually exist, or whether a gradual transition must be inter- polated, it is certain that the changes should always occur in the same way; for they merely represent the differential quotients of the neutralization curve, and should therefore, if this curve were hyper- bolic, show a successive decrease. The manifestly very irregular ' The sole object in employing this poison is to illustrate the two methoda of graphic representation. TOXIN AND ANTITOXIN; METHODS OF THEIR STUDY. 553 rise and fall of the differential quotients shows at once that a hyper- bolic curve is out of the question in the case pictured above. If we examine the poison spectrum, on the other hand, we find that this represents Madsen's poison entirely in accord with Ehrlich's views concerning the constitution of diphtheria poison. If toxin and anti- toxin unite firmly, and the course of the neutralization curve there- fore is a straight line, the irregular course is explained by the toxoid present in the poison and by the varying affinity of the poison con- stituents. The highest zone in the poison spectrum (zone c) indicates that at this point equal amounts of antitoxin cause the greatest 3 8 I- s 5 e 4 u Z 3 o g 2 I 1 a., PROTOTCJXOID 1 h., HEMITOX N c. PURE TO :iN TOXON 0.25 0.3 0.35 0.4 ANTITOXIN 0.45 Fig. 1. — Poison spectrum according to Ehrlich. decrease in toxicity. Hence this part of the poison must contain the least toxoids, or none at all, and we may therefore speak of this as pure toxin. It will serve as a unit for judging the degree of con- tamination with toxoid in the remaining portions. We should then speak of zone b as the hemitoxin, i.e., for each molecule of toxin there is one of toxoid. The sequence of the different zones corre- sponds to the different affinities of the components. Thus we see that the addition of a small amount of antitoxin (o) does not cause any decrease of toxicity whatever. And yet the antitoxin must have been bound. We conclude, therefore, that toxoids must here be present which possess a higher affinity than any other constituent of the poison. We are here dealing with the important prototoxoid zone which we encounter so frequently in diphtheria poison, abrin, ricin, crotin, etc. The hemitoxin zone which follows this is to be regarded as a deutero toxin in its affinity. The constituents of the -554 COLLECTED STUDIES IN IMMUNITY. poison can thus be arranged as proto-, deutero-, , tritotoxin, etc., after which finally comes the constituent possessing the weakest affinity, namely, the toxon. That this varied affinity does not arise when the toxoids are formed, but differentiates the undecomposed constituents of the poison from the outset, is demonstrated by the genesis of toxoid formation. Thus if one is in a position to .study a very pure poison in its various stages of decomposition, it will be found that there is a first phase which leads to the formation of hemitoxin, and that a later phase changes this into prototoxoid. If there were a change in affinity, however, we should have had a pure toxoid zone from the start. The prototoxoids proved a serious obstacle to Arrhenius and Madsen in the logical develop- ment of their views. According to their theory just the first amounts of antitoxin added should de- crease the toxicity the most. Nevertheless a number of experi- ments were published by these authors (Madsen, with diphtheria poison, and Madsen and Wal- baum, for ricin) in which the proto- toxoids and their development were only too apparent. And Arrhenius and Madsen seem to appreciate that they can no longer explain this contradiction by assuming that the prototoxoid zone is due to "change- ments minimes dans le milieu am- biant," or by saying that the proto- toxoid zone is "of little interest." In order, therefore, to eliminate these prototoxoids, so annoying for their formula, they have discarded the well-tried criterion for a fatal dose of diphtheria poison (death of the guinea-pig in 3 to 4 days), and now attempt to calculate the fatal dose in a new way. Their procedure is as follows: Retaining the definition of a fatal dose, they believe it possible to calculate the fraction or multiple of the fatal dose employed, from the time of the animal's death or even from the resulting loss of weight. Such 60 45 40 35 ieo > |20 IS 10 5 n K \ \ \ \ \ \ \ \ \ ^ ^ 0.05 0.1 0.1S 0.2 0.25 0.3 0.35 0.4 OM ANTITOXIM Fig. 2. — Neutralization curve accord- ing to Arrhenius and Madsen. TOXIN AND ANTITOXIN: METHODS OF THEIR STUDY. 555 a procedure, in order to possess any justification whatever, would have to be based on an enormous experience. But even aside from this it is amazing to see how a lot of experimental protocols, going back to 1897, are unhesitatingly used for their calculations. The old determinations of the lethal dose, in which death produced acutely in 3 to 4 days was the criterion, are very difficult to make use of owing to the individual variations in the animals. Certainly it re- quires some experience to know which animals should be discarded because .of over- or undersusceptibility. But how much more com- plex the conditions really are is at once apparent if one attempts to •determine J or J of a lethal dose from the clinical course of the disease. Hence it is not surprising to find that the lethal doses calculated by Arrhenius and Madsen represent the averages of figures which •often differ from each other by many times. The tedious work which these authors have undertaken may perhaps satisfy a mathe- matician ; to the biologist, however, it can only represent useless and dangerous playing with figures. It signifies nothing, therefore, if the figures recently obtained by this method by Arrhenius and Madsen with three poisons fail to show any prototoxoid zone.' For the same reason, also, we cannot regard certain other figures, which ■differ markedly in observation and calculation, as arguments against their views. However, we need neither confirmation nor controversion of their theory. For it has been found that the assumptions on which this theory is based have no existence whatever. We have already alluded to the fact that van Calcar has recently demonstrated the •existence of toxons. But it has also been shown by another method that diphtheria poison, as well as most other toxins, must contain "various constituents capable of binding the antitoxin. This method Lad its inception in the following considerations. Arrhenius and Madsen, as already stated, regard the union of toxin and antitoxin as a reversible reaction between two simple [einheitlich] substances. According to this view, therefore, the reaction is incom. plete, i.e., the two substances reacting (toxin and antitoxin) are never completely used up, a certain portion of both toxin and anti- toxin always remaining free beside the neutral toxin-antitoxin combi- nation. The equilibrium which exists between the three components ' We should not neglect to mention that the existence of the prototoxoid ■zone and its development from the hemitoxin phase has also been demonstrated dn diphtheria poison by so excellent a worker as Theobald Smith. 556 COLLECTED STUDIES IN IMMUNITY. will then be governed by the law of mass action formulated by Guldberg-Waage, namely, (toxin). (antitoxin) = A; (toxin-antitoxin), in which the brackets denote the concentration, and k the constant of equilibrium to be determined for each poison. ^ All the calculations of Arrhenius and Madsen are based on this formula, and their entire work stands or falls with the applicability of the formula to the sub- ject of toxins. The formula, however, is only then applicable if the reaction is really completely reversible, and this is not the case. Thus if mix- tures containing the same amounts of toxin and antitoxin are tested at the end of the reaction, it is easy to convince one's self that the toxicity is dependent not only on the amounts of toxin and anti- toxin, but on the manner of making the mixtures. If to the same amount of antitoxin we add at intervals fractional parts of the toxin, we shall find that the resulting end product is considerably more toxic than if the'same amount of toxin is mixed with the antitoxin at once. This holds true even if the toxin is added at the time corresponding to the addition of the last fraction in the former case. Von Dungem was the first to point out the significance of this experiment, in con- nection with an observation made by Danysz, for the question of reversibility. He showed that if this really was a completely reversible reaction between simple substances, as is assumed by Arrhenius and Madsen, we should expect that the same equilibrium should always ensue with the same total amounts of reacting substances, i.e., the toxicity of the end products should always be the same. Any devia- tion from this could occur in the fractioning process only during the course of the reaction ; and then, provided the deviation were a function of the reaction-time, this would be just the reverse of what is actually observed.2 Hence all those poisons in which this phenomenon of ' In their recent publications Arrhenius and Madsen assume that one mole- cule toxin combines with one molecule antitoxin, not to form two molecules of the toxin-antitoxin combination, as the above formula would show, but that two different substances are formed, toxinan and titoxin. To be sure as the equation then reads, (toxin) (antitoxin )= A; (toxinan) (titoxin), one objection to the above formula is done away with, but a new hypothesis, lacking all evi- dence whatever, is thus introduced merely for the sake of the formula. ^ The phenomenon in question therefore shows exactly the reverse of what Arrhenius and Madsen's theory demand. For this reason the limit of error need not be considered, although, owing to the enormous quantitative differences, it would play no r61e in judging the result. Nor can Arrhenius extricate him- self from the predicament by suggesting that we are dealing with slowly progress- TOXIN AND ANTITOXIN: METHODS OF THEIR STUDY. 557 increasing toxicity on the fractional addition of toxin can be demon, strated must at once be excluded from any mathematical analysis based on a formula of equilibrium derived from the law of mass action. In all of the cases ^ examined for the purpose (diphtheria poison, tetanolysin, ricin, staphylolysin, arachnolysin, rennin.and precipitin), this method has shown that the conception of Arrheniios and Madsen is entirely inapplicable. The phenomena observed, however, are very readily explained by the assumption of a plurality of combining groups in the poison solution. Thus if to an excess of antitoxin a small quantity of poison is added, as is done in the fractioning experiment, the result would be that even the constituents possessing a feeble afEnity and which are of no consequence so far as any toxic action is concerned, would be bound by the antitoxin. When then the second portion of poison is added, it will be impossible for the toxin molecules, al- though possessing a higher affinity, to crowd the previously bound constituents out of their combination with the antitoxin. The result is that a certain portion of toxia, which would have been neutralized by the antitoxin if all the poison had been mixed with the antitoxin at once, now remains free. That is to say, the fractional method of adding the poison has resulted in an increased toxicity, the Lf dose being reached with a smaller amount of poison. Furthermore it is possible, by means of suitable technique, to cause a reduction of the Lq dose, from which it follows that the Lq serum mixture contains free non-toxic constituents capable of binding antitoxin, and that these must possess still less affinity than the toxon. These are the so-called "epitoxonoids" of von Dungern. The discovery of the epitoxonoids also offers an easy explanation of the fact that it is possible to immunize with mixtures of toxin and antitoxin which are physiologically neutral. All this shows that a complete reversibility, even of the individual ing side reactions which do not interfere with the main reaction when one works rapidly. For, as was pointed out by von Dungern and Sachs, the increased toxicity is already demonstrable at a time when the union of toxin and antitoxin is not yet ended. The hypothetical "side reaction" would therefore proceed just as quickly as the main neutralizing reaction. ' The single exception met with, namely cobra venom, only proves the rule; for cobra venom (we are dealing with the hsemolytic portion which is activated by lecithin) is a simple substance with a strong affinity for the antitoxin, as can be seen from the course of the neutralization curve, which is a straight Hue. 558 COLLECTED STUDIES IN IMMUNITY. poison constituents, is out of the question. On the contrary we must assume that the union of these substances with the antitoxin is subse- quently tightened. This tightening is also borne out by other observa- tions, both old and recent. If the toxin-antitoxin reaction were reversible, it should be possible, by removing the supposedly free toxin residue, to constantly change the equihbrium, so that the toxin could all be recovered. Nevertheless, although toxin can be filtered through gelatine and antitoxin cannot, it is impossible either by gelatine filtration (Martin and Cherry) or by gelatine diffusion (van Calcar) to obtain free toxin from neutral toxin-antitoxin mixtiu-es.'- In addition to this one cannot help being surprised that the calcula- tions of Arrhenius and Madsen entirely ignore the cells' toxin-binding receptors which effect the poisoning. In accordance with their views, these receptors should represent an important element in the equilibrium; and yet they appear to have entirely overlooked this- fact. It would lead us too far to discuss all the arguments against the views of Arrhenius and Madsen. It will suffice to call attention to the serious objections which Nemst has raised regarding the prin- ciples involved, and to Koppe's criticism of their technique in making haemolytic test-tube experiments. This illustrates the danger of a one-sided mathematical study of biological problems. Even if one succeeds now and then in making the figures of observations and cal- culation tally, it is impossible at the present time for these mathe- matical expressions to explain the facts. To be sure they may be able to represent the resultants of the processes which bring about the phenomena, but in that case the formula is nothing more than an interpolation formula. Corresponding to this, therefore, we see that the formulas of Arrhenius and Madsen vary widely for the same poison, every new lot of poison of the same bacillary origin has a new constant of equilibrium. Hence the formula is applicable only to one particular case, and so, even if it were a correct interpolation formula, progress of biological science would in no way be furthered by it. ' It is perfectly evident that toxin can be obtained from fresh toxin-antitoxin; mixtures by diffusion through gelatine, and this has recently been demonstrated by Madsen and Walbaum. According to Morgenroth such mixtures require at least twenty-four hours for the union to become complete. Hence the state- ment by Madsen and Walbaum that the mixtures must be fresh in order to demonstrate what they regard as dissociation only confirms our view. TOXIN AND ANTITOXIN: METHODS OF THEIR STUDY 559' Biology does not content itself with a mere registration of phe- nomena; it seeks to discover their nature and their relation to one another. In fact the chief mission of biology is to attempt, by link- ing facts and theories and hypotheses, to satisfy the craving of the: thinking naturalist for an insight into causes. The following is a summary of the literature bearing on this subject. 1897. P. Ehrlich, Die Werthbemessung des Diptherieheilserums. Klinisches. Jahrbuch. The same, Zur Kenntniss der Antitoxinwirkung. Fortschritte der Medizin. 1898. The same, Uber die Constitution des Diphtheriegiftes. Deutsche med. Wochensohrift. 1899. Th. Madsen, Uber Tetanolysin. Zeitschr. fiir Hygiene, Vol. 32. 1902. S. Akrhenids and Th. Madsen, Physical chemistry apphed to toxins. and antitoxins. Festskrif t ved IndvielsenafStatens Serum Institut. 1903. The same, Anwendung der physikalischen Chemie auf das Studium der Toxine und Antitoxine. Ztschr. f. physik. Chemie, Vol. 44. P. Ehrlich, Uber die Giftcomponenten des Diphtherietoxins. Berl.. klin. Wochenschr. E. VON DuNGBEN, Bindungsverhaltnisse bei der Pracipitinreaktion. Centralblatt f. Bacteriol., Vol. 34. Th. Madsen. La constitution du poison diphthgrique. Centralblatt f. Bacteriol., Vol. 34. 1904. S. Arrhenihs, Die Anwendung der physikalischen Chemie auf die Serumtherapie. Arbeiten a. d. Kaiserl. Gesundheitsamte, Vol. 20. The same, Zur Theorie der Bindung von Toxin und Antitoxin. Berlin, klin. Wochenschr. P. Ehrlich, Bemerkungen zur Mitteilung von Arrhenius: Zur Theorie der Absattigung von Toxin und Antitoxin. Berl. klin. Wochenschr. E. von Dunqern, Beitrag zur Kenntniss der Bindungsverhaltnisse bei der Vereinigung von Diphtheriegift und Antiserum. Deutsch. med. Wochenschr. S. Arrhenius, Die Anwendung der physikahschen Chemie auf die serumtherapeutischen Fragen. Boltzmann Festschrift. H, Sachs, Uber die Constitution des Tetanolysins. Berl. klin. Wo chenschr. P. Ktes, Cobragift und Antitoxin. Berliner klin. Wochenschrift. Th. Madsen et L. Walbatjm, Toxines et Antitoxines. De la ricine et de I'antiricine. Centralblatt f. Bacteriol., Vol. 36. W. Nernst, Uber die Anwendbarkeit derGesetze des chemischen Gleich- gewichts auf Gemische von Toxin und Antitoxin. Ztschr. f . Electro- ehemie. Vol. X, No. 22. 560 COLLECTED STUDIES IN IMMUNITY, J. MoRGENROTH, UntersuchuDgen uber die Bindung von Diphtherie- toxin und Antitoxin, sowie iiber die Constitution des Diphtherie- giftes. Berlin, klin. Wochensclar. In detail in Zeitschr. f. Hygiene, VoL 48. H. KoppE, Zur Anwendung der physikalischen Chemie auf das Studium der Toxine and Antitoxine und das Lackfarbenwerden rother Blutsctieiben. Pfliiger's Archiv, Vol. 103. An address entitled "Die Serumtherapie vom physikalisch-chemischen Standpunkte," by Sv. Arrhenius. Discussion by Ehrlicb, Nernst, Arrhenius. Zeitschr. f. Electrochemie, Vol. X, No 35. E. VON DuNGERN, Bemerkung zum Vortrag von Professor S. Arrhenius: "Die Serumtherapie vom physikalisch-chemischen Standpunkte." Zeitschr. f. Electrochemie, Vol. X, No. 40. Th. Madsen, Toxins and Antitoxins. British Medical Journal. R. P. VAN Calcar, Uber die Constitution des Diphtheriegittes. Berl. klin. Wochenschr. S. Ahhhenids et Th. Madsen, Toxines et Antitoxines. Le Poison diphth&ique. Centralblatt f. Bacteriol., Vols. 36 and 37. H. Sachs, tJber die Bedeutung des Danysz-Dungemschen Kriterium nebst Bemerkungen uber Prototoxoide. Centralblatt f. Bacteriol., Vol. 37. Xi. MiCHAELis, A collection of studies on this question together with a critical review. Biochemisches Centralblatt, Vol. VI, No. 1. XL. THE MECHANISM OF THE ACTION OF ANTIAMBOCEPTORS.i By Prof. Paul Ehrlich and Dr. H. Sachs. Owing to closer investigations into the nature of immunity our conceptions regarding the relation between antibody and the sub- stances exciting the production of immunity (the antigen, as it is called) have undergone a certain modification. This consists in a more precise definition of the concept specificity. In the beginning it was assumed that an antibody produced by immunization acted only against the substance through which it was developed. Further observations, however, soon brought to light cases in which this law was apparently violated. A clear insight into this subject was finally made possible when the receptor was looked upon as the agent which excited the production of immunity. According to the side-chain theory, therefore, specificity of the antibodies always means " the specific relations between the individual types of antibodies and of receptors." ^ Since, therefore, the same receptor can be dis- tributed not only among different kinds of cells, and bodies of differ- ent functions all within the same animal species, but also among different species of animals, we see that it is impossible to speak of a specificity in a zoological sense, or of a specificity in respect to the Taorphological or -functional properties of the antigens. The anti- body is specific only for the receptor, i.e., for those elements possess- ing this fitting receptor. Of the various substances which excite the production of im- munity, a special place is occupied by the receptors of the third order: these, when free, constitute the amboceptors. As is well known, the amboceptors possess a double function. On the one hand they unite with the cytophile group of the cells^ and on the other with the ' Reprinted from Berliner klin. Wochenschrift, 1905, No. 19. ' P. Ehrlich and Morgenroth, Haemolysins. See page 88. 561 562 COLLECTED STUDIES IN IMMUNITY. complementophile group of the complement. Each of these two haptophore groups will therefore be able to excite the production of corresponding antibodies, a fact to which attention was called in the Croonian lecture, 1900.^ "The lysin, be it bacteriolysin or hsemolysin, possesses altogether three haptophore groups, of which two belong to the immune body and one to the complement. Each of these haptophore groups can be bound by an appropriate antigroup." Three 'antigroups' are thus conceivable, any one of which, by uniting with one of the haptophore groups of the lysin, can frustrate the action of the lysin." In other words, according to the amboceptor theory two different antiamboceptors are at once conceivable, either of which would inhibit the action of the amboceptor One would act by preventing the union of amboceptor and cell, the other by preventing the comple- ment from uniting with the amboceptor. Originally the antiambo- ceptors produced by immunization were regarded as being directed against the cytophile group.^ In view of this it was extremely de- sirable for the support of the amboceptor theory that the existence of antibodies for the complementophile group 'should be demon- strated. This has recently been done by Bordet,^ and it is strange to see that he employs his discovery in combating the receptor theory when it really is a very neat confirmation of this. Bordet finds that antiamboceptors can be produced not only by immunization with hsemolytic immune serum, but also with normal serum of the same species, even though this normal serum contains no corresponding amboceptors. He treated guinea-pigs with normal rabbit serum which contains no hsemolytic amboceptors for ox blood, and obtained an immune serum which yet was able to in- hibit the action of the amboceptors derived by immunizing with ox blood. That, certainly, is a discovery which cannot readily be ex- plained in harmony with Bordet's sensitization theory. According to Bordet, as we know, these immune bodies (his "sensitizers") possess the one property of combining with the susceptible cell and thus rendering this vulnerable to the action of the complement. This being the case it is incomprehensible how a serum which possesses ' P. Ehrlich, On Immunity, Proceedings Royal Society, 1900. 'Ehrlich and Morgenroth, VI. Communication, page 88. ' J. Bordet, Les propri^t^s des antisensibilatrices et les theories chimiques de I'immunit^. Anna!, de I'lnstit. Pasteur, 1904, No. 10. MECHANISM OF THE ACTION OF ANTIAMBOCEPTORS. 563 no sensitizers whatever for the species of cell in question can yet excite the production of antibodies directed against them. The matter takes on an entirely different aspect if we regard this phenomenon from the standpoint of the amboceptor theory. Ac- cording to what has been said above we at once see that two func- tionally different types of anti amboceptors are possible. In Eordet's case the normal rabbit serum possessed no amboceptors (i.e., no cyto- phile groups) for ox blood ; therefore the antibodies which are de- veloped cannot be antiamboceptors directed against the cytophile groups. Hence by exclusion one will already pronounce them anti- amboceptors of the com-plementopMle group. The facts brought for- ward by Bordet all go to confirm this. If such antiamboceptors are to be produced, the only requisite is that the serum used for immunization must contain the corre- sponding complementophile groups. Is this the case in normal rabbit serum? Every normal rabbit serum, as Eordet admits, con- tains a large number of different amboceptors. If, by immunizing with a given species of cell, a new specific amboceptor develops in the serum, the new element in the receptor apparatus is really only the q/tophile group, which is produced in response to immunization. The complementophile apparatus need not suffer the least change quali- tatively; in fact according to our conception it usually does not change markedly, there is merely an increase in the complemento- phile groups corresponding to the formation of the additional immune body. We have already expressed this opinion in a previous paper. ^ "In my judgment we shall arrive at a correct conception if we pro- ceed from the standpoint that in general the specific amboceptors exhibit a uniform structure so far as their complementophile portion is concerned, while their cytophile groups, which physiologically are concerned with the absorption of food, differ most widely." It must not be thought that this uniform constitution of the com- plementophile portion ^ contradicts the assumption of a multiplicity ' P. Ehrlich, Betrachtungen iiber den Meohanismus der Amboceptorwirkung und seine teleologische Bedeutung. Koch Festschrift, Jena, 1903. ' For the present we cannot say whether the complementophile complex is really uniform throughout or whether, perhaps, certain partial groups do not differ in the individual amboceptor types of the same animal species. Such a condition is easily conceivable. In any event we must assume that the com- plementophile apparatus of the amboceptors of a given species is identical at least in some essential part of its haptophore functions, and that this char- acterizes it as coming from the animal species in question. 564 COLLECTED STUDIES IN IMMUNITY. of complements. Naturally the different complements must have different complementophile groups corresponding to them. But, as was stated in the Sixth Communication on Hsemolysins,^ an immune body, in addition to a particular cytophile group, contains two, three, or more complementophile groups. In a later paper Ehrlich and Marshall offered experimental evidence for just this point; besides this, Bordet's experiments, according to which an amboceptor after having combined with cellular elements is able almost completely to rob a serum of its complement, also support this view.2 We must therefore conceive the amboceptor to be structurally a polyceptor, and assume further that the amboceptors of a distinct species are all supplied with a large number of complementophile groups which vary considerably in detail but in their entirety repre- sent a uniform complex. This complex is reproduced in all the amboceptors of the same serum. In general the amboceptors are different and specific only so far as the cytophile group is concerned. This being so it will at once be clear that antiamboceptors directed against the complementophile groups, and obtained through immuni- zation with any particular amboceptor, will act against all ambocep- tors of the same animal species no matter whether these ambo- ceptors are normally present in the serum or have been produced by immunization. For the complementophile amboceptor apparatus is the same for all types of amboceptors of the same species. As a result of this, an immune serum obtained through immunization with normal serum contains, thanks to the normal amboceptors in the serum, antiamboceptors directed against the artificially produced amboceptors of the same species. This explains also the earlier observations made by Pfeiffer and Friedberger ^ that antiamboceptors obtained by immunizing with cholera serum act also against typhoid serum;* it also explains the recent experiments made by Bordet. We ' Ehrlich and Morgenroth. See page 88. ' P. Ehrlich and H. T. Marshall, tJber die complementophilen Gruppen der Amboceptoren. Berl. klin. Wochenschr. 1902, No. 25. ' R. Pfeiffer and E. Friedberger, Weitere Beitrage zur Frage der Antisera und deren Beziehungen zu den bacteriolytischen Amboceptoren. Centralblatt f. Bacteriol. 1904, Vol. 37; also 1903, Vol. 34. * Naturally the statement made by Ehrlich and Morgenroth (Berl. klin. Wochenschr. 1901, No. 21) that "it seems improbable, unless in a given case a fortunate coincidence intervenes, that anti-immune bodies will be obtained directed against the bactericidal immune bodies" cannot apply to the antiambo- ceptors directed against the complementophile groups. That statement applies MECHANISM OF THE ACTION OF ANTIAMBOCEPTORS. 565 must call particular attention to the fact that the chief point in Bordet's study, the non-specificity of the antiamboceptors so far as the cytophile group is concerned, had already been published by Pfeiffer and Friedberger. These authors have explained the fact entirely in accordance with our views, as follows: "We are inclined to believe that the various immune bodies of one and the same animal species possess one group in common which in a way stamps them as coming from that particular animal organism. The antiserum must possess certain relations to this group." To this we would add that for the present it seems simplest to class this group or groups, specific for the animal species, with the complemento- phile group. In the amboceptor we differentiate a specific cytophile group and a large apparatus made up of complementophile groups. Aside from the- property of anchoring the cells, the latter groups exercise all the remaining functions of the amboceptor. Considering that the normal amboceptors and those produced by immunization are essentially similar (a point which we have always emphasized), it is perfectly obvious that one can produce the same antiamboceptors by immunizing with normal amboceptors. Hence what Bordet's study really brings forward is the actual experimental demonstration of what we had long expected was the case. Naturally we were able to confirm all of Bordet's statements of fact. We had at our disposal the serum of a goat which had been, immunized with normal rabbit serum, and could easily convince ourselves that this serum acts as an antiamboceptor againsl ambo- ceptors derived from rabbits by specifically immunizing with ox blood. Furthermore, we succeeded, by adding the antiamboceptor to previously sensitized blood-cells, to protect these against haemolysis by complement. The antiamboceptor acts just like a complementoid according to the conception of "complementoid-blo eking" described by one of us some time ago.^ It occupies the complementophile groups and so prevents the anchoring of the complement.^ only to the antibodies directed against the cytophile groups, since it is to be assumed that these cytophile groups, which have their natural counter-groups in bacterial cells, will not have these in the cells of higher animals. This limi- tation, however, does not apply to the antiamboceptors acting on the comple- mentophile complex. This, then, disposes of Bordet's objections to this point. ' Ehrlich and Sachs, "tJber den Mechanismus der Amboceptorenwirkung. Berl. klin. Wochenschrift, No. 21, 1902. ' We must not fail to mention that, in contrast to Bordet, we made these experi- ments without the addition of inactive guinea-pig serum, and were able, despite 566 COLLECTED STUDIES IN IMMUNITY. We were also able to readily confirm Bordet's statement that the antiamboceptor action is easily inhibited by normal rabbit serum, Naturally the normal amboceptors, whose complementophile groups excited the production of the antiamboceptor, will combine with this antiamboceptor and so be able to deflect it from the amboceptor acting in the given case. Since we regard the antiamboceptor in the sense of a complementoid, this phenomenon corresponds in prin^ ciple to that described by Neisser and Wechsberg as deflection of complement.^ The entire complex of phenomena just discussed shows most strikingly that our assumption harmonizes best with the observed facts. We assume that in Bordet's antiamboceptors we are dealing with antibodies directed against the complementophile groups. The existence of such antiamboceptors again demonstrates that the amboceptor theory is correct. According to Bordet's sensitization theory only such antiamboceptors are conceivable which prevent the amboceptor's union with the cell. But if there are other kinds of antiamboceptors, as the findings just discussed show, we must assume that the amboceptor has other affinities besides those for the cell, and this leads us at once to the conception which we have defined under the name amboceptor. The sensitization theory must therefore be abandoned. The next question which arises is whether or not it is possible by means of immunization with amboceptors to produce antiambo- this, to effect an inhibition of hsemolysis by subsequently adding antiambo- ceptor. It seems to us that this simplified procedure is more convincing, for it will hardly be claimed that the guinea-pig serum is a better suspending medium than physiological salt solution, and that it therefore, in contrast to the latter, leaves the blood-cells intact. Furthermore, inactive guinea-pig serum itself inhibits the haemolysis of ox blood by amboceptor and complement (guinea- pig). Hence when guinea-pig serum is present the question whether the ab- sence of haemolysis is due to an antiamboceptor or not is left undecided. ' In contrast to Bordet, however, we were unable by means of normal ambo- ceptor to effect the subsequent breaking of the union between antiamboceptor and sensitized blood-cells. It may be that in our case the union between anti- amboceptor and sensitized cells so rapidly became firm that it could no longer be dissolved by the normal amboceptor. Even Bordet admits that this dis- solution can be effected only for a certain period, and that then the union becomes very firm. We are pleased to note that Bordet accepts this conception of a gradual tightening of the union of these substances, a conception of the highest importance in the study of immunity reactions. MECHANISM . OF THE ACTION OF ANTIAMBOCEPTORS. 567 ceptors also against the cytophile group. We have therefore ex- amined another antiamboceptor serum, and compared its properties with those of the antiserum made by injections of normal rabbit serum. This serum, like the latter, was also obtained from a goat, but instead of using normal rabbit serum for immunization the goat had been treated with the serum of a rabbit previously immunized with ox blood. Our experiments, however, did not permit of a decision on this point. We are unable to say whether among the antiamboceptors excited by the injections of the immune serum there were any directed against the cytophile group. Tt is entirely con- ceivable that, despite the presence of the cytophile group, these are unable to exert any immunizing power, since the complementophile groups invariably encounter the corresponding counter-group in the organism and so are the only ones bound to the tissue receptors. In that case previous to injection one would attempt to destroy the complementophile group ( = cytophilic amboceptoids) or to neutralize it by means of a suitable antibody. The decision of this question must be left to_further detailed investigations. In the course of our experiments we met with a very curious phe- nomenon, one not only of some practical significance, but also of considerable theoretical interest. Our experiment showed exactly the opposite behavior which Bordet had found. That is to say, where Eordet found that the antiserum acts as an antiamboceptor on the amboceptor anchored to the cell, and that this action is overcome by normal rabbit serum, one of our cases represents the reverse of this. We see, therefore, that it can happen that the antiamboceptor as such does not act, but requires the addition of normal rabbit serum before exerting its action. We have constantly observed that in a "curative" experiment, i.e., after a previous binding of amboceptor and cell, large amounts of the antiserum produced by means of im- mune serum were unable to prevent haemolysis. The following proto- col may serve as an example: To each of a series of test-tubes, containing decreasing amounts of the antiserum, 1 cc. of ox blood was added. This blood, after having previously been sensitized with 0.003 cc. ( = 1J amboceptor •units) of an amboceptor obtained from a rabbit by immunization with ox blood, was freed from serum constituents by centrifuging and then used in the test. After digesting the mixtures for half an hour the blood-cells were centrifuged off and the sediments, to which 0.1 cc. guinea-pig serum was added as complement, were 568 COLLECTED STUDIES IN IMMUNITY. suspended in salt solution. The result of the experiment is shown in the following table: TABLE I. Amount of the Antiserum (derived from a goat by treatment with an am- boceptor, the result of Amount of HsemolyBis. immunizing a rabbit with ox blood) CO. 0.1 complete 0.05 well-marked 0.025 moderate 0.015 little 0.01 0.005 faint trace 0.0025 very little 0.0015 moderate 0.001 almost complete 0.0005 complete 0.00025 complete complete Here we see the curious result that with a certain excess of the antiserum there is no inhibition of haemolysis. This paradoxical phenomenon we observed only with the antiserum produced by immune serum injections, and then only in the "curative" experi- ment. If the antiserum was used for "protective" experiments, i.e., mixed with amboceptor previous to adding the blood-cells, or if the antiserum produced by injections of normal serum was employed, the course of the experiment was entirely uniform, an increase in the amount of antiserum causing an increase in the antilytic action. For the present we are imable to say whether we are here dealing with an essential difference between the antiserum produced by normal serum and that produced by immune serum, or whether we have to do with an individual fluctuation. So far as the mechanism of the phenomenon is concerned we were able to clear up at least one point, namely, that the essential factor in the experiment is the presence or absence of the very small quantities of normal rabbit serum which contains the amboceptor. Thus if the blood-cells are sensitized with amboceptor without subsequently removing the serum by centrifuging, it will be found that the course of the "curative" experiment is perfectly regular. There is no inhibition of the antilytic action with an excess of antiserum. The same holds true if we sepa- MECHANISM OF THE ACTION OF ANTIAMBOCEPTORS. 56& rate the sensitized blood-cells by centrifuge and replace the serum fluid with the corresponding amount of normal serum (in our cases 0.003 cc). The active substance contained in normal serum is thermostable at 56° C, but is destroyed by heating for half an hour to 100° C. The following experiment may serve as an illustration : The blood-cells which have been sensitized with 0.003 cc. serum and then separated by centrifuge are treated with a considerable- excess (0.5 cc.) of the antiserum. This amount corresponds to that, quantity which by itself is just able to overcome the antilytic action.. To this mixture are added decreasing amounts of normal rabbit serum which has been heated to 56° C. and to 100° C. After allowing the mixture to stand for half an hour the blood-cells are centrifuged off and suspended in salt solution to which 0.1 cc. guinea-pig serum (complement) is added. The result is shown in the following table: TABLE II. Amount of Normal Rabbit 1 CO. 5% Ox Blood (sensitized with 0.003 cc.)+0.5 cc. An tiserum+ Normal Rabbit Serum. Serum. Heated to 56° C. Amount of Esmolysis. b. Heated to lOO" C. Amount of Hsemolysis. 0.005 0.003 0.0015 0.001 0.0005 little moderate complete complete complete This shows us what a tremendous effect the presence or absence- of a small amount of normal serum can exercise. This of course at once explains the difference which manifests itself between the "curative" and the "protective" experiments. In the latter, it will. be recalled, the amboceptor and antiamboceptor are first mixed. All of the normal serum constituents, therefore, come into action; whereas in the "curative" experiment these are removed when the blood-cells are centrifuged. How are we to conceive the mechanism of this action? Phe- nomena in which an excess of a certain substance produces a- change in the character of the reaction are frequently due to the 570 COLLECTED STUDIES IN IMMUNITY. presence of other substances with different properties. In the case described above there is an absence of antilytic action with a certain excess of the antiserum. If we look at the subject from this stand- point, we shall have to assume that the antiserum contains two sub- stances,! one of which, of course, is the effective antiamboceptor. The other substance would then be the cause of the inhibition of the antiamboceptor action. Furthermore, since this inhibition is only brought about by large quantities of the serum, this substance would be present in the serum in much smaller amounts than the former. The simplest explanation of the action of this substance seems to be somewhat as follows: We must assume that this sub- stance's point of attachment is a complementophilic auxiliary group in the amboceptor. The occupation of this group so affects the amboceptor molecule that the simultaneous presence of antiambo- ceptor no longer prevents the combination with complement. Such a behavior would be analogous to an observation published by Ehr- lich and Marshall.^ At that time, by means of a differentiating method made available for one particular instance^ by Marshall and Morgenroth, it was shown that the amboceptor anchored to the cell, although it could deprive native guinea-pig serum of all its complement functions, was unable to absorb the non-dominant complements if the dominant complement had first been neutralized by the partial anticomplements of Marshall and Morgenroth. In other words, an anchoring of the non-dominant complements was only possible after the corresponding complementophile group of the amboceptor had combined with the dominant complement. In our case we would be dealing with an influence entirely similar in principle, except that here the influence is reversed, i.e., the affinity of the amboceptor to the antiamboceptor is reduced by the occupa- tion of the auxiliary group. We believe that we can show directly that the antiamboceptor is bound in either case, but that where the auxiliary group is occupied, the union of amboceptor and antiambo- ' We can of course assume a priori that an antiamboceptor serum directed against the complementophile groups will possess a multiplicity of partial antiamboceptors, for the amboceptors which take part in the immunization possess a large number of different complementophile groups, and against each of these a particular antibody is conceivable. ^ Ehrlich and Marshall, 1. c. ' H. T. Marshall and J. Morgenroth, Tiber Differenzierung von Comple- Tnenten durcb ein Partialanticomplement. Centralblatt f. Bact. 1902, Vol. 31, No. 12. MECHANISM. OF THE ACTION OF ANTIAMBOCEPTORS. 571 ceptor remains a loose one, while in the other case it becomes firm. The following diagram may help to make this clear. See Fig. 1. We shall designate the two complementophile groups of the ambo- ceptor as a and /?; the effective antiambo ceptor corresponding to group a is a, the antibody fitting group fi is b. In small quan- tities of antiserum, b can practically be disregarded owing to its slight concentration ; a therefore by occupying a prevents the comple- ment uniting with the amboceptor. In larger quantities of anti- serum, however, b comes into play, so that the occupation of group /J Loose tJnion Fig 1. — a and /?: Compleinentopliile groups of ihe amboceptor, a and b are Partial Substances of the Antiserum, a is the effective Antiamboceptor; h is the antibody which inhibits the action of the antiamboceptor. c is the Complement. changes the reactive capacity of group a in such a way that either a is not bound at all while the corresponding complement is, or so that, wh le a may still be bound, the union is such a loose one that the complement still has access. We shall see that the latter pos- sibility is the more probable. First, however, it will be necessary for us to understand clearly the manner in which , normal rabbit serum overcomes the influence of the antiserum constituent b. In view of what has been said this will not be difficult, for it is but a 572 COLLECTED STUDIES IN IMMUN-ITY. natural consequence for us to assume that normal rabbit serum con- tains the corresponding counter-group /? in such high concentration that even small amounts are able to neutralize b and so prevent its union with the amboceptor anchored by the cell. See Fig. 2. Coming now to the question whether, after group /? is occupied, group a no longer reacts with a, or whether, while the reaction takes place, the union remains a very loose one, we decided this according to the following considerations. If the latter assumption were cor- rect, it would follow that the loose union should subsequently become -^^ Fig. 2. — /?: Complementophile group of an amboceptor of normal serum. Otherwise as in Fig. 1. firm if in some way group b could again be freed from its combination with ^. In that case, evidently, the "curative" action of the anti- amboceptor a should become manifest. If, on the contrary, a has not been bound at all, this "curative" action should fail to appear on the removal of b. Owing to the presence of group /? in small amounts in normal rabbit serum the possibility is given of abstracting the antigroup b already bound to the sensitized cell. We have at once taken advan- tage of this fact, and attacked the question experimentally as follows: Sensitized blood-cells are digested with an excess of the antiserum MECHANISM OF THE ACTION OF ANTIAMBOCEPTORS. 573 (0.25 CO.). After centrifuging, decreasing amounts of inactivated normal rabbit serum are added to the sediments, and the mixtures again centrifuged. The blood-cells thus separated are suspended in 0.1 cc. salt solution containing 0.1 cc. guinea-pig serum. The result is shown in the following table: TABLE III. In active Normal Rabbit Serum. cc. Amount of Haemolysis. 0.01 0.006 0.003 0.0015 little to moderate complete This table, therefore, shows that sensitized blood-cells which have been treated with an excess of antiamboceptor and then freed from all free serum constituents by centrifuging can be deprived of a con- siderable portion i of the antiserum constituent b by subsequently digesting them with small amounts of normal rabbit serum, thus again allowing the antiamboceptor action to become manifest. It is permissible, therefore, to assume that the antiamboceptor a had been bound and that the union had remained a loose one owing to the occupation of group ^, Owing to the looseness of the -union a and a the complement was not prevented from combining with the amboceptor. We have gone into the analysis of this case with such detail because it again shows how complicated is the mechanism of amboceptors and yet how easy it is by means of the amboceptor theory to bring these apparently paradoxical phenomena into harmony. In this case we are certainly dealing with extraordinarily complex conditions, conditions in which Bordet's rudimentary sensitization theory is entirely helpless. The phenomenon just described possesses a certain practical significance in so far as it could easily lead to the erroneous assump- ' It is likely that the reason why the inhibiting action cannot be entirely brought out by this means is that the union of 6, once it is bound, rapidly be- comes firm, thus permitting only a partial dissolution by means of free /?. In any event this e.xperiment clearly exhibits, as already stated, exactly the re- verse behavior of that shown by Bordet's. 574 COLLECTED STUDIES IN IMMUNITY tion that the antiamboceptor acts only in "protective" experiments, but is unable to act on amboceptor already anchored by the blood- cells. In order to orientate ourselves concerning this last question, we would of course begin by using an excess of antiamboceptor, expecting very naturally, if the antiamboceptor exerts any influence whatever on the anchored amboceptor, that this influence will most likely become manifest with large amounts of antiamboceptor. Further- more, it can then happen that the conditions obtaining are those of the zone in which the curative action obtained with smaller doses is concealed, owing to the excess of antiamboceptor. This may perhaps account for Morgenroth's negative findings; i the antiambo- ceptor serum employed by us was also used by that author. The demonstration of the fact that the antiamboceptors pro- duced by immunization are usually directed against the complemento- phile groups calls for a correction of certain deductions based on our earlier conception of antiamboceptors as being directed against the cytophile group. We must therefore concede that Bordet is correct when he refuses to accept our method of differentiating partial amboceptors by means of antiamboceptors, a method which we pub- lished in the Sixth Communication on Hsemolysins.^ Our experi- ments at that time dealt with an amboceptor of an immune serum derived from a rabbit by treatment with ox blood. This amboceptor could be complemented either with guinea-pig serum or goat serum. In complementing with goat serum so much more amboceptor is necessary that the absence of the antiamboceptors' action must be ascribed to the antiantilytic action of the normal amboceptors present. But this correction does not signify that the conclusion as to the plurality of the amboceptors must be abandoned. On the contrary this con- clusion is confirmed by so many weighty arguments of a different kind that the existence of partial amboceptors must now be classed as one of the facts in immunity. We need only call attention to a point con- tained in our Sixth Communication, namely, that by mutual elective absorption we have shown that immunization of animals with ox blood results in the formation of two fractions of amboceptors, one of which acts only on ox blood, the other also on goat blood; and that immunization with goat blood has exactly analogous reverse ' J. Morgenroth, Deflection of Complement by Means of Haemolytic Ambo- ceptors. Centralblatt Bact. 1904, Vol. 35, No. 4. ^Ehrlich and Morgenroth. See page 88. MECHANISM OF THE ACTION OF ANTIAMBOCEPTORS. 575 results. The plurality of amboceptors is further demonstrated by the results of the isolysin experiments published by Ehrlich and Morgenroth,! for in these experiments the presence of antibodies acting against the complementophile group of the amboceptor can be excluded. The fact that we have drawn an incorrect conclusion from one single experiment certainly does not justify Bordet in deny- ing the existence of a plurality of antibodies (especially amboceptors) in a given immune serum ; the correctness of our view is established by a number of incontestable experiments. Bordet's arguments concerning deflection of complement by an excess of amboceptor may be answered in the same manner. Even granted that Morgenroth's view ^ is incorrect, namely, that the inhibi- tion of haemolysis on the addition of an amboceptor-antiamboceptor mixture is due to a deflection of complement, this would not in the least refute the results obtained by Neisser and Wechsberg with bactericidal sera. In these experiments absolutely no antiambo- ceptor is present ; there are merely bacteria, amboceptor, and comple- ment. Despite this, however, there is no bactericidal action when a certain excess of amboceptor is present. The only explanation for this is the one offered by Neisser and Wechsberg,^ namely, that the complement is deflected from the amboceptor combined with the cells by the free amboceptor. This explanation has also been accepted by Lipstein,* who controverted a number of objections which had been made by various authors. Bordet does not even attempt to controvert our explanation, but contents himself by saying: "Pour nous, la thSorie de la deviation du complement par I'ambocepteur est une legende." Needless to say this will have little effect on our view. It is thus seen that Bordet's recent experiments have furnished additional important confirmation of the amboceptor theory. Analysis of the antiamboceptor action clearly demonstrates the fact that the amboceptor possesses other affinities besides those of the cytophile group; and the circumstance that the occupation of these groups bars the action of the complement shows that they are complemento- phile in character. Bordet's attack on the receptor theory has thus ' Ehrlich and Morgenroth, Third Communication. See page 23. ' J. Morgenroth, 1. c. ' M. Neisser and Wechsberg. See page 120. * A. Lipstein, Centralblatt fur Bacteriologie, 1902, Vol. 31, No. 10; see also page 132 of this volume. 576 COLLECTED STUDIES IN IMMUNITY. failed utterly; his experiments, on the contrary, are to be welcomed as supplementing the arguments supporting the amboceptor theory .1 ' The mistake contained in our previous conception of antiamboceptors, that they were antibodies directed against the cytophile group, is essentially one regarding the situation of the point of attack. In this connection we may look upon certain chemical substitutions as furnishing ready comparison; for example, the different substances resulting when the benzole nucleus is substi- tuted in the ortho, meta, or para positions. Considering how difficult these problems are, it is not surprising that a statement concerning localization will now and then be made which subsequent deeper study shows must be corrected. Even so high an authority as Kekul6 once erred in defining a compound, and yet this did not in the least affect his fruitful hypothesis. In our case after the way had been cleared by the demonstration of the "blocking of complements" (the nature of which corresponds to an antiamboceptor action), and by the studies of Pfeiffer and Friedberger, it was an easy matter to arrive at a correct interpretation and transfer the site of the antiambocepter's action to the comple- mentophile group. It is at once clear that this merely fulfills an old postulate of the side-chain theory. It would therefore be interesting to see how Bordet could explain the facts according to his sensitization theory, and to have him show how the sensitizers, which he believes do not combine with the comple- ment, excite the production of substances whose constitution is just what would be demanded of immunization products of "complementophile groups." XLI. A GENERAL REVIEW OF THE RECENT WORK IN IMMUNITY.i By Paul Ehelich. Two years have elapsed since the appearance of my "Collected Studies in Immunity" in Germany, and now that the book is about to appear on the other side of the ocean it is a pleasure for me to review briefly the progress made in that time, naturally without pretending to give a complete resume of the literature. I may at once say, however, that very little really new has been added to the views formulated by myself and my collaborators, and that the stereochemical conception of the immunity reaction, despite numerous attacks, has proven itself able to dominate every phase of the subject. The arithmetical view of the toxin-antitoxin reactions and their analogues, which was introduced chiefly by Arrhenius and Madsen, has invariably shown itself to be untenable. It has led to a numer- ical science which is far removed from the principles of biological investigations and from the experimental results underlying these. On the other hand, so able an authority as Nemst at once recognized, that the laws of chemical equilibrium are not appUcable to mixtures of toxin and antitoxin. In addition to this von Dungern, Morgen- roth, and Sachs have collected considerable new experimental evi- dence which demonstrates absolutely that the toxin-antitoxin combination gradually becomes firm, although it may in some instances be quite loose in the first stage. The complex constitution of the poison solutions has thus been conclusively demonstrated; and I may also remind the reader that there can also no longer be any question as to the independent existence of toxons in diphtheria poison, for van Calcar has succeeded in a direct separation of these bpdies.2 ' This chapter is written expressly fof this American edition. * van Calcar effected this by means of an ingenious dialyzing procedure {Berlin, klin. Wochenschr. No. 39, 1904). Certain objections raised by ROmer 577 578 COLLECTED STUDIES IN IMMUNITY. In view of the extraordinary success which physical chemistry has scored, it is readily understood how tempting it was for so emi- nent a representative of this science as Arrhenius to apply its princi- ples to the new field of immunity. I have always emphasized the chemical nature of the reaction, and am glad therefore thai the attempt to apply these principles has been made. It has demon- strated anew that the phenomena of animate nature represent merely the resultants of infinitely complex and variable actions, and that they differ herein from the exact sciences, whose problems can be- treated mathematically. The formulas devised by Arrhenius and Madsen for the reaction of toxins and antitoxins explain absolutely nothing. Even in particularly favorable cases they can merely represent certain experimental results in the form of interpolation formulas. Neither do I beheve that the phenomena observed in toxins and antitoxins bear any relation to the processes of colloid chemistry. The attempt which has been made to interpret the immunity reaction from the standpoint of colloid chemistry, a sub- ject itself more or less obscure, is based on purely external analogies. I see absolutely no advantage in such a method, and I have grave fears that it will result in checking further progress along this line. Structural chemistry, on the other hand, has not only served to explain all the phenomena in immunity studies, but has also proved a valuable guide in indicating the lines along which further progress might be made. The limitations of colloid chemistry have already manifested themselves, and enthusiastic advocates of this science have been compelled to assume the existence of specific atomic groupings in accordance with my views. I therefore see no reason for abandoning the views expressed in my receptor theory, a theory in complete accord with the principles of synthetic chemistry. My decision finds additional support in the fact that the. studies in immunity are constantly bringing to light new observations best harmonized with the views of structural chemistry. Thus I may remind the reader that Morgenroth has recently very cleverly proved the postulate that the components of the neutral toxin-antitoxin combination can be restored. This author succeeded in completely recovering the two components of a neutral mixture of cobra venom (Berl. klin. Wochenschr. No. 8, 1905) have been effectually answered by van Calcar by means of some additional experiments, and by the demonstration that the membranes employed by Romer were unsuitable (Berl. klin. Woch. No. 43, 1905). A GENERAL REVIEW OF THE RECENT WORK IN IMMUNITY. 670 and antitoxin by means of an ingenious method. But even here we are not deahng with a reversible reaction, for it requires certain manipulations to disrupt the neutral combination; thus, in the case of cobra venom, the addition of hydrochloric acid is necessary. The neutral cobra-venom-antitoxin combination therefore behaves like a glucoside, which in itself is entirely stable, but is split up by the addi- tion of hydrochloric acid. Besides this, the interesting investigations recently published by Obermayer and Pick,i on the production of immune precipitins by means of chemically altered albuminous bodies, are of particular sig- nificance in connection with the chemical conception of the immunity reaction. These authors succeeded, by iodizing, nitrifying, and diazotizing animal albuminous bodies, in so changing them that, when introduced into the organism of the same or of different species, they excited the production of precipitins which lacked specificity. These precipitins, however, were strictly specific for their respective iodized albumins, xanthoproteids, or diazo-albumins, no matter from what animal species the albumins were derived. We see, therefore, that the introduction of a certain chemical group into the albumin molecule completely alters the latter's power to excite the production of antibodies. This certainly corresponds entirely to the view that the production of antibodies is dependent on the chemical constitution of the exciting agent, a view which finds expression in my receptor theory. The heuristic value of the receptor idea, the idea which underlies my side-chain theory, can best be appreciated by studying the devel- opment of our knowledge concerning the cytotoxins of blood serum. As a prototype of these substances the hsemolysins occupy a promi- nent place in this volume. The view that the hsemolytic immune bodies are amboceptors has been proven to be correct in every case, thus conclusively showing that Bordet's sensitization theory is un- tenable. To begin, the observations of M. Neisser and Wechsberg, that the action of bactericidal sera depends not only on the absolute but on the relative concentration of amboceptor and complement, presented conditions which could not be harmonized with Bordet's views. On the other hand, they were readily explained in accord- ance with the side-chain theory by assurning that the complement was deflected by an excess of amboceptor. But even if this expla- > Centralbl. f. Physiologie, Vol. XIX, No. 23. 580 COLLECTED STUDIES IN IMMUNITY. nation is not the correct one, as Gay has recently stated, it would in no way affect the soundness of the amboceptor theory. The exist- ence of amboceptors is confirmed by so many experimental consider- ations that it is no longer a postulate of the theory, but is practically the direct expression of observed phenomena. The term amboceptor, of course, is used merely to express the two-sided affinity, to the cell on the one hand and to the complement on the other. The affinity of the amboceptor to the cell was demonstrated by the com- bining experiments published by Morgenroth and myself; and the direct union of amboceptor and complement is confirmed by a host of decisive observations. Of these, it will suffice to mention the test-tube demonstration of complementoids which occupy the com- plementophile groups of the amboceptor. This demonstration has since been effected in other ways (Fuhrmann, Muir, Browning, and Gay), so that the existence of complementoids is no longer evidenced merely by the possibihty of producing anticomplements by means of inactivated serum, but is demonstrated primarily by the unmistak- able interference of the complementoids in hsemolytic test-tube experiments. It is not necessary that complementoids should always exert an inhibiting action on haemolysis ; for it is obvious that changes in affinity may occur in consequence of external influences, physical, chemical, or chronological in nature. I believe that changes in affinity, either positively or negatively, are of the highest importance in cor- rectly understanding the course of immunity reactions, although I do not deny the influence of certain catalytic factors on these proc- esses (von Behring, Morgenroth, Otto, and Sachs). However, no general rule can be laid down. Experiments are constantly bringing forth surprises, but by dihgent empiricism it is usually possible to bring the many different observations into harmony with a single point of view. The original assumption, that amboceptor and complement (at least in the case of hsemolysins) exist free side by side, and that the complement does not take part in the reaction until the amboceptor has been bound by the cell (owing to an increase in the affinity of the complementophile group), — this assumption has not proven ten- able in every case. In addition to the case described in a previous chapter by Sachs and myself, we now know of a number of combi- nations, discovered by Sachs, in which the amboceptor alone does not unite with the receptor of red blood-cells, or does so to only a ;slight degree. By combining with the complement, the amboceptor A GENERAL REVIEW OF THE RECENT WORK IN IMMUNITY. 581 has the affinity of its cytophile group increased, so that now it is able to unite with the cells. Thus far, such observations have been made only on normal amboceptors; and this fact explains why the numerous attempts of various authors to separate normal haemolysins, by means of absorption at low temperatures, have failed.^ The amboceptors obtained by immunization, on the other hand, regularly possess a, high affinity for the cell-receptor. This is easily understood if we consider their mode of origin, for we may perhaps see in this a selec- tion of the groups with the highest affinity. Certainly in this case the exception proves the rule; for the mere fact, that in some instances the amboceptor does not unite with the cell until it has first com- bined with the complement, at once shows that we cannot be dealing with a sensitization. On the contrary, this shows that the ambo^ ceptor is an interbody in the strict sense of the word. These condi- tions have been most clearly brought out by the experiments of Preston Kyes on cobra venom. The researches of Flexner and Noguchi, as we all know, showed that cobra venom by itself is no hsemolysin, but plays the r61e of amboceptor in haemolysis. The most important of the activators is the one discovered by Kyes, namely, lecithin. The relation between snake venom and lecithin is really the same as that between amboceptor and complement; but the former possess one great advantage for chemical analysis, — they are both stable substances, and thus contrast strongly with the highly susceptible substances found in blood serum. Hence what was impossible in the case of the latter could readily be effected with cobra venom. Kyes, it will be remembered, has demonstrated, ad ocular, the direct union of cobra amboceptor and lecithin comple- ment, and has furthermore succeeded in isolating the resulting com- bination, the cobra-lecithid, in pure form.^ Thus, for the first time, the conclusion was reached chemically ' In this connection I should also like to mention the interesting atypical behavior discovered by Donath and Landsteiner in the amboceptor reaction. These authors observed haemolytic autoamboceptors in the serum of a patient suffering from paroxysmal hsemoglubinaria. These autoamboceptors, how- ever, only united with the bloods at low temperature. ' Kyes has recently continued his studies at my laboratory, and has demon- strated the important fact that in this formation of cobra-lecithid there is a true chemical synthesis. The course of this synthesis is such that a fatty acid radical is split off from the lecithin molecule, whereupon the residual combina- tion, which corresponds to a monostearyllecithin, unites with the cobra ambo- 582 COLLECTED STUDIES IN IMMUNITY. which, as a result of biological experiences, I had always looked forward to. The correctness of the amboceptor theory formulated by Morgen- roth and myself is confirmed by another important link in the chain of evidence. As far back as 1900, in the Croonian lecture, I stated that, according to the amboceptor theory, three antilytic antibodies were possible. In addition to the substances which act as anticom- plements, we could conceive of antiamboceptors of two different kinds. One of these inhibits the action of the amboceptor by pre- venting the union of amboceptor and cell, the other by occupying the complementophile groups. So far as the confirmation of the ambo- ceptor theory is concerned, it is evident that the demonstration of antiamboceptors directed against the complementophile group is by far the most important; for, owing to the mode of origin, the devel- opment of cytophile groups of the amboceptor as reaction products of the specific counter-group (the cell-receptor) is self-evident. It was therefore particularly gratifying when I found that Bordet had recently furnished the demonstration that the antiamboceptor developed with an immune, or with a normal serum, is usually directed against the complementophile group. This discovery very prettily demonstrates that the mechanism of hsemolysin action proceeds according to the amboceptor theory. The error contained in our earlier conception, that anti-immune bodies were usually antibodies directed against the cytophile group, is practically only an error in the localization of the point of attack. This must now be corrected by regarding the complementophile group as the point attacked by the antiamboceptor. We know that it is possible to produce antiamboceptors by im- munizing with normal serum, and Pfeiffer and Friedberger have shown that the action of the antiamboceptor serum extends to all the amboceptors of the animal species whose serum was used for inmiunization. These facts are only apparently a contradiction of the specificity of amboceptors, for the specificity of the amboceptors applies only to the cytophile group. On the other hand, we must assume that all the amboceptors of the same animal species are at least partly similar in structure so far as the complementbphile ceptor. This of course destroys the foundations of Noguchi's calculations, which are based on the assumption that the reaction is reversible; it also disposes of certain statements naade by Bredig. A GENERAL REVIEW OF THE RECENT WORK IN IMMUNITY. 583 apparatus is concerned. In a way, therefore, the amboceptor bears the stamp of the animal species from which it is derived. In this connection I have already expressed my views in the article entitled " The Mechanism of the Amboceptor Action and its Teleological Sig- nifieance " (Koch Festschrift, 1903): "In general, the specific ambo- ceptors possess a uniform structure in their compleraentophile por- tions, whereas they differ to a high degree in their cytophile groups, whose physiological function is the absorption of foodstuffs." The studies of antiamboceptors have demonstrated that this con- ception is correct. We see, therefore, that the specificity of the com- plementophile group of the amboceptor, a specificity based on the animal species, at once leads to a difference in the amboceptors obtained from different species by means of the same immunizing material. In our Sixth Communication on Hsemolysins, Morgenroth and I published certain experiments showing that by means of an antiamboceptor we had been able to demonstrate the diversity of the amboceptors produced in different animal species by injections of ox-blood. This statement still holds good, and its direct conse- quence demands that in the practical application of bactericidal sera, we should mix immune sera derived from different animals. In view of Bordet's observation, however, we shall have to revise our interpretation in so far as the site of this differentiation is con- cerned; the difference is in the complementophile group instead of in the cytophile group. On the other hand, we must abandon the differentiation of partial amboceptors in one and the same serum by means of antiamboceptors, a differentiation which we proposed in the study on hsemolysins. It must not be thought, however, that the pluralistic conception of the amboceptor apparatus is thereby overthrown. This conception is supported by so many arguments of a different kind that the existence of partial amboceptors can be classed as one of the demonstrated facts in immunity. I may remind the reader that by means of mutual elective absorption it is possible to differentiate the strictly specific portion of an immune serum from the non-specific components which give rise to the group reac- tions. By this means the presence of different amboceptor fractions could be demonstrated in the same immune serum. The observa- tions made by Morgenroth and myself on isolysins also speak strongly in favor of a multiplicity of amboceptors. In these the possible presence of antibodies acting on the complementophile portion of the amboceptor is absolutely excluded. Finally, if we glance at the con- 584 COLLECTED STUDIES IN IMMUNITY. ditions existing among bacteria, we find the so-called group reactionsi showing that the receptor apparatus and the antisera possess a highly multiple constitution. This fact, as is well known, has here been of great practical value. We see, therefore, that the plurality of the amboceptors, so far as the cytophile group is concerned, is an assured, fact; the differentiation by means of antiamboceptors directed against the cytophile group can therefore very well be foregone. The production of antiamboceptors against the cytophile group seems; to encounter particular difficulties, for the complementophile group always .finds the corresponding counter group in the organism more- readily than does the cytophile group, and therefore is alone bound by the tissue receptors. It is possible that in order to successfully immunize with cytophile groups, it will be necessary to isolate these groups. The latter might be accomplished by neutralizing the com- plementophile group with the corresponding antibody, or by destroy- ing this group (=cytophilic amboceptoids). In any event these studies confirm the correctness of the ambo- ceptor theory, i.e., that there is a direct combination of amboceptor and complement. To repeat, therefore, the specificity of the ambo- ceptors apphes: (1) To the receptor employed in immunization, and this mani- fests itself in the configuration of the haptophore group ; and (2) To the animal species from which the amboceptor is derived. The latter kind of specificity shows itself in the structure of the com- plementophile apparatus, which, as we know, consists of a large number of individual complementophile groups. To this plurahty of the complementophile groups there corresponds a plurality of com- plements as can hardly longer be questioned. So far as the consti- tution of the complement is concerned, the fact that it is made up of a haptophore and a toxophore group is sufficiently proven by test- tube experiments. The indirect method first employed for the demonstration of the haptophore group, namely, by the production of anticomplements, can therefore be dispensed with. However, I am convinced that just as normal body-fluids so often contain anticomplements, it will also be found possible to produce these by immunization. But as Moreschi has well pointed out, the experiments by which it was sought to demonstrate the production of anticomplements are not absolutely conclusive. Recent studies by Gengou, Moreschi, and Gay have shown that in the immunization with serum, antibodies directed against the albuminous constituents A GENERAL REVIEW OF THE RECENT WORK IN IMMUNITY. 585 are formed which, by uniting with the corresponding albuniinous bodies, possess the property of exerting anticomplementary effects. In this case, therefore, the anticomplement action is brought about by the interaction of two components, one present in the serum of the immunized animal and the other in the serum of that animal species whose serum was used for immimization (Moreschi). It is clear, of course, that here the dissolved albuminous substances, not the complements, were the antigens. This being the case, the demon- stration of anticomplements produced by immunization becomes extremely difficult, and it must be left for future investigations to see whether it is at all possible to differentiate these substances from those antibodies against albuminous substances which exert an anti- complement action. So far as the mechanism of the described anti- complement action is concerned, I do not think that the observations of Moreschi and Gay, that absorption of complement is associated with precipitation, necessarily mean that precipitation and anticomplement have any causal relationship. In fact it seems reasonable to assume, in accordance with Gengou's first explanations, that the property of binding the complements is exercised by the albuminous bodies sen- sitized with the specific amboceptor. We would have to conceive this somewhat in this fashion, that just as when immunizing with cells, agglutinins and amboceptors are formed, so also when immuniz- ing with dissolved albuminous bodies two kinds of antibodies are formed, precipitins and amboceptors. If the latter, however, are really amboceptors in the sense of Ehrlich and Morgenroth, we must demand that they will have the same properties which we have always ascribed to the amboceptor type. As a matter of fact, the experiment shows that this is the case. These albumin amboceptors also, in order to react with the complements, must have the affinity of their com- plementophile apparatus raised, only in the present case this is effected by the combination of the amboceptor with the susceptible body, the albumin. We see, therefore, that this anticomplementary action cor- responds to the deflection of complement through an excess of im- mune body, first described by M. Neisser and Wechsberg. Only in this case the deflecting amboceptor is of a different kind, and needs first to react with the corresponding receptor. Through the researches of Wassermann and Schiitze and of Uhlen- huth, one class of antibodies against dissolved albumins, namely-, the precipitins, has been used, as is well known to differentiate albuminous bodies of various origin. These have thus come to be successfully 586 -COLLECTED STUDIES IN IMMUNITY. employed in the forensic demonstration of the origin of blood-stains. The same thing, of course, was possible in the case of the albumin, amboceptors. This fact has recently been taken advantage of by M. Neisser and Sachs,^ who have devised a procedure by which, by deflecting haemo- lytic complements by means of albuminous bodies loaded with am- boceptor, they diagnosticate human blood, etc. The study of im- munity thus furnishes two biological methods for deciding a point 6i vital importance in forensic medicine, namely, the origin of blood- stains. Considering the extreme importance of tests of this kind, I am convinced that hereafter it will be well to use this method in addition to the well-tried Uhlenhuth-Wassermann reaction. This brief r6sum4, I beheve, covers the chief points which have recently come up for discussion, and it is indeed gratifying to me that all the vital questions have been decided in favor of my views. I have gladly applied the results obtained in experimental investiga- tions to an extension of my views, for it is obvious, considering the rudimentary character of a new science, that any successful prosecu- tion of the work will also extend the theoretical conceptions. If then, in spite of this, all the facts brought to light fit naturally into the views formulated by me, I regard this as additional evidence that these views are not so riSuch a theory as a necessary abstraction of the observed facts, an abstraction which is necessary not only in order to obtain a clear and harmonious conception of all the various observa- tions, but also to furnish a scientific basis for a further successful development of the subject. ' Berlin, kiln. Wochenschr. No. 44, 1905, and No. 3, 1906. SHORT-TITLE CATALOGUE OF TEE PUBLICATIONS OF JOHN WILEY & SONS, New York. London: CHAPMAN & HALL, Limited. ARRANGED UNDER SUBJECTS. Descriptive circulars sent on application. 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A Manual for Steel-users x2mo, Patton's Practical Treatise on Foundations Svo, Richardson's Modern Asphalt Pavements. Svo, Richey's Handbook for Superintendents of Construction i6mo, mor., Rockwell's Roads and Pavements in France i2mo, Sabin's Industrial and Artistic Technology of Paints and Varnish Svo, Smith's Materials of Machines i2mo. Snow's Principal Species of Wood '. Svo, Spalding's Hydraulic Cement i2mo. Text-book on Roads and Pavements z2mo, Taylor and Thompson's Treatise on Concrete, Plain and Reinforced Svo, Thurston's Materials of Engineering. 3 Parts Svo, Part I. Non-metallic Materials of Engineering and Metallurgy Svo, 2 00 Part II. Iron and Steel Svo, 3 50 Part III. A Treatise on Brasses, Bronzes, and Other Alloys and their Constituents Svo, 2 50 Thurston's Text-book of the Materials of Construction Svo, 5 00 Tillson's Street Pavements and Paving. Materials Svo, 4 00 Waddell's De Pontibus. (A P«cket-book for Bridge Engineers.). . z6mo, mer., 2 00 Specifications for Steel Bridges z2m^o, i 25 Wood's (De V.) Treatise on the Resistance of Materials, and an Appendix on the Preservation of Timber Svo, 2 00 Wood's (De V.) Elements of Analytical Mechanics Svo, 3 00 Wood's (M. P.) Rustless Coatings: Corrosion and Electrolysis of Iron and Steel Svo, 4 00 RAILWAY ENGINEERING. 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